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Inhibition of Streptococcus Lactis in Milk by Fatty Acids1
INHIBITION OF STREPTOCOCCUS LACTI8 IN I~[ILK BY F A T T Y ACIDS 1 R A L P H N. COSTILOW AND M A R V I N L. S P E C K
Department of Animal l~dustry, No. Carolina State College, l~ale4gl;
The inhibitory action of rancid milk on the growth of Streptococcus lactis has been confirmed by Tarassuk and Smith (8). Costilow and Speck (2) have shown recently that such milk also inhibits the growth of a number of other microorganisms. The latter workers noted that the inhibition of bacterial growth was not due to slightly lowered pH, low surface tension of rancid milk or to any activity of lipase upon the bacterial cell. The inhibition of S. lactis was related directly to the degree of lipolysis which had occurred in milk and thus probably to some component of rancid milk. Owing to the increased concentration of free fatty acids in rancid milk, attention was directed to the effect of these compounds upon the growth of 8. /act/s. METHODS
The f a t t y acids used were purchased commercially in as chemically pure a form as possible. These were dissolved in ethyl alcohol and added to homogenized milk before autoclaving. The fatty acids were prepared in a concentration so that only 1 per cent ethyl alcohol was added to the milk to be cultured. One per cent of the alcohol alone was added to the control samples to compensate for any possible effects of residual alcohol in the sterile sample. The procedures for carrying the culture of 8. /acf/s H-l-10, plating the samples and the measurement of pH and surface tension have been reported earlier (2). RESUr,TS ~
DISCUSSIOS
All of the f a t t y acids known to occur in milk fat (5) plus linolenic acid were observed for their effect upon the growth of 8. lacfis by culturing this organism in milk containing the individual acids. Lacking information on the actual concentrations of these acids present in rancid milk, an arbitrary concentration of 0.1 per cent of each acid was used unless otherwise specified. This concentration of any one acid undoubtedly exceeded that present in rancid milk, but it was expected to reveal more clearly the toxicity or non-toxicity of the individual f a t t y acids. The acids which were observed to have no effect upon 8. lactis were oleic, butyric and 0.05 per cent caproic 2 (fig. 1, 2); linoleic, linolenic and arachidic (fig. 3, 4) ; palmitie (fig. 5) ; and 0.5 per cent stearic (fig. 6, 7). Thus, neither Received for publication J u n e 13, 1951. 1 Approved for publication as P a p e r no. 390 in the J o u r n a l Series of the N o r t h Carolina Agricultural Experiment Station. As 0.075 per cent caproi¢ acid caused coagulation of the m~lk upon autoclaving, the coneentration was reduced. Butyric aeld caused coagulation a t the 0.05 per cent level, but~, since i t is a water-soluble acid, a sterile solutinn was added aseptically to the sterilized milk. 1104:
1105
S. LACTIS INHIBITION
I 0
4
8
1Z
16 20 24 Z8 TIME IN HOURS
32
36
40
Fie. 1. The growth of S. lao~is in milk containing oleic acid (0.1%), butyric acid (0.1%)~ and ¢aproic aaid (0.05%).
the low-molecular-weight fatty acids (butyric and caproic) nor the acids of higher molecular weights (patmitic, stearie, oleic, linoleic and linolenic) showed any inhibitory effect. In contrast, the intermediate acids, caprylic, capric and lauric, inhibited the growth of S. tactis (fig. 5). The inhibitory effect of myristic acid was somewhat doubtful even at the 0.5 per cent level (fig. 6, 7). As the concentrations of capric and lauric acids were increased, their respective inhibitory effects also increased (fig. 6, 7). A concentration of 0.5 per cent capric acid almost completely inhibited growth of S. lactis, and the count actually
6.4~0 ~
6.Z
6.0
' RMALMILKI
5.8 5.6 pH 5.4
~
~
5.2 5.0 4.8
4.6 --4.4 0
4
8
12
16
20
24
Z8
32
36
40
T I M E IN H O U R S
l~I~. 2. The acid production by S. lac~is in milk containing oleic acid (0.1%), butyrle acid (0.1%) and caproic acid (0.05%).
1106
RALPH N. COSTII~W" AND MARVIN L. SPECK
9.$
I~Acmmc~]~'os.~..t.
mi..x ]
9.0
I!I
cl 8.5
o
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FIG. 3. acids.
4
|
8 12
i 16
.
i
,
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24 Z8 32 TIMEINHOURS ZO
I 36
t 40
The growth of 8. hwt/s in milk eontaln~ng 0.1% linoleie, linolenic and axaehldic
declined after 5 days; thi~ sample failed to coagulate on continued incubation. A concentration of 0.5 per cent laurie acid delayed the development of the culture almost entirely for 11 hr., after which time growth proceeded fairly rapidly, and the milk coagulated within 36 hr. Various reports dealing with the effects of fatty acids on the growth of different lactic acid bacteria in synthetic or semi-synthetic media have demonstrated toxicity of unsaturated fatty acids. However, the presence of lecithin, cholesterol or serum albumin eliminated the toxicity (3, 6, 9), and addition of oleic 64 ~
~Lxj
6"2I ~ 6.0
5.8 pH 5.6 5.4
44
•
0
J
4
8
1Z
16
~ O . 4. The acid production by B. ~ t ~ araehidie acids.
:~0
Z4
'
~
'
28
3Z
36
TIMEINHOURS
40
in milk eontsL~ug 0.1% ]inolei~, linolenic and.
1107
S. LACTIS INHIBITION
9.~
_=
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/.I ,,
F i
//
...t"
,
///
./ ....
t ///....-'0
4
8
1z
./ 16
zo 24 z8 TIME IN HOURS
32
36
40
~IO. 5. The growth of 8. lo~t~s in milk containing 0.1% of palmitie, laurie, eaprie and caprylie acids.
acid in a combined form was stimulatdry (1) or essential for growth in the absence of biotin (10). As milk contains cholesterol and lecithin, the observations that oleic, linoleic and linolenic acids were non-inhibitory for S. lacffs in milk were anticipated. There appears to be: a critical relationship between the carbon chain length and the degree of the inhibitory property. Thus, capric acid was the most inhibitory, caprylie acid next and laurie acid the least inhibitory of the three. Hardwick et aL (4) noted a similar relationship in the carbon chain length of
9.5~
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~
7 . 0 ~ = " - - ' ~ •. . . . . I 0 4 8
IN° ~ M ~
~ lZ
T 16
1 zo
i z4
M!L~I
~ ;'8
i 3z
-T 36
40
TIME IN HOURS
I~IG. 6. The growth of 8. lactis in milk containing 0.5% stearie, myristi¢, laurie and eapri¢ acids.
1108
RALPH N. COSTII~W A N D
L, SPECK
MARVIN
pH ;
•
"-%.
~,
s.~, ~'~ - I " ~ T ' ~
I
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I
0
4
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I
I
12
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I
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24 TIME IN HOURS 16
ZO
!
28
I
32
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36
I
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1~(}. 7. The acid productio,, b y ~g. ~a~t~s in milk contaJn;n~ 0.5% of stearie, myristic~ laurie and eapric acids.
fatty acids to their antisporulation and growth inhibition activity on certain bacteria, with the Clo to C14 fatty acids having the greatest effect. Salts of capric, laurie and myristie acids also have been found to be toxic to resting cells of Esche~ch~ coli (7). This toxicity for E. coil was considered to be the result of the accumulation of these diffnsable compounds within the cell resulting from the inability of the organism to use them anaerobically. Cells stored aerobically were unaffected. Tarrasuk and Smith (8) also noted the inhibition by laurie 51 50 49 48
~: 46 4s z 44
~-- . . . . . . .-~,..
43
/
4z 40
. . . . . . . . .-~"
39 ~: 3s 36 35
~ ' ~
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,
' '|
I
I
0 l
4
8
12
~ RANCID MILK l
.
[SURFACETENSION ADJUSTEDJ
I
I
I
I
16 20 24 28 T I M E IN H O U R S
I 32
I
.36
!
40
FIo. 8 The surface tension of cultures of 8. ~ct{~ i n normal mH~ rancid miIk~ normal milk confabbing " T w e e n " 40 a n d normal m~|k containing various f a t t y acids.
S. LACTIS II~HIBITIOI~
1109
acid and diglycol laurate of S. lactis in milk and attributed the inhibitory prope r t y of these compounds to their depression of the surface tension. I n the present study surface tension of the milk containing the f a t t y acids failed to correlate with the degree of inhibition of the growth of S. lactis (fig. 8). I n addition to data for milk cultures containing various f a t t y acids, results also are included for cultures in normal milk with the surface tension lowered with " T w e e n " 40, in rancid milk and in normal milk. Previous work (2) had shown that rancid milk was inhibitory to S. lactis, while normal milk with its surface tension lowered with " T w e e n " 40 to the level of that of the rancid milk was not inhibitory. F u r t h e r evidence of the independence of inhibition from surface tension can be noted f r o m the cultures containing f a t t y acids. Milk containing 0.1 per cent oleic acid, which showed no inhibition, had a surface tension as low or lower t h a n milk containing the same concentration of laurie or caprie acid, each of which showed marked inhibition of S. lactis. Milk containing 0.1 per cent caprylic acid showed definite toxicity, whereas the surface tension of this milk was e~sentially the same as normal milk. These and similar observations indicate that the inhibition of S. fact@ in milk by f a t t y acids is caused by some factor(s) other than the surface tension of the medium. SUGARY I n an effort to determine the cause of the inhibitory p r o p e r t y of rancid milk on S. lactis all of the f a t t y acids common to milk fat were tested for their effect upon the growth of this organism. Oaprylic, capric and laurie acids, inhibited growth of S. lactis; the degree of inhibition increased as the concentration of the acid increased. The effect of myristie acid was doubtful, and none of the other acids tested was f o u n d to inhibit the growth of this organism. The inhibitory p r o p e r t y of rancid milk and that observed in milk containing f a t t y acids is not the result of the low surface tension of the milk, but is due to some other unexplained toxic effect of the individual f a t t y acids.
(1)
(2) (3) (4) (5) (6) (7)
REFERENCES COLLINS,E. B., NELS01~, F. E.~ AND PAR~E~, C.E. Acetate and Oleate Requirements of the Lactic Group of Streptococci. J. Beet., 59: 69-74. 1950. CosITLow,R. N., AND SP]~C~r~M.L. Inhibitory Effect of Rancid Milk on Certain Bacteria. J. Dairy Sci., 34" 1104--1110. 1951. DAVIS, B. D., AND DUB0S, R. J. The Inhibitory Effect of Lipase on Bacterial Growth in Media Containing Fatty Acid Esters. J. Bact., 55: 11-23. 1948. HARDWICK,W, A., GUIaARD,B., AI~DFOSTER,J.W. Antisporulation Factors in Complex Organic ~/[edia. IL Saturated Fatty Acids as Antisporulation Factors. J. Baet., 61: 145-151. 1951. HrODITOH~T. P. The Gh~nival Gonst~t~tion of !~a~uraZ F ~ . 2n~ ed. John Wiley and Sons, New York. pp. 119-123. 1947. KODIC~.K,E., ~ D WORV~N, A. N. The Effect of Unsaturated Fatty Acids on La~obacit~s helw$~ou~ and other Gram-positive Micro-organisms. Bioehem.J., 39: 78-85. 1945. RITTENBERG,S. C., SILLIKER~J. Ho, AN]) WARD, M. E. Anaerobic Toxicity of Certain Fatty Acids for J~svhe~,~vh~a~o~. Beet. Proc., p. 145. 1950.
1110
RALPH N. COSTILOW AND MARVIN L. SPECK
(8) T~ASSUK, 1~. P., ~ SMITH, F . R . Relation of Surface Tension of Rancid Milk to its Inhibitory Effect on the Growth and Acid Fermentation of Streptococcus lact~. J. Dairy Sci., 23: 116~-1170. 1940. (9) WILLIAm,S, V. R., ~ m ~CEe~a, E . A . Further Studies on Lipide Stimulation of Lavtoba~llus casei. J. Biol. Chem. 170: 399-411. 1947. (10) WZLLIA~s, W. L., B~0QUIST, H. P., AND SNELL, E. E. Olei¢ Acid and Related Compounds as Growth Factors for Lactic Acid Bacteria. J. Biol. Chem., 170: 619--630. 1947.
Department of Animal l~dustry, No. Carolina State College, l~ale4gl;
The inhibitory action of rancid milk on the growth of Streptococcus lactis has been confirmed by Tarassuk and Smith (8). Costilow and Speck (2) have shown recently that such milk also inhibits the growth of a number of other microorganisms. The latter workers noted that the inhibition of bacterial growth was not due to slightly lowered pH, low surface tension of rancid milk or to any activity of lipase upon the bacterial cell. The inhibition of S. lactis was related directly to the degree of lipolysis which had occurred in milk and thus probably to some component of rancid milk. Owing to the increased concentration of free fatty acids in rancid milk, attention was directed to the effect of these compounds upon the growth of 8. /act/s. METHODS
The f a t t y acids used were purchased commercially in as chemically pure a form as possible. These were dissolved in ethyl alcohol and added to homogenized milk before autoclaving. The fatty acids were prepared in a concentration so that only 1 per cent ethyl alcohol was added to the milk to be cultured. One per cent of the alcohol alone was added to the control samples to compensate for any possible effects of residual alcohol in the sterile sample. The procedures for carrying the culture of 8. /acf/s H-l-10, plating the samples and the measurement of pH and surface tension have been reported earlier (2). RESUr,TS ~
DISCUSSIOS
All of the f a t t y acids known to occur in milk fat (5) plus linolenic acid were observed for their effect upon the growth of 8. lacfis by culturing this organism in milk containing the individual acids. Lacking information on the actual concentrations of these acids present in rancid milk, an arbitrary concentration of 0.1 per cent of each acid was used unless otherwise specified. This concentration of any one acid undoubtedly exceeded that present in rancid milk, but it was expected to reveal more clearly the toxicity or non-toxicity of the individual f a t t y acids. The acids which were observed to have no effect upon 8. lactis were oleic, butyric and 0.05 per cent caproic 2 (fig. 1, 2); linoleic, linolenic and arachidic (fig. 3, 4) ; palmitie (fig. 5) ; and 0.5 per cent stearic (fig. 6, 7). Thus, neither Received for publication J u n e 13, 1951. 1 Approved for publication as P a p e r no. 390 in the J o u r n a l Series of the N o r t h Carolina Agricultural Experiment Station. As 0.075 per cent caproi¢ acid caused coagulation of the m~lk upon autoclaving, the coneentration was reduced. Butyric aeld caused coagulation a t the 0.05 per cent level, but~, since i t is a water-soluble acid, a sterile solutinn was added aseptically to the sterilized milk. 1104:
1105
S. LACTIS INHIBITION
I 0
4
8
1Z
16 20 24 Z8 TIME IN HOURS
32
36
40
Fie. 1. The growth of S. lao~is in milk containing oleic acid (0.1%), butyric acid (0.1%)~ and ¢aproic aaid (0.05%).
the low-molecular-weight fatty acids (butyric and caproic) nor the acids of higher molecular weights (patmitic, stearie, oleic, linoleic and linolenic) showed any inhibitory effect. In contrast, the intermediate acids, caprylic, capric and lauric, inhibited the growth of S. tactis (fig. 5). The inhibitory effect of myristic acid was somewhat doubtful even at the 0.5 per cent level (fig. 6, 7). As the concentrations of capric and lauric acids were increased, their respective inhibitory effects also increased (fig. 6, 7). A concentration of 0.5 per cent capric acid almost completely inhibited growth of S. lactis, and the count actually
6.4~0 ~
6.Z
6.0
' RMALMILKI
5.8 5.6 pH 5.4
~
~
5.2 5.0 4.8
4.6 --4.4 0
4
8
12
16
20
24
Z8
32
36
40
T I M E IN H O U R S
l~I~. 2. The acid production by S. lac~is in milk containing oleic acid (0.1%), butyrle acid (0.1%) and caproic acid (0.05%).
1106
RALPH N. COSTII~W" AND MARVIN L. SPECK
9.$
I~Acmmc~]~'os.~..t.
mi..x ]
9.0
I!I
cl 8.5
o
O
8.0
o
,J
7.~ T.~e 0 I
FIG. 3. acids.
4
|
8 12
i 16
.
i
,
i
24 Z8 32 TIMEINHOURS ZO
I 36
t 40
The growth of 8. hwt/s in milk eontaln~ng 0.1% linoleie, linolenic and axaehldic
declined after 5 days; thi~ sample failed to coagulate on continued incubation. A concentration of 0.5 per cent laurie acid delayed the development of the culture almost entirely for 11 hr., after which time growth proceeded fairly rapidly, and the milk coagulated within 36 hr. Various reports dealing with the effects of fatty acids on the growth of different lactic acid bacteria in synthetic or semi-synthetic media have demonstrated toxicity of unsaturated fatty acids. However, the presence of lecithin, cholesterol or serum albumin eliminated the toxicity (3, 6, 9), and addition of oleic 64 ~
~Lxj
6"2I ~ 6.0
5.8 pH 5.6 5.4
44
•
0
J
4
8
1Z
16
~ O . 4. The acid production by B. ~ t ~ araehidie acids.
:~0
Z4
'
~
'
28
3Z
36
TIMEINHOURS
40
in milk eontsL~ug 0.1% ]inolei~, linolenic and.
1107
S. LACTIS INHIBITION
9.~
_=
t "o
/.I ,,
F i
//
...t"
,
///
./ ....
t ///....-'0
4
8
1z
./ 16
zo 24 z8 TIME IN HOURS
32
36
40
~IO. 5. The growth of 8. lo~t~s in milk containing 0.1% of palmitie, laurie, eaprie and caprylie acids.
acid in a combined form was stimulatdry (1) or essential for growth in the absence of biotin (10). As milk contains cholesterol and lecithin, the observations that oleic, linoleic and linolenic acids were non-inhibitory for S. lacffs in milk were anticipated. There appears to be: a critical relationship between the carbon chain length and the degree of the inhibitory property. Thus, capric acid was the most inhibitory, caprylie acid next and laurie acid the least inhibitory of the three. Hardwick et aL (4) noted a similar relationship in the carbon chain length of
9.5~
_
~
7 . 0 ~ = " - - ' ~ •. . . . . I 0 4 8
IN° ~ M ~
~ lZ
T 16
1 zo
i z4
M!L~I
~ ;'8
i 3z
-T 36
40
TIME IN HOURS
I~IG. 6. The growth of 8. lactis in milk containing 0.5% stearie, myristi¢, laurie and eapri¢ acids.
1108
RALPH N. COSTII~W A N D
L, SPECK
MARVIN
pH ;
•
"-%.
~,
s.~, ~'~ - I " ~ T ' ~
I
5.0
"....."-4 "~..~
"--..
4.6 4.4
I
0
4
I, 8
I
I
12
J
I
I
24 TIME IN HOURS 16
ZO
!
28
I
32
I~
36
I
40
1~(}. 7. The acid productio,, b y ~g. ~a~t~s in milk contaJn;n~ 0.5% of stearie, myristic~ laurie and eapric acids.
fatty acids to their antisporulation and growth inhibition activity on certain bacteria, with the Clo to C14 fatty acids having the greatest effect. Salts of capric, laurie and myristie acids also have been found to be toxic to resting cells of Esche~ch~ coli (7). This toxicity for E. coil was considered to be the result of the accumulation of these diffnsable compounds within the cell resulting from the inability of the organism to use them anaerobically. Cells stored aerobically were unaffected. Tarrasuk and Smith (8) also noted the inhibition by laurie 51 50 49 48
~: 46 4s z 44
~-- . . . . . . .-~,..
43
/
4z 40
. . . . . . . . .-~"
39 ~: 3s 36 35
~ ' ~
°~''qq''C
,
' '|
I
I
0 l
4
8
12
~ RANCID MILK l
.
[SURFACETENSION ADJUSTEDJ
I
I
I
I
16 20 24 28 T I M E IN H O U R S
I 32
I
.36
!
40
FIo. 8 The surface tension of cultures of 8. ~ct{~ i n normal mH~ rancid miIk~ normal milk confabbing " T w e e n " 40 a n d normal m~|k containing various f a t t y acids.
S. LACTIS II~HIBITIOI~
1109
acid and diglycol laurate of S. lactis in milk and attributed the inhibitory prope r t y of these compounds to their depression of the surface tension. I n the present study surface tension of the milk containing the f a t t y acids failed to correlate with the degree of inhibition of the growth of S. lactis (fig. 8). I n addition to data for milk cultures containing various f a t t y acids, results also are included for cultures in normal milk with the surface tension lowered with " T w e e n " 40, in rancid milk and in normal milk. Previous work (2) had shown that rancid milk was inhibitory to S. lactis, while normal milk with its surface tension lowered with " T w e e n " 40 to the level of that of the rancid milk was not inhibitory. F u r t h e r evidence of the independence of inhibition from surface tension can be noted f r o m the cultures containing f a t t y acids. Milk containing 0.1 per cent oleic acid, which showed no inhibition, had a surface tension as low or lower t h a n milk containing the same concentration of laurie or caprie acid, each of which showed marked inhibition of S. lactis. Milk containing 0.1 per cent caprylic acid showed definite toxicity, whereas the surface tension of this milk was e~sentially the same as normal milk. These and similar observations indicate that the inhibition of S. fact@ in milk by f a t t y acids is caused by some factor(s) other than the surface tension of the medium. SUGARY I n an effort to determine the cause of the inhibitory p r o p e r t y of rancid milk on S. lactis all of the f a t t y acids common to milk fat were tested for their effect upon the growth of this organism. Oaprylic, capric and laurie acids, inhibited growth of S. lactis; the degree of inhibition increased as the concentration of the acid increased. The effect of myristie acid was doubtful, and none of the other acids tested was f o u n d to inhibit the growth of this organism. The inhibitory p r o p e r t y of rancid milk and that observed in milk containing f a t t y acids is not the result of the low surface tension of the milk, but is due to some other unexplained toxic effect of the individual f a t t y acids.
(1)
(2) (3) (4) (5) (6) (7)
REFERENCES COLLINS,E. B., NELS01~, F. E.~ AND PAR~E~, C.E. Acetate and Oleate Requirements of the Lactic Group of Streptococci. J. Beet., 59: 69-74. 1950. CosITLow,R. N., AND SP]~C~r~M.L. Inhibitory Effect of Rancid Milk on Certain Bacteria. J. Dairy Sci., 34" 1104--1110. 1951. DAVIS, B. D., AND DUB0S, R. J. The Inhibitory Effect of Lipase on Bacterial Growth in Media Containing Fatty Acid Esters. J. Bact., 55: 11-23. 1948. HARDWICK,W, A., GUIaARD,B., AI~DFOSTER,J.W. Antisporulation Factors in Complex Organic ~/[edia. IL Saturated Fatty Acids as Antisporulation Factors. J. Baet., 61: 145-151. 1951. HrODITOH~T. P. The Gh~nival Gonst~t~tion of !~a~uraZ F ~ . 2n~ ed. John Wiley and Sons, New York. pp. 119-123. 1947. KODIC~.K,E., ~ D WORV~N, A. N. The Effect of Unsaturated Fatty Acids on La~obacit~s helw$~ou~ and other Gram-positive Micro-organisms. Bioehem.J., 39: 78-85. 1945. RITTENBERG,S. C., SILLIKER~J. Ho, AN]) WARD, M. E. Anaerobic Toxicity of Certain Fatty Acids for J~svhe~,~vh~a~o~. Beet. Proc., p. 145. 1950.
1110
RALPH N. COSTILOW AND MARVIN L. SPECK
(8) T~ASSUK, 1~. P., ~ SMITH, F . R . Relation of Surface Tension of Rancid Milk to its Inhibitory Effect on the Growth and Acid Fermentation of Streptococcus lact~. J. Dairy Sci., 23: 116~-1170. 1940. (9) WILLIAm,S, V. R., ~ m ~CEe~a, E . A . Further Studies on Lipide Stimulation of Lavtoba~llus casei. J. Biol. Chem. 170: 399-411. 1947. (10) WZLLIA~s, W. L., B~0QUIST, H. P., AND SNELL, E. E. Olei¢ Acid and Related Compounds as Growth Factors for Lactic Acid Bacteria. J. Biol. Chem., 170: 619--630. 1947.