Differentiation of Bifidobacterium longum subspecies longum and infantis by quantitative PCR using functional gene targets.

BACKGROUND: Members of the genus Bifidobacterium are abundant in the feces of babies during the exclusively-milk-diet period of life. Bifidobacterium longum is reported to be a common member of the infant fecal microbiota. However, B. longum is composed of three subspecies, two of which are represented in the bowel microbiota (B. longum subsp. longum; B. longum subsp. infantis). B. longum subspecies are not differentiated in many studies, so that their prevalence and relative abundances are not accurately known. This may largely be due to difficulty in assigning subspecies identity using DNA sequences of 16S rRNA or tuf genes that are commonly used in bacterial taxonomy. METHODS: We developed a qPCR method targeting the sialidase gene (subsp. infantis) and sugar kinase gene (subsp. longum) to differentiate the subspecies using specific primers and probes. Specificity of the primers/probes was tested by in silico, pangenomic search, and using DNA from standard cultures of bifidobacterial species. The utility of the method was further examined using DNA from feces that had been collected from infants inhabiting various geographical regions. RESULTS: A pangenomic search of the NCBI genomic database showed that the PCR primers/probes targeted only the respective genes of the two subspecies. The primers/probes showed total specificity when tested against DNA extracted from the gold standard strains (type cultures) of bifidobacterial species detected in infant feces. Use of the qPCR method with DNA extracted from the feces of infants of different ages, delivery method and nutrition, showed that subsp. infantis was detectable (0-32.4% prevalence) in the feces of Australian (n = 90), South-East Asian (n = 24), and Chinese babies (n = 91), but in all cases at low abundance (<0.01-4.6%) compared to subsp. longum (0.1-33.7% abundance; 21.4-100% prevalence). DISCUSSION: Our qPCR method differentiates B. longum subspecies longum and infantis using characteristic functional genes. It can be used as an identification aid for isolates of bifidobacteria, as well as in determining prevalence and abundance of the subspecies in feces. The method should thus be useful in ecological studies of the infant gut microbiota during early life where an understanding of the ecology of bifidobacterial species may be important in developing interventions to promote infant health.

Dietary Effects on Plasma Glycerophospholipids.

OBJECTIVE: Human milk provides a complex mixture of animal lipids, whereas the fat supply of most modern infant formula is based on vegetable oils. We studied the effects of breast-feeding and of feeding infant formula either without or with dairy goat lipids on the composition of infant plasma glycerophospholipids. METHODS: Healthy-term infants were randomized double blind to feeding with infant formula based on whole goats' milk (GIF, approximately 60% milk fat and 40% vegetable oils) or a control cows' milk infant formula based on vegetable oils (VIF) from 2 weeks after birth. A reference group of fully breast-fed infants was also followed. At the age 4 months, blood samples were collected and plasma glycerophospholipids were analyzed with liquid chromatography coupled to triple quadrupole mass spectrometry. RESULTS: The group of breast-fed infants showed significantly higher contents of glycerophospholipid species containing sn-2 palmitic acid [PC(16:0/16:0) and PC(18:0/16:0)] and significantly higher contents of glycerophospholipid species containing long-chain polyunsaturated fatty acids than infants in both formula groups. The GIF group demonstrated significantly higher glycerophospholipid species containing myristic acid [LPC(14:0), PC(14:0/18:1), PC(16:0/14:0)] and palmitoleic acid [LPC(16:1), PC(16:0/16:1), and PC(16:1/18:1)] than the VIF group. CONCLUSIONS: We conclude that breast-feeding induces marked differences in infant plasma glycerophospholipid profiles compared with formula feeding, whereas the studied different sources of formula fat resulted in limited effects on plasma glycerophospholipids.

Nutritional adequacy of goat milk infant formulas for term infants: a double-blind randomised controlled trial.

The safety and nutritional adequacy of goat milk infant formulas have been questioned. The primary aim of the present study was to compare the growth and nutritional status of infants fed a goat milk infant formula with those of infants fed a typical whey-based cow milk infant formula. The secondary aim was to examine a range of health- and allergy-related outcomes. A double-blind, randomised controlled trial with 200 formula-fed term infants randomly assigned to receive either goat or cow milk formula from 2 weeks to at least 4 months of age was conducted. A cohort of 101 breast-fed infants was included for comparison. Weight, length and head circumference were measured at 2 weeks and 1, 2, 3, 4, 6 and 12 months of age. Nutritional status was assessed from serum albumin, urea, creatinine, Hb, ferritin, and folate and plasma amino acid concentrations at 4 months. Z-scores for weight, length, head circumference and weight for length were not different between the two formula-fed groups. There were differences in the values of some amino acids and blood biomarkers between the formula-fed groups, but the mean values for biomarkers were within the normal reference range. There were no differences in the occurrence of serious adverse events, general health, and incidence of dermatitis or medically diagnosed food allergy. The incidence of parentally reported blood-stained stools was higher in the goat milk formula-fed group, although this was a secondary outcome and its importance is unclear. Goat milk formula provided growth and nutritional outcomes in infants that did not differ from those provided by a standard whey-based cow milk formula.

Comparison of the compositions of the stool microbiotas of infants fed goat milk formula, cow milk-based formula, or breast milk.

The aim of the study was to compare the compositions of the fecal microbiotas of infants fed goat milk formula to those of infants fed cow milk formula or breast milk as the gold standard. Pyrosequencing of 16S rRNA gene sequences was used in the analysis of the microbiotas in stool samples collected from 90 Australian babies (30 in each group) at 2 months of age. Beta-diversity analysis of total microbiota sequences and Lachnospiraceae sequences revealed that they were more similar in breast milk/goat milk comparisons than in breast milk/cow milk comparisons. The Lachnospiraceae were mostly restricted to a single species (Ruminococcus gnavus) in breast milk-fed and goat milk-fed babies compared to a more diverse collection in cow milk-fed babies. Bifidobacteriaceae were abundant in the microbiotas of infants in all three groups. Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium bifidum were the most commonly detected bifidobacterial species. A semiquantitative PCR method was devised to differentiate between B. longum subsp. longum and B. longum subsp. infantis and was used to test stool samples. B. longum subsp. infantis was seldom present in stools, even of breast milk-fed babies. The presence of B. bifidum in the stools of breast milk-fed infants at abundances greater than 10% of the total microbiota was associated with the highest total abundances of Bifidobacteriaceae. When Bifidobacteriaceae abundance was low, Lachnospiraceae abundances were greater. New information about the composition of the fecal microbiota when goat milk formula is used in infant nutrition was thus obtained.

Association of infant feeding practices in the general population with infant growth and stool characteristics.

This was a prospective cohort study of 976 infants from birth to 12 months of age. Infants were fed breast milk, goat infant formula, cow infant formula, or a combination of formula and breast milk during the first 4 months of age. Data on type of milk feeding and infant growth (weight and height) were collected at birth and at 4, 8, and 12 months during routine clinical assessment. The number and consistency of bowel motions per day were recorded based on observational data supplied by the mothers. Infants fed breast milk or goat or cow infant formula during the first 4 months displayed similar growth outcomes. More of the infants fed cow infant formula had fewer and more well-formed bowel motions compared with breast-fed infants. The stool characteristics of infants fed goat formula resembled those of infants fed breast milk.

The effect of formulated goats' milk on calcium bioavailability in male growing rats.

BACKGROUND: There are two main proteins in milk; whey and casein. Casein contains casein phosphopeptides (CPP), which are released on digestion of the milk. These may increase calcium solubility by binding calcium in the small intestine. Thus increasing casein in the diet may help to stimulate bioavailability of calcium and increase bone density. The present study tested this hypothesis in growing male rats fed diets containing three different concentrations of casein from goat milk. RESULTS: Rats fed the diet containing no casein had significantly lower calcium absorption when compared to rats fed the diets that contained 80% and 57% of goat milk protein as casein; however, no significant difference was observed between rats fed diets with 80% and 57% casein. The varying amounts of casein had no effect on mineral uptake or retention in the femur. Biomechanical testing and mineral analysis of the femurs showed no differences between diet groups. The mechanism to explain this lack of retention remains unclear. CONCLUSION: The diets containing 80% and 57% of goat milk protein as casein delivered increased calcium absorption compared to the diet containing no casein, suggesting a minimum level of casein is needed to optimize calcium absorption from goat milk.

Amino acid composition determined using multiple hydrolysis times for three goat milk formulations.

The amino acid composition of goat milk formulations with varying protein and carbohydrate concentrations were determined. Proteins in goat milk infant formula, goat milk growing-up formula and goat whole milk powder were hydrolysed using multiple hydrolysis time intervals. A least-squares non-linear regression model was used to predict the free and protein bound amino acid concentrations. The amino acid composition of goat infant formula was compared with human milk reference values. There was good agreement between the multiple hydrolysis and single 24-h hydrolysis methods for approximately one-half of the amino acids. Tryptophan, aspartic acid, threonine, tyrosine, isoleucine, valine, serine and alanine contents were underestimated by 10.6, 5.6, 5.6, 4.7, 4.4, 3.7, 3.7 and 3.6%, respectively, by the single 24-h hydrolysis. The study provides accurate reference data on the amino acid composition of goat milk powders. Goat milk infant formula has amino acids in amounts similar to human milk reference values, when expressed on a per-energy basis.

Composition of the non-protein nitrogen fraction of goat whole milk powder and goat milk-based infant and follow-on formulae.

The non-protein nitrogen fraction of goat whole milk powder and of infant and follow-on formulae made from goat milk was characterized and compared with cow milk powder and formulae. Goat milk infant formula contained 10% non-protein nitrogen, expressed as a proportion of total nitrogen, compared with 7.1% for cow milk formula. Goat follow-on formula contained 9.3% and cow 7.4% non-protein nitrogen. Urea, at 30%, was quantitatively the most abundant component of the non-protein nitrogen fraction of goat milk and formulae, followed by free amino acids at 7%. Taurine, glycine and glutamic acid were the most abundant free amino acids in goat milk powders. Goat milk infant formula contained 4 mg/100 ml total nucleotide monophosphates, all derived from the goat milk itself. Goat milk has a very different profile of the non-protein nitrogen fraction to cow milk, with several constituents such as nucleotides at concentrations approaching those in human breast milk.