Digestive bacterial theory. The first theory propounded was that of Duclaux,[197] who in 1887 advanced the idea that this change was due to that type of bacteria which is able to liquefy gelatin, peptonize milk, and cause a hydrolytic change in proteids. To this widely-spread group that he found in cheese, he gave the generic name Tyrothrix (cheese hairs). According to him, these organisms do not function directly as ripening agents, but they secrete an enzym or unorganized ferment to which he applies the name casease. This ferment acts upon the casein of milk, converting it into a soluble product known as caseone. These organisms are found in normal milk, and if they function as casein transformers, one would naturally expect them to be present, at least frequently, if not predominating in the ripening cheese; but such is not the case. In typical cheddar or Swiss cheese, they rapidly disappear (p. 168), although in the moister, softer varieties, they persist for considerable periods of time. According to Freudenreich, even where these organisms are added in large numbers to the curd, they soon perish, an observation that is not regarded as correct by the later adherents to the digestive bacterial theory, as Adametz and Winkler.
Duclaux's experiments were made with liquid media for isolation purposes, and his work, therefore, cannot be regarded as satisfactory as that carried out with more modern technical methods. Recently this theory has been revived by Adametz,[198] who claims to have found in Emmenthaler cheese a digesting species, one of the Tyrothrix type, which is capable of peptonizing the casein and at the same time producing the characteristic flavor of this class of cheese. This organism, called by him Bacillus nobilis, the Edelpilz of Emmenthaler cheese, has been subjected to comparative experiments, and in the cheese made with pure cultures of this germ better results are claimed to have been secured. Sufficient experiments have not as yet been reported by other investigators to warrant the acceptance of the claims made relative to the effect of this organism.
Lactic-acid bacterial theory. It has already been shown that the lactic-acid bacteria seems to find in the green cheese the optimum conditions of development; that they increase enormously in numbers for a short period, and then finally decline. This marked development, coincident with the breaking down of the casein, has led to the view which has been so ably expounded by Freudenreich[199] that this type of bacterial action is concerned in the ripening of cheese. This group of bacteria is, under ordinary conditions, unable to liquefy gelatin, or digest milk, or, in fact, to exert, under ordinary conditions, any proteolytic or peptonizing properties. This has been the stumbling-block to the acceptance of this hypothesis, as an explanation of the breaking down of the casein. Freudenreich has recently carried on experiments which he believes solve the problem. By growing cultures of these organisms in milk, to which sterile, freshly precipitated chalk had been added, he was able to prolong the development of bacteria for a considerable period of time, and as a result finds that an appreciable part of the casein is digested; but this action is so slow compared with what normally occurs in a cheese, that exception may well be taken to this type of experiment alone. Weigmann[200] inclines to the view that the lactic-acid bacteria are not the true cause of the peptonizing process, but that their development prepares the soil, as it were, for those forms that are more directly concerned in the peptonizing process. This they do by developing an acid substratum that renders possible the more luxuriant growth of the aroma-producing species. According to Gorini,[201] certain of the Tyrothrix forms function at high temperatures as lactic acid producing bacteria, while at lower temperatures they act as peptonizers. On this basis he seeks to reconcile the discrepancies that appear in the experiments of other investigators.
Digestive milk enzym theory. In 1897 Babcock and the writer[202] showed that milk underwent digestive changes spontaneously when bacterial activity was suspended by the addition of such anaesthetics as ether, chloroform and benzol. The chemical nature of the by-products produced by this auto-digestion of milk resembles quite closely those found in ripened cheese, except that ammonia is not produced as is the case in old cheese. The cause of the decomposition of the casein, they found to be due to the action of a milk enzym which is inherent to the milk itself. This digestive ferment may be separated from fresh milk by concentrating centrifuge slime extracts by the usual physiological reagents. This ferment, called by them galactase, on account of its origin in milk, is a proteolytic enzym of the tryptic type. Its activity is destroyed by strong chemicals such as formaldehyde, corrosive sublimate, also when heated to 175° F. or above. When such extracts are added to boiled milk, the digestive process is started anew, and the by-products produced are very similar to those noted in a normal cheese.
Jensen[203] has also shown that the addition of pancreatic extracts to cheese accelerated the formation of soluble nitrogenous products.
The action of galactase in milk and cheese has been confirmed by Freudenreich[204] and Jensen,[205] as well as by American investigators, and this enzym is now generally accepted as one of the factors concerned in the decomposition of the casein. Freudenreich believes it is able to change casein into albumose and peptones, but that the lactic-acid bacteria are chiefly responsible for the further decomposition of the nitrogen to amid form.
Failure before to recognize the presence of galactase in milk is attributable to the fact that all attempts to secure sterile milk had been made by heating the same, in which case galactase was necessarily destroyed. A brief exposure at 176° F. is sufficient to destroy its activity, and even an exposure at lower temperatures weakens its action considerably, especially if the reaction of the medium is acid. This undoubtedly explains the contradictory results obtained in the ripening of cheese from pasteurized milk, such cheese occasionally breaking down in an abnormal manner.
The results mentioned on page 172, in which cheese failed to ripen when treated with disinfectants,—experiments which were supposed at that time to be the foundation of the bacterial theory of casein digestion—are now explicable on an entirely different basis. In these cases the casein was not peptonized, because these strong disinfectants destroyed the activity of the enzyms as well as the bacteria.
Another important factor in the breaking down of the casein is the pepsin in the rennet extract. The digestive influence of this agent was first demonstrated for cheddar cheese by Babcock, Russell and Vivian,[206] and simultaneously, although independently, by Jensen[207] in Emmenthaler cheese. In this digestive action, only albumoses and higher peptones are produced. The activity of pepsin does not become manifest until there is about 0.3 per cent. acid which is approximately the amount developed in the cheddar process. These two factors undoubtedly account for by far the larger proportion of the changes in the casein; and yet, the formation of ammonia in well ripened cheese is not accounted for by these factors. This by-product is the main end product of proteid digestion by the liquefying bacteria but their apparent infrequency in cheese makes it difficult to understand how they can function prominently in the change, unless the small quantity of digestive enzyms excreted by them in their growth in milk is capable of continuing its action until a cumulative effect is obtained. Although much light has been thrown on this question by the researches of the last few years, the matter is far from being satisfactorily settled at the present time and the subject needs much more critical work. If liquefying bacteria abound in the milk, doubtless they exert some action, but the rôle of bacteria is doubtless much greater in the production of flavor than in the decomposition of the curd.