33 



111 



substances must therefore have chalk added, and this is the more necessary, since the 

 proteolytic enzymes of lactic acid bacteria can only excejitionally be active in acid liquids. 



Only Streptococcus liquefaciens, and some few tetracocci (micrococci and sarcinæ) 

 split up casein in sour milk, but they do so to a far greater degree wiien the acid is partly or 

 entirely neutralised. 



By way of measuring the degree to which the protein decomposition takes place in 

 the milk, we have, as in previous works, determined the quantity of soluble nitrogen 

 (SN) and decomposition nitrogen (D.V) both expressed as % of the total nitrogen present. 

 The former is simply the nitrogen in the liltrate, after the casein, albumin and globulin 

 have been precipitated by boiling with a small quantity of acetic acid. As we work with 

 sterilised milk, however, where the albumin and globulin are already coagulated, the 

 boiling is generally unnecessary, and where the casein has been separated off by the lactic 

 acid fermentation (the casein is precipitated despite the addition of the chalk) it is like- 

 wise superfluous to add the acid, so that as a rule. i'A' will mean the nitrogen in the liltrate 

 of the culture. This fdtrate is also used for determination of D.V. by which is understood 

 the nitrogen which is not precipitated by addition of sulphuric acid and phosphotungstic 

 acid in certain definite proportions^). As with the determinations of acid content, where 

 we always subtracted the degree of acidity in the control tubes, so also here we subtract 

 the SA' and DA' of the original milk (sterilised with chalk). AVhat we want to accertain 

 is, of course, the quantities formed by the bacteria. These quantities may quite well be 

 negative, provided the organisms in question consume more than they give off, and D\ 

 may also be slightly in excess of SA', in cases where any of the original SA' in the milk 

 has also become broken down. In peptone broth, where all the nitrogen is found in a state 

 of solution, the determination of SA' will no longer be required; we can, however, on the 

 other hand determine the amount of formol-titratable nitrogen according to S. P. L. 

 Sorensen's method 2). It will nevertheless be necessary first to distill off the ammonia in 

 the cultures with barium carbonate, and then remove the phosphoric acid with barium 

 chloride and baryta. From the quantity of formol-titratable nitrogen found, we have 

 throughout subtracted the amount of formol-titratable nitrogen in the non-inoculated 

 broth, and by FA', therefore, we understand the formol-titratable nitrogen produced by 

 the bacteria. In some cases, we have noted the first stage of the formol titration separately 

 (i. e. prior to addition of formalin, from neutral point of litmus to faint red with phenolph- 

 talein) and calculated the quantity of nitrogen corresponding thereto as a percentage of 

 the total nitrogen content. According to Henriques and Gjaldb.ck^). this stage will 

 as a rule be the less, the farther the proteins have been decomposed. The figures thus 

 found should then be inversely proportional to DA', which is also to some extent found 

 to be the case (See Table X). In conformity with the fact that the true lactic 

 acid bacteria are unable to live on ammonia salts or single amino acids, 

 we find that they are also incapable of splitting up amino acids. They 



' Ô0 cm/' of the milk filtrate — 30 cm ^ sulphuric acid of 25 " „ ■ 20 cm.' phosphopungstic acid of 10 "(o 

 are filled up to 250 cm and left to stand for 12 hours before filtration. For determination of nitrogen 

 by Kjeldahl s method, 50 cm.' of the new filtrate is used, answering to 5 cm.' milk. 



-) Meddelelser fra Carlsberg Laboratoriet 1907, VII. p. 1 



3) Zeitschr. f. physiol. Chemie 1911, Bd. 75, p. 363. 



D. K. l). Vidensk. S«lsk, Skr., naturvidensk. og niatheni. Afd. . 8. Raekke. V. 2. 15 



