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copy "CHE LECITHIN CONTENT OF BUTTER AND ITS POSSIBLE RELATIONSHIP ie TO THE BISHY FLAVOR

A THESIS

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL

OF CORNELL UNIVERSITY FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

BY

GEORGE CORNELL SUPPLEE

{Reprint from the Cornell University Experiment Station Memoir 29, November, 1919]

ANDER eter)

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foe PEGCIHIN CONTENT OF BUTTER AND ITS POSSIBLE RELATIONSHIP VOVIIE FISHY FLAVOR

A THESIS

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL

OF CORNELL UNIVERSITY FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

BY

GEORGE CORNELL SUPPLEE

[Reprint from the Cornell University Experiment Station Memoir 29, November, 1919]

9. OF w. we 8 7999.

CONTENTS

Pete IOUS Wn eS ben IONS 20 fc gee on oe ok een bea te wks wee 101 Lecithin decomposition in butter as a possible cause of the fishy flavor 194 Chemical constitution, properties, and distribution of lecithin. ... 104

Ticoremcah mistuisslane..- spa 6.c pase oe ty hO dla PsbS vs seo bas 106 MG eS O a VOM WORK 2 pes. aloe Gas, Seer ee aoe sO ECE Gewese bs 108 Qualitative determination of lecithin in butter................. 108 Amount ot leqithimim BUGter oN ic wasn one ee Sok wade ve tee an o 110 Trimethylamine salts of the fatty acids.....................0. 111 Effect of working trimethylamine salts of the fatty acids into CLUE E cs ee ea RR Oe ee . bs Quantitative estimation of trimethylamine in fishy butter...... 123 Development of fishy flavor in experimental butters............ 126 Variation in acid value of experimental butters.............0.. 129 Trimethylamine and ammonia content of experimental butters... 134 [ESR tera Paikores tere | US} 0 ho’ ane aeRO en A eee ee a 136 BCLeU Ge ANALY Sis yc. seed Deo ee Oconmiees Himba See Soares x 4 « 137 Inoculations for the purpose of developing the fishy flavor. . 137 Longevity of Bacterium ichthyosmius in butter...........000.00. 141 Further studies with Bacterium ichthyosmius.. 0.000.000.0000 00% 142 Trimethylamine and ammonia production by Bacteriwm ich- thyosmius in milk and cream.............. 0000 c cece ee eee 144 Production of trimethylamine from lecithin and choline by TOA er HAS 2s he Ge SPE, secede cgrgts & Sea iee ae a ae bial . 145 Bacterial inoculations into butterfat......................04. 146 aeRO Say be MOE AG, ny a 2 ee BOR ced a Sw OV Be sats 148 Pe enor ISOM Mia Svs Je. us-A6 ds ks eek = shed eld ae coco Ao Seeds 149 Lb) (Ol G{¢9 924) Came en Re 9 ene em ee 150

97

THE LECITHIN CONTENT OF BUTTER AND ITS POSSIBLE RELATIONSHIP TO THE FISHY FLAVOR

THE LECITHIN CONTENT OF BUTTER AND ITS POSSIBLE RELATIONSHIP TO THE FISHY FLAVOR!

GEORGE CORNELL SUPPLEE

The commercial value of butter is based to a great extent on its quality, which in turn is determined *by its flavor. The commercial grading of butter on this basis, and the recognition of certain characteristic defects, have resulted in the establishment of certain terms more or less descriptive of the flavors found. Among the terms commonly applied to the flavors in butter are such words as metallic, fishy, oily, rancid, tallowy. Since the presence of any of these flavors carries with it a reduction in commercial value of the butter, considerable effort has been made to determine their causes and prevent their development. Unfortunately most of these efforts have not met with a high degree of success. This may be ascribed to several reasons, among which are the following: lack of positive identification of the same flavor by different investigators; lack of adequate chemical methods for the isolation and measurement of the small amount of sub- stance capable of producing the flavor; and lack of cooperation between the chemist, the bacteriologist, and the experienced butter judge.

Fishiness in butter, with which this investigation is primarily con- cerned, is usually described as a flavor resembling that of salmon or mackerel, altho the names of other varieties of fish are occasionally used to describe the flavor more explicitly. While the typical fishy flavor in butter is readily recognized by experts, it is often accompanied by a more or less oily condition which tends to create differences of opinion as to its exact nature. But if the opinion of butter judges of long experience is to be considered as trustworthy, it may be said that the true fishy flavor is entirely distinct. from the oily flavor even tho the oily condition may precede or accompany it.

PREVIOUS INVESTIGATIONS

The earlier investigations bearing on fishiness in dairy products have been largely confined to milk and butter. One instance is recorded, how- ever, in which this condition was observed and studied in evaporated milk.

1 Also presented to the Faculty of the Graduate School of Cornell University, December, 1918, as a major thesis in partial fulfillment of the requirements for the degree of doctor of philosophy.

101

102 GEORGE CORNELL SUPPLEE

Harding, Rogers, and Smith (1900)? report the fishy flavor in a sample of milk brought to the New York State Agricultural Experiment Station at Geneva in 1900. The source was traced to a single animal in the herd, but investigation failed to locate any pathological condition or any irregularity in the feeding which might cause the trouble. Attempts to reproduce the flavor by inoculating milk with bacteria isolated from this eow’s udder also failed. The same authors mention also an instance brought to their attention by W. E. Griffith, in which a peculiar flavor developed in June butter after storage at 18° to 22° F. This flavor was described by butter experts as fishy.

Piffard (1901) discusses the fishy flavor in dairy products, and suggests the possible relationship between certain algae found in stagnant water, and fishiness in milk and butter. His theory is supported by the fact that the flavor is often produced in water by the growth of algae and diatoms, and he believes, therefore, that cows having access to such water may transmit the condition to milk. Referring to the flavor in butter, he considers the idea that salt may be responsible and mentions the ability of Sait to absorb flavors and odors of materials stored near it.

Harrison (1902), discussing butter defects at about the same time, states that the characteristic off-flavors of butter — fishy butter being specifically mentioned — are caused by the growth of undesirable bacteria in the cream.

O’Callaghan (1902) published certain observations on fishiness in Australian butter. He states that he has found this condition in butter only two hours old. From his investigations he concludes that Oidium lactis is the causal agent. Later (O’Callaghan, 1908) he elaborated on his former views, concluding that Oid/um lactis associated with the lactic- acid bacteria in eream will usually produce a fishy flavor in the butter. He mentions the presence of the defect in unsalted butter, and recom- mends the improvement of sanitary conditions in the creameries, and pasteurization, as a remedy. His conclusions have not been confirmed by other investigators. ;

Rogers (1909), after a rather exhaustive study of the occurrence and ‘ause of fishy butter, confirms many observations commonly noted in connection with this trouble but is unable to confirm the observations of O’Callaghan. He also seemingly eliminates the theory that trimethyl-

? Dates in parenthesis refer to Bibliography, page 150,

—

THe LeEcITHIN CONTENT OF BUTTER 103

amine is directly responsible for the flavor. After studying the effect. of high-acid cream, overworking, and the consequent increased oxygen con- tent of the butter, and by conducting bacteriological investigations, he concludes that high-acid cream is essential to bring about the condition, altho he points out that not all butter made from such cream develops the fishy flavor. In this respect he states (page 20 of reference cited) that “ fishy flavor may be produced with reasonable certainty by over- working the butter made from sour cream.’ From his viewpoint the probability that microorganisms are the cause falls into disfavor by the advancement of the opinion that “‘ fishy flavor is caused by a slow, spon- taneous, chemical change to which acid is essential and which is favored by the presence of small amounts of oxygen” (page 20 of reference). More recently the same author (Rogers, 1914, a and b) points out that fishiness in butter may be preceded by an oily or a metallie flavor, and reiterates his views that the evidence is against the theory that the fishy condition is of a bacterial nature. He also states (1914b) that ‘ fishy flavor is said to occur rarely or not at all in unsalted butter and it is possible that the salt furnishes certain conditions which are essential to the development of the fiavor.”’

Reakes, Cuddie, and Reid (1912) find no significant differences in the bacterial flora of fishy and of high-grade butter, and, in agreement with Rogers, state that “‘ the development of fishy flavour in butter arises as a result of a chemical change inducing a splitting-up of some of the con- stituents into compounds possessing this peculiar character of smell and taste, the factors responsible for such change being apparently a degree of high acidity of the cream and overworking.”’

Hunziker (1916) states that high pasteurization temperatures (185° F.) when used on sour cream tend to produce a very poor quality of butter, which often has a disagreeable oily taste suggestive of fishiness. He points out that this is particularly true when cows are on green pasture and the butterfat contains a rather high percentage of olein, which may be oxidized with relative ease in the presence of high temperatures and high acid.

Hammer (1917) reports that he found a can of evaporated milk which possessed a marked fishy flavor and odor and from which he was able to isolate an organism heretofore undescribed. He gives to this organism the name Bacterium ichthyosmius, which was suggested by Dr. A. W.

104 GEORGE CORNELL SUPPLEE

Dox. The description of the organism seems to indicate that it is closely allied to the Proteus group. By inoculation experiments Hammer was able to reproduce the flavor in milk and cream under both aerobic and anaerobic conditions. He noted that the intensity of the odor was in- ereased by the addition of alkali to the milk after the incubation period. He was unable, however, to produce fishiness in butter by direct inocula- tion or by inoculating the cream before churning. Bacteria counts at various intervals during the storage period showed an immediate decrease in numbers in salted butter, and an increase during the first few days in unsalted butter followed by a pronounced decrease.

Washburn and Dahlberg (1918), while studying the influence of salt on storage butter, found that salted butter was more likely to turn fishy in storage than was unsalted butter, and furthermore that there appeared to be a tendency toward a progressive development of the flavor thru metallic to oily and finally to fishy.

LECITHIN DECOMPOSITION IN BUTTER AS A POSSIBLE CAUSE OF THE FISHY FLAVOR

CHEMICAL CONSTITUTION, PROPERTIES, AND DISTRIBUTION OF LECITHIN

Lecithin, which stands in close relation to the fats, belongs to a more or less definite group of substances known as phosphatides, or phos- phorized fats. These bodies appear to be a group of esters containing nitrogen, phosphoric acid, and fatty-acid radicals. Lecithin, which is the best known of the phosphatides, contains two fatty-acid radicals and the nitrogenous base choline, eombined with glycerophosphorie acid. According to the kind of fatty acid present in the molecule, it is possible to have various types of lecithin, such as stearyl, palmityl, and oleyl. A number of investigators seem to agree that every true lecithin con- tains at least one oleic-acid radical. There seems to be uncertainty as to whether choline is the only base present in lecithin. MacLean (1909) was able to get only 42 per cent of the theoretical amount from lecithin isolated from heart muscle, and 65 per cent from lecithin of egg yolk. Other investigators have found the same to hold true of lecithin from different sources.

Lecithin has certain properties in common with the fats, particularly with respect to its solvents. It differs, however, by being less soluble

Tue LeciraHin Content or BUTTER 105

in ether and more soluble in alcohol. It is precipitated from alcoholic solution by acetone; in water it swells to a colloidal mass which on micro- scopic examination appears as oily drops and threads. It saponifies with alkalies and baryta water, yielding the corresponding soaps, salts of elycerophosphoric acid, and choline. Hammarsten and Hedin (1915) state that it is slowly decomposed by dilute acids and enzymes (lipase). Barger (1914) states that Bactertum prodigiosus produces trimethylamine from choline and lecithin; he also cites references to show that lecithin is decomposed during putrefaction, yielding fatty acids, glycerophosphoric acid, choline, and ultimately trimethylamine. Hasebroek (1888) claims that methylamine, ammonia, methane, and carbon dioxide may be finally produced from choline during putrefaction. On being heated with strong caustic soda or potash, lecithin yields trimethylamine, which has a dis- tinct fishy odor, this being one of the characteristic qualitative tests for it. Leathes (1913), in citing the work of various investigators, seems to think that lecithin is rather unstable. He thinks this property is due to the unsaturated oleic-acid radical contained, and offers this as the reason why the substance gives Pettenkofer’s reaction. The work of Long (1908), however, seems to indicate that lecithin is more stable than has been generally believed. Koch (1902-03) has shown that various salts will cause lecithin to precipitate as a gelatinous mass, and that acids, if sufficiently dissociated (0.005 M_ sulfuric), will accomplish the same thing.

Lecithin seems to be widely found in nature, being present in many plant cells and animal fluids. It is particularly abundant in the brain, the nerve tissue, and the yolk of egg. It is also reported as existing in blood corpuscles, blood plasma, lymph, milk, and bile. Since the methods used for the quantitative estimation of lecithin depend on the deter- mination of phosphorus in alcoholic or alcohol and ether extracts, it is doubtful whether the figures given are absolutely correct due to the fact that other phosphatides are extracted and also because the empirical formula used in the calculation may be inaccurate for the particular lecithin involved. Altho there have been conflicting statements as to whether milk contains lecithin, there seems to be sufficient evidence that it does. The results obtained by Nerking and Haensel (1908) are sub- mitted in table 1:

106 GEORGE CORNELL SUPPLEE

TABLE 1. Tuer Lecrrarn Content or Various MiLkKs

(From Nerking and Haensel)

Percentage of lecithin Isind of milk

Highest Lowest Average

EUuieet lO SATADICS 4 ts.c feu eicee 2 Ne Veer ea roar 0.080 0.024 0.050 Cow /s; 1 7-seriples: ssh be aaes seco is eek eee eee 0.116 0.036 0.063 ABS 6 ORATINOIORS oy 8lm 4 osteo alee xa elo ecm EN 0.0389 0.006 0.016 weld; Aisa lGhin on64 sta te bea eee Lee ees 0.167 0.051 0.083 Goate hE) samples: 7./. 2.405% oe eek. 28 kee eer 0.075 0.036 0.049 Mares, S SAtANles sdk 5 se ver. 2-3 vans 04 e oe eee 0.017 0.007 0.011

Glikin (1909), studying the lecithin and iron content of milk, reports 0.0515 per cent lecithin in whole milk, 0.05 per cent in cream, and 0.1329 per cent in human milk. fetzer (1911), studying the lecithin content of milk under pathological conditions, finds that it is lower in milk from cows suffering with mastitis than in milk from normal cows. He finds also that the lecithin content decreases as the fat decreases. The work of Bordas and De Raezkowski (1902) indicates that the amount of lecithin varies with the lactation period. They find that it is at the maximum at the beginning, and gradually deereases during the remainder of the period. Their observations were from seven cows.

THEORETICAL DISCUSSION

Trimethylamine as a decomposition product of lecithin was brought to the attention of the writer as a possible cause of the fishy flavor in butter about three years ago, when he was working with lecithin isolated from brain tissue. Altho there seem to be no published data concerning trimethylamine in relation to this subject, and Rogers (1909) claims that it can be worked into butter in large amounts without producing the fishy flavor, it is nevertheless believed by many that this substance is in some way responsible. On boiling lecithin isolated from brain tissue and egg yolk with strong caustic soda, the writer has been able to obtain a distinet oily and fishy odor which was asserted by many to be typical ef the odor of fishy butter. The only possible source of such an odor in this case was the trimethylamine derived from the lecithin. This result, together with the fact that there seems to be good evidence that lecithin is present in milk, led to the assumption that the substance

THe LEcITHIN CONTENT OF BUTTER 107

may exist also in butter and that by its decomposition it can slowly liberate trimethylamine with the consequent production of the fishy flavor and odor. ;

Provided that lecithin can be shown to exist in butter, the above assump- tion is supported by several facts. It is well known that enzymes are capable of bringing about many chemical decompositions which result from the action of acids and alkalies on organic substances. It also appears to be a fact that butter may contain enzymes derived from the udder, and from.the action of bacteria in the milk or the cream before it is made into butter, and furthermore that the activity of such enzymes is not entirely stopped at the temperature at which butter is stored. Hammar- sten and Hedin (1915) state that lecithin is decomposed by dilute acids and enzymes. The citations of Hasebroek (1888), Barger (1914), and others show that lecithin is decomposed by bacteria yielding choline, which finally yields trimethylamine. It is also a well-known fact that this substance in very small amounts possesses a distinct fishy odor, but in concentrated solution it has a strong ammoniacal odor. Speaking of the former property, Davis (1912), quoting Taylor, states that the ‘‘ odour (referring to the peculiar fishy odor suggestive of herring brine] is grad- ually developed by adding lime to a solution of the base, but requires some time to reach its maximum intensity.”

In addition to the foregoing facts the writer has observed certain fea- tures that may have some bearing on this problem. In inspecting butter used in the Navy, it has been noticed that certain samples of cream evolved a peculiar fishy odor on the addition of alkali used for titration. This phenomenon was first brought to the attention of the writer by A. M. Besemer, and has since been confirmed by a number of men, some of whom have wide reputation as butter judges. Since trimethylamine is a base which is liberated from its acid combinations by alkalies, it is quite possible that the odor mentioned above was due to this substance’s having been liberated from its acid combination in the cream. If such were the case, it is conceivable that butter made from such cream might, during storage, give up its trimethylamine thru the action of enzymes. In this connection it has been noted that certain samples of old butter, which were not scored as fishy, when brought into contact with a warm solution of soap powder would give off a strong herring-like odor. This phenomenon might also be explained as in the case of the cream. In addition to these features it has been noted that certain samples of fishy

i08 GEORGE CORNELL SUPPLEE

butter may lose their characteristic flavor after a period of time. This has also been observed by other investigators. It is possible that this characteristic may be explained by the fact that, since trimethylamine is extremely volatile, it may pass off, or that the instability of the acid combination changes so that the conditions are not right for its manifes- tation. The writer has noted a very strong fishy odor in partially decom- posed ege yolk held at refrigerator temperature, which had entirely dis- appeared two weeks later.

On the basis of the foregoing facts and observations and the evident lack of contradiction of most of them with what is known about fishy butter, the following experimental work was planned with the object of determining the possible relationship of trimethylamine to this flavor. In calling attention to the lecithin, it may be stated that the writer is cognizant of the fact that trimethylamine may be produced from other substances. This material has been chosen as the object of study pri- marily because there is exact knowledge concerning its cleavage and some of the agencies bringing this condition about.

INVESTIGATIONAL WORK QUALITATIVE DETERMINATION OF LECITHIN IN BUTTER

The first experimental work undertaken was to demonstrate the pres- ence of lecithin in butter, since there appeared to be no reports on this point in the literature. One hundred grams of melted butter was thoroly mixed with sufficient anhydrous calcitum sulfate (about one kilogram) so that the mixture retained its dry powdered form to such an extent that it could be readily sifted between the thumb and the finger. The inixture was transferred to a specially constructed percolator and extracted for 48 hours with 95-per-cent alcohol at 60° C. The alcoholic extract was evaporated down and the residue was treated with a small amount of ether, which took up the fat, the fatty acids, and part of the lecithin. The part insoluble in ether was again taken up with warm alcohol, and what may be termed the lecithin fraction was precipitated by thoroly cooling the aleoholie solution. The substance thus obtained precipitated in the form of small, wart-like masses, which clung tenaciously to the sides of the beaker. On this material, which presumably contained a high proportion of lecithin, various observations were made and qualita- tive tests applied. The following characteristics were noted:

Tue LECITHIN CONTENT OF BUTTER 109

On drying at ordinary temperature and pressure the material appeared as a semi-amorphous and oily substance of a pale, dirty yellow color. It was entirely soluble in alcohol but was partially thrown out of solu- tion by the addition of an excess of ether. The precipitate formed in this manner was finely granular and was white in color. In water it formed a semi-colloidal solution which on microscopic examination ap- peared as minute oily drops. When the watery suspension was heated, the particles would cohere to form a sticky mass which changed to a distinct brown color. Both the dry substance and the watery suspension, when heated with strong caustic soda, gave off a marked fishy odor resembling sometimes dried herring and sometimes salmon oil. This observation was in the great majority of cases confirmed by a number of colleagues. The fishy odor obtained from the material in this manner seemed to furnish positive evidence that lecithin was present. To further strengthen this belief, Pettenkofer’s test with sugar and sulfuric acid was -applied to the dry material with positive results. The above observations were confirmed with lecithin extracted from fresh butter, salted and unsalted, and from other miscellaneous samples of normal butter.

Altho the evidence that lecithin exists in butter in detectable quanti- ties seemed conclusive, it was decided to determine, if possible, the pres- ence of choline, which, as already pointed out, is one of the components of the lecithin molecule. This was accomplished by boiling the residue of the first alcoholic extract with baryta water, which removed all fat, fatty acids, and fatty-acid radicals of the lecithin in the form of barium soaps. After the barium soaps were filtered off, the excess barium was removed with carbon dioxide, the barium carbonate filtered off, and the filtrate containing choline and barium glycerophosphate evaporated to a sirupy consistency. This residue was then treated with absolute alcohol, in which choline is soluble but barium glycerophosphate is insoluble. On evaporation of the absolute alcohol a small amount of sirupy sub- stance remained. To this material qualitative tests for choline were applied. The most characteristic of such tests is the periodide test described by Stanék (1905), which is made by adding a small amount of strong iodine solution (153 grams of iodine and 100 grams of potassium iodide in 200 grams of water) to an aqueous solution of choline. <A posi- tive test is indicated by the formation of a brown precipitate of choline periodide, which on microscopic examination in the presence of the reagent appears as dark brown refractive and notched prisms or rhomboidal

110 GEORGE CORNELL SUPPLEE

leaflets. On evaporation of the reagent the crystals lose their shape and appear to liquefy, forming brown, oily droplets which again assume their erystalline structure on the addition of more reagent. On the application of this test to the choline obtained from butter lecithin, * was found that the results conformed in all respects to the descriptions of this periodide. The accompanying plate of photomicrographs (Plate VI) shows the characteristic crystals and oily droplets of the periodide formed by the choline from butter. In addition to this test it was shown that the small amount of choline obtained would give off a slight but distinct fishy odor on being heated with solid caustie soda.

AMOUNT OF LECITHIN IN BUTTER

Since the qualitative tests seemed to leave no room for doubt as to the presence of lecithin in butter, the next step was the quantitative estimation of this substance. In view of the evident difficulty in securing an absolutely pure lecithin free from other phosphatides, the estimations were based on the phosphorus content of extracts and the amount. of lecithin calculated according to the formula of the distearyl type. The results of such determinations on various types of butter made from different lots of cream are shown in table 2:

TABLE 2. Lecirain Content in Various Burters*

arnt Lecithin, g Type of cream from eae P.O; distearyl Sample F ; butter |, which butter was made awe) | (per cent) type (days) (per cent) 1 OR oo RN ee eet ir Raw sweet............. 6 0.0127 0.0723 ) re kee oe re ae, ee Pasteurized sweet....... 6 0.0127 0.0723 SF ivi ty etek ee Raw ripened............ 6 0.0122 0.0693 Le of Senate 2 ee ee eee Pasteurized ripened...... 6 0.0075 0.0433 Berna) ehee tate on tee eee Raw sweet............. 48 0.0052 0.0522 Ie yarn ante borates ere Pasteurized sweet....... 48 0.0120 0.0682 1 niudse ceQeee ee ee eee Raw ripened............ 48 0.0086 0.0488 ISu.5 ik < 6 ee Oe eee Pasteurized ripened...... 48 0.0083 0.0471 I Fe ey me Raw sweet............. 72 0.0111 0 063 Le bicces See ee e Pasteurized sweet. ...... (e. 0.0089 0.0505 Lae 2. sacle Ene Bie ee Raw ripened............ 12 0.0083 0.0471 Le, caeke, oe es: See Pasteurized ripened... ... 72 0.0095 0.0540

* These figures were furnished by J. T. Cusick, chemist for the State Department of Agriculture, located at Cornell University.

Memorr 29

Puate VI

& veo . ys Ne - % Fal 4 é ‘ S & - / & H & , i ee @ Ll i ee ES | <3 ® Snowy Ae acne i ¢ f : é ee Ee) a és a >

CHOLINE PER!ODIDE CRYSTALS OBTAINED FROM CHOLINE OF BUTTER LECITHIN Photomicrographs, X 380

Tue LECITHIN CONTENT OF BUTTER 111

A study of table 2 shows a fairly constant lecithin content in butter from various lots of cream and in different types of butter made from the same lot of cream. There is one feature, however, which is worthy of note, and this is that in most instances there is a tendency toward a lower lecithin content in the ripened-cream butter than in that made from unripened cream. This may be significant in the light of the statement by Hammarsten and Hedin (1915), that lecithin is decomposed by dilute acids and enzymes. This fact applied to these results might indicate that the acidity of the cream slowly decomposed the lecithin, and its decomposition products, particularly the glycerophosphorie acid, were washed out with the buttermilk. If such were the case it would be very easy to account for the lower phosphorus content in sour-cream butter.

TRIMETHYLAMINE SALTS OF THE FATTY ACIDS

It was decided that before an attempt was made to correlate trimethyl- amine with the fishy flavor of butter, this substance should be prepared in a pure state and those characteristics determined which might have a bearing on this particular problem. Trimethylamine was made by heating 50 grams of ammonium chloride and 440 grams of a 40-per-cent solution of formaldehyde in the autoclave at 122° C. for thirty minutes. Any excess formaldehyde was then expelled and the trimethylamine liberated from its hydrochloride by distilling from an alkaline solution. A 10-per-cent solution was easily obtained at the ordinary temperature and pressure. The trimethylamine thus procured was combined with lactic, butyric, oleic, and stearic acids, and also with the mixed soluble and insoluble fatty acids obtained from butter according to the procedure outlined by Browne (1899). While the properties of these acid addition products seemed to offer an interesting field for study, only such of their characteristics are recorded here as might have a direct relationship to the fishy flavor in butter, namely, their stability, volatility, and behavior in the presence of sodium chloride.

The lactic-acid combination with trimethylamine proved to be a rela- tively stable oily liquid possessing no characteristic taste other than that shown by many common salts. The odor, especially after the liquid had been standing in a stoppered bottle, seemed to be slightly fishy. Evidence on this point is not conclusive because it is possible that this was due to excess trimethylamine added at the time of neutralization and

1s, GEORGE CORNELL SUPPLEE

not shown by the indicator used. Heating seemed to intensify the odor to some extent, which would indicate instability at high temperatures.

The butyric-acid combination with trimethylamine was a substance extremely volatile at ordinary temperatures. The odor greatly resembled fish oil; the taste resembling this product was manifest only when very small amounts were used, and then not regularly.

Oleic acid and trimethylamine formed a soft soap which was very unstable as evidenced by the liberation of the trimethylamine; the greater the dilution, however, the less this condition was manifested. This soap could not be obtained entirely free from water, even at the ordinary temperatures, because of the simultaneous giving-off of trimethylamine and water. This liberation was such that nothing remained but the free acid. Furthermore, on the addition of sodium chloride to its water solution, the sodium ion readily replaced the trimethylamine radical, with the consequent precipitation of the sodium soap and the formation of trimethylamine hydrochloride.

The trimethylamine stearate showed the same characteristics as the oleic soap, but to an even greater extent. Trimethylamine was con- stantly given off in large quantities, and the only way in which it could be handled as a soap was in a mixture of alcohol and water in a tightly stoppered bottle.

The mixed soluble and insoluble fatty acids combined with trimethyl- amine showed the same general characteristics as the butyric and stearic combinations, respectively.

The instability of the combinations of fatty acid and trimethylamine ean undoubtedly be accounted for by the fact that they are addition products in which the trivalent nitrogen of the latter substance changes to the pentavalent condition in the presence of an acid. The fact that these are weak acids with relatively large molecules is probably also significant. It was observed that the hydrochloride was more stable than the above salts, and that the sulfate was even more stable than the hydrochloride. This instability 8f the fatty-acid combinations and their reaction in the presence of sodium ehloride may have an impor- tant bearing on the relation of trimethylamine to fishiness in butter, and may be of particular significance in explaining why the flavor is usually found in salted butter. As yet, however, the relationship is not clear.

Tue LecITHIN CONTENT OF BUTTER 113

EFFECT OF WORKING TRIMETHYLAMINE SALTS OF THE FATTY ACIDS INTO BUTTER

The character of the trimethylamine salts of the fatty acids in pure state seemed to justify the following series of experiments, in which these salts are incorporated into various types of butter for the purpose of determining the possibility of their producing the fishy flavor in the presence of butterfat. In view of the desirability of incorporating the trimethylamine in logical amounts, the following plan was adopted:

The largest quantity of lecithin reported in cow’s milk by Nerking and Haensel (1908) was used as the basis of calculation. These authors report 0.116 per cent as the largest amount found in seventeen samples. For the calculations of this experiment, this was assumed to be lecithin of the distearyl type, and it was further assumed to be pure lecithin with the empirical formula assigned to the type named. Granting these assumptions, this amount would yield on complete decomposition the equivalent of 85 parts per million of trimethylamine. This substance alone or in acid combination was therefore added to cream, wash water, or butter on this basis. It is very evident that because of the volatility of some of the materials and because of mechanical loss, none of the samples of butter when completed would contain 85 parts per million of trimethylamine. The method of arriving at the quantity to be added seemed to afford a uniform basis and to approximate in a logical manner the amount of this substance that might be produced in butter. When the fatty acids were used aione they were added in quantities equivalent to the amounts added in the corresponding trimethylamine salts. The addition of the acids was merely for the purpose of checking against the trimethylamine.

In tables 3 to 9 inclusive are shown the comments of various Judges on different types of butter containing trimethylamine added as already indicated and incorporated by various means. Because of the great importance of the personal factor in judging butter, an effort was made in all cases to get a number of men familiar with the various flavor defects of the product. In all cases the samples were so labeled that the judges had no knowledge of their contents. They were instructed to comment on the flavor and to work independently of one another, and it is believed that this injunction was carried out. In presenting the results in tabular form the author has intentionally omitted comments having no direct bearing on the fishy flavor.

114 GEORGE CORNELL SUPPLEE

TABLE 3. Errect on THE FLAVOR OF Butter, oF ADDING TRIMETHYLAMINE AND Fatry Acips to Raw Sweer Cream wits 0.23 per Cent Acip at THE Rate or 85 Parts PER MILLION OF THE FORMER

(S indicates salted butter)

Comments by judges Material added

No. 1 | No. 2 | No. 3 No. 4 | No. 5 | No.6

NO GHITIO Ss biyxcw.n's wayne we ee Kae eae een |e Oily Oily ire VOUUI Ws 65 Ge Ceara ee ea ance 1. iirerd ra eaeep wre Oily|......

| mp > & 3 l.

id)

mM

Trimethylamine lactate. . <2 35..4 | eee waliscet s'nce laces seca |e ale ae eee nen Trimethylamine lactate........| Fishy} Oily Fishy| Fishy|.......|......

9

Trimethylamine oleate......... Fishy 7s tats ees Wiehy}.32 yee Trimothylamine oleate <.5. 6c). s<lias coos |paes cos pss Seelen 2 ee eee Sr ae

> Pe fall re Pe

TQ

Triméthylamine Stearate v.i.c.c | (SHY |s sac <5.¢| evs 14 volley b-aecee eee ene ee Trimethylamine stearate... .... High ys. o¢ 044 lnc <4 a4) os sok ee

oon Fe WwW NN Fe

> PS wm

Trimethylamine and soluble fatty

WOLUSLy Oh la. Fu a ued te eee a eee Fishy} Fishy|....... Fishy Trimethylamine and soluble fatty

MOQUE gay feusty Paes ee nine Fishy} Wishy} Oily |... ..<..|).2 20 eee

ao QD =

7 AS | Trimethylamine and_ insoluble

LOGY (HGIOB..i aera wes sacra me ileedie ered Fishy} Fishy] Fishy|....... Fishy TA Trimethylamine and insoluble

Cai hy Hobie. kes iach ilies eR OY bos san ooh’ a4 va th oe Oily

BS } SBLGSACICLL G 4.0 vs oes Wa wet ee ee ee Fishy} Fishy} Fishy].......]......

A A AS | Butyric acid........0.<:4.s26; BiAny | 5.ce < oi }ae << co's us che peer | A A A Oleic SGIG 6 4's ass Jee x Sed] a5 has eed eel era 4 cae baetere | yore

11 AS. | Soluble fatty acids... 245.265 3hdii chi eee - 54] e032 eae ba te ete o) aeeoke en 11 A Soluble'fatty acide. 2) 2.403401: lawned fei lean ea eee ee sales eee | aye ee

12. AS” | Insoluble fatty aoide. . cs« es cc ad}c vis ee ellae ye eee | oles 6 orn ae ee eee ere en 12A Insoluble fatty acids... ..6¢2ldcic% bal eee e ae yh tee ss athe te ert leap | ee

5556565656. (52666 OOOO

THe LEcITHIN CONTENT OF BUTTER 1t5

TABLE 4. Eprrect on THE FLavor or Burrer, oF ADDING TRIMETHYLAMINE AND Fatty Acips To Raw Sweet Cream wit 0.23 per Cent Actp aT THE Rate or 8&5 PARTS PER Mrnrion AND THEN WASHING THE BurreR In WaTeR ConTAINING THE SAME CONCEN- TRATION OF THE VARIOUS SUBSTANCES

(S indicates salted butter)

ee

Comments by judges

Sample Material added

No. 1 | No. 2} No. 3 | No. 4] No. 5 | No6 A SMNIMIN CUNT Die eels a 4 sou see. wes n< 34(S042bedlae as.oce lates Oily = Osby Bere ere B ONDINE ty desicce fps Sc so eras He ots oa2 ¢ OUNy Is ceoatla ce oes Oily Io ose 1 BS ; Trimethylamine...............J....... Fishy|....... Fishy| Oily | Fishy 1B PUTING GOV IRIS 255 xise e:+ suse | < sa se |id ae eo Fishy}....... Fishy]... ... 2BS | Trimethylamine lactate........)....... Oily }....... Fishy} Oily | Fishy 2B Trimethylamine lactate........ Fishy} Oily Fishy] Fishy].......)...... 3 BS | Trimethylamine butyrate.......} Fishy} Oily Fishy].......)....... Fishy 3B Trimethylamine butyrate.......| Fishy].......)....... Fishy|....... Fishy 4BS | Trimethylamine oleate.........} Fishy|......./....... Fishy|.......]...... 4B Trimethylamine oleate......... HASEY) | eer secs | re ere | ecto | ene ee Fishy 5 BS | Trimethylamine stearate.......] Fishy} Oily Fishy} Fishy]....... Fishy 5B Trimethylamine stearate.......] Fishy].......]....... BIShy| 2 ce 3a Fishy

6 BS | Trimethylamine and soluble fatty BMneere fea a yeas tie ee Fishy} Fishy]....... Fishy] Oily | Fishy

6B Trimethylamine and soluble fatty GIS eee wen aly OR Ge cs sae RY | Meee el emercees : |'aneas see | et Fishy

7 BS | Trimethylamine and __ insoluble PetDy MOLORG tee tri a hc ke Oe eS Fishy| Oily Fishy| Fishy}....... Fishy

7B Trimethylamine and _ insoluble fatby acids: ..:5..220.28403%> HISD V Trae aren | lane spade ISH peso ee] oe oe Sussm isactic acid... 2.5.5: 28256055 05% PUSHY ee Make alee eaten han Sets es oe ees 8B MOP OUIG AGI se sae ye see os fo oe COTE fate at ci ertick ites: || ae Oe CUO At le | ie ge ge PRET DV EIGIACIO S's cae oi set yey avi as c ose a lbens «|e tare a. 4-ti| fees Conss O71 er mere [arene 9B 1B} DG tains Cee ce .0) (6 eee eu a ate | DP ee Fors eee Oliva TA da elle 2 NOM PO IGIGIACIG). ©. ¥ s.c sa vacds< 8 see ee e|ieees ¥ 3.3 ibys || eee BYSHV|\o- ee Olive 10B I STOMAGIO ees SMa 6 09 5 cic aor le wee oes HOST IE, Dee lee cen lhe ee HME mMOMIDIG La bGy AGIOS. 0155 A6.8,3.0..4 2.2 |b h.2.0.5 oGlleRis eb Sel] ho aie ancl] eSeeera oe]. wearer ce octhae 11 B Sits lea bby AGI Ne sted ae os alia dean. del |'d gees stele be eatro| qancettod | oa a aera Sterne eee Sen saline techy ACIS pets aca + casea| x wicceates ancl) bcdeeeehsvare| fe a Sr airnesh| atee sie. am | shop ateneracs ls" 6 aad

12B IMR MDIS TEU LY CIOS iw. arses avril ove Parco all lee schiectvre ail okie: =r cecal leds cereue Gila ate.a. = fine acters

116 GEORGE CORNELL SUPPLEE

TABLE 5. Errecton THE FLAvor oF Burrer, oF ADDING TRIMETHYLAMINE AND Farry Acips to Raw Sweet Cream wits 0.23 per CENT AcID AT THE Rate oF 85 PARTS PER MILLION AND THEN WorkING THE SuBSTANCES DIRECTLY INTO THE BUTTER AT THE Same RATE

(S indicates salted butter)

Comments by judges

Sample , Material added

{ No. 1 | No. 2 | No. 3 | No. 4 | No. 5 | No. 6 SP INOURIB aise aces veo ae © 05 422s slaeese se a eeee tp) ve eabees Oily Oily “|see oes Cc WGLDInE .2nnmaatien ether Ve ens hence LLY Ae et all sees Oily Slee LCS: | “Trimethylimines i) 365 25 eas Vishy| Fishy} Tishy} Fishy} Fishy} Fishy He Trimethy liming. :.7 see he bawdy e ce ah IST | 5 ere Fishy} Fishy) Vishy 2CS | Trimethylamine lactate........| Fishy; Tishy| Vishy|....... Fishy} Fishy Ze Trimethylamine lactate. ....... Vishy| Vishy| Fishy} Fishy} Fishy, Fishy 3CS | Trimethylamine butyrate....... Fishy| Fishy} Fishy} Fishy]....... Fishy 3C Trimethylamine butyrate.......| Fishy} Fishy| Fishy|....... Fishy] Fishy 4CS | Trimethylamine oleate.........]......-|.0.5--- Fishy}, Fushi}; 2 sccaee| eee 4C Trimethylamine oleate......... Fishy| Fishy} Fishy} Fishy}.......]...... 5 CS | Trimethylamine stearate....... Pishy | 2ea:5's |e sae Fishy} Fishy} Fishy §C Trimethylamine stearate. ...... BUSHY | hes coetl tse pe Hishy | 2.54 Fishy

6CS | Trimethylamine and soluble fatty 21] kOe eg Ce Fishy|....+. Rishy |. uo eaie Fishy|......

6C Trimethylamine and soluble fatty BAS cis at hota aie ob tens BiG Yl wk tecars FIShy|4.f2 032 Fishy. ee

7CS | Trimethylamine and __ insoluble Paty Olea ck i aoe st Fishy|....... Fishy} Fishy] Fishy} Fishy

7C Trimethylamine and insoluble fatty GOs: 2.5 ress si ee sawt BSH y fuitistcnareteaie ae Fishy} Fishy) Fishy BOS} Laehe acid (40a baa eee Ony Wea tae Bishy |... 295 ll¢eaw4 =e $C Lactic acid’: ie eer sc 258 Re Sead |W Sx oa bases Ow ol eee ee OGs °|) Bittyrig aeds.2,..e7e0eee antes Fishy) “Hishy}es ss ss) sie sts |e ee 9C BieTIG BIG a nyes eos ae i yhlis'4 Pom ri CRE ce Wishy}|'<:./4.527ie| qecee TO'OS. «| Olea RG. ia a 2k. 2s a eee eel ee Fishy} Fishy].......)...... 10 C CUeIGECIG « = Knilo ah Wx coe eterna eral oe Fishy] fishy) 2... eee 11-CS | Sohtble fatty geidss 4. cliiees<s ectcetea a heneanee|% ae tis] ee eee) one 11 C Soluble fatty acids. ss. ieus e544 | Same Joa a6 pee] sae eed lesa te nee neon 12 CS | Insoluble fatty acids.......... COLT Whee. 1.171 Stoll pales Iie eee Oily |...... 12C Insoluble fatty acids........... COTES oH) ic6c%a ya Deil voss osoe ete a oe a |e eee

Tue LectTHiIn Content oF BUTTER a7

TABLE 6. Errect on THE FLAvor or Burren Mape rrom PastEuRIZzED RIPENED CreAM WITH 0.32 PER CrEeNnT Actin, WorKING TRIMETHYLAMINE AND Farry Acips DIRECTLY INTO THE BuTTerR at THE Rate or 85 Parts peR MILLION

Sample

Dn an PR WH ND He N

Oo oO OG OU CO ee

7 DS

(S indicates salted butter)

Comments by judges Material added

No. 1 | No. 2 | No. 3 | No. 4 | No. 5 | No. 6

Fishy Fishy

Fishy Fishy]......

Fishy|......

Minimethyiamine AAChALG +...) F< cess aceessalecs eee ofacee adhere ce wales cere @rmncmyinimine lactate 256632 s 23s) eee e clecenais slates Gssbock wee rr Trimethylamine butyrate....... Kash yi eevee: Trimethylamine butyrate.......].......

Trimethylamine stearate....... Trimethylamine stearate.....

Trimethylamine and soluble fatty RS Oo e ag St eG Raa he ae

Trimethylamine and_ insoluble TAUUY HOLS et is gine yee 25 lis ates Trimethylamine and insoluble PELEDVSACIUS tel geste ye ashen ae. e.a0erei|ie kin ote

[LET OUNISRES (0 Le ene ge coe aa DELS CERCA ete etc to cs, cis ac wisin' [191 vein eva o' | ia es ores, Paeecen toe

STIL YIG CIOs antes 5 auteetey bond a PUTO ACIO one ater so bedliakoes.!

Fishy].......

tS) 1,14 [eee (eee / |) ar err

(CLESTOS GT ee eae er pie ee eee (ae ean (er Sea MO etter Clee hs ht A atcts of save 9 4+ ciaes'| i yursea ere

Soluble fatty acids.............]....... Soluble fatty acids.............,.......

Oily

Insoluble fatty acids.......... Oily

Insoluble fatty acids

118 GEORGE CORNELL SUPPLEE

TABLE 7. Errect on THE FLAvor oF ButrerR Mabe FROM PASTEURIZED SWEET CREAM with 0.16 per Cent Actip, WorKING TRIMETHYLAMINE AND Farry Acips DrrecrLy INTO THE BuTTEeR AT THE RATE OF 85 PARTS PER MILLION

(S indicates salted butter)

Comments by judges

Sample Material added ee ee eee No. 1 | No. 2 | No. 3 | No. 4] No. 5 | No. 6 OSs | ANGER ES. cc cae eee tee tee Sere |v ewe ie or esc erel| eee y fee cee ne |e E INO GHEE. «2 o-25 lw ora ee ale lik tom re ery Vinee ie. ves | oe Tome a |e esas rere | 1ES | Trimethylamine............... Fishy| Fishy} Fishy) Fishy|....... Fishy 1E Trimethylaming 45 «ins tte a ss Bisby) Pishy|. s.0:2<|..se00¢ |e Fishy 2ES | Trimethylamine lactate........ Fishy} Fishy} Fishy} Fishy}....... Fishy ‘2E Trimethylamine lactate........]....... Fishy|'s;. cs. ecle cers 3 | eee Fishy 3 ES | Trimethylamine butyrate.......} Fishy] Fishy} Fishy| Fishy} Fishy] Fishy 3 E Trimethvlamine butyrate.......). 2.2... INS) oc 7 5211s a<a cee eee Fishy 4 ES | Trimethylamine oleate......... Pisny| — EisShy|ii< aes: 4|- 24 2s [epee |e 4E Trimethylamine oleate........./....... Fishy] 222246: scum |e eee Fishy 5 ES | Trimethylamine stearate.......]....... PUSHY | seve aien Pishy¥lie <a en Fishy 5 E Trimethylamine stearate.......|....... Oily Fishy| Fishy] Fishy! Fishy

6 ES | Trimethylamine and soluble fatty PANNA 1de, eater he in cinerea Fishy] Fishy] Fishy} Fishy} Fishy| Fishy

6E Trimethylamine and soluble fatty ROIUB 0 x Ge nN are eeNa ot ey ee Fishy| Fishy] Fishy|.......)......

7 ES | Trimethylamine and_ insoluble LEERY UACIUS 1 ey as oenee ea ia 8 Wishy|!: IMshiy|..ss7slecseeee Fishy]......

7E Trimethylamine and _ insoluble THULE OLUS 4 eels ok ae ices a1 ASH Y |e ceiyiney Lae eee Fishy|...... SHS! |) EaGue agian eres ese ewes Slee eee Wishy|.. 22.5). 2ves oa | cone 8E DACUIO ACIAS. 2.75 atealey.4 ali Fa F9 tN a oles om Fishy} | :23'sa:+| « & ys ce aera OES. | Butyricacid.. ui... 4... 4s ceny BISHnVin ve sys HISHY | cv ae) eee Fishy 9E PAR VELG*ENICL 5 ois Sey a alanis eine cise Fishy|....... Fishy} Fishy} Fishy|...... LQ. ||} IG ROIs cc. saa eS aa Pelee od nee te dl ee eee Fishy!, 2 25 er || 10 E Olesai BGI, ¢oinca cs Me Sie Oe Ll eee Oe ee reales HASH] nance eee LD Ss) | Sotnblevstiviacldss. sce aiceest| eee ae ee eee Fishy|\ 2 acre eee 1LE Bpoluble fatty Aids s.0.05 Y5. sk es ee | eae Belgie 9 Fishy|......

12 ES | Insoluble fatty acids........... ODO oss 56 25S a Seeeell eee Oily

12E Insoluble fatty acids........... OU V iia e tA os cer BAlliee aee dle eee

Tue LecitHIn CoNnTEeNT OF BUTTER 119

TABLE 8. EFrrect on THE FLAvor or Butter Mapk FRoM PastreuRIZED RIPENED CREAM witH 0.38 per Cent Acip, WorKING TRIMETHYLAMINE AND Farry Acips DIREcTLY INTO THE BUTTER AT THE RATE oF 40 Parts PER MILLION

(S indicates salted butter)

Comments by judges

Sample Material added No. 1 No.2 | No.3 | No. 4 me unin ee eek ork at 5d sk A eee Ae eee ee ile tole Rar een Weds. cat F IN O10 ATA OO eRe eRe a PN eR eco ea nr, Kick Saat Tl (eee Ce Same MR Sey |p crimethylamine. ....<s..0c+ esac casts wea ae Oily Oily Bishi |). occ. uD MPI UY MING. < sia «+ <% oa eee vce esas Oily Fishy | Oily |........ 2FS | Trimethylamine lactate.................. Rishiy® ||. eves e. WISH ys |(je'sy ete s 2F ‘Trimethylamine lactate. .............22+.[..-e004 TSI tere eee] et 3 FS | Trimethylamine butyrate................. Fishy Fishy Fishy | Fishy 3F Trimethylamine butyrate.................[.....25. Fishy Fishy Fishy 4FS | Trimethylamine oleate..............020.0)e cece ee cfec cece sleccccccclececcees 4F Trimethylamine oleate................... 7h aoe Seer ane) reed meNe | oie Primethylamine SteaPate: <<: . <a. sass ~ Oe] e.csveslize:cceeels cates tls nneeds 5F PPrUME ay AMIN BUCATAUG: oa. c.s shat cee cele y sade e|a os esas |asecs e valleact tence 6 FS | Trimethylamine and soluble fatty acids ....]........]........] Fishy |........ 6F Trimethylamine and soluble fatty acids.....| Oily |........J.....0.. Fishy 7 FS | Trimethylamine and insoluble fatty acids...}........)........)........).0...00. (ald Trimethylamine and insoluble fatty acids...|........)......../........ Fishy UEC WCAG her kita a ee ssc. Nereis tere gelearer: oto [aoe are ate erin ee ee rae 8F Ses BTW Ta Ie a nrc a ESR gir am err Aree mea ese (een gears ees De sary | er eae SPE UG VIIC ACs a,c ee 4c os dca ceacte et raley cee wales ces: sapteeece.dlecsesaa. 9F EMEC LCI Ere hee a tial oot iho ner rites Metlis oS act wes [ie tere ka ee cient | ere eee MO EIG ELUTC Stearn gene, Mee ce fs clectncty emere ve sla oto ial et miceer ese Savane line weer 10 F ODEETEG Era IR re aol RR Sree 3 ie te al hoe Pn eer sae) ee

120 GEORGE CORNELL SUPPLEE

TABLE 9. Errect on THE ILAVoR or Burren Mapes From PAstTEURIZED RIPENED CREAM WITH 0.28 per Cent Acip, OF WorkKING TRIMETHYLAMINE AND Farry ACIDS DIRECTLY INTO THE BuTTER AT THE Rate or 40 Parts PER MILLION

GS indicates salted butter)

Comments by judges

Sample . Material added No. 1 No.2 | No.3 | No.4 GSwl Wothing:< <. (ok Sosa sdscnd Sees eee bebe lew eee Pelee eee eae G Nothin ae oo Ged cas = sy sears Oe ey eo ue elles aloe alls 2 vata oe ee | LGS.| (rimethylaminercs sine yseeeay sec. aeer Fishy Vishy Fishy. ||).2y sere - ae LTimGth yamine rns 4c He cine dfe cree De alow ts a vee ces owl Seen 2GS | Trimethylamine lactate.................. Fishy Vishy Bishy |e, eee 2G Trimethylamine 1nGtate gis. ds es oa hein vib ea ye e 8h 42d o> <> Gay esau ole 3GS | Trimethylamine butyrate................. Vishy Fishy Fishy |........ 3G Trimethylamine butyrate. ssc.) ees Seabee peed ese Vishy Fishy | Fishy 4GS | Trimethylamine oleate................... BUSHY: Ny aaa as Oily 3|@aeeenaes 4G ‘Trime@bivlarmne Olebte cs <siss’s 0 s'i/es oye sabe 2 Fae | rai ape eo | 5 GS: |. Trimethylamine stearate .. <i c.4he +t hisley ce ew tulsa eo |eeeeed eee 5G Thimethy laming stearate, isa sist vas s ive elodes vets |iea sean e| hacen epales ean 6GS | Trimethy!lamine and soluble fatty acids... .]......../........ Fishy sacs 6G Trimethylamine and soluble fatty acids. ...}......../........{ Fishy Fishy 7 GS | Trimethylamine and insoluble fatty acids...}........)....... 0)... 0.0 0c fee eee eee Ge Trimethylamine and insoluble fatty acids...|........[......../..0.eccelee cece 8 GS? |) Linotig 60id a3). tases dine le tedsas © cou lisaiiahe | Cones es ed ys aice en 8G LeGtie BO ho tae eda} Sacco er. 8 eee Arete gio ee cle clea fae ee el 9 <5) | Beye Geel cee eee tune ou aire ete ov “ORY Vecy 0x5 dice le 9G Btityrio golds ei oc: iiGwewaa sles us oe neg a x 5) ecb iel e ee oe ee a ee 10'GS: Glee gods). 5 2ii-gs0cuauns aie bet dees} eal naire mead cae e ce teen ee) 10G Oleia Reid i ove hii aa saa aurnnacea Vie hare priate ears fa a0) 004s alae

All of the samples of butter represented in tables 3 to 9 inclusive were scored from three to five days after making. They were then placed in storage for different lengths of time and rescored by two or more judges. The results of this examination are shown in table 10. In this table are listed only those samples showing a fishy flavor by unani- mous opinion of the persons judging them.

Tue Leciruin Content oF BUTTER 121

TABLE 10. PRresENcE oF FisHy FLAVOR AFTER STORAGE, IN BuTTER TO WHICH TRIMETHYLAMINE Hap BEEN ADDED AT THE TIME OF MAKING

Comments by judges

The results obtained from these experiments bring out some very interesting facts. While there are several conflicting opinions as to the presence of the fishy flavor in any particular sample, it is neverthe- less evident that the greatest number of positive comments is found in the samples containing trimethylamine in one form or another. It will also be noticed that usually the greatest uniformity of such comments is found in the samples containing trimethylamine in unstable form. This is particularly true as to the samples to which trimethylamine was added alone, in combination with butyric acid, or in combination with the mixed soluble fatty acids of butter. These results are in harmony with the volatility, the taste, and the odor of the compounds in pure state. The lack, in a few instances, of a majority opinion with regard to the samples containing trimethylamine oleate, trimethylamine stearate, and trimethylamine with the mixed insoluble acids, might be explained on the basis that, since these substances were so extremely unstable, the trimethylamine had nearly all volatilized before the time of scoring. The greater number of positive comments from the salted: butter is also worthy of note, and, from what is generally known regarding the occur- rence of the fishy flavor in such butter, it might tend to strengthen the trimethylamine theory of this flavor. Another feature found in this series of experiments is that a greater number of fishy-flavored samples were found where the acidity of the cream was the lowest. This condi- tion is in harmony with the chemistry involved, for the reason that butter made from low-acid cream contains less lactic acid when fresh than is

122 MEORGE CORNELL SUPPLEE

found in butter made from high-acid cream. It is therefore conceivable that trimethylamine given off by the unstable compounds which were added could not be taken up by the excess lactic acid in the butter to form the more stable trimethylamine lactate. The finding of numerous fishy-flavored samples where trimethylamine lactate had been added to low-acid butter might be explained on the basis that certain conditions were present, possibly particular enzymes, which were capable of bringing about the more rapid dissociation of the trimethylamine lactate, and that due to the absence of sufficient free lactic acid to hold the trimethyl- amine it passed into the free state. This explanation is borne out by the fact that in the butter from high-acid cream there were a smaller number of samples showing fishiness where the lactate was added. It might be stated further that one would naturally expect to find a greater variety of enzymes capable of bringing about the above-described decomposition in unripened than in ripened cream.

The evidence obtained from the various samples of butter to which trimethylamine butyrate was added, indicates a striking relationship between this substance and the fishy flavor. This seems to be true regardless of the type of butter, and to a certain extent regardless of the presence of sodium chloride. The extremely volatile nature of this substance and its characteristic odor in pure state easily account for the results obtained. The fact that there were more positive comments on the butters containing butyric acid alone than there were on butters containing the other acids alone, indicates that this substance may be a contributing factor in the development of the fishy flavor under natural conditions. This feature indicates also that the fishy flavor may be due to a definite balance between a decomposition yielding trimethylamine and one yielding butyric acid, with the consequent formation of trimethyl- amine butyrate. This associative action would be entirely possible in storage butter, Judging from what is known regarding these fermentations.

In the foregoing discussion the writer has called attention to certain theoretical possibilities which might correlate the findings with what is generally known regarding the various types of normal and of fishy- flavored butter. It may be said, however, that the evidence points strongly toward trimethylamine as a contributing factor in fishy-flavored butter. The amount of this substance responsible for the flavor described as fishy in these experiments is very small. It would be a hazardous

Tue LeEcITHIN CoNTENT OF BUTTER |

guess to assign a definite quantity, other than to say that in all cases there was less than 85 parts per million.

QUANTITATIVE ESTIMATION OF TRIMETHYLAMINE IN FISHY BUTTER

The results obtained by working trimethylamine into normal butter warranted an attempt to isolate this substance from samples of fishy- flavored butter found on the market. One of the first difficulties met with in this connection was the lack of a method which would accurately measure the small amounts of trimethylamine that would be found. The method that was finally worked out consisted of a combination and modification of the methods of Folin and Macallum (1912) for ammonia and of Budai [Bauer] (1913) for trimethylamine. The adap- tation of these methods for this purpose was as follows:

The material in which trimethylamine and ammonia were to be deter- mined was concentrated to a volume not exceeding 15 cubic centimeters. This material was then placed in the proper tube of the Folin apparatus, 10 grams of anhydrous potassium carbonate was added, and the mixture was covered with a thin layer of kerosene to prevent foaming. This mixture was aspirated for five hours. The ammonia and trimethylamine set free by the potassium carbonate was collected in N/10 hydrochloric acid. The excess acid was titrated with exactly N/100 alkali, methyl red being used as the indicator. The results of this titration gave the total amount of the mixture of ammonia and trimethylamine. To this titrated mixture 10 cubic centimeters of a neutral 40-per-cent formalde- hyde solution was added. The ammonium chloride present reacted with the formaldehyde to form hydrochloric acid and hexa-methylene-tetramine according to the equation

6 HCOH and 4 NH;CL = (CH:),N:, 4 HCL, and 6 H.O. The hexa-methylene-tetramine being neutral, the hydrochloric acid liberated from the ammonium chloride was titrated and the ammonia vas calculated from this titration. Since the trimethylamine hydro- chloride present was not affected by the formaldehyde, the trimethyl- amine was calculated by differences. Since this is essentially a micro method, the technique involved is of the utmost importance. In all cases the volume of the solution to be titrated was kept as nearly con- stant as possible, and the same amount of indicator was used for each titration. A check on the standard acid and alkali was made with each

124 GEORGE CORNELL SUPPLEE

determination, and the end-points of all neutralization processes were compared colorimetrically with the standard neutral color. In table 11 are shown the results obtained by this methed from mixtures of known amounts of trimethylamine hydrochloride and ammonium chloride:

TABLE 11. Erricrency or tHe Mopiriep Micro Metuop ror ESTIMATING TRIMETHYLAMINE AND AMMONIA

Actual amount of

(CH3)3N and NH3 as Amount recovered Derceniacn Sample hydrochlorides (milligrams ) 8 (milligrams) (CH;);N | NH; | (CHs)sN NH; | (CH:);N | NH; ) ewe Oe ae ede ee 6.66 6.66 6.64 6.63 99.70 99.55 Dignen tea aen eee 6 66 6.66 6.51 6.63 97 .75 99.55 et OSS DRA os ores 2730) 2.30 2 25 2.278 97.83 99 04 WT srg wy Gan xeh 1 S4 1.84 1.79 1.820 97.28 98 91 Mi eae Sea ee 0.92 0.92 0. S85 0.935 96.19 101.63 SR oe eee 0.92 0.92 0.914 0.9385 99 35 101.63 y eee eee ee eee 0.46 0.46 0.442 0.476 96.09 103.48 a Ne a Fe el Pe 0.46 0.46 0.476 0.476 103.48 103.48 Wi sips acer 0 276 0.276 0.295 0.323 106.88 wales X05: LOT ea eee eran 0.276 0.276 0.295 0.297 106.88 107.61 ELS ce eos eee arasiie 0.276 0.276 0.295 0.297 106.88 107.61 eae fe cd tac ies b Ball a oes: relay ea C250) type 104.71 Es ee ae re rere te 1 St sete ia E20 | 2 eee 98.91 Lass eye ese al) Oe Ls 0.92 ATT USES) soar 99.78 cae Vee cates Soe SAS ie O.46- || «cv sence QO. 8505) > caqaue 99.78 EY oasis econ alas ee vale ee tale O04 | Ll wiecns O264631 ..0+<dee 100.31 lh ree eee ec eat A exh. 3 eee tt aaa Ae OO Ht. eae 114.13

The results shown in table 11 having justified the reliability of the micro method for measuring small amounts of trimethylamine, a number of fishy-flavored samples of butter were subjected to analysis. The butter was thoroly washed in a separatery funnel five times with equal volumes of water acidified with hydrochloric acid at the rate of 25 cubic centimeters of normal acid to the liter. The wash water was then evapo- rated to a small volume as quickly as possible, and the trimethylamine and ammonia were determined as outlined above.

In table 12 are shown the trimethylamine and ammonia results obtained from fishy butter appearing in commerce and procured from widely different localities. The ammonia results are shown as a matter of

\

C=) heen.

Tue LecitHin ContTENT oF BUTTER 125

interest but they probably have no direct bearing on this particular problem. Since the experiments with artificially produced fishy flavor seemed to indicate the importance of acidity, the acid values of the samples _are also included. The acidity is expressed as cubic centimeters of N/10 sodium hydroxide used to neutralize 20 grams of butter in boiling alcohol. Trimethylamine and ammonia are expressed in parts per million.

TABLE 12. TRIMETHYLAMINE AND AMMONIA CONTENT AND AcID CONTENT OF MiIscELLANEOUS SAMPLES OF FISHY-FLAVORED BUTTER

Sample Trimethylamine Ammonia Acid

fe : (parts per million)|(parts per million)| value

1S, AS ee 30.4 12 3.8-

eR Pe cet sh i pelea tees ek: 35.4 14.4 5.7

Sneee “Eqn MEETS oe ee re 28.8 15.2 5.5

Ae SOTO ee ees 27.3 26.1 5.4

SOc ae eee eye lake chat stetne ts cheek 14.0 18.3 6.8

(1 oR a a None 11.9 3.5

To AS RS ne ee ee None 20.0 2d

Ee Ste Pi sj art pease ceek 26.0 5p.0 3.8

AT A Ne eS cheers he Gs fue ae No analysis No analysis 3.8

TOL Sik ae aoa eee No analysis No analysis 3.0 Uk). a ere No analysis No analysis inl)

—

The data submitted in table {2 are of considerable interest in view of the history of some of the samples. On arriving at the laboratory, all of the samples, with the exception of samples 5 and 11, were scored as fishy by several judges. These two exceptions were samples of butter sent from a distance and were presumably scored as fishy when shipped but could not be so judged when received. It will be noted that in both cases there was a higher acid value than in any of the other samples, and also that the trimethylamine content of sample 5 is low. With these exceptions the acid value appears to be relatively constant, as does the trimethylamine content with the exception of samples 6 and 7, in which no trimethylamine whatever was found. The majority of these results would seem to point to a definite trimethylamine-acid relationship, as referred to elsewhere in this paper. It is to be regretted that in three instances the sample of butter submitted was too small to warrant analysis. The available data, however, point to trimethylamine as one of the causal agents in fishy-flavored butter.

126 GEORGE CORNELL SUPPLEE

DEVELOPMENT OF FISHY FLAVOR IN EXPERIMENTAL BUTTERS

In order that the development of the fishy flavor might be more care- fully studied, three series of experimental butters were made with the object of determining the influence of pasteurization, of acidity developed during ripening of the cream, of adding lactic acid to the cream, of inocu- lating butter with lactic-acid bacteria, and of salt. The procedure fol- lowed in each of these series consisted of making nine different types of butter, salted and unsalted, from the same original lot of eream. The different series were made at intervals of from three to six weeks. The description of each of the different types of butter in each of the series, and the designation of the samples, are shown in table 13:

TABLE 13. Description oF Types or Butrer MapbgE To Stupy THE DEVELOPMENT or Fishy FLAVOR

Name of sample

Treatment of cream : or butter Salted Unsalted

Raw sweet cream......... ASRS BSRS

CSRS ASR BSR CSR

Pasteurized sweet cream. ..| ASHS BSHS CSHS ASH BSH CSH Raw cream ripened with

starters... ).452;000 Hare ARRS BRRS CRRS ARR BRR CRR Pasteurized cream ripened

with starter............ APRS BPRS CPRS APR BPR CPR taw cream ripened natu-

Raw sweet cream with Bac-

terium lactis acidi worked

into butter............. ASRBS | BSRBS |} CSRBS | ASRB BSRB CSRB Pasteurized cream with

Bacterium lactis — acidi|. .

worked into butter......| ASHBS | BSHBS | CSHBS | ASHB BSHB CSHB faw sweet cream acidified

with lactic acid. ........ ASRLS | BSRLS | CSRLS | ASRL BSRL CSRL Pasteurized cream acidified

with lactic acid. ........ ASHLS | BSHLS | CSHLS |} ASHL BSHL CSHL

EP

The samples indicated in table 13 were placed in storage at a tempera- ture of 0° F. or lower, and were scored by three or four judges at various intervals. The results of these scorings are given in table 14. Non- characteristic flavors are purposely omitted from this table.

Tue LeciraH1n CoNnTENT OF BUTTER

127

TABLE 14. Comments oF JUDGES ON THE DIFFERENT TyPEs oF EXPERIMENTAL Butters AFTER VArRtous LENGTHS OF TIME IN STORAGE AT 0° F.

Acid Days Comments by judges ‘Sample eee in (per cent) | “78° | oy No. 2 No. 3 No. 4 ste ore 0.27 45 | Fishy ee ee eee eae eee eee Pakda- a : 0.27 45 | Fishy ecu haeoe baleen asta Piasaeeeues Mee ares: 0.27 130 | Fishy ee eed eres Seer eee eee Sevases 25 0.27 285 | .......... | Fishy Fishy Sehr a ee no, 0.18 45 | Oily eee... | Oily ares Ce nee 0.18 45 | Fishy te errs Othe eer ee 0.18 Drs he ey en |e aren Leet ee | SLL; peeer Gt 0.162 AB oe itacaeay |e by ee eee eee on ee: 0.162 OSA To esta No ewkecen ees | ey Fishy 1S) 0.16 45 | Metallic | .......... Metallic ..| ......... See ress 0.144 TAO crease ear Olly eee eee hee ones (O51: ae 0.144 AFL OVALE Meme Neer eee em | Mrecomeratete 2 iat PA lig 26a: eae BONEGE. <5 mi oss 0.68 45 | Metalic Wetalligu ye) een eke cess ARRS....- ae 0.68 45 | Metallic | .......... Metallic | ......... ARRS 0.68 OVD e.¥e. toler seers Metallic Oty ESOS 0 iste st aor PATEUI ee 6 hes 4. 0.68 130 | Metallic Oily Metallic.” |) lear. 8% PAR Shea cs wos 0.68 285 | Metallic Metallic Bishi We) ||) celeaes:- BIRD ess cc ss 0.567 AN | Ramee ee ears Wiarsliicenn|| (gaeeaci tet || esewsca cs RRS. . os ..2.. 0.567 0) 4 a, aortas ch Metallic Wetaliie 06) G.s oe eee CRRS......... 0.567 285 | Fishy Oily Metallic Fishy ee. Ss 0.52 Resitd 2s a ee eee eek OLY ee oe shed 5 0.52 130 | Metallic ee, See Ws sot, cee EL anit its shames POPS. 5c 05s 0.66 Das. e002 s+ Fishy Metallic | ......... APRS......... 0.66 STE Dee tals | Sige Ee See Fishy IIPS. sans a Q252 Oe eee Metallic: “<"l).-42cacteceilh Reece > LISTS ere 0.52 TSO} eMietallice - | 2208. eigen, IP ccc ne pS’ (years ees BER Ge. oe: de3.. 0.52 CT Xa ree ican Metallic: )) se sSieteaear IN Sanaa ts IOPRS. .. v.55. 0.562 STON (A O01 i ML re epee eee Oilye” 4 Mh betetcie atgtane ARRNS....... 0.675 10 eee Oily Bishyir @ Nit. seca: PURIN Seas. 0.576 SPD it aW es tee sage qens ee Give . Woetaste BRRNS....... 0.576 Bey Wil chain’ |e anc cadv-n'|l ara scorte® | knitioes IBRIRINS:«. 2%. 0.576 90 | Fishy Pishy OUR ZA 11} cy gaan Parmer ton BRRNS....... 0.576 285 |- Fishy Vishy Vishy Oily CRENS......: 0.600 90 | Fishy | .....-.-.. Oily, HSRY «is Secs oe 1A) 54 0.27 AP WISH. || cpvtes seg sae ||) eo seae ecepeccsates |] esim ethane wes ASRBS 0.27 ROR A hada oP vices are allt cos Scar ASRBS 0.27 130 | Fishy PIGHY, Wiluesenen mc || ne cecieee BORB........- 0.18 Hs eg >, cies Wa aleae is ay” y > Nie aged CSRBS 0.162 45 | Oily, fishy Bishiy~ © |) asset eee: Nyro ores CSRBS, «oe. 40: 0.162 OOS a teat | Seed obauies (251 SRG) | | eres eaecse cy eS) 0.162 DM kuin din td | Oily Metallic, | ob. icauds

128 GEORGE CORNELL SUPPLEE TABLE 14 (concluded) Acid Dave Comments by judges in De Sample cream Sin

(per cent) a No. 1 No. 2 No. 3 No. 4 ial sis ee 0.189 130 | Fishy Oily’) "| Sin ate ae ASHBS....... 0.189 OT ee Ses be ee ee Fishy Metallic CSHBS aie ete 0.144 45 | Metallic Metallic Metallic | ......... OSHBS. 22.05 rs 0.144 Q( Ws ems | dee ts eee ee 0.6388 45°) Bishy’ 9 eis ee BG. || ore cae ceed ASRLS........ 0.638 OU ice eee poet Aa es eee Kishy’ - ) e eee ASRLS........ 0.638 it {0 0 ee ere ee Fishy Bishy- | eee ree ASHUS . 4 cons 0 6388 285 | Fishy Fishy Fishy Fishy BSRLS;...5. 2 0 605 to eae Wish” — “ol! san salon ete | en BSRLS.. 2.6. 0.665 01 Roe eae ee Fishy? > [5 saScd eee need ee BSRUS......~. 0.665 Peay | at eeser eee Fishy Fishy Metallic CSRS. =... .2:- 0.472 AS Vos Leavin hae Metallie:. |)". 5 . ee eee Art elvis ale vows 0.472 Pa ee Metallic Fishy Oily ASHES: cnet. 0.690 (FS (i) Ae ee Metallic Metallic | ......... AS RULey White 0.690 Beh) a ieee tee Fishy Fishy | ......... BSHUELS) i 50.42 0.594 45 | Vishy Oily Wi tec ceva ee Beis acesera * 0.594 MOU ATY agena gee Bishty. . | “ll duvaco-d eaeaveee |e COS ibis ies 4 0.504 ABo) Hishty 9 | pes $4.42 ol) a oe eee (Ocul. peer 0 504 45°) Fishy sf! ssa sy a os oe ota ee pe OSHS, «8: 0.504 SAY || Oral ee Sa en eer Fishy Oily

In considering the results from the different types of experimental butter, it is evident that there is considerable diversity of opinion among the judges as to the presence or the absence of the fishy flavor in certain samples. It is also evident that there is some relationship between the metallic, oily, and fishy flavors, particularly. when these flavors are not sufficiently pronounced to be distinctive as was the case in these samples. This would seem to indicate that there are possibly certain fundamental conditions which are common to the development of each of these flavors.

‘ven tho there is difference of opinion as to the presence of the char- acteristic flavors, certain conclusions may be drawn from these experi- ments. Probably one of the most significant is the presence of the fishy or the metallic flavor in the salted butters. Of a total of 105 character- istic comments, 93 are found in the samples containing salt. Another conclusion which may be drawn from the relative agreement of the judges, is that the fishy flavor appears oftener in the butter made from

THE LecirHiIn ContTent or Burrer 129

high-acid cream than in that from low-acid cream, there being little difference whether the acid was developed by the use of starter, by ripen-

ing naturally, or by the addition of lactic acid to raw sweet cream.

These findings, compared with the results obtained from pasteurized cream either churned sweet, ripened with starter, or acidified with lactic acid — all of which showed fewer fishy samples than did raw cream — clearly indicate that the fundamental cause of this butter defect is primarily biological, not brought about by a spontaneous chemical change in which such agencies do not play a part. While it is evident that acid plays an important rdle in the development of the fishy flavor, it is equally

clear that there are other important contributing factors. Just what

these factors are, is unknown. The variable results obtained from the same type of butter in the different series would indicate that the original cream or milk possessed the unknown factors which in the presence of lactic acid determined the development of the flavor. From the fact that pasteurization tends to reduce the occurrence of the fishy flavor, it is quite probable that these agencies are bacterial enzymes which are only partially inactivated by heat; or it may even be possible that certain microorganisms which are incorporated in the butter from the cream, either in a living or in a dead condition, could on autolysis liberate the enzymes capable of supplying the determining factor. It may also be added that pasteurization may kill certain enzymes and not others, the particular ones that are important being among those killed.

These contentions are further supported by the fact that in the butters made from raw sweet cream there is a suggestion of fishiness after the first storage period which is not found after the longer periods, the dis- appearance or lack of further development of the flavor being due to the absence of the proper acid condition. It is clear that large numbers of Bacterium lactis acidi added directly to butter without their usual accom- panying by-products are not the cause of any characteristic change in flavor.

VARIATION IN ACID VALUE OF EXPERIMENTAL BUTTERS

The importance of acidity in the manifestation of the fishy flavor by trimethylamine, and the relatively constant acid value of the miscel- laneous samples of fishy butter found on the market, emphasized the importance of studying this factor in the experimental butters described

130 GEORGE CORNELL SUPPLEE

above. The variation in acid value of the different types of butter in each of the three series is shown in tables 15, 16, and 17. Results are expressed as cubic centimeters of N/10 alkali necessary to neutralize 20 grams of butter in boiling neutral alcohol.

TABLE 15. Vartation in Acip VALUE OF EXPERIMENTAL BuTTers oF A SERIES AFTER VARIOUS STORAGE PERIODS

Acid value after various storage periods

Sample 43 days 86 days | 128 days | 310 days ARSE. (i: 145 sea beniaca a oeiay'ds one hace maT 2 8.8 10:5 10.8 13.0 Pol ice EE ee eee eee 7.4 8.0 8.2 10.0 PARES oi 2 he ons 45 0e-ica Seeeta ae 5.8 8.6 10.0 y ho ASUS sa Ge es eid ye red dir steees esate 5.7 5.9 5.7 y ge PO : See Ek eee te RE ea ee pre 8.2 8.5 9.3 10.3 Pd ee ek oe eee ee ee ee ee PE rere 8.7 8.4 8.6 10.3 WP Re Nieves Seca ee ahs mandir teed wlan Se ct 7.9 8.2 9.8 BRERA, ssc vena e in aba sks era eeee 8.1 7.8 8.0 9.6 ATU said epapeesctys eeiaeee recs spe bltn 8.9 8.6 2 10.5 AERING if ckice red exte ost op owe weer aes 8.8 8.4 8.7 10.5 ASH ie dia CLs eamere yas ere eee Fu 9.7 10.4 Lies 15,0 PTO i hess rawinte ones kee eaeeaar ieee | 7.4 C0 7.8 9.8 ABELBs 25 ocak yee e te Sha oe tear vag V7 8.6 9.5 14.3 ABER sss eu epeeeeies niente 5.8 5.9 5.8 9.7 AB Hi bvxs¢ iy ava eet aes saete 1 gw eee 8.9 8.8 8.8 10.3 ASHis 43.4 bette eeee ra eve seen er een 8.9 8.9 9.0 10.0 ABHI..v¥uteva generis eames eran eae 7.0 74 7.2 8.0 ARTEL Gate ious Bales yen Weer iriaeah ek peatins 7.4 6.5 Wail 8.5

The data presented in tables 15, 16, and 17 show many interesting features, some of which are worthy of discussion in connection with this preblem. It may be stated in the beginning that the variations in acid value of the different types of butter point to biological agencies as the cause of those variations. The lower acid value obtained in nearly all instances from salted butter indicates a preservative action by the salt, a function which is well known. The greatest increase in acidity is

THe LecITHIN ConTENT oF BUTTER 1ot

shown in the butter made from raw sweet cream. It is interesting to note that very few of the samples were scored as fishy. When such a condition was suggested, it is to be noted that it occurred after the first storage period, when the acid value was lowest.

TABLE 16. Vartation 1n Actp VALUE oF EXPERIMENTAL Butters or B SERIES AFTER VARIOUS STORAGE PERIODS

Acid value after various storage periods

Sample 20 days 88 days | 126 days | 286 days IB Ee ey We AU oe, 28 cia ae acs Rye el eee 13.9 14.6 ee 7-5 IBYS RUS) OO oe tee See ere err 9.7 9.5 10.0 10.6 IBISI EL ah Oe ois) 0 A eee 723 9.0 10.6 13.4 TESTI SRS SS eee eee ee ee ee 6.3 6.5 6.8 ‘Oe6 Le Me Re sige fash, teks cleo s% + 10.1 10.2 '10.8 11.9 TBULRYL RY) Sy ea Mer nee 10.1 10.1 10.4 13 [BSL Poe ka OR a ae 10.1 10.1 10.0 ih le [BETS ae 8 eee ee 9.8 10 9.6 11.0 1 BiLRSI RUIN Ft ee Sea 11.3 Lig7 126 14.3 151 Rv1 RVI S eer a A 11.2 1h / 11.8 13.8 su se a rss 2 oe oe ene oie SS akanrt oo 13.6 135 14.3 15.4 Evens lerttiete) he. te Bs tess o ane bb aRiuends « Sina case aa: 10.6 10.8 10.8 12.0 SEER tere ate Syed gioe sayse5.5 oe aang shea 0S 8.1 8.4 9.0 9.2 LAST RULES y fet ee ee ee 6.8 6.6 6.8 8.0 Eleven MM crate eet laia Frits de herp Sie aes 11-5 ila Us 11.6 12.9 TSI RIL Ss ce Ree eee eee ee 11.5 is all 11.6 12.6 TESS UL ok Mea eel 9.4 9.4 9.5 11.0 153915 0 US) 5 ee rer 9.5 9.6 9.6 1

With reference to the samples from pasteurized cream as compared with those from raw sweet cream, it will be noticed that pasteurization has tended to cause a lowering of the acid value but has not entirely prevented its gradual increase. This would be in accord with possi- bilities already stated regarding bacterial enzymes. The data show also a retarding action exerted by the acid originally in the cream. This is evident in the butter made from both raw and pasteurized cream ripened

lag GEORGE CORNELL SUPPLEE

with starter, from raw cream ripened naturally, and from both raw and pasteurized cream to which lactic acid has been added. In comparing the results from these samples it will be observed that cream ripened naturally shows the greatest increase in acid value, raw cream ripened

TABLE 17. Variation In Acip VALUE OF EXPERIMENTAL BuTTERS OF C SERIES AFTER VARIOUS STO2AG3 PERIODS

Acid value after various storage periods

Sample 6 days 48 days 90 days | 272 days ELE, Searaics atvaiinadl «Ge ne ade avi 7.8 10.4 11.0 13.5 RUSCINS. 2a Aad Gane Cane: cote 7.0 7.9 8.0 ett COE ce ote ee oe ee genes 4.4 7.6 11.0 12.0 OSES rxtcn b> eat bane eetciaas Ae eeae d 4.4 4.7 4.4 5.2 211 er ee ee eee oer ae 7.6 9.3 10.4 12.8 CORE th Sys ce oe Lees cette ds con ree 7.5 8.2 8.4 10.0 CSIR so onvipen kh He et a ee Sy dS al 6.6 Yio 8.9 10.0 COP dso: se Grace ks 6.5 70 7.0 8.2 CUHINE eee ee ee Be ihre ae es ehly ans 8.5 5 10.0 12.9 GRINS: i4a5 5308 h0 8.2 9.0 9.1 Tigo CBRNE ycapce cot cme at cae: aaa es eres 7.8 10.2 11.8 13.5 CRIBS gas. 2aeeas wicker dace nee e er aeen 6.9 7.4 7.9 9.0 CELE: i Bas ba ear LG eee ay oak awed 1 4 6.5 nat enn CSEBB tra Pepe k tere 4.4 4.4 4.4 5.2 OORivic-ns 6:5 pie ee abe Cre ed cae eo fran 8.3 8.1 8.0 9.8 OBR yk a Ve UneLie eee nay sd Mee es ube 8.4 8.5 8.4 9.5 CHRIS. Cy eave ey ea ee 5.8 6.0 5.9 6.7 COLE ot cere eet 6.9 7 5.6 6.3

with starter a little less increase, pasteurized cream ripened with starter a still less increase, and eream to which lactic acid has been added the least increase of any in the group. The low acid value caused by the addition of lactic acid might possibly be explained in one of two ways: either the addition of the acid in pure form has tended to inactivate the enzymes, or the lactic acid retained in the butter has been changed to butyric acid during storage — which is entirely possible by enzymatic

i]

Tue LecitHtn ContTeENT OF BUTTER 133

action. If such a change as the latter did take place, a lowering of the acid value would be manifested because of the formation of a weaker acid which probably has resulted from the splitting and condensation of two parts of the stronger lactic acid. The possibility of butyrie acid being formed in this way might be supported by the fact that the other samples from high-acid cream showed a lower acid value than those from sweet cream but had a higher value than those to which pure lactic acid was added. Even tho this change did take place, however, it is improbable that it could entirely account for the low value indicated. It is more probable that the lactic acid acts as an inhibiting agent. A study of the tables will show that the increase in acid value of the sour- cream butters seems to be greater when the amount of pure lactic acid is lowest in the cream. In using the phrase “ pure lactie acid,” reference is made to that which was added and also to that developed by bacteria, it being logical to assume that the greatest amount so developed is found in pasteurized cream ripened with starter and the least amount in the raw cream ripened naturally. The condition mentioned above also sup- ports the theory of devitalized enzymes, altho it is more difficult to explain why approximately the same degree of commercial lactic acid has a more marked effect than the acid produced by bacteria. It would seem that the structure of the particular lactic acids involved produced different results in this respect, or that the other acids produced by the bacteria are less.inhibitive than the lactic.

Regardless of what the explanation for the variations may be, the data seem to indicate that there may be a relationship between the acid value and the fishy flavor, not so much by a constant condition as by a proper balance between the progressive development of the acid value and some other contributory cause. The most favorable condition would seem to be a very gradual increase’ in acid value, and one that would be in proper harmony and relationship to some other important and transient factor. If these views can to any degree serve as a basis of explanation, it is comparatively easy to see how an improper balance of any one of the conditions would determine the presence or the absence of the fishy favor. It might also be conceivable that the intensity of the true flavor would be in inverse proportion to the degree in which these factors were out of equilibrium. Such a conception could explain the occurrence and the disappearance of the flavor in the same sample of butter at different

134 GEORGE CORNELL SUPPLEE

times, why the fishy, the metallic, and the oily flavors seem to be closely related, and possibly why trimethylamine can be detected in some fishy butters and not in others.

TRIMETHYLAMINE AND AMMONIA CONTENT OF EXPERIMENTAL BUTTERS

The micro method already described was used to ascertain the tri- methylamine and ammonia content in the experimental butters after different lengths of time in storage. The results of these determinations are shown in tables 18, 19, and 20. Amounts of the substances are expressed in parts per million.

TABLE 18. TrimmetHyLaAMIne AND AMMONIA CONTENT OF EXPERIMENTAL Burrers IN A SERIES AFTER VARIOUS STORAGE PERIODS

After 128 days After 347 days Sample ; Pit GEL oe RII PS PGS hia © rt a's None 36.0 None 17.6 este eer een wha Siri: <;.2.0ee ae pulse None 17.2 None 8.4 tNS13 Ses Pee ee en eee ee ae None 23.18 None 16.6 SLE, rate eieatee Ce ice Page. 6. Vises None 15.0 None 6.8 Pare. ees Sek es a a eu as ee en None OD 22 None 30.8 ECE rant icy «ar Anh Gee Park eo aii 16.8 9.4 18.2 PUR Higt (stern Sar De ree (ie sy Se eee Aa None 34.0 None 24.4 SATA RR, Pig eter erat Sth fs we th es ee None 20.7 None 24.0 NIFLEEINS UGS caste terre Lee hor ee ae None 25.3 None iat) INAS IRING Go be Okc a pa heen es ear eae ae 13.5 994 None 6.8 ALE, 7icckan ee eer an is 49 es | TER None 18.4 None 24.4 PRR ous ye ee odie ie «wee ates None 22.1 None 12.8 RAE tet bie thy cee eae PLE Bae Sr Pt oe None 23.1 11.8 10.8 BS 50.3.2 Oe ee ek aes See es None 17.3 15.3 9.4 ASR. <b.t fsck See esters cue 4s None 14.2 6.0 10.7 ADE Liveries Sc heer ene ae Taw oN Le oka None 16.3 None LZe2 yt ee, ee Ok eer None 19.3 None 8.8

The results shown in tables 18, 19, and 20, altho erratic, are of impor- tance as indicating the variations in decomposition in the same and in different lots of cream, and further emphasize the complexity of a problem of this nature. The same general results with respect to enzymatic activity in salted and in unsalted butter from raw, pasteurized, and ripened cream are found here as were found in connection with the acid

values of the same butters. Altho the trimethylamine results are some-

what discordant, a tendency is shown for the presence of this substance

=

Tue LectrH1n Content or BUTTER ia

to harmonize with the samples scored as fishy, metallic, or oily. Of 21 samples. in which trimethylamine was found, 15 were assigned one of the characteristic flavors by one or more of the judges at some time during the storage period. In 5 of the remaining instances, it is to be noted that, while trimethylamine was found in the same type of butter of the same series, its presence did not harmonize with the characteristic flavor in the salted or the unsalted sample. On the other hand, there were 10 samples of different types of butter which were indicated as having a characteristic flavor by one or more of the judges at some time during the storage period, in which trimethylamine could not be detected.

TABLE 19. TrimeTHYLAMINE AND AMMONIA CONTENT OF EXPERIMENTAL Burters IN B

D

SERIES AFTER 323 DAYS IN STORAGE

Sample (CH3)3N NH; Seger ta es ds nt ed ele ad oa abut wae nis None ata ay ee te Eh ky Pa tand > po dipasdiae feed andes None 18.0 Let I De kg Bc aac fot ee ee ee carea| Swacecsa | Soamcades FESS TBIS OS SAP ESC oe tre ee a 7 ee 53.0 10.2 VESTRC TRS SU 2s 5 AR AR a None 5 Aes TBURU ES. = 2 0h GASES pear re eee 8.2 21.6 aE Meroe ee oe coat Se cp Sia s bees Jom beads wn ces 10.6 23.8 ERs ol cp mad ce hss ss deaesaadede cases bape caneasen 9.4 25a0) TRIBUNE a A a re ee 4.7 27.0 PECTIN MIE Pong dca Soe a hee ak see he cep ee eauee devoudecs cat None ay TRIS TRL Bi = 0c 6 5 aa nen air eae ang gn oc 35.4 29.4 TESS IPE SS i = ee ae ey ae None 20.0 ye ee he ct edge ni edactesaees te 5.9 23:0 TESTOUB Ss 8 ee) SR ae ee a None 10.4 IBVSIR IDG 2 op gyn eR a ee re None 23.0 TORS PALS). © = Seether oe Par a ae None 17.0 TE}STE Eo.) © RS ae a None 7.4 TE}STSELL|S 2 © © sie ab eet een (ag None 11.4

While these results are not absolutely conclusive, there is nevertheless an indication that trimethylamine may be one of the contributing factors in the development of the true fishy flavor. It has been shown that this substance is capable of producing a flavor described by butter Judges as fishy, this being particularly true in the presence of butyric acid. Furthermore, it has been shown that trimethylamine may be present in fishy-flavored butter. Therefore it would not appear to be beyond the

136 GEORGE CORNELL SUPPLEE

realm of possibility that the results shown in this paper point to a definite trimethylamine and acid relationship as being the cause of that flavor in butter which resembles the flaver of herring or mackerel brine, and that non-typical flavors resembling other fish products, or the metallic

TABLE 20. TrRIMETHYLAMINE AND AMMONIA ContTENT OF EXPERIMENTAL BuTTERS IN C SERIES AFTER VARIOUS STORAGE PERIODS

After 90 days After 310 days Sample

OSH aa eae 4c oe ale hes oe eee None 30.2 8.2 Be CORE eine ave Soe aie Genes wing 2 Renee ee he None 20.0 None 28.2 O'S )5 ee A ee ret eee POT None 32.4 ea era OSHS. os mectc ved eee eb isd SSA ee None 19.7 None ge Ci Ree oe on Livin ke sieink a Vise nies dale helt None 36.3 None 20.6 GHRS ohh ose eee tee ee east kes aye None AIS None 36.2 OPER ge chiieaot ce apo ea se Pe a lead None 34.7 None 29.2 DPS mee ees. seca arenes None See 14.0 27.4 CORSeaaa.) Set aat le Sua lw eees thee ee eee. None 37.0 18.8 SL CLUE Cae Si eR ET Oa SO Rl None 270 8.2 29.8 Chie Cees eee ou reie eee ees None 33.3 13.0 33.6 Ga Baseecee act aes ak i eae eee None 23:1 5.8 18.6 COREE rine pikes > anne SP! "vet aravin, None 31.6 None 31.6 CSEUBEiGuaceee fo tee Paw esa ee Se eee : None 34.0 17.6 15.4 CS rule ees idle AY oo a oe ee - None 21.4 None 14.8 (O04 Fee ee eee de = ee : None 14.2 None 10.8 GSE «ober sea a tc bas ieee : None 38.7 5.8 20.6 (OSE a. ee eee ie TES Secu, | 1 None 1st

and the oily flavor, may be due to an unbalancing of this relationship, the occurrence of these flavors being due to factors in which the presence of trimethylamine in detectable amounts is in no way contributory.

BACTERIOLOGICAL STUDIES

In an effort to correlate the preceding observations with the biological aspects of the problem, certain bacteriological studies were carried out. These ineluded a bacterial analysis of fishy- and non-fishy-flavored butters, and inoculations with pure and mixed cultures into choline, lecithin, butterfat, and cream for the purpose of finding, if possible, an organism or a group of organisms which in some way might contribute to the develop- ment of the fishy flavor.

CO SS ae

——

Tue LeEcttTHIN CoNTENT OF BUTTER HEYE

Bacterial analysis

Bacteriological examinations of fishy- and of normal-flavored butter from various sources seemed to show no characteristic differences in flora, neither were the quantitative results consistent. Even tho the samples examined did not appear to possess marked differences in flora, cultures of the predominating type were isolated from the fishy samples for the purpose of determining a condition under which they might contribute to the characteristic flavor. The types of bacteria found included a number of acid-producing varieties, both coccus and rod forms. Among the species commonly found were Micrococcus lactis acidi, Mice. lactis al- bidus, Bacterium lactis brevis, Bact. aerogenes, and Bact. lactis flocculus.

The bacteria content of certain samples of fishy- and of non-fishy- flavored butter is given in table 21:

TABLE 21. Numsper or Bactrerta FouND IN VARIOUS SAMPLES OF FIsHY- AND NON-FISHY-FLAVORED BUTTER

Character Bacteria Sample of per flavor gram UE MOE eI ok cteys a wivcch GteTeecd sh wie ers Baek Strong 1,200,000 SN et 6 See a.) aye i ougvd & apd Abend aye G-2 auine Oth Fishy 1,400 ,000 ST INT 56.2 nat. sc. Son, oce 98 PE esd tS sates Ts Fishy 8,000 , 000 EPR sk. 8d alee Ge pr a's os 04 terete oye 4b ar’ Fishy 21,600 , 000 ST ey Me SSR bee ne fg Gin 5 gis, 4 Gis as wy tie insane heats | Oily 30,000 ,000 Pe Nees he SovS Sua aed oe vanes, so ndin? Bab acto 4 Fishy 60,000 Et a a EVE re Eo 25. 3 gars 3d Be A RSs oie ge Fishy 135,000 MOS ee Sole sa 2om tocrs tnd tare oe bin) SGA Hin Oily 3,900 ,000 8 Oe Ae eS a oc ung vhs eed oaes ade sus Strong 760,000 MO) 5 ee, Ste ea ee Fishy 23,500 A rer oe, e228 Se aah Gana Ge eee Sores a aa es Fishy 8,300 ,000 ee re Ns (enh Sie 8 SiS s aces Se LSE OTe hoes Aaa Strong 1,600 ,000 a eg is nc 2M Tey Salt ens see oe. Bo Fishy 340 000 MOO ces Ei GB. SS cc ds Sree a ok nates od Storage 2,600 IGS, ea ne ne Vishy 350 ,000 fn SS OR ee em | cet ens Scns Fishy 465 ,000

Inoculations for the purpose of developing the fishy flavor

The results of previous investigations show that little success has been attained in the attempt to develop the fishy flavor in butter by inoculating the butter itself or by inoculating the cream just prior to churning. If the enzymatic idea as already stated in this paper is to be

138 GEORGE CORNELL SUPPLEE

upheld, such negative results can be explained by the fact that organisms inoculated into the cream just prior to churning, or into the butter, do not have the opportunity for growth and consequent production of by-products which they would have if allowed to grow in the milk or the cream before it is made into butter. There seems to be good evidence that there is a rapid dying-off of the bacteria in butter after the first few days of storage. Furthermore, it is well known that low storage temperatures do not entirely prevent enzymatic activity. Rogers (1909) shows that, while low temperatures delay the development of the fishy flavor, they do not entirely prevent it. In accordance with these facts it is conceivable that certain enzymes which would be produced by the growth of organisms in the cream, and carried into the butter, would there continue their activity, with the consequent manifestation of certain changes in flavor. Results obtained in the present investigation indicate also the importance of a definite acid relationship. With these factors in mind, the inoculation experiments undertaken in connection with this problem were carricd out in a manner that would allow for the manifestation of the possibilities indicated.

Nine organisms, all of which were isolated from samples of fishy butter and Bactertum ichthyosmius — which Hammer (1917) found would produce the fishy flavor in milk — were used in these experiments. The same original lot of cream was divided into six parts, and each of these parts was further divided into ten parts, each of which was moculated with a specific organism. Different methods of handling the six groups of ten inoculations each were carried out in such a way that the effect of acid in conjunction with the specific organism could be determined. Adequate checks were made from uninoculated cream. Pasteurized sweet cream was used as the basis for all inoculations. When the cream was neutralized the acidity was reduced to 0.18 per cent. All samples were made up both salted and unsalted, and were scored after 231 days in storage at a temperature of 0° F. or lower. The results of the experiment as regards salted butter are given in table 22.

It is characteristic of the results of this experiment that none of the samples of unsalted butter showed any of the characteristic flavors and are therefore not included in table 22. It is believed that the results shown in the table clearly confirm the opinion that biological agencies, particularly bacterial enzymes, are responsible to a great degree for the flavors indicated; also, that the fundamental condition necessary for the

Tue LEcITHIN CONTENT OF BUTTER 139

TABLE 22. Errect on THE FLAvorR OF SALTED Bu7vrer, or INOCULATING CREAM WITH SPECIFIC ORGANISMS UNDER DIFFERENT CONDITIONS

| Acidity

Comments by judges

; of cream* Sample Treatment of cream 1(+)|2(4+)| No.1 No. 2 No. 3 No. 4

1 Raw sweet.............-)....-. CRIS oe arerola ces lve ataasiang (eee ee gr sete wees tisere 2 Pasteurized sweet.......}...... CL ene ee yee | fe een eee lly cee oreiersey | aires ses 3 Pasteurized sweet, ripened]... ... OSC oo ncubt: 1h. 96 a5 Ee Rice acess otek eae 4 Pasteurized sweet, ripened

and neutralized. ...... (0 ).275101 Rk Oe 0 | Rea ne aon ge ar, | Oa Pa di Pe Ie 5 Pasteurized sweet, acidi-

fied with lactic acid... .}...... 0.54) Fishy Vishy Oily Fishy 6 Pasteurized sweet, acidi-

fied with lactic acid and

neutralized........... 4. Fe? (ipa ese anes se elie 2a tee eea el | needa ee |e ee ae DBI | Pasteurized sweet, in-/...... SIS ersere y seetecee | eae St eee |= D 100 oculated with specific)... ... (OF 5i Fae ra ae (eae eae ed oer Metalic D 23 organisms and churned)]...... (OU eee x el aes cee Seen, Oe Palle | Soe 1D SUGUCCMNER = § WP ine ce 0.13) Tallowy)...... bet | eee Ree aa Tallowy WORSE 0.13] Metallic).........)......... Oily IDA |S Se [eee RIS Eg eeetrsce cae | Steere cence cirsasle ts aol eee Dt | (alee ole eeenn sleep Oily 1D) ALES |) 0 | elie wa chaatesaut |cavancvaleua sesne | mappa ineseer ay ler denice Gane 1D) 211) rr | (Pc DIS eevee: olla eee <dnte | ee ree td | a NH os | 0 ed verona dag ain edo een | eden ee 8 (5 omens E BI .| Pasteurized sweet, in-|...... CUTE erento ee A cn ce a Ue eee 2. | is E 100 oculated with specific]...... eer eae Reb Ooi Bvesleh riee Tishy Tallowy E 23 organisms and held 24)...... 0 A eee ee Fishy Metallic] Oily E 12 hours before churning |...... CORA ee PON conc osx alice, ata tel | ad ree WreSey ee eee 7 Gia is SRD | rece | PRON tae cdc RR eee & 1D) 1) a |e CAS aie eh cath etree accel ewectank yee Mam tne ERS ieee OEE G aera area (Piecree Genrer rae) [nee rar ae earl eae ane ee Mm ee Ne sued UP Eee eee Oily Oily Fishy ee | 0 a a ke) A a a rd LPP ae 1S OS | cee Ce) We cet teens a ore aero eee te ceatel| ne peateaetae K BI Pasteurized sweet, in-| 0.45) 0.18} Metallic}......... Vishy Fishy K 100 oculated with specific!) 0.41) O.18).........).....-..- [eee eee ele eee eee Ke organisms, held 24 0.41) O.18).........).........]......... Oily K 12 hours, and neutralized] 0.43} O.18)......... Butyric| Butyric]........ K 18 before churning UAB MCS else faa all eyesore aon gare [lati i ocala ore ae K 19 CASS Ra las ie ee citnve tere ee eave lari eye taual eee ce KS (0) OTK U8 bol eure (nie Pare [Fnriven ears omen | | are remoren K 11 0.43) O.18)......... Tallowy|.........|........ K 21 CRS aD elie eean gaelic ts tensa praca teat ae endl Reta K 10 GeO) HORUS sive docile s nes are eke ENS V5 eae ye

*1 (+) =acidity at time of neutralizing. 2(+)=acidity at time of churning.

140 GEORGE CORNELL SUPPLEE

TABLE 22 (concluded)

| Acidity Pike Comments by judges

Sample Treatment of cream a

1(+))2(+)| No.1 No. 2 No. 3 No. 4 N BI Pasteurized sweet, in-| 0.45) 0.32/........./.........]........./........ N 100 oculated’ with: ‘specitis|’\ 0.41) (O236)\....6.2- 1:52... +2). eee ee N 23 organisms, held 24 0.41) 0.35).........].........].........|.-.ese0e N 12 hours, neutralized, and} 0.43) 0.37|.........|.........|.........|.....--. N 18 ripened O45) -O 88h og allo: |= + < acld a pee ee ee N 19 UF: SES (ene | N8 O,4¢) O238l oy pee. 244 |. coy den lho cd een N ll 043) -°0529).. 225 62a lies Siena dle ee eee N 21 O245) “O.89) 2...) vents e xs a0 oe. 3 oa ee N 10 299) OSH) 24g waa al hietie.c os eellee eee R BI Pasteurized sweet, in-| 0.36) 0.18) Fishy Oily Vishy Fishy R 100 oculated with specific) 0.26) 0.18).........].........].........|...0.00- Ro organisms, held 24, 0.38) O.18].........].........]......... Oily R 12 hours, neutralized,} 0.31] 0.18) Butyric].........].........]........ R18 ripened, and again neu-| 0.33) -0.18|.........)...<...2.]:.ca0ceccteeteunee R19 tralized Drag SUS caer be calen ea ca Rancid | >. a. ee R8 0,20) > OF18!5s tcc eas lina ioe ee Fishy Rll OBO) TOTS sy au, 0 vy ar eee ee eee Vishy ii 21 Oio2) OIBU wine esa: oes > «ello es eee ee R 10 O29) OV1Sl ss ncsa easter eee X BI Pasteurized sweet, in-|...... 0.29) Metallic) Metallic! Tallowy| Fishy X 100 oculated with specific)...... ren) ter eee | eens vp Oily X 23 organisms, held 24)...... 0.40} Metallic}! Metallic) Fishy Metallic X 12 hours, andripened ~—s |... 0.39} Butyric | Butyric|......... Strong SL e we me Sie ee We he oe OUSA ya a2 6 sollte cig dy a & oon a et LUA aT ©, Meee f O: 86bs fc5 5b Saha ceceeu | ee xo | > Deh 2 Mele, pa O84) 0s ose ne ca] so a ee 0 9/0 ore cee aa! A See ee. ieee 2 O30). < 23 cry 8] on bee oho ee a Sees. 5 Ce ee | al ate O36) 5 «odie easel srs oe oo =| elec ee er Se i of © CER Pra rs OB6) i ies 8 polls s © ns 3 Olea oe

*1 (+) —acidity at time of neutralizing. 2(+)—=acidity at time of churning.

manifestation of these flavors is greatly enhanced by the growth of the organisms in the cream; and furthermore, that a definite acid condition is essential for the development of these flavors, which are potentially possible from the specific bacteria or enzymes. In this experiment. it is appreciated that the results are obtained by an associative action with the organisms in the starter and those surviving pasteurization; this fact, however, does not depreciate the specificity of the particular organisms that were inoculated. In reviewing the data from this experi-

Tue LecirHIn CoNTENT OF BUTTER 141 ment, it is interesting to note that the sweet pasteurized cream to which lactic acid had been added and which was uninoculated, developed the fishy flavor. The description of the flavor in the same sample of butter by different judges again calls attention to the fact that there seem to be some conditions common to the fishy, the metallic, and the oily flavor.

The most consistent comments from specific organisms seem to be from cultures BI and 23. The former is Bact. ichthyosmius, which was obtained from Hammer; the latter is an organism isolated from raw- ripened-cream butter which developed the fishy flavor after two months and retained it for nearly twenty months.

Butter samples EK 23 and X BI were analyzed for trimethylamine and ammonia. None of the former substance was found. Sample X BI showed 35.2 parts per million of ammonia, and sample E 23 showed 17 parts per million.

LONGEVITY OF BACTERIUM ICHTHYOSMIUS IN BUTTER

Preliminary experiments with Bacterium ichthyosmius indicated that this organism might produce the fishy flavor in butter. It seemed desirable, therefore, to determine its longevity in butter made from the inoculated cream. Results of the quantitative determinations of the bacteria in salted butter containing this organism are shown in table 23; results are given only for those samples to which a characteristic flavor was assigned.

TABLE 23. Bacrerra ContTENT oF SALTED Burren MapgE FROM CreEAM INOCULATED WITH BACTERIUM ICHTHYOSMIUS AND STORED AT A TEMPERATURE OF 0° F. on LowEer* _

Number of bacteria per gram Age of sample (days) ee ee K BI RBI X BI

ee re ie er ng Meee 232 E23 23,400 , C00 10; 4007000 F 4.2. tec nccan 24 | 2a Oa aa Se 14,400,000 10, 100,000 34, 250 , 000 Ee A he a yD ae Weta foo Bute 10,450 , 000 8,300,000 28 ,000 , 000 Ee eS eed cana Co So, aul Sis rs SAREE © 8,500 , 000 6,700 ,O00 17,400 ,000 (Oy), OR tae ee ee 6,300 , 000 6,350 , 000 10,150,000 eas Ae iy YS eS ornate es 3 1,800,000 4,000,000 11,500,000 (Oe oY a Os a ee ee 1,150,000 1,350,000 11,200,000 TS MES oe as Pe 8a paigees o & Rast tb 8 890,000 | ............ 6,400 000 UPAR Ae eee i gree Sirs hes uke ectine 4 To, CUO" ese neues 5,400 ,000 ee ret tens Urs Bre Se 2 BmanSve. Sh ME anthgwi a Ragis. avs 740,000 | ............. i... lA bene corre meta eee S59 O00) o.aaae edocs ai ee Ae Sf eee pe aaae ss 37 ,500 4,600,000

* These results were furnished by J. T. Cusick.

142 GEORGE CORNELL SUPPLEE

The decrease in bacteria content of the salted butter containing Bact. ichthyosmius shows that, altho this organism may contribute to the development of the fishy flavor in butter, it does not do so by active multiplication in that medium.

FURTHER STUDIES WITH BACTERIUM ICHTHYOSMIUS

The results obtained with Bactertwn ichthyosmius seemed to warrant a further study of its relationship to the fishy flavor in butter. The following experiment was carried out with the purpose of determining the conditions in butter under which the development of the flavor could be accelerated. Pasteurized sweet cream was inoculated with this organism and held for two days at room temperature. The butter made from this cream was divided into twelve parts, and to each of these parts a different substance was added. The cream at the time of churning contained 0.23 per cent acid. The treatment of this butter, and the time of occurrence of the fishy flavor as determined by two or more Judges, are shown in table 24:

TABLE 24. Fisny Fiavor as DeveLopep In Burrer Waich Was Mabe FroM CREAM

INocuLATED witH BACTERIUM ICHTHYOSMIUS AND TO WuIcH VaRIoUS SUBSTANCES Were AppED

Days in storage Sample} Substance added to butter

52 O4 136 175 | 328

BI | IND RET 2 Bate 5d tare 5. A: pha Ages tae Fishy Tishy Not scored BLZ Berkfeldt filtrate from milk |

culture of Bact. wchiliiosmtish: =<. =. yl caw we | eeu wes thal oes: ule eee BI 3 Choline, 0.0118 per cent... . OILY. fs cece eve wll. Seas SU oa 8 2 BI 4 (alent Gaseinates « 6-1. (ais all fsa bee ts 1a aiv eee a sdillins crenata BLS Lactic acid, 0.117 per cent Fishy Fishy Fishy Not scored BL 6G Lecithin from butter........ data Stile putes Pele ee Ve rae Fishy BIZ Lactie acid and choline... . . Fishy) jh) sia). e40544 ls da4 41 re BI8 Bact, lactis neids starter. 0. 2). esi s He va bo Alone eneci Nena ee Slightly fishy BI 9 Berkfeldt iltrateand caseinate: . 2... f)esiee siabetee asad [yea ys el BI 10 | Berkfeldt filtrate and choline..|....... 1 en Penny Ae BI 11 | Berkfeldt filtrate, lactic acid,

BH: CHOUNE. oo. caints 5x |oce & aoe Ve < oaks c ban A \ Weare ell Wee ee an BI 12 | Berkfeldt filtrate made alkaline}... ..... Oily Fishy Not seored

All of the butters indicated in table 24 possessed a very disagreeable flavor and odor when fresh but they seemed to improve in quality during storage. The development of the fishy flavor in certain samples shows some very interesting features. In reviewing the results of this experi-

— ——

ae Se Ee ae

THe LEcITHIN CONTENT OF BUTTER 143

ment it must be borne in mind that the cream from which they were made contained the products of two days growth of Bact. ichthyosmius.

The development of the fishy flavor in the sample to which nothing was added, is therefore significant. The earlier occurrence of the flavor in the sample to which lactic acid was added is significant in that. it confirms certain observations already noted. The development of the flavor in the sample containing the alkaline Berkfeldt filtrate seems to be about simultaneous with its development in the sample to which nothing was added. The lack of development of the flavor in the samples to which the filtrate was added alone or in combination with other sub- stances, might appear to be contradictory to the enzymatic idea pre- viously expressed. It,is believed, however, that this is more than offset by the other data, which point to the necessity of a definite set of con- ditions that must be met in order to produce the flavor. Such being the ease, the absence of the flavor when the filtrate was added may be explained on the basis that the proper equilibrium had been disturbed. The final occurrence of fishiness in the sample containing lecithin is of importance as indicating that this may be the mother substance of the material causing the flavor. Other scattering results do not merit particular discussion at this time.

The trimethylamine and ammonia content of the samples shown in table 24, and their acid value, are given in table 25:

TABLE 25. TRIMETHYLAMINE AND AMMONIA CONTENT AND Acrp VALUE oF BurTER

SampuLes Wutcu Were MADE FROM Cream INOCULATED WITH BACTERIUM ICHTHYOSMIUS AND TO WatcH OTHER SuBSTANCES WERE SUBSEQUENTLY ADDED

Age of (CH3)3N NH,

Sample sample (parts per | (parts per Acid

(days) million) million) value Eel EPO hs, a 2 Dats ieee sa & 136 7.2 20.7 8.7 HEWN ame eee yet 0) ies ewan Gof sol Fie Spends taut 328 9.4 30.2 9.5 Earn Peet! ae es Bos iets se Sal 5 328 5.8 28.4 8.5 15S oC a A re eee 328 4.6 26.8 7.8 ES eM coal eas abe acne le As 5} 94 ah 12.7 8.2 AA GMRN eee aN gtepy. Fete 2ij. <7.G 5 Be S eFiseelh eles 328 9.4 25.4 8.0 LHL, 7 p.e" clea Ans 8c eo eee 328 8.2 24.0 3:9 (BAL, §o0hP Noh ware og oa ea oe 328 6 22.4 8.5 fe ET oo cree SA aia o> tes 328 5.8 DOS) A See , oe ne 5 Fae LG) ee A 55 aee geht a oo nace 245 9 ree ee | Ors See 9.2 POUND ee. «fad ole Pal ba ees oe 328 Ys 9.4 Se a os ciel ean 4 136 11.8 17.8 9.3

144 GEORGE CORNELL SUPPLEEB

The relatively constant trimethylamine results shown in table 25 indicate strongly that this substance has been produced in the cream by Bact. ichthyosmius.

TRIMETHYLAMINE AND AMMONIA PRODUCTION BY BACTERIUM ICHTHYOSMIUS IN MILK AND CREAM

In order to determine the trimethylamine production by Bacterium ichthyosmius in milk and cream, 50-cubic-centimeter quantities of these substances, sterilized, were inoculated with the organism alone and in combination with a lactic-acid starter. The inoculations were held for forty hours at 30° C., and the trimethylamine and ammonia were then determined in 20-cubic-centimeter quantities. The results of these. determinations are shown in table 26: ;

TABLE 26. Amount or TRIMETHYLAMINE AND AMMONIA PRODUCED IN SKIMMILK AND IN Cream BY BACTERIUM ICHTHYOSMIUS

(CH3)3N NH: Inoculation Material inoculated | (parts per | (parts per million) million)

Bacterium ichthyosmius and starter ......... Dkimmille 2 ..+ oc. oe None 84

Bact. ichthyosmius and starter ............. COAT «4a geist? 204.0 88 Back. cnthyosmius:..2. = 2s sas5 S¥eiee os ve oes CMMI. eis eu gee 94.4 125 Batt. 1chihajosmis, oxaa<ciccadessiesvecas COEATI ah wath a tN 74.7 78

The results presented in table 26 are of great interest as showing beyond a doubt that the fishy flavor produced in milk and cream by Bact. ichthyosmius is due to trimethylamine. This being the case, it is obvious that this substance would be carried into the butter, and there, under proper conditions which have already been pointed eout, be responsible for the characteristic flavor in that material. With respeet to the pro- duction of trimethylamine in cream and in milk by this organism, it is desirable to again call attention to the observations of the author, in which the evolution of a fishy flavor was noted on the addition of alkali to sweet cream.

These results are of further importance in that the cream inoculated with starter and Bact. ichthyosmius contained a greater amount of trimethylamine than did the cream inoculated with the organism alone. This indicates that an acid condition is most favorable for this particular

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Tue LecirHin Conrent or Burrer 145 fermentation, which would be in harmony with the idea that lecithin furnishes the source of the trimethylamine produced by the organism. The results are supported also by the fact that lecithin is largely asso- ciated with the fat, and that according to Hammarsten and Hedin (1915) lecithin is decomposed by dilute acids. Such being the ease, it is readily seen that this fermentation brought about by Bact. ichthyosmius would be greatly enhanced by the presence of acid. The presence of trimethylamine in skimmilk inoculated with the organism alone might be explained on the basis that the organism was able to produce this substance from proteins as well as from lecithin. Certain data not included in this paper, however, indicate that there is a certain amount of lecithin present in skimmilk. Just why there is no trimethylamine in skimmilk inoculated with the starter and the organism, is more difficult toexplain. It may be that the greater acidity in the skimmilk has inhibited the particular factor responsible for trimethylamine production.

PRODUCTION OF TRIMETHYLAMINE FROM LECITHIN AND CHOLINE BY BACTERIAL ACTION

In order to determine, if possible, whether certain organisms found in milk and in butter were capable of decomposing lecithin or choline into trimethylamine, a series of inoculation experiments were carried out. Lecithin alone in 0.3 per cent concentration, and in the presence of lactic acid and salt, was inoculated with a number of organisms, some of which were obtained from milk, some from fishy butter, and some from decomposed egg yolk which had developed the fishy flavor. The following known species were also used: Bacter‘wm lactis acidi, Bact. aerogenes, Bacillus prodigiosus, B. proteus, Bacterium ichthyosmius, Pseudomonas

_liquefaciens fluorescens, Oidium lactis. All organisms were inoculated

singly and in various combinations, and the cultures were held at 20° C. for approximately nine months. At the end of that time the cultures were tested for the presence of trimethylamine by heating with alkali. Negative results were obtained from all of the lecithin inoculations tested. Unfortunately, many of the cultures were contaminated with mold, and, since the results could not be considered trustworthy, they were discarded.

The same series of experiments was repeated by inoculating 0.1-per- cent choline alone and in the presence of lactic acid and salt. These cultures were held under the same conditions as were the lecithin inocula-

146 GEORGE CORNELL SUPPLEE

tions. Trimethylamine was found where Bacteriwn ichthyosmius was inoculated alone, in combination with Oidzwm lactis, and with Bacterium lactis acidi. The presence of salt did not seem to prevent the production of trimethylamine. Two organisms which were isolated from milk gave a pronounced test from the choline inoculation, but gave negative results in the presence of lactic acid and salt; Bacillus prodigiosus gave a positive reaction from the choline alone; and Bacteriwn aerogenes gave a non- typical test under the same conditions, as did Pseudomonas liquefaciens fluorescens. All other inoculations gave negative results.

It would appear from the results of the inoculation experiments that. since trimethylamine is produced from choline by Bacterium ichthyosmius and certain organisms found in milk, it is quite possible that the fishy flavor and odor found in milk and in butter may be due to this substance’s having been produced from the choline of the lecithin molecule. The fact that the two organisms isolated from milk gave a positive reaction and that they were selected at random, indicates that such a fermentation might be found fairly often. These results would therefore seem to point to bacterial agencies as the cause of the fishy flavor, its manifestation in butter being dependent on conditions previously mentioned. :

BACTERIAL INOCULATIONS INTO BUTTERFAT

A further attempt was made to produce the fishy flavor by bacterial inoculations into a medium in which all the constituents were fairly definitely known. Pure sterile butterfat from which the phosphatides had been extracted was used as the basis of such a medium. Four series of inoculations were made, using, with a few exceptions, the species that were inoculated inte lecithin and choline. These inoculations were stored at a temperature of 0° F. or lower for two hundred and nineteen days, at the end of which time they were examined for the fishy flavor by four judges. Negative results were obtained from all inoculations, not one of the judges pronouncing any of the 120 samples to be fishy in flavor. Whether any of the samples had possessed the flavor at some time during the storage period is difficult to say. All of the samples had a disagreeable tallowy flavor and odor. The composition of each of the four series, and the variation in acid value caused by the inoculations, are shown in table 27. All samples contained from 10 to 12 per cent of moisture and 2 per cent of salt.

Tue LeciraHin Cowrenr oF BuTTER 147

TABLE 27. Composition oF ARTIFICIAL BuTTER AND VARIATIONS IN AcID VALUE CaUsED BY BacTeRIAL INOCULATIONS

Composition and acid value

Butterfat, asl Organism or source Butterfat Butterfat Berkfeldt ple Butter- | and 0.15 and filtrate from fat per cent ere starter, and of lecithin | “trate from | 0.15 per cent starter of lecithin 1 | Bacterium lactis acidi......... 5.2 6.1 6.5 8.8 2\ Ordium lactis...........2.5. 7.8 9.2 7.8 <5 3 | Bacterium aerogenes.......... 7.1 6.8 5.8 (ae: 4| Bacterium ichthyosmius....... o.2 6.8 Gut 7.1 5| Pseudomonas liquefaciens flwor- 2h a ee Sere eee 6.8 rial fe rea 6| Bacillus prodigiosus.......... 8.4 6.5 ria) re! 7 | Bacillus proteus............. 6.1 5.8 6.5 6.8 Si bphashy PUMET. oo < wk. ig | y oe 7.5 rie: Oi) Misha WUAOE. . x 5.G. ieee sess 6.1 9.1 6.5 7.5 ae Nishi, butter. ...<5 <4 set = 65 6.5 7.8 (po! a Misha Utter, 2.52 esa ae ea 5.8 6.1 7.8 8.8 12) Mishy butter: . 4.6 ssa -4-- 7.1 6.1 8.8 8.1 Pee WOUCL: 6. Senge we Foes ee 7.1 6.5 8.4 8.1 Pt Wee yolk... 8 eee t ae es 7.1. 6.5 8.8 OZ 15 | Strong butter............... 9.1 6.8 8.4 7.8 16 | Strong butter............... 8.4 8.1 8.4 8.4 7a), Normal Dutier...c sot. 25-225. (ies CoD 8.4 8.4 1Soyhishy butter... 2s. +. 2... ..o + 8.8 6.8 8.1 8.8 19 | Fishy butter................ S21 7.8 7.8 9.7 20 | Fishy butter.............-. 8.1 6.1 8.1 10.4 UM Bishi PUPIL, os. ood eae 8 6.5 al 8.1 11.0 OP lehashy DUGG: oF 20 6c oso ve 8.4 8.1 9.4 7.8 23 | Fishy butter................ 10.4 Ges 8.4 7.8 OA Mee yollesrn.. nn nce nee. eee. OCy, 7 | 8.4 Fieal iG) Sa) | i or 8.8 7.1 6.5 irs) Zh (1 1 a a ee ree 6.8 6.1 (es 7.8 ese 5 0438 ws deem ia ey Os a 6.5 el 9.1 S| BD 1 ea nn 7.8 6.8 6.1 10.0 BA Ios ets «xia < os vv ie ps ates 6 - 6.5 8.1 Sell 5.8 310) Vel @1 cic) le ne ene ee eee 5.8 5.9 8.1 7.4

The results shown in table 27 are of interest only to the extent that they show the variation in acid value caused by different species of bacteria. Inasmuch as the samples were placed in stcrage immediately after being inoculated, it is probable that the changes are the result of bacterial enzymes liberated by autolysis, because it has been repeatedly shown that little or no growth takes place during storage.

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SUMMARY

_ The data presented in this paper show beyond a doubt that there is in normal butter a sufficient amount of lecithin to yield, on decom- position, small quantities of trimethylamine, and it is shown also that small quantities of this substance are essential for the manifestation of a fishy odor. Iurthermore, it is shown that when this substance is worked into butter under the proper conditions, it produces a flavor deseribed as fishy. These results are most uniform when trimethylamine butyrate is used. An associative fermentation in butter or in cream, with the ultimate formation of this substance, is quite possible. As to whether or not this or some other volatile and unstable combination of trimethylamine is the cause of the natural fishy flavor, remains to be shown more conclusively. Certain data do indicate that trimethylamine is found in some samples of fishy-flavored butter but not in others. Altho it is possible that its presence is incidental in such samples, that is not believed to be the case. In this connection it is worth while to call atten- tion to the confusion between the fishy, oily, and metallic flavors when they are present to only a slight degree. It seems possible that the initiation of the development of these flavors depends on a common fundamental factor. Whether or not any particular one of them develops to its typical flavor would depend on the presence of certain conditions which were specific for that flavor. With this possibility in view, it would be logical to assume that trimethylamine is responsible for the typical herring, or mackerel, flavor and odor in butter, and that the absence of this substance would result in the manifestation of similar but non-typical flavors.

There seems to be no doubt that the presence of a definite acid con- dition in the butter is essential for the development of the fishy flavor. This condition is best obtained when butter is made from cream con- taining lactic acid, regardless of whether this is developed by bacteria or added to the cream in the form of the commercial product. Further- more, the results indicate that, while a definite acid condition is essential, it must be accompanied by some other equally important factor. The data show that this factor is determined by biological agencies. It appears that both these factors must exist in a definite and delicate relationship, and that if the proper equilibrium is disturbed, the characteristic flavor is not manifest. Numerous results and observations indicate that the unknown transient factor is trimethylamine.

THe LecirHin Content or BuTrreR 149

The bacteriological aspects of the problem seem to involve the deter- mination of the relationship already mentioned. It is shown that the acid value of butter is to a certain extent regulated by biological factors, probably enzymes. It is shown also that trimethylamine may be produced in milk and in cream, probably to some extent from lecithin, with the consequent production of the fishy flavor in those products. Furthermore, it is shown that Bacteriwm ichthyosmius, which produced the flavor in those substances, would produce the flavor in butter also under certain conditions. It would therefore seem possible that other species of microorganisms might bring about the same type of change. It seems highly probable that the growth of bacteria in the cream before it is made into butter determines the conditions necessary for the later mani- festation of the fishy flavor.

The data dealing with lecithin as the source of trimethylamine in milk products are too meager to warrant definite conclusions at this time. However, the results presented herein, taken together with what is known regarding this substance, indicate that this is one of the most logical sources.

\ ACKNOWLEDGMENTS

The author wishes to express his keen appreciation and gratitude to Dr. E. 8. Guthrie, Mr. W. E. Ayres, and Mr. H. C. Jackson, of the Depart- ment of Dairy Industry at Cornell University, to all other members of this Department who have been kind enough to aid in the scoring of butter, and to all who have furnished samples for study.

150 GEORGE CORNELL SUPPLEE

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NERKING, J., AND Hawnsen, FE. Der Lecithingehalt der Milch. Biochem. Ztschr. 13 :348-353. 1908.

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Memoir 24, A Study of the Plant Lice Injuring the Foliage and Fruit of the Apple, the fifth preceding number in this series of publications, was mailed on October 7, 1919.

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Memoir 26, The Dry Root-Rot of the Bean, the third preceding number in this series of publications, was mailed on October 23, 1919.

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