STUDIES ON THE PROTEOLYTIC BACTERIA OF MILK

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1 STUDIES ON THE PROTEOLYTIC BACTERIA OF MILK III. ACTION OF PROTEOLYTIC BACTERIA OF MILK ON CASEIN AND GELATIN WILLIAM C. FRAZIER AND PHILIP RUPP From the Research Laboratories, Bureau of Dairy Industry, United States Department of Agriculture Received for publication April 1, 1928 In Paper II of this series (1928) the action of 229 cultures of proteolytic milk organisms on milk was discussed and the organisms were roughly grouped according to that action. A study of the action of these same organisms on casein and gelatin has also been undertaken to aid further in their grouping. The tests and methods used in the present work are the same as those used in the work on milk and are as follows: The bromine test for tryptophane, the Salkowski test for indol-acetic acid, the Thomas test for ammonia, and the formol titration for increase in amino nitrogen. In addition, since the casein and gelatin solutions were clear, the "amino-ph number" was determined as an indication of increase in amino nitrogen. The method for this determination has been described elsewhere by one of the authors (Frazier, 1926). Berman and Rettger (1918) reported that the bacteria which they used required the presence of simpler and more available nitrogen compounds to start the breaking down of purified casein. Therefore, most of the media tried in this work contained in addition to the casein or gelatin small quantities of simpler nitrogenous compounds, such as beef infusion, peptone, or sterile milk. The basic casein medium was made as follows: Casein (according to Hammarsten) grams N/1 NaOH cc. K2HPO grams KCI grams Distilled water to 1000 cc. 187

2 188 WILLIAM C. FRAZIER AND PHILIP RUPP This medium was modified by the addition of one or more of the following: peptone (Bacto), 1 gram; double strength beef infusion, 10 cc.; glucose, 0.1 gram; or sterile milk, 10 to 100 cc. As much as 20 grams of casein per liter were tried, and the glucose was varied between none and 3 grams per liter in different experiments. The gelatin medium contained the following: Bacto gelatin, 10 grams; NaH2PO4-2H20, 1.2 grams; KCl, 5 grams; peptone, 0.1 gram; beef infusion, 10 cc., glucose, 0.1 gram; and distilled water to make 1000 cc. In one experiment the glucose, peptone, and beef infusion were omitted. The gelatin agar plate method used is described in another paper (Frazier, 1926). Because of erratic results with M. varians, a casein medium was prepared in which lime water was used instead of sodium hydroxide in order that calcium caseinate would be present instead of sodium caseinate. Except that the formula was trebled, the medium was prepared in the same way as the casein solution used for the casein-agar plates and described in Paper I of this series (1928). First 10.5 grams of casein (according to Hammarsten) are soaked in 100 cc. of water. Then 216 cc. of saturated lime water are added and the mixture is shaken until nearly all the casein is in solution. After the addition of 1.05 grams of potassium citrate the shaking is cont*ued until all the casein has dissolved. Then 10 cc. of double strength beef infusion are added and the solution made up to 500 cc. with distilled water. To this solution are added 15 cc. of a 3 per cent solution of calcium chloride and 15 cc. of a phosphate solution of ph 6.7 (2.5 grams of Na2HP04 2H20 and 2.2 grams of KH2PO4 in 100 cc. of water). The solution is then made up to 1 liter. DATA In table 1 is shown the action of the cocci and in table 2 the action of the rods in a casein medium containing 0.01 per cent glucose, 0.1 per cent peptone, and 10 cc. of beef infusion. It will be observed that not all the organisms which give an increase in amino-nitrogen in milk can break down casein in the synthetic

3 STUDIES ON PROTEOLYTIC BACTERIA OF MILK medium used. All cultures of M. perfiavus, M. varians, M. ureae, M. freudenreichii and Str. liquefaciens were non-caseolytic. It was thought that the failure of these organisms to decompose the casein might be due to the use of a faulty medium. Therefore the casein medium was varied in a number of ways-the quantity TABLE 1 Action of cocci on casein (Incubated ten days at 300C.) 0~~~~~ ORGANISM = 0 0 Ps CC. Control o _ M. citreus M. perflavus Si M. varians _ M. casei (yellow) M. percitreus SI. Si M. luteus Si M. cereus _ M. subftavescen Sl M. casei (white) S Staph. albus Si M. freudenreichii _ M. ureae _ P P t 6.2 P Str. liquefaciens _ Str. bovis SI * Increase in amino-n expressed as cubic centimeters of N/'10 NaOH per 100 cc. of medium. Sl. = slight. of casein and of glucose was varied or small quantities of sterile milk were added. Experiments were made to determine whether the addition of metals would help in the decomposition of the casein. An increase in sugar seemed to cause caseolysis by Str. liquefaciens, but none of these modifications of the medium caused any of the other non-caseolytic organisms to split the

4 190 WILLIAM C. FRAZIER AND PHILIP RUPP casein. Calcium and magnesium seemed to help to some extent the action of organisms which were able to split casein. It will be shown ini a following paper that these organisms which are _ TABLE 2 Action of rods on casein (Incubated ten days at 30 C.) ~~~~~1 E4 W 0 ~~~~105 z z _ Control _ Flavobacterium synxanthum Flavobacterium lactis SI Flavobacterium tremelloides P Achromobacter coadunatum S1. - SI Achromobacter liquefaciens SI Achromobacter delictatulum Proteus vulgaris Alcaligenes bookeri P 107 (Escherichia) Serratia ruber Serratia indica Red B. albokactis B. cereus Strain "A" B. cereus, Strain "B" B. vulgatus B. subtilis B. simplex S1. S1. S B. mesentericus SI B. cohaerens B. tumescens B. nmegatherium B. ruminatus B. macerans P P _ S * See footnote to table CC. proteolytic in milk, yet do not split casein in synthetic media, are able to break down lactalbumin. M. varians was caseolytic according to the casein agar plates

5 STUDIES ON PROTEOLYTIC BACTERIA OF MILK and according to the bromine test in milk but did not split casein in the sodium caseinate media. Therefore, the organim were inoculated into a calcium caseinate medium which was the same as the casein agar medium described in Paper I of this series except that the agar was omitted. This decidedly opalescent medium was cleared up by the M. variars cultures, and a slight positive bromine test was obtained, although the medium contained such a small quantity of casein that the increase in amino-nitrogen was almost within the limit of error of measurement. The extreme delicacy of the bromine test for free trypto- TABLE 3 Action of organi8ms on calcium caseinate medium ~OPALESCENT BH RO- TITRAT- AMINo-N ORGANISM ~ OR CLEAR ph N&MINE ABLETT (FORMOL)t O~~~ ACIADITT8 ~ ~~~~~~~TB Control... Opalescent M. varians... Clear M1. citreus... Clear 7.0 S X 3.2 Staph. albus... Clear M. perfiavus... Opalescent M. freudenreichii. Opalescent o0.7 0 Flavobacterium synxanthum... Clear Achromobacter liquefaciens... Clear B. cereus... Clear B. albolactis... Clear * Increase in acidity expressed as cc. of N/10 NaOH to neutralize 100 cc. of medium to phenolphthalein. t See footnote to table 1. phane as a test for caseolysis is shown by the fact that in this case an increase in formol titration of only 0.03 to 0.07 cc. of N/10 NaOH per 5 cc. of medium was detected in a 1 cc. sample. When the quantity of casein in the medium was trebled, a measurable increase in amino-nitrogen, as well as a distinct positive bromine test, was obtained with M. varians. The action of M. varians and of some of the other organisms on this calcium caseinate medium is shown in table 3. Some of the organisms can apparently split calcium caseinate more readily than sodium caseinate, but with most of the organisms tried there was little 191

6 192 WILLIAM C. FRAZIER AND PHILIP RUPP difference in caseolysis in the two media. The calcium caseinate medium has the advantage that, because of its opalescence, caseolysis can readily be detected by the appearance of the medium. In casein media the bromine test for free tryptophane is apparently a clear-cut and reliable test for proteolysis. As is shown in tables 1 and 2 the bromine test is positive in all cases of caseolysis except with the three cultures of M. cereus, an organism which has given very erratic results on both milk and casein. ORGANISM TABLE 4 Comparison of casein media containing various amounts of glucose NO GLUCOSE 0.01 PER CENT GLUCOSE 0.1 PER CENT GLUCOSE Ii ~~ Z Z r~ ~Z P 2 a a Control _ _ M. citreus S M. percitreus S1. S _ M.casei S O Str. liquefa.. ietns S Achromobacter coadunatum SI. - SI Proteus vulgaris SI B. albolactis B. cereus The Salkowski test for indol-acetic acid practically parallels the bromine test but does not seem quite so reliable. The test for ammonia also parallels closely the bromine test in this casein medium, which contains only 0.1 per cent glucose. The aminoph numbers are shown to correspond roughly to the respective formol titration figures. In table 4 are shown the results of the action of some of the representative cultures on casein media containing 0, 0.01 and 0.1 per cent of glucose respectively. The presence of fermentable

7 STUDIES ON PROTEOLYTIC BACTERIA OF MILK sugar up to 0.1 per cent does not seem greatly to affect the amount of proteolysis or the bromine or Salkowski test, but does nullify the ammonia test in the case of the acid cocci. In these experiments increasing quantities of glucose seemed to cut down caseolysis to some extent in the case of M. percitreus and to TABLE 5 Action of cocci on gelatin SOLUTION ~~~~* GELATIN STAB ORGANISM Z L GELATIN-AGAR PLATE IQUE 2,TCTI ~ z LIUEFACTON HgCl2 [ Tannic acid _ z._ I _ Control _ - M. citreus SI Precipitate M. perfiavus Precipitate M. varians (slow) + Precipitate M. casei (yellow) to sl. + Precipitate M. percitreus S (slow) + Precipitate M. luteus M.cereu Precipitate M. subflave8cens M. ca8ei (white) S to sl. + Precipitatle Staph. albus ls. to + + Precipitate M. freudenreichii or + + Precipitate M. ureae _ - P Ring P Precipitate Str. liquefaciens Heavy precipitate Str. bovis P o0.0 - * See footnote to table 1. t Plus marks refer to width of clear zone about colony. increase it in the case of Str. liquefaciens. It will be noted that B. albolactis and B. cereus, which resemble each other very closely except that the former ferments lactose, have practically the same caseolytic action when only small quantities of fermentable sugar are present. The organisms which are able to decompose casein with an increase in amino-nitrogen fall into two distinct groups: a high 193

8 194 WILLIAM C. FRAZIER AND PHILIP RUPP amino-nitrogen group and a low amino-nitrogen group. The "high" group might include all organisms which required 20 cc. or more of N/10 NaOH per 100 cc. of media (see tables 1 and 2) B. cereus, Strain "B" B. vulgatus. B. subtilis. B. simplex. B. mesentericus. B. cohaerens. B. tumes. B. megatherium. B. ruminatus. B. macerans. P67. P285. TABLE 6 Action of rods on gelatin INGOLTIN ORGANISM II 7.0 II 7.6 II 7.2 II GELATIN-AGAR PLATE _.. Z~ gprecip- ta Tannic acid E prciitnt ~~. ~ "4 tanttp reiptat.~~~p Control Flavobacterium synxanthum Flavobacterium lactis p Flavobacterium tremelloides : I. 4.6 p a 17.0 P I 5.E Achromobacter coadunatum Achromobacter liquefaciens ( I 14.3 p D 9.4 Achromobacter delictatulum I D) 29.4 Proteus vulgaris Alcaligenes bookeri. 8.2 I P 107 (Escherichia) I Serratia ruber. 7.2 I1 p O.C +.1- I Serratia indica B. albolactis B. cereus, Strain "A" It IL 6.5;c 23.5 A + S1. pi II III I * See footnote to table 1. t Plus marks refer to width of clear zone about colony. L Precipitate Precipitate Precipitate Precipitate Precipitate and the "low" group those organisms which required 10 cc. or less of NaOH. There were no intermediate organisms among those studied.

9 STUDIES ON PROTEOLYTIC BACTERIA OF MILK The action of the proteolytic organisms on gelatin solution, gelatin stab cultures, and gelatin agar plates is shown in tables 5 and 6. It is evident that the organisms may be subdivided into a number of different groups on the basis of their action on gelatin media. This will be discussed in a later paper on classification. As with the casein, however, the organisms may be divided into a negative group, a low amino-nitrogen group, and a high amino-nitrogen group. It is interesting to note that the lactose-fermenting cocci, M. perfiavus and M. freudenreichii, which do not split casein (table 1), also do not break down gelatin. Evidence will be presented in a following paper to show that these organisms are, however, able to decompose lactalbumin. SIUMMARY The action of 229 cultures of proteolytic bacteria from milk on casein and gelatin was studied. Four species of cocci and four of rods were found to cause no increase in amino-nitrogen in casein or gelatin media. The remaining organisms fell into either high or low amino-nitrogen groups in casein and gelatin solutions. The bromine test was found to be positive in all cases of caseolysi.r except with M. cereus. M. varians was apparently unable to split sodium caseinate but did break down calcium caseinate. Some of the other organisms seem to split calcium caseinate more readily than sodium caseinate. Increasing amounts of fermentable sugar up to 0.1 per cent did not seem to affect appreciably the amount of caseolysis by most organisms. With M. percitreus, however, the caseolysis seemed to decrease with increasing quantities of glucose. Caseolysis by the acid-forming Streptococcus liquefaciens seemed to be aided by more fermentable sugar. A study of the proteolytic milk organisms on casein and gelatin media is of value in the grouping of these organisms. 195

10 196 WILLIA C. FRAZIER AND PHILIP RUJPP REFERENCES BERMAN, N., AND RETTGER, L. F Further studies on the utilization of protein and non-protein nitrogen. Jour. Bact., 3, FRAZIER, W. C A method for the detection of changes in gelatin due to bacteria. Jour. Infectious Diseases, 39, FRAZIER, W. C., AND Rupp, P Studies on the protolytic bacteria of milk. I. A medium for the direct isolation of caseolytic milk bacteria. Jour. Bact., 16, 57. FRAZIER, W. C., AND RuPP, P Studies on the proteolytic bacteria of milk. II. Action of proteolytic bacteria of milk on milk. Jour. Bact., 16, 65. Downloaded from on October 8, 2018 by guest