vided with their natural environment, they will multiply when the medium is in a frozen condition. The experimental work which has been done in the past has used chiefly the usual laboratory media and pure cultures of laboratory-grown bacteria. It is perfectly possible that such conditions, since they are not comparable with those naturally prevailing, will lead to some erroneous results, and in view of recently demonstrated facts it becomes necessary to attack the problem of bacterial development in flesh foods from this point of view before the assumption can be accepted that temperatures below freezing guarantee a freedom from bacterial activity.

Observations on the growth of bacteria under the conditions of the very low temperature cold-storage houses, such as almost universally prevail in the United States, are entirely lacking. What information is to be had on the growth of bacteria in flesh when cold-stored comes from abroad, chiefly from Germany, where the temperature of the "Kühlhaus" rarely reaches 0° C. and is commonly several degrees above it. Exposed to such temperatures there is a unanimity of opinion regarding the "ripening" of the flesh, and the tenderness and flavor acquired in the course of it. To what this maturation is due, however, is not so well settled. Glage would ascribe much of the flavor to “aroma-producing" bacteria which develop best at low temperatures; Müller, on the other hand, believes that the process is essentially dependent upon the enzymes of the flesh itself. He holds that the temperature of the chilling room prevents putrefaction, and, therefore, all those poisonous properties dependent upon putrefaction, while it assists natural autolysis. Neither does he consider that the changes in the ripening of meat are the early stages of putrefaction.

The observations made under commercial conditions he has reinforced by a study of freshly killed, bichlorid-washed fish, which was kept at 0° C. After 5 days there was an unpleasant taste and a characteristic odor, both of which appeared in 2 days when kept at 12° C. Schmidt-Nielsens kept a carp packed in ice for 14 days, and, though bacteria-free, it had, at the expiration of the above period, so unpleasant a taste that it was unfit for food. Müller determined the amount of nitrogen soluble in water in both mammalian and fish muscle kept at 0° C., concluding therefrom that an autolysis proceeds. He found that muscle loses its elasticity, becomes tender, and the clear red color changes to an opaque, dark red. The odor after 3 days at 25° C. or at 0° C. after 14 days is strongly acid.

Zts. Fleisch- Milchhygiene, 1900-1901, p. 131.

Der Reifungsprozess des Fleisches, Zts. Fleisch- Milchhygiene, 1904, 14: 217 and 337.

c Archiv Hyg., 1903, 47: 127.

In 1897 Gautier published the account of a chemical and histological examination of frozen flesh, chiefly of that sent to France in a frozen condition from South America and the United States. He stated that the flavor of the frozen muscle was never quite as good as the fresh. That the juice when exuded was always more abundant than from the fresh and that it contained globulins, albumins, peptones, and organic and inorganic constitutents. Among the most recent chemical studies of such flesh is that made by Rideal," who determines the ratio of nitrogen to the total solids in both chilled and fresh meats and, finding them the same, excludes decomposition. Artificial digestion gave identical results with frozen and fresh muscle. He states that there were no signs of incipient decomposition. Neither bacteriological nor histological examinations were reported. However, this statement is made: "The tenderness of meat which has been frozen has been attributed to the slow action of sarcolactic acid, and the loosening of the intermuscular tissue promotes rapid decomposition."

Martel, in a recent article on the cold storage of foodstuffs, favors cold rooms rather than a temperature sufficiently low to freeze, believing the latter only necessary when transportation for long distances is to follow. He states that bacteria do not readily penetrate the muscle, about 10 days being required to carry them within. 1 cm of the surface, but that there is a marked autodigestion of the cell contents, due to the action of the cell ferments, which proceeds easily at 2° to 3° C., though below zero such action is stopped. As to the quality of the meat after storage he believes it to have an improved flavor if kept at 2° to 3° C. and not allowed to freeze. The cause of this improved flavor is ascribed to the aroma bacteria, which he considers desirable.

Accompanying the change in taste there are, according to Martel, alterations in the muscle fiber which consist in a change from translucent to opaque and from brilliant to dull, as well as from tough to tender. The reaction of the fresh muscle is neutral, but becomes acid as coagulation proceeds. In cold storage at 2° to 3° C. about 8 hours are required for a strong acidity to develop, and an odor, aromatic but not at all putrefactive, makes its appearance at the same time. When muscle is fresh it is very difficult to extract any fluid. After 3 days in storage flesh yields much juice. Microscopic examination shows an annihilation of the striations and abundant granulations.

a Les viandes fraiches et congelées.

b Cold Storage, 1907.

c Conservation et maturation des viandes emploi du froid industriel, L'Hygiene de la viande et du lait, Vol. 1, Nos. 1 and 2.

DISCUSSION OF RESULTS OBTAINED IN THE BUREAU OF CHEMISTRY.

a

HISTO-CHEMICAL CHANGES IN COLD-STORED CHICKENS.

Gautier states that the muscle fibers of cold-stored flesh remain unaltered except, perhaps, for a slight pulling apart of the individuals. The marked and deep-seated changes, the occurrence of which has been demonstrated in connection with the study in this laboratory, of chickens preserved by cold are, therefore, of interest, and it seems probable that the solving of the problem of the cause, sequence, and ultimate result of such changes would throw much light on the whole question of the various alterations undergone by flesh at low temperatures. While the various dyestuffs which have been used to differentiate the tissues preparatory to microscopic study have not, as yet, served as exact microchemical reagents in the sense of classification of compounds, they have very positively indicated an alteration in the chemical character of certain morphologically distinct elements.

The characteristic green of the normal, fresh muscle fiber, when stained as previously described, is very decidedly altered by long keeping at low temperatures, so much so that finally it is almost entirely replaced by dirty yellow and brown greens, or even by orange tints. The white fibrous connective tissue, staining a brilliant blue in the fresh chicken, in the cold-stored takes on a greenish tint. The material which exudes from the muscles after keeping for even a comparatively short time has, at first, almost the same staining reaction as the fiber itself, but it changes gradually, until it is a dirty brownish or bluish green. The irregular staining of the fiber would also indicate the presence of a progressive chemical alteration. The rupture of the sarcolemma and the extrusion of the muscle substance are not merely mechanical alterations due to freezing and thawing. If such were the case the progressive effects which are so clearly traced in fowls kept for periods of varying lengths of time would be wanting. The differences between the chicken muscle frozen for 48 hours and that of the fresh bird lies chiefly in the size of the spaces between the fibers or bundles of fibers, and deep-seated changes

are not seen.

The histological alterations which have been traced are confirmed by the differences noted between stored and fresh chickens in the distribution of the protein nitrogen. It would seem probable that for the chickens stored 2 years and 4 years, respectively, the greater part of the change is due to enzyme action, not only because the tissue shows but few bacteria either living or dead, nor because the histological degeneration proceeds differently from that observed. when ordinary temperatures prevail, but also because of the changes

a Loc. cit.

in the relative distribution of the protein nitrogen. The fowls in storage 14 months give indications of more marked changes, chemically, than did the others examined, considering the length of the storage period, but such changes may be due to the number of organisms which were found in a living condition in the tissues and which may have been present when the fowl was stored. Because these chickens have an unknown history preceding their entrance into the cold-storage warehouse it is impossible to say whether the numbers found represented an increase or a decrease during the storage period. Such questions can only be answered by the study of chickens of known and of a strictly comparable history.

Neither can this report deal comprehensively with the question of the migration of organisms from the intestine into the edible portions. Judging from the ravages undergone by the walls of the gut during storage it could offer but a slight barrier to active bacteria. From an investigation which is now in progress to determine the resistance of intestinal organisms to cold when in their natural environment it appears that they do remain alive in large numbers. They are, however, of the varieties which develop best at 20° C. instead of body heat, agreeing in this with the behavior of the naturally occurring milk organisms when kept under like circumstances." The fact that the organisms which have been found are not gas producers in dextrose media would argue against possible migration. The location of the bacteria in the tissues, on the other hand, would indicate its possibility since there are fewer in the muscles of the breast than in the inner thigh muscles, which in the chicken lie closely adherent to the body wall.

CHANGES IN FAT.

The decomposition of the fat of the chicken is much more pronounced than is the decomposition of the protein, and in the tracing of the changes which occur in this tissue not only bacteria and enzymes must be taken into account, but light and air must also have due consideration.

It has been held that the splitting of fat into acid and glycerol is the cause of rancidity. More recent studies would indicate that, while such a splitting ordinarily accompanies the condition known as "rancid," it is not the real cause of it, but that rancidity is due to the action of air and light on fats which have been previously split by enzymes acting in the presence of moisture. The part played by bacteria in the decomposition of fats in their natural environment is not a minor one, though the rôle to be assigned to them regarding

a Pennington, loc. cit.

Lewkowitsch, Chemical Technology and Analysis of Oils, Fats, and Waxes, 3d ed., 1904, p. 22.

the production of rancidity is still a mooted question. Since bacteria will not propagate in pure, dry fat, but must. have present suitable nitrogenous foodstuffs as well as moisture, the conditions for their activity, as pointed out by Lewkowitsch, are the same as for the activity of enzymes, hence it is exceedingly difficult to say where the action of either the one or the other ceases, but it would seem probable that fat splitting can be ascribed to both enzymes and bacteria, whereas the decomposition of the free fatty acids or of the glycerol must be referred to bacteria alone.

The researches of Kastle and Loevenhart on lipase a have established the wide distribution of the enzyme in nature and its stability as well as its adaptability to its environment. It was kept dry and moist, and at room temperatures and in cold storage, retaining its activity for months. It hydrolyses most rapidly at 40° C., at which temperature 11.29 per cent of fat splitting was noted; at 10° C. the amount was 3.89 per cent; at 0° C., 2.26 per cent, and at - 10° C., 0.70 per cent, a quantity which, given weeks and months in which to augment, might easily reach notable proportions. The enzyme lipase shows a decided preference for fats of higher molecular weight, wherein it differs from the usual acids used to induce hydrolysis. It is also able, under favorable conditions, to reverse its action, and synthesizes ethyl butyrate, for instance, from alcohol and butyric acid. Hanriot' states that lipase can form monobutyrin from butyric acid and glycerin and the higher the molecular weight of the acid the more easily is the synthesis accomplished. Such facts indicate a most important and far-reaching physiological rôle for the enzyme, as is discussed at length by Loevenhart, who finds it in considerable quantities wherever fat synthesis is taking place in the living tissue, as in the subcutaneous fat and in the secreting mammary gland. Between the enzyme studied by Loevenhart, in the milk gland, and that occurring in milk, Gillet finds a most important difference, namely, that the latter splits monobutyrin but not ordinary fats. In accord with such a train of thought comes the work of Connstein, Hoyer, and Wartenberg, who found a great variation in the ability of lipase to split fats from different sources. Butter is very resistant, owing, probably, to its high percentage of acids of low molecular weight, since triacetin gave 0.4 per cent, tributyrin 9.5 per cent, and triolein 50.6 per cent of free fatty acid when all were treated under like conditions. It is also noteworthy that the reaction is much more a Concerning Lipase, the Fat Splitting Enzyme, and the Reversibility of its Action, Amer. Chem. J., 1900, 24: 491.

с

Sur la reversibilité des actions diastatiques, Comptes rend. soc. biol., 1901, p. 70. c Amer. J. Physiol., 1901-2, 6: 351.

d Existe-t-il une lipase dans le lait?-J. physiol. pathol. general, 1903.

The Enzymic Decomposition of Fat, Abs. J. Soc. Chem. Ind., 1902, 21: 1541.