[Infant formulas supplemented with prebiotics: intestinal microbiota and immune responses]. / I prebiotici nei latti per lattanti: ruolo nello sviluppo dell'ecosistema microbico intestinale e nella modulazione del sistema immunitario.

It is well known that the type of feeding influences the composition of the gut microflora after birth. Human milk favours the growth of a ''bifidus flora'' which, according to several evidences, may activate the immune system and defend from pathogens. Breast milk oligosaccharides, which are involved in many functional effects both at local and systemic level, are thought to stimulate the growth of health promoting microbes, such as bifidobacteria, and may ultimately influence the immune system. In accordance with this current working hypothesis, dietary modulation of the gut microbiota to obtain a ''bifidus flora'' also in bottle-fed infants may be a useful way to stimulate immunological functions and to harbour a biological barrier against pathogens. In several clinical trials prebiotic oligosaccharides have been used to mimic the beneficial effects of breast milk oligosaccharides. A mixture of oligosaccharides has shown its efficacy in stimulating the establishment of a ''bifidus flora'', with stools closer to those found in breast-fed infants. Several experimental data also indicate that oligosaccharides might modulate the immune system and contribute to the improvement of the protective properties of infant formulas.

Acidic oligosaccharides from pectin hydrolysate as new component for infant formulae: effect on intestinal flora, stool characteristics, and pH.

OBJECTIVES: To come even closer to the functional composition of human milk, acidic oligosaccharides (AOS) from pectin were added to well known neutral prebiotics (galacto-oligosaccharides (GOS) and long-chain fructo-oligosaccharides (FOS)). The effect of AOS and GOS/FOS/AOS on intestinal flora, stool characteristics as well as acceptance and tolerance was investigated. METHODS: Human milk contains 75% to 85% neutral and 15% to 25% acidic oligosaccharides. In this prospective, randomized, double blind study, a mixture of 80% neutral oligosaccharides (from long-chain galacto- and long-chain fructo-oligosaccharides) with 20% acidic oligosaccharides derived from pectin hydrolysis was investigated. Forty-six term infants were fed a standard formula supplemented with either maltodextrin as control (n=15), or with 0.2 g acidic oligosaccharides (n=16), or with the latter plus 0.6 g neutral oligosaccharides (mixture of galacto- and fructo-oligosaccharides; n=15). Fecal flora using plating technique and pH were measured. Stool characteristics and possible side effects (crying, vomiting, and regurgitation) were recorded. RESULTS: There was no difference in the bifidobacteria counts between the control and the group supplemented with acidic oligosaccharides alone (8.75+/-0.50 vs. 8.58+/-0.94 log colony forming units [CFU]/g stool). In infants fed the combination of acidic and neutral oligosaccharides, bifidobacteria were increased (9.61+/-0.70 log CFU/g stool; P<0.01). The same pattern was observed with lactobacilli. Stool consistency was softest in infants fed the complete oligosaccharide mixture, but also in those fed formula supplemented with acidic oligosaccharides alone, the stool consistency was significantly softer compared with the control group. Fecal pH increased in the controls, remained constant in acidic oligosaccharides alone, and decreased in the complete mixture of oligosaccharides group. CONCLUSION: There was no difference in growth, crying, vomiting, and regurgitation patterns between the groups. In summary, acidic oligosaccharides from pectin hydrolysate are well tolerated as ingredient in infant formulae but do not affect intestinal microecology.

Prebiotics in infant formulas.

BACKGROUND: The intestinal flora of breast-fed infants is an important physiologic factor in the function of the gut and in the development of the immune system. The current research is part of a group of studies performed to answer the question whether a bovine milk formula supplemented with a prebiotic mixture from galactooligosaccharides and fructooligosaccharides can stimulate an intestinal flora similar to that of breast-fed infants. METHODS: The prebiotic effect of the oligosaccharide mixture was tested in preterm and term infants by measuring fecal flora using plating as well as fluorescent in situ hybridization techniques. The effect of the oligosaccharides on the bacterial metabolism was studied by measuring short-chain fatty acid production in vitro and the short-chain fatty acid pattern in the stools of a group of term infants. RESULTS: The oligosaccharide mixture increases significantly the number of bifidobacteria and reduces the number of pathogens in term as well as in preterm infants when compared with a group of infants fed an unsupplemented formula. Using a concentration of 0.8 g oligosacchrides/100 mL formula, the amount of bifidobacteria is similar to that typical of breast-fed infants. In vitro, the short-chain fatty acids produced by the mixture of oligosaccharides under study were similar to those produced by the human milk oligosaccharides fraction. In clinical trials the pattern of fecal short-chain fatty acids in infants fed the oligosaccharide mixture was similar to that of breast-fed infants but was significantly different from that of a group of infants fed with an unsupplemented formula. Additionally, the fecal pH was significantly higher in the group fed an unsupplemented formula than in the groups fed either breast milk or a supplemented formula. CONCLUSION: The data obtained indicate that the prebiotic mixture under study is able to stimulate the development of a microbial flora similar to that of breast-fed infants. Several biota, whose growth is enhanced by this prebiotic mixture, represent important factors in the postnatal development of the immune system. On this evidence it can be suggested that prebiotics may play a role as modulators of the postnatal development of the immune system.

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.

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.

"Minor" feeding problems during the first months of life: effect of a partially hydrolysed milk formula containing fructo- and galacto-oligosaccharides.

BACKGROUND: Colic, regurgitation and constipation are common feeding problems in formula-fed infants that might benefit from dietary treatment. A formula containing fructo- and galacto-oligosaccharides, partially hydrolysed proteins, low levels of lactose and palmitic acid in the beta position and higher density has been tested to reduce the occurrence of these symptoms. The aim of this prospective study was to describe the effects of such a formula in infants with minor gastrointestinal disorders. METHODS: An observational prospective trial involving practising Italian paediatricians was performed. Formula fed-infants up to 90 d of age with minor gastrointestinal problems such as infantile colics and/or regurgitation and/or constipation were enrolled in the study from January 2001 to May 2001. The study was completed within 14 d of treatment. On days 1, 7 and 14 the infants were visited by the paediatricians. Parents were given a structured diary to record daily episodes of colic, regurgitation and type and number of stools. RESULTS: Of the 932 infants enrolled, 604 completed the study. Of the 214 infants with colic, 169 (79%) demonstrated a reduction in frequency of colic from 4.1 +/- 2.0 per day at the beginning of the study to 2.0 +/- 1.8 at the end of the study (I.C. 95%: 1.72-2.39; p < 0.005). A reduction in the number of episodes of colic of 1.8 per day at the beginning of the study (I.C. 95%: 1.49-2.11; p < 0.05) was recorded between day 1 and day 7, and of 0.26 (I.C. 95%: 0.15-0.37; p < 0.05) between day 7 and day 14. Of the 201 infants with regurgitation problems, 141 (70%) demonstrated a reduction of frequency of the symptoms from 4.2 +/- 2.0 per day at the beginningof the study to 2.1 +/- 2.2 at the end of the study (I.C. 95%: 1.75-2.35; p < 0.005). A reduction of 1.87 in the number of regurgitation episodes was reported between day 1 and day 7 (I.C. 95%: 1.57-2.16; p < 0.05) and of 0.18 (I.C. 95%: 0.06-0.31; p < 0.05) between day 7 and day 14. Of the 232 infants with constipation, 147 (63%) demonstrated an increase in the daily number of stools of 0.42 (I.C. 95%: 0.5-0.3; p < 0.005). An increase in stool frequency of 0.41 (I.C. 95%: 0.51-0.23; p < 0.05) was reported between day 1 and day 7, and of 0.04 (I.C. 95%: 0.22-0.14; p = ns) between day 7 and day 14. Parents' evaluation of the formula was 7.9 +/- 1.8 (score 0-10); 550 parents (91%) gave a positive judgement (score > 6). The evaluation by the paediatricians of the improvement in symptoms after the treatment was 8.2 +/- 1.5; 574 (95%) a positive effect (score > 6). CONCLUSION: This study shows that the majority of infants followed by paediatricians for minor gastrointestinal symptoms improve within 2 wk of feeding with this new formula. Further double-blind, controlled studies are needed to confirm whether the amelioration of symptoms observed in this trial is in fact due to the new formula.

[Protein quality and quantity in infant formulas. A critical look]. / Le proteine nei latti per lattanti. Quali e in che misura?

The ideal quantity and quality of dietary proteins in milk formulas employed for infant nutrition is still a matter of controversy and debate. During the last years, there has been a tendency to lower the protein content in response to new estimations of protein requirements based on protein intakes and growth rates of breastfed infants. In most infant formulas the protein content is in the range of 1.4-1.8 g/100 ml. The lower limit of this range appears to be adequate for the growth and metabolic needs of the healthy infant. The safety of a further reduction of this limit to 1.2 g/100 ml (~1.8 g/100 kcal), which has been recently proposed, needs additional scrutiny and observation. In addition, the real benefits of this choice have still to be proven. Adjustments for protein digestibility and quality have recently been introduced. The removal from the whey protein fraction of the glycomacropeptide (GMP), with a consequent reduction of excessive threonine intakes, is a novel approach and a further step in the development of infant formulas closer to the model of hu-man milk.

Efficient otoacoustic emission protocols employed in a hospital-based neonatal screening program.

Within the context of a hospital-based newborn hearing screening program, we have studied the application of two OAE protocols (TEOAE and DPOAE) on a group of 250 well babies. The main goal of this study was to evaluate the performance of DPOAE protocol in a relatively large population sample. using a preset number of five tested frequencies, in comparison with a default TEOAE screening protocol. The data were collected on the second day of life and during spontaneous sleep. The TEOAE recordings were acquired with linear protocols using click stimuli of 70-75 dB SPL and were used as indicators of normal cochlear function. The cubic distortion product DPOAE responses were evoked by an asymmetrical 75-65 dB SPL protocol, with a frequency ratio of 1.22. Five frequencies (referring to F2) were tested at 1.5, 2.0, 3.0, 4.0 and 5.0 kHz. The data from the DPOAE responses show a similar pass rate (similarity = 0.98) to the linear TEOAE protocol. The data presented suggest that a DPOAE cochlear evaluation, at 5 pre-selected frequencies, has clinical potential.

[Food allergies in early childhood. 2. Food choices]. / Le allergie alimentari nella prima infanzia. 2. Le scelte nutrizionali.

The incidence of allergic diseases appears to be on the increase in industrialized societies. In infants at high risk for atopic diseases prevention in early life seems to be a high priority at this time. However, many questions remain to be answered because, in the absence of breast feeding which should be encouraged in all infants, substitute formula employed for prevention should guarantee the following requisites: optimal nutrition, reduced allergenicity or better hypoallergenicity, good palatability and low cost. Current concepts in the field of prophylaxis and diet therapy of food allergy are reviewed. Special emphasis is given to the biochemical, nutritional and antigenic properties of the different formulas employed in the management of these conditions.

[Food allergies in early childhood. 1. General concepts, etiopathogenesis, and main clinical features]. / Le allergie alimentari nella prima infanzia. 1. Concetti generali, eziopatogenesi e principali quadri clinici.

Abnormal immune reactions to food antigens are a rather common event during infancy. Adverse reactions to milk proteins occur in 2.5% of suckling infants. Both IgE and non IgE-mediated allergic mechanisms may be involved in the pathogenesis of food allergy. IgE mediated allergic responses are the most dramatic and the most often diagnosed types. Non IgE mediated food allergy is usually more difficult to diagnose and its clinical course is more chronic. Food induced allergic reactions mediate a variety of symptoms, involving the gastrointestinal and respiratory tract and the skin. A limited number of foods are responsible for the vast majority of allergic reactions: cow milk proteins, egg, fish and peanuts are the main causes. The vast majority of infants with formula-protein intolerance will outgrow their symptoms by the third year of age. Eliminating the food allergens is the only means of dealing with the problem. In part I of this series, immunopathogenic mechanisms and clinical disorders are described.

[Distortion product otoacoustic emission (DPOAEs) and newborn hearing screening: a feasibility and performance study]. / Otoemissioni Acustiche dei Prodotti di Distorsione (DPOAEs) e screening audiologico neonatale: studio di applicabilità e performance.

In view of the great psychological effect deafness has on one's social life, the scientific community has long sought the best way to define the hearing function. The development of new technologies in this field has set "early intervention" as the primary target for screening. For this reason, within the contest of a program of audiological newborn hearing screening, the Authors have used data from a selected group of 1250 newborns to (a) compare the clinical feasibility and performance of three different DPOAE protocols and (b) establish the scoring criteria defining "pass" or "fail" responses. All subjects participating in this study were randomly selected and their normal hearing was verified by linear TEOAE recordings. The test was carried out, using an Otodynamics ILO92 Analyser version 5.60, on the second day of life or later (in case of extended recovery), during natural sleep and after feeding. The DPOAE recordings were elicited using asymmetric stimuli with L1 > L2 and a frequency-ratio of 1.22 following three different SPL protocols: 60-50 dB (PR1), 65-55 dB (PR2) and 75-65 dB (PR3). Five frequencies of the cubic distortion product (referring to f2) were tested with an ILO macro at 1.5 KHz, 2.0 KHz, 3.0 KHz, 4.0 KHz and 5.0 KHz. The statistical evaluation of differences in the signal-to-noise ratio (S/N) between the PR1 and PR2 protocols showed no significant differences. On the other hand significant differences were found between the PR3 and the PR2 S/N ratios, the former providing the best performance (higher values). The scoring criteria were defined by minimum free distribution tolerance intervals of the S/N ratios at the five tested frequencies. In conclusion this study confirms the feasibility of DPOAE recordings in a un iversal newborn hearing screening program and shows the appropriate pass-fail parameters to be used for this purpose. It should be noted that this approach enables acquisition of frequency-specific information which might further improve audiological diagnosis.

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 = 12 mg, copper = 1.4 mg and iodine = 145 microg. 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 microg. 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 20mg zinc sulfate, 2mg copper sulfate and 116 microg 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, 90d 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.

[Evaluation of a linear TEOAE protocol in hearing screening of neonates: feasibility study]. / Valutazione di un protocollo TEOAE lineare nello screening audiologico neonatale: studio di fattibilità.

The audiological screening of newborns based on recording TEOAEs--the expression of anatomical-functional integrity of the cochlea--has unanimously gained importance. The prevalence of severe of genetic or congenital hearing loss in the healthy infant population and in the population at risk for audiological disorders, as well as the consequent psycholinguist and socialization problems such children have to deal with, have led the authors to set up a preliminary protocol for the audiological screening of neonates. The purpose of this protocol is to improve the feasibility of applying such a program. To this purpose, the preliminary protocol called for the recording of the TEOAE in both non linear (NL) and linear (L) modes. Examination was performed in 347 newborns (30% of all the children born during 1997) the second day of life and during spontaneous sleep. The ILO 92 was used for the screening. The results obtained with the two methods were statistically compared using the 9 parameters considered response indicators. Using the linear method of a function-window and improving the signal-to-noise ratio, the study showed a significant difference in the quality of the TEOAE using the linear method as compared with the non linear method, although this did not modify response reliability. This lead to the definition, through free distribution statistical analysis, of broader than normal criteria by which to evaluate the responses evoked with the L method. All this is aimed at increasing system specificity and reducing the number of false positives which feeds family anxiety.

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 mother's 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.