RESEARCH
PAPERS
Influence of Consuming Nonfermented Milk Containing Fecal F l o r a o f H e a l t h y Males I
Lactobacillus acidophilus o n
S. E. GILLILAND 2 , M. L. SPECK, G. F. NAUYOK, JR., and F. G. GIESBRECHT Departments of Food Science & Statistics N. C. State University Raleigh 27607 ABSTRACT
ences 22, 23). Studies have shown that Lactobacillus acidophilus included in the diet will become established in the intestinal tract of man (11, 19, 20, 21, 24). Related investigations have established that L. acidopbilus produces antagonistic actions toward undesirable bacteria whose growth or actions in the intestinal tract can produce various forms of gastroenteritis (9, 25, 26). For many years, L. acidophilus was available to the consumer in the form of cultured acidophilus milk. This product, however, had an unpleasant taste and was not accepted well. Furthermore, the culture lost viability rapidly when the product was stored under refrigeration (12, 13). Myers (19) described an "unfermented acidophilus milk" which had the taste of fresh whole milk. The product had high numbers of viable L. acidopbilus suspended in it. Consumption of the milk (.95 liter/day) resulted in the implantation of L. acidopbilus in the human intestinal tract within 2 days. Duggan et al. (2) described the preparation of a frozen concentrated culture of L. acidopbilus which contained the "daily requirement" of viable cells stored in 5- or 10-ml quantities. They recommended that the concentrate be thawed and added to .47 or .95 liters of whole milk before consumption. They suggested that the culture could be taken with a glass of milk but stressed the need for consuming .95 liters of milk daily. Results showing the effect on the intestinal flora of consuming such a culture in nonfermented milk were not given. Development of procedures for producing frozen concentrated cultures (3) have made it possible to prepare milk commercially to which large numbers of viable cells of L. acidopbilus have been added, and the product can be distributed through conventional channels for pasteurized milk. Most research done on the effect(s) of consuming L. acidopbilus has involved the use
A milk beverage was prepared by adding a commercially available concentrated culture of Lactobacillus acidopbilus to pasteurized low-fat milk. The culture did not alter the flavor of the milk. Consumption of the milk by healthy adult men increased facultative lactobacilli in their feces. No significant effects were observed on numbers of coliforms or anaerobic lactobacilli including bifidobacteria. After consumption of the milk was stopped, the numbers of facultative lactobacilli in the feces decreased. However, in some cases the test subjects retained considerably higher numbers than they had prior to consuming the milk containing Lactobacillus acidopbilus. This was particularly true for those whose feces contained small numbers of the facultative lactobacilli before the feeding periods. INTRODUCTION
Eli Metchnikoff suggested in 1908 that man should consume milk fermented with lactobacilli capable of living in the intestines (16). This was to control microbes in the intestine that might produce a toxic effect on the host. Since then, much research has shown the importance of lactobacilli in maintaining a balanced intestinal flora (for reviews see refer-
Received May 24, 1977. 1Paper number 5154 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina. The u s e o f trade n a m e s in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the products named nor criticism of similar ones not mentioned. 2Department of Animal Science, Oklahoma State University, Stillwater 74074. 1978 J Dairy Sci 61:1--10
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GILLILAND ET AL.
of fermented L. acidopbilus milk. In the one study (19) of nonfermented milk, the product contained high numbers ( * 3 x 108/ml of added cells of L. acidopbilus), and the test subjects consumed .95 liters per day. The objective of this study was to determine the effect of consuming nonfermented milk containing different concentrations of L. acidophilus on the number of fecal lactobacilli in healthy men. The influence on coliforms also was determined.
M A T E R I A L S A N D METHODS Source and Maintenance of Cultures
Frozen concentrated cultures (125 ml cans) of L. acidopbilus were obtained from the Marschall Division of Miles Laboratories (Madison, WI). The frozen concentrates were shipped to us in dry ice and upon receipt were stored in liquid nitrogen until needed. The fermentation pattern and DNA base composition were determined as described by Gilliland et al. (4). Preparation of Milk
Each frozen concentrated culture was thawed by submerging the can in approximately 6 liters of tap water at about 30 C until thawing was complete. The exterior of the can was sanitized with ethanol prior to opening. Nonfermented milk containing cells of L. acidopbilus was prepared by adding the required amount of thawed concentrated culture to cold, pasteurized low-fat milk. The milk was stored at 5 C and was analyzed by plating on lactobacillus selection (LBS) agar (BBL) and on LBS agar plus .15% crude sodium taurocholate (LBSB) obtained from Sigma Chemical Company. The plates were incubated in CO2 at 37 C for 72 h (4), and the colonies on the two m e d i a were recorded as LBS-CO2 and LBSB-CO2 counts. Fresh milk for the feeding trials was prepared weekly. Feeding Trials
Three feeding trials were performed to determine the influence of consuming nonfermented milk containing L. acidopbilus on the fecal flora of healthy adult men. The test subjects ranged in age from 22 to 60 and were not e x p e r i e n c i n g gastrointestinal disturbances. Journal of Dairy Science Vol. 61, No. 1, 1978
None of the subjects had consumed L. acidophilus prior to the study. No attempt was made to control their diet .other than consumption of the milk during the experimental period. In the first feeding trial the test subjects were divided randomly into three groups (four men/group). Each person was instructed to consume .47 liters of the low-fat milk daily during the test feeding period. The L. acidopbilus in the milk for one group was 2 × 107/ml. A second group received milk containing 8 × 108/ml. The milk for the third group was uninoculated (control). Fecal samples were collected for microbial analyses from each test subject O, 6, and 11 days before the feeding period. Additional samples were collected on days 8, 14, 35, and 51 during the period the milk was consumed. A final sample was obtained from each subject 13 days after the milk feeding was stopped. The second feeding trial, which also included three groups (4 to 5 men/group), was conducted in a similar manner except that the feeding period was reduced to 23 days. Fecal samples were collected 0, 6, and 11 days before feeding, on days 8, 16, and 23 during feeding, and on the l l t h day after feeding was stopped. One group of five men was used in the third feeding trial. During the test feeding period all five test subjects consumed milk containing 5 × 106 L. acidopbilus/ml (.47 liters daily). Fecal samples for microbial analyses were collected from each test subject 0, 3, 7, and 10 days before consuming the milk and on days 7, 11, 14, and 18 during the period the milk was consumed. Additional samples were collected from each 7, 11, 14, and 17 days after consumption of the milk was stopped. Microbial Analyses of Fecal Samples
Stool samples were collected by the test subjects in unused 227 g plastic cottage cheese cartons and stored at 4 C for no longer than 2 h prior to analysis. Serial dilutions were prepared with 99-ml dilution blanks containing 1% peptone. The initial dilution (1:100) was prepared by adding 1 g (wet wt) of feces to a 99-ml dilution blank. The 1-g portion was removed from the interior of the stool with a sterile wooden tongue depressor. All plates containing the required dilutions were poured with the desired agar media. After solidification, an
L. acidopbilus EFFECTS ON FECAL FLORA
3
colonies on LBSB-CO2 (before, during, and after feeding) were picked into LBS broth (BBL). The tubes of broth were incubated at 37 C in CO2 following the procedure used for the LBS-CO2 and LBSB-CO2 plates. The isolates were subcultured in lactobacilli MRS broth (Difco) at 37 C, then Gram stained, and checked for catalase and for the ability to grow at 15 C as described (5).
overlay using the same medium was added to each plate. Coliforms were enumerated on violet red bile agar (VRBA) (BBL). The plates were incubated 24 h at 37 C. Total facultative lactobacilli (LBS-CO2) and bile-resistant facultative lactobacilli (LBSBCO2 ) were enumerated by procedures described for analysis of the milk containing L. acidophilus. The samples also were plated on LBS agar and incubated anaerobically in a GasPak system (LBS-GP counts); bifidobacteria and anaerobic lactobacilli were the predominant colonies developing under these conditions (4). Colonies for all types of counts were counted with the aid of a Quebec colony counter and the results reported as toglo of count per gram wet weight of feces.
R ESU LTS
Statistical Analyses
The data from each feeding trial were analyzed separately. To eliminate the effects of any outliers or spurious counts, the data from each type of microbial count were screened by fitting a linear model incorporating effects due to individual test subjects and feeding regimen to all the data by least squares (7, 8). All observations (counts) having obsolute normed residuals (deviations from the fitted model) larger than 2 were rejected. The remaining observations then were subjected to a standard least square analysis for estimates of effects due to consumption of milk and to individual test subjects. The screening procedure provided protection against possible spurious bacterial counts at the cost of a slight increase in the variance of the estimates in cases of no outliers. Isolation of Predominating Lactobacilli
In the third feeding trial predominating
The L. acidophilus culture in this study was a catalase negative, spore-forming, straight, Gram-positive rod. It fermented amygdalin, cellobiose, dextrose, galactose, lactose, maltose, mannose, melebiose, raffinose, salicin, sucrose, and trehalose but not arabinose, inositol, mannitol, melezitose, rhamnose, sorbitol, or xylose. Esculin was hydrolyzed, and ammonia was not produced from arginine. It grew at 45 C but not at 15 C. The DNA contained 38% guanine plus cytosine. These characteristics are consistent with those of L. acidopbilus as described in (1). The LBS-CO2 and LBSB-CO2 counts of the nonfermented low-fat milk containing L. acidophilus remained unchanged during the periods that the product was used in the feeding trials. The milk to which the two amounts of L. acidophilus had been added possessed no discernable off flavor as determined by trained taste specialists. Milk containing 8.1 × 10S/ml had to be stored at about 1 C to prevent acid development. The lower counts presented no problem with acid development under normal refrigeration. Microbiological analyses of the fecal samples prior to the feeding period from all test subjects and in all trials revealed wide variation in the numbers of the types (Table 1). The widest range (2.28 to 8.19/g) was for the facultative
TABLE 1. Variability of the fecal floral from 25 test subjects prior to consuming milk containingLactobacillus
acidopbilus. L°gl o of count/g Average
Type count
Medium
Rangea
Coliform Facultative lactobacilli Anaerobic lactobacilli plus bifidobacteria
VRBA LBS-CO 2 LBS--GP
4.80 - 8.53 2.28 - 8.19 4.18 -- 8.86
6.79 5.27 7.56
aEach count in the ranges represents the average logI 0 count/g obtained by examining three samples from one test subject. Journal of Dairy Science Vol. 61, No. 1, 1978
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GILLILAND ET AL.
lactobacilli. The anaerobic lactobacilli plus bifidobacteria generally were in highest numbers followed, in order, by coliforms and facultative lactobacilli. The numbers of coliforms for the test subjects who consumed milk containing the high L. acidopbilus decreased during the feeding period although the average decrease was not significant (Table 2). The variation among individuals was significant in two of the three groups. The numbers of anaerobic lactobacilli plus bifidobacteria were not affected significantly by milk containing either concentration of L. acidopbilus (Table 3). Some individual test subjects did exhibit considerable increases (I-G and I-L). However, these subjects had fairly few bacteria of this group initially as compared to the majority of the other subjects. One subject (I-F) exhibited a considerable decrease. Test subjects in both groups consuming the L. acidopbilus exhibited highly significant increases in the numbers of facultative lactobacilli in their feces (Table 4). While the variations among test subjects were highly significant for all three groups, the counts for each subject in the control group remaiaed fairly constant before, during, and after the feeding period. The test subjects having low counts before the feeding period (I-F and l-A) exhibited the greatest increases during the period they consumed the milk containing L. acidopbilus. The numbers of facultative lactobacilli decreased after the milk feeding was stopped. However, most of the test subjects who had received L. acidopbilus in the milk retained considerably higher counts than they had before feedings. The results from the second feeding trial, which was conducted in a manner similar to the first one, were essentially the same as those in the first one. Again, the increases in the numbers of facultative lactobacilli in the feces of test subjects who consumed both concentrations of L. acidopbilus were highly significant. No changes were significant for the other groups of microorganisms. In the third feeding trial, the test subjects consumed milk containing less L. acidopbilus (5 × 106/ml) than was consumed in the first two trials. Consumption of the milk had no significant effect on the numbers of coliforms or anaerobic lactobacilli. The numbers of facultative lactobacilli increased significantly as a Journal of Dairy Science Vol. 61, No, 1, 1978
result of the milk containing the lower L.
acidopbilus (Table 5). The increase in the numbers of bile resistant facultative lactobacilli (LBSB-CO2) was even more significant (Table 6). Again, increases were greater in those test subjects who had small populations of the facultative lactobacilli prior to feeding. The predominating colonies isolated from LBSB-CO2 plates in the third feeding trials were all catalase-negative, Gram-positive, rodshaped bacteria. All those isolated prior to the feeding period grew at 15 C (Table 7). This indicates that they were not L. acidopbilus. During the period the milk was consumed, 83% of the isolates of lactobacilli failed to grow at 15 C suggesting that there was a definite shift in the predominating type of lactobacillus. Presumably the shift was due to increased numbers of L. acidopbilus. The numbers failing to grow at 15 C decreased after feeding was stopped. DISCUSSION
Some variation in bacterial counts of fecal samples may have resulted from comparing the counts on a wet rather than dry basis. However, it is highly unlikely that this would have had much effect on the statistically significant increases in numbers of facultative lactobacilli resulting from the consumption of milk containing L. acidopbilus. The average increases in numbers of facultative lactobacilli ranged from about 10 to 1000 fold (Tables 4, 5, 6), which would not have been affected by the differences in dry weight of the samples of feces. On the other hand, conclusions might have been different concerning the effect of consuming L. acidopbitus on the numbers of coliforms and anaerobic lactobacilli had counts been compared on dry weight bases. The observed numerical relationships of the groups of bacteria enumerated in the fecal flora agree with (5, 15, 17). Gorbach et al. (5) also observed significant variation among test subjects in numbers of various types of fecal bacteria. In our study, higher counts were obtained routinely when LBS agar was incubated anaerobically (GasPak) than when plates of the same medium were under CO2. While LBS agar is highly selective for enumerating lactobacilli in feces, we have shown (4) that incubation in GasPak system resulted in higher counts than incubation in a CO2-enriched
TABLE 2. Effect of consuming n o n f e r m e n t e d low-fat milk containing Lactobacillus acidopbilus on coliforms in feces of healthy m e n (Trial 1). Probability of observing larger differences b y chance Population of
L. acidopbilus in milk 0/ml (control group)
Sampling period
Test subject
Before
During
After
I-D I--E I--I I--K Average
5.3 a 6.8 7.0 6.9 6.5
4.8 b 7.3 7.1 6.6 6.3
4.5 c 7.5 6.9 7.2 6.5
I--B I--F I--G l-J Average
6.7 7.0 6.4 5.7 6.8
7.2 6.4 6.7 7.3 6.9
5.6 4.0 5.8 7.5 5.5
I--A I-C I--H I--L Average
8.5 6.8 8.4 8.2 8.0
7.7 6.6 8.3 7.2 7.5
8.7 6.5 8,0 5.9 7.3
Among test subjects
As result o f drinking t h e milk
.0004 .52 r3
2 X 107/ml (low group)
=
8 X 108/ml (high group)
aAverage log, 0 of c o u n t / g of three samples per test subject taken before c o n s u m i n g milk. <
bAverage log, 0 of c o u n t / g of four samples per test subject taken during period in which milk was consumed. CL°ga 0 of c o u n t / g for one sample per test subject taken 10 days after milk c o n s u m p t i o n stopped.
Z
o
7~
c/)
O Z .76 .10
(3 > Q
.0006 .10
O, e-
.q'
TABLE 3. Effect of c o n s u m i n g n o n f e r m e n t e d milk containing Lactobacillus acidopbilus on anaerobic lactobacilli plus bifidobacteria in feces of healthy m e n (Trial 1).
o=
Probability o f observing larger differences by chance
< o Population of
z o
L. acidopbilus in milk
O/ml (control group)
2 X 107/ml (low group)
8 X 10 s / m l (high group)
Test subject
Before
Sampling period During
After
I--D I--E I--1 I--K Average
8.2 a 8.4 8.6 5.9 8.5
8.2 b 8.0 8.2 7.3 8.3
7.7 c 8.1 7.3 6.1 8.0
1-B I--F
7.9 8.8
8.0 7.0
I--G
5.4
7.0
I-J Ave rage
4.8 6.7
5.5 7.1
7.9 8.2 4.9 4.6 6.4
I--A I-C I--H I--L Average
8.5 8.2 8.6 5.9 7.8
8.6 8.6 9.3 7.3 8.4
8.3 8.9 8.0 6.1 7.8
aAverag e l°gl 0 of c o u n t / g of three samples per test subject taken before c o n s u m i n g milk. bAverag e l°gt 0 of c o u n t / g of four samples per test subject taken during period in which milk was consumed. CL°gt 0 of count/g for one sample per test subject taken 10 days after milk c o n s u m p t i o n stopped.
Among test subjects
As result o f drinking the milk
t~ .0028 .07
.0004 .58
,0001 .019
t" > Z t~ ~q >
TABLE 4. Effect of c o n s u m i n g n o n f e r m e n t e d low-fat milk containing on Lactobacillus acidopbilus
facultative lactobacilli in feces of healthy m e n (Trial 1). Probability of observing larger differences by chance As
Population of
L. acidopbilus in milk 0/ml (control group)
2 X 107/ml (low group)
Test subject
Sampling period During
Before
After
I--D I--E I--I I--K Ave rage
2.5 a 7.8 5.1 7.3 5.7
2.9 b 7.8 5.2 7.2 5.8
2.0 c 8.5 4.4 7.0 5.5
I--B I--F I--G I-J Average
6.8 3.6 5.1 4.8 5.1
7.3 6.0 6.9 6.2 6.6
7.9 4.7 4.8 4.6 5.5
I--A I-C I--H I--L Average
2.0 6.4 7.1 4.3 4.9
8.0 8.5 8.6 6.4 7.9
4.9 7.6 7.9 5.9 6.6
Among test subjects
result of drinking the milk
.0001 .73
O Z .0001 .OOOl
e-
8 × 10 a/ml (high group)
=
aAverag e l°gt o of c o u n t / g of three samples per test subject t a k e n before consuming milk.
<
bAverag e l°gl 0 of c o u n t / g of four samples per test subject taken during period in which milk was c o n s u m e d .
O
CL°gl 0 of c o u n t / g for one sample per test subject taken 10 days after milk c o n s u m p t i o n stopped. z
O
-a 00
o~
> tO >
.0002 .O0Ol
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GILLILAND ET AL.
TABLE 5. Effect of consuming nonfermented low-fat milk containing Lacto bacillus acidopbilus (5 × 106/ml) on facultative lactobacilli in feces of healthy men (Trial 3). Probability of observing larger differences by chance As
Test subject
Before
III--A III--B III-C III--D III--E Average
7.3 a 3.5 3.4 5.8 8.6 5.7
Sampling period During 7.9 6.5 5.7 5.4 8.0 6.6
After 7.8 2.5 3.4 5.2 8.6 5.5
Among test subjects
result of drinking the milk
.0001 .012
aAverage log1 0 of count/g of four samples per test subject taken before, during, and after the consumption of L. acidopbilus.
a t m o s p h e r e (LBS-CO2). In these studies, bifidob a c t e r i a were isolated f r o m t h e LBS-GP plates. Such findings i n d i c a t e t h a t c o u n t s o b t a i n e d b y plating fecal samples o n LBS agar a n d i n c u b a t ing t h e plates in a GasPak (H2-CO2) s y s t e m include b i f i d o b a c t e r i a as well as a n a e r o b i c lactobacilli. T h e p r e s e n t s t u d y shows t h e necessity o f i n c u b a t i n g LBS agar plates u n d e r CO2 to s t u d y t h e e f f e c t o f ingesting L. acidopbilus o n t h e f a c u l t a t i v e fecal lactobacilli. F e e d i n g L. acidopbilus has b e e n s h o w n in a n u m b e r o f i n s t a n c e s to alter t h e i n t e s t i n a l flora so t h a t a m o r e f a v o r a b l e b a l a n c e exists (22). Most of these i n s t a n c e s have involved t e s t subjects w h o were suffering f r o m s o m e f o r m o f
g a s t r o e n t e r i t i s caused b y b a c t e r i a such as ent e r o p a t h o g e n i c Escbericbia coli. In o u r s t u d y feeding L. acidopbilus significantly increased t h e n u m b e r s o f f a c u l t a t i v e lactobacilli in t h e feces, b u t t h e c o l i f o r m s were n o t significantly affected. This was p r o b a b l y because all t h e test subjects were h e a l t h y a n d n o t e x p e r i e n c i n g i n t e s t i n a l disorders. C o m p a r e d to p r e v i o u s rep o r t s (5, 15, 17) n o n e of t h e test s u b j e c t s h a d e x c e p t i o n a l l y high c o l i f o r m counts. T h e decline in the n u m b e r s of lactobacilli in feces a f t e r c o n s u m p t i o n o f L. acidopbilus h a d s t o p p e d is similar t o (21, 24). T h o s e test subjects w h o s e feces c o n t a i n e d relatively low n u m b e r s of f a c u l t a t i v e lactobacilli p r i o r to
TABLE 6. Effect of consuming nonfermented low-fat milk containing Lactobacillus acidopbilus (5 × 106/ml) on bile-resistant facultative lactobacilli in feces of healthy men (Trial 3). Probability of observing larger differences by chance As
Test
Sampling period
subject
Before
During
After
III--A III--B III-C III--D III--E Average
3.2 a 3.0 2.5 3.8 5.2 3.4
4.9 6.2 5.1 4.9 5.4 5.2
5.0 2.7 2.8 3.4 6.0 3.9
Among test subjects
result of drinking the milk
.005 .002
aAverage l°gt 0 of count/g of four samples per test subject taken before, durir,g, and after the consumption of L. acidopbitus. Journal of Dairy Science Vol. 61, No. 1, 1978
L. acidopbilus EFFECTS ON FECAL FLORA
9
TABLE 7. Effect of consuming nonfermented low-fat milk containing Lactobacillus acidopbilus (5 X 106/ml) on the types of bile-resistant facultative lactobacilli in feces of healthy men (Trial 3). Sampling period
Total isolates
Positive
Growth at 15 C Negative
% Negative
Before During After
67 77 88
67 13 73
0 64 15
0 83 17
consuming the milk containing L. acidopbilus retained higher numbers than were initially present. Literature has indicated that large numbers of viable cells of L. acidophilus must be ingested for the organism to become established in the intestinal tract (10). Myers (19) fed a group of human volunteers .95 liter daily of an unfermented milk product containing an average of 3.4 × 108 viable cells of L. acidophilus/ml. All volunteers exhibited increased numbers of lactobacilli in their feces. In the first two feeding trials in our study, the test subjects receiving the milk containing the high L. acidophilus (8 × 108/ml) were consuming a total number of L. acidophilus equivalent to that reported by Myers (19). However, results from the groups receiving the lower L. acidophilus (2 x 107/ml) in these trials and the group i n the third trial receiving milk containing (5 x 106/ml) show that consumption of a much lower population than that used by Myers (19) could increase successfully the lactobacillus content of the feces. The increased numbers of lactobacilli in the fecal material as a result of consuming L. acidophilus was accompanied by a shift in the type(s) of lactobacilli. This shift was due presumably to increased numbers of L. acidophilus in the intestinal contents during the period the milk was consumed. However, other lactobacilli were in the samples prior to and after consuming the milk containing L. acidophilus. Additional work is needed to characterize the lactobacilli of the intestinal tract further and to evaluate the effect(s) of consuming L. acidophilus on the distribution of these lactobacilli. While analyses of the fecal flora is often used to measure the effect(s) of consuming L. acidophilus on the intestinal flora of man, it is
possible that this may not provide a complete picture of their importance. Gorbach et al. (6) have shown that the small intestine contains lactobacilli, streptococci, staphylococci, and fungi whereas the flora of the colon is composed primarily of bacteroides, anaerobic lactobacilli, and coliforms. Moore et al. (17) have confirmed this, and in a later study Moore et al. (18) showed that L. acidophilus was in the human small intestine. Lerche and Reuter (14) also reported that L. acidophilus was abundant in the upper intestine. In summary, our results show that a nonfermented low fat milk product prepared using a commercially-available, frozen, concentrated culture can provide a source of L. acidophilus that will survive in the intestinal tract. The taste of the product is the same as that of pasteurized low fat milk. Consumption of the milk generally resulted in significantly increased numbers of facultative lactobacilli in the feces. Further studies are planned to clarify additional relationships of L. acidopbilus to the overall well being of the intestinal tract.
ACKNOWLEDGMENT
This work was supported in part by funds from the National Dairy Council.
REFERENCES
1 Rogosa, Morrison. 1974. Lactobacillus acidopbilus (Moro) Hansen and Mocquot 1970, 326. In Bergey's manual of determinative bacteriology. R. E. Buchanan and N. E. Gibbons, ed. 8th ed. The Williams & Wilkins Co., Baltimore, MD. 2 Duggan, D. E., A. W. Anderson, and P. R. Elliker. 1959. A frozen concentrate of Lactobacillus acidopbilus for preparation of a palatable acidophilus milk. Food Technol. 13:465. 3 Gilliland, S. E., and M. L. Speck. 1974. Frozen concentrated cultures of lactic starter bacteria. A Journal of Dairy Science Vol. 61, No. 1, 1978
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Journal of Dairy Science Vol. 61, No. 1, 1978
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