R Chierici

Intestinal microflora in early infancy: composition and development.

The neonatal intestinal microbiota is a complex ecosystem composed of numerous genera, species and strains of bacteria. This enormous cell mass performs a variety of unique activities that affect both the colonic and systemic physiology. Its primary activities include nutritive, metabolic, immunological and protective functions. Most studies of infants have been based on faecal samples using the classical plating techniques with culturing on specific media. The limitations of these methods must be taken into account when evaluating the varying results of the different studies. The establishment of the gut microbial population is not strictly a succession in the ecological sense; it is rather a complex process influenced by microbial and host interactions and by external and internal factors. The climax intestinal flora is attained in successive stages. The foetal intestine is sterile and bathed in swallowed amniotic fluid. Following delivery, multiple different antigens challenge the intestine of the newborn. The maternal intestinal flora is a source of bacteria for the neonatal gut. The bacterial flora is usually heterogeneous during the first few days of life, independently of feeding habits. After the first week of life, a stable bacterial flora is usually established. In full‐term infants a diet of breast milk induces the development of a flora rich in Bifidobacterium spp. Other obligate anaerobes, such as Clostridium spp. and Bacteroides spp., are more rarely isolated and also enterobacteria and enterococci are relatively few. During the corresponding period, formula‐fed babies are often colonized by other anaerobes in addition to bifidobacteria and by facultatively anaerobic bacteria; the development of a “bifidus flora” is unusual. In other studies the presence of a consistent number of bifidobacteria in infants delivered in large urban hospitals has not been demonstrated, whether the babies were bottle fed or exclusively breastfed. The predominant faecal bacteria were coliforms and bacteroides. According to these studies, environmental factors may be more important than breastfeeding in gut colonization after delivery. Environmental factors are indeed extremely important for the intestinal colonization of infants born by caesarean section. In these infants, the establishment of a stable flora characterized by a low incidence of Bacteroides spp. and by the isolation of few other bacteria is consistently delayed. In extremely low‐birthweight infants, hospitalization in neonatal intensive care units, characterized by prolonged antibiotic therapy, parenteral nutrition, delayed oral feedings and intubation seems to affect the composition of the intestinal microbiota. The gut is colonized by a small number of bacterial species; Lactobacillus and Bifidobacteria spp. are seldom, if ever, identified. According to the few studies so far performed, the predominant species are Enterococcus faecalis, E. coli, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus epidermidis and Staphylococcus haemolyticus. Hygienic conditions and antimicrobial procedures strongly influence the intestinal colonization pattern.

Supplementation of an adapted formula with bovine lactoferrin: 1. Effect on the infant faecal flora

The development of the infant faecal flora was studied over the first three months of life in infants receiving breast milk, a modern adapted formula and adaptations of this formula. Breast‐fed infants developed a flora rich in Bifidobacterium sp. Facultative anaerobes were ubiquitous, but in relatively small numbers within the diet group. Other obligate anaerobes, such as Clostridium sp. and Bacteroides sp. were rarely isolated. Standard formula produced a flora rich in bifidobacteria, but the growth of facultative organisms was not suppressed by this diet. Clostridium sp. and Bacteroides sp. were more common in this feeding group. After the addition of lactoferrin at 10 mg/100 ml to the formula diet, a flora similar to that of the standard formula‐fed babies was achieved. Lactoferrin at 100 mg/100 ml was able to establish a “bifidus flora” in half of the babies given this formula, but only at age three months. Clostridium sp. and Bacteroides sp. were common faecal isolates from babies receiving both the lactoferrin diets.

Dietary supplements for the lactating mother: influence on the trace element content of milk.

Milk production is a complex process where nutritional factors interact with structural hormonal and behavioural influences. In recent years important advances have been made in understanding the role of the nutritional status of lactating women on the outcome of breastfeeding. Many questions remain unanswered about the exact requirement of trace elements for lactating mothers. The effect of dietary zinc, copper and iodine supplements on the milk concentration of these micronutrients was studied. The supplementation trial employed a specific balanced nutritional supplement prepared for the nursing mothers. The study was carried out on women living in Ferrara and its surrounding area. The population under study was healthy Italian mothers, of good socioeconomic status, and their normal infants. In total, 32 women were enrolled in the study and 22 completed it. The infants (9F, 13M) were full‐term, healthy singletons and were put to breast within 12 h of birth. All women who finished the study completed a 3 d dietary record. Nutrient analysis revealed the following mean daily dietary trace element intake in the lactating mothers: zinc = 12mg, copper = 1.4 mg and iodine = 145 μg. The zinc and copper dietary intake was in agreement with the daily intake proposed for nursing Italian mothers, while the daily intake of iodine was below the recommended intake of 200 μg. The breastfeeding mothers were placed in 2 groups, with 7 primiparas and 4 multiparas per group: lactating women eating a traditional Italian diet without vitamin and mineral supplements, and lactating women enrolled in the nutrification programme and given a nutritional supplement to their traditional diet. The supplement (PerMamma Abbott) provided 20 mg zinc sulfate, 2 mg copper sulfate and 116 μg potassium iodide. These quantities cover about 60‐90% of the recommended intake for nursing Italian mothers. Samples of 10 ml of milk were collected at 3, 30, 90 d postpartum. Zinc milk concentrations declined significantly over the study period for all lactating subjects, without differences in the rate of decline between the women who started supplementation during lactation and those who did not. Copper did not change during the first month of lactation, then declined at day 90 in supplemented and unsupplemented women, without significant differences between the two groups. An early sharp decline in milk iodine occurred in all lactating subjects, independently of iodine supplementation. After the first month of lactation breast milk iodide levels remained stable in all subjects under study. No significant differences between the two study groups were observed. The lack of correlation between the iodide level in breast milk and maternal dietary intake of iodine is not in agreement with previously published reports. The present results indicate that in healthy, well‐nourished lactating Italian women, whose diet is adequate, the levels of zinc, copper and iodine in milk are not influenced by short‐term supplementary intakes and that the milk levels of the trace elements studied are maintained over different levels of intake. Further research and examination by longitudinal studies are needed to establish the exact relationship between the amount of iodine furnished to the nursing mother and the iodine content of human milk. The role of compensatory homeostatic mechanisms which act during lactation needs further consideration and closer scrutiny.

Supplementation of an adapted formula with bovine lactoferrin. 2. Effects on serum iron, ferritin and zinc levels

Breast milk provides an excellent supply of most nutrients for newborn infants. Infant formulae should be nutritionally comparable to breast milk especially with regard to critical nutrients like iron and other trace elements. Infant formulae supplemented with various amounts of bovine lactoferrin were given to two groups of infants. These infants were compared with infants receiving unsupplemented formula and breast‐fed infants. The effects of these diets on levels of haemoglobin, haematocrit, serum iron, ferritin and zinc were examined for a study period of 150 days. At birth, concentrations of iron, haemoglobin, haematocrit and zinc were comparable in all four feeding groups. The fact that the serum zinc level was not altered by lactoferrin supplementation appears to rule out an in‐vivo effect of lactoferrin on zinc nutrition of infants. Ferritin levels of breast‐fed infants were significantly higher than in non‐supplemented formula‐fed infants at day 30 and day 90. This difference was seen only at day 30, when comparing breast‐fed infants to lactoferrin‐supplemented formula‐fed infants. Comparing the infants receiving formulae, the formula supplemented with the higher amount of bovine lactoferrin induced significantly higher serum ferritin levels compared to the unsupplemented formula at day 90 and day 150. These observations favour the idea that lactoferrin may be involved in iron absorption. Since this effect was pronounced only after 90 days, it has to be discussed as to whether this effect is a convincing argument for supplementing infant formulae with bovine lactoferrin.

Advances in the modulation of the microbial ecology of the gut in early infancy.

It is now generally accepted that the microbiota of the human gut may influence health and well‐being. Lactic acid bacteria are the most important microorganisms associated with these beneficial effects and the elevated bifidobacterial count may be one of the greatest advantages that breastfed infants have over infants fed with milk formulas. Several studies relative to the selective growth stimulation of bifidobacteria, both in vitro and in vivo, are reported in this review. Over the years, diverse human milk components have been identified as the specific factors able to modulate the growth of bifidobacteria. Even if there is a certain agreement that the bifidogenic activity of human milk may be based not on single growth substances, but on a complex set of interacting factors, the present state of knowledge indicates that the use of non‐digestible but fermentable carbohydrates may be an easy and reliable method to influence the growth of lactic acid bacteria. In this context, some of the characteristics of the major physiological effects of inulin‐type fructans, of galacto‐oligosaccharides, but also of lactoferrin, a milk whey protein fraction with purported bifidogenic activity, are briefly examined.

Fecal flora measurements of breastfed infants using an integrated transport and culturing system

Sir, It is generally recognized that bacterial colonization of the intestine with indigenous flora is an important component in development of the intestinal mucosal barrier function in the human infant (1). There is accumulating evidence that the intestinal flora plays an important role in the postnatal development of the immune system (2) and promotes the absorption of nutrients in the colon (3). As a consequence, the artificial manipulation of the intestinal ecosystem by including preor probiotics in feeding formulas to generate a predominantly bifidobacterial flora even in bottle-fed infants has been attempted (4). Detailed knowledge of the microbial ecology of the human colon is essential to obtain reliable information on the effects of preor probiotics and on the influence of diet on the intestinal microflora. Technical difficulties are still a major barrier in studying the gut flora from stool samples and may represent a limiting factor in studies on the effect of early diet on the establishment of the intestinal bacterial ecosystem in the newborn infant. We therefore studied an integrated system of stool sampling, transporting and culturing in order to investigate the number of bifidobacteria and lactobacilli in stool samples of infants of up to 3 mo of age. Particular attention has been paid to the possible influence of storage time. Fifteen exclusively breastfed term infants were enrolled in the study. The Ethics Committee of the University Hospital approved the protocol of the study and informed parental consent was obtained for each infant before enrolment in the study. The infants were examined on the first day of full breastfeeding, i.e. 120 ml kg 1 d 1 (examination day 1; mean age: 5.3 2.4 d) and were re-examined after a 6 to 8-wk period of breastfeeding (examination day 2). On each examination day, the stool samples were collected in the hospital. Two samples were taken for evaluation of a possible influence by a circadian rhythm. When more than two stools were available for each infant, the first and second samples were collected at intervals of 12 h, the 24-h sampling period starting at 06.00 h. To account for data not normally distributed, the data on the microflora as well as stool frequency and consistency were described as medians and interquartile ranges (IQR, 25th–75th percentiles). The influence of the duration of feeding or the influence of duration of storage on these parameters was investigated using nonparametric tests. A linear regression analysis was performed to investigate the relationship between the two different samples from the same examination day. All tests were performed on an alpha-level of 5%. Pvalues 0.05 were considered as significant. The software used was StatView 5.0 (SAS Institute Inc.). On the second examination day, one stool of the first 5 infants was divided into four portions. Each portion was separately transferred to the transport medium, homogenized and stored at 80°C. The samples were analysed after different storage times. The first portion was analysed within 1 mo, the second after 3 mo, the third after 5 mo and the fourth portion after 8 mo. A portion of a fresh faecal sample (0.2 g) was

Lactoferrin in infant formulae

The physiological significance and the specific role of various nutrients in human milk are still not known completely. Particularly intriguing is the role of lactoferrin (Lo, one of the major components of the whey protein fraction of human milk, which together with secretory IgA (sIgA) accounts for 30-40% of total breast milk proteins ( I ) . Lactoferrin may play a role, still controversial, in host defence, iron absorption, immune responses and cell proliferation (2). An issue that has generated much interest recently is the level of nutritional utilization of this protein (3). The finding that a fraction of some proteins of human milk, such as Lf and sIgA, is not digested and can pass through the gut of the infants almost intact and in an active form has questioned the common belief that these proteins are available completely for nutrition (4, 5). It has been calculated that about 10% of the total protein intake and 1-2% of the Lf ingested from breast milk can survive in the gut (3,4). The extent to which Lf escapes digestion and the effect of pH are, however, not known completely. Experimental evidence has indicated that the proteins found in the faeces are probably not secretory products from intestinal cells but undigested proteins of human milk (3, 4). Recent data suggest that dietary Lf may be hydrolysed minimally in the stomach of preterm infants (6). There are indications also that undegraded Lf of maternal origin is absorbed by the gut and excreted in the urine of preterm infants fed with human milk (7, 8). Goldman et al. have postulated that the fragments of Lf found in the faeces of very low birthweight infants fed with human milk could be produced in vivo by proteolysis (9). The close similarity between Lf fragments present in the stools and urine suggests that the urinary Lf fragments can originate in the gastrointestinal tract (9). It remains unclear whether these fragments were derived from ingested Lf or were produced partly by the infant, induced by human milk feedings (9). Although this possibility exists in theory, it is unlikely that some factors in human milk can stimulate the local synthesis

Experimental milk formulae with reduced protein content and desialylated milk proteins: influence on the faecal flora and the growth of term newborn infants

We have assessed the growth, tolerance and the faecal flora composition in healthy infants on different feeding regimens. Four groups of infants were fed exclusively on mothers milk, a standard formula and two experimental formulae. The first experimental formula consisted of a milk with a reduced protein content (1.2 g/100 ml), the second in a formula with the same protein content and with milk proteins desialylated by mild acid hydrolysis. The aim of the study was to test whether lowering the protein content and/or modifying the proteins by desialylation would favour the development of a bifidus flora. A bifidus flora was detected in 60% of breastfed infants at 1 month of life. All formulae employed during the study failed to induce a prevalence of colonization with bifidobacteria at 1 month of age. The two experimental milk formulae were well tolerated, but the infant growth rate was slightly lower as compared to the breastfed infants and the infants fed the standard formula. The presence in milk formulae of pre‐digested and desialylated proteins can offer some advantages in term of digestibility and mimic a physiological intestinal mechanism of the infant.

Milk formulae for the normal infant. III. Lipids and trace elements

The role of fa ts in infant nutrition Lipids represent a major component of milk formulae, and imbalances in their composition and content can have long-term deleterious effects on the health of the infant. The lipid classes of mature human milk consist of triacylglycerols (TGs) (98%), phospholipids (1.3%), cholesterol (0.4%) and traces of other lipids (1). Triacylglycerols are esters of glycerol where all three hydroxyls groups are esterified with a fatty acid (FA). Milk composition changes through the period of lactation, especially during the transition from colostrum to mature milk, and fat is certainly one of the most variable nutrients in human milk. Wide variations in the FA content of breast milk have been reported frequently (2). These differences appear to be primarily the consequence of variations in the mother’s dietary fat intakes and pose considerable problems in the attempt to establish the “normal” FA intake of the breast-fed infant (2). In line with variations in dietary FA intakes, the fat composition in human milk has changed slightly during the years (3). However, notwithstanding differences in the FA profile, there is always a certain consistency in the fat composition of human milk (4). In particular, it is worth emphasizing that, despite wide cultural differences in feeding practices, the quantity of long-chain polyunsaturated fatty acids (LCPs), which are major constituents of brain structures, is relatively constant in breast milk (5). The following functions are peculiar to dietary lipids (2, 6-8):

Milk formulae for the normal infant. II. Recommendations, energy, physical characteristics and protein composition

Modern infant formulae try to be as close as possible to human milk and to approximate its macronutrient composition (l), even if, up to the present time, it has not been possible to mimic breast milk exactly. Although it seems sensible to use the analysis of breast milk as a model, it must be remembered that the composition of breast milk varies markedly because it is dependent on the time postparturition, the diet of the mother and the stage of feeding. Thus individual samples of human milk show consistent variations from one another in volume, energy value and nutrient composition (2). In addition, there are physiological variations during a feed (“hind” milk and “fore” milk) and between different feeds in the same mother (2). Therefore, the figures relative to the composition of human milk must be taken as representative and not as absolute values. The best available guide remains the “average composition of human milk”, which is the basis for the calculation of the infant requirements and for the preparation of formulae (3) .