Biodiversity of Beneficial Bacteria in Infant Feces and Their Potential Probiotic Functions

Abstract

Gut colonization of bifidobacteria in early infancy is essential for the well- being of the infant. Several factors have involved in bifidobacterial colonization, such as delivery mode, infant age, antibiotic treatment and feeding type. Factorial experimental design was applied to evaluate the factors including delivery modes (natural born and Cesarean born), feeding types (breast milk, formula milk and combination milk) and ages of infant (1 week, 1 month, 2 months, 3 months, 4 months and 5 months) on the bifidobacterial population and composition of infant feces. Among 6 healthy newborn infants, infant age and feeding type were the most influential factor for the population of bifidobacteria, clostridia, bacteroides and eubacteria, whereas feeding type and delivery mode were the main factors influencing total bacteria population. The amount of bifidobacteria rapidly increased from first week to 2 months of infant age and stable then after 2 months in all of infants. Moreover, the amount of clostridia, bacteriodes and eubacteria significantly increased from the first week up to 5 months of ages in all of infants. Feeding type was also main factor influencing on population of bifidobacteria, clostridia, bacteroides, eubacteria and total bacteria. The exclusive breast-feeding greatly supported the bifidobacterial growth. The level of bifidobacteria in both naturally born, exclusive breast fed infant (BF1-D) and Cesarean born, exclusive breast fed infant (BF2-E) was significantly higher than other infants. However, the highest level of bifidobacteria was found in naturally born, exclusive breast fed infant (BF1-D) from first week to 5 months of ages (8.79 to 9.83 log CFU/g feces). In addition, the amount of bifidobacteria in naturally born, formula fed infant (FF1-F) greatly increase (8.82 to 9.45 log CFU/g feces) when received the exclusive formula milk supplemented with B. lactis at 2 months to 5 months of age. Using antibiotic (amoxicillin) in Cesarean born, combination fed infant (CF2-C) was significantly decreased the amount of bifidobacteria (9.21 to 8.77 log CFU/g feces) at 3 months of age. Moreover, formula milk seemed to enhance the growth of clostridia and bacteroides. The highest level of the clostridia was found in Cesarean born, exclusively formula fed infant (FF1-F) (9.22 log CFU/g feces) when received exclusive formula milk supplemented with FOS and GOS at 2 months of age. However, the amount of clostridia was significantly decreased when received the exclusive formula milk supplemented with B. lactis. Conversely, low level of clostridia was found in both naturally born, combination fed infant (CF1-A) and naturally born, exclusively breast fed infants (BF1-D). In addition, the amount of bacteroides was significantly increased (8.59 to 9.19 log CFU/g feces) in Cesarean born, formula fed infant (FF2-H) at the first week to 5 months of age. The amount of total bacteria in naturally born, exclusively breast fed infant (BF1-D) and naturally born, exclusively formula fed infant (FF1-F) was significantly higher than another infants. PCR-DGGE analysis demonstrated that there was change in the bifidobacterial community composition associated with feeding type and delivery mode. The high numbers of bifidobacteria band were observed in both naturally born, exclusively breast fed infant (BF1-D) and naturally born, combination of breast and formula fed infants (CF1-A). However, the highest number of bifidobacterial species was observed in both naturally born, exclusively formula fed infant (FF1-F) and naturally born, combination of breast and formula fed infants (CF1-A). Several species of bifidobacteria including Bifidobacterium adolescentis, Bifidobacterium sp. Bifidobacterium longum, Bifidobacterium catenulatum and Bifidobacterium breve were found persistently from the first week to 5 months of ages in naturally born, both of breast fed (BF1-D) and combination fed infants (CF1-A). The most common bifidobacterial species found in healthy infants were Bifidobacterium sp. and B. longum. Feeding type in infancy is the most significant determinant in shaping the profile of intestinal microbiota at early life. The population level and diversity of lactic acid bacteria (LAB) present in the feces of infants were compared between breast-, formula- and combined-feeding types. There were two infant formulae with FOS-GOS and inulin-GOS supplementations. The number of fecal LAB from the infants fed with the combination diet of breast milk and a formula containing FOS- GOS (CF1-A infant) and inulin-GOS (CF2-C infant) was significantly higher than that from both exclusively breast-fed (BF1-D infant and BF2-E infant) and exclusively formula fed ones (FF1-F infant and FF2-H infant) (P<0.05). According to PCR-DGGE analysis, the band richness of LAB population in infants with combination (CF1-A infant and CF2-C infant) and exclusive formula feeding (FF1-F infant and FF2-H infant) and the exclusively breast-fed infants (BF2-E) was not different, while the number of LAB in exclusively breast fed infant (BF1-D) was lower than another. Type of feeding had not a significant influence on LAB diversity. Fourteen species of LAB were found from 6 infants and each infant presented 4-6 species of LAB and the most number of LAB species was found in the infant with the exclusive breast fed infant (BF2-E infant) and exclusive formula fed diet supplemented with FOS-GOS (FF1-F infant). Moreover, uncultured bacteria were commonly found in the commensal intestinal microbiota in Thai infants, independently on feeding type. They were detected abundantly in feces of all infants. Combination feeding was able to support the growth of L. plantarum and Leu. mesenteroides. The exclusive feeding with formula supplemented with FOS-GOS enhance growth of L. rhamnosus and L. gasseri, while the formula supplemented with inulin-GOS sustained growth of L. gasseri, L. fermentum and L. paracasei. In addition, exclusive breast milk feeding was capable to promote the growth of L. gasseri, L. crispatus, L. plantarum and L. helveticus. Thirty strains of lactic acid bacteria (LAB) and four strains of bifidobacteria isolated from healthy infant feces and breast milk were evaluated for cholesterol- lowering activity through various mechanisms. Enterococcus feacalis (10), Enterococcus feacium (6), Lactobacillus plantarum (7), Lactobacillus casei (2), Lactobacillus rhamnosus (4), Lactobacillus paracasei (1), Bifidobacterium longum (2) and Bifidobacterium bifidum (2) were determined for the ability to assimilate cholesterol from growth media, bile salt hydrolase activity and cholesterol binding property. All of 34 strains were able to assimilate cholesterol in the 0.3 % oxgall bile containing medium in the range of 14.39-65.57 μg/ml using O-phthalaldehyde method. L. plantarum 108 displayed the highest assimilation. The bile salt hydrolase (BSH) activity was only confined among nine strains of Ent. faecalis and five strains of Ent. faecium by showing precipitation zone on MRS agar supplemented with 0.5% (w/v) sodium salt of taurodeoxycholic acid (TDCA). The cholesterol binding ability of the resting cells (live cells) and dead cells (heat-killed cells) was exhibited within all LAB and bifidobacteria range of 7.62-19.49 mg/g dry weight and 3.48-13.65 mg/g dry cell weight. Furthermore, Ent. faecium EMA410.4, Ent. faecalis M134 and Ent. faecalis EM17.3 strongly adhered to Caco-2 and HT-29 cell lines. According to this study, Ent. faecium EMA410.4, Ent. faecalis M134 and Ent. faecalis EM17.3 had greatly ability to produce BSH enzyme, remove cholesterol from the media during growth, remove cholesterol via binding to cellular surface of resting and dead cell and strongly adhered to Caco-2 and HT-29 cell lines. Therefore, these strains can be potentially applied as a good probiotic candidate for lowering cholesterol level.