Elsevier

Food Research International

Volume 89, Part 1, November 2016, Pages 679-688
Food Research International

The influence of breast milk and infant formulae hydrolysates on bacterial adhesion and Caco-2 cells functioning

https://doi.org/10.1016/j.foodres.2016.09.022Get rights and content

Highlights

  • Enzymatic hydrolysis of milk protein increases causes an increase of BCM7 concentration.

  • BCM7 increases cause an increase of IL-8 secretion by intestinal epithelial cells.

  • Human milk hydrolysates of human milk did not alter quantitative profile of adhering bacteria.

  • IF hydrolysates stimulated adhesion of Bifidobacterium from healthy (H) infants.

  • IF hydrolysates reduced adhesion of enterobacteria from H and allergic infants.

Abstract

The aim of the study was to determine the concentration of BCM7 in human milk and infant formulae (IF) before and after eznymatic hydrolysis, and to evaluate the effect of obtained hydrolysates on interleukin-8 (IL-8) secretion and on proliferation of enterocytes in the in vitro model (Caco-2 cells). This study evaluates also the effect of hydrolysates on the adhesion of intestinal microbiota isolated from faeces of both healthy (H) and allergic (A) infants.

In the study we investigated breast milk delivered by mothers of healthy (‘healthy milk’; HM) and allergic (‘allergic milk’; AM) infants. Three infant formulae were investigated: from hydrolysed cow casein (IF1), from hydrolysed cow whey (IF2) and from whole cow milk (IF3). Intestinal bacteria: Bifidobacterium, lactic acid bacteria, Enterobacteriaceae, Clostridium and Enterococcus were isolated from faeces of five healthy and five allergic infants. Mixtures of bacterial isolates and bacteria adhering to Caco-2 cells were characterised qualitatively with PCR-DGGE, and quantitavely with FISH.

Concentration of BCM7 in breast milk and infant formulae was 1.6 to 8.9 times higher after enzymatic hydrolysis in comparison to undigested samples. The presence of this peptide resulted in alteration of intestinal epithelial proliferation and increase in secretion of IL-8. The quantitative profile of adherred bacteria applied as a mix of all isolates from healthy infants (H-MIX) was unchanged in the presence of HM hydrolysate and was modulated (increased number of beneficial Bifidobacterium and reduced commensal Enterobacteriaceae) in the presence of all IF hydrolysates. The presence of IF hydrolysates affected the profile of adhering isolates obtained from allergic infants (A-MIX) and reduced the adhesion of Enterobacteriaceae; the IF2 and IF3 hydrolysates decreased also the total number of adhering bacteria (TBN). However, a stimulating effect of AM hydrolysate on A-MIX adhesion (increased TBN) was observed.

Introduction

Breast milk provides infants with all nutritional requirements and is therefore an ideal source of optimal quality and quantity of nutrients for neonates. Numerous biologically active substances control, compensate and support infant digestion, absorption, metabolism and immunity, and breast milk based nutrition provides primary prevention of many diseases, including allergies. Children are fed infant formulae in maternal milk absence, insufficiency or physiological disorders. The main ingredient of formula is cow milk, which is processed by appropriate technology, including hydrolysis, used in such a way to obtain a nutritional value close to the reference material. Unfortunately, setting a proper conditions for hydrolysis process sets majority of obstacles, thus causing difficulties in obtaining a final product having universal properties (Andreas et al., 2015, Salvatori et al., 2014).

In addition to the nutritional components, breast milk and infant formulae, both contain biologically active compounds which influence growth and maturation of the infant immune system and digestive tract. These biologically active ingredients are present in the milk or they result from milk-protein enzymatic hydrolysis (Hernandez-Ledesma et al., 2007, Sidor et al., 2008).

Most dietary compounds are potentially biologically active and therefore have beneficial or harmful effects on bowel function and general human health (Świątecka, Świątecki, Kostyra, Marciniak-Darmochwał, & Kostyra, 2010). Accordingly, studies have intensively examined opioid peptides released from milk proteins. The main group of these compounds are β-casomorphins (BCMs); fragments of the β-casein chain confirmed in breast milk, cow milk, dairy products and infant formulae. Casomorphins can also infiltrate mammary glands and be transmitted in maternal milk (Kostyra, Sienkiewicz-Szłapka, Jarmołowska, Krawczuk, & Kostyra, 2004). Current literature describes β-casomorphin-7 (BCM7) effects on digestive, immune, cardiovascular and nervous systems resulting from its interaction with the μ opioid receptor (MOR) (Fiedorowicz et al., 2014). BCMs involvement is suspected in apparent life threatening event (ALTE) and sudden infant death syndrome (SIDS) (Wasilewska et al., 2011), autism (Sokolov et al., 2014), allergies and atopic dermatitis (Fiedorowicz et al., 2014, Kurek et al., 1996, Reddi et al., 2012).

The neonatal gastrointestinal tract is affected by postpartum microbiota, and in conjunction with an immature immune system, this disposes intestine to increased permeability and macromolecular infiltration (Houghteling & Walker, 2015). Neonates initially encounter the external environment during childbirth. In addition to maternal genital tract microbiota, they assimilate maternal skin and gastrointestinal microbiota during breastfeeding and also environmental bacteria. The first bacteria detected in infants include Enterococcaceae and Enterobacteriaceae family representatives. Following anaerobic stabilization, breast-fed infant microbiota is approximately 90% dominated by bifidobacteria with attendant Enterococcus, and Streptococcus population. In contrast, formula-fed children have a more complex micobiota profile; more reminiscent of adult microbiota; not dominated by a single group of bacteria but containing more numerous Bacteroides, Clostridium coccoides, Desulfovibrio, Enterococcus faecalis and enterobacteria than breast-fed infants (Harmsen et al., 2000). Furthermore, Clostridium difficile is recognized more frequently and in higher numbers in formula-fed infants. Childhood gastrointestinal tract microbiota alters with changed diet, and the varied diets introduced in the second year of life produce microbiota similar to those seen in the adult (Satokari, Vaughan, Akkermans, Saarela, & de Vos, 2001).

The aim of the study was to determine the concentration of BCM7 in human milk and infant formulae (IF) before and after eznymatic hydrolysis, and to evaluate the effect of obtained hydrolysates on interleukin-8 (IL-8) secretion and the proliferation of enterocytes in the in vitro model (Caco-2 cells). This study evaluates also the effect of hydrolysates on the adhesion of intestinal microbiota isolated from faeces of both healthy (H) and allergic (A) infants.

Section snippets

Control and study groups

Our study involved 51 breast-feeding women recruited in the Gastroenterological Clinic of Hansa Medical Centre and the Third Department of Pediatrics at the Medical University of Białystok. The control group consisted of 24 women (age range 25 to 32) whose children were fed only on breast milk and registered no symptoms of food allergy or atopic disease. Milk collected from these women is labelled HM (‘healthy milk’) in the text. All mothers in the control group used the overall diet

Degree of protein hydrolysis in breast milk and infant formulae

The results are presented in comparison to content of free amino groups in undigested sample, which was designated as 100% (Fig. 1). The free amino group content in hydrolyzed HM and AM was 163% and 158%, respectively, compared to raw milk (p < 0.001). While no free amino group increase was noted from starting product levels in the hydrolysed IF1 and IF2, the IF3 hydrolyzed sample contained 127% free amino groups compared to the undigested formula (p < 0.01).

β-casomorphin-7 content in breast milk and infant formulae

The presence of BCM7 in samples is

Discussion

While breastfeeding is considered the “gold standard” and the best method of infant nutrition, infant formula administration is required in special cicumstances. This article differentiates the health effects of control (HM) and study group (AM) breast milk and three infant formulae (IF1, IF2, IF3) digested with pepsin and pancreatin in in vitro conditions. Enzymatic hydrolysis provided a designed system to examine food degradation in the human digestive tract, and the crucial factor in this

Conclusions

Hydrolysed breast milk and infant formulae changed functions of the enterocytes by influencing the process of adhesion of bacteria to their surface. Additional consequences were alterations of intestinal epithelial proliferation and secretion of pro-inflammatory cytokine (IL-8), which may result in the disorders in the digestive system and development of food allergy in infants. These, all together may significantly impact the status of the intestinal barrier and by that influence the overall

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Acknowledgments

Work was supported by the Ministry of Science and Higher Education, number N N 407153939. The authors would like to thank sincerely all the children and parents who participated in this study.

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