Phytate Hydrolysate Differently Modulates the Immune Response of Human Healthy and Cancer Colonocytes to Intestinal Bacteria.

(1) Phytic acid (PA) is a component of cereal seeds and legumes, therefore its consumption is much higher in a vegan and vegetarian diet compared to a conventional diet. The diet is the main driver of metabolic activity of gut microbiota, therefore, the ability to degrade phytates by the microbiota of vegans significantly exceeds that of the gut microbiota of omnivores. The aim of the study was to investigate the early phase of the immune response of colonocytes treated with an enzymatic hydrolysate of phytic acid (hPA120) and gut bacteria. (2) Cell lines derived from healthy (NCM460D) and cancer (HCT116) colonic tissue and fecal bacteria from vegan (V) and omnivorous (O) donors were investigated. Fecal bacteria were grown in mucin and phytic acid supplemented medium. Cultured bacteria (BM) were loaded onto colonocytes alone (V BM and O BM) or in combination with the phytate hydrolysate (V BM + hPA120 and O BM + hPA120). After a treatment of 2 h, bacterial adhesion, secretion of cytokines, and the expression of genes and proteins important for immune response were determined. (3) All bacteria-treated colonocytes increased the expression of IL8 compared to controls. The significant increase of the secreted IL-8 (p < 0.01) in both cell lines was observed for O BM and O BM + hPA120. The increase of TNF, IL-1ß, and IL-10 secretion in healthy colonocytes (V BM alone and with hPA120 treatments; p < 0.05) and for TNF and IL-10 in cancer cells (treatments except O BM + hPA120 and V BM, respectively; p > 0.05) were stated. A comparison of solely the effect of hPA120 on bacteria-treated colonocytes (BM vs. BM + hPA120) showed that hPA120 decreased expression of NFkB1 and TNFR (p < 0.001) in healthy colonocytes. In cancer colonocytes, the expression of TLR4 and IL1R increased after BM + hPA120 treatment, whereas the secretion of IL-8 and MYD88 and TNFR expression decreased (p < 0.01). (4) The investigated hPA120 showed a differentiated modulatory activity on the immune response of healthy and cancer human colonocytes. Especially when analyzed independently on the gut bacteria origin, it reduced the proinflammatory response of HCT116 cells to gut bacteria, while being neutral for the bacteria-treated healthy colonocytes.

Microbiological Biodiversity of Regional Cow, Goat and Ewe Milk Cheeses Produced in Poland and Antibiotic Resistance of Lactic Acid Bacteria Isolated from Them.

(1) Unique sensory values of traditional and regional dairy products made them more and more popular among consumers. Lactic acid bacteria naturally occurring in these products can express antibiotic resistance and be a reservoir of antibiotic resistance genes (ARG) in the environment. The aim of the study was to characterize the microbial diversity of twenty regional cheeses produced from non-pasteurized cow, goat and ewe milk, and investigate the phenotypic and genotypic antibiotic resistance (AR) of lactic acid bacteria isolated from these products. (2) Conventional microbiological methods were applied for the enumeration of lactic acid bacteria (lactobacilli and lactococci) and their isolation, and for the enumeration of Enterococcus, Staphylococcus, Enterobacteriaceae and spores. The disc diffusion method was applied for phenotypic AR. The PCR-based methods were used for strain identification, microbiological diversity of cheeses (PCR-DGGE), and for AR gene detection. (3) Among 79 LAB isolates the most frequent species were L. plantarum (n = 18), Leuc. lactis (n = 17), Lc. lactis (n = 11), Leuc. mesenteroides (n = 9) and L. pentosus (n = 8). Additionally, by using the PCR-DGGE method, DNA of L. casei was found in nine products. Lactobacilli (5.63-8.46 log cfu/g) and lactococci (6.15-8.41 log cfu/g) predominated over Enterococcus (max. 4.89 log cfu/g), Staphylococcus (max. 4.18 log cfu/g), and Enterobacteriaceae (mostly up to 4.88 log cfu/g). Analysis of phenotypic resistance to tetracycline (30 µg), erythromycin (15 µg), and chloramphenicol (30 µg) showed that 29% of LAB isolates were resistant to one antibiotic, 8%-to two, and 12%-to all tested antibiotics. Antibiotic resistance genes (AGR) for tetracycline (tet(M), tet(L), tet(W)), erythromycin (erm(B)) and chloramphenicol (cat-TC) were detected in 30 (38%), 29 (36.7%) and 33 (43.4%) LAB isolates, respectively. Among 31 LAB isolates phenotypically susceptible to all tested antibiotics, only 5 (16%) had no ARGs. (4) The results obtained in our work shed light on the potential threat posed by the widespread presence of ARGs in LAB present in regional cheeses.

Phytate and Butyrate Differently Influence the Proliferation, Apoptosis and Survival Pathways in Human Cancer and Healthy Colonocytes.

The colonic epithelium is never exposed to a single factor, therefore studies on the effect of combinations of factors naturally and persistently present in the intestines are of special importance for understanding the phenomena occurring at this place. The aim of the study was to investigate the combined effect of 1 mM phytate and 1 mM butyrate (PA1B1) on cell lines derived from cancer (HCT116 and HT-29) and healthy (NCM460D) human colonic epithelium. Colorimetric and flow cytometry methods were used to determine the proliferation rate, cell cycle, and apoptosis. Selected markers of proliferation, inflammatory, and survival pathways were investigated at the mRNA and/or protein level. The combination of phytate and butyrate disturbed the cell cycle and triggered apoptosis and/or death in both studied cancer colonocytes to a higher extent compared to healthy colonocytes. Moreover, in healthy colonocytes, phytate activated the survival pathway without stimulation of inflammatory response. This may indicate that the response of healthy colonocytes to phytate protects colonic epithelium from the loss of integrity and tightness that would occur if inflammation developed. Based on the obtained results we postulate that studies on both cancer and/or healthy colonocytes should be carried out in the presence of butyrate as the permanent component of colonic contents. This should be of special importance when anti-proliferative/pro-apoptotic activity or inflammatory status of colonocytes is to be investigated.

Effect of Low-Immunogenic Yogurt Drinks and Probiotic Bacteria on Immunoreactivity of Cow's Milk Proteins and Tolerance Induction-In Vitro and In Vivo Studies.

There is no effective therapy for milk allergy. The role of lactic acid bacteria (LAB) and probiotics in protection against allergy-related outcomes is still under investigation. The aim of the study was to evaluate the immunomodulative and therapeutic potential of yogurt drinks in cow's milk allergy (CMA) management. We compared immunoreactivity of α-casein (α-CN), ß-casein (ß-CN), κ-casein (κ-CN), α-lactalbumin (α-LA), and ß-lactoglobulin (ß-LG) in 27 yogurt drinks fermented with different basic yogurt cultures, or yogurt cultures enriched with Lactobacillus plantarum and/or Bifidobacterium lactis strains, by competitive ELISA assay. Drinks with the lowest antigenic potential were used as allergoids for CMA therapy. BALB/c mice were sensitized via intraperitoneal injection of α-CN + ß-LG mixture with aluminum adjuvant, and gavaged with increasing doses of selected low-immunogenic drinks (YM-basic, or YM-LB-enriched with L. plantarum and B. lactis) to induce tolerance. Milk- or phosphate-buffered saline (PBS)-dosed mice served as controls. Compared to milk, the immunoreactivity of proteins in drinks increased or decreased, depending on the bacterial sets applied for fermentation. Only a few sets acted synergistically in reducing immunoreactivity. The selected low-immunogenic drinks stimulated allergic mice for profiling Th2 to Th1 response and acquire tolerance, and the effect was greater with YM-LB drink, which during long-lasting interventional feeding strongly increased the secretion of regulatory cytokines, i.e., IL-10 and TGF-ß, and IgA and decreased IL-4, IgE, and anti-(α-CN + ß-LG) IgG1. The studies revealed variations in the potency of yogurt bacteria to change allergenicity of milk proteins and the need for their strict selection to obtain a safe product for allergy sufferers. The YM-LB drink with reduced antigenic potential may be a source of allergoids used in the immunotherapy of IgE mediated CMA, but further clinical or volunteer studies are required.

Increased prevalence of eating disorders as a biopsychosocial implication of food allergy.

INTRODUCTION: The study evaluates the impact of biopsychosocial factors involved in food allergy (FA) on the prevalence of eating disorders (ED). For the 5-year follow-up studies, 75 participants (aged 1-14 years) with early-onset FA and 81 healthy peers were included. METHOD: Participants were diagnosed with FA using antibody/cytokine content immunoassay tests. Medical history, including BMI z-scores, was completed using data obtained in response to a validated allergic questionnaire that incorporated the SCOFF and EAT-8 screening questionnaires for ED. FA was confirmed if total IgE was elevated, specific sIgE to food allergens exceeded 0.7 kUA/L and if manifestations were observed. Screening for ED was considered positive if two or more SCOFF and EAT-8 items were confirmed. RESULTS: In the FA+ group, 50% of female participants and 6.7% of their healthy female peers reported ED. An ED+ result was more frequent in FA+ individuals than in their healthy peers (p = 0.046) although the association is weak. In the FA+/ED+ group, 25.3% of the participants were underweight, and 14.7% were overweight compared to their peers where this reached respectively 4.2% and 2.8% (p<0.005). 74% of the FA+/ED+ individuals reported elimination diet implementation and only 15% declared it was medically consulted. The prevalence of ED in the FA+ male group was consistently correlated with lack of confidence in FA issues (r = 0.5424) and in the FA+ female group with applied medical procedures (r = 0.7069; p<0.005). CONCLUSION: These findings suggest that participants with FA especially struggling with lack of confidence in FA issues and those following an uncontrolled, restrictive elimination diet are more prone to food aversion and ED than their healthy peers. Applied procedures are necessary, and their neglect is associated with FA deterioration; however, the possibility of ED and biopsychosocial implications development should not be underestimated.

The Effect of Oligofructose-Enriched Inulin on Faecal Bacterial Counts and Microbiota-Associated Characteristics in Celiac Disease Children Following a Gluten-Free Diet: Results of a Randomized, Placebo-Controlled Trial.

Celiac disease (CD) is associated with intestinal microbiota alterations. The administration of prebiotics could be a promising method of restoring gut homeostasis in CD. The aim of this study was to evaluate the effect of prolonged oligofructose-enriched inulin (Synergy 1) administration on the characteristics and metabolism of intestinal microbiota in CD children following a gluten-free diet (GFD). Thirty-four paediatric CD patients (mean age 10 years; 62% females) on a GFD were randomized into two experimental groups receiving Synergy 1 (10 g/day) or placebo (maltodextrin; 7 g/day) for 3 months. The quantitative gut microbiota characteristics and short-chain fatty acids (SCFAs) concentration were analysed. In addition, side effects were monitored. Generally, the administration of Synergy 1 in a GFD did not cause any side effects. After the intervention period, Bifidobacterium count increased significantly (p < 0.05) in the Synergy 1 group. Moreover, an increase in faecal acetate and butyrate levels was observed in the prebiotic group. Consequently, total SCFA levels were 31% higher than at the baseline. The presented trial shows that Synergy 1 applied as a supplement of a GFD had a moderate effect on the qualitative characteristics of faecal microbiota, whereas it stimulated the bacterial metabolite production in CD children.

Seasonal variances in bacterial microbiota and volatile organic compounds in raw milk.

The aim of this study was to define the composition of microbiota and the volatile organic compounds (VOCs) in samples of raw milk collected for 22 months between 2012 and 2014 originated from north-eastern region of Poland. The results revealed that the VOCs profile changed with respect to the season of milk collection, and milk collected in autumn was characterized by a higher content of acetic acid (C2), propionic acid (C3) and valeric acid (C5), whereas spring was characterized by a frequent presence of acetone (Ac), ethanol (Et) and ethyl acetate (EtAc). Bacterial species composition changed considerably within the tested period and some bacterial species/groups occurred seasonally, e.g. L. helveticus (summer), L. casei (winter). The results show usefulness of the applied techniques (PCR-DGGE and HS-GC) and data analysis (PCA, correlation coefficients) methods in characterizing the raw milk quality intended for dairy production.

A simultaneous two-colour detection method of human IgG- and IgE-reactive proteins from lactic acid bacteria.

Whole cell extracts of two Lactobacillus strains were tested with primary antibodies from two pooled sera from allergic patients. Fluorescently labelled anti-human IgG and anti-human IgE secondary antibodies applied in Western blotting, together with an appropriate image acquisition protocol facilitated imagining bacterial proteins that reacted with human IgG and IgE.

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

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.