Palmitic acid (PA) is the qualitatively and quantitatively major saturated fatty acid (FA) in human milk. Unlike that of other FAs, the PA concentration is highly conserved in human milk, regardless of the ethnic origin or nutritional status of the woman; PA comprises 17% to 25% of the total FAs, and approximately 70% to 75% of the PA is esterified in the sn-2 (β) position of triglycerides (TGs) (β-palmitate) (1). In contrast to human milk fat, most PA from several vegetable sources (ie, palm oil) is esterified in the sn-1 and sn-3 positions (2), whereas the sn-2 position is usually occupied by unsaturated FAs. Because palm oil is commonly used in infant formulas, the position of PA in the TGs of most commercialized infant formulas differs from its position in human milk TGs.
During TG digestion, the FAs esterified in the sn-1 and sn-3 positions are released, whereas those esterified in the sn-2 position remain unhydrolyzed (3). Human milk PA is absorbed as an sn-2 monoacylglycerol (4) and is conserved as such through the digestion, absorption, and chylomicron TG synthesis (5); however, after digestion, the free PA molecules solidify in the intestine because of their high melting temperature, creating insoluble and indigestible complexes with dietary minerals (eg, calcium) (6), and causing hard stools (7). Clinical studies on term and preterm infants have shown up to a 20% increase in the PA absorption with β-palmitate–enriched infant formulas compared with standard palm oil formulas (8,9). Compared with standard palm oil, the energy absorbed increased by approximately 0.2 g · kg−1 · day−1 (5%) when using β-palmitate (8–12). The beneficial effects of β-palmitate as compared with standard palm oil include increased calcium absorption (8–12) and bone strength (11,13).
The intestinal microbiota is an essential “organ” that serves numerous important functions for the human host (14–16). The intestine is rapidly colonized after birth. Unlike the stable microbial flora of adults, during the first 2 years of life the intestinal microbiota is continuously modified. Therefore, the critical stages of gut colonization during infancy are probably after birth and during weaning (17). The composition and diversity of the intestinal flora in infants are influenced by nutrition and environmental and genetic factors (15,18,19). Indeed, differences in the fecal flora have been observed between breast-fed (BF) and formula-fed infants; the reduced risk of gastrointestinal infection in BF infants has been attributed to their intestinal microbiota (20). It is believed that human milk is an important factor in the initiation, development, and composition of the intestinal microbiota. Human milk provides a source of microorganisms such as lactic acid bacteria and bifidobacteria (21) as well as components such as oligosaccharides that act as substrates for the growth of beneficial bacterial strains and/or the inhibition of other microorganisms (19,22).
There is a particular interest in modulating the composition of the intestinal microbiota to possibly influence the future health status of the individual. This may be achieved through supplementation with probiotics and prebiotics (19). We hypothesize that increasing the amount of PA esterified in the sn-2 position in infant formula (thereby making it more similar to human milk) influences the microbiota profile of formula-fed infants. Therefore, we studied the effect of infant formula enriched with β-palmitate on the microbiota profile.
Study Design and Participants
Healthy term infants (born at gestational age [GA] ≥37 weeks), who were appropriate for GA and younger than 7 days, were eligible for participating in a 2-center, randomized, double-blind study. Infants were eligible for inclusion in the study if the mother had unequivocally decided not to breast-feed (for formula-fed infants) or to breast-feed (for BF infants). Infants were excluded from the study if they experienced congenital or chromosomal disorders, neonatal morbidities, or metabolic illnesses, or if they (or their mothers) received antibiotics around the time of birth. Infants who received antibiotics at any time throughout the study period were not included in the data analyses.
Formula-fed infants were randomized (using sealed envelopes) in blocks of 4 with stratification. According to the study protocol, twins were assigned to the same group, and thus received the same formula. Stratification was also conducted for twins. To avoid a possible bias, the second born of each set of twins was removed from the dataset and not included in the data analyses.
The feeding effect was examined during 6 postnatal weeks. BF infants constituted the nonrandomized reference group. Formula-fed infants were randomly assigned to 1 of 2 formulas: an infant formula with a high β-palmitate (HBP) content, in which 44% of the PA was esterified to the sn-2 position of the formula TG (HBP group), or an infant formula with a standard vegetable oil mix (low β-palmitate [LBP] content), in which 14% of the PA was esterified to the sn-2 position of the formula TG (LBP group). Both formulas were produced by the same manufacturer (Materna Laboratories, Kibbutz Maabarot, Israel) and under the same conditions, using identical ingredients. The fat composition of each formula was a vegetable oil mix consisting of palm kernel oil, rapeseed oil, sunflower oil, and either a structured palm oil (InFat, Advanced Lipids AB Sweden, Karlshamn) or a standard unmodified palm oil (control formula). The fat of both formulas differed mainly in their FA structure: the HBP formula was enriched with β-palmitate (InFat), and the LBP formula was enriched with the standard unmodified palm oil. Supplementary Table 1 (http://links.lww.com/MPG/A185) lists the compositions of both formulas.
The study was conducted according the principles of the Declaration of Helsinki and the Good Clinical Practices. The protocol was approved by the ethics committees of the Meir Medical Center, Kfar-Saba, Israel; the Bnai-Zion Medical Center, Haifa, Israel; and the Israeli Ministry of Health. Both parents and/or legal guardians of the infants were provided with written informed consents.
The formula consumption data were obtained from follow-up diaries filled by the parents or legal guardians; the time of feeding and the amount of formula consumed were documented.
Growth parameters were measured at enrollment and at 6 postnatal weeks. The measurements included the following variables: body weight (the average of 3 measurements by a Model 20 Tabletop Infant Scale, Olympic Medical, Seattle, WA), body length (the average of 2 measurements of recumbent crown-heel length to the nearest 0.1 cm by a O’Leary Preemie LengthBoard, Ellard Instrumentation Ltd, Monroe, WA), and fronto-occipital head circumference (using a standard 1-cm-wide measuring tape to the nearest 0.1 cm).
Intestinal Flora Analyses
A stool sample (∼1 g) was collected from each infant at enrollment and at 6 postnatal weeks. The analyzed microorganisms included the total aerobic/facultative aerobic bacteria, lactobacilli, bifidobacteria, coliforms, enterococci, staphylococci, clostridia, Escherichia coli, and pseudomonas. Stool samples were collected in sterile bottles, kept in a 4°C refrigerator, and analyzed within 24 hours. The cold stool samples were aseptically weighed and suspended in an anaerobic sterile saline (0.85% NaCl) to obtain a 1:10 dilution. Samples were treated in a stomacher at high speed for 1 minute. The colony-forming units (CFU) were determined by first plating different dilutions of the stool samples on a series of specific agar plates and then incubating the plates anaerobically or aerobically according to the manufacturers’ recommendations. The selective media used in the present study included plate count agar (Oxoid Ltd, London, UK), for total aerobic/facultative aerobic bacterial counts; Man-Rogosa-Sharpe agar (MRS) (Oxoid) containing L-cysteine/0.05% HCl (Merck, Darmstadt, Germany), for lactobacilli; MRS-cysteine (described above) supplemented with LiCl (1 mg/mL) (Merck) and dicloxacillin (0.5 μg/mL) (Sigma-Aldrich, Rehovot, Israel) (MRS+++), for bifidobacteria; and sulfite-polymyxin-sulfadiazine agar (Difco, Laboratories, Detroit, MI), for clostridia. The incubation time was 5 days for MRS+++; 24 hours for MacConkey, Tryptone Bile X-Glucuronide medium, and Pseudocent, and 48 hours for all remaining media. All stool samples were plated in duplicates. It should be noted that several studies have shown that the media composition affects the total number of recovered bacteria. The commercial media used in the present study were chosen from the numerous media described in the literature (23).
The baseline (BL) characteristics of the mothers and infants from the 2 formula groups were compared using a pairwise t test for scale outcomes and a pairwise χ2 test for nominal outcomes. The bacterial mean counts were compared with the Poisson regression and the generalized estimating equations (distribution = Poisson and link = log) using the SPSS version 18 software (SPSS Inc, Chicago, IL). The model was rerun adjusting for BL levels to account for BL differences. A subset analysis was performed on infants born by vaginal delivery to avoid any effects caused by this type of delivery. BF infants served as the reference group; therefore, all parameters were compared with the BF infants. A P < 0.05 was considered to be statistically significant with no correction for multiple testing.
Thirty-six term infants were enrolled in the study. Fourteen infants were exclusively BF (BF group), and 22 infants were randomly enrolled either to the HBP (n = 14) or LBP formula-fed groups (n = 8). Six infants (16.67%) dropped out during the study period (Fig. 1). The statistical analyses excluded data obtained from the second born twin.
Demographic Characteristics of Mothers and Infants
No significant differences in maternal characteristics were observed between the formula-fed groups. Maternal education was higher in the BF group than in the HBP group.
Infants in the HBP group were enrolled at a significantly lower GA than infants in the LBP group (38.8 ± 1.2 vs 39.9 ± 1.0 weeks, respectively, P = 0.045). Birth weight was not significantly different between the 2 groups. Further details are available in supplementary Table 2 (http://links.lww.com/MPG/A186).
At BL, no significant differences were found in the body weight, length, and head circumference between the HBP and LBP formula-fed groups. At 6 weeks, no significant differences in weight and length were observed between the 2 formula-fed groups; however, the head circumference was lower in the HBP group than in the BF group (37.1 ± 0.8 vs. 38.3 ± 1.1 respectively, P = 0.019). Further details are available in supplementary Table 3 (http://links.lww.com/MPG/A187).
Infant growth was also analyzed using z scores for weight, length, and head circumference obtained from the World Health Organization Child Growth curves (http://www.who.int/childgrowth/software/en). All z score parameters were found to be within the normal growth range (data not shown).
At 6 weeks, there were no significant differences between the HBP and LBP groups in the amount of formula consumed. No supplementary feeding was reported during the study.
Figure 2 shows the average counts of lactobacillus (Fig. 2A) and bifidobacteria (Fig. 2B) at BL and at 6 postnatal weeks. At BL, the bifidobacteria counts were much higher in the LBP group than in the HBP and BF groups (P < 0.05), whereas the Lactobacillus counts did not significantly differ between the 2 formula-fed groups.
At 6 weeks, both the Lactobacillus and bifidobacteria counts of the HBP and BF groups were significantly higher than the LBP group (P < 0.01). Additionally, there was no significant difference in Lactobacillus counts between the HBP and the BF groups (Fig. 2).
Because of differences in the BL counts of bifidobacteria, statistical analyses were performed controlling for BL levels, and all the results remained statistically significant.
All analyses also were performed for the subcohort of infants who were born by vaginal delivery. Similar to all completers, both the Lactobacillus and bifidobacteria counts of the HBP and BF groups were significantly higher than the LBP group at 6 postnatal weeks (P < 0.01) (data not shown).
The mean values of 6 additional microbial counts (ie, Clostridium, Staphylococcus, coliforms, E coli, Enterococcus, and Pseudomonas) are presented in Table 1. The results demonstrate that at 6 weeks, the HBP group had a pattern of bacterial counts that is comparable with that of the BF group. The resemblance between the BF and HBP groups was maintained in the subgroup infants born by vaginal delivery (data not shown). Furthermore, the ratios of the detected bacteria were also comparable in the HBP and BF groups. For example, the ratios of the bifidobacteria to clostridia counts were 4.86 × 106, 3.14 × 105, and 1630, and the ratios of the Lactobacillus to clostridia counts were 2.76 × 107, 2.23 × 105, and 3510 in the BF, HBP, and LBP groups, respectively. Furthermore, the ratios of the coliform to clostridia counts were 2.90 × 106, 2.45 × 105, and 260 in the BF, HBP, and LBP groups, respectively, and the ratios of the clostridia to Staphylococcus counts were only 1350 in the BF group and 3160 in the HBP group, but 3.29 × 105 in the LBP group.
In the present study, we measured the microbiota composition of infants in the first and sixth postnatal week following the administration of infant formulas that differed in their fat composition. We found that HBP content in the infant's diet affects the microbiota at 6 weeks; bifidobacteria and Lactobacillus species differed significantly between the HBP and LBP groups. Our results revealed that both the HBP and BF groups exhibited a notable increase in the counts of beneficial bacteria from BL to the sixth postnatal week. The LBP group, however, exhibited a smaller increase in the Lactobacillus counts and a significant decrease in the bifidobacteria counts.
Human milk provides a natural and optimal nutrition for infants until the age of 6 months (24). Despite the well-known advantages of breast-feeding, there are mothers who cannot or choose not to breast-feed and use infant formulas as a substitute. The challenge is to provide adequate nutrition that supports proper infant development and leads to a beneficial gut microbiota.
The human diet plays a major role in the gut microbiota composition and development (15,18,22). Following the first days after birth, the flora composition rapidly changes under the influence of the infant's diet, and consequently it is different between BF and formula-fed babies (18,20). Within 1 week after birth, bifidobacteria becomes the predominant species in the intestine of BF infants, whereas formula-fed infants have a more diverse flora without any prevalent microorganism (20,25,26). Our study showed that infants fed the HBP formula did not statistically differ from the BF infants in the Lactobacillus counts. This result emphasizes the importance of fat structure in infant formulas. In addition to bifidobacteria, other microorganisms such as Streptococcus and Lactobacillus were found to be associated with breast milk feeding (20), whereas clostridia, Staphylococcus, and E coli were found to be more prevalent in the intestines of formula-fed infants (20). Our data (Table 1) show that enrichment of infant formula with PA at the sn-2 position is associated with a profile of microorganisms that is similar to that found in BF infants.
The role of each bacterial strain in the intestine is not completely known yet; however, there is some evidence suggesting several beneficial effects of bifidobacteria and Lactobacillus for the human host. These bacteria are known to protect infants from pathogenic intestinal microorganisms (27), possess synergistic activity with other beneficial bacteria (28), and decrease the incidence of infantile diarrhea (29). They produce vitamins such as thiamine (B1), riboflavin (B2), biotin (B7), folate (B9), and cobalamin (B12) (28), and are associated with a higher production of short-chain FAs (ie, acetic and lactic acids), a source of energy for colonocytes. Furthermore, strains of bifidobacteria and Lactobacillus influence gut maturation processes in infants and have anti-inflammatory effects (30). The prevalence of these microorganisms in the intestine may also have long-term benefits on irritable bowel syndrome, Crohn disease, lactose intolerance, and cancer (31,32).
The effect of FAs on gut microbiota has been shown for polyunsaturated FAs (PUFA). The PUFAs and the gut microbiome interactions have been reported in several studies: the effect of PUFAs on bacterial adhesion, proliferation, and microbiota composition, and the effect of bacterial activity on PUFA absorption (33,34). Our study demonstrates that fat structure may be an important factor in the development of beneficial microbiota and it may be one of the factors contributing to the effect of human milk on gut microbiota.
It has been shown that cesarean deliveries reduce early bacterial exposure and thus delay the establishment of a stable and functionally active intestinal microbiota (25). To account for the effects of delivery type on the flora composition in early infancy (18,22,35), and in view of a significant difference in the percentage of vaginal deliveries between the HBP and LBP groups, we analyzed a subgroup of vaginally delivered infants. The beneficial effect of HBP was even more pronounced in the vaginally delivered subgroup, emphasizing the significance of HBP for the establishment of favorable flora composition.
Although the mechanism by which HBF affects the microbiota composition is unclear, the effect may be attributed to direct or indirect mechanisms. The compound 2-monopalmitate, which is the digestion product, may directly induce the adhesion and proliferation of bifidobacteria and lactobacilli and/or inhibit other competitive bacterial species in the gut. Alternatively, the indirect mechanism may be through nonabsorbed vegetable oil products (ie, calcium-palmitate insoluble complexes) that reduce the growth of the beneficial bacteria in the intestine, or through fat degradation products that activate metabolic pathways in the intestine, thereby affecting bacterial growth. Further studies are presently being performed to explore the possible mechanisms.
There are several limitations in our pilot study. First, the study had a small sample size. Second, significant differences in the BL characteristics and bacterial counts were present despite proper approaches to allocation concealment. The statistical analyses did take these differences into account. Third, using a 6-week follow-up period, the long-term effects of the HBP on the microbiota remain unknown.
The strength of the present study was the randomized, double-blind design. Earlier studies demonstrated the beneficial effects of β-palmitate on stool hardness, bone strength, and calcium and fat absorption (8–12), and to the best of our knowledge, the association between TG structures in infant formula and the gut microbiota has never been investigated. A beneficial effect was demonstrated in a study examining the effect of infant formula containing β-palmitate, casein, and oligosaccharides (36), compounds known to be beneficial to gut microbiota; thus, the beneficial effect could not be attributed exclusively to β-palmitate.
In conclusion, our study demonstrates that 2 infant formulas, differing in the position of PA in the TGs, have different effects on the intestinal microbiota of infants. These findings suggest that β-palmitate may affect the intestinal microbiota composition during the first weeks of life by increasing Lactobacillus and bifidobacteria abundance in the stool, and thus may provide beneficial effects for the health and well-being of formula-fed infants. These findings further emphasize the importance of fat structure in infant formulas and suggest that part of the effect on the gut microbiota may be attributed to the lipid structure.
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