LABORATORY STUDIES
FOR
BREWING STUDENTS
FERMENTATION ORGANISMS :
A LABORATORY HANDBOOK.
BY ALB. KLOCKER.
Translated by G. E. ALLAN, B.Sc., and , H. MILLAR, F.I.C.
With 146 Illustrations in the Text. Svo, i2s. net.
THE CHEMICAL CHANGES AND
PRODUCTS RESULTING FROM
FERMENTATIONS.
BY R. H. ADERS PLIMMER.
Svo, 65. net.
LONGMANS, GREEN. AND CO.,
39 PATERNOSTER Row, LONDON, E.C.
I t
LABORATORY STUDIES
FOR
BREWING STUDENTS
A SYSTEMATIC COURSE OF PRACTICAL WORK IN
THE SCIENTIFIC PRINCIPLES UNDERLYING
THE PROCESSES OF MALTING AND
BREWING
ADRIAN J. BROWN, M.Sc.
DIRECTOR OF THE SCHOOL OF BREWING, AND PROFESSOR OF THE BIOLOGY AND CHEMISTRY
OF FERMENTATION IN THE UNIVERSITY OF BIRMINGHAM
EXAMINER IN BIOLOGICAL CHEMISTRY TO THE INSTITUTE OF CHEMISTRY, ETC.
WITH 36 ILLUSTRATIONS
LONGMANS, GREEN, AND CO.
39 PATERNOSTER ROW, LONDON
NEW YORK AND BOMBAY
1904
PREFACE.
SOME years ago, when it fell to the author's lot to
arrange a course of instruction in the principles
of brewing for his students at the University of
Birmingham, an examination of the literature of
the subject showed that there was no book in exist-
ence which could be used as a systematic guide
to practical work in the laboratory, and as the author
recognised that a sound knowledge of the principles
of brewing must be based on work of this nature,
it became necessary to draw up a course of
laboratory studies for the special use of his students.
This course, subject to alterations and additions
suggested by experience and by the progress of
knowledge, has now been in use for several years,
and as it has been found to fulfil its requirements
in a satisfactory manner, the author now ventures
to publish it in the hope that it may contribute
in some measure towards filling a gap in the
literature of brewing.
The work is essentially a student's laboratory
vi PEEFACE
guide, and must not be regarded in any way as a
text-book of the scientific principles of brewing
as it confines itself mainly to descriptions of ex-
perimental work. It is intended for use under
the supervision of a competent instructor, and it
is assumed that the student is able to attend
lectures on the subjects upon which he is working.
The chief difficulty of a study of the scientific
principles underlying brewing practice lies in the
fact that as it so often touches the limits of our
present knowledge there are many questions
which have to be studied about which there exists
considerable uncertainty and difference of opinion.
This naturally raises difficulties for the teacher,
for, on the one hand, it is well recognised that
some amount of dogmatism in teaching is necessary
when introducing a new subject to the student,
and, on the other, the state of our knowledge of
certain of the questions dealt with in these studies
does not justify dogmatic treatment. The position
may be illustrated by a consideration of the very
important and difficult problems of the constitution
of the starch molecule and its transformation by
diastase. A large amount of knowledge on these
points has been accumulated, and many varied
views have been advanced by different investigators
concerning them, but none of these views have
met with general acceptance, even of a provisional
PREFACE vii
nature. This is due no doubt partly to the ex-
ceptional difficulties which surround the study of
starch and its transformation by diastase, and
partly to a militant spirit which appears to emanate
from the starch molecule and influence the minds
of most of its investigators.
How is this state of affairs to be met by the
teacher ? An attempt to lay before the student
at the commencement of his studies all the
different views concerning starch and its trans-
formation products must result in reducing his
mind to a state of chaos. It appears to the author
that the only satisfactory course open is to teach
those views which lend themselves best to ex-
planation and demonstration, and, when the student
is sufficiently advanced, to encourage him to
criticise such views and compare them with others.
This is the method of teaching attempted by
the author, and it is followed in this book with
regard both to experimental studies, and to a con-
sideration of the analytical processes employed
in the brewery laboratory, many of which cannot
be regarded as above criticism.
The course of studies might perhaps have been
advantageously lengthened, but it had to be borne
in mind that the majority of students are unable
to devote more than one year to such studies. A
good worker who has previously had a sound
viii PREFACE
chemical training is able to work through the
whole course in this time ; but, for the benefit of
those who have had a less complete preliminary
training or who are less expert workers, some of
the experiments are printed in small type to
indicate that they may be omitted.
The references given in this book to original
papers are not intended to be exhaustive, but have
been selected in order to encourage the student
to refer to original sources for information. In
making the selection regard has been paid to those
sources which are likely to be readily available.
The author desires to express his indebtedness
to Mr. J. H. Millar for very valuable assistance in
planning the studies of the carbo-hydrates, and also
to Mr. Thomas H. Pope for kindly reading over
the proof-sheets of this work, and for giving many
useful suggestions.
SCHOOL OF SHEWING,
UNIVERSITY OF BIRMINGHAM,
June, 1904.
CONTENTS.
PAGE
PREFACE v
SECTION I. BAKLEY AND MALTING.
PART I. A STUDY OF THE BARLEY CORN 1
The General Characteristics of a Grain of Barley .... 1
Ear of Ripe Barley and Spike of Barley when in Flower compared 3
The Flower of Barley 3
Ovary 4
Anthers 5
Lodicules 5
The Flower after Fertilisation 6
Barley and Wheat compared 6
Ears of Two-rowed and Six-rowed Barley compared ... 7
Characteristics of Sub-species of Barley 9
Ears of Chevalier and Goldthorpe Barley compared ... 10
Basal Bristles of Chevalier and Goldthorpe Barley ... 11
Corns of Chevalier and Goldthorpe Barley compared . .11
Transverse Furrow of Goldthorpe Barley 11
Hordeum vulgare and H. liexastichum compared .... 12
PART II. ANATOMY OF THE BARLEY CORN 13
Endosperm and Embryo of the Barley Corn .... 13
Cutting and Preparing Sections for the Microscope ... 14
Anatomy of the Embryo 16
Anatomy of the Endosperm 17
The True Skins of the Barley Corn 19
Examination of Green Six-rowed Barley 19
Position of Starch Granules in the Cells of the Endosperm . 20
Demonstration that the Starch-containing Cells contain Proto-
plasmic Matter .20
ix
x CONTENTS
PAOB
PABT III. EXPERIMENTS CONNECTED WITH THB TECHNICAL STODY
OF BARLEY AND OTHER CEREALS 22
Microscopic Appearance of the Commoner Kinds of Starch . 22
Preparation of Starch from Barley 22
Determination of Moisture in Barley 23
Determination of the " Vitality " or Genninative Power of Barley 24
The " Weight " of Barley 24
Specific Gravity of Barley 25
Technical Examination and Valuation of Barley for Malting
Purposes 26
PART IV. THE CHANGES IN BARLEY DURING GERMINATION . . 27
Determination of Moisture in Steeped Barley .... 27
Study of the Changes in Barley during Germination ... 27
Growth of the Embryo when Separated from its Endosperm . 30
The Embryo of the Barley Corn secretes Diastase during its
Development 31
Generation of Carbon Dioxide during the Growth of the Barley
Corn 33
Oxygen is Absorbed during Growth 34
Preparation of Malt Diastase 34
Action of the Enzyme Cytase 3&
Loss in Weight during the Conversion of Barley into Malt . . 36
Technical Examination of Malt 37
1. General Appearance 37
2. "Modification" 37
3. "Condition" 37
4. Flavour 37
5. Regularity of Growth 37
6. Damaged Corns 38
7. Mouldy Corns 38
The "Sinker" Test 38
Specific Gravity of Malt 3&
PART V. THE CHEMICAL ANALYSIS OP MALT 39
Moisture in Malt 39
Ash 39
Determination of the Extract of Malt 39
Determination of the Specific Gravity of Liquids ... 40
Preparation of the Malt Mash (Heron's Method) ... 42
Extract of the Boiled Wort 44
Calculation of Dry Extract 44
Calculation of Weight of Dry " Grains " .... 45
Influence of Grinding on Extract 46
Criticism of the Method Employed 46
CONTENTS xi
PAQB
Determination of Extract by Weighing the Mash ... 47
The " Full Theoretical " Extract 49
Extract of " Flaked " and Prepared Grain 50
Extract of Black and Brown Malt 51
Extract of Raw Grain 51
Acidity of Malt 52
Determination of the Diastatic Power of Malt (Lintner's Method) 53
Diastatic Power of Barley 55
" Non-coagulable Albuminoids " of Malt ..... 55
" Ready-formed Carbo-hydrates " of Malt 59
Soluble Ash and Colour of Malt 61
" Saccharification " Test 62
SECTION II. PRINCIPLES OF THE MASHING PROCESS.
PABT I. A COURSE OF EXPERIMENTS CONSTITUTING A STUDY OF
SOME OF THE CARBO-HYDRATES CONCERNED IN WORT PRO-
DUCTION, AND INTRODUCING THE STUDENT TO THE SPECIAL
METHODS EMPLOYED IN THEIR EXAMINATION .... 64
Action of Water on Starch 64
Preparation of Soluble Starch 65
Hydrolysis of Starch by Acid 65
Preparation of Dextrose 67
Quantitative Study of the Hydrolysis of Starch by Acid . . 68
Determination and Use of the " Solution Weights " of Carbo-
hydrates 69
Solution Density of Dextrose 71
Introduction to the Use of the Polarimeter in Carbo-hydrate
Work 72
The Specific Rotation of Dextrose determined by means of the
Sodium-light Polarimeter 75
The Specific Rotation of Dextrose determined by means of the
Half-shadow Polarimeter 75
Use of a Factor other than the True Solution Factor in Deter-
minations of the Specific Rotations of Carbo-hydrates . 76
Determination with the Polarimeter of the Amount of Sugar
present in a Solution when the [a] D of the Dissolved
Sugar is known 76
" Mutarotation " of Dextrose and other Carbo-hydrates . . 77
The Cupric Oxide Reducing Method of Estimating Sugars . . 78
Preparation of Fehling's Solution 78
Determination of the Reducing Power of Dextrose . . 79
Preparation and Properties of Phenyl-glucosazone ... 81
xii CONTENTS
PAGE
Cane-Sugar or Saccharose 82
Cane-Sugar does not possess Cupric Oxide Reducing Power 82
Cane-Sugar does not Combine with Phenyl-hydrazine to
form an Osazone 82
Inversion of Cane-Sugar 82
Inversion with Acid 83
Inversion with Yeast 83
Determination of the Cane-Sugar present in Malt ... 85
Levulose 86
Specific Rotation 86
Reducing Power . . .86
Preparation of Osazone 86
PAST II. STCDY OP THE HYDBOLYSIB OP STABCH BY DIASTASE, AND
OP THE PRODUCTS OP HYDBOLYSIS 87
Preparation of Cold-water Malt Extract 87
Action of Diastase on Starch 87
Action of Diastase is Destroyed at 100 88
Preparation of Maltose 88
Specific Rotation of Maltose 90
Reducing Power of Maltose 91
Preparation and Properties of Phenyl-maltosazone ... 91
Hydrolysis of Maltose by Acid 92
Maltose is not Hydrolysed by the Action of Invertase ... 92
Dextrin 93
Preparation 9G
Specific Rotation 93
Reducing Power 98
Preparation and Quantitative Examination of a Low Starch Con-
version 94
Influence of Heat on the Hydrolysis of Starch by Diastase . . 98
Influence of Heat in Restricting Starch Transformation is Due to
Modification of the Diastase 99
Experiment Showing that Although a Restricted Starch Trans-
formation may be Represented as a Mixture of Maltose
and Dextrin, much of the apparent Maltose cannot be
Fermented by Ordinary Yeast and Exists as Malto-
dextrin 99
Preparation of the Starch Conversion 100
Determination of the Free Maltose Present .... 101
Calculation of the " Apparent " Maltose present which exists
as Malto-dextrin 102
Determination of the "Apparent" Dextrin existing as
Malto-dextrin . . . . . . . . 102
Constitution of the Malto-dextrin found .... 104
Determination of the Stable Dextrin 104
CONTENTS xiii
PAQK
PART III. STUDIES BEARING DIRECTLY ON THE TECHNOLOGY OP
BREWING 106
Study of the Influence of the Mashing Temperature on the
Optical Activity of Malt Worts 106
Experiments on the Fermentation of Boiled and Unboiled Worts
Illustrating the Different Methods of the Brewer and the
Distiller or Vinegar Maker 107
Study of the Rise hi Temperature observed when mixing Dry
Malt with Cold Water 108
Analysis of a Fermented Wort or Beer 110
1. Determination of the " Original Gravity " and Alcohol . 110
2. Analysis of the Unfermented Matter .... 114
Free Maltose 114
" Apparent " Maltose Combined as Malto-dextrin . . 115
" Apparent " Dextrin Combined as Malto-dextrin . . 115
Stable Dextrin 116
Residual Reducing and Rotatory Powers . . . 116
PART IV. ANALYSIS OP BREWING SUGARS 117
Analysis of Invert-Sugars 117
Ash 117
Albuminoids 118
Brewer's Extract and Water 118
Reducing Sugars 119
Cane-Sugar 120
Unfermentable Matter 121
Example 122
Analysis of Glucose or Starch Sugar 125
SECTION III. FERMENTATION.
INTRODUCTION 126
PART I. THE PHYSIOLOGICAL ASPECT OP FERMENTATION . . . 127
Determination of the Amount of Alcohol and Carbon Dioxide
produced during the Fermentation of Sugar by Yeast . 127
Glycerin a Secondary Product of Alcoholic Fermentation . . 180
Chemical Composition of Yeast 131
Water 131
Ash 132
Nitrogen 132
Use of the Haemacytometer 133
Nature of the Food Requirements of the Yeast Cell . . . 137
xiv CONTENTS
PAGE
Multiplication of Yeast Cells in a Nutritive Solution not Directly
Dependent on the Number of Cells Originally Introduced 138
Multiplication of Yeast Cells in a Nutritive Solution not Directly
Dependent on the Amount of Yeast Food Present . . 189
Removal of Nitrogen from Malt Wort during Fermentation . 140
Influence of Temperature on the Development and Fermenta-
tive Power of Yeast 140
Actions of some of the Enzymes Present in the Yeast Cell . . 142
1. Zymase 142
2. Invertase 142
3. Maltase 142
Auto-digestion of Yeast 143
PAST II. A STUDY OF THE MORPHOLOGY AND LIFE HISTORY OF
SOME OF THE MORE IMPORTANT MICRO-ORGANISMS OF
FERMENTATION WHICH INTRODUCES THE STUDENT TO THE
SPECIAL METHODS EMPLOYED IN THE STUDY OF THE
FERMENTATION ORGANISMS 144
Preparation of Culture Media 145
Hopped Malt Wort 145
Yeast Water 145
Meat Extract 146
Wort Gelatin . 146
Preparation of Flasks and Test-Tubes Containing Malt Wort
and Gelatin Malt Wort 147
Sterile Water 148
A. The Saccharomycetes and Lower Fungi
Study of the Morphology of a Yeast Cell .... 148
Growth of Yeast in a " Drop Culture " 151
Study of Some of the Well-recognised Races of Yeasts and
Torula 153
Study of Some of the more Common Species of Moulds . 154
Preparation of Spore Cultures of the Saccharomycetes . 155
Film Formation of the Saccharomycetes .... 158
Preparation of Pure Cultures of Yeasts and Moulds . . 159
Gelatin Plate Method 159
Hanson's Single Cell Method 160
Analysis of Mixed Cultures of Yeast 162
Gelatin Plate Method 163
Fractional Culture Method 164
Method of Testing Pure Cultures of leasts obtained by
Previous Methods of Analysis for Technical Purposes 165
Experiments on the Powers of Hydrolysis and Fermentation
of Different Species of Yeasts 166
CONTENTS xv
PAQK
B. The Schizomycetes or Bacteria
Morphology of a Bacterium (Bacillus subtilis) . . . 166
Making Stained Preparations 168
Spore Formation 169
Preparation of a Drop Culture of Spores . . . 169
Study of Some of the Well-recognised Species of Acid-pro-
ducing Bacteria 169
Examination of Beer Sediments 170
The " Forcing " Test 171
Micro-organisms Present on Barley, Malt and Hops . . 171
Biological Examination of Air for Technical Purposes . . 172
Eoll Culture Method 172
Petri Dish Method 173
Biological Examination of Water for Technical Purposes . 173
Biological Examination of Water for Hygienic Purposes . 174
SECTION IV. THE HOP.
The Hop Plant 176
The Female Flower of the Hop 176
The Hop-Cone 178
Bracts of the Hop-Cone 179
The Stem or " Strig " of the Hop 179
Lupulin Glands of the Hop 181
Comparison of the Strig of Different Varieties of the Hop . . . 182
Chemical Examination of Hops 183
The Soft and Hard Resins 183
Moisture 185
Detection of Sulphur 185
Tannic Acid 186
TABLE I. Solution Factors for Carbo-hydrates at Various Densities . 187
TABLE II. Divisors for the Transformation Products of the Hydro-
lysis of Starch 188
TABLE III. Reducing Values of Varying Quantities of Dextrose,
Levulose and Invert-Sugar 189
TABLE IV. Reducing Values of Varying Quantities of Maltose . . 191
TABLE V. Spirit Indication Table 192
TABLE VI. Table for Ascertaining the Correction for Acid in Original
Gravity Determinations 193
b
ILLUSTRATIONS.
FIG.
1. Barley Flower page 4
2. Lodicules of Barley Flower facing page 4
3. Ovary of Barley Flower page 4
4. Diagram' of a Transverse Section through the Ovary of a
Barley Flower ,5
5. Photomicrograph of Anthers and Lodicules from
an Undeveloped Barley Flower . . . facing page 6
6. Photomicrograph of Transverse Section of a Fer-
tilised and Developing Ovary .... 6
7. (a) Six-rowed Barley ; (6) Two-rowed Barley . . . page 7
8. (a) Hordeum distichum ; (b) Hordeum zeocriton . . . 10
9. (a) Bachis of Goldthorpe Barley; (b) Bachis of Chevalier
Barley ( 10
10. (a) Basal Bristle of Goldthorpe Barley ; (b) Basal
Bristle of Chevalier Barley .... facing page 11
11. Hordeum vulgare page 12
12. Hordeum hexastichum ,,12
13. Diagram of a Longitudinal Section through the Germ End
of a Barley Corn 15
14. Diagram of a Transverse Section through a Barley Corn . 18
15. Photomicrograph of Transverse Section through
a Barley Corn, showing the Funicle . . facing page 19
16. Photomicrograph of Section of Skins and Endo-
sperm of Barley Corn (highly magnified) . 19
17. Experiment Demonstrating the Secretion of Diastase by the
Growing Embryo of a Barley Corn page 32
18. Apparatus for Demonstrating that Carbon Dioxide is Gener-
ated during the Bespiration of Germinating Barley . 33
19. Apparatus for the Determination of Carbon Dioxide and
Alcohol Generated during Alcoholic Fermentation . . 128
20. Figure of Thoma's Hsemacytometer 133
21. Freudenreich Flask 147
22. Holder for Sterile Water " 147
xvii
xviii ILLUSTRATIONS
Fio.
23. Moist Chamber page 151
24. Multiplication of Top Yeast ,,153
25. Gypsum Block ,,156
26. Sporulation of Saccharomyces Cerevisice 157
27. Saccharomycetes which form Ascospores . . . ,, 157
28. Squared Cover-glass ,,161
29. Object Marker ,,161
30. Clostridium butyricum 167
31. Female Inflorescence of the Hop ,, 177
32. Diagram of a Female Flower of the Hop 177
33. Bracts of Hop-cone (a) Stipular Bract ; (b) Bracteole . . 178
34. Axis or " Strig " of Hop-cone 179
35. Group of Floral Axes on Hop Strig (much enlarged) . . ,, 179
36. Apparatus for Extraction of Hop Resins , 183
LABORATORY STUDIES
FOB
BREWING STUDENTS.
SECTION I.
BAKLEY AND MALTING.
PAET I.
A STUDY OF THE BAELEY COEN.
Examine a Grain of ordinary English Chevalier
Barley. Note that the grain is spindle-shaped or
fusiform, and is about one-third of an inch in
length. On closer inspection it will be observed
that one end of the grain, which is somewhat
sharper than the other, shows signs of having been
fractured. This is the end which was originally
attached to the ear of barley previous to threshing,
and is consequently the lower end of the grain.
Observe that a deep, narrow furrow runs down
the more convex side of the grain. This furrow is
termed the ventral furrow, and the side of the grain
on which it is situated is termed the ventral side.
1
2 STUDIES FOE SKEWING STUDENTS
The other side which is flatter than the ventral
side is termed the dorsal side.
Examine the ventral furrow towards the lower
end of the grain, and notice that within it there
lies a thin bristle. Remove the bristle with the
point of a dissecting needle, and examine it under
the microscope with a low-power (1 inch) lens.
It will be found that it is covered with fine lateral
hairs. When removing the bristle, usually termed
the basal bristle, from the ventral furrow, notice
that it is attached to the base of the grain close
to the point where the grain was originally con-
nected with the ear. The basal bristle, or rachilla,
is merely the prolongation of the axis or point
from which the corn was originally developed. It
has no physiological importance, but its appearance
is sometimes useful in assisting to discriminate
between different kinds of barley.
Examine a Grain of Barley when softened by
soaking in Cold Water for about forty^eight hours,
Observe that it is covered by a thick skin. A
careful examination will show that this skin is not
continuous round the grain, but consists of two
separate portions, one closely adhering to the dor-
sal and the other to the ventral side of the corn,
and that the skin on the dorsal side overlaps the
edges of the skin covering the ventral side. With
care the two skins may be removed entire, and it
will then be noticed how much their general struc-
ture resembles that of a grass leaf the dorsal skin
A STUDY OF THE BAKLEY COEN 3
especially showing conspicuous longitudinal veins,
or vascular bundles, similar to those in a grass
leaf.
Compare the Barley under Examination with an
Ear of Unthreshed Barley, It will be noticed that
the furrowed or ventral side of a grain of barley is
the side which is turned inward towards the stem,
or rachis. It will also be noticed that the awn, or
beard, of unthreshed barley is merely a prolongation
of the skin which covers the dorsal side of the
barley corn, and that it is broken off during the
threshing process.
Compare an Ear of Ripe Barley with a Spike of
Barley when in Flower. A general similarity be-
tween the two is apparent. The flowers are ar-
ranged alternately on the stem or rachis, and the
outer covering of the flower with its long awn at
once suggests that this covering is the same as the
thick skin of the barley corn.
Detach a Flower from the Spike. Notice that
the outer covering of the flower is composed of the
same two leaf -like structures which were found en-
closing the barley corn. The one which partially
wraps round the other and is terminated by the
awn, is called the palea inferior, and the other the
palea superior.
Open the Flower and Remove the Palea Inferior.
-The organs of the flower will then be observed
lying within the fold of the palea superior. These
consist of :
1*
4 STUDIES FOB BBEWING STUDENTS
(a) An ovary. (The female organ.)
(b) Three pollen-bearing anthers, each supported
on a hair-like filament. (The male organs.)
(c) Two minute transparent leaflets called lodi-
cules which are situated at the base of the ovary and
embrace it. (The lodicules are sometimes regarded
as abortive petals of the flower.)
FIG. 3. Ovary of Barley
Flower.
FIG. 1. Barley Flower
with Palea Inferior
removed exposing the
Ovary and Anthers.
Remove the Ovary with a Dissecting Needle and
Examine it under a low Microscopic Power. It is
roughly heart-shaped and is surmounted by two
hairy stigmata adapted for the purpose of retaining
pollen grains. On the inner side of the ovary (the
side nearest to the rachis), a furrow will be seen
which is retained during development into a barley
corn and thus represents the ventral furrow.
FIG. 2. Lodicules of Barley Flower.
A STUDY OF THE BAELEY COEN 5
A practical study of the structure of the ovary is too difficult
for the ordinary student to attempt, as it is not easy to cut
sections of it which are suitable for microscopical examination.
The ovary is mainly composed of soft, thin- walled cells, or par-
enchyma, which enclose a single ovule surrounded by double
walls or integuments (see Fig. 4). The interior of the ovule is
occupied by a very large cell called the embryo-sac. Note that
the ovule lies free within the ovary except at one point called
the funicle, where it is attached to the inner walls of the ovary.
The thick walls of the ovary are almost colourless with the
FIG. 4. Diagram of a Transverse Section through the Ovary of a Barley
Flower, (a) Embryo-sac ; (b) Integuments of Ovule ; (c) Layer of Cells
Containing Chlorophyll ; (d) Point of Attachment ot Ovule.
exception of one layer of cells coloured bright green by chloro-
phyll ; this layer may be easily seen in a transverse section of
a fresh ovary.
Remove an Anther from the Flower. Mount it
on a slide with water and cover with a thin glass.
Observe under the microscope the pollen grains con-
tained within the anther.
Dissect out the Lodicules at the Base of the
Ovary* Note their exact position within the palese
with a view to tracing them subsequently in the
ripe barley corn.
Mount the Lodicules in Water. Examine them
under the microscope, noting their structure and
6 STUDIES FOR BREWING STUDENTS
the thin transparent hairs with which they are
covered.
Again Examine the Spike of Barley in Flower.
Observe that on either side of a fully developed
flower, and springing from the same internode, is
an undeveloped flower sheath about one-third of
an inch in length. Close examination will show
that this sheath consists of two incompletely de-
veloped palese. They enclose merely an abortive
trace of an ovary, but occasionally fully developed
anthers and lodicules are found within the flower
sheath.
Examine a Spike of Barley collected a few days
after Fertilisation. Note how rapidly the ovaries
are increasing in size and tending to fill the space
enclosed by the flower sheaths.
Fertilisation of the barley flower is effected when pollen
grains shed by the anthers come into contact with the stigmata
of the ovary. A minute tube carrying a nucleus is extended
from a pollen grain into the conducting tissue of the ovary, and
after finding its way through the micropyle of the ovule to the
embryo-sac, pierces it, leading to fusion of the nuclei of the
embryo-sac and pollen grain. After fertilisation has been effected
in this manner, the cells within the embryo-sac commence to
divide and subdivide, eventually producing the embryo and en-
dosperm of the barley corn to be referred to later on. Fig. 6 is
a micro-photograph of a fertilised and developing ovary ; note
the enlargement of the embryo-sac and the compression of the
surrounding walls of the ovule and ovary. Eventually these
walls become the thin skins of the barley corn.
Compare a Spike of Ripe Two^rowed Barley
FIG. 5. Photomicrograph of Anthers and Lodicules from an Undeveloped
Barley Flower.
FIG. 6. Photomicrograph of Transverse Section of a Fertilised and
Developing Ovary.
A STUDY OF THE BAKLEY COEN
with a Spike of Ripe Wheat. Observe in the case
of barley that the ovary has increased in size until
it has completely filled the space enclosed by the
palese, and that the latter adhere firmly to the grain,
forming the outer covering or false skin of the
grain. In the case of wheat, note that the palese
enclosing the grain do not adhere, and that the
seed is therefore naked after threshing, the palese
being separated as chaff.
a to b
FIG. 7. (a) Six-rowed Barley ; (b) Two-rowed Barley.
Compare a Spike of Ripe Six^rowed Barley (Hoiv
deum vulgare) with a Spike of Two^rowed Chevalier
Barley (H. distichum). In the two-rowed barley
it will be noticed that on either side of each corn
there is an undeveloped flower, but in the six-rowed
barley all the flowers have developed into corns,
thus making three rows of corns on each side of
the spike. The difference between six-rowed and
two-rowed barley consists therefore in all three
flowers of the six-rowed barley being fully developed
and fertile, whereas in two-rowed barley only the
8 STUDIES FOE BKEWING STUDENTS
central flower of the three is fully developed and
fertile.
Observe as a consequence of the mode of de-
velopment of six-rowed barley that the side corns
on each spikelet have a twisted form. This un-
symmetrical shape is readily observed if the lateral
grains are detached from the spikelet and examined
on the ventral side. If two-rowed barley and the
central corns of six-rowed barley are examined in
a similar manner it will be found that the corns
are developed symmetrically. After making these
observations it is easy for a student to determine
whether a sample of threshed barley belongs to the
two-rowed or six-rowed class.
The student has now studied the more conspicu-
ous characteristics of the barley corn, the develop-
ment of the barley corn from the barley flower, and
the leading features which distinguish the two-rowed
from the six-rowed barleys. It is now desirable
that he should study the distinguishing character-
istics of the four leading types or sub-species of
barley which are met with in ordinary technical
experience. He should commence by making him-
self familiar with the different varieties of culti-
vated barley mentioned in the following table. Six
sub-species are described in the table, but the two
sub-species, Hordeum intermedium and H. dedpiens,
are not met with in commerce and do not demand
special study :
A STUDY OF THE BAELEY CORN
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10 STUDIES FOE BEEWING STUDENTS
Compare Heads of Ripe Barley of Chevalier
and Goldthorpe Types. A marked difference will
be perceived. The head of the Chevalier barley is
much narrower than that of the Goldthorpe. This
is due to the corns on the head of the Chevalier
a
FIG. 9. (a) Rachis of Gold-
thorpe Barley ; (b) Rachis
of Chevalier Barley.
FIG. 8. (a) Hordeum disticlmm, or Chevalier
Type of Barley ; (b) Hordeum zeocriton,, or
Goldthorpe Type of Barley.
barley forming a more acute angle with the stem
or raehis than is the case with the Goldthorpe.
Another distinction is that the corns on the head
of the Chevalier are situated on the raehis at a
greater distance apart than those of the Gold-
thorpe. This is rendered more apparent by strip-
FIG. 10. (a) Basal Bristle of Goldthorpe Barley; (b) Basal Bristle of
Chevalier Barley.
A STUDY OF THE BAELEY CORN 11
ping the heads and examining the bare rachis.
The internodes, or joints, from which the corns
spring are thus exposed, and it will be noticed that
there are from six to seven internodes in one inch
of the rachis of the Chevalier, and in the Goldthorpe
from nine to ten. 1
Remove the Basal Bristle from a Corn of GolcU
thorpe Barley and Examine it with a Pocket Lens
or a low Microscopic Power, It will be found to
be covered with long hairs, a characteristic which
all barleys of the Goldthorpe type possess. On
the other hand, the basal bristle of a barley of the
Chevalier type (H. distichum) may be covered with
either very short hairs or long ones. This depends
on the variety of the type. The true Chevalier
variety possesses very short hairs ; on the other
hand the Archer variety and some others possess
long hairs somewhat resembling those of the Gold-
thorpe type (H. zeocriton).
Compare a Sample of Threshed Goldthorpe
Barley with a Sample of Chevalier Barley, The
skin of the Goldthorpe is somewhat " greasy" in
appearance, and does not adhere so closely to the
corn as in the Chevalier.
Examine the Lower Ends of Goldthorpe Corns
on the Dorsal Side. A small dimple or transverse
furrow may be noticed in the skin near the ex-
1 Samples of threshed barley frequently contain portions of
rachis, and these fragments will often assist the observer in
determining the nature of the samples.
12 STUDIES FOB BREWING STUDENTS
tremity of the lower end of some of the corns.
This dimple is characteristic of the Goldthorpe
type of barley, but is not always observed owing
to fracture of the corn during the threshing process
at the point where the dimple is situated.
FIG. 11. Hordeum vulgare.
FIG. 12. Hordeum hexastichum.
Compare a Head of H. vulgare with H.
hexastichum. Both are six-rowed barleys, but
the head of H. vulgare is narrower than that
of H. hexastichum. It will be noticed that the
internodes from which the corns spring are more
widely separated in H. vulgare than in H. hexa-
stichum. Note that the general appearance of
the head of H. hexastichum is somewhat square
ANATOMY OF THE BAELEY COKN 13
in appearance, hence the name " square-headed
barley" sometimes applied to it. (See Figs. 11
and 12.)
Remove the side rows of corns from heads of H. vulgare
and H. hexastichum, leaving the middle rows undisturbed,
thus converting the six-rowed heads into two-rowed heads.
Compare the two artificially produced two-rowed heads
with heads of Chevalier and Goldthorpe barley.
It will be noticed that the general features of the head of
H. vulgare are markedly similar to those of the Chevalier, and
that the head of H. hexastichum bears a strong resemblance to
that of the Goldthorpe. It is believed that these resemblances
may indicate a near kinship in species of Chevalier and H.
vulgare, and of Goldthorpe and H. hexastichum. (See Munro
and Beaven, Journal of the Royal Agricultural Society, 1900 y
p. 185.)
PAKT II.
ANATOMY OF THE BAELEY COEN.
Bisect a soaked barley corn longitudinally
through the ventral furrow with a knife. Observe
that the greater bulk of the grain is composed of
a white, starchy mass, called the endosperm, and
that at the base of the corn there is a small,
yellowish bud. The bud is the embryo, or living
germ of the corn, which eventually grows into the
young barley plant. Carefully remove the thick
outer skin from another well-soaked corn, and note
that below the thick false skin there is an extremely
14 STUDIES FOE BREWING STUDENTS
thin skin, which completely envelops both the em-
bryo and the endosperm of the grain. This is the
true skin of the corn, and consists of several layers,
these being the remains of the walls of the ovary
and ovule, which contained the embryo-sac from
which the corn has developed. (See Figs. 4
and 6.)
Note also that at the base, or germ end, of the
corn, the two lodicules previously observed in the
barley flower at the base of the ovary are now
found compressed between the thick palese and the
thin true skin of the corn, thus further demonstrat-
ing the relation of the barley corn to the barley
flower.
Remove the Thin Skins enveloping the Embryo
and Endosperm of a Barley Corn, and Observe that
the Embryo may be Easily Detached from the Endo*
sperm without Injury. This indicates that there
is no direct connection between the embryo and
endosperm, but that they merely adhere together.
This observation is of much importance in con-
nection with the germination changes of the barley
corn which will be studied later on.
HISTOLOGY OF THE BAELEY COEN.
Cut thin Longitudinal Sections of a Barley Corn
which has been Soaked in Water for about twenty^
four hours, in the Plane of the Ventral Furrow of
the Corn. This may be done with a sharp razor,
HISTOLOGY OF THE BAELEY COEN 15
the grain being held in a piece of cork or in a
hand microtome ; it is, however, difficult to obtain
good sections without a considerable amount of
practice. Sections for permanent mounting are
best obtained by employing a freezing microtome,
l?ia. 13. Diagram of a Longitudinal Section through the Germ End of a
Barley Corn, (a) Palea Superior ; (b) Palea Inferior ; (c) Pericarp ; (d)
Testa ; (e) Plumule ; (/) Radicle ; (g) Root-cap ; (h) Scutellum ; (i) Ab-
sorptive Epithelium of Scutellum ; (k) Emptied and Compressed Cells
of Endosperm ; (m) Aleurone Cells of Endosperm ; (n) Funicle ; (o)
Starch-containing Cells of Endosperm; (p) Basal Bristle.
the barley having been previously soaked in gum
and water.
In order to render sections of barley transparent
for examination with the microscope, they should
16 STUDIES FOR BREWING STUDENTS
be dehydrated by means of absolute alcohol, and
cleared by transferring to clove oil. They can then
be examined in the latter medium, or mounted
permanently in xylol and Canada balsam.
Examine the prepared sections under the micro-
scope, first with a low-power objective (1 or 2
inch), and afterwards with a higher power of & or
inch. 1
[N.. It is very desirable when a student is
commencing the microscopic examination of an
object which is new to him, that he should first
employ a low-power objective in order to famili-
arise himself with the general features of the object
before proceeding to study its more minute features
with high power lenses.]
First note that the thick outer covering of the
palese of the grain and the two thin true skins,
called the pericarp and testa, enclose both the em-
bryo and endosperm. Then study the embryo of
the barley corn, which is the future young plant
lying dormant in the seed, using Fig. 13 as a guide.
Observe the appearance and position of the plum-
ules, which eventually develop into the leaves of
the young plant. Note the position and appear-
ance of the radicles or undeveloped roots, and also
the root-caps situated at the ends of the radicles.
Identify and study the part of the embryo called
1 See H. Brown and Morris, " Eesearches on the Germina-
tion of some of the Gramineae " (Structure of a Grain of Barley),
Journ. Chem. Soc., 1890, Ivii., p. 461.
HISTOLOGY OF THE BAELEY COEN 17
the scutellum, and specially note that part which
is known as the absorptive epithelial layer. It
is the layer of elongated cells which bounds
the scutellum on the side which is pressed
against the starchy endosperm of the barley
corn. The cells of this layer, sometimes called
the palisade cells, have most important functions
and play a leading part in the feeding of the
young embryo when it commences to develop into
a young plant.
Study the Endosperm. It will be noticed that
it is composed of two distinct types of cells.
Nearest the skin will be observed a triple layer
of thick-walled, square-shaped cells. These are
called the aleurone cells. The rest of the endo-
sperm is composed of much larger cells of irre-
gular shape bounded by very thin walls. These
are the starch-containing cells, and under a high
power and proper illumination the starch granules
will be observed lying closely packed within
the cell walls. Later on, when the student ex-
amines stained and specially prepared transverse
sections of barley, he will learn more regarding
both the starch-containing cells and the aleurone
cells.
Attention should also be given to a layer of
compressed and empty cells which lie between the
scutellum and the endosperm, but which form an
integral portion of the latter. During the early
development of the corn they contained starch
18 STUDIES FOB BEEWING STUDENTS
granules, but during its later development the starch
was absorbed by the embryo.
Cut Transverse Sections of a Barley Corn in
a Similar Manner to the Longitudinal Sections al*
ready made. These sections should be cut as thin
as possible, and it is desirable to prepare a dozen
or so and preserve them in chloroform water for
future use.
FIG. 14. Diagram of a Transverse Section through a Barley Corn, (a)
Palea Superior ; (b) Palea Inferior ; (c) Pericarp ; (d) Testa ; (e) Funicle ;
(/) Aleurone Cells ; (g) Starch-containing Cells.
Dehydrate a Section in Alcohol and Examine
it under the Microscope in Clove Oil or when
mounted in Balsam. The thick outer coverings, or
palese, enclosing the grain will now be seen in cross
section. The points at which the palea on the
dorsal side of the grain overlaps the palea on the
ventral side will be recognised. The five little
ridges which run along the dorsal side of a barley
corn and give it a slightly angular appearance will
also be noticed as being due to five vascular bundles,
FIG. 15. Photomicrograph of Transverse Section through a Barley Corn,
showing the Funicle.
FIG. 16. Photomicrograph of Section of Skins and Endosperm of Barley
Corn (highly magnified).
HISTOLOGY OF THE BARLEY COEN 19
or veins, in the palea inferior, which is now seen
in cross-section.
The two true skins, the pericarp and testa, are
also very clearly shown in the transverse section.
Note how they continue round the grain until they
arrive at the ventral furrow where they appear to
be merged together and lost in a conspicuous dark
brown spot called the funicle. This spot represents
the point where the ovule was originally attached
to the ovary of the barley flower before it developed
into a corn (see Fig. 4, p. 5).
Within the thin skins of the transverse section,
the triple layer of aleurone cells already noticed in
the longitudinal section will be again observed.
Within these lie the thin-walled starch-containing
cells, forming the greater bulk of the endosperm.
In general arrangement these cells will now be
seen to radiate from near the centre of the grain.
Cut a Transverse Section of one of the Green Corns
which are usually abundant in Samples of Six-rowed
Barley. Dehydrate the section, mount it in clove oil or bal-
sam, and examine it under the microscope. Observe that the
contents of the aleurone cells are coloured blue. The colour of
the aleurone cells seen through the yellow outer skin of the
corn is the cause of the green appearance of the corn originally
observed. A green colour is a natural characteristic of certain
varieties of two-rowed and six-rowed barleys and is not an in-
dication of unripeness.
The student, after working through the studies
-described above, which should have given him a
20 STUDIES FOE BEEWING STUDENTS
good general knowledge of the structure of the
barley corn, must now proceed to study some special
points in the anatomy of the corn.
Note the Position in which Starch Granules
occur in the Endosperm of Barley. Transfer a thin
transverse section of barley to a very dilute solution
of iodine in a watch-glass, and allow it to remain
in the liquid until the section is stained a distinct
blue colour. Mount the section in water on a
glass slide, cover with a thin glass, and examine
first with a low and afterwards with a high power.
The position of the blue-stained starch granules is
now distinctly seen. It will be noticed that the
aleurone cells contain no starch granules, and that
these occur only within the thin-walled cells which
compose the larger part of the endosperm. In
these cells the starch granules lie very closely
packed together.
Show that the Starch-containing Cells contain
Protoplasmic Matter enveloping the Starch Gran^
ules. In order to demonstrate this clearly, the
starch granules must be removed without disturbing
the protoplasm. This can be done by means of
the action of saliva, as this secretion contains an
enzyme ptyalin which is able to dissolve starch,
but has no action on protoplasmic matter. Prepare
3 or 4 c.c. of saliva by washing out the mouth with
a very little water. (As the secretion appears to
be more active before meals it is better to obtain
it then.) Filter the solution into a test-tube, place
HISTOLOGY OF THE BARLEY CORN 21
several thin transverse sections of barley in the
liquid, and keep it at a temperature of 46 C. for
three or four hours by immersing it in a vessel of
water maintained at the desired temperature. (If
the solution is kept for more than a few hours,
which may be necessary, a little chloroform water
should be added to prevent the growth of micro-
organisms.) When the action of the ptyalin is
complete the original white appearance of the sec-
tion, due to the presence of starch granules, will
have disappeared. Now, place one of the starch-
freed sections in a very dilute solution of eosine to
stain the protoplasm, transfer the section to a slide,
and examine it under the microscope. It will be
noticed that the thin-walled cells are filled with a
network of red-stained protoplasmic matter which
originally enclosed the starch granules. The
cavities in which the starch granules formerly lay
will be clearly seen. A permanent mounting of
the section in balsam should be made after first
very gradually dehydrating the section with alcohol
of increasing concentration.
22 STUDIES FOE BEEWING STUDENTS
PAET III.
EXPERIMENTS CONNECTED WITH THE TECHNICAL
STUDY OF BARLEY AND OTHER CEREALS.
Study the Microscopic Appearance of Some of
the Commoner Kinds of Starch. The starches of
barley, wheat, potato, rice and maize are suitable
for this study.
Transfer a little of the starch to a drop of water
on a glass slide, cover with a thin glass and exa-
mine with a high power. Observe the charac-
teristic appearance of the different kinds of starch
granules and make a drawing to scale of each. If
the starch used has been obtained direct from the
grain it is desirable, after examining in water alone,
to stain with a little iodine solution ; as the starch
granules alone are stained blue, this differentiates
them from other organic matter which may be
present. By this means also the mixture of very
small granules of starch with much larger ones, as
in the case of barley starch, will become more
evident.
Prepare a Sample of Starch from Barley or
Other Grain, Grind about 100 grms. of the grain
to a very fine powder and mix with cold water.
Separate the starch from the husk and tissue of
the grain as far as possible by filtering the milky
liquid through very fine muslin. Allow the starch
to subside from the filtrate, pour off the liquid, and
THE TECHNICAL STUDY OF BAELEY 23
purify further by repeated washing with cold water
and decantation. Digest the starch with 0*5 per
cent, caustic potash solution for twenty-four hours,
wash well, and drain the starch on a filter. Trans-
fer to an unglazed porcelain plate and air-dry at
the temperature of the room.
Determination of the Moisture in Barley and
Other Cereals, This determination must be con-
ducted on a ground sample of the corn. First clean
the mill by grinding through it some of the corn
to be tested. Reject this sample. Then grind
from 3 to 5 grms. of the corn and transfer the
whole to a stoppered drying-tube the weight of
which has been previously ascertained. Weigh the
tube and its contents, transfer to a water-oven,
remove the stopper and dry at 100 for four or five
hours. Replace the stopper, transfer to a desiccator
until cool and weigh. Return the tube to the
water-oven for an hour or two, cool and weigh
again, as before. These operations must be re-
peated until the weight remains constant within
one or two milligrams. Now calculate from the
weights obtained the percentage of moisture lost.
A more rapid estimation of the moisture may be obtained
by drying in a hot-air oven kept at a constant temperature of
102 C., but care must be taken that this temperature is not
exceeded.
The student should determine the moisture in
kiln-dried as well as undried samples of barley, in
order to demonstrate that an apparently perfectly
24 STUDIES FOE BEEWING STUDENTS
dry barley contains from 10 to 12 per cent, of
moisture. It is important that he should recognise
that this amount of moisture is natural to a barley
in perfect " condition," and that it is the presence
of moisture in excess of this amount which pro-
duces the different degrees of " want of condition "
in barley.
Determination of the " Vitality " or Germinative
Power of Barley, The student should experiment
with Coldewe's, or other form of germinator, on
various samples of barley. The germinative power
of barley is often regarded from two points of view
its germinative " energy " and its germinative
" capacity ". Germinative energy is expressed by
the percentage number of corns which vegetate
in a definite time, generally taken as three days.
Germinative capacity is expressed by the per-
centage number found capable of germinating,
irrespective of time.
Determination of the "Weight" of Barley.
In this country the term " weight " as applied
technically to barley is understood to refer to the
weight in pounds of a bushel measure of the grain.
The student can gain some experience in the
laboratory of the bushel weights of different kinds
of barley by weighing them in the miniature bushel
of the instrument known as the " chondrometer ".
But the results obtained must be regarded more
as comparative values than reliable determinations
of the actual bushel weight of the corn.
THE TECHNICAL STUDY OF BAKLEY 25
Another meaning of the expression " weight "
as applied to barley refers to the weight of a
certain number (usually 1,000) of corns. This
method of comparing the weight of different bar-
leys, is employed more frequently on the Continent
than in this country, and has the merit of giving
the true average weight of the corns, which is not
expressed accurately by the bushel weight. The
latter is much influenced by the shape of the corns,
and, consequently, by the different manner in which
they arrange themselves in the measure.
Count out 200 average corns from several
samples of different barleys, including English
Chevalier and Smyrna, or other " light foreign "
barley, and weigh them. Express the results as
weights in grams of 1,000 corns. Compare the
weights of the English and Smyrna barleys, and
note that the relation they bear to each other
differs very considerably from the relation of their
bushel weights found by the chondrometer.
Determination of the Specific Gravity of Barley. In
a well-matured, mealy barley its white friable endosperm is
permeated with minute spaces containing air, which diminish its
specific gravity. In a badly matured, steely barley the hard en-
dosperm contains fewer air-spaces, and consequently its specific
gravity is greater than that of the mealy endosperm. A com-
parison of the specific gravities of the endosperms of different
barleys is therefore a measure of their relative tenderness. But
in technical work it is not practicable to obtain a sufficient num-
ber of skinless endosperms in order to determine their specific
gravity, and recourse must therefore be had to a determination
26 STUDIES FOE BEEWING STUDENTS
of the specific gravity of the whole barley corns. This may be
effected by observing their displacement-volume in a liquid like
toluene which does not readily penetrate the interior of the corn.
Weigh out accurately about 50 grms. of the barley, and after
noting the exact weight, transfer the corns to a dry 100 c.c. flask.
From an accurately graduated burette run toluene slowly into
the flask, shaking it to disentangle any air which may be con-
fined in the mass of barley corns. When the toluene reaches
the 100 c.c. mark, note the volume of toluene which has been
run into the flask. If the temperature of the toluene in the flask
and in the burette is kept constant, the difference between the
volume of toluene run into the flask and 100 c.c. represents the
displacement volume of the barley. Divide the weight of this
volume, considered as water, into the weight of the barley taken,
and the specific gravity of the barley will be obtained. But it
should be noticed that the specific gravity of the barley obtained
in this manner is influenced by the air confined within the skins
of the barley and that the proportion in volume of this air is
liable to vary in different types of barley. Hence the above
method of experiment is not reliable if used for comparing diffe-
rent types of barley, and can only be used with advantage for the
purpose of comparing the relative mealiness of samples of barley
of the same type.
As toluene is a volatile, easily inflammable liquid, experi-
ments with it must not be conducted near a flame.
Technical Examination and Valuation of Barley
for Malting Purposes. This is a convenient time
for the student to gain some experience in the
valuation of barley for malting purposes. Personal
instruction by an expert is absolutely necessary.
BAELEY DURING GERMINATION 27
PART IV.
THE CHANGES IN BAELEY DUEING GEEMINATION.
Steep about 100 grms. of English barley in
water for forty-eight hours, and change the steep
water twice each day.
Keep the first highly coloured steep water in a
beaker, and note how rapidly it commences to de-
compose on standing. Water extracts from barley
matter which readily putrefies, and hence arises the
necessity to change the steep water in the malt-
house frequently.
After the barley has been steeped for about two
days, examine the corns. If they are moistened
throughout, but not saturated with water, the
barley is sufficiently well steeped.
Determination of Moisture in the Steeped Barley.
-Weigh about 10 grms. of the steeped barley in a
drying bottle. Place in a water-oven and dry very
slowly with the door of the oven open until most of
the moisture is evaporated. Finally dry at 100*
until the weight is constant. Calculate the per-
centage of water lost. This will give some idea
of the amount of water retained by barley when
steeped in the malt-house.
Study of the Changes in Barley during Ger^
mination. Spread the rest of the steeped barley
(see above) in a thin layer in some suitable form
of germinator to start it into growth. Proper
28 STUDIES FOE SKEWING STUDENTS
attention must be given in order to keep it under
conditions of temperature and moisture which
favour regular and slow growth.
Observe from day to day the appearance of the
growing corn and make a series of sketches show-
ing the gradual development of the rootlets and the
acrospire. Note also how the starchy part of the
endosperm is modified in character as the growth
of the embryo proceeds, and observe that this mo-
dification commences in the part of the endosperm
which is nearest to the embryo, and gradually
spreads towards the far end of the grain.
At intervals during the germination of the bar-
ley make longitudinal sections of a corn for micro-
scopical examination. A freezing microtome should
be employed, if possible, for making the sections.
Prepare the sections for examination in the manner
already described for preparing sections of barley
(p. 14).'
Note that the first sign of the action of the
growing embryo on the endosperm is the solution
of the compressed layer of emptied cells adjacent to
the scutellum (Fig. 13). This action is produced by
the enzyme cytase which is secreted by the scutellum
of the growing embryo. Cytase transforms the in-
soluble cellulose of the cell- walls into a soluble sugar.
1 See H. Brown and Morris, " Kesearches on the Germina-
tion of some of the Graminese" (The Visible Changes which
occur in the Embryo and Endosperm during Germination),
Journ. Chem. Soc., 1890, Ivii., p. 466.
BAELEY DUBING GEKMINATION 29
As the development of the embryo proceeds,
note the disappearance of the cell-walls of the
starch-containing cells of the endosperm which are
nearest to the embryo, and the consequent libera-
tion of the starch granules. Observe how the ac-
tion spreads through the endosperm as the growth
of the embryo proceeds. Note that it is this action
which transforms the hard barley corn into the
friable grain of malt.
Remove with the point of a needle a trace of
the starch nearest to the scutellum of a partially
grown barley corn, and examine it in water with a
high-power lens. Note that many of the starch
granules are " pitted " i.e., show signs of being
partially dissolved. This is due to the action of
the enzyme diastase, secreted by the scutellum of
the growing embryo. Diastase converts the in-
soluble starch granules into soluble sugar, and it
is mainly by its action that the growing embryo
obtains its necessary carbo-hydrate food from the
endosperm.
Eemove carefully the thick and thin skins from a well- grown
barley corn, and note that the layer of aleurone cells can be
readily detached from the rest of the endosperm. In a non-
germinated barley corn this is not the case. The readiness with
which the aleurone cells separate in a germinated corn is due to
the fact that the aleurone cells, as well as the scutellum of the
embryo, 1 secrete cytase, and the action of this enzyme dissolves
1 See H. Brown and Escombe, " On the Depletion of the
Endosperm of Hordeum vulgar e during Germination," Proc. of
Eoyal Soc., vol. Ixiii., 1898.
30 STUDIES FOE BKEWING STUDENTS
the walls of the starch-containing cells adjacent to the aleurone
layer, and so ruptures the connections between it and the starch-
containing cells of the endosperm. Hence the aleurone cells, as
well as the cells of the epithelial layer of the scutellum, contribute
towards the modification of the endosperm.
Demonstrate that the Embryo will Grow to a
Limited Extent when Separated from its Endo^
sperm and Supplied with Water only, and that it
will Grow Freely under the Same Circumstances
if it is Supplied with Suitable Food. 1 Soak some
barley corns (preferably six -rowed Chilian or
Smyrna) 2 in water for twenty-four hours, and re-
move the embryos from about a dozen of the corns
without injuring them. This may be easily done
by turning back the skin of the corn at the germ
end and lifting the exposed embryo with the point
of a blunt knife.
Sterilise two small Petri dishes, each containing
a flat piece of porous unglazed porcelain, about 2
square inches in area. Also prepare some sterilised
water, and a sterilised 3 per cent, solution of cane-
sugar. Pour sufficient sterilised water into one of
the dishes to cover the bottom and thoroughly
moisten the unglazed porcelain. Repeat this opera-
tion with the other dish, using the cane-sugar
solution. Now place four or five of the excised
1 See H. Brown and Morris, " Kesearcb.es on the Germination
of some of the Graminese " (Culture of Embryos of Barley on
Water), Journ. Chem. Soc., 1890, Ivii., p. 482.
2 The embryos of these barleys are not so tender as those of
two-rowed barleys.
BAELEY DUKING GEEMINATION 31
barley embryos on the flat surface of each of the
pieces of porcelain with the scutella of the embryos
resting on the moistened surface of the porcelain.
The embryos are then in a position to obtain the
moisture they require in order to start them into
growth. Place the covers on the dishes, and keep
them in a moderately cool place. In about twenty-
four hours signs of growth in the embryos will be
noticed in both experiments. Protrusion of the
roots is first observed, and shortly afterwards the
plumules commence to elongate. During the first
two or three days the growth of the embryos pro-
ceeds in both experiments at about the same rate,
showing that water alone, as well as cane-sugar
solution, stimulates germination. But by-and-bye
the growth of the water-fed embryos ceases owing
to lack of food, whilst the sugar-fed embryos con-
tinue to increase to a considerable size, and the
young plants commence to develop chlorophyll.
If the cane-sugar solution contains suitable
mineral nutriment and a little nitrate of potash,
perfect plants can sometimes be reared.
When conducting the above experiments note
that every precaution must be adopted to prevent
the growth of moulds and bacteria, which readily
attack the exposed embryos and check their de-
velopment.
Demonstrate that the Embryo of the Barley
Corn secretes Diastase during its Development-
Prepare 100 c.c. of a 1 per cent, solution of soluble
32 STUDIES FOE BKEWING STUDENTS
starch in water. Add to the solution when cold
7 grms. of gelatin, and heat gently in a water-bath
until the gelatin is dissolved. Sterilise the solution
in a steam steriliser, and when it is still hot pour
it into a sterilised Petri dish, which is sufficiently
deep to hold easily to an inch of the liquid.
Place the cover on the dish and allow the gelatin
solution to cool until it commences to show signs
of setting. Now take several excised barley em-
bryos which have been previously prepared, and
place them on the surface of the setting jelly so
that their scutella are in perfect contact with it.
FIG. 17. Experiment Demonstrating the Secretion of Diastase by the
Growing Embryo of a Barley Corn.
Put the covered dish in a cool place, and in three
or four days the embryos will have developed con-
siderably. Remove the cover from the dish, and
with a knife make two long parallel cuts in the
jelly, one on each side of an embryo almost touch-
ing it, and place the thin slice of jelly obtained in
this manner on a white porcelain plate, and brush
over it a solution of iodine in iodide of potassium.
The iodine will stain most of the gelatin a deep
blue colour, owing to the soluble starch present,
but in the portion of the jelly situated immediately
below the scutellum of the embryo a cup-shaped
colourless part will be noticed. This is due to the
BAKLEY DUKING GEKMINATION
33
soluble starch originally contained in this portion
of the jelly having been converted into sugar by
the action of the diastase secreted by the scutellum
of the embryo.
If the jelly below another embryo is examined
in a similar manner, but at a later date, it will be
noticed that the action of the diastase gradually
extends as the embryo develops.
The growth of liquefying bacteria must be
guarded against as far as possible in this experi-
ment, or the success of the experiment may be
interfered with.
Demonstrate that Carbon Dioxide is Generated
during the Growth of the Barley Corn. Arrange
four flasks as in the accompanying illustration so
FIG. 18. Apparatus for Demonstrating that Carbon Dioxide is Generated
during the Respiration of Germinating Barley.
that the first contains a solution of caustic soda,
the second a solution of barium hydrate, the third
some germinating barley corns, and the fourth a
solution of barium hydrate. Connect an aspirator
to the fourth flask, and draw a current of air slowly
through the flasks. As the air enters the first flask
34 STUDIES FOE BEEWING STUDENTS
it is deprived of the carbon dioxide it contains, and
this is shown by the solution of barium hydrate
in the second flask remaining clear. But as the
current of air passes through the flask containing
the germinating barley, it mixes with the carbon
dioxide generated by the respiration of the growing
corn and conveys it to the fourth flask, where its
presence is indicated by the formation of a copious
white precipitate of barium carbonate.
Demonstrate that Oxygen is Absorbed during
the Growth of the Barley Corn. Introduce a quan-
tity of actively germinating barley into a large,
wide-mouthed stoppered bottle, and place in the
bottle a small dish containing a strong solution of
caustic soda in order to absorb the carbon dioxide
generated by the respiration of the growing corn.
Close the bottle with the stopper and allow it to
remain overnight in a warm room. Kemove the
stopper and introduce a lighted taper into the
bottle. Observe that the taper is extinguished,
owing to the oxygen originally present in the bottle
having been removed by the respiration of the ger-
minating barley.
Preparation of Malt Diastase. Digest 100
grins, of air-dried or pale-dried malt with 250 c.c.
of 20 per cent, alcohol for four hours and then
filter. Add strong alcohol to the filtrate so long
as a flocculent precipitate of diastase is formed.
Allow the precipitate to subside and pour off the
supernatant liquid. Wash the precipitated diastase
BAELEY DURING GERMINATION 35
by decantation with a little strong alcohol, and
afterwards transfer the precipitate to a hardened
filter. Wash the precipitate on the filter repeatedly
with small quantities of absolute alcohol and trans-
fer both the filter and precipitate to a vacuum
desiccator. By dehydrating the diastase in this
manner it may be obtained as a light granular
powder.
In order to test the activity of the prepared
diastase dissolve about 0*1 grm. of it by rubbing it
in a small mortar with about 5 c.c. of water, and
transfer the mixture to 50 c.c. of a 1 per cent,
solution of soluble starch. Keep the mixture at
a temperature of 60 (140 F.). The activity of
the diastase will be shown by the gradual disap-
pearance of the iodine starch reaction, due to the
transformation of the starch into sugar.
When preparing diastase it is desirable to carry
on the process as rapidly as possible, since prolonged
contact of the diastase with alcohol tends to destroy
its activity. The first precipitate of diastase may
be redissolved in water and reprecipitated by al-
cohol if a purer preparation is desired.
Action of the Enzyme Cytase. Prepare an extract of
air-dried malt, or of oats, by digesting 25 grins, when finely
ground with about 70 c.c. of cold water for three or four hours.
Filter the extract, which contains the enzyme cytase. Im-
merse in the extract several very thin transverse sections of a
barley corn, and place the vessel containing the solution in an
incubator at a temperature of 25 to 30. In four or five hours
examine one of the sections with a microscope, and it will be
3*
36 STUDIES FOE BKEWING STUDENTS
observed that the walls of the starch-containing cells are swell-
ing and gradually dissolving away. A complete disintegration
of the cell walls should be obtained in about twelve hours, but
if the experiment is continued for this length of time it is advis-
able to add a little chloroform water to the cytase extract in
order to prevent the growth of micro-organisms.
If malt is employed for the purpose of preparing the extract
used in this experiment, it is essential that it should not have
been kiln-dried, for cytase is destroyed by the heat of the kiln.
For a similar reason kiln-dried oats should not be used.
An Experiment to Determine the Loss in Weight
occurring during the Conversion of Barley into
Malt. The loss in weight which occurs in the
malting of any special steeping of barley is often
difficult to determine in the ordinary routine of
malt-house work, as it entails the weighing-up of
the finished malt under conditions which are
usually inconvenient. A measure of the loss may,
however, be made in the laboratory in the follow-
ing manner :
Average samples of the barley, as steeped, and
of the screened malt made from the barley, are
required.
Count out 1,000 corns from each sample, and
weigh them accurately. The figures thus obtained
express the weight of the barley and the weight
of the malt obtained from a similar weight of bar-
ley. Calculate from these figures the yield of malt
from 448 Ibs. (or 1 qr.) of the barley. Calculate
also from the same figures the percentage loss in
weight of the barley when malted.
TECHNICAL EXAMINATION OF MALT 37
Note that the loss determined is only in part
due to a loss of solid matter during malting, be-
cause barley always contains considerably more
moisture than finished malt. In order to estimate
the true loss of solid matter due to root-growth,
respiration, and extraction by steeping water dur-
ing the malting process, determine the moisture
in the samples of barley and malt. From the
figures thus obtained and those previously deter-
mined, the amount of solid matter lost is readily
calculated.
TECHNICAL EXAMINATION OF MALT.
This is a convenient time to study the ordinary
technical methods of examining and valuing malt.
Practical experience under the guidance of an ex-
pert is essential. As a general guide to the student
his attention is directed to the following points :
1. The general appearance of the sample, and
the kind and character of the barley from which
the malt has been made.
2. The " modification " or relative tenderness of
the sample.
3. The " condition " or relative dryness of the
sample.
4. The flavour of the sample.
5. The regularity of growth. (In order to prac-
tise the eye in judging this character it is useful to
separate 200 corns from a sample of malt into four
classes of " still corns," half-grown, three-quarters
38 STUDIES FOR BREWING STUDENTS
grown and " grown out," and express the results as
percentages.)
6. The broken corns present.
7. The mouldy corns present.
The " Sinker " Test. This test is extensively
used in the technical examination of malts, and gives
valuable information when employed intelligently.
Count out 200 corns and place them in a glass
full of water at a temperature of about 15 (60 R).
After moistening the corns by gentle agitation, re-
move at once all the corns which float. Note the
position of the corns which have sunk ; some will
probably rest upright, indicating that the corns are
steely ended ; others may lie flat, which usually
indicates dead or very steely corns. Remove the
"sinkers," count them and examine each corn
separately after dividing it longitudinally with a
knife. Class the corns as (1) dead ; (2) steely ; (3)
steely-tipped ; (4) vitreous ; (5) corns which have
sunk through damage to skin, etc.
In cases of importance it is desirable to take 500 or 1,000
corns for this test, as 200 is too small a number on which to base
a satisfactory judgment. It is very important that the malt
should be in good condition when subjected to the " sinker "
test, as the specific gravity of malt is raised very considerably
when it becomes " slack ".
Determination of the Specific Gravity of Malt. The
specific gravity of malt may be determined by the toluene
method, as already described for barley (see p. 25). Some
idea may be gained of the relative tenderness of malts of similar
nature by this means, but the results are not very reliable.
THE CHEMICAL ANALYSIS OF MALT 39
PAET V.
THE CHEMICAL ANALYSIS OF MALT.
Determination of the Moisture in Malt. This
determination is carried out in a similar manner to
the determination of moisture in barley, described
on p. 23. As malt takes up moisture from the
air very rapidly it should be exposed as little as
possible, particularly after grinding. Subject to
the complete drying of the malt, the drying process
should be carried on for as short a time as possible,
as after prolonged heating malt is inclined to gain
slightly in weight.
Determination of the Ash of Malt. Place about 5
grms. of the ground malt in a weighed platinum dish, and
re-weigh in order to determine the quantity of malt taken.
Heat the dish gently with a Bunsen flame until the malt is
completely charred. Transfer the dish to a muffle furnace, and
heat until the ash is colourless. Weigh and calculate the
percentage of ash obtained. The ash should be examined quali-
tatively for potassium, phosphoric acid and silica, of which it
principally consists.
Determination of the Extract of Malt. Before
this very important determination is attempted the
student must make himself familiar with the or-
dinary method of determining the specific gravity
of liquids by means of the specific gravity bottle
and the balance.
40 STUDIES FOE SKEWING STUDENTS
Determination of the Specific Gravity of Liquids.
A 50 c.c. specific gravity bottle with a perforated
stopper and a counterpoise are required.
Clean the bottle thoroughly, and after washing
with distilled water, rinse it out with a little strong
alcohol. Warm the bottle gently over a flame and
suck air through it by means of a glass tube until
it is quite dry. Allow it to cool in a desiccator.
Ascertain by means of a balance which turns dis-
tinctly with -0005 grm., if the bottle together with
its stopper is of the same weight as the counter-
poise. If not, the counterpoise must be accurately
adjusted by adding to, or subtracting from, its
weight.
The capacity of the bottle must now be deter-
mined by filling it with distilled water at the
standard temperature of 15'5 (60 F.), and weigh-
ing. The following is a convenient and rapid way
of carrying out this operation :
Take about 70 to 80 c.c. of distilled water in a
100 c.c. flask, introduce a thermometer with a wide
degree-scale into the water, and bring the water
exactly to the temperature of 15 '5 (60 F). Pour
the water rapidly into the specific gravity bottle,
which must be held by the neck during this and
the following operations. Close the bottle with its
stopper, so as not to include any air bubbles, and
at once wipe the top of the stopper, which must
not afterwards be touched. Dry the bottle with a
soft cloth as quickly as possible, and place it at
THE CHEMICAL ANALYSIS OF MALT 41
once on the pan of the balance and weigh quickly.
In order to obtain manipulative skill in this process,
the student should repeat the operations of filling
the bottle with water, and weighing until he can
obtain consecutive weights varying by not more
than *001 grm.
If it is found that the bottle contains exactly
50 grms. of water, the specific gravity of any other
liquid may now be obtained by multiplying by
20 the weight of the liquid it will contain, for the
capacity of the bottle is equal to that of 50 grms.
of water at 15 '5, and 50 * 20 = 1,000, the usual
standard to which the specific gravity of liquids is
referred. It rarely happens, however, that a specific
gravity bottle contains exactly 50 grms. of water.
Although it is possible to make a bottle correct
by grinding the stopper, this operation is very
tedious, and it is preferable, if the error of a
bottle does not exceed '02 grm., to use the error
as a constant correction and, as the case may be,
either add it to or subtract it from every weighing
of the bottle. 1 The specific gravities of the solu-
tions likely to be met with by the student do not
differ sufficiently from that of water to introduce
1 The error should be redetermined at frequent intervals.
Also the student should be careful to employ the same ther-
mometer in all his determinations, as ordinary laboratory
thermometers are often very inaccurately graduated. If the
same instrument is always used, even considerable inaccuracy in
graduation will not introduce any appreciable error.
42 STUDIES FOE BEEWING STUDENTS
an appreciable error when adopting this convenient
plan.
If the error exceeds '02 grm. the bottle should
be exchanged for a more accurate one, or, if pre-
ferred, the weight of the water contained by the
bottle may be used as a divisor for the weight of
the contents of the bottle when filled with any
other liquid whose specific gravity is required.
This method gives accurate results with a bottle of
any capacity, but the calculation involved becomes
troublesome when many specific gravity deter-
minations have to be made.
The method of determining specific gravities recommended
above is sufficiently accurate for ordinary purposes of technical
analysis, but for special work it is desirable to use a Sprengel
tube, or a pyknometer with a thermometer stopper and side
tube.
Preparation of the Malt Mash (Heron's Method).
Weigh out roughly about 55 grins, of malt and
grind it through a mill to a moderately fine meal.
Transfer the whole of the ground malt to a balance,
and weigh 50 grms. accurately. (This method of
weighing ground malt should always be adopted
in order to obtain an average sample. The weigh-
ing must be done rapidly, as ground malt is very
hygroscopic.)
Transfer the 50 grms. of malt to a 500 c.c.
copper beaker, and mash it with 350 c.c. of water at
a temperature of 69 (156 F.). Cover the beaker
THE CHEMICAL ANALYSIS OF MALT 43
with a clock-glass and place it in a water-bath
regulated by a thermostat, so that the mash is kept
at a constant temperature of 66 (151 F.). After
one hour transfer the whole of the mash to a 515 c.c.
graduated flask by means of a wide-necked copper
funnel, taking care that the water used for washing-
in the " grains " does not raise the volume above
the 515 c.c. mark. Cool the contents of the flask
to 15-5 (60 F.), and make up the volume to the
515 c.c. mark by the addition of water. Mix the
solution thoroughly by agitation, and filter about
250 c.c. of the wort through a dry filter into a dry
vessel.
Determine the specific gravity of the filtered
wort by the method already described, and cal-
culate the pounds of extract derived from 1 qr.
(336 Ib.) of the malt.
The calculation is made in the following man-
ner : The total volume of the mash is made up to
515 c.c. on the assumption that the volume dis-
placement of the residue of "grains " is 15 c.c., and
therefore that the total volume of the wort is 500
c.c. As 50 grms. of malt were mashed, the extract
from this weight of malt is contained in 500 c.c. of
the wort. Hence the extract from 10 grms. of malt
is present in 100 c.c. of the wort. Assuming that
a specific gravity of 1027*5 has been found for
a wort, it is evident that 100 c.c. of this wort
weighs 10275 grms. Now, this volume of wort
contains the extract from 10 grms. of malt, and
44 STUDIES FOB BEEWING STUDENTS
therefore the solution weight of the extract, i.e.,
the " wet " extract from 10 grms. of malt, amounts
to 10275 - 100, or 275 grms. It is obvious that
the extract from 10 Ib. of malt must be 275 lb.,
therefore the proportion 10 : 275 : : 336 : 92'4
indicates that 92*4 lb. extract has been obtained
from 336 lb. or 1 qr. of the malt.
Extract Determined on the Boiled Wort.
Measure accurately 100 c.c. of the filtered wort
already prepared, in a 100 c.c. flask, and transfer it
to a beaker. Rinse the flask with a little water
and add the rinsings to the beaker. Boil and note
the " breaking " of the wort due to the coagulation
of the precipitated albuminous matter. After the
volume of the liquid is reduced by boiling to about
70 c.c., pour it back into the 100 c.c. measure,
washing the beaker carefully and transferring the
washings to the flask. Take care that the wash-
ings do not raise the volume above the 100 c.c.
mark. Cool the liquid to 15 -5 (60 F.), make up
to 100 c.c. and filter through a dry filter. Take
the specific gravity of the wort and calculate the
extract as before. Compare the extract found
with that of the unboiled extract and note the
small difference caused by the removal of the co-
agulable albuminous matter.
Calculation of the Dry\ Extract from the observed
" Wet" Extract. One hundred c.c. of a solution
containing 1 grm. of dry malt extract weighs
100 '40 grms., indicating that 1 grm. of malt extract
THE CHEMICAL ANALYSIS OF MALT 45
weighs 0'4 grm. when in solution. If, therefore,
the weight of 100 c.c. of a wort minus 100 grms.
is divided by 0*4, the weight of dry malt extract
present in 100 c.c. of the wort is obtained. 1 For
instance, the specific gravity of the unboiled malt
extract already referred to was 1027*5, and there-
fore 100 c.c. weighs 10275 grms. If the weight
of the extract when in solution, viz., 275 is divided
by 0*4, the quotient 6*875 is the weight of dry
extract present in 100 c.c. of the wort. As the
dry extract was derived from 10 grms. of malt it
follows that the malt yielded 6875 per cent, of
dry extract when mashed.
Calculation of weight of Dry " Grains". From
the calculated dry extract it is possible to calculate
the amount of dry " grains " left after mashing
the malt. 100 grms. of malt have been shown to
yield 6875 grms. of soluble dry extract, there-
fore 100 grms. - 6875 grms. = 31*25 grms. not
accounted for. This weight represents the insoluble
" grains," and the moisture in the malt previous to
mashing. If the moisture amounted to 3 per cent.,
the approximate amount of dry grains left after
mashing 100 grms. of malt would be 31*25 - 3 =
28*25 per cent, of dry grains. The weight of
" grains " produced during the mashing of 1 quarter
(336 Ib.) of malt can then be calculated if desired.
1 The divisor for wort solids varies slightly with the specific
gravity and nature of the wort ; but a constant factor of '4 is
sufficiently accurate for the purposes of the experiment.
46 STUDIES FOE BEEWING STUDENTS
It should be observed, however, that the above method of
calculating the weight of dry " grains " yields a result which is
a little too low, because a small fraction of the weight of the
dry extract used in the calculation is not derived from the malt,
but is due to fixation of water during the hydrolysis of the starch
of the malt.
Influence of Fine and Coarse Grinding on Ex-
tract. In order to study this effect two samples of
the same malt, one ground to a coarse meal, and
the other as finely as possible, should be mashed
under similar conditions, and the extracts of both
determined. The finely ground sample will be found
to yield the higher extract, and the difference will
be greater with a hard " steely " malt than with
a tender sample. This experiment indicates the
desirability of establishing standard conditions of
grinding for the purpose of systematic malt ex-
amination. 1
Criticism of the Method of Determining Extracts
described above. The method just described is the
one most commonly employed in this country for
determining extracts, but although it is a very use-
ful method it is open to the objection that the
means by which the measure of the volume of the
wort is obtained is not very accurate. It has
already been mentioned that it is based on the
assumption that the insoluble " grains " from 50
grms. of malt occupy a volume of 15 c.c., and,
therefore, if the total volume of the mash is made
1 A thoroughly satisfactory malt mill has yet to be introduced.
THE CHEMICAL ANALYSIS OF MALT 47
up to 515 c.c. the volume of the wort is 500 c.c.
But the displacement volume of the dry grains
from all malts is not the same, because the propor-
tion of the insoluble husk and cellular tissue to the
soluble matter in malts varies. The difference is
not great, and has little or no influence on the
result when the method is used for comparing the
relative yield of extract from malts of a similar
type, but when the extracts of malts of widely
differing types, such as Smyrna and English, are
compared, the results may be affected appreciably.
No doubt also a more accurate volume than 15 c.c.
might be selected with advantage to represent the
average displacement of the " grains " from 50
grms. of malt, but at present it is advisable to
follow the plan most commonly adopted, as uni-
formity of method in the determination of malt
extracts for ordinary technical purposes is very
desirable.
Determination of Extract by Weighing the
Mash. The method of mashing employed is similar
to that used in the previous process, but the volume
of the wort obtained is calculated from the weight
of the water used in mashing and the specific
gravity of the wort obtained.
Weigh the empty beaker in which the mash is
to be made, and then mash in it 50 grms. of the
ground malt with about 350 c.c. of water at the
same temperature and for the same time as before.
Cool the mash and ascertain the total weight of
48 STUDIES FOE BEEWING STUDENTS
the beaker and the mash. Subtract the weight of
the beaker and malt from the total weight, and the
difference represents the weight of the water used
in mashing. Filter the mash and determine its
specific gravity in the usual manner.
The method of calculating the extract obtained
will be seen in the following example :
Weight of empty beaker = 102-0 grms.
of malt (containing 4 % moisture) = 50 -0
152-0
Total weight of beaker and mash = 532-0 grms.
and malt = 152-0
Water used in mashing = 380*0
Moisture in 50 grms. malt = 2'0
Total water in mash = 382-0
Sp. gr. of wort found = 1035-5. Therefore 100 c.c. weighs
103-55 grms. As 0-4 is the weight of 1 grm. of malt extract in
3*55
solution, - - = 8-875 grms. dry extract in 100 c.c. Therefore
103-55 - 8-875 = 94-675 grms. water in each 100 c.c. wort.
Then if 94*675 grms. water = 100 c.c. of wort,
382-0 (total water in mash) x 100 = m . 5 WQrt ^ magh<
94-675
Now 100 c.c. wort contains 3'55 grms. wet extract ; therefore
403-5 x 3-55 = 14 . 324 8> t extract f rom 50 grms. of malt.
100
Therefore 14 ' 824 x 386 = 96-2 Ib. extract obtained from the
50
malt.
THE CHEMICAL ANALYSIS OF MALT 49
The accuracy of this method of determining the
extract of a malt turns mainly 1 on the degree of
accuracy with which the factor 0*4 represents the
solution weight of 1 grm. of dry malt extract.
This factor is not quite correct in all cases, as
the true factor varies slightly for malt worts of
different gravities and of varying composition, but
the error introduced by its use is very small, and
may be ignored in technical analysis.
A method of determining the percentage of dry
extract yielded by a malt which is very generally
employed on the Continent is based on the process
just described.
Two methods of determining extracts of malts other than
those already described are sometimes used. One consists in
mashing the malt in the usual way, filtering and washing out,
the extract to a known volume. A description of this process,
will be found in L. Briant's Laboratory Text-Book for Brewers,
second edition, p. 123. The second process,- advocated by Dr. A.
L. Stern, is described in the Journal of the Federated Institutes of
Brewing, vol. i., p. 448.
Determination of the " Full Theoretical " Extract.
Those methods already described are employed to determine the
extracts of malts when mashed under conditions approximating
to those in a brewery ; but the whole of. the possible extract is
not obtained by such means, because even in the case of tender
malts a small amount of the starch present is not hydrolysed,
whilst with hard malts a large amount often escapes conversion.
A method frequently employed to obtain the total available,
or " full theoretical " extract consists in treating the ground
1 Probably a small error is introduced when attempting to
correct for the moisture in the malt.
4
50 STUDIES FOE BKEWING STUDENTS
malt previous to mashing with a cold water infusion of oats.
Eipe oats (not kiln-dried) contain a considerable amount of the
enzyme cytase, and a cold water infusion containing this enzyme
is employed to act on the malt for the purpose of liberating
the starch contained in the unmodified portions of the malt by
dissolving the cell walls enclosing the starch granules.
Digest 50 grms. of oats, rather finely ground, with 250 c.c.
of cold water, for three or four hours, and filter. Mix 100 c.c.
of the filtrate with 50 grms. of the ground malt experimented
with, and allow the mixture to stand at the room temperature for
from eighteen to twenty-four hours. Mash with 250 c.c. hot
water, so as to obtain an initial heat of 66 (151 F.), and keep at
this temperature for one hour. Transfer the mash to a 515 c.c.
flask, and after cooling, make up to the mark and filter, and
proceed to determine the specific gravity of the wort in the
usual manner. Before calculating the extract a correction must,
however, be made for the specific gravity of the oat extract used
in the experiment. Determine the specific gravity of the oat
extract used, and subtract one-fifth of its excess gravity above
1,000 from the specific gravity of the malt extract. The extract
derived from the malt can then be calculated in the usual manner.
If the ordinary laboratory extract of the same malt has
been determined, the difference between the two extracts is a
measure of the modification of the malt. The so-called "co-
efficient of modification " may be obtained by calculating the
ordinary extract as a percentage on the " full theoretical "
extract. For instance, if the ordinary extract of a malt is
87*6, and the " full theoretical " extract 95-7,
,, 87-6 x 100 Q _
then g^ = 91-5,
the "co-efficient of modification " of the malt, and the value is
comparable with the " co-efficient of modification " of any other
malt found in a similar manner.
Determination of the Extract of " Flaked " and
Prepared Grain, A determination of the extract
THE CHEMICAL ANALYSIS OF MALT 51
of "flaked" or other forms of grain, prepared for
use in the mash tun without previous conversion,
may be made in the following manner :
Mash 16 '66 grms. of the prepared grain with
33 '33 grms. of pale malt in the usual manner. Also
mash 50 grms. of the pale malt alone. Determine in
the usual manner the extract obtained from 336 Ib.
of the malt alone, and from 336 Ib. of the mixture
of malt and prepared grain. Now subtract two-
thirds of the extract derived from the malt alone
from the extract of the mixed malt and grain, and
it is obvious that the remaining extract is derived
from one-third of a quarter (112 Ib.) of the grain. 1
Multiply the amount by 3 to obtain the extract
derived from 336 Ib. of the grain.
Determination of the Extracts of Black and Brown
Malts. The extracts of these kinds of malt may be obtained by
the same method as the one used for prepared grain. The colour
of the malt should be measured by means of the Lovibond
' tintometer," in accord with some determined standard of
dilution.
Determination of the Extract of Raw Grain,
Weigh out 16*66 grms. of the finely ground grain,
and 33-33 grms. of ground pale malt of which the
extract is known, and put the grain and the malt
x The extract of the malt alone must be determined very
carefully, for any error accumulates on the determination of the
extract of the prepared grain. This remark also applies to the
calculation of brewery extracts when mixed mashing materials
have been used.
4*
52 STUDIES FOE BEEWING STUDENTS
into separate beakers. Add 250 c.c. of cold water
to the raw grain, slowly raise the heat of the mash
in a water bath to 100 (212 F.), and keep at this
temperature for thirty minutes. Cool the contents
of the beaker to 70 (158 F.), and add about 5 grms.
of the ground malt. Stir the mixture well and
allow it to stand for ten minutes in order that the
gelatinised starch may be liquefied by the diastase
of the malt. Raise the heat of the mash slowly
over a gas flame to the boiling point, and boil for
one hour with frequent stirring. Add 100 c.c. cold
water to the boiled mash, reduce its temperature
to 69 (156 F.), and mash in the rest of the malt.
Keep the temperature at 66 (151 F.) for one hour
and then proceed as before (p. 51) to determine the
extract yielded by the raw grain.
It is desirable to use a beaker of Jena glass for
boiling the mash in the above experiment.
When determining the extract of very hard grain it is some-
times advantageous, in place of boiling the mash, to digest it in
an autoclave at a pressure of 7 to 10 Ib. The operation should
be conducted in a wide- mouthed stoppered bottle, with the
stopper tied down in order to prevent loss of the mash during
cooling in the autoclave, which is otherwise likely to occur.
Determination of the Acidity of Malt, Digest
50 grms. of the ground malt with 300 c.c. of cold
distilled water for three hours, stirring the mash
occasionally. Filter, and estimate the acidity in
150 c.c. of the filtrate by means of ^ ammonia
THE CHEMICAL ANALYSIS'; OF MALT 53
solution, using litmus-paper as an indicator. Cal-
culate the acidity as lactic acid, and express the
result as acid found in 100 grms. of malt.
This method is the one usually adopted in malt
analysis, but it is a very unsatisfactory one, as the
acid* reaction of most malts is not due to, free acid
but chiefly to acid phosphates for which litmus is a
very insufficient indicator. .
KH 2 P0 4 is acid to litmus until about half the 'salt is changed
to K 2 HP0 4 , when the mixture is acid to blue and alkaline to red
litmus ; i.e., its action is amphoteric. Consequently, as malt
contains both acid and secondary phosphates, titration with
litmus as an indicator gives no reliable measure of any form
of acidity, and the value of the determination, if it possesses
any, is solely empirical.
The acidity of malt due to acid phosphates (and free acid,
if it contains any) may be determined in a fairly satisfactory
manner with caustic soda, using phenolphthalein as indicator.
(See Fernbach, " Phosphoric Acid in Barley and Malt," Journal
of the Federated Institutes of Brewing, vol. ii., 1896, p. 128.)
Determination of the Diastatic Power of Malt
(Lintner's Method).
(1) Digest 25 grms. of the ground malt with
500, c.c. of distilled water for four hours at 18
(65 F.), stirring every half-hour. Filter about 50
c.c. of the malt extract perfectly bright.
(2) Prepare 100 c.c. of a 2 per cent, solution
of soluble starch (see p. 65) in distilled water.
Measure 10 c.c. of this solution into each one of a
series of eight test-tubes which have been recently
washed and well drained.
54 STUDIES FOE BREWING STUDENTS
(3) Measure by means of a pipette exactly 01
c.c. malt extract into the first of the tubes. Into
the second tube measure 0'2 c.c., into the third
0'3 c.c., and so on, until all eight tubes of starch
solution contain malt extract in regularly increas-
ing amounts. Note the exact time when the malt
extract is added, and place the tubes in a water
bath kept at 21 (70 F.), having previously shaken
them in order to thoroughly mix the added malt
extract with the starch solution. Allow the tubes
to stand for exactly one hour, during which time
the amount of the soluble starch transformed is
directly proportional to the quantity of malt extract
present. Now add to each tube 5 c.c. of Fehling's
solution (see p. 78), mix by shaking, and then heat
the tubes in a boiling water bath for ten minutes.
Remove the tubes and allow them to stand until
the precipitated cuprous oxide has settled. Ex-
amine the tubes and ascertain by means of their
colour the amount of malt extract which has pro-
duced enough sugar in one hour to exactly reduce
all the copper in 5 c.c. of Fehling's solution. Then,
assuming that 01 c.c. malt extract corresponds to
a diastatic power of 100, and x equals the quantity
of extract determined by experiment,
0-1 x 1QQ = the diastatic power of the malt.
X
But a correction for the reducing sugars present in
the malt extract must be made. If this is not
determined directly (which is rarely necessary), 1*5
THE CHEMICAL ANALYSIS OF MALT 55
should be subtracted from the diastatic power
found.
When pale malts are examined, the malt ex-
tract should be added in quantities of '10, '15, *2,
25 c.c., etc., as subdivision into the larger amounts
mentioned above is not sufficiently accurate for
malts of high diastatic power.
When the student is learning this process he
should always make his experiments in duplicate,
as the manipulation necessary to obtain good re-
sults requires a considerable amount of practice.
Special attention must be given to the cleansing of
the test-tubes, which should be treated shortly
before use with a little strong nitric acid and then
thoroughly washed with water. The tubes should
be drained by inverting them in a clean beaker ;
they must not be placed on pegs, which are always
liable to be dirty. The soluble starch used must
be perfectly free from acid. All the minor details
in connection with this process must receive very
careful attention, or reliable results will not be
obtained.
Determination of the Diastatic Power of Barley.
Ungerminated barley contains a form of diastase which hydro-
lyses soluble starch, but, unlike malt diastase, acts very slowly
on ordinary starch paste. The diastatic power of barley is
determined in a similar manner to that of malt, and experiments
should be made with two or three different kinds of barley.
(See Baker, Journ. Chem. Soc., 1902, Ixxxi., p. 1177.)
Determination of the " Non^coagulable Albu^
minoids " in Malt. The Kjeldahl method of esti-
56 STUDIES FOE BREWING STUDENTS
mating nitrogen in organic substances is employed
in this determination, and if the student is not
already familiar with the process he must practise
it before proceeding with the analysis of malt.
The method consists first in converting the
nitrogen of the substance under examination into
ammonia by treatment with strong sulphuric acid,
and then in estimating the ammonia formed by
distilling it into a known volume of standard acid.
Standard solutions of 20 sulphuric acid and am-
monia are required. Prepare a normal solution of
sulphuric acid in the usual manner. Dilute 25 c.c.
of this solution to 500 c.c. with ammonia-free water,
thus making a 20 sulphuric acid. To prepare a 20
ammonia solution add 3 c.c. strong ammonia to
500 c.c. water, titrate the solution with ^o a cid and
dilute until correct. Methyl orange should be used
as indicator.
Determine the Nitrogen in Asparagin. Dissolve
0'30 grms. of finely powdered crystals of asparagin,
dried by pressing between layers of filter-paper, in
a little water, and make up the volume of the solu-
tion to 50 c.c. Measure 5 c.c. of the solution into
a 100 c.c. Jena glass flask or beaker, and evaporate
it to dryness on a water bath. Add 10 c.c. pure
strong sulphuric acid and 5 grins, potassium sul-
phate to the dried residue, and heat the mixture
very strongly until the solution is decolourised.
Transfer to the distilling flask of a Kjeldahl dis-
THE CHEMICAL ANALYSIS OF MALT 57
tilling apparatus with 200 c.c. ammonia- free water.
Add as rapidly as possible a strong solution of
caustic soda (sp. gr. 1300) to the solution until it
is distinctly alkaline, and at once connect the dis-
tilling flask with the distilling apparatus. Proceed
to distil the solution into a flask containing 25 c.c.
20 sulphuric acid. When about half the volume of
the solution has distilled over the operation is
complete. The contents of the flask containing the
20 sulphuric acid must now b'e titrated with 20 am ~
monia solution, using methyl orange as an indicator.
The difference of the volume of the ammonia
solution used, from the original 25 c.c. ^ acid taken,
represents the volume of 20 acid neutralised by the
ammonia distilled over from the mixture in the
flask. Before attempting to calculate the amount
of nitrogen in the asparagin originally taken, it is
necessary to make a correction for the ammonia
which may have been present in the reagents used.
An experiment with the reagents alone is made in
a similar manner to the one already described, and
the amount of 20 ac id neutralised by the ammonia
present in the reagents is used as a correction.
When fresh reagents are employed it is always
necessary to re-determine the correction.
To calculate the amount of nitrogen found in
the asparagin taken, the number of c.c. of 20 ac id
neutralised, less the correction for materials, is
58 STUDIES FOR BREWING STUDENTS
multiplied by '0007, the nitrogen equivalent of 1
N
c.c. 20 sulphuric acid. As 0*03 grm. of asparagin
was taken, the percentage of nitrogen obtained is
readily found. The known amount of nitrogen
in crystallised asparagin is 18 '67 per cent., and
the result obtained by experiment should be within
0'2 per cent. If the result is not satisfactory the
experiment should be repeated until accuracy is
obtained.
Determination of the " Non-coagulable Albumin-
oids " in Malt - - Digest 25 grms. of the finely
ground malt in 250 c.c. distilled water for three
hours at about 15'5 (60 F.), stirring the mixture
occasionally. Filter, and measure 100 c.c. of the
filtrate into a suitable beaker or flask, and boil for
about twenty minutes to throw out of solution all
the coagulable nitrogenous bodies. Transfer the
solution and precipitate to a 100 c.c. measuring
flask and, after cooling, make up to the original
volume of 100 c.c. Filter, and measure 10 c.c.
of the filtrate into a Jena glass flask or beaker.
Evaporate to dryness on the water bath, and add
10 c.c, sulphuric acid and 5 grms. potassium sul-
phate. Heat the mixture, at first gently, until the
first violent action is over, and afterwards strongly,
until it is decolourised. Transfer to a distilling
apparatus, and proceed as already described in
the analysis of asparagin. The nitrogen found,
after making the necessary correction, is the
amount of nitrogen from 1 grm. of malt; from
THE CHEMICAL ANALYSIS OF MALT 59
this the amount from 100 grms. of the malt is at
once obtained. On the assumption that all the
nitrogen found is combined as albuminoids, this
number should then be multiplied by the factor
6-3.
The factor 6 - 3 is based on the consideration that the average
amount of nitrogen contained in proteids is about 16 per cent. ;
but the use of the factor 6'3 should be considered as a conven-
tional arrangement, for the nitrogen in proteids varies very
considerably, and, moreover, much of the soluble nitrogen of
malt exists in combination as amides and bodies other than
proteids, concerning which we have very little exact knowledge
at present. This, however, does not detract from the value of
the determination of " non-coagulable albuminoids " when used
comparatively in the analytical examination of malts.
When the student is learning the process de-
scribed above he should conduct his experiments
in duplicate in order to check the accuracy of his
manipulation.
Determination of the " Ready ^formed Carbo^
hydrates " of Malt. Digest 25 grms. of the finely-
ground malt with 250 c.c. distilled water for three
hours at 15 to 18 (60 to 65 R). Filter, and
measure 100 c.c. of the filtrate into a suitable
beaker or flask, and boil it for about twenty
minutes to throw out of solution all the coagulable
bodies. Transfer the solution and precipitate to a
100 c.c. measuring flask, and after cooling make
up to the original volume of 100 c.c. Filter, and
determine the specific gravity of the filtrate. Cal-
culate the amount of dry solids in 100 c.c. of the
60 STUDIES FOE BKEWING STUDENTS
filtrate by using the factor 0'386, which is assumed
to represent the weight of 1 grm. of these solids
when in solution. 1 The amount of dry solids ob-
tained is derived from 10 grms. of malt, and there-
fore ten times the amount represents the total
solids derived from 100 grms. of malt.
The method usually adopted to determine the
amount of " ready-formed carbo-hydrates " present
in the total solids is as follows : It is assumed that
the total solids are a mixture of " non-coagulable
albuminoids," soluble ash, acid, and " ready-formed
carbo-hydrates". Then, if the albuminoids, ash,
and acid have been determined experimentally, the
sum of the quantities found is subtracted from
the total solids, and the difference is supposed
to represent the " ready-formed carbo-hydrates "
of the malt.
If the albuminoids, ash, and acid are not de-
termined experimentally, an average correction for
these constituents of the total solids may be made
in the following manner : If the total solids are
18 '5 per cent., 3 '5 is subtracted, and for each
variation of 07 per cent, of solids from this
amount 01 is added to, or subtracted from, 3*5
previous to its employment as a correction.
The results of determinations of " ready-formed carbo-
hydrates " obtained by this method must not be regarded as
accurate measures of quantity, but they are of value in malt
is the factor for cane-sugar, but a portion only of the
"ready-formed carbo-hydrates " of malt is cane-sugar.
THE CHEMICAL ANALYSIS OF MALT 61
analysis for purposes of comparison. The same purposes would,
however, be attained equally well and by simpler means if the
" total solids " only were determined, but as the method of
estimating the "ready-formed carbo-hydrates" just described is
very generally adopted, it is better at present to conform to
common usage for the sake of uniformity.
It should also be noticed that in the preparation of the cold
water extract of malt for the above process it is assumed that
diastase has no action on starch during the three hours in which
the malt is soaking in cold water. This is not quite accurate,
for a slight but perceptible action on the starch takes place. If
the cold water mash is made feebly alkaline the action of the
diastase is arrested, and a more accurate measure of the " total
solids " is obtained, but it is not usual to employ alkali.
Determination of the Soluble Ash and the
Colour of Malt, Both these determinations may
be made on the 10 per cent, cold water extract
prepared for the determination of the "ready-
formed carbo-hydrates ".
1. Determination of the Soluble Ash. Evapo-
rate in a weighed platinum dish 25 c.c. of the boiled
and filtered solution prepared for the " ready-
formed carbo-hydrate " determination. Ignite the
dry residue gently with an ordinary Bunsen flame
until it is thoroughly carbonised, and afterwards
heat in a muffle furnace until the residual ash is
quite white. Cool in a desiccator and weigh.
Calculate the amount of ash found as a percentage
on the original malt.
2. Determination of the Colour of the Malt.
Determine the colour in the one-inch cell of a
Lovibond " tintometer " by means of the series of
62 STUDIES FOB BREWING STUDENTS
yellowish-brown tinted glasses known as Series No.
52. Reflected light from the porcelain reflector of
the tintometer should be employed.
Some analysts prefer to determine the colour of malt in an
ordinary hot mash of known specific gravity, and refer it by
calculation to a standard gravity of 1055-5 (20 lb.).
Analyse the colour of the malt by means of the
standard red and yellow glasses of the tintometer,
and express the result in terms of red and yellow.
The " Saccharification " Test. This test is intended to
measure the time in which complete Saccharification of a malt
mash takes place when the mash is made under normal con-
ditions.
Ten grms. of the ground malt are mixed with 100 c.c. of
water at 68 (154 F.) and kept in a water bath at 66 (151 F.),
the mash being stirred occasionally. In fifteen minutes about
5 c.c. of the mash are withdrawn and filtered through a small
filter, and the filtrate, after cooling, is tested with iodine solu-
tion for the presence of starch. If starch is found the test is
repeated at intervals of five minutes until the reaction of iodine
with starch is no longer observed. The time taken for the
complete Saccharification of the mash is then noted.
When the student is familiar with the methods
of malt analysis described above he should analyse
several samples of malt of different character, mak-
ing the following determinations :
Extract.
Moisture.
Diastatic power.
Non-coagulable albuminoids.
Acidity.
THE CHEMICAL ANALYSIS OF MALT 63
Soluble ash.
Colour.
Ready-formed carbo-hydrates.
At the same time he should make a physical
examination of the malts as described on p. 37.
The results of the analyses should be tabulated by
the student and submitted to his instructor for
criticism.
SECTION II.
PEINCIPLES OF THE MASHING PEOCESS.
PAET I.
The Following Course of Experiments Constitutes a
Study of Some of the Carbo-Hydrates Concerned
in Wort Production, and Introduces the Student
to the Special Methods Employed in their Ex-
amination.
Action of Water on Starch. --The student
during his previous course of work has examined
starch granules from various sources under the
microscope (p. 22). He should now study the
action of water of gradually increasing temperature
on starch.
Mix about 4 grms. of potato starch with 100
c.c. of cold water in a Jena glass beaker, and heat
the mixture slowly, keeping it constantly stirred
with a thermometer. Note that the starch granules
commence to swell as the temperature of the water
approaches 65 (149 F.), and as the temperature
rises further they gelatinise completely and mix
with the water, forming a semi-transparent viscid
mixture called starch paste.
64
PRINCIPLES OF THE MASHING PROCESS #5
Boil the starch paste and note that it still re-
mains viscid. Cool the paste, and its viscosity is
much increased.
Boil 1 or 2 c.c. of the starch paste with 5 c.c.
of Fehling's solution. No red precipitate of sub-
oxide of copper is formed, showing that starch
paste does not reduce cupric oxide.
Preparation of Soluble Starch, Introduce about
50 grins, of potato starch into a 500 c.c. flask, and
half fill the flask with a 7*5 per cent, solution of
hydrochloric acid made by diluting 125 c.c. of the
concentrated acid to 500 c.c. with distilled water.
Allow the starch to digest with the dilute acid at
the ordinary room temperature for seven or eight
days. The acid should then be poured off and
the starch washed repeatedly with distilled water
by decantation until the granules no longer give
an acid reaction when placed on blue litmus-paper.
One or two drops of dilute ammonia should then
be added, and the starch again washed until every
trace of ammonia is removed. Drain the starch
thoroughly on a filter, and spread it on filter-paper
to air-dry at a temperature of about 25 (77 F.).
Examine the starch granules under the micro-
scope, and note that in general appearance they
are very similar to granules not treated by acid.
Dissolve about 3 or 4 grms. in 100 c.c. of boiling
water, and note that the solution differs from an
ordinary starch paste in being limpid.
Study the Action of Dilute Sulphuric Acid on
66 STUDIES FOR BREWING STUDENTS
Starch. Prepare 200 c.c. of a 3 per cent, starch
paste. When preparing starch paste note that the
starch must not be introduced into boiling water
as a dry powder, for under these conditions it
forms lumps which do not gelatinise properly.
The dry starch should first be mixed with a little
cold water to form a thin cream, and the mixture
should then be run slowly into boiling water with
constant stirring until gelatinisation is complete.
When the starch paste is boiling gently, add
2 grms. of strong sulphuric acid diluted with about
5 c.c. of water, and continue heating the mixture.
The first noticeable change is the very rapid lique-
faction of the starch paste owing to its conversion
into " soluble starch ".
At intervals of five minutes from the addition
of the acid to the starch paste transfer two quan-
tities of 5 c.c. each into two test-tubes. Cool one
tube and add just enough of a dilute solution of
iodine to develop its full colour. Add 10 c.c. of
Fehling's solution to the other tube and heat it in
a boiling water bath for ten minutes. Arrange
the tubes in consecutive series, and note that, as
the hydrolytic action of the sulphuric acid on the
starch paste proceeds, the iodine-blue reaction of
the original starch gives place to purple, reddish-
purple, red, and finally to no colour ; at the same
time the precipitate of red oxide of copper in the
series of tubes containing Fehling's solution in-
creases rapidly in amount.
PEINCIPLES OF THE MASHING PKOCESS 67
This experiment indicates that the hydrolytic
action of sulphuric acid on starch paste is first to
change it into a more soluble form (soluble starch),
and afterwards to form a compound which gives a
red reaction with iodine (erythro-dextrin), which
is subsequently changed to a body which gives no
colour with iodine. The increasing amount of red
oxide of copper in the tubes containing Fehling's
solution indicates the gradual production of a re-
ducing substance (dextrose) as the starch is hydro-
lysed to its final point.
Prepare the Sugar, Dextrose, which is the Final
Product of the Acid Hydrolysis of Starch. Mix
100 grms. of potato starch with a little cold water
to a thin cream and pour it slowly into 1,000 c.c.
of boiling water in which 50 grms. of oxalic acid
have been previously dissolved. When the starch
is poured slowly into the boiling acid solution it is
rapidly converted into soluble starch, and there is
no difficulty in preparing a 10 per cent, solution in
this manner.
Digest the solution for ten hours at 100 (212
F.) in a steam steriliser to completely hydrolyse
the starch to dextrose. Neutralise the hot acid
solution by adding an excess of calcium carbonate,
and so remove the oxalic acid from the solution
as insoluble oxalate of calcium. Filter, and con-
centrate the filtrate to a thick syrup on a water
bath. Dissolve the syrup in twice its volume of
93 per cent, alcohol and allow the solution to cool
5*
68 STUDIES FOE BEEWING STUDENTS
slowly. If a syrup falls out of solution on cooling,
the alcohol is too strong, and a few drops of water
should be added and the solution again heated to
redissolve it. When the cooled solution no longer
deposits any syrup add a crystal of dextrose and
set aside to crystallise.
After crystallisation is complete, which may
take six or seven days, drain the crystals of dex-
trose and dry by spreading them on a porous
earthenware plate. To recrystallise the dextrose,
dissolve the dried crystals in half their weight of
water and add to the resulting syrup twice its
volume of boiling 93 per cent, alcohol. Set the
alcoholic solution aside to crystallise and dry the
resulting crystals as before.
Study the Hydrolysis of Starch by Acid quan^
titatively, and show that 100 parts of Starch yield
111*1 parts of Dextrose when completely Hydro^
lysed, according to the Equation, C 6 H 10 O 5 + H 2 O =
C 6 H 12 O 6 . Dry 6 grms. of pure potato starch in a
weighing bottle at a temperature of 80 (176 R),
gradually rising to 110 (230 F.), until the weight
is constant. Mix the known weight of dry starch
with a little water to a thin cream, and carefully
transfer the whole of the mixture to a beaker con-
taining 70 c.c. of boiling water in which 2 '5 grms.
of oxalic acid have been dissolved. Boil gently
until solution of the starch is complete. Then
wash the whole of the solution into a flask, taking
care that the total volume of the solution closely
PEINCIPLES OF THE MASHING PROCESS 69
approximates to 100 c.c., thus ensuring that the
solution contains the proper strength of acid, viz.,
2*5 per cent.
Digest the solution in a steam-steriliser at 100
(212 F.) for fourteen hours. When the digestion
is completed, neutralise the hot solution with
calcium carbonate, filter, and wash the precipitate
thoroughly. Evaporate the filtrate and washings
to about 70 c.c., transfer to a 100 c.c. flask, cool
and make up to 100 c.c.
Determine the specific gravity of the solution,
and its opticity in the 100 mm. tube of a polari-
meter. Calculate from each of the above deter-
minations separately the amount of dextrose which
has been obtained from 100 parts of dry starch.
(At present the methods of quantitatively de-
termining carbo-hydrates by means of their solution
density and optical activity have not been studied,
but they are described in the following pages, and
the student should have had time to become familiar
with them before the termination of the experiment
just described.)
Determination and Use of the " Solution
Weights " of Carbohydrates* In the ordinary
course of experimental work with carbo-hydrates
it is often necessary to ascertain the amount of
substance present in a solution ; but the usual
chemical method of evaporating a known volume of
the solution to dryness and weighing the residue
can rarely be employed, owing to the obstinate
70 STUDIES FOR BREWING STUDENTS
manner in which most carbo-hydrates retain water,
and to their liability to decompose on long-con-
tinued heating. In order to dry them without
decomposition they must be submitted to very
special and lengthy processes. 1
To avoid these difficulties, the specific gravity
of a solution of a carbo-hydrate may be used
to determine the weight of substance present,
if the solution weight of the carbo-hydrate is
known.
Experiment. Dry some pure cane-sugar in a
water-oven at 100 (212 F.). Weigh out exactly
10 grms. of the sugar, and dissolve it in about 50
c.c. of water in a small beaker. Transfer the solu-
tion to a 100 c.c. flask, and after cooling to 15*5
(60 F.), make up the volume to the mark with
water.
Determine the specific gravity of the solution
(which should prove to be 1038 '64). The weight
of the solution in excess of 1,000, viz., 38 '64, is the
solution weight of 10 grms. of sugar, and therefore
the solution weight of 1 grm. is 3 '864. It is ob-
vious, therefore, that the use of the factor 3*864 as
a divisor for the specific gravity of the solution
less 1,000, determines the weight of dry cane-sugar
in 100 c.c. of the solution. It should be noted,
however, that different carbo-hydrates possess dif-
1 See Brown, Morris and Millar, " Examination of the Pro-
ducts of Starch-Hydrolysis by Diastase," Journ. Chem. Soc.,
1897, Ixxi., p. 76.
PEINCIPLES OF THE MASHING PEOCESS 71
ferent solution factors, and that the solution factors
vary slightly with the concentration of the sugar
solutions. The factors for different concentrations
of well-known carbo-hydrates met with in ordinary
technical work will be found on referring to Table
L, p. 187.
As an exercise, dissolve a known weight (4 or
5 grms.) of pure dry cane-sugar in about 50 c.c. of
water, and make up the volume of the solution to
100 c.c. at 15*5 (60 R). Determine the specific
gravity of the solution, and, after referring to the
table for the correct solution factor for the specific
gravity found, calculate the amount of cane-sugar
in 100 c.c. of the solution, and compare with the
known weight of cane-sugar originally taken.
Determine the Solution Density of Dextrose. This
sugar cannot be dried in the oven in the ordinary manner, as it
does not part with the whole of its water of crystallisation under
such conditions. Special means for drying in a vacuum must be
employed.
Introduce 10 grms. of the pure sugar into the tared flask of
a " Lobry de Bruyn " apparatus. 1 Dry in a vacuum, first at a
temperature of 70 (158 F.) until it has lost most of its moisture,
and then raise the heat slowly to 105 (221 R). When the
weight has become constant, dissolve the sugar in the flask in
about 90 c.c. of water. Note the weight of the solution in the
flask, and after determining its specific gravity, calculate its
volume. As the volume of the solution and the weight of the
dry dextrose it contains are now known, calculate the weight of
dextrose in 100 c.c.
1 See note, p. 70.
72 STUDIES FOE BKEWING STUDENTS
Then - - = the solution factor of dextrose for
grms. per 100 c.c.
the concentration of the solution experimented with. Compare
the result with the solution factor given in Table I. for a dex-
trose solution of similar specific gravity.
Introduction to the Use of the Polarimeter in
CartxvHydrate Work. The student should have at
his disposal a Laurent sodium-light polarimeter,
and a Schmidt and Haensch half-shadow polari-
meter.
The specific rotation of an optically active carbo-
hydrate is the angle through which a ray of po-
larised light of definite refrangibility is rotated
when it traverses a column 1,000 mm. in length of
a solution of the carbo-hydrate containing 10 grms.
in 100 c.c. of solution. But although a column of
1,000 mm. is adopted as the standard of length to
which specific rotations are referred, such a length
is very rarely used experimentally ; columns of
100 and 200 mm. in length are generally em-
ployed, for which the proper corrections are
readily made, since the rotation of a ray of polar-
ised light varies directly with the length of the
column through which the ray passes.
Moreover it is unnecessary to observe the rota-
tion of a 10 per cent, solution of a carbo-hydrate
in order to determine its specific rotation, for the
rotation of a ray of polarised light varies directly
with the concentration of the solution through
which it passes. Therefore, if the rotatory power
PEINCIPLES OF THE MASHING PKOCESS 73
of a column of any known length of a solution
containing any known amount of substance is ascer-
tained, the specific rotation of the substance may
be calculated in the following manner :
Example. A solution known to contain 5 '50
grms. of cane-sugar in 100 c.c. is observed in a
sodium-light polarimeter in a column of 100 mm.,
and its rotation is found to be 3 '66.
Then 3-66 x 10 = 36-6 rotation in 1,000 mm. Therefore
36-6 x 10 grms. = 6Q , QO = the specific ro t a tion of cane-sugar.
5-5 grms.
The rotation of a ray of polarised light by an
optically active substance varies with the refrangi-
bility or colour of the ray ; it is therefore necessary
to indicate the character of the light used when
expressing the specific rotation of a substance.
The standard light employed is the yellow light
derived from the D lines of the sodium spectrum,
and specific rotations determined by this light are
denoted by the sign [a] D .
When the light employed is the so - called
" medium yellow " of the spectrum, specific ro-
tations are denoted by the sign [a],-.
The relation of [a] D to [a], is in the ratio of
1 : 1111; therefore to convert [a] D into [a], the
expression [a] D x 1*111 is employed; to convert
[a]f into [a] D the expression $L applies. Specific
rotations should always be expressed as [a] D , but
it is necessary to grasp the relation of [a] D to [a]>
74 STUDIES FOE SKEWING STUDENTS
in order to understand the at one time frequent
use of [a],- in literature concerning the carbo-
hydrates. 1
The scale of the Laurent sodium-light polarimeter
is graduated in degrees of arc, and therefore read-
ings with this instrument can be used directly for
the purpose of determining [a] D , as described above.
But the Schmidt and Haensch half-shadow instru-
ment differs from the Laurent inasmuch as the
scale is subdivided into divisions of an arbitrary
character. The full scale of 100 divisions with
which it is furnished expresses the rotation of a
solution of cane-sugar of a specific gravity of 1,100
in a column 200 mm. in length such a solution
containing 26*048 grms. of cane-sugar per 100 c.c.
at 17'5 (63*5 F.). When using the instrument
there is, however, no necessity to take any ac-
count of this, for the divisions of the scale of the
instrument bear a fixed relation to the degrees of
a sodium-light polarimeter. Each single division
is equal to 0*3459 of a degree of the Laurent
instrument for the ordinary carbo-hydrates, except-
ing cane-sugar, for which each division of the scale
is equal to 0*3469 of a degree. When, therefore,
the half-shade instrument is employed to determine
the specific rotation of a carbo-hydrate, multiply
by 0*3459 for all carbo-hydrates other than cane-
sugar, in order to convert the observed scale
1 See Brown, Morris and Millar, Journ. Chem. Soc., 1897,
Ixxi., pp. 84 et seq.
PKINCIPLES OF THE MASHING PROCESS 75
divisions into angular degrees for sodium light ;
in the case of cane-sugar multiply by 0*3469.
In order to convert divisions of the half-shade instrument
into degrees [a]_, multiply by 0-3843 for all carbo-hydrates other
than cane-sugar ; in the case of cane-sugar multiply by 0-3854.
Determine the Specific Rotation, or [a] Dt of
Dextrose by means of the Laurent Sodiunvlight
Polarimeter. Dissolve 7 or 8 grms. of dextrose in
about 100 c.c. of water, and boil the solution for
a few minutes. Cool the solution, determine its
specific gravity, and calculate by means of the
proper solution factor (see Table I.) the weight
of dextrose in 100 c.c. of the solution. Now ob-
serve the rotatory power of the solution in the
200 mm. tube of the Laurent instrument, and
calculate the [a] D of dextrose from the observed
angle of rotation, and the known amount of dextrose
in the solution. Compare with the known specific
rotation of dextrose, [a] D = 52*8.
Determine the Specific Rotation, or [a] D , of
Dextrose by means of the Half^shadow Instru*
ment* Transfer the 200 mm. tube of dextrose
solution used in the previous experiment to the
half-shadow instrument, and observe the number
of divisions of the scale expressing the rotation of
the sugar solution.
Compare the observed reading in divisions with
the reading in degrees found when using the Laurent
instrument, and calculate the value of a division
expressed as the fraction of a degree. Compare
76 STUDIES FOE BEEWING STUDENTS
the result with the known value one division =
0-3459.
Convert the observed number of divisions into
degrees by means of the factor 0'3459, and calculate
the [a] D of dextrose as in the previous experiment.
Compare the results of the two determinations,
which should be in close agreement.
The Use of a Factor other than the True So^
lution Factor in Determinations of the Specific
Rotations of Carbohydrates. In the previous
experiments on the determination of the specific
rotation of dextrose, the true solution factor has
been employed in the calculations. Occasionally,
however, when calculating specific rotations of the
carbo-hydrates, it is desirable to employ factors
which differ to some extent from the true solution
factors. If specific rotation is determined in this
manner the solution factor employed should be
expressed after the sign [a] D or [a^. Thus
[a] D3 . 86 indicates that 3 '86 is the solution factor
which has been employed in determining the par-
ticular specific rotation to which the sign refers.
Experiment. - - Calculate the [a] D3 . 86 and the
Mtf-se of dextrose from the results of the pre-
vious experiment.
Determine by means of the Polarimeter the
Amount of Sugar present in a Solution when the
[a] D of the Dissolved Sugar is Known, Prepare
a solution of pure cane-sugar of unknown strength
(5 to 15 grms. in 100 c.c.), and determine its rota-
PKINCIPLES OF THE MASHING PKOCESS 77
tory power in the 100 or 200 mm. tube of the
Laurent polarimeter.
From the result obtained and the known speci-
fic rotation of cane-sugar [a D ] 66*6, calculate the
amount of cane-sugar present in 100 c.c. of the
solution. Check the result by means of the solu-
tion density method.
" Mutarotation " of Dextrose. When the optical activity
of a freshly prepared solution of dextrose in cold water is deter-
mined, it is found that the [a] D of the dissolved sugar is much
higher than 52'8, the recognised [a] D of dextrose; but on stand-
ing at room temperature the optical activity of the solution
slowly decreases, until after ten or twelve hours the value [a] D
52 '8 is finally reached. The explanation of this phenomenon
lies in the fact that dextrose, when first dissolved in cold water,
exists temporarily as a modification which possesses about twice
the rotatory power of the ordinary stable form of dextrose. This
bi-rotatory or "a" form is, however, rapidly converted into the
stable " ft " form on raising its solution to the boiling point.
Experiment. Dissolve 6 or 7 grms. of dextrose in 100 c.c.
of cold water and divide the solution into two portions. Allow
one portion to remain at the ordinary temperature. Eaise the
other portion to the boiling point for two or three minutes and
cool it again. Observe the difference in rotatory power of the
two solutions when examined in the polarimeter. Allow the
solutions to stand for two or three hours and examine them
again. Note that the rotatory power of the boiled solution
remains constant, but that the rotatory power of the other has
decreased.
This experiment demonstrates that a freshly prepared solu-
tion of dextrose must be raised to the boiling point, or its bi-
rotation otherwise destroyed, before determining its optical
activity.
The phenomenon of mutarotation is exhibited by other
78 STUDIES FOB BKEWING STUDENTS
sugars than dextrose. Among these, maltose and levulose are
sugars with which the student will frequently be brought into
contact in the course of his experiments. A freshly prepared
solution of maltose containing the "a" modification has a lower
specific rotation than that of the stable "/8" modification. On
the other hand, the " a " modification of levulose, like that of
dextrose, has a higher specific rotation than the " ft " or stable
modification. It will be evident from these remarks that freshly
prepared solutions of both maltose and levulose must be treated
in a similar manner to those of dextrose previous to examination
in the polarimeter. 1
The Cupric Oxide Reducing Power of Dextrose,
Introducing the Cupric Oxide Reducing Method of
Estimating Sugars*
Preparation of Fehling's solution. Two solu-
tions are required :
(1) Coppei* Sulphate Solution, prepared by dis-
solving 69*2 grms. of the pure salt in distilled
water and making up the volume of the solution
to 1,000 c.c.
(2) Alkaline Tartrate Solution, prepared by dis-
solving 346 grms. of Rochelle salt (sodium potassium
tartrate) and 130 grms. of anhydrous sodium hy-
drate in distilled water and making up the volume
of the solution to 1,000 c.c.
The solutions must be stored in separate bottles,
and when required for use must be mixed in equal
volumes.
1 For recent views concerning the mutarotation of dextrose,
see H. E. Armstrong, " Studies on Enzyme Action," Journ.
Chem. Soc., 1903, Ixxxiii., p. 1310.
PKINCIPLES OF THE MASHING PKOCESS 79
Determination of the Reducing Powei* of Dex-
trose. 1 Weigh out about 5 grms. of dextrose,
dissolve it in 100 c.c. of water, and determine
the concentration of the solution from its specific
gravity. (See Table I. for divisor.)
Prepare the Fehling's solution required by mix-
ing 25 c.c. of the copper sulphate solution with
25 c.c. of the alkaline tartrate solution in a No. 5
spout beaker, and dilute the mixed solution with
such a quantity of water that, with the dextrose
solution, to be added subsequently, the total
volume is 100 c.c. In other words, the Fehling's
solution used is diluted with its own volume of
liquid.
Cover the beaker containing the diluted Fehling's
solution with a clock-glass, and heat in a boiling
water-bath. After a few minutes, add to the
Fehling's solution an accurately measured or weighed
volume of the dextrose solution, and continue the
heating of the solution for exactly twelve minutes.
Filter off the red precipitate of cuprous oxide
which is formed through a Soxhlet tube connected
with a water pump, and wash it with at least 200
c.c. of boiling water, and afterwards with about
10 c.c. of strong alcohol. Place the Soxhlet tube
in a water or hot-air oven and dry at 100 (212 F.).
When dry, reduce the cuprous oxide in the tube
to metallic copper by gently heating the tube with
1 The standard method proposed by H. Brown, Morris and
Millar is adopted. (See Journ. Chem. Soc., 1897, Ixxi., p. 278.)
80 STUDIES FOE SKEWING STUDENTS
st gas flame in a current of dry hydrogen. Allow
the tube to cool in a desiccator and weigh.
Refer to Table III. to ascertain the amount of
dextrose corresponding to the weight of copper
found, and compare the weight found with the
known weight of dextrose used in the experiment.
Calculate the weight of dextrose found as a per-
centage on the weight of dextrose taken.
When judging the proper amount of dextrose
to be taken for the reduction experiment just de-
scribed, some points must be borne in mind :
(1) The amount of copper weighed should be
not less than 0'15 grm., and not more than 0*35 grm.
As dextrose reduces approximately twice its weight
of copper, the weight of dextrose taken should be
from 0*07 to 017 grm. Two c.c. of a 5 per cent,
solution would therefore be about the right amount
to take.
(2) When it is necessary to use less than 2 c.c. of
a sugar solution, the experimental error of measure-
ment from a pipette has an appreciable influence
on the accuracy of the result. In such case it
is advisable to weigh the desired amount of solu-
tion accurately, and calculate the volume of the
solution used from its weight and specific gravity.
(3) As Fehling's solution almost always gives a
slight precipitate on heating due to spontaneous
reduction, it is necessary to make a blank deter-
mination upon every fresh quantity of Fehling's
solution prepared, and correct for this in all experi
PKINCIPLES OF THE MASHING PROCESS 81
ments in which the solution is used. The amount
usually varies from 1 to 3 milligrams of copper.
Preparation and Properties of PhenyLGluco*
sazone. Dissolve 1 grm. of dextrose in 50 c.c. of
water, and add to the solution 2 grms. of phenyl-
hydrazine dissolved in 2 grms. of 50 per cent.
acetic acid. Heat the mixture in a boiling water
bath, and observe that the glucosazone slowly
separates from the hot solution as a dense yellow
precipitate. The action is complete in one hour.
Examine the precipitate under the microscope
with a J-inch lens, and note that it is composed
of needle-shaped crystals, some of which may occur
in fan-shaped aggregates. Make a drawing of
the crystals. Filter off the precipitated osazone,
wash with hot water, and dry at 100 (212 F.).
Note that glucosazone is very insoluble in boil-
ing water ; this characteristic assists in its identi-
fication.
The reaction of phenyl-hydrazine with the hexoses is as
follows : If one molecule of phenyl-hydrazine is allowed to act
on one molecule of a hexose, a normal hydrazone is formed :
Dextrose. Phenyl-hydrazine. Hydrazone.
CH 2 .OH(CH.OH) 4 .CHO + C 6 H S NHNH 2 = CH 2 .OH(CH.OH) 4 .CH + KjO
II
N-NH.C 6 H 5
But if two molecules of phenyl-hydrazine are used, an
osazone is obtained :
CH 2 .OH(CH.OH) 3 .C - CH=N - NH.C 6 H 5
N
NH.C 6 H 5
Glucosazone.
6
82 STUDIES FOE BKEWING STUDENTS
Cane'Sugar or Saccharose. An experiment
has been made (p. 73) on the optical activity of
cane-sugar.
Solution Density of Cane^Sugar. The solution
factors for cane-sugar in solutions of varying con-
centration are given in Table I.
Cane^Sugar does not possess Cupric Oxide
Reducing Power. Dissolve about 1 grin, of pure
cane-sugar in a little water, and mix the solution
with Fehling's solution under the standard condi-
tions already described (p. 79). Heat the solu-
tion for twelve minutes. No reduction takes
place.
Cane^Sugar does not Combine with Phenyl-
Hydrazine to Form an Osazone. Dissolve 1 grm.
of cane-sugar in 50 c.c. of water and treat the
solution with phenyl - hydrazine as described for
dextrose (p. 81). Note that no osazone is formed
until after prolonged heating, when a little glucos-
azone may be formed owing to slight inversion of
the cane-sugar by the acetic acid in the solution.
Inversion of Cane^Sugar. -- Cane-sugar is a
disaccharide, and when hydrolysed (inverted) by
the action of dilute acids or that of the enzyme
invertase, is resolved into a mixture of equal parts
of dextrose and levulose (invert-sugar), according
to the equation :
Cane-sugar. Dextrose. Levulose.
The change may be demonstrated quantitatively
PRINCIPLES OF THE MASHING PEOCESS 83
by both the optical and the reducing properties of
the invert-sugar formed.
Inversion of Cane- Sugar with Acid. Dissolve
an accurately weighed quantity of about 10 grms.
of dry cane-sugar in 50 c.c. of water, and transfer
the solution without loss to a 100 c.c. flask, taking
care that the volume of the solution and wash
water does not exceed 90 c.c. Add 5 c.c. of strong
hydrochloric acid, and make up the volume of the
solution nearly to the 100 c.c. mark. Heat the
flask in a boiling water-bath for thirty minutes,
when inversion of the cane-sugar should be com-
plete. Cool the solution, and make up the volume
to 100 c.c.
(1) Determine the rotatory power of the solu-
tion at a temperature of 20 (68 F.), and calculate
the amount of invert-sugar present in 100 c.c. of
the solution from the known specific rotation of
invert-sugar, [a] D - 19*6 (at 20).
(2) Determine the cupric oxide reducing power
of the solution, using about 1'5 c.c. for reduction,
and calculate the amount of invert-sugar present
in 100 c.c. of the solution from the weight of re-
duced copper obtained.
Acid inversion of cane-sugar does not give such satisfactory
quantitative results as inversion with yeast (described below), as
the levulose formed is slowly acted upon and decomposed by
the acid employed.
Inversion of Cane-Sugar with Yeast. The in-
verting power of yeast is due to the enzyme inver-
6*
84 STUDIES FOE BREWING STUDENTS
tase contained in the yeast cells. Two methods of
experiment may be adopted when yeast is employed
as an inverting agent :
(1) Prepare a solution of a known weight of
cane-sugar in a 100 c.c. flask as in the previous ex-
periment, but in place of hydrochloric acid, add 1
grm. of washed pressed yeast, and digest the solution
in a water-bath at 50 (122 F.) for six hours. A
temperature of 50 is employed because the fer-
mentative power of yeast is arrested at this tem-
perature, but its power of inversion is still retained.
When the digestion is completed, raise the solution
to the boiling point and cool to 15 '5 (60 F.).
Add a little alumina cream to facilitate clarifica-
tion, make up to 100 c.c., and filter. Determine
the rotatory power of the solution as in the previous
experiment, and calculate the percentage yield of
invert-sugar from cane-sugar which has been ob-
tained. Compare with the calculated amount
according to the equation for the inversion of cane-
sugar (p. 82).
(2) Measure 50 c.c. of a solution of cane-sugar of
known concentration into a flask doubly graduated
to 50 and 55 c.c. and add 0*5 grm. of pressed yeast
and three or four drops of chloroform. Cork the
flask tightly, place it in an incubator at a tem-
perature of about 30 (86 F.), and allow it to
remain overnight. Chloroform is added because
it arrests the fermentative power of yeast but
does not influence its power of inversion. Remove
PEINCIPLES OF THE MASHING PROCESS 85
the cork, place the flask in a boiling water-bath and
allow it to stand there until free from the smell of
chloroform. Removal of the chloroform is neces-
sary, as it reduces Fehling's solution and would
affect the estimation of the invert-sugar by the
reduction method if it was allowed to remain in
the solution. Cool the solution, add a little
alumina cream to facilitate clarification, and make
up the volume to the 55 c.c. mark with water.
Filter, and determine the reducing power and
rotatory power of the solution, as before. When
making the requisite calculations, note that a
correction for the dilution of the solution from
50 c.c. to 55 c.c. is necessary.
Determine the Amount of Cane^Sugar present
in Malt by Inversion with Yeast. Weigh out 50
grms. of finely ground malt, introduce it into a flask,
and add 200 c.c. of water. Make the mixture very
faintly alkaline with caustic soda, and allow it to
stand over night. The solution is made slightly
alkaline in order to prevent the invertase which may
be present in the malt from acting on the cane-
sugar during the process of extraction. Filter 100
c.c. of the solution and render it very faintly acid
with dilute acetic acid. This is done in order to
restore the solution to a condition in which invertase
is active. Observe the reading of the solution in
the 100 mm. tube of the polarimeter. Now place
50 c.c. of the solution in a 50-55 c.c. flask, add
0*5 grm. pressed yeast and a few drops of chloro-
86 STUDIES FOE BEEWING STUDENTS
form, and proceed according to the second yeast-
inversion method described above. Make a second
reading of the solution in the 100 mm. tube, and
after correcting for the dilution of the solution
(50 c.c. to 55 c.c.), calculate from the readings
made before and after inversion the amount of
cane-sugar in 100 grms. of the malt.
Levulose (C 6 H 12 6 ). Pure levulose may be obtained from
Schering's levulose (originally prepared from invert-sugar by
means of the insoluble calcium compound of levulose), by re-
crystallisation from absolute alcohol. Pure levulose may also
be obtained by the hydrolysis of inulin with dilute acid. (For
method of preparation see abstract of paper by A. Wohl in
Journ. Chem. Soc., 1890, vol. Iviii., p. 1087.)
Specific Rotation of Levulose. [a] D - 92-0 at 20 (68
F.). Dissolve about 5 grms. of pure levulose in 80 to 100 c.c.
water, heat to boiling (see p. 78), cool and determine the con-
centration of the solution by means of its specific gravity. (See
Table I. for solution factors for levulose.) Determine the rotatory
power of the solution in the 200 mm. tube at the different tem-
peratures of 15-5 (60 F.), 20 (68 F.) and 30 (86 F.), and
calculate the [a] D of levulose for the three temperatures.
These observations will demonstrate that the optical activity of
levulose is very sensitive to alterations of temperature, and that
it is necessary when determining the rotatory power of solutions
of levulose and invert-sugar to take special care with regard
to the temperature of the solutions at the time of observation.
Determination of the Cupric Oxide Reducing Power
of Levulose. Determine the reducing power of about 2 c.c. of
the solution of levulose according to standard conditions, and
compare the result with the reduction figures for pure levulose
given in Table III.
Preparation of the Osazone of Levulose. Dilute about
20 c.c. of the levulose solution to 50 c.c. with water, and heat in
87
the usual way with 2 grms. of phenyl-hydrazine and acetic acid
(p. 81). An osazone separates during heating with'the charac-
teristics of glucosazone. Under the microscope the crystals
appear similar to those of glucosazone, and in fact the osazone
of levulose is identical with glucosazone. Study the constitution
of levulose and dextrose with regard to the formation of the
same osazone from both.
PAET II.
The Following Course of Experiments Constitutes a
Study of the Hydrolysis of Starch by Diastase,
and of the Products of Hydrolysis.
Preparation of Cold-water Malt Extract* A
cold-water malt extract supplies a solution of
active diastase for the experiments described below.
Mix one part by weight of finely ground pale
dried malt with two and a half parts of cold water,
allow the mixture to stand four or five hours, and
filter. When cold-water malt extract is referred to
in the following studies it is intended to be pre-
pared by this method unless otherwise stated.
Study the Hydrolysing Action of Diastase on
Starch, Prepare 200 c.c. of a 3 per cent, starch
paste (see p. 66). Cool to 60 (140 R), and add 10
c.c. of cold-water malt extract, the temperature of
the starch paste being kept constant. Observe the
rapid liquefaction of the starch paste due to its
conversion into soluble starch.
88 STUDIES FOR BREWING STUDENTS
At intervals of five minutes from the addition
of the malt extract to the starch paste, remove two
quantities of 5 c.c. each of the solution to two
test-tubes, and immerse them at once in boiling
water for a few minutes to stop further action of
the diastase. Proceed to treat the solutions in
the tubes with iodine and with Fehling's solution,
in the manner described for the acid conversion of
starch on p. 66. Changes similar to those observed
during the acid hydrolysis of starch will be noticed.
But although the earlier chemical changes of starch
hydrolysis by acid and by diastase are very similar,
the final reducing sugar produced by the action of
acid is dextrose, and that produced by the action
of diastase is maltose.
Show that the Hydrolysing Action of Diastase
is Destroyed at 100 (212 F.). Boil 10 c.c. of malt
extract for a few minutes, cool the solution, and
add it to 50 c.c. of starch paste at a temperature of
60 (140 F.). Observe that neither liquefaction nor
hydrolysis of the starch paste takes place.
Preparation of Maltose (C^H^On), the Reducing
Sugar formed by the Action of Diastase on Starch.
-Weigh out 200 grms. of potato starch, and divide
it into three approximately equal portions. Gela-
tinise one portion in about 1,200 c.c. of boiling
water in the usual manner. (If a beaker is em-
ployed for this experiment it should be of Jena
glass.) Cool the starch paste to 60 (140 F.), and
liquefy it by the addition of 1 c.c. of malt extract.
PBINCIPLES OF THE MASHING PEOCESS 89
Raise the liquefied starch solution to the boiling
point by immersing the vessel containing it in
boiling water, and gelatinise the second portion of
starch, as before.
Cool again to 60, and liquefy a second time by
the addition of 1 c.c. of malt extract. Raise the
solution again to the boiling point and gelatinise
the final portion of starch. A solution containing
about 15 per cent, of soluble starch is thus pro-
cured.
Cool the solution to 55 (131 R), add 40 c.c.
of malt extract, and allow the mixture to stand
over night in order to obtain the maximum conver-
sion of the starch into maltose and dextrin. Boil,
filter, and evaporate the filtrate in a porcelain dish
on a water-bath until a fairly thick skin forms on
the surface of the syrupy solution. Pour the syrup
while hot into a flask containing 500 c.c. of boiling
93 to 95 per cent, alcohol. Part of the syrup-
maltose dissolves, and part dextrin mixed with
maltose is precipitated. Connect the flask con-
taining the mixture with an inverted condenser,
and keep the alcohol boiling gently in a water-bath
for five or six hours in order to dissolve out as
much of the maltose from the dextrin as possible.
Repeated agitation at this stage of the process aids
the solution of the maltose.
Allow the mixture to cool and then decant
the alcoholic solution of maltose from the gummy
residue of dextrin into another flask. Reserve the
90 STUDIES FOE BKEWING STUDENTS
flask containing the residue for future experiment
(see p. 93).
Proceed to connect the flask containing the
alcoholic solution of maltose with a distilling ap-
paratus, and remove the alcohol as completely as
possible from the solution by heating the flask in
a water-bath. Pour the thin syrupy residue into
a beaker, and when cold add a little crystallised
maltose. In twenty-four hours the syrup should
set to a semi-solid crystalline mass of impure
maltose which should then be spread on a plate of
porous earthenware to dry. In order to purify the
crude product by recrystallisation, ascertain its
'weight, and for every 100 grms. found, add 25 c.c.
of water and heat until a syrup is obtained. Dis-
solve the syrup in 250 c.c. of hot 88 per cent,
alcohol (sp. gr. 0*828) and note that at this point
it may be necessary to boil the alcoholic solution
with animal charcoal in order to remove colouring
matter. Add a little crystallised maltose to the
filtered alcoholic solution and allow it to stand.
Drain the maltose which crystallises out from the
alcoholic solution, wash on a filter with 95 per
cent, alcohol, and dry on a porous earthenware
plate.
The amount of maltose obtained may be in-
creased by again extracting the original dextrinous
residue with boiling 85 per cent, alcohol, and pro-
ceeding as described above.
Specific Rotation of Maltose [a] D 138-0. Dis-
PRINCIPLES OF THE MASHING PROCESS 91
solve about 10 grms. of pure maltose in 100 c.c. of
water, and boil the solution for a few minutes.
(See p. 78 on the mutarotation of maltose.) Deter-
mine the strength of the solution by means of its
specific gravity. (See Table I., for solution fac-
tors for maltose.) Determine the rotatory power
of the solution and calculate from the observed
angle the [a] D of maltose. Compare with the known
[a] D of maltose.
Determination of the Cupric Oxide reducing
Power of Maltose. Determine the reducing power
of a known volume of the solution of maltose by
the standard method previously employed (p. 79).
Maltose reduces about its own weight of copper, so
that a suitable quantity of the solution for the ex-
periment is readily determined. Compare the result
of the reduction experiment with the results for
maltose given in Table IV.
Preparation and Properties of Phenyl'MaltoS'
azone. Dissolve 2 grms. of maltose in 50 c.c. of
water, and add to the solution 2 grms. of phenyl-
hydrazine dissolved in 2 grms. of 50 per cent,
acetic acid. Heat the mixture for one hour in a
boiling water-bath.
Observe that the solution, when hot, remains
clear in a manner very different from a solution of
glucose under similar conditions (see p. 81), but on
allowing the solution to cool, a yellow precipitate
of an osazone is formed. Examine the precipitate
under the microscope and note that the crystals
92 STUDIES FOE BKEWING STUDENTS
of the osazone are in the form of long flat plates.
Compare with the crystalline form of glucosazone,
(p. 81).
Filter off the maltosazone precipitate, wash
with cold water, and dry.
Hydrolysis of Maltose by Acid. Maltose is a di-
saccharide which yields dextrose on hydrolysis. (Compare
with the acid hydrolysis of the disaccharide cane sugar which
yields a mixture of dextrose and levulose, p. 83.)
Dissolve about 10 grms. of pure maltose in 150 c.c. of water,
and determine the concentration of the solution by the specific
gravity method. Measure exactly 100 c.c. of the solution into a
suitable flask and add 2-5 grms. of oxalic acid. Heat the solu-
tion for fourteen hours in a steam steriliser. When digestion
is complete, neutralise the hot solution with calcium carbonate,
filter, and wash the precipitate thoroughly. Evaporate the filtrate
and washings to about 70 c.c. ; transfer to a 100 c.c. flask, cool
and make up to 100 c.c. Determine the amount of dextrose
from its rotatory power. Note that 100 parts of maltose yield
105*26 grms. of dextrose on hydrolysis, according to the equa-
tion :
C 12 H 22 O n + H 2 = 2C 6 H 12 6 .
Maltose. Dextrose.
Confirm the presence of dextrose by the preparation of its
osazone.
Maltose is not Hydrolysed by the Action of In-
vertase. Prepare a solution of maltose of known concentration
and proceed according to the second method described for the
inversion of cane-sugar by yeast (see p. 84). Observe by means
of the polarimeter that the maltose in the solution has not been
hydrolysed by the invertase of the yeast.
Although this experiment demonstrates that the invertase
of yeast has no action on maltose, since cane-sugar is inverted
under similar conditions, it will be found later on when study-
PEINCIPLES OF THE MASHING PROCESS 93
ing yeast (p. 142), that the cells contain another enzyme, maltase,
which under certain conditions hydrolyses maltose to dextrose.
Dextrin : Preparation of Dextrin. The residue
insoluble in alcohol which was left during the pre-
paration of maltose (see p. 90) is employed.
Dissolve the residue in about half its weight of
hot water, and add boiling alcohol of 90 per cent,
strength to the solution until a permanent precipi-
tate of dextrin is formed. Allow the solution to
cool, pour off the mother-liquor, and extract the
precipitate with boiling 80 per cent, alcohol to
remove as much of the remaining maltose as
possible. The precipitate now consists mainly of
dextrin, but it will still retain some maltose. This
can only be removed completely by a tedious pro-
cess which includes fermentation and fractional
precipitation ; the process is too lengthy for the
student to attempt. 1
Specific Rotation of Dextrin. This is usually
considered to be [a] D 202.
Non^reducing Property of Dextrin. Dextrin
is generally considered to be a non-reducing carbo-
hydrate.
Although it is usual to consider that the [a] D of dextrin is
202 and that it has no reducing power, there is good reason to
believe that stable 'dextrin possesses a feeble reducing power,
or E, of about 5-5, compared with the reducing power of maltose
1 See H. Brown and Millar, " The Stable Dextrin of Starch
Transformations," Journ. Chem. Soc., 1899, Ixxv., p. 317.
94 STUDIES FOE BEEWING STUDENTS
reckoned as 100, and that its specific rotation is [a] D 197-198.
These results have been obtained by H. Brown and Millar (see
" The Stable Dextrin of Starch Transformations," Journal of the
Chemical Society, 1899, Ixxv., pp. 331 et seq.), who consider that
the molecule of stable dextrin is probably represented empirically
(0 H O ^ "k
by the formula ^ A -a- n r> an ^ * na ^ ^ 8 reducing power is due
^ J 6 a -l2 (J 6 J
to the single C 6 H 12 6 , or glucose group, in the molecule. In
analytical work connected with the carbo-hydrates the very
small reducing power of dextrin is, however, generally ignored,
and an [a] D of 202 employed. This course is adopted in the
following studies.
Preparation and Quantitative Examination of a
Low Starch Conversion. Starch paste when acted
upon by diastase under the most favourable condi-
tions for conversion is hydrolysed to a mixture of
maltose and stable dextrin, the carbo - hydrates
recently studied.
Prepare 100 c.c. of a 5 per cent, starch paste
in a small beaker, cool to 55 (131 R), and add
10 c.c. of malt extract (for preparation, see p. 87).
Heat the solution in a water-bath to 55 (131 R),
for thirty minutes, when conversion will be com-
plete if the malt extract used is sufficiently active.
While the starch conversion is in course of
preparation, measure 10 c.c. of the malt extract
used for the conversion into a 100 c.c. flask, dilute
with water nearly to the mark, and heat the solu-
tion to 55 (131 R) in the same water-bath as the
starch conversion, and for a similar length of time.
The solution is made in order to correct the specific
PEINCIPLES OF THE MASHING PEOCESS 95
gravity, rotatory power, and reducing power of the
starch conversion for the malt extract used in the
conversion. It is treated in a similar manner to
the starch conversion as it is liable to undergo
change, which renders it desirable that the con-
ditions of experiment should be similar in both
cases.
Both the starch conversion and the check solu-
tion should now be boiled, and the reduced volume
of each made up to 100 c.c. after cooling to 15 '5
(60 F.). After nitration, determine the specific
gravity, rotatory power and reducing power of the
two solutions by the methods already described.
(The reducing power is determined on 5 c.c. of
each solution.) Now correct the different experi-
mental values found for the starch conversion by
subtracting the corresponding experimental values
found for the check solution.
After making the required corrections, proceed
to calculate the weight of starch conversion pro-
ducts present in 100 c.c. of the solution from the
specific gravity of the solution by employing the
appropriate solution factor for "Low Starch Con-
versions," which will be found on referring to
Table II.
Then calculate the [a] D of the conversion pro-
ducts in the usual manner from the weight of
starch conversion products found and the observed
rotatory power of the solution. The value found
should approximate closely to [a] D 150*3, the value
96 STUDIES FOE BKEWING STUDENTS
for a complete conversion of starch into maltose
and dextrin.
From the reducing power of the solution cal-
culate the R, or percentage weight of maltose in
the conversion products, which should approxi-
mate closely to 80'8, the value of E- for a complete
conversion.
Also calculate from the [a] D found the per-
centage weight of maltose and dextrin present in
the starch conversion, employing the values [a] D
138 for maltose and [a] D 202 for dextrin. Com-
pare the amount of maltose found in this manner
with the amount found by the copper reduction
experiment. The two amounts should be in close
agreement.
The transformation products in a starch con-
version possessing an [a] D 150*3 consist of :
Maltose - 80 '8 per cent.
Dextrin - 19-2
100-0
The optical activity of a starch transformation
effected by unrestricted diastase falls rapidly from
[a] D 202, representing the original soluble starch, to
[a] D 150*3 representing a (so-called) complete con-
version, and when it reaches this stage the velocity
of the transformation change is checked. [a] D
150*3, therefore, represents a well-defined point in
the hydrolysis of starch.
An equation representing this change, and com-
PRINCIPLES OF THE MASHING PKOCESS 97
monly called the " No. 8 " equation, is given
below :
[5(C 12 H 20 10 )J + 4H 2 = 4C 12 H 22 O n + C 12 H 20 10 .
Starch. Maltose. Stable Dextrin.
It will be noticed that this equation represents
that four-fifths of the starch molecule is converted
into maltose and one-fifth into stable dextrin ; but
the amount of maltose in the starch transformation
products is not of course four-fifths the weight of
these products, owing to fixation of water during
hydrolysis, hence the proportion 80*8 maltose to
19 - 2 dextrin, in the products of a complete con-
version.
The equation representing the transformation of starch given
above expresses the action in its simplest form, but the starch,
molecule is probably much larger than [5(C 12 H 20 10 )].
Brown and Millar l bring forward evidence to show that the
molecule of stable dextrin is [20(C 12 H 20 10 )], and therefore that,
the molecule of soluble starch must be at least five times aa
large. According to this view, the conversion of soluble starch
into maltose and dextrin may be represented as follows :
(C 12 H 20 O 10 ) 20 .
(O 12 H 20 O 10 ) 20 .
(C 12 H 20 10 ) 20 + 80H 2 = 80C 12 H 22 O n + [20(C 12 H 20 10 )j.
(C 12 H 20 10 ) 20 . Maltose. Stable dextrin.
Soluble starch.
The so-called " stable " dextrin of a starch conversion,
although it strongly resists the action of diastase, is eventually
hydrolysed to maltose and dextrose if the action of the diastase
is very prolonged ; but the velocity of the action is exceedingly
1 See reference to Brown and Millar, p. 93.
7
98 STUDIES FOE BEEWING STUDENTS
slow as compared with the velocity of hydrolysis of starch to mal-
tose and dextrin.
Influence of Heat on the Hydrolysis of Starch
by Diastase. When starch paste is acted on by
diastase under the most favourable conditions for
conversion it has been shown that the mixed pro-
ducts of hydrolysis possess an [a] D 150 '3, corres-
ponding to a mixture of 80*8 per cent, maltose
and 19 - 2 per cent, dextrin. If, however, the action
of diastase on starch is restricted by heat, the [a] D
of the transformation products does not fall so low
as 150'3, indicating the presence of less maltose
and more dextrin in the conversion products.
Prepare 100 c.c. of a 5 per cent, starch paste, as
in the previous experiment, but instead of cooling
to 55 (131 R), cool only to 68 (154'5 F.). Add
10 c.c. of malt extract previously heated to 68, and
keep the mixture in a water-bath at 68 for thirty
minutes.
At the same time prepare a check solution of
malt extract as in the previous experiment and
submit it to the same conditions as the starch
conversion.
Boil the starch conversion and check solution,
cool and make up the reduced volume of each to
100 c.c. Filter, and determine, as in the previous
experiment, the specific gravities, rotatory powers
and reducing powers of the two solutions. Correct
for the check solution, and after determining the
solids present in the starch conversion by means
PBINCIPLES OF THE MASHING PROCESS 99
of the appropriate divisor, calculate the [a] D of
the transformation products.
From the observed [a] D calculate the relative
amounts of maltose and dextrin present, and check
the result by means of a cupric oxide reducing
determination.
It will be found that the [a] D , and therefore
the apparent amount of dextrin, in this experiment
is much higher than in the experiment conducted at
the lower temperature of 55 (131 F.).
Experiment Showing that the Influence of Heat in
restricting Starch Transformation is Due to Modifica-
tion of the Diastase. Digest about 50 c.c. of malt extract in
a water bath at a temperature of 68 (154-5 F.) for thirty minutes.
Note that the solution becomes turbid owing to matter being
thrown out of solution. Cool the solution.
Prepare 100 c.c. of a 5 per cent, starch paste, cool to 55
(131 F.), as in the "low" starch conversion experiment, and
add 10 c.c. of the previously heated malt extract. Proceed with
the conversion exactly as in the "low" starch conversion ex-
periment, making a check solution, as before. Determine the
[a] D of the solution, and compare with the [a] D of the low starch
transformation (p. 95). It will be observed that although both
transformations have been carried on at the same temperature
favouring complete hydrolysis (55), the [a] D of the transformation
with malt extract previously heated indicates that its activity has
been permanently restricted by this treatment.
Experiment Showing that although a Restricted
Starch Transformation may be Represented as a
Mixture of Maltose and Dextrin, much of the
apparent Maltose cannot be Fermented by Ordinary
Yeast and Exists as Malto'Dextrin. This experi-
7*
100 STUDIES FOE BEEWING STUDENTS
ment also introduces a method of analysing high
starch transformation products.
Prepare 500 c.c. of a 5 per cent, starch paste,
and after cooling it to 66 (151 F.), add 50 c.c. of
malt extract previously heated to 66. Keep the
solution in a water-bath at 66 (151 F.) until it is
found on testing that the iodine blue reaction of
starch has disappeared but a red erythro-dextrin
reaction remains. Then check the action of the
diastase by rapidly heating the solution to the boil-
ing point. Boil until the solution is reduced in
volume to less than 500 c.c., and after cooling
make up the volume again to 500 c.c. and filter.
At the same time prepare a check solution by
diluting 25 c.c. of malt extract to 250 c.c. with
water, and proceed as with the starch conversion.
Determine the specific gravities, reducing powers
and rotatory powers of the two solutions. From
the results obtained calculate :
(1) The total solids per 100 c.c., using the ap-
propriate divisor for high starch transformations.
(2) The [a] D of the solids of the starch conversion.
(3) The weight of maltose present in 100 c.c. of
the conversion on the assumption that the whole of
the reducing power is due to maltose.
From the results of the experiment at this
stage, it will be noticed that a high starch con-
version has been obtained, and that the amount
of the reducing matter of the transformation cal-
culated as maltose is known ; but at present there-
PKINCIPLES OF THE MASHING PEOCESS 101
is no means of determining how much of the re-
ducing matter is free maltose and how much is
"apparent" maltose, existing as malto-dextrin or
reducing dextrin.
Demonstrate qualitatively the presence of free maltose by
heating 25 c.c. of the conversion for one hour with 1 grm. of
phenyl-hydrazine dissolved in 1 grm. of 50 per cent, acetic acid.
Note the formation of crystals of maltosazone when the solution
cools. Malto-dextrins do not form crystalline osazones.
Determine the Amount of Free Maltose present
in the Conversion. Measure 350 c.c. of the con-
version into a large flask, add about 3 grms. of
washed pressed yeast, in order to ferment the
maltose, and keep the solution at a temperature of
about 26 (79 F.), until fermentation is complete.
(This part of the experiment usually takes about
forty-eight hours.) As a correction, ferment at
the same time 100 c.c. of the check solution of
diluted malt extract (see p. 100).
When fermentation is complete, boil the two
solutions in order to expel the alcohol formed, and
make them up to their original volumes with water
and a little alumina cream. 1 Filter and determine
the reducing power of each solution. Calculate
the amount of "apparent" maltose present in 100
c.c. of the fermented conversion after correcting
for the check solution.
Note that during fermentation of the starch
conversion the free maltose it contained is de-
1 The alumina cream is employed to aid clarification.
102 STUDIES FOE BREWING STUDENTS
composed, and the " apparent " maltose existing
as malto-dextrin is not fermented ; hence the
difference between the amounts of maltose found
before and after fermentation represents the free
maltose present in the original conversion. Cal-
culate the amount of free maltose in 100 c.c. of
the original conversion.
Demonstrate [qualitatively that free maltose is no longer
present in the fermented conversion by treating 25 c.c. with
phenyl-hydrazine, as before. Note that no crystalline maltos-
azone is formed.
Calculate the Amount of " Apparent " Maltose
in the Starch Conversion which exists as Malto-
dextrin. It has already been shown that the free
maltose originally present in the conversion has
been removed during fermentation, therefore the
reducing power of the fermented conversion, after
correcting for the malt extract used, is due to
"apparent" maltose existing as malto-dextrin.
Calculate the amount of " apparent " maltose
found in 100 c.c. of the conversion.
Determine the Amount of "Apparent" Dextrin
existing as Malto-dextrin. It has already been
shown that the stable dextrin of a low starch
conversion is not hydrolysed (or is only hydro-
lysed with extreme slowness) by malt extract (p.
96), but a marked property of the " apparent"
dextrin of a high starch transformation is the
readiness with which it is hydrolysed to maltose
by unrestricted diastase ; or, expressed in another
PKINCIPLES OF THE MASHING PKOCESS 103
manner, a high starch transformation produced by
restricted diastase is readily hydrolysed to the low
starch transformation (No. 8 equation), containing
maltose and stable dextrin only, by the further
action of active diastase.
The following example explains the general character of the
action during a restricted conversion :
Soluble Starch. Maltose.
100(C 12 H 20 10 ) + 60 H 2 = 40C 12 H 22 O n
Malto-dextrin. Stable Dextrin.
On the further addition of unrestricted diastase to the restricted
conversion the malto-dextrin only is acted on :
Malto-dextrin. Maltose.
Ol 2 H 22 O n
O
u 1
As a final result, therefore, the original restricted conversion is
brought down to the low starch transformation, [a] D 150'3, repre-
sented by :
80C 12 H 22 O n + 20(C 12 H 20 lp )
Maltose. Stable Dextrin.
To 200 c.c. of the fermented conversion, add
10 c.c. of malt extract and digest the mixture in
a water-bath at 55 (131 F.) for one hour. The
malto-dextrins present will thus be converted into
free maltose.
Make a check solution by adding 5 c.c. of malt
extract to 100 c.c. of water, and treat in a similar
manner to the conversion.
Boil, cool, and make up the two solutions to
200 c.c. and 100 c.c. respectively with water.
104 STUDIES FOE BKEWING STUDENTS
Filter and determine the reducing power of both
solutions. The reducing power of the degraded
solution corrected for the malt extract used, less
the reducing power of the solution previous to
degrading (not corrected for malt extract), is due
to maltose derived from the "apparent" dextrin
which existed as malto-dextrin.
Calculate the amount per 100 c.c., first as mal-
tose, and afterwards as the equivalent amount of
dextrin, according to the equation :
Dextrin. Maltose.
C 12 H 2<Ao + H 2 = C 12 H 2211
Constitution of the Malto-Dextrin Found. As
the amounts of "apparent" maltose and dextrin
existing as malto-dextrin in 100 c.c. of the starch
conversion have now been determined, it is now
possible to express in a simple form the proportion
in which they appear to be combined (e.g., 1'5 mal-
tose, 1 dextrin). The proportion is sometimes re-
garded as indicating the type of malto-dextrin found.
Note that the proportion of " apparent " maltose to " ap-
parent " dextrin found by the above method of experiment may
only express the proportion of a mixture of malto-dextrins of
varying reducing power.
Determine the Amount of Stable Dextrin present
in the Conversion. Measure 150 c.c. of the degraded
conversion (see above) into a flask, add about 1*5
grms. of washed pressed yeast, and allow the solution
to ferment at a temperature of 26 (79 F.), until the
whole of the maltose present is decomposed. Boil
PKINCIPLES OF THE MASHING PKOCESS 105
the solution to expel the alcohol formed, cool and
make up to the original volume of 150 c.c. Filter
and determine the rotatory power of the solution.
Measure 100 c.c. of the fermented solution into
a small flask, add 5 c.c. of malt extract and about
1 grm. of pressed yeast, and keep the flask at a
temperature of 26 (79 F.) until all signs of fer-
mentation have disappeared. Also make a check
solution of 100 c.c. water, 5 c.c. malt extract and
1 grm. pressed yeast, and ferment as above.
Note that in this experiment stable dextrin, which
is not fermented by yeast under ordinary conditions,
has been submitted to the joint action of diastase and
yeast. Neither of these agents alone is able to hy-
drolyse stable dextrin, the resisting power of which is
however overcome by the joint action of the two, with
the result that the dextrin is fermented.
Boil the fermented conversion and the check
solution to expel the alcohol formed, cool and
make both up to the original volume of 100 c.c.
Filter, determine the rotatory power of both solu-
tions, and correct the starch conversion result for
the malt extract used. The stable dextrin may
now be calculated from the loss in rotatory power
of the conversion during fermentation in the pre-
sence of malt extract.
When the analysis of the high starch trans-
formation is completed, express the results as
amounts of maltose, malto-dextrin, and stable
dextrin in 100 c.c. of the conversion.
106 STUDIES FOE BEEWING STUDENTS
PAET HI.
Studies Bearing Directly on the Technology of
Brewing.
A Study of the Influence of the Mashing Tenv
perature on the Optical Activity of Malt Worts.
Take two beakers, each containing 250 c.c. of water
heated to 148 F. and 161 F. respectively, and
mash into each beaker 50 grms. of ground pale
malt (from the same sample). The mashes should
then have "initial" heats of 144 F. and 157 F.,
and they must be kept at these temperatures for
one hour by being placed in separate water baths
heated to the required temperatures. Cool, filter,
and determine the specific gravities and rotatory
powers of the two worts. Use the 3 '86 factor to
determine the weight of solids present, and cal-
culate the [a] D of the two worts.
Note that the specific rotation of the wort from
the high temperature mash is higher than that from
the low, indicating the presence of more dextrin in
the former than in the latter. The reason for this
will be found on considering the results of the ex-
periments already made with high and low starch
transformations.
Measure 100 c.c. of each of the two worts into
separate flasks, boil for twenty minutes to destroy
the diastase present, cool, and make up the volumes
again to 100 c.c. Add about 0'5 grm. of pressed
PBINCIPLES OF THE MASHING PEOCESS 107
yeast to each solution and ferment in a warm place.
When fermentation is complete, filter, and deter-
mine the specific gravity or "attenuation" of the
fermented worts. Note that the specific gravity
increases with the mashing temperature and [a] D
of the original wort, indicating that the high
temperature mash contains a larger amount of
unfermentable matter than the low temperature
one. Consider this in the light of the experiment
made with a high starch conversion.
The divisor 3 - 86 is employed in the above experiments as it
is customary to use it in determinations of the [a] D of brewery
worts. The divisor (3 - 86) was originally obtained from experi-
ments with cane sugar and the divisor 4 '00 is more accurate for
malt wort solids, but custom at present decides in favour of 3 '86,
and it is employed here for this reason. As the chief value of
determinations of the [a] D of worts lies in their use for purposes
of comparison, the employment of the incorrect 3 '86 factor is of
little moment so long as its use is indicated in the proper manner,
6'9'> WD3-86-
The student is often surprised to find, on first determining
the [a] D of a malt wort, that it is much lower than the [a] D of a
low starch transformation (150-3), although it represents a con-
version restricted by heat. The low [a] D is occasioned by the
presence in malt wort of bodies other than starch transformation
products which, as a whole, possess little or no rotatory power.
Experiments on the Fermentation of Boiled and
Unboiled Worts, Illustrating the Different Methods
of the Brewer and the Distiller or Vinegar Maker.
Mash 100 grms. of ground pale malt in a beaker
with 500 c.c. of water at a temperature of 154 F.
and keep the mash at a temperature of 150 F. for
108 STUDIES FOE BEEWING STUDENTS
one hour. Filter, and measure out two samples of
100 c.c. of the wort into two flasks. Boil one
sample for twenty minutes to destroy the activity
of the diastase present ; cool, and make up to the
original volume of 100 c.c. Add 0'5 grm. of pressed
yeast to both the boiled and unboiled worts, and
keep them at a temperature of about 26 (79 F.)
until fermentation is complete. In the case of the
unboiled wort the fermentation may take three or
four days. Filter the fermented worts and de-
termine their specific gravities ("attenuations").
Note that the specific gravity of the boiled and
fermented wort is much higher than that of the
unboiled one. It has already been shown by a
previous experiment (p. 105) that the malto-dex-
trins and stable dextrin are fermented by yeast in
the presence of active diastase ; during the fermen-
tation of the unboiled wort this action has gone on,
leading to the fermentation of a larger proportion
of malt wort solids than in the boiled wort in which
the malto-dextrins and stable dextrin remain un-
fermented. Fermentation of an unboiled wort,
therefore, tends to yield the highest possible
amount of alcohol the object of the distiller and
vinegar maker. Fermentation of a boiled wort,
on the other hand, yields a smaller amount of
alcohol, but a liquid containing malto-dextrin and
dextrin the object of the brewer.
A Study of the Rise in Temperature observed
when mixing Dry Malt with Cold Water. Weigh
PEINCIPLES OF THE MASHING PKOCESS 109
25 grms. of finely ground, well-dried malt in a dry
100 c.c. beaker, and cover the beaker with a watch-
glass. At the same time measure 60 c.c. of cold
water into another beaker of similar size. Place
the two beakers side by side, and allow them to
remain so for about an hour in order that the con-
tents of both may attain the temperature of the
laboratory.
Note that the quantities of malt and water
taken are in the same proportion as those com-
monly employed in technical mashing operations,
and that the malt and the water before mixing are
at the same temperature.
Ascertain the temperature of the water with
a chemical thermometer, and then pour the water
into the beaker containing the dry malt and mix
thoroughly, using the thermometer for stirring. As
the mixing proceeds notice the rise of temperature
taking place in the mash, and continue stirring the
mash until the maximum temperature is reached,
which will probably be observed in two or three
minutes.
If the sample of malt experimented on is very
dry, the observed rise in temperature may be as
much as 8 F. ; if the malt is very slack the rise
may possibly not exceed 4 F. The rise in tem-
perature is due to a loose molecular combination
of the water with the starch and other constituents
of the malt. Note that the evolution of heat ob-
served in a cold mash must take place equally
110 STUDIES FOE SKEWING STUDENTS
during the preparation of a hot mash in the
brewery, and therefore that the relative dryness
of malts must exert a marked influence on the
" initial " heats obtained in brewery mashing
operations.
Analysis of a Fermented Wort or Beer. The
operations involved in an analysis of a fermented
wort or beer may be subdivided into two parts :
1. A determination of the " original gravity,"
and the amount of alcohol present in the sample.
2. An analysis of the unfermented matter in the
sample.
1. The term " original gravity " as applied to a
fermented wort or beer means the specific gravity
of the original wort before fermentation.
The method very generally employed in this
country for the purpose of determining the original
gravity of fermented liquids is one originated by
the excise authorities and recognised by the excise
laws. The method is based on the following con-
siderations. :
During the fermentation of a wort the ferment-
able matter it contains is decomposed into alcohol
and carbon dioxide, the former remaining in the
fermented liquid, the latter being evolved as a gas.
As a consequence, the specific gravity (or original
gravity) of the liquid diminishes during fermenta-
tion, for not only does the liquid lose matter which
originally contributed towards its weight, but this
PEINCIPLES OF THE MASHING PEOCESS 111
matter is replaced by alcohol a liquid having a
less density than water which further tends to
diminish the specific gravity of the fermented wort.
The specific gravity of a fermented wort represents
therefore the weight in solution of neither the
unfermented matter present nor the alcohol, for,
since the density of the one is greater than water
and that of the other is less, the joint influence
of the two tends to obscure the weight of both.
If, however, the alcohol in a measured volume of
the solution is separated by distillation from the
non-fermented matter and the volumes of both the
distillate and the non-volatile residue are made
up to the original volume, then the specific gravity
of the distillate expresses the weight in solution of
the alcohol present, and that of the residue ex-
presses the weight in solution of the unfermented
matter present.
It will now be evident that if the solution
weight of the fermented matter can be deter-
mined from the weight of the alcohol, this weight
added to the solution weight of the unfermented
matter found will express the original gravity of
the wort before fermentation. Although the solu-
tion weight of the fermented matter cannot be
determined satisfactorily from the alcohol by direct
calculation, it may be arrived at by means of a
special table based on the results of a series of
direct experiments on fermenting wort in which
the solution weights of fermented matter corres-
112 STUDIES FOE BEEWING STUDENTS
ponding to varying amounts of alcohol have been
determined (see Table V.).
Determination of the Original Gravity of a Fer-
mented Wort or Beer. If the beer contains car-
bon dioxide, remove it by agitation or nitration.
Measure 100 c.c. of the beer in a graduated flask
at the standard temperature of 60 F. Pour the
measured volume into the distilling flask of an
original gravity distilling apparatus, and afterwards
rinse the measure with about 40 c.c. of water and
transfer the washings to the distilling flask. Distil
the fermented wort, collecting the distillate in a
100 c.c. measure until 80 to 90 c.c. have passed
over, when the whole of the alcohol will be present
in the distillate. Make up the distillate with water
to 100 c.c. Cool the residue in the distilling flask
and transfer it together with the washings of the
flask to a 100 c.c. measure, and makeup the volume
with water to 100 c.c. Determine accurately the
specific gravity of the two solutions at 60 F.
The specific gravity of the residue represents
the weight in solution of the unfermentable matter
in the original wort. The specific gravity of the
distillate represents the fermented matter expressed
as the weight of a mixture of alcohol and water.
In order to find the amount of fermented matter,
subtract the specific gravity of the alcoholic distil-
late from 1,000 and the difference is the so-called
"spirit indication" number. Refer to Table V.,
and find by means of the " spirit indication " num-
PKINCIPLES OF THE MASHING PEOCESS 113
ber the number of " degrees" of specific gravity
lost during fermentation. Add this number to the
specific gravity of the unfermented matter, and the
sum is supposed to represent the original gravity of
the fermented wort.
It should be noted, however, that this method
requires correction if the acidity of the beer ex-
perimented on exceeds 0*1 per cent, calculated as
acetic acid. It is assumed, somewhat incorrectly,
that any excess of acidity over O'l per cent, is
formed at the expense of the alcohol in the beer,
and that consequently the original gravity will ap-
pear too low if the excess of acid is not allowed for.
To correct for the acidity of a beer, determine
the amount of acid present by titration with deci-
normal ammonia solution and litmus-paper, and
calculate as acetic acid. Subtract O'l per cent.,
which is allowed for in the ordinary spirit indication
table, and refer the difference to Table VI., which has
been constructed for the purpose of indicating the
correction due to the excess acid found. Add the
number found to the spirit indication figure, and
proceed with the calculation as before.
The method of determining original gravities
just described, although adopted officially as the
standard method for this country, is not very accu-
rate. When it is employed for the purpose of
ascertaining the original gravities of finished beers
of medium gravity the results obtained are usually
about two degrees too low, and a correction for
114 STUDIES FOE BKEWING STUDENTS
the error should be employed. With fermenting
worts containing yeast and little alcohol the re-
sults are, however, fairly accurate.
Determination of the Amount of Alcohol in a,
Fermented Wort or Beer. Refer the specific
gravity of the distillate (p. 112) to alcohol tables
showing the weight of alcohol corresponding to
varying specific gravities of aqueous spirit, and from
the weight found calculate the weight of alcohol
present in 100 c.c. of the original beer.
2. Analysis of the Matter remaining Unfer-
tnented in a Fermented Wort or Beer. - - The
analysis is conducted in a similar manner to that
of a high starch conversion (p. 99), but is subject
to correction for the optical activity and reducing
power of certain constituents other than maltose,
malto-dextrin and dextrin.
Boil 500 c.c. of the fermented wort until its
volume is reduced to about one-half in order to
expel the alcohol present. Cool, and make up the
residue to its original volume with water. Deter-
mine the specific gravity of the solution, and cal-
culate the grms. of solid matter present in 100 c.c.,
using the 4*0 factor.
Determine the rotatory power (in 100 mm.) and
reducing power of the solution, and express the
reducing power as grms. maltose in 100 c.c.
Determine the Amount of Free Maltose in the
Solution. Ferment 250 c.c. of the solution with
2 grms. of pressed yeast. Boil the solution to
PEINCIPLES OF THE MASHING PEOCESS 115
expel alcohol, and make up to the original volume
with water and a little alumina cream. Filter,
and determine the reducing power of the solution.
Calculate the reducing power as maltose, and ex-
press as grms. maltose in 100 c.c.
The difference in the amount of maltose found
in 100 c.c. of the solution before and after fermen-
tation represents the free maltose in 100 c.c. of the
solution. (Note that possibly some of the maltose
found may have existed as low type, easily fer-
mentable, malto-dextrin. )
Determine the Amount of "Apparent" Maltose
combined as Malto- Dextrin. The reducing power of
the fermented solution, less the correction for the
reducing bodies other than sugar, is due to "appa-
rent " maltose combined as malto-dextrin. Express
as grms. combined maltose in 100 c.c. (Note that
the correction for the reducing bodies other than
combined maltose is determined at a later stage of
the experiment.)
Determine the Amount of "Apparent" Dextrin
combined as Malto-Dextrin. Treat 200 c.c. of the
fermented solution with 10 c.c. malt extract at 50
(122 F.) for one hour. Also make a check solu-
tion of 5 c.c. malt extract with 100 c.c. water,
and treat in a similar manner to the fermented
solution. Proceed as in the analysis of a high
starch conversion (see p. 102), and calculate the
.amount of "apparent" dextrin as malto-dextrin in
100 c.c. of the solution.
8*
116 STUDIES FOE BEEWING STUDENTS
Also calculate the constitution of the malto-
dextrin found (see p. 104).
Determine the Amount of Stable Dextrin. Fer-
ment 150 c.c. of the degraded conversion in order
to remove the free maltose present ; boil, cool and
make up to the original volume.
Determine the rotatory power of the solution in
a 100 mm. tube.
To 100 c.c. of the solution, add 5 c.c. of malt
extract, and ferment with about 1 grm. of freshly
pressed yeast to decompose the stable dextrin
present. Also prepare a check solution with 5 c.c.
of malt extract in 100 c.c. water, and ferment as
above. When fermentation is complete in both
solutions, boil to expel the alcohol present, and
make up in each case to 100 c.c. after adding a
little alumina cream. Filter, and determine the
rotatory powers (in 100 mm.) and reducing powers
of both solutions. Correct the rotatory power and
reducing power of the fermented solution for those
of the malt extract used.
Note that the remaining reducing power is due
to constituents other than maltose and must be
used as a correction in the determination of the
"apparent" maltose combined as malto-dextrin
(see p. 102).
The stable dextrin may now be determined from
the difference in rotatory power of the solution
before and after fermentation in the presence of
malt extract.
ANALYSIS OF BEEWING SUGARS 117
Express the results of the analysis in grms. per
100 c.c., the difference between the weight of total
solids and the sum of the weights of maltose,
malto-dextrin and stable dextrin found being ex-
pressed as " undetermined matter ".
PAET IV.
Analysis of Brewing Sugars.
The most satisfactory method at present avail-
able for the purpose of analysing brewing sugars is
one suggested by Dr. Gr. H. Morris (Journal of the
Federated Institutes of Brewing, 1898, vol. iv., p.
162). The method is described below, but the
student is recommended to consult Dr. Morris's
original paper for full information.
Analysis of Invert'Sugars. The analysis in-
cludes determinations of the ash, albuminoids,
water, dextrose, levulose, cane-sugar, unferment-
able matter and the brewer's extract.
Ash. Weigh out about 5 grms. of the sample
in a 50 c.c. platinum dish, add 2 c.c. of strong
sulphuric acid, and heat gently over a Bunsen
burner ; when the first violent intumescence has
subsided, place the dish with its charred contents
in a muffle furnace, and heat until the residue is
quite white. Cool in a desiccator, and weigh.
Calculate the percentage amount of sulphated ash
found.
118 STUDIES FOK BKEWING STUDENTS
Albuminoids. Weigh about 2 grms. of the
sample in a 100 c.c. Jena glass beaker or flask, and
treat with 20 c.c. concentrated sulphuric acid.
Add 5 grms. of potassium sulphate and heat the
mixture, at first very gently, and afterwards
strongly, until decolourisation is complete. Pro-
ceed subsequently to determine the nitrogen pres-
ent according to the usual Kjeldahl method, and
calculate the percentage of albuminoids found,
using the 6*3 factor (see p. 59).
Brewer's Extract and Water. Dissolve 25 grms.
of the sugar in boiling water, cool the solution and
make up to 250 c.c. at 15 '5 (60 F.), Determine
the specific gravity of the 10 per cent, solution thus
obtained.
Calculate the Brewer's Extract. The specific
gravity, by the removal of the decimal point one
place to the left, indicates the weight of 100 c.c.
of the solution, and the excess weight of this volume
above 100 grms. indicates the weight in solution of
10 grms. of the sugar.
From the amount found calculate the weight in
solution of 224 grms. of the sugar, and express the
result as pounds of brewer's extract derived from
224 Ib. (or 2 cwt.) of the sugar.
Calculate the Water. Determine the solid
matter present in 100 c.c. of the 10 per cent, solu-
tion from its specific gravity by means of the 3*86
divisor. The amount of solids found requires,
however, to be corrected for the high solution
ANALYSIS OF BEEWING SUGARS 119
density of the ash present, for the ash of sugars has
a solution density rather more than twice that of
the carbo-hydrates, i.e., the divisor for the ash is
about 8. The most convenient way of correcting
for the ash is to multiply the solid matter found in
100 c.c. by 10, in order to convert it into a per-
centage, and then to deduct from the amount the
percentage of ash already found. The percentage
weight of solid matter obtained in this manner, de-
ducted from 100, gives the percentage weight of
water in the sugar.
Reducing Sugars. Dilute 10 c.c. of the 10 per
cent, sugar solution used in the above experiment
to 50 c.c., and take 10 c.c. of the diluted solution
for an estimation of the reducing power, the opera-
tion being conducted in accordance with the usual
standard conditions (see p. 79). In this way the
amount of copper reduced by 0*2 grms. of the sugar
is obtained. Calculate from the amount found the
quantity reduced by 100 grms. of the sugar by
multiplying by 500. This value then requires cor-
recting for the reducing power possessed by the
unfermentable residue of the sugar, which is subse-
quently determined ; it then expresses the reducing
power of the dextrose and levulose in 100 grms. of
the sugar.
The rotatory power of the 10 per cent, sugar
solution is then determined in the half-shadow
instrument at a temperature of 20 (68 F.), and
is expressed in divisions, care being taken that the
120 STUDIES FOE BEEWING STUDENTS
bi-rotation of the sugar solution has been previously
destroyed. But the value found requires correcting
for the opticity of the cane-sugar and that of the
unfermentable matter present in the sugar, which
are subsequently determined. The corrected value
in divisions of the half-shadow instrument is then
employed to calculate the [a] D of the sugar on the
basis that the solution contains 10 grms. in 100 c.c.
The percentages of the dextrose and levulose
in the sugar are now calculated by means of a
pair of simultaneous equations in which :
xD = the gram, value of dextrose expressed as Cu.
xLi = the gram, value of levulose expressed as Cu.
a = the Cu. reduced by 100 grms. of the sugar.
b the specific rotatory power ([a] D ).
The equations then are :
xD + xlj = a
4- [a] D L = b X 100.
Cane-Sugar. In order to determine the amount
of uninverted cane-sugar, if any, present in the
invert-sugar, 50 c.c. of the 10 per cent, solution are
measured into a double-marked 50 to 55 c.c. flask
at 15-5 (60 F.), and digested with 0'5 grm. of
pressed yeast for six hours at 50 (122 F.). The
solution is then cooled to 15 '5 (60 F.), a little
alumina cream added, made up to 55 c.c. and
filtered. The rotation of the solution in the 200
mm. tube is then determined at 20 (68 F.), in
divisions of the half - shadow polarimeter. The
ANALYSIS OF BKEWING SUGAKS 121
reading is increased in the proportion of 55 to 50
in order to correct for the dilution of the solution,
and is then subtracted from the original reading of
the solution (in a 200 mm. tube) before inversion.
The difference, divided by 5*02, gives the cane-
sugar ; and this, multiplied by 10, gives the per-
centage of cane-sugar in the sugar analysed.
The divisor 5*02 used in the above experiment is the num-
ber of divisions of the half-shadow polarimeter which a solution
of 1 grm. of cane-sugar in 100 c.c. of water, read in a 200 mm.
tube, loses on being converted into invert-sugar.
Un fermentable Matter. 50 c.c. of the 10 per
cent, solution of the sugar are placed in a 100 c.c.
flask, and sterilised by boiling for a few minutes.
After cooling, 2 grins, of washed and pressed yeast
are added, and the mixture is allowed to ferment
at about 24 (75 R). When fermentation is com-
plete, which will probably be in about forty-eight
hours, a little alumina is added to the fermented
liquid and its volume is made up to 100 c.c. at 15 '5
(60 R).
After nitration, the reducing power is deter-
mined on 25 c.c. of the solution, and the rotation
read in the 200 mm. tube. The values so obtained
are then expressed in the same manner as those of
the original solution for the purpose of correcting
the reducing sugar values as already described (see
p. 119).
The percentage weight of the unfermentable
matter in the invert-sugar is usually obtained by
122 STUDIES FOE BKEWING STUDENTS
difference ; i.e., by subtracting from 100 the total
weight of the sugars, ash, albuminoids and water
found.
The following example illustrates the method of
analysing an invert-sugar described above :
Analysis of an Invert'Sugar.
Ash. A direct estimation showed the presence
of 1*54 per cent, of ash.
Albuminoids. A determination by Kjeldahl's
method, as described (p. 118), gave 0*24 per cent,
of albuminoids.
Water. The specific gravity of the 10 per cent,
solution of the sugar was 1031*06, which indicates,
by the use of 3 '86 as a divisor, the presence of 8*046
grms. of solids per 100 c.c. Multiplying this by 10
gives 80*46 as the percentage weight of solid mat-
ter in the sugar. But this amount has to be
corrected for the solution density of the ash (see
p. 119). To do so, the percentage weight found,
1*54, is subtracted from 80*46, leaving 78*92 as
the corrected percentage weight of solids in the
sugar. Then 100 - 78*92 = 21*08, the percentage
weight of water in the sugar.
Cane-Sugar . The 10 per cent, sugar solution
before inversion gave a reading of - 5*25 divisions
of the half-shadow polarimeter in the 200 mm. tube.
After inversion and treatment as described above,
the reading corrected for dilution was - 5*77 divi-
sions in the 200 mm. tube. Hence the difference
in the readings before and after inversion is 0*52
ANALYSIS OF BKEWING SUGAES 123
division, due to the conversion of cane-sugar into
invert-sugar. Then
052 x 10
= 1'03 per cent, cane-sugar. 1
Unfermentable Residue. After fermentation of
the 10 per cent, sugar solution and treatment as
described above (p. 121), the reading in the 200
mm. tube was - O'lO division, and 25 c.c. reduced
0-032 grm. of Cu.
Redwing Sugars. Ten c.c. of the 10 per cent,
solution of the invert-sugar were diluted to 50 c.c.,
and 10 c.c. of the diluted solution reduced 0'2759
grm. Cu. The rotation in the 200 mm. tube of the
10 per cent, solution was - 5 '25 divisions.
The reduced copper, 0'2759 grm., multiplied by
500 gives the amount of copper which would be re-
duced by 100 grms. of the original invert-sugar,
viz., 137 '95 grms. Cu. But this amount has to be
corrected for the amount of copper reduced by the
unfermentable residue. Twenty-five c.c. of the
half-diluted fermented solution reduced 0'032 grm.
Cu. Therefore 0'032 x 80 = 2'56 grms. Cu. re-
duced by the unfermentable matter in 100 grms.
of the sugar.
Hence 137 '95 - 2'56 = 135*39 grms. Cu. reduced
by the dextrose and levulose in 100 grms. of the
invert-sugar.
The observed rotatory power of the 10 per cent,
solution in the 200 mm. tube has now to be corrected
1 See p. 121.
124 STUDIES FOR BREWING STUDENTS
for the rotatory power of the cane-sugar and the
unfermentable matter present. 0103 grm. of cane-
sugar were found in 100 c.c. of the solution, and
the calculated rotation for this amount in the 200
mm. tube is 0*39 division ; the corrected reading
found for the unfermentable residue was -0*2 divi-
sion in the 200 mm. tube. Hence the rotation due
to the cane-sugar is added to, and that of the
unfermentable residue is subtracted from, the ori-
ginal reading (- 5*25), leaving a corrected reading
of - 5 '44 divisions, which gives on calculation an
[a] D - 9-44.
On referring to Table III., it is found that with
a reduction of 0*2759 grm. of Cu. the grm. value of
dextrose is 1*975, and that of levulose 1*814; the
simultaneous equations for calculating the per-
centages of dextrose and levulose in the original
invert-sugar then become :
1-975 D + 1-814 L =135-39 } f Dextrose, 38-65 p.c.
52-8 D + ( -92-OL) = - 9-44 x 100 J = (Levulose, 32-44
The result of the complete analysis of the invert-
sugar is therefore :
Dextrose - - 38'65 per cent.
Levulose 32-44 ,,
Cane-sugar - 1-03 ,,
Ash 1-54
Albuminoids - 0-24,,
Water - 21-08 ,,
Unfermentable matter (by difference) 5 - 02 ,,
100-00
ANALYSIS OF BEEWING SUGAKS 125
Analysis of Glucose or Starch Sugar. The
analysis is carried out in the same manner as the
analysis of invert-sugar described above, but the
determination of the cane-sugar is omitted as this
sugar does not occur in sugars prepared from
starch. When calculating the reducing sugars
present the constant for maltose is substituted for
that of levulose.
Thus, in an analysis of a glucose, in which 1-45
per cent, of ash, 0*97 per cent, of albuminoids and
1570 per cent, of water were obtained, the Cu.
reduced by 100 grms. of the glucose was found to
be, after due correction, 121*97 grms., and the cor-
rected opticity [a] D 41 *35. The simultaneous equa-
tions for calculating the percentage amounts of
dextrose and maltose then become :
1-979 D + 1-089 M = 121-97 \ _ f Dextrose, 57'16 p.c.
52-8 D + 138-0 M = 41-35 x 100 J " \Maltose, 8-09
The result of |the complete analysis of the
glucose is therefore :
Dextrose 57 '16 percent.
Maltose - 809
Ash 1-45
Albuminoids - 0'97 ,,
Water - 15-70
Unfermentable matter (by difference) 16-63
100-00
SECTION III.
FEEMENTATION.
INTRODUCTION.
THE following studies are divided into two parts-
one concerned more especially with the physiologi-
cal aspect of fermentation, and the other with the
morphology and life history of the more important
micro-organisms of fermentation and with the
special methods employed in their examination ;
but the division is a somewhat arbitrary one, and
many points relating to the first part are of neces-
sity studied in the second.
In the earlier courses of study described in this
book it was found necessary to enter into detail
regarding much of the experimental work, as
there was no text-book covering the whole of the
required ground to which the student could be
referred for information ; but there are several
text-books on fermentation available, and in the
following studies it is proposed to refer to these
whenever possible in order to avoid enlarging the
present work unnecessarily. For this reason the
space devoted to the study of fermentation may
126
FERMENTATION 127
appear limited when compared to that devoted to
other studies, but the student must not be misled
by this and underrate the length of time required
for his fermentation work, for he will perhaps find
it necessary to devote more time to it than to any
of his previous studies.
PAET I.
The Physiological Aspect of Fermentation.
It is not practicable for a student in the time
usually at his disposal to study the physiological
aspect of fermentation in the laboratory in any-
thing like a systematic manner, for the subject is
a very large one, and experiments connected with
it take up much time. The following short course
of experiments must therefore be regarded as merely
an introduction to the experimental side of the
subject. It is expected that the student will have
become familiar by means of lectures and books
with the modern views of fermentation and the life
history of fermentation organisms before com-
mencing the experiments described ; following on
this, the short course of experimental work will
then put him in touch practically with the general
bearings of the subject.
Determination of the Amount of Alcohol and
Carbon Dioxide produced during the Fermentation
of Sugar by Yeast. Prepare a small, light flask
of about 200 c.c. capacity with a perforated rubber
128 STUDIES FOE BKEWING STUDENTS
stopper furnished with a chloride of calcium tube
and a side tube as represented in the annexed
figure, and provide the side tube with a removable
stopper made with rubber tu-
bing and a small piece of glass
rod. When constructing the
apparatus, note that it must
be made of such a size as to
admit of it being placed on
the pan of a chemical balance.
Weigh the apparatus accu-
rately on the balance, and
then introduce about 8 grms.
of powdered pure dry cane-
sugar into the flask and re-
weigh to ascertain the weight of
sugar taken. Introduce about
90 c.c. of yeast water (for pre-
paration of yeast water, see p. 145), and shake the
flask gently until the sugar is dissolved. Add to the
solution about 0'5 grm. of pressed yeast rendered
liquid with a little yeast water, and re-weigh the
apparatus immediately. After weighing, place the
apparatus in an incubator kept at a temperature of
about 24 (75 F.). Active fermentation will com-
mence in a few hours, and it is advisable in the
earlier stage of fermentation to note if there is any
danger of the frothy " head " rising into the neck
of the flask. If it appears likely for this to occur
the apparatus should be removed to a cooler place
FIG. 19. Apparatus for the
Determination of Carbon
Dioxide and Alcohol Gen-
erated during Alcoholic
Fermentation.
FEKMENTATION 129
until the more active stage of fermentation is past.
When fermentation of the sugar in the flask is com-
plete, which is usually the case in about sixty hours,
remove the cap from the side tube and attach some
form of aspirator to the drying tube of the apparatus
by means of rubber tubing, and draw a current of
air slowly through the liquid in the flask in order
to expel the carbon dioxide present. Disconnect
the aspirator and weigh the apparatus. Again draw
more air through the flask and again weigh, and,
if required, repeat the operation until a constant
weight is obtained, showing that all the carbon
dioxide has been removed. When this operation
is complete, the difference between the weight of
the apparatus before and after fermentation rep-
resents the weight of carbon dioxide liberated
during the fermentation of the cane-sugar origin-
ally taken.
In order to determine the amount of alcohol
formed during fermentation, transfer the fermented
solution and rinsings of the flask to the flask of an
original gravity still, and distil over about 90 c.c.
into a 100 c.c. flask. Make up the volume of the
distillate to 100 c.c. at 15*5 and determine its
specific gravity accurately. Refer the specific
gravity found to a table 1 giving the densities of
mixtures of alcohol and water, and calculate the
weight of alcohol present in the distillate. The
weight found represents the amount of alcohol
1 See Hehner's alcohol tables.
9
130 STUDIES FOR BREWING STUDENTS
formed during fermentation of the cane-sugar
originally taken.
Calculate the percentage weights of carbon
dioxide and alcohol obtained from cane-sugar, and
compare with those obtained by Pasteur :
Carbon dioxide - - 49'42 per cent.
Alcohol 51-11
The amounts found by the method of experi-
ment described above usually agree with Pasteur's
figures within 0*5 per cent.
The student should now calculate the percentage
amounts of carbon dioxide and alcohol which would
be obtained from cane-sugar if it fermented com-
pletely according to the equation :
C 12 H 22 O n + H 2 = 4C0 2 + 4C 2 H 5 HO.
Determination of Glycerin as a Secondary Product
of Alcoholic Fermentation. The residual liquid obtained in
the previous experiment after removal of the alcohol may be
used for this determination, but the result obtained with such
a small volume of liquid is not usually very satisfactory. It
is better to ferment 25 grms. of cane-sugar dissolved in about
250 c.c. of yeast water for the special purpose of the determina-
tion.
Evaporate the fermented solution in a porcelain dish on a
water bath to a volume of about 50 c.c. Add 20 grms. of pow-
dered animal charcoal to the liquid, mix well and evaporate the
mixture to dryness at a temperature of about 70 C. Transfer
the whole of the dried residue to a mortar, add 20 grms. of
quicklime, and triturate the mixture until it is reduced to a fine
grey powder. The object of adding the lime is to thoroughly
dry the mixture, and also to neutralise and render insoluble the
FEEMENTATION 131
succinic acid present. Transfer the whole of the powder to a
small dry flask, and add about 80 c.c. of specially dried ethyl acetate
free from alcohol. Shake the mixture well for several minutes,
and after allowing the precipitate to settle, decant off the clear
supernatant liquid and pass it through a filter. Add another
80 c.c. of ethyl acetate to the residue, repeat the shaking and
transfer the whole of the mixture to the filter. After the liquid
has drained through, wash the precipitate with a little more
ethyl acetate. The mixed filtrates now contain all the glycerin.
Transfer to a small flask and distil off the ethyl acetate by heating
in a water bath until about 30 c.c. of solution are left. Transfer
to a small tared porcelain dish, and evaporate in an air bath at
60 C., until the weight is constant. The residue, glycerin, should
be of a light yellow colour, and possess a pure sweet taste.
Calculate the percentage weight of glycerin formed during
the fermentation of cane-sugar.
Chemical Composition of Yeast, Freshly
pressed brewer's yeast, if it is sufficiently dry
to crumble, may be used for the following experi-
ments ; but if ordinary pressed yeast in good
condition is not obtainable, liquid yeast may be
strained on a cloth filter, and the filter and pasty
yeast placed in the folds of a strong cloth and
submitted to pressure by twisting the ends of the
cloth in opposite directions with considerable force.
By this means yeast may be obtained as a dry
crumbly mass. Pressed yeast may be regarded as
a mass of yeast cells separated from the liquid in
which they were originally contained.
1. Determination of Water in Yeast. Weigh
about 075 grm. of finely crumbled pressed yeast
in a drying tube. Transfer the tube to a drying
132 STUDIES FOE BREWING STUDENTS
oven, raise the temperature very slowly to 100,
and continue drying at this temperature until the
weight is constant. From the weights obtained
before and after drying calculate the percentage
weight of water in the yeast.
2. Determination of the Ash of Yeast. Weigh
10 grms. of pressed finely crumbled yeast in a
platinum dish, and dry it slowly on a water-bath.
When dry, ignite the yeast gently with a Bunsen
flame until the residue is thoroughly carbonised,
and afterwards ignite more strongly in a muffle
furnace until a very light grey ash is obtained.
Weigh, and calculate the percentage weight of ash
in the pressed yeast taken. Also calculate the
percentage weight of ash in dried yeast, employing
for this purpose the result obtained in the previous
experiment.
Test portions of the ash qualitatively for the
presence of phosphoric acid, potash and magnesia,
of which it is principally composed.
3. Determination of Nitrogen in Yeast (Kjel-
dahl's method, see p. 55). Weigh very accurately
about 0*25 grm. of pressed yeast in a suitable Jena
beaker or flask. Heat with sulphuric acid and
potassium sulphate, and proceed in the usual
manner of a Kjeldahl determination. From the
amount of nitrogen obtained calculate the percent-
age weight present in the yeast both in the pressed
state and also when dried. Note that nitrogen is
an important constituent of yeast, which must,
FEBMENTATION
133
therefore, require a considerable amount of nitro-
genous food during its growth.
The Nature of the Food Requirements of the
Yeast Cell. Before commencing this study it is
necessary for the student to learn the use of the
hsemacytometer for the purpose of counting yeast
cells.
1. Use of the Hcemacytometer. The form of
hsemacytometer recommended is the one known as
Thoma's (Fig. 20).
A
e c d> "ft
\ \ / i /
.HftSSSKS'S - ^ I I'ltiMsSSS?
FIG. 20. Figure of Thoma's Haemacytometer.
A is a thick glass microscope slide on which a
square of glass (a) 0'2 mm. thick with a circular
hole in the middle is cemented. A circular glass
(c) 01 mm. thick is cemented centrally in this hole,
leaving an annular space (d) between (a) and (c).
In the middle of (c) two sets of equi-distant parallel
lines are etched, cutting each other at right angles.
There is thus formed a large square with a side of
1 mm. subdivided into 400 small squares each
134 STUDIES FOE BREWING STUDENTS
having a side of 0'05 mm. If a drop of liquid is
placed on the square and enclosed by the cover
glass (b), the depth of the layer of liquid thus
formed is O'l mm. As the large square has an
area of 1 square mm. and the column of liquid
above it is 0*1 mm. in depth, the volume of the
liquid prism above the large square is thus O'l
cubic mm. ; and as the large square is subdivided
into 400 small squares, the prism of liquid above
each small square is wire, or '00025, of a cubic mm.
If now a liquid placed in the cell contains
yeast cells, these after standing a short time will
settle on the surface of (c), and the cells in the
prism of liquid over the large etched square will
settle on the square. If the cells resting on the
large square are then counted under the micro-
scope the number found will represent the number
contained in O'l cubic mm. of the original liquid.
The same remarks apply also to the small squares,
each of which represents x<nnr of a cubic mm. of
liquid.
The above description indicates very briefly the
nature of Thoma's hsemacytometer and the manner
in which it is used ; for fuller information the
student is referred to Klocker's Fermentation Or-
ganisms, p. 126. But it should be understood that
when the student is learning the use of the hse-
macytometer and other special operations con-
nected with fermentation work referred to later on,
he must work under the direction of a competent
FEKMENTATION 135
instructor, for personal guidance in the details of
such work is necessary for all but the most ex-
perienced students.
Experiment. - - Weigh out 1 grm. of pressed
yeast in a small beaker, and mix it with a little
water to a thin liquid. Transfer the liquid yeast
and the washings of the beaker to a 100 c.c. flask,
and dilute with water to a volume of 100 c.c.
Shake the flask violently to disseminate the
yeast cells evenly throughout the liquid. If this
cannot be done sufficiently well owing to the
limited capacity of the 100 c.c. flask, transfer the
liquid to a dry flask of larger capacity. Imme-
diately after shaking the flask, transfer 50 c.c. of
the liquid by means of a pipette into another flask
and dilute with 50 c.c. of water. Again shake
violently and remove as rapidly as possible a small
drop with a capillary glass tube or pointed glass
rod to the hsemacytometer, and cover it at once
with the glass (b). Note that the drop taken must
be of sufficient size to touch the lower surface of
the glass (b), but must not be so large as to
run into the annular space (d). If the size of the
drop does not conform to these conditions, it must
be wiped off, and the experiment repeated. When
a satisfactory drop is obtained, transfer the hsema-
cytometer to the stage of a microscope placed in an
upright position, and allow a few minutes for the
yeast cells to settle. Then proceed to count the
cells under a J-inch objective. It will usually be
136 STUDIES FOE BEEWING STUDENTS
found convenient to count the number of cells in a
perpendicular column of twenty of the smaller
squares ; but before commencing to do so it is
necessary of course to decide with regard to those
cells which may happen to touch the lines bound-
ing the column of squares. If all those touching
the lines bounding the left hand and top of the
column are counted, those touching the right hand
and bottom lines must be omitted ; either this or
a reverse method must be adopted, or a correct
number will not be obtained. The cells in at least
five columns of the smaller squares, (100 squares),
must be counted in order to obtain a fair average.
After making one count of the cells, the hsema-
cytometer should be cleaned and the cells counted
in a fresh drop. If the numbers obtained agree
closely, the determinations may be regarded as
satisfactory ; if not, further countings of other
preparations must be made until close agreement
is obtained.
The average number of cells obtained for a
small square represents the number present in j^
or *00025 of a cubic mm. of the liquid. Calculate
the number of cells present in the original 1 grm.
of pressed yeast taken, bearing in mind that the
original preparation was diluted to one-half its
volume before counting.
In order to assist in realising the vast number
of cells obtained, calculate the distance a single
row of the cells would cover if placed in a straight
FEKMENTATION 137
line with the cells just touching each other, assum-
ing that the average diameter of a cell is 6/a. A /*,
or micro millimeter, equals ^ or -001 of a milli-
meter, and is the unit employed in microscopic
measurement.
2. The Nature of the Food Requirements of the
Yeast Cell. Three flasks, of similar shape, with a
capacity of about 250 c.c. are required for this
study.
Introduce into flask (a) 100 c.c. of a 10 per cent,
solution of pure cane-sugar in distilled water.
Introduce into flask (b) 100 c.c. of a 10 per cent,
solution of cane-sugar in Pasteur's nutritive mineral
solution. This solution is prepared by dissolving
the following constituents in 1000 c.c. of water :
K 2 HP0 4 - 2-0 grms.
MgS0 4 0-2
Ammonium tartrate - 10 '0 ,,
Calcium phosphate - 0'2
Introduce into flask (c) 100 c.c. of a 10 per cent,
solution of cane-sugar in yeast water.
Weigh out 5 grms. of pressed yeast, and after
mixing it thoroughly with a little water, dilute to
100 c.c. with more water, and agitate the mixture
violently. Now add 5 c.c. of the diluted yeast to
each of the flasks (a), (b) and (c), so that the con-
tained solutions are yeasted with an equal number
of cells in an equal volume of liquid. Close the
flasks with cotton-wool stoppers, and place them
in an incubator at a temperature of about 20. At
138 STUDIES FOE BKEWING STUDENTS
the same time add 5 c.c. of the diluted yeast used
in the above experiments to 100 c.c. of water, and
after agitation count the cells in the hsemacyto-
meter. Note that the number thus obtained for
the standard volume of ^QQ cubic mm. is the number
of cells present in the same volume of the different
solutions in the three flasks at the commencement
of the experiment. Allow the three flasks to re-
main in the incubator for about forty-eight hours
to admit of cell multiplication being complete, and
then count the yeast cells present in each solution
in tjie usual manner. The solutions (b) and (c)
may require dilution before counting (see Klocker,
p. 127). Compare the new numbers found for the
standard volume of ^QOO cubic mm. in each experi-
ment with those originally obtained, and note that
yeast supplied with cane-sugar as its only food
supply exhibits little or no signs of multiplication,
but that yeast supplied with cane-sugar and Pasteur's
mineral solution, or cane-sugar and yeast water
multiplies freely.
The Maximum Number to which Yeast Cells
Multiply in a Nutritive Solution is not Directly De-
pendent on the Number of Cells Originally Introduced.
Two flasks (a) and (b), of 250 c.c. capacity, each containing
100 c.c. of a boiled and filtered malt wort of a specific gravity of
about 1055, are prepared ; a mixture of 5 grms. of pressed yeast
in 100 c.c. water is also prepared as in the previous experiment.
Add 5 c.c. of the diluted yeast and 5 c.c. of water to flask (a), and
10 c.c. of the diluted yeast to flask (b). It will be noticed that
flask (b) then contains twice as many yeast cells in a given volume
FERMENTATION 139
as are present in flask (a). Place the flasks in an incubator heated
to about 20, after shaking them to mix the yeast. As a check
experiment add 10 c.c. of the diluted yeast to 100 c.c. of water, and
count the number of cells present.
When yeast multiplication is complete (usually in forty-eight
hours), proceed to count the yeast cells present in (a) and (b), noting
that the number of cells present will probably necessitate dilution
of the wort with water to four or five times its volume previous to
counting. After correcting for dilution, compare the number of
cells found per standard volume in both flasks, and note that the
numbers found are very similar, although the number of cells with
which (a) was yeasted was only half that of (b).
From the counting experiment made as a check at the com-
mencement of the study, the number of yeast cells per standard
volume present in (a) and (b) when the experiments were started
may be ascertained. Compare the numbers with the numbers
found for (a) and (6) at the close of the experiment, and note
that the multiplication of each original cell in (a) has been about
twice as great as in (6).
The Maximum Number to which Yeast Cells
Multiply in a Nutritive Solution is not Directly De-
pendent on the Amount of Yeast Food Present. Prepare
two flasks (a) and (6) of 250 c.c. capacity, (a) containing 100 c.c.
of a malt wort of specific gravity 1075, and (&) 100 c.c. of the same
malt wort diluted with water to a specific gravity of 1050. Add
to (a) and (b) 10 c.c. of a mixture of yeast and water, prepared as
in the previous experiment, and, after shaking the flasks well,
place them in an incubator heated to about 20. When yeast
growth is complete, remove the flasks from the incubator and
count the number of cells present in the usual manner. Observe
that although the amount of fermentable matter and yeast food
present in (a) exceeds the amount in (b) to the extent of 50 per
cent., the numbers of yeast cells found in (a) and (b) are very
similar.
The student's attention is specially called to the technical
bearing of the last two studies.
140 STUDIES FOE BEEWING STUDENTS
Removal of Nitrogen from Malt Wort during
Fermentation. Prepare a boiled, hopped malt wort
of specific gravity 1055. Measure 100 c.c. of the
wort into a 250 c.c. flask, and add about 0'25 grms.
pressed yeast. Close the flask with a cotton-wool
plug, and place it in an incubator at 20 to ferment.
Evaporate 10 c.c. of the same wort and proceed
to determine the amount of nitrogen it contains,
using Kjeldahl's method.
As soon as the fermentation in the flask is
complete, filter some of the fermented wort until
it is quite bright. Evaporate 10 c.c. and determine
its nitrogen contents.
Compare the amount of nitrogen found in 100
c.c. of the wort before and after fermentation, and
note that the amount which has disappeared has
been assimilated during yeast growth. Compare
this result with the determination of nitrogen in
yeast made previously (p. 132).
Influence of Temperature on the Development
and Fermentative Power of Yeast. Prepare three
flasks (a), (b) and (c) of 250 c.c. capacity, each
containing 100 c.c. of malt wort of a specific
gravity of about 1055, and add to each flask 5 c.c.
of a mixture of yeast and water containing 5 grms.
pressed yeast in 100 c.c.
Put (a) in a cool cellar at a temperature of
about 12 (54 F.), and (b) in an incubator at a
temperature of about 30 (86 F.). Raise the tem-
perature of (c) to 60 (140 F.) in a water-bath for
FEEMENTATION 141
fifteen minutes, and afterwards place it in the incu-
bator with (#). At the same time add 5 c.c. of the
diluted yeast to 100 c.c. of water, and count the
number of cells present in the standard volume of
jooo cubic mm.
In eighteen to twenty-four hours add 0*1 grm.
of salicylic acid to each of the flasks to arrest fer-
mentation, and, after shaking violently, count the
number of cells present in ^ cubic mm. of each
solution. Filter about 70 c.c. of the wort in
each flask and determine its specific gravity or
" attenuation ".
Compare the number of cells found with the
number originally introduced, and note that the in-
crease in (b) is much greater than in (a), thus indi-
cating that a temperature of 30 is much more
favourable to yeast multiplication than a tempera-
ture of 12. Observe also that the " attenuation "
of (b) is much lower than (), showing that fer-
mentation has proceeded more rapidly at the higher
temperature.
On the other hand, the number of yeast cells
in (c), and the " attenuation " of the wort have
remained unaltered from the commencement of the
experiment, indicating that the short period of
heating to 60 has destroyed the multiplying power
and the fermentative power of the yeast originally
introduced. Note that this experiment illustrates
the principle of the technical process known as
" Pasteurisation ".
142 STUDIES FOE BREWING STUDENTS
Actions of Some of the Enzymes Present in the
Yeast Cell.
1. The power possessed by the yeast cell of
breaking down or fermenting sugar into alcohol
and carbon dioxide has been shown to be effected
by an enzyme, zymase, but the experimental diffi-
culties associated with a demonstration of this
prevent it at present from becoming a student's
laboratory experiment.
2. Invertase. The student has already become
acquainted with the actions of this enzyme when
studying the inversion of cane-sugar (see p. 83).
3. Maltase. The presence of this enzyme in the
yeast cell, unlike invertase, cannot be demonstrated
without special preparation of the yeast.
Spread a very thin layer of about 20 grms. of
finely crumbled, well-pressed yeast on a porous
plate, and dry it in a vacuum desj6icatpr__oiyer/
sulphuric acid for two or three days. Powder the
dry mass very finely in a mortar and transfer to
an air-bath, the temperature of which must be
raised very slowly (in about two hours) to 50, at
which point it must be kept for one hour.
To demonstrate the presence of maltase in the
prepared yeast, add about 0*5 grm. of the powder
to 100 c.c. of a solution of about 5 per cent, of
maltose of known rotatory power containing 0'5
c.c. of toluene as an antiseptic (chloroform must not
be used as it prevents the action of maltase). Cork
the flask containing the solution and keep it at a
FERMENTATION 143
temperature of 35. In three or four hours filter
and examine the filtered solution in the polarimeter.
A considerable fall in the rotation of the solution
will be observed, indicating the conversion of maltose
into dextrose. From the fall in rotation calculate
the amount of maltose converted into dextrose.
Confirm the presence of dextrose by preparing its
osazone (see p. 81).
AutoDigestion of Yeast. Crumble about 1 Ib.
of freshly pressed yeast as finely as possible, and
after heaping it together on a sheet of filter-paper,
introduce the bulb of a chemical thermometer into
the centre of the mass. Observe the temperature
of the mass from time to time, and note that it
tends to increase owing to the respiration of
oxygen by the yeast cells and the combustion of
reserve material.
Pack into a broad-mouthed 10 or 20 oz. bottle
some freshly pressed brewer's yeast, as firmly as
possible, until the bottle is about three-parts filled.
Close the bottle by tying a piece of filter-paper
over its mouth, and put it aside in some place
where it will be kept at a temperature of 20 to 25.
Observe from day to day that the mass of yeast
gradually liquefies until in the course of several
weeks it consists of a dark muddy liquid. Note
that the change is not due to putrefaction, for the
liquid has no unpleasant smell and few or no living
organisms are to be found in it. The change may
be regarded as an auto-digestion of the yeast by its
144 STUDIES FOE BKEWING STUDENTS
own proteolytic enzymes. If the liquefied yeast is
kept for some months small concretions of tyrosine
will usually be found in the deposit which have
resulted from the breaking down of the proteids of
the yeast.
The liquid expressed from the auto-digested
yeast may be utilised as a source of invertase if re-
quired. 1
PAKT II.
The Following Course of Experiments Constitutes a
Study of the Morphology and Life History of
Some of the More Important Micro- Organisms
of Fermentation, and Introduces the Student
to the Special Methods Employed in the Study
of Fermentation Organisms.
In the following studies description of detail
has been avoided as far as possible, for the student
can obtain much of the information he will require
from the books to which references are given in
the text. But books alone are an insufficient guide
for a beginner, and it is essential that he should
carry on his studies under the supervision of an
instructor who is able to assist him in many details
of his work which require, personal demonstration.
It is assumed that the student is able to work
1 See C. O'Sullivan and Tompson, Journ. Chem. Soc., 1890,
Ivii., p. 869.
FEEMENTATION 145
in a laboratory properly equipped for the special
character of his studies, and that he already pos-
sesses some knowledge of the principles and methods
of bacteriology.
Preparation of the Culture Media Commonly
Employed in the Study of Fermentation Organisms.
It is desirable for the student to prepare stocks
of culture media when commencing the following
studies, in order to acquire a practical knowledge
of the methods of preparation and sterilisation
usually adopted. Subsequently it will save much
time if he is supplied with the media ready pre-
pared.
Hopped Malt Wort (see Klocker, p. 71). Boiled
hopped wort from a brewer's copper may be used
with advantage. The wort should be diluted with
water to a specific gravity of about 1050. The wort
must be filtered until it is quite bright, and should
remain free from deposit after sterilisation. The
wort may be stored in flasks, closed with cotton-
wool plugs.
Prepare 2 liters of wort and store in four flasks
after sterilising in the usual manner.
Yeast Water. Heat a liter of tap water in a
2 liter flask over a gas flame, and when it is boiling
briskly add gradually about 70 grms. of pressed
yeast. Boil the mixture for fifteen minutes, and
afterwards allow it to digest on a boiling water
bath for one hour. Filter until quite bright and
store in the same manner as malt wort.
10
146 STUDIES FOR BREWING STUDENTS
Meat Extract. Mince finely 500 grms. of lean
meat, and mix it with a liter of water in a beaker.
Keep the beaker over-night in an ice cupboard
or cold cellar. Strain the liquid from the meat
through a cloth, and boil the filtrate to remove the
albuminous bodies present. Add 5 grms. of sodium
chloride and 10 grms. of peptone to the liquid, and
filter. Render the solution very feebly alkaline
with sodium carbonate, and make up its volume to
1000 c.c. with water. Divide into two equal
volumes, and store in two flasks after sterilisation
in the usual manner.
For other Liquid Media sometimes used see
Klocker, pp. 79, 80.
Wort Gelatin. Soak 100 grms. of best French
gelatin in cold water in a large beaker for an
hour. Pour away the water, and replace with a
liter of the malt wort already prepared. Transfer
the beaker to a boiling water bath, and heat until
the gelatin has completely dissolved in the wort.
Cool the wort to about 50, and add to it half the
white of an egg, previously diluted and well mixed
with a little of the wort. After mixing the solu-
tions thoroughly, raise the mixture to the boiling
point and boil for two or three minutes. Filter
the solution through flannel, and the wort gelatin
will be sufficiently bright for many purposes. For
some purposes, however, perfectly clear wort gela-
tin is required. To prepare it, filter about 500 c.c.
of the wort gelatin when hot through a paper-filter
FEKMENTATION
147
placed in a hot-water funnel. Store the wort gela-
tin in flasks, and sterilise in the ordinary steam
steriliser ; note that an autoclave must not be used
for this purpose, as the high temperature to which
the wort gelatin is exposed injures its setting power
(see Klocker, pp. 81-85).
Preparation of Freudenreich Flasks and Test*
Tubes Containing Malt Wort and Gelatin Malt
Wort, Sterilise in the usual manner a number of
FIG. 21.
Preudenreich Flask.
FIG. 22. Holder for Sterile Water.
Freudenreich flasks and test-tubes, plugged with
cotton wool, in the hot air steriliser at 150 (302 F.).
When cool measure into the flasks from a burette
or pipette, 5 c.c. of malt wort, or malt wort gelatin
rendered liquid by heat. Measure into the test-
tubes 10 c.c. of the same media. When filling
the flasks and test-tubes avoid wetting the necks
10*
148 STUDIES FOR BREWING STUDENTS
of the vessels with the culture media. Sterilise
with steam in the usual manner, noting that wort
gelatin should be heated for only sufficient time
to ensure perfect sterilisation, as prolonged heat-
ing affects its setting property.
Sterile Water. A supply of sterile water is
frequently required during work with micro-organ-
isms. A holder containing a supply of sterile water
should be prepared. For description see Klocker,
p. 67.
A. THE SACCHAEOMYCETES AND LOWEK FUNGI.
Study of the Morphology of a Yeast Cell. A
sample of ordinary matured brewer's yeast is re-
quired. Place a small drop of water on a micro-
scope slide and add to it a minute drop of yeast,
so that the appearance of the water drop is slightly
milky. Cover with a clean cover-glass and examine
under the microscope with a J-inch objective.
Observe that the yeast cells are spherical or
slightly elliptical in shape. Like most living cells,
they consist of protoplasm, or living matter, en-
closed by a membrane the cell wall.
To render the cell wall easily visible, remove the
slide from the microscope and press the centre of
the cover-glass gently, in order to burst some of
the yeast cells. Examine again under the micro-
scope and the walls of the burst cells will now be
visible as very thin transparent membranes.
Ex&mine anpther preparation of yeast in water,
FEBMENTATION 149
and note that the contents of the cells are not
homogeneous, but that one or two round, clear
spaces of considerable size are visible in each cell
which have the appearance of being of less density
than the protoplasm. These spaces are called
vacuoles, and are filled with cell sap.
Examine the protoplasm carefully and it will
be noticed that a number of dense particles lie
embedded in it, giving it a mottled appearance.
These particles, or granules, are composed of fatty
matter enclosed in a layer of albuminous material.
The granules situated in the protoplasm are without
motion, but granules may be frequently observed
within the vacuoles which possess a Brownian
movement indicating the fluid condition of the cell
sap in which they are floating. (See Technical
Mycology, Lafar, vol. ii., p. 145, concerning the
"Anatomy of the Yeast Cell".)
The protoplasm of the yeast cell, like that of
all living cells, is composed of a central body
the nucleus surrounded by cytoplasm ; but the
nucleus of the yeast cell is only rendered visible
by certain staining processes which are somewhat
difficult of execution (Klocker, p. 89).
Dry, stain and mount a preparation of the
yeast in the usual bacteriological manner (Klocker,
P-
Having now studied the general anatomy of the
mature yeast cell, the student should proceed to
150 STUDIES FOE BEEWING STUDENTS
observe the changes which take place in the ap-
pearance of the cell during the various stages of
its growth.
Add a little of the mature yeast already ex-
amined to some hopped malt wort in a small flask,
and place the flask in an incubator kept at a tem-
perature of about 25. Proceed to examine the
yeast cells from time to time under the microscope,
at first at intervals of one hour, and afterwards at
longer intervals. Note the changes which rapidly
take place in the general appearance of the cells.
The granular appearance of the protoplasm dis-
appears and the cells become very transparent ;
the walls of the cells also appear to become
thinner.
Note also the changes in appearance of the
vacuoles. Observe that the first signs of repro-
duction of the yeast cell by budding is evidenced
after two or three hours by a slight protrusion on
one side of the cell. The bud gradually enlarges
until a full-sized cell is formed from the mother
cell ; but this method of reproduction will be fol-
lowed better when employing the drop culture
method described below. Observe as fermentation
draws to a close that the protoplasm of the yeast
cells becomes more granulated, and the cells return
to the original appearance of the mature yeast with
which the present study was commenced.
Determine the average size of the yeast cells by
means of the micrometer eye-piece of the micro-
FEKMENTATION 151
scope, and express in micromillimeters (1 /& or
micromillimeter equals '001 mm.)
Growth of Yeast in a "Drop Culture*'. Re-
quirements for the preparation of a " drop culture ".
Thin glass rods in a tin box with a glass cover.
A glass plate and glass bell jar or beaker to act as a
cover.
Microscope slides and Bottcher's moist chambers (see
Klocker, p. 69), or hollow-ground slides.
Microscope cover-glasses in small Petri dish (for
cleaning cover- glasses, see Klocker, p. 87).
Freudenreich flasks of sterilised wort.
Vaseline.
Forceps.
FIG. 28. Moist Chamber.
Sterilise the box containing glass rods, and the
Petri dish containing cover-glasses, in the hot air
steriliser. Sterilise the glass plate and cover with
a Bunsen flame, and afterwards sterilise the micro-
scope slides and moist chambers in the same
manner, holding them in the flame with the forceps.
Place the slides and moist chambers after sterili-
sation on the glass plate underneath the cover.
Preparation of a Drop Culture. In special work
the preparation of a drop culture is conducted in
a sterile chamber (see Klocker, p. 21), but this
precaution is not necessary in the ordinary course
of a student's work, and therefore reference to this
part of the process is omitted here.
152 STUDIES FOE BREWING STUDENTS
Take a glass slide from below the cover with
the forceps, and lay it on the top of a Petri dish
which has been sterilised by being passed through
a Bunsen flame. Place three drops of sterile wort
from a Freudenreich flask side by side on the glass
slide by means of a sterile glass rod. A trace of
the yeast to be examined is now added by means
of the glass rod to one of the drops of water and
the whole well mixed. A little of the first drop
is then added to the second by means of a second
sterile rod. After mixing, a little of the second
drop is added to the third by means of another
sterile rod. A small drop of the final mixture is
now placed in the centre of a cover-glass, and the
glass is then rapidly inverted and fixed on the ring
of a moist chamber, the edge of which has been
previously smeared with vaseline. A small drop
of wort from the Freudenreich flask should be
placed on the bottom of the moist chamber previous
to the fixing of the cover-glass, in order to keep
the air of the chamber moist.
Examine the drop culture under the microscope,
which must be in an upright position. The yeast
cells present should be so few in number that not
more than one or two are visible in each field of
view. If the cells are too numerous, another drop
culture must be made with further dilution of the
culture.
Study the cells from time to time as they
multiply by budding, and make drawings. The
FEEMENTATION
153
culture when not under examination should be
kept in an incubator at 20-25 (68-77 R). If it
is desired to follow the multiplication of one
particular cell it must be carefully marked, or the
slide must be fixed on the microscope, and the
latter placed in an incubator or other suitably
warm place. (For mode of propagation of yeast
by budding, see K locker, p. 191.)
PIG. 24. Multiplication of top yeast : I., ^, 7 P.M. ; II., 8 A - M - 5 ni -
9 A.M. ; IV., 1<H A.M. ; V., 12 noon ; VI., 8J P.M. ; VII., 8 P.M. ; VIII.,
V, 8 A.M. ; IX., 10 A.M. ; X., 11 A.M. ; XI., 1 P.M. ; XII., ^, 8 P.M. ;
XIII., V- J XIV., |, 12 o'clock. (After Mitscherlich.)
A Study of Some of the Well^recognised Races
of Yeasts and Torula. When the student is
familiar with the appearance and ordinary mode
of reproduction of brewer's yeast, he should then
proceed to study some of the well-recognised races
and species of yeasts and torula. Inoculations
154 STUDIES FOE BKEWING STUDENTS
from pure cultures of these should be obtainable
in the laboratory in which the student works, and
should at first be grown in tubes of malt wort.
The general appearance of the organisms should be
studied under the microscope and also their modes
of development in drop culture, careful drawings
being made at all times. A selection from the
following list of organisms is recommended :
Saccharomyces cerevisiae. Top fermentation race.
,, Saaz and Logos ; low fermentation
races. (See Klocker, p. 220.)
Pastorianus L, II., and III. (See Klocker, pp.
254-256.)
ellipsoideus I. and II. (See Klocker, pp. 257-
259.)
,, Marxianus. (See Klocker, p. 261.)
,, anomalus. (See Klocker, p. 262.)
Schizo-saccharomyces Pombe. (See Klocker, p. 269.)
octosporus. (See Klocker, p. 270.)
Saccharomyces (?) apiculatus. (See Klocker, p. 294.)
Mycoderma vini. (See Klocker, p. 297.)
Torula. (See Klocker, p. 289.)
A Study of Some of the more Common Species
of Moulds, The close relationship of the saccharo-
mycetes to the moulds, and the important ferment-
ation changes induced by many species of moulds,
renders it most desirable for the student to study
some of the more important types of these or-
ganisms.
Inoculations from pure cultures of the moulds
should be made in malt wort and on wort gelatin,
and the modes of growth of the organisms studied.
FERMENTATION 155
At the same time drop cultures should be made
with spores or mycelia of the moulds, and the
different stages in the growth and development
of the organisms carefully followed under the
microscope. Drawings should be made throughout
these studies.
A selection from the following moulds is re-
commended :
Mucor racemosus. (See Klocker, p. 179.)
Mucor (Amylomyces) Eouxii. (See Klocker, p. 181.)
Penicillium glaucum. (See Klocker, p. 278.)
Aspergillus glaucus. (See Klocker, p. 274.)
Monilia Candida. (See Klocker, p. 298.)
Oidium lactis. (See Klocker, p. 303.)
Dematium pullulans. (See Klocker, p. 305.)
Preparation of Spore Cultures of the SaccharO'
mycetes.
Requirements :
A pure culture of yeast twenty -four hours old.
Sterilised gypsum blocks in covered glass dishes. (See
Klocker, p. 64.)
Sterilised water.
Sterilised pipettes. (These may be made by drawing
out thin glass tubing in a gas flame.)
In order to induce sporulation in yeast, the
culture employed must be young and vigorous
(see Klocker, p. 122).
Inoculate a small flask or test-tube of sterile
wort from a pure culture of top fermentation yeast,
and keep it in an incubator at 25 (77 F.) for
twenty-four hours, in which time a sufficient deposit
156 STUDIES FOE SKEWING STUDENTS
of freshly formed yeast for the purpose of the
experiment will probably be found.
Sterilise the gypsum blocks and dishes in the
hot-air steriliser for an hour at 110 to 115 (230 to
FIG. 25. Gypsum Block.
239 F.). The temperature must not rise above
120 (248 F.) or the blocks may be spoiled by
dehydration of the gypsum.
The small pipettes should be wrapped separately
in filter-paper and sterilised at about 150 (302 F.).
To prepare the spore culture, pour away the
supernatant fermenting liquid from the yeast cul-
ture, and take out a small quantity of the sedi-
mentary yeast with a pipette and spread it in a thin
layer on a gypsum block. Pour water from the
sterilised water-holder into the dish in which the
gypsum block is placed until it is about two-thirds
filled. During these manipulations uncover the dish
for as short a time as possible. Place the covered
dish in an incubator at 25 (77 F.). Examine the
culture after twenty-four hours under the microscope
for the formation of spores, removing for this pur-
pose a little of the yeast with a sterilised needle
and mounting it in water on a glass slide (see
FEEMENTATION
157
Klocker, p. 122). If spores are not found, examine
at intervals of six or eight hours, and note the
time when spore formation is first observed.
Fia. 26. Saccharomyces cerevisiaz I.,_Hansen. First stages of development
of the spores. i$JL. (After Hansen.)
FIG. 27. Saccharomycetes which form ascospores. 1. Sacch. cerevisicB I.
2. Sacch. Pastarianus I. 3. Sacch. Pastarianus II. 4. Sacch. Pastori-
anus III. 5. Sacch. ellipsoideus I. 6. Sacch. ellipsoideus II. a, Cells
with septa ; b, cells with more than normal number of spores ; c, cells
distinctly beginning to sporulate. About ^f 4 . (After Hansen.)
Make spore cultures of the various species of
yeasts which are being studied, and note the differ-
ent times at which spore formation is first observed
(see Klocker on spore formation, pp. 202 et seq.).
158 STUDIES FOB SKEWING STUDENTS
Observe the character of the spores in the different
species.
Prepare a drop culture of the spores of a yeast
in malt wort, and observe the germination of the
spores (see Klocker, pp. 205 et seq.).
To make permanent stained preparations of
sporulating yeast, distribute the spores mixed in a
small drop of water on a clean cover-glass, and dry
and fix in the usual manner (see Klocker, p. 88).
Place the cover-glass in a watch-glass containing
carbol fuchsine solution (see Klocker, p. 92) and
boil gently for a few minutes. Wash in water, and
afterwards for a very short time in dilute hydro-
chloric acid. Rinse in water, dry, and mount in
Canada balsam. The spores of the yeast cells
should then appear red under the microscope, and
the remaining parts colourless. Double staining
can be attempted, if desired.
Film Formation of the Saccharomycetes. Inoculate
pure cultures of the different species of yeast which are being
studied into small flasks half-filled with sterilised wort and
lightly stoppered with cotton wool. Allow the cultures to stand
at room temperature in a place where they will not be dis-
turbed in any way. It is essential for the formation of film
growths that air should have free access to the cultures. The
time of development of the film formation of a yeast varies
with the species, and with the temperature at which the culture
is kept (see Klocker, p. 125 and p. 194). Note the characteristics
of the film growths of the different species of yeast, and the
manner in which the forms of the cells usually vary from those
of the original submerged cultures.
FEKMENTATION 159
Preparation of Pure Cultures of Yeasts and Moulds.
Gelatin Plate Method of Culture.
Bequisites :
Tubes of sterile wort gelatin.
Sterilised Petri dishes.
Freudenreich flasks containing sterile water.
Inoculating needles.
Sterilised pipettes and glass rods.
Transfer with a sterilised needle a small inocu-
lation of brewer's yeast to a Freudenreich flask
containing sterilised water, and shake well in order
to distribute the yeast cells evenly throughout the
volume of the liquid. Melt the wort gelatin in
three test-tubes by placing them in water at about
30 (86 F.), and burn the surface of the cotton-
wool plugs of the tubes. Remove the plug from
the first tube and add with a pipette 10 drops of
the diluted yeast. Mix thoroughly, and with a
sterilised rod or wire transfer one drop of the
mixture to the second tube. Mix, and transfer one
drop of the mixture from the second tube to the
third tube. When agitating the tubes to distribute
the yeast, take care not to cause the formation of
foam in the liquid wort gelatin. Pour the con-
tents of the three tubes into three Petri dishes,
keeping them uncovered for as short a time as
possible. Mark the dishes with the dilution used,
and place them on a level surface until the gelatin
is set. Keep the cultures in an incubator at a
temperature of about 20 (68 F.) until the yeast
160 STUDIES FOE BKEWING STUDENTS
colonies develop. Note the relative size and number
of the colonies obtained in the three cultures.
To make a wort culture from any one of the
yeast colonies selected, sterilise a small piece of
platinum or brass wire by holding it in a gas flame
in forceps, and when cool dip it into the selected
colony so that some of the yeast adheres to the
wire. Transfer the piece of wire to a flask or tube
containing sterile wort, and allow the culture to
grow at a suitable temperature (see Klocker, pp.
105 et seq.).
The gelatin plate method of culture is specially
suitable for the preliminary separation of mixed
cultures of yeast, but there is no guarantee when
employing the method that each colony found is
a pure .growth, owing to the possibility of two or
more cells of different species of yeast being close
together when the original gelatin culture was
made. In order to obviate this difficulty and
perfect the process, Hansen introduced his single
cell method.
Hansen 's Method of Pure Yeast Culture from a
Single Cell.
Requisites :
A sterilised glass plate and bell jar or other cover.
Sterilised glass rods.
Sterilised Bottcher's chambers.
Sterilised cover-glasses divided into numbered squares,
or plain for use with cell-marker.
Freudenreich flasks of sterilised wort gelatin, and of
sterilised water.
FEBMENTATION
161
Mix a drop of fresh yeast with sterilised water
in a Freudenreich flask, shake well and dilute still
further by transferring a drop of the mixture to a
second flask of water. Again mix by shaking, and,
if the liquid then appears slightly opalescent, the
right dilution has probably been obtained. Transfer
a drop of the mixture to a Freudenreich flask con-
taining liquefied wort gelatin, and mix thoroughly.
Then spread a drop of the wort gelatin mixture
PIG. 28. Squared Cover-Glass.
PIG. 29. Klonne and Miiller's
Object Marker.
in a thin layer on a cover-glass by means of a glass
rod, and place the glass on the glass plate under-
neath the bell jar and leave until the gelatin has
set. Prepare a Bottcher chamber by placing a
small drop of water at the bottom of the cell and
smearing the edge of the ring with vaseline. Then
reverse the glass with the gelatin film and adjust
it to the ring of the chamber. The preparation
should then be transferred to the microscope for
examination. The lowest power objective with
which the yeast cells can be distinctly seen should
11
162 STUDIES FOR BREWING STUDENTS
be employed. Those cells are chosen for the pur-
pose of obtaining colonies which are several milli-
meters apart from other cells, and their position
must be carefully recorded. If a cover-glass divided
into squares is employed, a diagram should be made
indicating the position of the cells chosen ; if a
plain cover-glass is used, the cells must be marked
with the cell-marker (see Klocker, p. 33).
After marking the position of several cells, keep
the culture at a temperature of about 20 (68 F.),
and examine it from day to day with the micro-
scope as the cells multiply, in order to be sure that
no cells in the immediate vicinity of the colonies
have been overlooked. When the colonies are
large enough, a pure culture in wort may be
obtained from each colony by inoculation in the
manner described for gelatin plate cultures (see
above).
Modifications of Hansen's Method of Pure Yeast Culture
from a Single Cell known as ''Lindner's Droplet Culture," and
" Schonfeld's Method " are sometimes employed. For description
of these processes see Klocker, pp. 110, 111.
Analysis of Mixed Cultures of Yeast. It is
desirable when the student has become familiar
with the ordinary biological methods employed in
the study of the saccharomycetes, that he should
attempt to separate and identify the forms in a
mixed sample. For this purpose he should be
supplied with a mixture of two or more well de-
nned forms of yeast by his instructor, who alone
FEEMENTATION 163
knows what forms are in the mixture. Two
methods of analysis may be employed the gela-
tin plate method, and the " fractional culture "
method. The former is the easier, and is recom-
mended to the inexperienced student for his first
attempt. But it has certain disadvantages, as, for
instance, some species of yeast develop very slowly
in wort gelatin, and in cases of yeasts which are
present only in small quantities in a mixture, they
are more apt to be overlooked than when the
" fractional culture " method is employed.
Gelatin Plate Method. Dilute the mixed culture,
which should be a freshly grown one, with steril-
ised water, and proceed to make plate cultures
as described above (p. 159). As long a time as
possible should be allowed for the development
of the yeast colonies, for, as before remarked,
some yeasts develop more slowly than others in
gelatin.
When the colonies are well developed, examine
their general appearance with a pocket lens, and
note any characteristic differences which may be
evidenced. Examine cells removed from a number
of different colonies under the microscope. Inocu-
late the colonies which exhibit differences into
sterilised wort, and observe the manner in which
they develop. Study the spore formation of the
different yeast cultures obtained. Endeavour to
identify the forms separated (see Klocker, pp. 133
et seq.}.
11*
164 STUDIES FOE BEEWING STUDENTS
Fractional Culture Method (see Klocker, p. 101).
Requisites :
Freudenreich flasks containing 5 c.c. sterilised water.
malt wort.
Small stoppered bottle, sterilised.
Hsemacytometer.
Sterilised pipettes.
Two or three drops of the mixed yeast culture are transferred
to a Freudenreich flask of sterile water, and well mixed. The
diluted culture is then transferred to a small sterilised glass-
stoppered bottle and well shaken in order to separate the yeast
cells very thoroughly. A small drop is then transferred to the
haemacytometer, and the number of cells in 1 c.c. of the liquid
determined (see p. 133). Dilute the liquid with sterilised water,
so that 1 c.c. contains about 1,000 cells, and one drop of the
liquid (0'05 c.c.) will then contain about fifty cells. Add 0'05
c.c. (or one drop) of this dilution to a Freudenreich flask con-
taining 5 c.c. of sterilised water, and shake the mixture well.
This mixture should now contain approximately one yeast cell
in two drops of the liquid. Fifty Freudenreich flasks of sterilised
wort are then inoculated, each with one drop of the mixture added
by means of a sterile pipette, and kept in a moderately warm place
for two days where they will not be disturbed in any way. The
flasks are then examined, and those containing a single yeast
colony are separated from the rest. After further development
of the cultures in these flasks, they are examined microscopic-
ally and separated into groups according to their characteristics.
Further examination is conducted as with the gelatin plate
method described above.
When analysing yeasts in order to determine the presence
of wild yeasts, which may be present only in very small quantity,
it is often necessary, previous to analysis, to cultivate the original
yeast in a cane-sugar solution containing tartaric acid (see Klocker,
p. 135).
FEBMENTATION 165
A Method of Testing the Pure Cultures of
Yeasts obtained by Previous Methods of Analysis
for Technical Purposes. Inoculate the pure
cultures to be tested into flasks containing about
half a liter of sterilised wort, and incubate at 25
(77 F.) for two days. Sterilise a number of glass
cylinders of about 1500 c.c. capacity by burning a
little alcohol in them, and pour into each cylinder
a liter of sterilised hopped wort of known specific
gravity (about 1050). Inoculate the wort in the
different cylinders with the cultures of yeast which
have accumulated from the fermentations in the
flasks. After covering the cylinders with two or
three layers of sterilised filter-paper, allow the
fermentations to proceed at about 20 (68 F.). As
the fermentations proceed, observe the nature of
the different yeasts regarding their high or low
fermentation characteristics. Note the behaviour
of the yeasts towards the close of the fermentation
as to whether they separate quickly, or remain
floating in the liquid. Note the flavour and smell
of the completely fermented worts. Determine
the "attenuation" of the fermented worts, and
also the amount of alcohol present.
By means of the above experiments information
of technical value can be often obtained in the
laboratory regarding the properties of pure yeast
cultures. (See Jorgensen's Practical Management
of Pure Yeast, p. 37.)
166 STUDIES FOR BREWING STUDENTS
Experiments on the Varying Powers of Hydrolysis
and Fermentation possessed by Different Species of
Yeasts. Experiments should be made by growing suitable
species of yeasts in solutions of such carbo-hydrates as cane-
sugar, dextrose, maltose and lactose, in yeast water, in order to
study their powers of selective fermentation. One hundred c.c.
of a 3 per cent, solution of the carbo-hydrate is usually a con-
venient amount to employ in these experiments. The solutions
should be sterilised in small flasks plugged with cotton- wool,
and inoculated in the usual manner. Examination of the flasks
during the course of the experiments is often sufficient to indicate
if fermentation is proceeding, but in some cases, which should
suggest themselves to the student, it is desirable to determine
the nature of the sugars present in the solutions when fermenta-
tion is proceeding. The means he should employ for this pur-
pose will be already familiar to him. During the continuance
of the fermentation experiments the flasks should be kept in an
incubator at 25 (77 F.).
Yeasts recommended for experiment are :
Sac. cerevisiae.
Sac. anomalus (see Klocker, p. 263).
Schizo-saccharomyces octosporus (see Klocker, p. 271).
B. THE SCHIZOMYCETES OK BACTEEIA. (See Klocker,
p. 271.)
The Morphology of a Bacterium. Prepare a
culture of Bacillus subtilis (the hay bacillus). In-
fuse some dry hay in water for two or three hours
at the temperature of the room. Pour the infusion
obtained into a flask, and close it with a plug of
sterilised cotton-wool. Boil the infusion gently
for five minutes, allow it to cool, and place it
in an incubator at 25 to 30 (77 to 86 R). In
FEBMENTATION 167
twenty-four hours a growth of B. subtilis should
develop on the surface of the infusion. Transfer
with a needle a little of the surface growth to a
drop of water on a glass slide, and examine it
under a cover glass with a high power objective.
Observe that the film is composed of long cylindri-
cal rods or cells. These cells, like those of the
saccharomycetes, consist of protoplasm enclosed
by a cell wall. Also, as with yeast cells, granules
and vacuoles occur in the protoplasm, but owing
FIG. 30. Clostridium butyricum, Prazmowski. Butyric acid bacterium
with flagella. a, Vegetative motile cell ; b, sporulating motile cell.
J^. (After Alfr. Fischer.)
to their small size are only seen with very high
magnification. (See Lafar's Technical Mycology,
vol. i., p. 42 ; also Klocker, p. 312.)
Note the transverse partitions or walls sub-
dividing some of the cells. These indicate the
ordinary way in which the cell multiplication of
bacteria takes place. In the cell of a fully de-
veloped bacterium one or more transverse septa
form, dividing it into two or more separate cells ;
these may remain united as a chain of cells, or
separate into individual cells. This method of mul-
tiplication of bacteria by fission differs essentially
168 STUDIES FOE BEEWING STUDENTS
from multiplication by budding which characterises
the saccharomycetes. Note that the long chains
of bacteria in the culture are motionless, but that
some of the single cells possess an active swimming
motion. The motion is due to the action tf flagella,
fine hair-like processes, which are attached to the
cells (see Klocker, p. 313). The flagella are too fine
and transparent to be observed without preparing
and staining the cells by certain processes which
are somewhat difficult of execution.
Make a Stained Preparation of B. subtilis showing the
Flagella. Dry and fix the organisms in the usual manner on
a cover-glass. Prepare a mordant by mixing one volume of
extract of logwood (1-8) with two volumes of a 20 per cent
solution of tannin, to which a few drops of a saturated solution
of ferrous sulphate have been added. Place some drops of this
solution on the cover-glass preparation and warm over a flame
until steam is formed. Wash in water and stain with Loffler's
gentian violet solution (see Klocker, p. 92). Wash again in water
and mount in the usual manner. The preparation should be
examined under a -^ inch immersion lens.
Make a Stained Preparation of B. subtilis.
The preparation should be stained and fixed in
the same manner as yeast (see Klocker, p. 88).
For Gram's method of staining bacteria for the purpose of
identification see Klocker, p. 90.
Spore Formation. When the culture of B.
subtilis is two or three days old, highly refractive
spores will be observed in many of the cells.
These endospores, similar to the endospores of
FEKMENTATION 169
the saccharomycetes, are not easily stained by
dyes.
Make a cover glass preparation of the spore-
bearing bacteria, and stain with fuchsine or methyl
violet in the usual manner. Under these conditions
it will be observed that the spores remain un-
coloured, whilst the surrounding cell matter is
stained. To stain the spores see Klocker, p. 90.
Prepare a Drop Culture of the Spores of B. sub-
tills in Hay Infusion. The spores may be obtained
from a culture of B. subtilis several days old.
Study the development of the spores into rods
(see Klocker, p. 318).
A Study of Some of the Well-recognised Species
of Acid-producing Bacteria.- A selection from the
following list of organisms is recommended :
Bacillus viscosus. (Klocker, p. 341.)
Bacillus acidi-lactici. (Klocker, p. 342.)
Bacillus amylobacter. (Klocker, p. 343.)
Saccharobacillus Pastorianus. (Klocker, p. 343.)
Bacterium aceti. (Klocker, p. 336.)
Bacterium Pasteurianum. (Klocker, p. 337.)
Bacterium xylinum. (Klocker, p. 340.)
Sarcina and Pediococcus forms. (Klocker, p. 331.)
Inoculations from pure cultures of these organ-
isms should be supplied to the student. B. viscosus,
B. acidi-lactici, B. amylobacter and Saccharobacillus
Pastorianus may be grown in solutions of dextrose
(3 per cent.) in yeast water to which a little calcium
carbonate has been added. B. amylobacter must
170 STUDIES FOK BEEWING STUDENTS
be cultivated under anaerobic conditions. The
acetic bacteria should be grown preferably in
sterilised fermented wort, or in a mixture of two
parts of red wine (claret) and one part water, but
they will also grow in unfermented malt wort.
Sarcinse and Pediococci may usually be grown
with advantage in neutral meat extract containing
a little calcium carbonate. The media used should
be sterilised in small flasks or test-tubes. After
inoculation in the usual manner the cultures should
be kept in an incubator at 30 (86 R).
The general characteristics of the cultures
should be observed as the organisms develop, and
from time to time they should be examined under
the microscope. Permanent stained preparations
of the organisms should also be made. Many
special points connected with the life history and
chemical actions of the different organisms under
observation may be studied with advantage if the
student has time at his disposal, and he should
now be in a position himself to suggest the nature
of his work, and carry it on under the supervision
of his instructor.
Examination of Beer Sediments. See Matthews
and Lott (The Microscope in the Brewery, second
edition, p. 55). The sediments from as many sam-
ples as possible of freshly racked beer, matured
beer and bottled beer, should be examined under
the microscope, and also the sediments of unsound
beer, and fretting beer, if they can be obtained.
FEBMENTATION 171
The student should note that a special know-
ledge of this class of technical work is very im-
portant, and that he will derive much assistance
from working under the supervision of a skilled
instructor.
The " Forcing " Test as Applied to Beer. For
a full description of this process see Matthews and
Lott, p. 128. Note that in place of an open " forc-
ing tray " it is preferable to use an incubator.
When studying the " forcing " test it is desirable
for purposes of experience to obtain samples of ale
which will develop on forcing growths of the acid-
producing bacteria which are common to beer. As
it is sometimes difficult to obtain such samples,
about 0*5 grm. of calcium carbonate, previously
sterilised by heat, should be added to some of
the samples before forcing. In the presence of
calcium carbonate most samples of beer which
would otherwise remain " sound " on forcing
develop growths of pediococci and lactic bacteria.
The action of the calcium carbonate appears to be
due to neutralisation of the natural acidity of the
beer, which exerts a retarding influence on the
development of acid-forming bacteria.
Micro-organisms present on Barley, Malt and Hops.
Agitate strongly a sample of the barley or hops with well-
filtered and sterilised water. Pour off the water quickly, and
allow it to subside. Examine the sediment under the micro-
scope (see Matthews and Lott, p. 149, and plates 18 and 19).
Wort gelatin plate cultures of the organisms in the sedi-
ments may be made in the usual manner, if required.
172 STUDIES FOE BEEWING STUDENTS
Biological Examination of the Air for Techni^
cal Purposes. Both qualitative and quantitative
methods may be employed, but for most technical
purposes qualitative methods suffice.
1. Qualitative Method of Examining Air in
which Roll Cultures are Employed. Sterilise two
wide-mouthed 20 oz. bottles plugged with cotton-
wool. Place the bottles where the air is to be
examined, and remove the cotton-wool plugs, keep-
ing them in a sterilised covered vessel. Allow the
bottles to remain open for one hour, and then
replace the wool plugs. Liquefy a tube of wort
gelatin and one of meat extract gelatin. Into one
bottle pour the wort gelatin, and into the other
the meat gelatin, and replace the cotton-wool
plugs. Agitate the liquefied gelatin in order to
incorporate with it the dust collected in the bottles,
and prepare roll cultures in both bottles by hold-
ing them horizontally in a stream of cold water
and rotating them until the gelatin sets round
the sides of the bottles as a thin film. Place
the bottles in a dark cupboard and keep them
at room temperature until colonies of the various
organisms contained in the dust of the air have
developed. Note the appearance of the colonies,
and examine them under the microscope. Classify
them as moulds, bacteria and yeasts, or torula,
noting the number of each. Study the character
of the different species found. Note the difference
in number and kind of the organisms which have
FEKMENTATION 173
developed in malt wort gelatin and in meat gelatin ;
from a technical point of view those developing in
malt wort gelatin are of most importance.
2. Qualitative Method of Examining Air in
which Petri Dishes are Employed. Prepare in the
usual manner two Petri dishes, one containing wort
gelatin, the other meat extract gelatin. Place the
dishes where it is desired to examine the air, re-
move the covers and allow the dishes to remain
open for fifteen minutes. Replace the covers, and
allow the germs which have fallen on the surface
of the gelatin to develop at room temperature in
a dark cupboard. Examine the colonies which
develop, in the manner described in the previous
method (see Klocker, p. 154).
Mould colonies spread more rapidly when the
second method is employed than with the first,
and are therefore more apt to interfere with the
success of the Petri dish method than when the
roll culture method is used. 1
Hansen's Quantitative Method of Examining Air. (For
description of this process, see Klocker, p. 150.)
Biological Examination of Water for Technical
Purposes. It has been pointed out by Hansen
that the ordinary biological examination of water
conducted with meat extract gelatin is not suit-
able for technical purposes connected with the
1 The too rapid growth of mould colonies may be controlled
by touching them with a small drop of a strong solution of
perchloride of mercury in alcohol.
174 STUDIES FOE BKEWING STUDENTS
fermentation industries, for the nutrient medium
employed favours the development of a large
number of organisms which are incapable of
development in unfermented or fermented wort
and so obscures the main point of the examination,
which is the recognition of the special organisms
which might occasion trouble in technical practice.
For this reason it is advisable to use sterilised
wort or sterilised beer as nutrient media in the
examination of water for technical purposes. (For
a description of the method of examination, see
Klocker, p. 145, and also Hansen, Practical Studies
in Fermentation, chap, iv., p. 110.)
Biological Examination of Water for Hygienic
Purposes. It is desirable that the student should
have some experience of this process.
Examination of Ordinary Tap Water.
Requisites :
Tubes of gelatin meat extract.
Sterilised Petri dishes.
Sterilised 1 c.c. pipettes (graduated in tenths).
Melt three gelatin tubes, and transfer 0'5 c.c.
of the water to one gelatin tube, 0*3 c.c. to the
second and 01 c.c. to the third. Mix the water
and gelatin in the tubes by swinging them round
in the hand rapidly, but be careful to avoid frothing.
Pour the gelatin in the three tubes into separate
Petri dishes, and after marking each culture with
the dilution of water used, allow them to set on
a level surface. Incubate the cultures at about
FEEMENTATION 175
20 for three or four days, and count the colonies
formed, expressing the result as so many organisms
found in 1 c.c. of the original water taken. The
nature of the colonies found, whether they consist
of liquefying organisms, mould growths, etc., should
be carefully noted.
Examination of Polluted Water. If the water
to be tested is polluted with sewage or otherwise
contaminated, it will be necessary to use much
smaller volumes than those mentioned above for
the purpose of inoculation. In order to do this
the water must be diluted with sterile water or
meat extract. A description of the method usually
adopted will be found in any text-book of bacteri-
ology.
SECTION IV.
THE HOP.
THE hop (Humulus lupulus] is a dioecious plant,
that is to say, the male and female organs of the
plant are contained in separate flowers which are
borne by different plants. The female plant alone
is cultivated by the hop grower, as its flowers alone
develop into the well-known hop-cones. The male
hop is usually found as a self-sown plant growing
wild in the vicinity of hop gardens, and its sole
function so far as the formation of hop-cones is
concerned is to fertilise the flowers of the female
plant with pollen from the anthers of its male
flowers, and so lead to the development of seed
in the hop-cone. 1
Examine a Branchlet of a Hop Plant Bearing
Female Flowers. 2 In this state the plant is usually
described by the hop grower as being in "burr"
(Fig. 3).
1 For a comprehensive account of the hop plant, see Agri-
cultural Botany, by J. Percival, p. 322.
2 Specimens of the hop in flower may be preserved in spirit
for the purpose of examination.
176
THE HOP
177
It will be noticed that the flowers have the
appearance of small brushes proceeding from a
cone composed of small pointed bracts, or scales.
The brush-like appearance is due to long stigmata
FIG. 31. Female Inflorescence of the Hop.
proceeding from the ovaries of the flowers which
are concealed within the scale-like bracts. Each
ovary is surmounted by two stigmata (Fig. 32 c),
and has at its base a very small scale-like bracteole
(Fig. 32 ft).
FIG. 32. Diagram of a Female Flower of the Hop.
After the flowering stage is over, the stigmata
shrivel, the main axis of the flower lengthens, and
the scale-like bracts (Fig. 32 a) and smaller bracte-
12
178 STUDIES FOE BREWING STUDENTS
oles within these bracts (see Fig. 32 b) grow into the
leaf-like structures which compose the hop-cone.
Owing, however, to the small size and complexity
of the hop flower, a practical study of its minute
anatomy is too difficult for the ordinary student to
attempt, and he is referred to the accompanying
diagram (Fig. 32) for information concerning the
arrangement of its different parts.
Examine a Hop^Cone or Strobile. It will be
found to consist of a number of leaf-like bracts
arranged on a central axis and overlapping one
another. 1
b
FIG. 33. Bracts of Hop-Cone, (a) Stipular Bract; (6) Bractcole.
On examining the bracts closely it will be
noticed that they are of two different forms. One
form is more symmetrical and rather coarser in
1 For this study freshly gathered hop-cones, or those which
have been dried without pressing should be employed if possible.
If such cannot be obtained, some ordinary dried hops should be
placed in a closed vessel on a layer of damp sand for forty-eight
hours, when they will to a large extent recover their original
shape and elasticity. After such treatment fairly perfect hop-
cones, in a condition suitable for examination, may usually be
obtained.
THE HOP
179
texture and more thickly veined than the other.
This form is called a "stipular bract," and is de-
veloped from the pointed scaly bracts of the flower
already noticed (Fig. 32 a).
The other form of bract is usually rather longer
than the stipular bract, and possesses a finer
texture. Towards its base on one side it is folded
on itself and encloses the hop seed, if one has de-
veloped. This form of bract is called a " bracteole,"
and has developed from the small scale at the base
of one of the ovaries of the flower (Fig. 32 b).
Remove all the Bracts from a Hop^Cone and
Examine the Stem or " Strig " of the Hop. The
strig or main axis, which is thickly covered with
fine short hairs, has a zigzag form and it will be
observed that on the outer angle of each bend, and
FIG. 34. Axis or Strig of
Hop Cone.
FIG. 35. Group of Floral Axes on Hop
Strig (much enlarged).
therefore placed alternately on the strig, are groups
of short irregular branches (Fig. 34).
On examining a group of the branches with a
pocket lens it will be found to consist of four
12*
180 STUDIES FOE SKEWING STUDENTS
short axes radiating from a common point of
attachment, and that the two central axes are
rather longer than the side ones (Fig. 35). In the
early flowering stage of the hop-cone each axis
originally carried an ovary partly enclosed by a
small scale or bracteole growing at its base (Fig. 32,
c and b). After fertilisation the ovary developed
into a seed, and the small scale-like bracteole
situated at the base of the ovary developed into a
full-sized bracteole enclosing the seed. So in a
perfect hop-cone developed from a fertilised flower
each of the four minor axes composing a group of
branches on the hop strig carries a seed (or an
abortive ovary), enclosed by a bracteole.
Now examine very carefully the strig of the
hop immediately below the group of branches
which bear the seeds and bracteoles. Two small
scars will be observed to which small portions of
leaf are frequently attached (see Fig. 35, a a).
These are the points of attachment of two stipular
bracts. The stipular bracts are in reality a pair of
stipules belonging to a leaf which has not developed
its blade. Occasionally, however, a monstrous de-
velopment of the missing blade between the two
stipules is observed and the hop-cone appears
interspersed with small green leaves.
Now confirm the position of the two stipular
bracts and the four bracteoles by examining a
complete hop-cone, and note that the four short
branches carrying the bracteoles and seeds proceed
THE HOP 181
from a point in the axil of the true leaf-bract re-
presented by the space between the two stipular
bracts.
The hop-cone is, therefore, composed of groups
of two stipular bracts and four bracteoles placed
alternately on opposite sides of the main axis or
strig of the hop.
The Lupulin Glands of the Hop. These glands,
often called " lupulin," " condition " or " hop meal,"
are golden-yellow, pollen-like grains attached to
various parts of the hop-cone. Botanically they
are regarded as glandular hairs.
If the bracteoles of a hop-cone are examined
with a pocket lens it will be observed that a large
number of these glands are attached to their outer
surface and are especially numerous near their base.
The thin skin or perigonium, enclosing the seed, is
also thickly covered with them. Some also are
usually found on the inner surface of the stipular
bracts. 1 On the number and size of these lupulin
glands depends to a large extent the value of the
hop, as they contain the essential oil and resins
(" condition ") which are of so much importance to
the brewer.
Examine Lupulin Glands with the Microscope
when Attached to a Bract. It will be noticed that
they are spherical, very thin-walled cells, enclosing
an oily liquid. Their points of attachment to the
1 See Hops and their Botanical and Commercial Aspect, by
E. Gros, p. 23.
182 STUDIES FOR BREWING STUDENTS
leaf are very slender and consequently they are
readily broken off when touched.
Compare the Bared Strig of Different Varie*
ties of Hops. It will be noticed that the distance
between the groups of branches is variable. A
numerical expression of the variability may be
obtained by counting the number of groups of
branches in three-quarters of an inch of strig. The
number found may vary from about five to about
eight, and the expression " density number " is
often applied to it, as it expresses the relative
compactness of the bracts of the hop-cone. The
density number of the hop-cone taken in conjunc-
tion with the shape of its bracts is of great use in
helping to classify or identify the different varieties
of hops. 1
The student should now receive instruction in
the commercial methods of judging and valuing hops.
It is essential that this instruction should be given
personally by a skilled instructor, who should be
provided with a large number of samples of different
kinds and qualities of hops for purposes of illus-
tration. The student should recognise, however,
that attainment of the knowledge necessary to
form a sound judgment of the value and qualities
of hops can only be gained by extensive experience.
1 For much valuable information concerning the different
varieties of hops, see a paper entitled " The Hop and its Eng-
lish varieties," by J. Percival, Journal of the Boyal Agricultural
Society, 1901, vol. Ixii.
THE HOP
183
Chemical Examination of Hops. The chemical
examination of hops is usually confined to deter-
minations of the resins and moisture, and to the
detection of sulphur.
Determination of the Soft and Hard Resins.
The method is based on the consideration that both
the soft and hard resins are soluble in ether, but that
the soft resins alone are soluble
in light petroleum spirit. 1
Weigh accurately about 3
grms. of an average sample of
the hops to be examined, place
them in a cone of filter-paper,
and transfer to a 100 c.c. Soxh-
let extractor. Connect the ap-
paratus by means of a well-fit-
ting cork with a wide-mouthed
flask containing about 100 c.c.
of petroleum ether. The pet-
roleum ether should possess a
constant boiling point of about
125 F., and should be fraction-
ally distilled, if necessary. The
top of the Soxhlet extractor
should be connected by means
of a cork with an efficient form of reflux condenser
(see Fig. 36).
The flask containing the petroleum ether is
1 For full information concerning this method see Laboratory
Text-Boole for Brewers, by Lawrence Briant.
FIG. 36. Apparatus for
Extraction of Hop Resins.
184 STUDIES FOE BREWING STUDENTS
immersed in a water bath, which is kept at a
temperature of about 155 F. During the extrac-
tion process the soft resins of the hop are only
slowly removed by the petroleum ether, and the
operation must be continued for about twenty-four
hours. After extraction is complete, the flask is
disconnected from the apparatus, and its contents
filtered through a small paper filter into a small
wide-mouthed tared flask. The petroleum ether is
then distilled off, and the flask containing the soft
resins placed in a water oven at a temperature of
about 140 F. until its weight is constant. The gain
in weight of the flask represents the amount of soft
resins obtained.
After removal of the soft resins from the hops
with petroleum ether, the hops are again extracted
with ordinary ethylic ether for the purpose of re-
moving the hard resins. About 100 c.c. of ether
is used, and the temperature of the water bath
surrounding the flask containing the ether should
be kept at about 135 F. Extraction is complete
in about twelve hours, when the ethereal extract
is filtered as in the previous operation, and evapor-
ation and drying also carried on as before.
The amounts of soft and hard resins found may
be calculated as percentages on the hops used, or
as percentages on the dried hops when the amount
of moisture in the original hops is known.
Special precautions must be taken when con-
ducting the above experiments to guard as far as
THE HOP 185
possible against the danger of explosions, as the
vapour of the liquids employed is highly inflam-
mable.
Determination of Moisture. This can be done
with a sufficient amount of accuracy for ordinary
purposes by drying about 3 grms. of the hops in a
water oven at 212 F. until the weight is constant.
Most of the essential oil of the hops is driven off",
but as the total amount is small the error involved
is but slight.
Detection of Sulphur. This method refers to
the detection of free sulphur which may have gained
access to the hops during the operation of treating
the growing hops with finely divided sulphur for
the prevention of mould.
Weigh out 5 grms. of hops and place them in a
beaker with 250 c.c. distilled water. Add about
5 grms. of pure freshly slaked lime, and boil the
mixture for twenty minutes. Filter a portion of
the solution, cool and proceed to test it at once
with a freshly prepared dilute solution of nitro-
prusside of sodium. The test may be conducted
by adding a few drops of the nitro-prusside solution
to about 5 c.c. of the extract in a test-tube, or by
bringing drops of the two solutions in conjunction
on a white porcelain tile. The formation of a red
colour indicates the presence of sulphur, due to the
reaction of the nitro-prusside with hydro-sulphide
of calcium formed by the action of calcium hy-
droxide on the free sulphur present in the hops.
186 STUDIES FOE BEEWING STUDENTS
The relative intensity of the colours found when
comparing samples of hops indicates to some extent
the relative amounts of sulphur present. When
conducting this test the hop extract must be tested
immediately after preparation, and it should be
noted that the colour reaction with nitro-prusside
is transient. As small quantities of sulphur are
very generally found in English hops, the student
should examine samples of Continental or Pacific
hops for a negative reaction if required. These
hops rarely show the presence of sulphur.
As a confirmatory test for the colour reaction,
the student should add a few drops of sulphuric
acid to a little of the hop decoction, and, after heat-
ing it, note the presence of sulphuretted hydrogen
by its characteristic odour.
Estimation of Tannic Acid. This estimation is rarely made.
For information the student is referred to a paper by J. Heron
on the tannin of hops (Journal of the Federated Institutes of
Brewing, 1896, vol. ii., p. 162).
TABLE I.
Solution Factors for Carbo-hydrates at Various Densities.
Specific Gravity at 15-5 C. (From Brown, Morris and
Millar, Journal of the Chemical Society, 1897, vol. Ixxi.,
p. 72.)
SPECIFIC
GRAVITIES 1,010
1,020 1,030 1,040 1,050 1,060 1,070 1,080 1)090
3-800
3-810
3-820
3-830
3-840
3-850
3-860
3-870
3-880
3-890
3-900
3-910
&3-920
3-930
E3-940
3-950
3-960
3-970
3-98C
3-990
4-000
4-010
4-020
4-030
4-040
4-050
4-060
188 STUDIES FOK BEEWING STUDENTS
TABLE II.
Divisors for the Transformation Products of the Hydrolysis of
Starch corresponding to [a] D and E. (Brown, Morris and
Millar, Journal of the Chemical Society, 1897, vol. Ixxi.,
p. 84.)
M
E.
Sp. Gr. 1010.
Sp. Gr. 1020.
Sp. Gr. 1030.
Sp. Gr. 1040.
198-8
5
4-038
4-031
4-023
4-015
195-6
10
4-033
4-027
4-020
4-013
192-4
15
4-029
4-023
4-017
4-010
189-2
20
4-024
4-019
4-013
4-008
186-0
25
4-019
4-014
4-010
4-005
182-8
30
4-014
4-011
4-006
4-001
179-6
35
4-009
4-005
4-002
3-997
176-4
40
4-004
4-001
3-997
3-993
173-2
45
3-999
3-996
3-992
3-989
170-0
50
3-994
3-991
3-987
3-985
166-8
55
3-989
3-986
3-983
3-979
163-6
60
3-984
3-980
3-977
3-974
160-4
65
3-979
3-975
3-972
3-969
157-2
70
3-973
3-970
3-966
3-963
154-0
75
3-968
3-964
3-961
3-957
150-8
80
3-962
3-958
3-955
3-951
147-6
85
3-957
3-952
3-948
3-945
144-4
90
3-951
3-946
3-942
3-938
141-2
95
3-945
3-940
3-935
3-931
138-0
100
3-939
3-934
3-929
3-924
STUDIES FOR BREWING STUDENTS 189
TABLE III.
Keducing Values of Varying Quantities of Dextrose, Levulose
and Invert- Sugar under Standard Conditions. (From
Brown, Morris and Millar, Journal of the Chemical Society,
1897, vol. Ixxi., p. 281.)
Dextrose.
Levulose.
Invert-Sugar.
O
"S
I
a
E
O
d
O
E
O
d
d
Cu. Corresponding
to 1 Griu. Dextrose.
Levulose. Grms.
GO
O
3
o
5
6
u
boaJ
fl OT
.3 o
13^
p
o
oo
SrH
00
Invert-Sugar. Grms.
03
1
E
O
o
1
E
Cs
O
O
050
1030
1289
2-060
050
0923
1155
1-846
050
0975
1221
055
1134
1422
2-061
055
1027
1287
1-858
055
1076
1349
060
1238
1552
2-063
060
1122
1407
1-870
060
1176
1474
065
1342
1682
2-062
065
1216
1524
1-871
065
1275
1598
070
1443
1809
2-061
070
1312
1645
1-874
070
1373
1721
075
1543
1935
2-058
075
1405
1761
1-873
075
1468
1840
080
1644
2061
2-055
080
1500
1881
1-875
080
1566
1963
085
1740
2187
2-046
085
1590
1993
1-871
085
1662
2084
090
1834
2299
2-038
090
1686
2114
1-873
090
1755
2200
095
1930
2420
2-033
095
1774
2224
1-868
095
1848
2317
100
2027
2538
2-027
100
1862
2331
1-862
100
1941
2430
105
2123
2662
2-024
105
1952
2447
1-859
105
2034
2550
110
2218
2781
2-020
110
2040
2558
1-855
110
2128
2668
115
2313
2900
2-012
115
2129
2669
1-851
115
2220
2783
120
2404
3014
2-003
120
2215
2777
1-846
120
2311
2898
125
2496
3130
1-997
125
2303
2887
1-843
125
2400
3009
130
2585
3241
1-990
130
2390
2997
1-840
130
2489
3121
135
2675
3354
1-981
135
2477
3106
1-834
135
2578
3232
140
2762
3463
1-973
140
2559
3209
1-828
140
2663
3339
145
2850
3573
1-964
145
2641
3311
1-822
145
2750
3448
150
2934
3673
1-956
150
2723
3409
1-815
150
2832
3546
155
3020
3787
1-948
155
2805
3517
1-811
155
2915
3655
160
3103
3891
1-940
160
2889
3622
1-806
160
3002
3764
165
3187
3996
1-931
165
2972
3726
1-803
165
3086
3869
170
3268
4098
1-922
170
3053
3828
1-799
170
3167
3971
190 STUDIES FOE BKEWING STUDENTS
TABLE III. continued.
Dextrose.
Levulose.
Invert-Sugar.
be oJ
boai
O &
09
i
1
O
1
i
II
i
1
03
II
a s
E
C
(
t
5
3
8^
j
i
i
a> .
&
5
o
,j
d
Js
8
P
d
O S
a
d
9
2
1
o
o
QO
1
o
a
P
o^
3
03
a
t-H
175
3350
4200
1-914
175
3134
3930
1-793
175
3251
4076
180
3431
4302
1-906
180
3216
4032
1-787
180
3331
4177
185
3508
.4399
1-896
185
3297
4134
1-782
185
3410
4276
190
3590
4501
1-890
190
3377
4234
1-777
190
3490
4376
195
3668
4599
1-881
195
3457
4335
1-773
195
3570
4476
200
3745
4689
1-872
200
3539
4431
1-769
200
3650
4570
205
3822
4792
1-863
205
3616
4534
1-765
205
3726
4672
The sugar values for weights of Cu or CuO lying between
any of the weights given in the above table must be arrived at
by calculation.
Example. The amount of dextrose corresponding with -2385 grm.
Cu is required. On referring to the table -2313 grm. Cu corresponds
with -115 grm. dextrose ; and -2404 grm. Cu with -120 grm. dextrose.
Hence -2404 - -2313 = '0091 grm. Cu ; and -120 - -115 = -005 grm.
dextrose. Therefore -0091 grm. Cu = -005 grms. dextrose in the portion
of the table used. Now the difference between the amount of Cu found,
2385, and the nearest lower amount in the table, -2313 grm. is -0072 grm.
Hence : -0091 : -005 : : '0072 : -004. Therefore -115 + '004 = -119 grm.
dextrose corresponding to -2385 grm. Cu.
STUDIES FOE SKEWING STUDENTS 191
TABLE IV.
Beducing Values of Varying Quantities of Maltose under
Standard Conditions. (From Brown, Morris and Millar,
Journal of the Chemical Society, 1897, vol. Ixxi., p. 100.)
Maltose
Grms.
Cu.
Grms.
CuO.
Grms.
Cn. Corre-
sponding to
IGrm.
Maltose.
Maltose.
Grms.
Cu.
Grms.
CuO.
Grms.
Cu. Corre-
sponding to
IGrm.
Maltose.
070
0772
0966
1-1029
190
2072
2593
1-0953
075
0826
1034
1-1026
195
2126
2661
1-0949
080
0880
1102
1-1023
200
2180
2729
1-0946
085
0934
1169
1-1020
205
2234
2797
1-0943
090
0988
1237
1-1017
210
2288
2865
1-0940
095
1042
1305
1-1013
215
2342
2933
1-0937
100
1097
1373
1-1010
220
2397
3000
1-0933
105
1151
1441
1-1007
225
2451
3068
1-0930
110
1205
1509
1-1004
230
2505
3136
1-0927
115
1259
1576
1-1001
235
2559
3203
1-0924
120
1313
1644
1-0997
240
2613
3272
1-0921
125
1367
1712
1-0994
245
2667
3340
1-0917
130
1422
1779
1-0991
250
2722
3407
1-0914
135
1476
1848
1-0988
255
2776
3475
1-0911
140
1530
1916
1-0985
260
2830
3543
1-0908
145
1584
1983
1-0981
265
2884
3610
1-0905
150
1634
2051
1-0978
270
2938
3678
1-0901
155
1692
2119
1-0975
275
2992
3747
1-0898
160
1747
2186
1-0972
280
3047
3814
1-0895
165
1801
2254
1-0969
285
3101
3882
1-0892
170
1855
2323
1-0965
290
3155
3950
1-0889
175
1909
2490
1-0962
295
3209
4017
1-0885
180
1963
2458
1-0959
300
3264
4085
1-0882
185
2017
2526
1-0956
305
3318
4154
1-0879
192 STUDIES FOR BREWING STUDENTS
TABLE V.
Spirit Indication Table showing Degrees of Gravity lost in
Malt Worts during Fermentation. Distillation Process.
(Graham, Hoffmann, and Eedwood.)
s^
1S|
H ^ *
<D 'S.-2
Q 02
o
1
2
3
4
5
6
7
8
9
3
6
9
1-2
1-5
.1-8
2-1
2-4
2-7
1
3-0
3-3
3-7
4-1
4.4
4-8
5-1
5-5
5-9
6-2
2
6-6
7-0
7.4
7-8
8-2
8-6
9-0
9-4
9-8
10-2
3
10-7
11-1
11-5
12-0
12-4
12-9
13-3
13-8
14-2
14-7
4
15-1
15-5
16-0
16-4
16-8
17-3
17-7
18-2
18-6
19-1
5
19-5
19-9
20-4
20-9
21-3
21-8
22-2
22-7
23-1
23-6
6
24-1
24-6
25-0
25-5
26-0
26-4
26-9
27-4
27-8
28-3
7
28-8
29-2
29-7
30-2
30-7
31-2
31-7
32-2
32-7
33-2
8
33-7
34-3
34-8
35-4
35-9
36-5
37-0
37-5
38-0
39-6
9
39-1
39-7
40-2
40-7
41-2
41-7
42-2
42-7
43-2
43-7
10
44-2
44-7
45-1
45-6
46-0
46-5
47-0
47-5
48-0
48-5
11
49-0
49-6
50-1
50-6
51-2
51-7
52-2
52-7
53-3
53-8
12
54-3
54-9
55-4
55-9
56-4
56-9
57-4
57-9
58-4
58-9
13
59-4
60-0
60-5
61-1
61-6
62-2
62-7
63-3
63-8
64-3
14
64-8
65-4
65-9
66-5
67-1
67-6
68-2
68-7
69-3
69-9
15
70-5
71-1
71-7
72-3
72-9
73-5
74-1
74-7
75-3
75-9
STUDIES FOR BREWING- STUDENTS 193
TABLE VI.
Original Gravity Determination. Table for Ascertaining the
Correction for Acid.
S3 .
O> O (-1
Corresponding Degrees of Spirit Indication.
ill
00
01
02
03
04
05
06
07
08
09
o
02
04
06
07
08
09
11
12
13
1
14
15
17
18
19
21
22
23
24
26
2
27
28
29
31
32
33
34
35
37
38
3
39
40
42
43
44
46
47
48
49
51
4
52
53
55
56
57
59
60
61
62
64
5
65
66
67
69
70
71
72
73
75
76
6
77
78
80
81
82
84
85
86
87
89
7
90
91
93
94
95
97
98
99
1-00
1-02
8
1-03
1-04
1-05
1-07
1-08
1-09
1-10
1-11
1-13
1-14
9
1-15
1-16
1-18
1-19
1-21
1-22
1-23
1-25
1-26
1-28
1-0
1-29
1-31
1-33
1-35
1-36
1-37
1-38
1-40
1-41
1-42
13
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