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TABLE 2.-Results of trials with different bacterial strains.

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A priori one could hardly expect any exact parallelism between the bacterial content of the cheese milk on the one hand, and the rapidity of ripening as expressed by the amount of soluble nitrogen at certain intervals, on the other, but the figures given in Table 2 nevertheless show as clearly as could be desired that unmistakable connection does exist between these two factors.

We find from the table that an increase in the acidity of the milk which took place when the milk was allowed to stand and ripen (being thus due to an increase in the bacterial content) always caused an increase in the amount of soluble nitrogen formed within a certain time; this must presumably be regarded as the chemical expression for a shorter ripening time. By special trials, which are not yet finished, we have found that the acidity itself, which of course, rises with the content of bacteria in the cheese milk, has no effect on th shortening of the ripening time, but that this shortening must really be attributed to the increase in bacterial cells.

If now on the basis of these results it can be upheld as probable that the rapidity of the ripening of the cheese during the first stages is more or less directly dependent on the bacterial content of the cheese milk (and by bacterial content we mean the content of lactic acid bacteria) then it can also be understood how the foregoing trials with bacterial strains of relatively different casein-splitting powers (Table 1) could give an absolutely negative result. This naturally depended on the fact that in these experiments no account was taken of the total bacterial content of the cheese milk at the time of curdling. It is clear that in order that a bacterial strain possessing a greater power of splitting casein should induce a correspondingly more rapid ripening, the bacterial content of the cheese milk should be of the same magnitude as in the milk to which a less powerful casein splitter was added. Otherwise the superior splitting power might conceivably be counteracted by a lower bacterial content.

The manner in which the cheese milk is soured may also possibly have some effect on the rapidity of ripening, for in an ordinary starter a large proportion of the lactic acid bacteria are certainly so firmly embedded in the coagulum that when the culture is mixed with the cheese milk they do not come into direct contact with the milk, and therefore not with the newly precipitated paracasein. Thus these bacteria could not be considered as having any real influence on the cheese-ripening process. If, however, the cheese milk is allowed to ripen, then the bacteria in it will be free and loose, and when the coagulation takes place, each bacterial cell will from the outset be in direct contact with the paracasein, and can form a colony. More over, in a starter which is coagulated when used, quite a number of cells will undoubtedly be already dead, but in a ripening milk it can safely be assumed that the number of dead cells will be quite negligible.

We are now occupied with more exhaustive investigations of ques. tions such as these, which will be published in due course. In the meantime, we considered that it might be of interest to set out at this stage the most important of the results obtained in the trials made up to the present by us.

SUMMARY.

One of us (Barthel) previously showed that the ordinary lactic acid bacteria belonging to the Streptococcus lactis group could decompose casein at temperatures usually employed in storing cheese (14 to 20° C.) and that, therefore, a far more important part must be ascribed to the action of these bacteria in the ripening process of hard cheeses than has hitherto been done. Moreover, these lactic acid bacteria are, during the first few months of ripening at any rate, absolutely predominant in the bacterial flora of the cheeses in question.

Attempts to influence the rapidity of the ripening of the cheese by the addition to the cheese milk of starters consisting of cultures of lactic acid streptococci having different powers of splitting casein were not successful. A probable explanation of this result lies in the circumstance that in these trials, although equal quantities of starter were added of the different strains, yet we had no knowledge of the actual total number of bacteria added. It certainly seems probable that the rapidity of the ripening of the cheese is directly dependent on the number of bacteria in the cheese milk at the moment of adding rennet. In order that the above trials with different bacteria should be really comparable with one another, it was, of course, necessary that the number of bacteria in the cheese milk at the moment of adding rennet should be the same in all cases.

In the first place, it was necessary to find out how a definite connection could be demonstrated between the total number of bacteria in the cheese milk on coagulation, on the one hand, and the rapidity of ripening of the cheese on the other. We also made such investigations, and showed that a connection of this kind undoubtedly exists. By bacterial content we naturally mean the content of lactic-acid bacteria. The rapidity of ripening of the cheese was determined in these trials by means of determinations of soluble

nitrogen expressed in percentages of the total nitrogen, carried out on samples taken at definite intervals.

That the rapidity of the ripening of cheese thus appears to be fairly directly dependent on the number of bacteria in the cheese milk is confirmed by practical experience, according to which it is considered possible to influence the course of ripening by adding starters to the cheese milk or by letting the milk ripen to a certain degree of acidity. In both of these operations, of course, a large number of lactic acid streptococci are added. As we found the abovementioned connection to exist, we proposed to return to the investigation of the question as to how far it is possible by the addition of strains of lactic acid bacteria of different casein-splitting powers to the cheese milk (pasteurized to 63° C. for 30 minutes) to influence the rapidity of the ripening of the resulting cheese.

REFERENCES.

1. BARTHEL, CHR. Das kaseinspaltende Vermögen von zur Gruppe Streptocor.

Cus lactis gehörenden Milchsäurebakterien. Centbl. Bakt. Abt. 2, Bd. 44,

no. 1/4, S. 76. Jena, Aug. 7, 1915. 2. BARTHEL, CHR., und SANDBERG, E. Weitere Versuche über das Kaseïn

spaltende Vermögen von zur Gruppe Streptococcus lactis gehörenden Milchsäurebakterien. Centbl. Bakt. Abt. 2, Bd. 49, no. 14/17. S. 392.

Jena, Oct. 22, 1919. 3. SAMMIS, J. L., SUZUKI, S. K., and LAABS, F. W. Factors controlling the

moisture content of cheese curds. U. S. Dept. Agr. B. A. I. Bull. no. 122,

p. 29. May 31, 1910. 4. HUCKER, G. J. The relation of the number of bacteria in milk to the quality

and yield of cheese. New York Agr. Exp. Sta. Bull. 486. Geneva, Jan., 1921.

THE USE OF SELECTED LACTIC FERMENTS IN THE MANUFAC

TURE OF HARD-PRESSED CHEESE. REYWICK HUTSON LEITCH, M. A., B. Sc., professor of dairying, dairy research department, West of Scotland Agricultural College, Kilmarnock, Scotland.

In order to secure desirable flavor and texture in cheese the cheese maker resorts to the use of a starter. A starter is simply a milk culture of certain lactic acid bacteria which grow quickly in milk and which produce a good-flavored dairy product. The starters in common use at the present time are more or less pure cultures of Streptococcus lacticus of which there are two distinct though closely related species: (1) Str. lacticus (diplococcus form) and (2) Str. lacticus (streptococcus form). The biological characteristics of these forms are so well known that they do not call for a detailed deseription in this paper. The first organism—the Streptorocrus lactis of Jensen's recent classification-is common to cow's milk in North Britain and predominates in naturally soured milk. As a pure-culture starter this organism gives a nice, mild-flavored butter. It produces cheese of a fine soft texture but the flavor is inclined to become sharp after the third month of curing. It might be used with advantage in the manufacture of quick-ripening cheese like the English Derby. Its employment in cheese making must be limited because it is not very resistant and in practice is apt to become impure after a fortnight's use.

Strains used by us in our experimental work did not produce in pure culture the sour smell and unpleasant flavor noted by Jensen. The other form of Streptococcus lacticus, named by Jensen Streptococcus cremoris occurs typically in characteristic chains, and is the starter organism extensively used in British dairying. Hansen's lactic ferment contains this bacterium. When used as a starter this organism gives satisfactory results. It grows well between 15° and 25° C., is a fairly rapid acid producer, remains vigorous and pure for a considerable period, and is easily propagated. (But it tolerates B. subtilis and many members of the coliform group.) When clean milk is used it gives a good-flavored cheese; the cheese keeps well for four or five months, after which time the flavor is inclined to become sharp. This organism has distinct caseolytic properties and the ripening of cheese in North Britain is in large measure due to its activity.

In the laboratory, it is possible to keep these organisms alive for a lengthened time in solid media of a suitable nature, but the sugar content should be low, and the medium well buffered. A good liquid medium is a 4 per cent solution of milk sugar in water, the reaction of which is adjusted by lactic acid to pH 3.0. Such a medium will preserve the lactic acid bacteria for a lengthened time without the necessity of transference, but the culture should be held in lightprotected bottles. In such a medium, one finds that the typical Str. cremoris temporarily loses its chaining habits and becomes a diplo form.

These ordinary lactic acid bacteria occasionally exhibit acquired characteristics. Some forms will produce a burnt flavor in milk, in butter, and in cheese. This peculiar property, which we have encountered on several occasions in starters, is only a temporary acquisition, and usually disappears when the organism is repeatedly subinoculated. In such a condition, the organisms are rapid acid producers, and the fermented milk has a sharp taste. We have found by experiment that Cheddar cheese made with such material acidifies rapidly during the making process, and exhibits a short texture when cured. Again many pure cultures will become slimy in milk. We have had repeated experience of this, but what has not been clearly recorded is that a slimy milk culture may exhibit a moderately high degree of acidity. Thus we have frequently found an acidity of 0.8 per cent lactic acid in a s!imy milk culture of Str. cremoris. This sliminess is stated by some investigators to be a juvenile characteristic: it may sometimes be induced by repeatedly growing the subcultures at low temperatures; occasionally it is caused by the biological condition of the milk.

It should be observed that the value of a starter in dairying can not be correctly estimated by the morphological character of the typical organism, or by the common biological tests of the laboratory. It becomes increasingly clear that strain counts for more than anything else. Organisms that have apparently the same differentiating bacteriological features may have quite different values as starters. The value of strain has been clearly proved in the case of Conn's bacillus 41 in American dairying. In our own work, a strain of Str. cremoris isolated from a sample of goat's milk gave an unusually fine flavor to butter and cheese. The ability of a starter

to remain pure under ordinary dairy conditions is also a most important feature; this is largely a matter of strain also. Some strains of the ordinary lactic acid bacteria possess this property in a high degree.

In marked contrast to the Streptococcus lacticus type are the lactic acid organisms of the B. bulgaricus type. Representatives of this group which we have made the basis of experiments are B. bulgaricus Massol, B. freudenreich, B. bulgare (from Pasteur Institute), Bastonicini lactici coagulanti Gorini, B. bulgaricus Samarani, Streptothrix dadhi Chatterjee, Streptobacterium casei, 11 and 32 (Jensen), Bacillus acidophilus Browning, bacillus of Tarkhana, and the bacillus of Laban Ra-yeb. These organisms, which are all closely allied, are distinguished by their morphology, by their staining properties, by the characteristic form of their colonies, by the kind and amount of lactic acid they produce, and by their strong caseolytic power. As a group, these organisms belong to the lactobacilli of Löhnis, or in the more recent classification of Jensen, to the thermobacteria and the streptobacteria.

The first representative of this group to which we directed attention in our technical work on cheese was the B. bulgaricus of Massol. Its main distinguishing characteristics are as follows: It is a grampositive streptobacillus varying from 3.5u. to 104 in sterilized milk, but often reaching from 304 to 50in raw or Pasteurized milk. It makes little growth at temperatures below 37° C.: it grows rapidly at 40° C. It does not succeed on ordinary media, but on special whey agar made with casein peptone and additioned with autolyzed yeast extract it produces very characteristic colonies which are minute in size (one-half millimeter after 48 hours' growth) and in appearance resemble a mass of tangled hairs. The pH of the medium should range from 5 to 6. With methylene blue the organismi shows differential staining, some parts of the protoplasm becoming more deeply impregnated than others. In B. bulgaricus these volutin-staining bodies are in successive rectangular blocks rather than in round beads. B. bulgaricus is nonspore bearing, but it is possessed of great vigor in milk cultures, and can be held over for much longer periods than Str. lacticus without the necessity for reinoculation. It has a rapid caseolytic effect at 37° C., which is slower but still quite distinct at 20° C. B. bulgaricus is not adapted for butter making, as it imparts a distinct cheesy flavor to the product, but when employed in the manner about to be described it produces a superb quality of cheese.

In our first experiments with B. bulgaricus Massol as a starter, we followed the usual technique in Cheddar cheese making, and employed the temperatures and acidities common to the Dunlop method. (The Dunlop cheese, which is of Scottish origin, is a white, softtextured, meaty cheese, made after the Cheddar principle, but wrought at lower critical temperatures and acidities.) "While cheese of a good quality and a special flavor emerged, the process of manufacture was unduly long, and a small proportion of ordinary starter had to be added to the cheese milk to induce a normally working curd. It was soon made apparent that temperature was a controlling factor, and in a subsequent batch of cheese the making temperatures

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