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Fermentation of milk by Lactobacillus bifidus. Brown, Charles Dwight 1970

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FERMENTATION OF MILK BY Lactobacillus bifidus by CHARLES DWIGHT BROWN B.S.A., University of British Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE m AGRICULTURE in the Department of Food Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1970 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree tha permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of British Columbia Vancouver 8, Canada ABSTRACT Bacteria, belonging to the Lactobacillus bifidus group, were isolated from rectal swabs obtained from healthy babies receiving breat milk. The fermentation properties of these bacteria in cow's milk were studied. Strains potentially suitable for fermentation in a food product were selected according to their a b i l i t y to produce lactic acid and no gas from lactose, as well as adhering to char-acteristics common to the bif idus group (<3;ram positive, catalase • negative and blanching morphology). The five strains chosen for intensive study produced 1.14 to 1.25% lactic acid and less than 0.05% volatile acid. The latter organisms could be cultured readily and coagulated milk in less than 24 hr when using a 5% inoculum. Repeated subculture resulted in a change from the blanched to the unbianched bacterial morphology. This transformation, which is not unusual among b i f i d bacteria, was accompanied by a change in a b i l i t y to produce lactic acid, but no gas was produced by btanched or unbianched strains. A great deal of v a r i a b i l i t y in morphology, growth consistency, and acid production was observed among the more than 500 bacterial strains isolated. However, the b i f i d populations from Caucasian babies was not noticeably different from those of native Indian babies. A l l of the five strains selected u t i l i z e d mnulin, dextrin, mannitol, and melezitose. A 24 hr culture of fermented milk yielded a product with a mild pleasant natural flavour which could conceivably be altered, i f desired, by adding sweeteners or flavouring agents. TABLE OF CONTENTS Page List of Tables i List of Plates i i Acknowledgements i i i Introduction.. 1 Literature Review 3 Methods and Materials Organisms 9 Medium 9 Acid Determinations 9 Catalase Test. 10 Subculturing.... 10 Sugar Fermentation. 11 Lactobacillus bif idus isolation. 11 Results and Discussion 14 Bibliography 25 Appendices Colony Morphology. 31 Bacterial Morphology 33 - i -LIST OF TABLES Page TABLE I Bacteria isolated from the r e c t a l svzabs from 1 to 5 day old infants receiving breast milk incubated in modified Gyorgy's medium (17) 15 TABLE II Isolation pattern f o r the selection of desirable strains of bacteria from a r e c t a l swab of a baby receiving human milk. A l l i n -cubations were done in broth cultures of mod-i f i e d Gyorgy's medium (17) 17 TABLE III Lactic a c i d produced by branched and unbranched strains of b i f i d bacteria i n modified Gyorgy's medium (17) 19 TABLE IV Change in amount of l a c t i c acid produced with changing morphology in Lactabacillus b i f i d u s grown in broth cultures of modified Gyorgy's medium (17) 21 TABLE V Fermentation of d i f f e r e n t sugars by unbranched b i f i d bacteria isolated from nursling infant r e c t a l swabs 23 - X I -LIST OF PLATES Page Plate 1 Forty-eight hour culture showing colony v a r i a b i l i t y . .32 Plate 2 Forty-eight hour culture showing colony v a r i a b i l i t y .32 Plate 3 Strain AXIg in its branching morphology.34 Plate 4 Strain mi$ i n i t s branching morphology. . 34 Plate 5 Strain AV^ in its branching morphology..35 Plate 6 Strain AVTItfin i t s branching morphology. .35 Plate 7 Strain AI f i in its branching morphology..36 Plate 8 Strain AXIg ^  i t s unbranched morphology. . 36 Plate 9 Strain A/TIg in its unbranched morphology. .37 Plate 10 Strain AVjj^ i t s unbranched morphology. . 37 Plate 11 Strain A l l ^ i n i t s unbranched morphology. . 38 Plate 12 Strain A I 6 in its unbranched morphology.38 ACKNOWLEDGEMENT I wish to express my appreciation to Dr. P. M. Towns ley for his i n s p i r a t i o n , enthusiastic interest and constructive c r i t i c i s m throughout this investigation. I also g r a t e f u l l y acknowledge the valuable suggestions and assistance given by Dr. W. K i t t s , Dr. T. H. Blackburn, and Dr. D. M. McLean. I would also l i k e to thank Dr. D. M. Black and the st a f f of the Vancouver C i t y Health Department and Metro-politan Health Services for supplying the r e c t a l swabs. Thanks are also directed to the Fraser Valley Milk Producer's Association of B r i t i s h Columbia for f i n a n c i a l support given t h i s project. - 1 -INTRODUCTION Man has made use of the fermentation process for cen-turi e s to a l t e r , enhance, concentrate and preserve food, not only increasing p a l a t a b i l i t y and v a r i e t y so that one food, can be made into several products, but also in some instances contributing unique n u t r i t i o n a l worth to the food product. Recently, s c i e n t i s t s have begun to r e a l i z e that the process of breakdown of organic matter and i t s subsequent ass-i m i l a t i o n into the body can be effected by the type of microflora inhabiting the in t e s t i n e . Undesirable bacteria, once established in the gut can cause very severe digestive upsets. A f t e r o r a l administration of heavy doses of a n t i -b i o t i c s , the alimentary canal may be l e f t e s s e n t i a l l y s t e r i l e , thus requiring reinoculation with a desirable microflora. The intestines of healthy breast-fed babies are inhabited almost exclusively by Lactobacillus b i f i d u s . This organism has demonstrated n u t r i t i o n a l value in i t s e l f as i t w i l l break down many disaccharides and other sugars that cannot be assimilated beforehand. The end products of mic-r o b i a l growth include vitamins (especially the B complex) and l a c t i c a c i d . The presence of l a c t i c acid lowers the pH of the i n t e s t i n e , thereby s t a b i l i z i n g i t against many pathogenic, acid s e n s i t i v e invaders. The b i f i d bacteria are known to es t a b l i s h themselves in the gut of the nursling infant, r e-siding in the mucus layer around the inner l i n i n g of the - 2 -intestine. Many newborn babies who are hyper-sensitive to their own mother's milk and elderly or middle aged people with digestive disorders may benefit by having a food product which when eaten would inoculate their alimentary tract with a desirable microflora. There are several requirements that Lactobacillus  bifidus must meet i f this organism is to qualify as a commercially useful bacterium. The growth rate of the organism must be rapid, yet dependable. The culture must maintain a high degree of palatability; thus accumulation of acetic acid and undesirable flavour compounds must be minimized. Gas production by the bi f i d bacteria must be minimal because the presence of large amounts of gas in the intestine causes considerable discomfort in the person who is receiving the bifidus milk product. The nutritional requirements of the bacteria must be such that these organisms can be cultured in whole or skimmed milk without expensive nutrient sup-plements in the form of vitamins, co-factors and so on. In the current investigation, five strains of Lactobacillus  bifidus have been isolated and described which w i l l ferment cow's milk in less than 24 hr yielding a conceivably marketable product. - 3 -LITERATURE REVIEW Tissier (50), in 1908, was the f i r s t to notice the presence of a unique microflora in the intestines of healthy breast-fed babies. The latter investigator surmised their physiological significance in the prevention of intestinal diseases and lower infant mortality in breast-fed as compared to bottle-fed infants. Since that time researchers (32) have claimed the presence of Lactobacillus bif idus increased the effectiveness of medicinal preparations containing live non-pathogenic coliforms used to reseed the gut. Adaptation of the intestinal flora to L. bifidus apparently enhanced nitrogen retention and increased by 400% the vitamin con-tent in the stools (59). Recently, i t has been reported (23) that a bifidus milk fed to premature and newborn babies created a bifidus microflora in these infants scarcely d i f -ferent from that of the breast-fed child. These investigators concluded that there seemed to be a distinct protective effect of bifidus milk. It has also been reported that with increasing b i f i d bacteria populations in the intestine, there was a marked decrease in the toxic products in the faeces (47). The latter researchers have shown that the number of harmful bacteria.decreased in the presence of the b i f i d bacteria, and that white rats were protected from several diseases of the diarrhoea type. In 1950, an attempt was made (1) to adapt baby feeding formulae to favour the intestinal growth of L. bifidus and i t was shown that addition of cystine, lactose and maltose to the formulae was necessary to get subsequent growth of this bacteria in the intestine. It was later found that a specific, selective growth stimulator for the b i f i d bacteria was present in human milk and also in other biological materials such as blood group A, B and 0, saliva, mucin and pancreas tissue (18) or partial hydro-lysates of these, but could not be found in the milk of cows, sheep goats or guinea pigs (17). Many researchers have since tried to adapt cow's milk to have more of the qualities of human milk, thereby encouraging growth of b i f i d bacteria in the bottle-fed child's intestine. Reports vary quantitatively and qualitatively as to the best additives and procedures to use. Hea€ decomposition products of lactose when added to the baby formula were reported to stimulate the growth of L. bifidus (48). Certain specific chemicals (for example, lactose and lactulose in a particular ratio to protein (49) were found to'be effective in promoting the growth of the b i f i d bacteria. It was demonstrated that several N-substituted sugar amines (N-octanoyl-N-benzoyl-D-glucosamine, N-carboethoxy-D-glucosamine (62) and ji-methyl glucosides of N-acetyl-fS-D-glucosamine (19)) could serve as enhancer compounds promoting potential growth of L. bifidus especially var. pennsyIvanicus in amounts equal to or greater than that of human milk. A l l the latter investigations were - 5 -aimed at increasing the bifidus populations of the gut. Support for the value of L. bif idus in promoting the health and well being of humans and other higher mammals have also been reported (10, 11, 37, 38, 40, 41, 46, 50). Confusion exists in the literature as to the exact nomenclature and classification of the b i f i d bacteria. Weiss and Rettger (57) who thoroughly reviewed the literature con-cluded that L. bifidus and L. acidophilus are closely related, in fact, probably variants of the same species. Orla-Jensen (et al.) (33) disagreed with this claim because their strains, unlike those of Weiss and Rettger, were branched. Subsequently, Orla-Jensen concluded that his Bacterium bifidum should be included among the Actinomycetales. Eggerth (9) described two types of Bacteroxides bif idus and Olsen (34) proposed that the name should be changed to Corynebacterium bifidum. Much of the confusion arose because of the lack of a suitable, selective growth medium and also because of the bacteria's vast a b i l i t y to vary. Previous literature has evidenced a great diversity in nutritional requirements, growth con-ditions and end products of growth of L. bififus (14, 17, 28,. 29, 31, 52, 58, 61). The Teply and Elvehjem medium supplemented with f o l i c acid, sorbitan, mono-oleate, pancreatin, ascorbic acid, and vitamin Bj_2 was satisfactory for isolation and propagation of L. bifidus as a branched organism from the stools and intestinal contents of breast-fed infants (31). Unbranched strains derived from the branched organism were found to occur frequently. These researchers also demonstrated that their bacteria, when grown on solid medium required small amounts of CO2 and would tolerate up to 3% O2. When grown in broth tubes the organisms tolerated atmospheric O2 and did not require the addition of CC^. None of the b i f i d strains produced 602. However, a l l with the:; except ion of one of the unbranched strains and three of the"*five ATCC strains of L. bif idus produced large amounts of CO2 under these conditions. Dextro-rotary lactic acid was formed by the b i f i d strains and optically inactive lactic acid by the unbranched and ATCC strains. The relative amount of volatile acid produced by the branched strains was greatly in excess of that formed by the unbranched and ATCC strains, but in both cases the volatile acid appeared to be acetic. Under the above conditions, the growth rates were very low and the colony size was small. As repeated attempts were made to group these organisms into one nutritional category, i t be-came increasingly clear that the microflora from nursling infants had a very complex nutritional requirement. L. bifidus isolated in some laboratories only required biotin, pantothenic acid and cysteine as specific growth factors whereas L. bifidus in other laboratories were found to re-quire in addition to the latter compounds small amounts of - 7 -riboflavin and preformed pantethine. Certain strains of b i f i d bacteria were dependent on purine and pyrimidine bases (14). Other researchers (58) have been unsuccessful in their attempts to grow freshly isolated strains on a simp-l i f i e d medium. In 1952, Gyorgy (17) isolated a variety of L. bif idus which he called var. pgnnsvlvanicus that showed no growth in the medium usually satisfactory for most strains of this organism. He demonstrated that human milk contained the bifidus factor pre-requisite to the growth of this bacteria, and that cow's milk had only 1/30 to 1/100 of the growth promoting activity of human milk. Although some strains of b i f i d bac-teria do not apparently require this growth factor, its presence in infant feeding formulae has definitely reflected increased L. bifidus populations in the intestines of formulae recipients. A vast number of publications on the nutrition (8, 16, 20, 21, 22, 26, 30, 39, 51, 53, 54), metabolism (2, 3, 7, 24, 25, 37, 42, 44, 55, 62) and clas-sif i c a t i o n (12, 13, 31, 35, 36, 45, 52, 56) of the b i f i d bacteria can be found in the literature. Bifid bacteria commonly w i l l change morphology with repeated subculture. Typically, they w i l l be branched, lobed or involuted upon i n i t i a l isolation. As they are passed repeatedly onto fresh media, these bacteria w i l l tend to lose this branch or ' b i f i d ' morphology. Some w i l l become straight rods, single or in chains while others form short, bent or pleomorphic cells with various degrees of - 8 -clumping. These unbranched forms have alternately been called contaminants and mutational adaptations, but i t now appears that the morphology change from branched to unbranched can be reversed by certain univalent a l k a l i metallic cations (24). These results indicate that neither contamination nor mutation is responsible for the morphological change. Accompanying the transformation from branched to unbranched c e l l shape, there has been reported dramatic changes in the metabolism of the bacteria. Gyllenberg (13) showed that the straight rod type of L. bifidus became capable of aerobic growth, did not ferment lactose, or produce lactic acid, fermented glycerol to produce propionic acid, were catalase positive, hemolytic and weak acid producers. Branched forms were found to be the opposite in a l l these aspects. These results do not correlate well with those discussed earlier (31). The purpose of the present investigation was to capitalize on the va r i a b i l i t y of bi f i d bacteria in an attempt to find strains which could be successfully cultured in cow's milk in a desirable fermentation rendering a product f i t for human consumption. An approach which has been used in the past (11, 47, 48, 49) is to add the bifidus factor to baby food to encourage growth of the bi f i d bacteria in the in-testine. The present approach would make either the fer-mented product or a pure culture a massive inoculum of L. bifidus and as such would not rely on natural contamination and selection for the presence of the bacteria in the human gut. - 9 -METHODS AND MATERIALS ORGANISMS: Strains 11146 arid 11147 of Lactobacillus bifidus were obtained from the American Type Culture Collection (ATCC). Rectal swabs obtained from healthy breast-fed babies (15 days to 5 months old), were received from the Kerrisdale Community Center Health Unit No. 2, 2112 West 42nd Avenue, Vancouver, B.C. MEDIUM: The most acceptable medium was found to be that reported by Gyorgy (17) and was modified by substituting Bacto-Casitone (Difco) for Casein Hydrolysate (NZ case, Sheffield). This was chosen rather than a tryptic digest because the latter has been shown to cauae significant morphological changes (44). This medium supported the growth of stock and isolated strains. Raw milk was skimmed by centrifugation for 20 min. at 1400 x g and then autoclaved at 15 lb. psi for 15 minutes. A l l sterile media were stored at 5° C. Incubations were carried out in a Gaspak anaerobic jar (BBL 5) which provided the necessary anaerobic con-ditions for primary culture. Isolated b i f i d cultures were maintained in 10 ml of modified Gyorgy's broth medium. ACID DETERMINATIONS: The method of Barker and Summerson (4) was used for - 10 -the lactic acid determinations. Volatile acids were determined by the following method. A small aliquot of fermented milk was weighed and washed into a micro-Kjeldahl steam d i s t i l l a t i o n apparatus. One drop of antifoam (Dow Corning Antifoam AF Emulsion) was added and the acidity increased to pH 1.0 with 10% H2SO^. Fi f t y ml of d i s t i l l a t e was collected and titrated with 0.05N NaOH using the in-dicator phenolphthalein. Using this method known amounts of acetic acid added to skimmed milk could be recovered quant itat ively. CATALASE TEST: Catalase was measured by a modified version of the Merchant and Packer method (27). To 0.5 ml of bacterial sediment from a 24 hr culture was added 10 ml of milk containing 1 ml of 3% ^2^2* The screw-capped tube was f i l l e d to the top with d i s t i l l e d water, capped, inverted in a rack and incubated at 37° c for 3 hr. The caps were not on tight in order that milk displaced by evolving oxygen could flow out the bottom of the tube. Catalase activity was determined by the presence of trapped gaseous O2 in the inverted tubes. SUBCULTURING: With a st e r i l e pipette, 0.1 ml of sediment from a 48 hr culture was transferred to fresh medium. After this - 11 -culture had incubated for 24 hr and the bacteria were actively dividing, this bacterial suspension served as the source of inoculum for a l l further subcultures. To minimize carry over of the modified Gyorgy's medium for experiments on sugar u t i l i z a t i o n , the cells were centrifuged aseptically at 300 x g for 10 min., washed once in 5 ml ste r i l e physiological saline and f i n a l l y suspended in 5 ml of saline. Inoculation of 0.5 ml of this suspension constituted a 10% inoculum. SUGAR PERMENTATION: Modified Gyorgy's medium without added lactose was prepared containing the carbon source at a f i n a l concentration of 1.75% and then autoclaved at 15 lb psi for 15 min. Following inoculation the medium was incubated for 48 hr at 37° c. Total acidity was measured by titr a t i o n with 0.1N NaOH using phenolphthalein as indicator. Growth was es-timated by visual turbidity. Lactobacillus bifidus ISOLATION: I n i t i a l tests were done on rectal swabs obtained from newborn breast-fed infants ranging in age from 1 to 5 days. Specimens were cultured under different conditions as outlined in Table I. Data presented in Table I represents average values for two isolation experiments. Anaerobic conditions were maintained by either using the Gaspak anaerobic jar &r - 12 -introducing CO2 into the atmosphere above the Betri dishes in a Torbal (Model AiI-2, The Torsion Balance Co/, Clifton, N.J.) anaerobic jar. To see the effect of added breast milk, cultures were incubated with 2% breast milk (skimmed by the same procedure as with the cow's milk) in the agar medium. Stock cultures of L. bifidus obtained from the American Type Culture Collection grex* well in this medium whether in the Gaspak or CO2 anaerobic jars and with or without added human milk. Ten slides from each plate in each treatment were gram stained and observed under the microscope. Bacteria were then categorized as: Gjram positive rods (possible b i f i d bacteria), Gram negative rods and cocci as shown in Table I. The cocci were not sub-divided into G;ram positive and G;ram negative cocci because the purpose <^  of the selection method was to locate b i f i d bacteria. As this procedure failed to locate a significant number of L. bif idus. the following procedure was used with somewhat older babies. The faecal bacteria on the rectal swabs from healthy breast-fed babies ranging in age from 15 days to 5 months , A 5 were given the appropriate dilution (10 to 10 ) in s t e r i l e physiological saline and cultured in modified Gyb'rgy's medium using:the standard plate count method (43) in a Gaspak anaerobic jar. A total of 31 rectal swabs were treated in this manner. Strains ATCC 11146 and 11147 grew - 13 -w e l l under these conditions. A l l incubations were for 48 hr at 37° C. Twenty colonies were picked at random from the plate cultures of each faecal sample and inoculated into broth cultures containing bromo-cresol green. Bromo-cresol green was found to indicate a c i d i t y in the proper range (pH 3.8 to 5.4) without noticeable t o x i c i t y (61). Gas production was measured with Durham Tubes. Five desirable strains of bacteria were selected during the 3rd to the 7th subculture on the basis of a positive gram s t a i n , a b i f i d morphology, on the a b i l i t y to coagulate * skimmed s t e r i l i z e d milk, and on the a b i l i t y of produce non-v o l a t i l e organic acids (e s p e c i a l l y l a c t i c acid) and no gas from lactose. The f i v e strains were subcultured an a d d i t i o n a l 20 times and changes in morphology and acid production were noted. Lac t i c acid determinations were done when the bacteria had the branching morphology and also a f t e r they had lost t h e i r a b i l i t y to branch. V o l a t i l e acid content, catalase a c t i v i t y and fermentable sugars were done only on the l a t t e r . These strains were then c l a s s i f i e d by the sugars they coulduuse according to Bergey's Manual (5). In t h i s t e s t , lactose, dextrose, galactose, sucrose, arabinose, xylose, maltose, inuMin* dextrin, starch, mannitol and melezitose were used. Stock cultures of the f i v e strains have presently been kept for several months i n l i q u i d nitrogen and s t i l l maintain a very high degree of v i a b i l i t y . Working cultures can be e a s i l y maintained by storing a 24 hour culture at 5° Q for three weeks. - 14 -RESULTS AND DISCUSSION Results of b a c t e r i a l s e l e c t i o n from 1 to 5 day old breast-fed infants, as presented in Table I did not reveal any bacteria of the b i f i d morphology on primary i s o l a t i o n . The data shown in Table I represents average values of two i s o l a t i o n experiments. However, two stock ATCC strains of L. bifidus r e a d i l y developed colonies under these con-d i t i o n s . It was therefore surmised that perhaps the r e c t a l swab i t s e l f contained no L. B i f i d u s . Microscopic examination of a f r e s h l y received swab showed no Gram po s i t i v e , branching bacteria. Two suggestions are therefore presented to attempt to explain t h i s lack of b i f i d microflora in these newborn babies. Many mothers are injected with quite large doses of a n t i b i o t i c s post-partum to aid i n the healing of torn tissues or wounds which occured during the b i r t h process. It i s well established that a n t i b i o t i c s administered to the mother would soon be passed on to the c h i l d v i a the breast milk, and the presence of these a n t i b i o t i c s i n the infant's in t e s t i n e could c e r t a i n l y hamper normal development of a b i f i d u s population. However, the bacteria on the r e c t a l swabs may have a c t u a l l y represented a normal f l o r a of mic-roorganisms which lose way to more adaptive bacteria once the c h i l d moves to the home environment. Secondly, many of the samples came from babies 1, 2 or 3 days of age and i t is therefore possible that the s e l e c t i v e powers of mother's milk did not have time to encourage small numbers of L. b i f i d u s TABLE I Bacteria isolated from the rectal swabs from 1 to 5 day old infants receiving lareast: milk incubated in broth cultures of modified Gyorgy's medium (17)* No. colonies per plate ATCC 11146 of L. bifidus + No. colonies per plate on rectal swabs* % <3;ram positive rods % tram negative rods i cocci C 0 2 and 2% human milk 832 474 20 0 80 Gaspak and 2% human milk 101 243 0 10 90 CCv, and no human milk 656 472 0 30 70 Gaspak and no human milk 480 74 0 10 90 * Ten colonies, which included a l l representative colony morphologies were studied from each plate. * x Numbers represent average values of two isolation experiments. + The same number of ATCC 11146 (L. bif idus) bacteria were i n i t i a l l y plated in each of the four t r i a l s and a l l bacteria in this experiment isolated from rectal swabs received the same i n i t i a l dilution. - 16 -bacteria entering the alimentary tract to increase in numbers to the point of dominance over the other microorganisms. In any case, a new source of samples was sought: one in which the babies were living at home, being exposed to heavy contamination by many species of bacteria. Under these conditions, almost 100% of the bacteria isolated were of the b i f i d morphology and stained Gjram positive. The primary culture of the b i f i d organisms in modified Gyorgy's medium showed a variation in colony size and mor-phology. As can be seen by Plates 1 and 2, the colonies varied in size from 0.7 to about 12 mm. in diameter and from grey translucent through opaque to white in color. The rate of growth of the colonies on the plates reflected their growth rates in the broth tubes. Data in Table II shows a typical isolation pattern including subculturing and discarding of a strain depending on its acid production and growth rate in broth culture. Since the purpose of this procedure was to select b i f i d bacteria which grew rapidly and lowered theppH quickly, morphological studies were done only on those strains that grew excellently and markedly decreased pH. As can be seen from Table II there was considerable variation in the a b i l i t y of a culture to produce acid. Although pH may not indicate quantitatively the amount of acid present, (some acids may be present as undissociated or partially un-dissociated acids) pH value was used as an index of acid content (TABLE II Isolation pattern for the selection of desirable strains of bacteria from a r e c t a l swab of a baby receiving human milk. A l l incubations were  done in broth cultures of modified Gyb'rgy's medium (17). 1st 2nd 3rd Tube No. Primary Isolation subculture subculture Subculture, (strai n ) pH Growth Morphology pH Growth pH Growth pH Growth AVIlJ 5.8 G* Discarded 4" AVII2 5.8 G Discarded AVI I3 5.5 G Discarded AVII4 5.2 Ex branched 5.2 Ex 5.4 Ex 5.1 Ex AVII5 5.2 Ex branched 5.2 Ex 5.3 Ex 5.2 Ex AVIIg 5.5 G Discarded AVII7 5.8 G Discarded A V I I s 5.1 Ex branched 5.2 Ex 5.2 Ex 5.1 Ex AVII9 5.0 Ex branched 6.0 P Discarded A V I I 1 0 5.2 Ex branched 5.7 Ex 5.4 Ex 5.3 Ex A V I I n 5.8 G Discarded AVII12 6.0 G Discarded A V I I 1 3 6.0 G Discarded A V I I 1 4 5.5 G Discarded AVIIJ5 5.6 G Discarded A V I I 1 6 6.0 G Discarded AVIl ! 7 5.5 Ex branched 6.0 Ex 5.2 Ex 6.0 Ex A V I I 1 8 6.0 G Discarded AVII19 5.3 Ex branched 5.2 Ex 5.3 Ex 5.2 Ex AVII20 5.5 G Discarded ^Growth measurements a r b i t r a r i l y made by v i s u a l comparison Ex. = excellent; G = good; P = poor +Cultures discarded due to low acid production and undesirable growth rate. Morphological c h a r a c t e r i s t i c s were observed on primary i s o l a t i o n , only on those bacteria selected for further study. *AVII indicates the number of the r e c t a l swab and the Arabic Numeral subscript indicates each p a r t i c u l a r isolate from that r e c t a l swab. - 18 -s o a c i d p r o d u c i n g s t r a i n s o f b i f i d b a c t e r i a c o u l d b e q u i c k l y d i f f e r e n t i a t e d . A c i d p r o d u c t i o n w a s n o t n e c e s s a r i l y a f u n c t i o n o f g r o w t h r a t e . S t r a i n A V I I j y , f o r e x a m p l e , g r e w e x c e l l e n t l y t h r o u g h a l l s u b c u l t u r e s , b u t t h e a m o u n t o f a c i d p r o d u c e d v a r i e d . S t r a i n A V I I g , o n t h e o t h e r h a n d , g r e w v e r y w e l l a n d a l s o w a s a c o n s i s t e n t p r o d u c e r o f l a r g e a m o u n t s o f a c i d . I n a l l c a s e s t h e b a c t e r i a s t a i n e d G r a m p o s i t i v e , a n d n o n e o f t h e s t r a i n s p r o d u c e d g a s . A l l s t r a i n s w e r e a l s o c a t a l a s e n e g a t i v e . M o r p h o l o g y , g r o w t h r a t e a n d a c i d p r o d u c t i o n o f t h e b a c t e r i a v a r i e d f r o m i n f a n t t o i n f a n t a n d among b a c t e r i a l i s o l a t e s f r o m t h e same i n f a n t . A p p r o x i m a t e l y 1/3 o f t h e r e c t a l s w a b s came f r o m C a n a d i a n I n d i a n s * n u r s l i n g b a b i e s a n d 2/3 f r o m C a u c a s i a n b a b i e s . T h e r e a p p e a r e d t o b e n o m a r k e d d i f f e r e n c e s b e t w e e n t h e r a c e s w i t h r e g a r d t o b i f i d m o r p h o l o g y , g r o w t h c o n s i s t e n c y , o r a c i d p r o d u c t i o n . L a c t i c a c i d a n a l y s e s o f t h e b i f i d c u l t u r e s i n d i c a t e d t h a t t h e a m o u n t o f a c i d p r o d u c e d w a s n o t a f u n c t i o n o f b a c t e r i a l m o r p h o l o g y , a s b o t h b r a n c h e d a n d u n b r a n c h e d s t r a i n s p r o d u c e d b o t h l a r g e a n d s m a l l a m o u n t s o f l a c t a t e a s s h o w n i n T a b l e I I I . I t w a s e v i d e n t ; h o w e v e r , t h a t w h e n a m o r p h o l o g i c a l c h a n g e d i d t a k e p l a c e i n c u l t u r e ( i . e . f r o m b r a n c h e d t o u n b r a n c h e d ) t h e r e w a s a n a c c o m p a n y i n g c h a n g e i n a c i d p r o d u c t i o n . T h e r e s u l t s s h o w n i n T a b l e I V i n d i c a t e t h a t w h e n t h e t h r e e b r a n c h e d s t r a i n s c h a n g e d t o p l e o m o r p h i c o r b e n t r o d s , t h e r e w a s a n i n c r e a s e i n l a c t a t e p r o d u c t i o n . I n c r e a s e s o f a b o u t 60 t o 2257<> l a c t i c a c i d - 19 -TABLE III Lactic acid produced by branched and unbranched strains of the b i f i d bacteria in modified Gybrgy's medium (17). Strain Morphology % Lactate* VIII 6 Branched 0.58 V l l l g Branched 0.50 v u i 1 4 Branched 1.46 v i n 1 6 Branched 1.44 x i n l 7 Branched 1.65 A I I 1 3 Some branching 1.52 A I I n Short, bent rods 1.54 AV1 7 Granular pleomorphic rods 0.52 AVI 1 3 Granular pleomorphic rods 0.57 AVI 2 0 Granular pleomorphic rods 0.59 x i 5 Pleomorphic rods 1.67 X I 1 1 Straight feods 1.86 X I 2 0 Pleomorphic rods 1.70 AIIi-5 Short, bent rods 1.45 * % Lactate refers to % Lactic acid by volume in the spent culture medium. - 20 -were seen in strains AXIg, AVIIg, and AV-^ 7 whereas the strains o f L* bif idus remaining in the unbranched form actually de-creased slightly in their a b i l i t y to produce acid. The trend to decreasing acid production seemed quite evident in particular with strain All^g which i n i t i a l l y produced 1.52% lactate after the 5th subculture (Table III) and decreased to 1.38% and 1.14% lactate with additional subculturing (Table IV). A l l five strains, however, produced acidities as low as pH 4.0 to 4.5 within four days of incubation. This would indicate that the bacteria continued to produce acid for more than 48 hours and also that they remained alive at relatively low pH values. Plates 3 to 7 show strains AXIg, AVIIs» A v i 7 , AIIi_g., and Alg respectively in their branching morphologies in modified Gyorgy's medium and Plates 8 to 12 respectively show these in their present morphology. There appeared to be no nutritional requirement difference between the two forms as both grew well in modified Gyorgy's medium and in cow's milk. This is in disagreement with other reports (13, 14, 31, 58) in which different nutritional needs and end products were apparent between the branched and unbranched strains of L. bifidus. It might be noted that although no volatile acid measurements were taken while the bacteria were s t i l l in the branched form, differences in volatile acid content were not noticeable organoleptically between the two forms. The quantity of TABLE IV Change in amount of lactic acid produced with changing morphology in  Lactobacillus bifidus grown in broth cultures of modified Gyorgy's medium (17). Stra i n Morphology on 7th transfer % lactate Morphology on 20th transfer % lactate % v o l a t i l e acid AXI 6 highly branched 0.79 short, bent rods 1.24 0.05 AVIIg highly branched 0.54 pleo. & bent rods 1.26 0.05 AV 1 7 pleo. & branched 0.60 pleo. & bent rods 1.20 0.05 i A I I 1 3 bent rods, chains 1.38 bent rods 1.14 0.05 A I 6 pleo. rods 1.47 pleo. & bent rods 1.23 0.05 1 % Lactate refers to % l a c t i c acid by volume in the spent culture medium. - 22 -volatile acids formed by these strains after loss of branching was very low, as seen in Table IV. Here again, the strains isolated in this investigation were quite different from those reported in the literature as the latter have been stated to produce 18 to 25% of the total acids as volatile acids (5). The bacteria isolated in this investigation complied closely with the b i f i d bacteria on the basis of fermentable sugars. Bacteria u t i l i z e d a l l sugars tested (Table V) with the exception of strain AVIIg which could not ferment starch. Other strains of Lactobacillus and G;ram positive bacteria found in the intestinal tract of infants generally do not fer-ment a l l these sugars. Arabinose, xylose and mannitol are not fermented by Lactobacillus acidophilus (5). Streptococcus  faecalis w i l l not u t i l i z e arabinose or :lriulim and rarely w i l l ferment sucrose or mannitol (5). Thus, although the bacteria isolated in this study vary somewhat from the group of b i f i d bacteria in that the branched and unbranched strains grew equally well in the same medium and the amount of volatile acids produced is very low, they do conform to the group classified as L. bif idus in several ways including morphology, G;ram stain, catalase negativity, sugars fermented and production of lactic acid. Additional evidence comes from the fact that they'vwere originally isolated by selective methods from rectal swabs of healthy nursling infants. The relative merit of L. bifidus bacteria for the production TABLE VF> Fermentation of different sugars by unbranched b i f i d bacteria isolated from nursling infant rectal swabs. Carbon source AXI 6* AVIIg AVI 7 A I I 1 3 A I 6 Growth Gas Acid Growth Gas Acid Growth GasSAcid Growth Gas Acid Growth Gas Acid Lactose +++ - 14.1 +++ - 6.6 +++ - 5.8 +++ mm 14.3 +++ - 12.8 Dextrose +++ - 13.3 ++ - 2.3 +++ - 9.2 +++ mm 13.9 +++ -»• mm 13.8 Galactose +++ - 11.5 ++ - 3.5 +++ - 6.2 +++ mm 12.3 +++ - 12.0 Sucrose +++ - 10.5 + - 0.1 +++ - 0.5 ++ - 0.7 +++ - 5.7 Arabinose ++ - 2.0 + - 1.3 -H-+ - 6.5 ++ - 1.8 + - 1.8 Xylose ++ 0.3 + - 0.3 +++ - 4.0 ++ mm 0.9 ++ - 1.0 Maltose +++ - 6.1 +++ - 5.5 +++ - 2.9 +++ - 5.7 +++ - 2.4 Inulin +++ - 10.9 ++ - 0.4 + - H - - 2.8 ++ - 0.7 +++ - 9.8 Dextrin ++ 1.6 ++ - 0.5 +++ - 0.7 +++ - 0.5 + 4 + - mm 0.7 Starch ++ 0 - - 0 +++ - 0.6 ++ mm 0 +++ - 0.7 Mannitol +++ - 2.3 ++ - 0 +++ - 0.5 ++ - 0.3 +++ mm 1.8 Melezitose + - 0 + - 0 •+++ - 9,5 +++ - 15.4 ++ mm 0 * The amount of acid is given as ml. of 0.1N NaOH required to ti t r a t e corrected against an uninoculated medium. 10 ml. of spent medium - 24 -of a fermented milk product would depend on t h e i r ease of handling, t h e i r culture v i a b i l i t y , t h e i r acceptable flavour c h a r a c t e r i s t i c s in the marketed product, t h e i r consistent growth requirements and end products, and t h e i r n u t r i t i o n a l worth as inhabitants of the human in t e s t i n e . Investigators have reported that the b i f i d bacteria are fastidious and quite d i f f i c u l t to grow, however, t h i s study has shown that the above f i v e strains grew r e a d i l y , indeed, in both modified Gyorgy's medium and in cow's milk. Isolated strains have coagulated both autoclaved and fresh raw milk even at temperatures lower than 37°c. V i a b i l i t y of the bacteria may be maintained for at least 4 days at 37 i n which case the pH decreases to below 4 . 5 . The f i n a l stages of the incubation under i n d u s t r i a l conditions could be ca r r i e d out with the product already in the container s i m i l a r to the manufacture of yogurt. The flavour of the b i f i d milk product can be described as clean, mildly sour with a s l i g h t taste of d i a c e t y l . However, i f desired, the organoleptic q u a l i t i e s could be changed with added ingredients such as f r u i t or f r u i t flavouring compounds. Strains AVUg, AV.^, and A I I l 3 in p a r t i c u l a r u t i l i z e sucrose to a very limited extent for acid production (Table VO and would not l i k e l y a l t e r added sucrose-containing flavouring compounds. Thus, the product contains an acceptable very mild natural flavour which could be altered with various sweeteners or flavour additives according to market demand. - 25 -BIBLIOGRAPHY 1. Adam, A. A r t i f i c i a l nutrition approaching the natural alimentation of the nursling. Creation of an a r t i f i c i a l bifidum flora. Monatsschr. Kinderheilk 97, 500 - 507, 1950. 2. Bailey, R. W. and Clarke, R.T.J. Bacterial dextranase. Biochem. J. 72, 49 - 52, 1959. 3. Bailey, R. W., Hutson, D.H. and Weigel, H. Action of Lactobacilli on a branched dextran. Biochem. J. 80. 514 - 519, 1961. 4. Barker, S.B. and Summerson, W.H. The colorimetric determination of lactic acid in biological material. J. Biol. Chem. 138, 535 - 554, 1941. 5. Breed, R.S., Murray, E.G.D., and Smith, N.R. Bergey's Manual of Determinative Bacteriology. The Williams and Wilkins Co., Baltimore, 1957. 6. Brewer, H.J. and Allgeier, D.L. Self-contained carbon dioxide - hydrogen anaerobic system. Appl. Microbiol. 14, 985 - 988, 1966. 7. Cummins, C.S., Glendening, O.M. and Harris, H. Composition of the c e l l wall of Lactobacillus bifidus. Nature 180, 337 - 338, 1957. 8. De Vries, W. and Southamer, A.H. Fermentation of glucose, lactose, galactose, mannitol and xylose by Bifidobacteria. J. Bacteriol. 96, 472 - 478, 1968. 9. Eggerth, A. H. The gram positive non-spore bearing anaerobic b a c i l l i of human faeces. J. Bacteriol. 30 . 277 - 299, 1935. 10. El Akkad, I. and Hobson, P.N. Effect of Antibiotics on some rumen and intestinal bacteria. Nature 209. 1046 - 1047,1966. 11. Fujita, C. Influence of the bifidus factor and Lacto-bacillus bifidus on the intestinal flora and fecal pH. Toho. Igakkai Zasshi 8, 1206 - 1238, 1961. 12. Gyllenberg, H. G. The development of the "straight rod" type of Lactobacillus bifidus. J. Gen. Microbiol. 19. 371 - 384, 1958. - 26 -13. Gyllenberg, H.G. S p e c i f i c contaminants i n cultures of Lactobacillus b i f i d u s . Acta, Pathol. Microbiol. Scand. 4 4 , 293 - 2 9 8 , 1958. 14. Gyllenberg, H. G. and Carlberg, G. The n u t r i t i o n a l c h a r a c t e r i s t i c s of B i f i d Bacteria (Lactobacillus b i f i d u s ) of infants. Acta. Pathol. Microbiol. Scand. 4 4 , 887 - 8 9 2 , 1 9 5 8 . 15. Gyllenberg, H. G. and Carlberg, G. The dominance of a s p e c i f i c n u t r i t i o n a l type of Lactobacillus bi f i d u s in breast-fed infants. Acta. Pathol. Microbiol. Scand. 4 2 , 3 8 0 - 3 8 4 , 1958. 16. Gyllenberg, H. G., Rossander, M. and Roine, R. Growth i n h i b i t a t i o n of Lactobacillus bi f i d u s by ce r t a i n f a t t y acids. Acta. Chem. Scand. J3, 133 - 134, 1954. 17. Gyorgy, P. A hitherto unrecognized biochemical difference between human milk and cow's milk. P e d i a t r i c s . 1 1 . 98 - 1 0 8 , 1952. 18. Gyorgy, P., Kuhn, R. and Z i l l i k e n , F. Food products containing N-acetyl-D-glucosamine. U.S. 2, 7 1 0 , 806 June 14, 1955. 19. Gyorgy, P., Kuhn, R., and Z i l l i k e n , F. Food products containing glucosides of N-acetyl-D- glucosamine. U.S. 2, 7 1 0 , 8 0 7 , June 14, 1955. 2 0 . Gybrgy, P. and Rose, C.S. Microbiological studies on the growth factor for Lactobacillus bifidus var. pen-nsylvanicus. Proc. Soc. Exptl. B i o l . Med. 9 0 , 2 1 9 -2 2 3 , 1955. 2 1 . Hassinen, J . B., Durbin, G.T., Tomarelli, R.M., Bernhart, F.W. The minimal n u t r i t i o n a l requirements.of Lacto-b a c i l l u s b i f i d u s . J . B a c t e r i o l . 6 2 , 771 - 7 7 7 , 1 9 6 1 . 2 2 . Huhtanen, C.N. Pantethine and casein hydrolysate in the growth of certa i n L a c t o b a c i l l i . Proc. Soc. Exptl. B i o l . Med. 88. 311 - 3 1 2 , 1955. 2 3 . Kaloud, H. and Stogmann, W. C l i n i c a l experience with a bifid u s milk feed. Arch. Kinderheilk 1 7 7 , 2 9 - 3 5 , 1968. 2 4 . Kogima, M., Suda, S., Hotta, S. and Hamada, K. Induction of pleomorphism in Lactobacillus b i f i d u s . J . B a c t e r i o l . 9 5 , 710 - 7 1 1 , 1968. - 27 -25. Lambert, R., Saito, Y. and Veerkamp, 3 . H. Incorporation of labelled derivatives of 2-deoxy-2-amino-D-glucose into the c e l l walls of Lactobacillus b i f idus var, .pen-nsylvanicus. Arch. Biochem. Biophys. 110. 431 - 345, 1965. 26. Lambert, R. and Z i l l i k e n , F. Novel growth factors for Lactobacillus b i f i d u s var. pennsylvanicus. Arch. Biochem. Biophys. 110, 544 - 550, 1965. 27. Merchant, I.A. and Packer, R.A. Etiology, diagnosis and control of infectious bovine m a s t i t i s . Burgess Pub. Co., Minneapolis, 1944. 28. Meyer, J.B., Tewes, G. and Dittman, J . Metabolism of Lactobacillus b i f i d u s : II. Formation of l a c t i c acid in Tomarelli's s o l u t i o n . Z. Kinderheilk 90, 368-373. 1964. 29. Meyer, J. B., Tewes, G. and Dittman, J. Metabolism of Lactobacillus b i f i d u s : VI. Lactic acid content of several cultures. Z. Kinderheilk 92, 274 - 278, 1965. 30. Nashizawa, Y., Komada, T., Seki, Y. and Nogami, K. Physiological action of Lactobacillus b i f i d u s . Arch. Pediat. 76, 97 - 109, 1959. 31. Norris, R.F., Flanders, T., Tomarelli, R.M. and Gyorgy, P. Isolation of Lactobacillus b i f i d u s : a comparison of branched and unbranched s t r a i n s . J . B a c t e r i o l . 60. 681 - 686, 1950. 32. Nouvel, L. Medicinal compositions containing a n t i b i o t i c r e s i s t e n t coliform bacteria. Belg. 644,455 August 27, 1964, Fr. 33. Orla-Jensen, S., Orla-Jensen, A.D. and Winter, 0. Bacterium bifidum and Thermobacterium i n t e s t i n a l e . Zentr. Bakt. Parasitenk. II, 93., 321 0 343, 1936. 34. Olsen, E. Studies on the i n t e s t i n a l f l o r a of infants. Erjnar Munksgaard, Copenhagen. 35. Pine, L. Fixation of carbon dioxide by Actinomycetes and Lactobacillus b i f i d u s . Proc. Soc. Exptl. B i o l . Med. 93, 468 - 472, 1956. 36. Pine, L. and Howell, J r . , A. Comparison of physiological and biochemical c h a r a c t e r i s t i c s of Actinomycetes Spp. with those of Lactobacillus b i f i d u s . J . Gen. Microbiol. 15, 428 - 446, 1956. ~ - 28 -37. Sachiko, K., Makajima, T. and Tamura, Z. Purification of a new bifidus factor from carrot root. Ghem. Pharm. Bull. (Tokyo) 13, 1262 - 1 2 6 3 , 1 9 6 5 . 38. Semenikhina, V.F. Acid forming and antibiotic activity of Lactobacillus bif idus. Moloch. Prom. 2JS, 17 - 20, 1 9 6 7 . 39. Sheggs, H.R., Spizzen, J., and Wright, R.D. Competitive antagonism of ribonucleic and deoxyribonucleic acid in -the nutrition of Lactobacillus bifidus. J. Amer. Chem. Soc. 72, 811 - 813, 1950 . 40. Shimomura, R. Bacterial synthesis and destruction of thiamine: I. Synthesis of thiamine by Lactobacillus  Bifidus. Vitamins (Japan) 10, 191 - 1 9 4 , 1956 . 41. Shimomura, R. Bacterial synthesis and destruction of thiamine: II. Synthesis of thiamine by Lactobacillus  bif idus in the caecum of rats. Vitamins (Japan) 10. 195 - 1 9 9 , 1956 . 42. Springer, G.F., Rose, C.S., and Gyorgy, P. Blood group mucoids: distribution and growth promoting properties for Lactobacillus bifidus var. pennsylvanicus. J. Lab. C l i n i c a l Med. 43, 532 - 5 4 2 , 1954 . 43. Standard Methods for the examination of Dairy Products 1 2 t h ed. Published by the American Public Health Assoc. Inc., 1 9 6 7 . 4 4 . Sundman, V. and Af Bjorksten, K. The globular involution forms of the Bi f i d Bacteria. J. Gen. Microbiol. 1 9 . 491 - 496, 1 9 5 8 . 45* Sundman, V., Af Bjorksten, K. and Gyllenberg, H.G. Morphology of the Bifid Bacteria (organisms previously incorrectly designated Lactobacillus bif idus) and some related genera. J. Gen Microbiol. 21, 371 - 384, 1959. 46. Taisho Pharmaceutical Co. Ltd. Dried pharmaceutical preparations containing lactic acid bacillus. Japan 10,900 August 10, 1966. 47. Taisho Pharmaceutical Co. Ltd. Chemical compounds to improve milk. Neth. Appl. 6 , 5 0 1 , 124 ( c l . A. 23c) August 2 , 1 9 6 5 . Japan) 48. Tervalon, N.V. Additives for baby foods. Dutch 82,509 August 15, 1956 . - 29 -49. Tervalon, N.V. Bi f i d u s - a c t i v e food preparations. Neth. 97,911 May 15, 1961. 50. T i s s i e r , H. Recherches sur l a f l o r e i n t e s t i n a l e normale des enfants ages d'un an a cinq ans. Ann. Inst. Pasteur 22, 189 - 297, 1908. 51. Tomarelli, R.M., Norris, R.F., and Gyorgy, P. I n a b i l i t y of vitamin Bj2 t o replace the deoxyribonucleic acid requirement of Lactobacillus b i f i d u s . J . B i o l . Chem. 179. 485 - 486, 1949. 52. Tomarelli, R.M., Norris, R.F. Gyorgy, P., Hassinen, J.B. and Bernhart, F.W. N u t r i t i o n of variants of Lacto-b a c i l l u s b i f i d u s . J . B i o l . Chem. 181, 879 - 888, 1949. 53. Tomarelli, R.M., Eckardt, E.R. and Bernhart, F.W. B i o l o g i c a l l y active material f o r promoting the growth of Lactobacillus b i f i d u s . U.S. 2, 792,389 May 14, 1957. 54. Tomarelli, R.M., Norris, R.F., Rose, C.S. and Gyorgy, P. The e f f e c t of f a t t y acids on the growth of strains of Lactobacillus b i f i d u s . J . B i o l . Chem. 187, 197 - 204, 1950. 55. Veerkamp, J.H., Lambert, R. and Saito, Y. Composition of the c e l l walloof Lactobacillus b i f i d u s var. pen-nsylvanicus. Arch. Biochem. Biophys. 112. 120 - 125, 1965. 56. Wang, M., Steers, E. and Norris, R.F. E x t r a c e l l u l a r polysaccharide of mucoid Lactobacillus b i f i d u s . J . Ba c t e r i o l . 86, (5) 898 - 903, 1963. 57. Weiss, J.E. and Rettger, L.F. Lactobacillus b i f i d u s . J . B a c t e r i o l . 28, 501 - 521, 1934. 58. Williams, N.B., Norris, R.F. and Gyorgy, P. Antigenic and c u l t u r a l relationships of Lactobacillus b i f i d u s and Lactobacillus parabifidus. J . Infect. Dis. 92. 121 - 123, 1953. 59. Wolf, H. Ef f e c t of bifi d u s colonization on the metabolism of f a t , protein and vitamin B. Ernaehrungsforschung 10, 174 - 178, 1965. 60. Yanai, T. Thiamine syntheses by b a c t e r i a l c e l l s : I. Syntheses of thiamine by washed c e l l s of Lactobacillus  b i f i d u s . Vitamins (Kyoto) 19, 391 - 394, 1960. -30-61. Yoshioka, M., Yoshioka, S., Tamura, Z. and Ohta, K. Growth responses of Bifidobacterium bifidum to coenzyme A, its precursors and carrot extract. Japan J. Microbiol. 12., 395 - 402, 1968. 62. Z i l l i k e n , F.W. Formulations for infant feeding. U.S. 3,274,003 (61. 99 - 54) September 20, 1966. 63. Zillikem, F., Smith, P.N., Rose, C.S. and Gyorgy, P. Synthesis of 4-()-B-D-galactopyranosyl-N-acetyl-D-glucosamine by intact cells of Lactobacillus bifidus var. pennsylvanicus. J. Biol. Chem. 217. 79 - 82, 1955. APPENDICES APPENDIX I Colony Morphology - 32 -Plates 1 and 2: Forty-eight hour cultures of b i f i d bacteria showing the colony morpgological v a r i a b i l i t y . ( x 0.85 ) - 33 -APPENDICES (cont'd) APPENDIX II Bacterial Morphology - 34 -Plate 3: Strain AXI^ in its branching morphology (x 1,430) Plate 4: Strain AVII in its branching morphology o (x 1,430) P l a t e 3 P l a t e 4 - 35 -„ Plate 5: Strain in its branching morphology (x 1,430) Plate 6: Strain A I I ^ in i t s branching morphology (x 1,430) P l a t e 5 P l a t e 6 - 36 -Plate 7: Strain Alg in its branching morphology (x 1,430) Plate 8: Strain AXIg in its unbranched morphology (x 1,430) - 37 -Plate 9: Stra i n AVIIg in i t s unbranched morphology (x 1,430) Plate 10: Strain AV|7 i n i t s unbranched morphology (x 1,430) P l a t e 9 P l a t e 10 - 38 -Plate 11: St r a i n A I I ^ i n i t s unbranched morphology (x 1,430) Plate 12: St r a i n AI^ in i t s unbranched morphology (x 1,430) Q P l a t e || P l a t e 12 

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