Study protocol: The role of milk matrix lipids in programming the immunoreactivity of proteins derived from lactic acid bacteria.

Food allergy is widely recognized as a significant health issue, having escalated into a global epidemic, subsequently giving rise to the development of numerous additional complications. Currently, the sole efficient method to curb the progression of allergy is through the implementation of an elimination diet. The increasing number of newly identified allergens makes it harder to completely remove or avoid them effectively. The immunoreactivity of proteins of bacterial origin remains an unexplored topic. Despite the substantial consumption of microbial proteins in our diets, the immunologic mechanisms they might induce require thorough validation. This stands as the primary objective of this study. The primary objective of this study was to evaluate the effects of bacterial proteins on the intestinal barrier and immune system parameters during hypersensitivity induction in both developing and mature organisms. The secondary objective was to evaluate the role of lipids in the immunoreactivity programming of these bacterial proteins. Notably, in this complex, comprehensively designed in vitro, in vivo, and ex vivo trial, the immunoreactivity of various bacterial proteins will be examined. In summary, the proposed study intends to address the knowledge gaps regarding the effects of Lactobacillus microbial proteins on inflammation, apoptosis, autophagy, and intestinal barrier integrity in a single study.

Recent Advances in Fermented Milk and Meat Products-Quality, Nutritional Value and Safety.

Products of animal origin have always been very popular among consumers due to their nutritional and sensory value [...].

The Manifold Bioactivity and Immunoreactivity of Microbial Proteins of Cow and Human Mature Milk in Late Lactation.

(1) Human milk (HM) is a source of many microorganisms, whose structure contains microbial protein (MP). In addition to the known health-promoting properties of HM, many activities, including immunoreactivity, may result from the presence of MP. Cow's milk (CM)-derived MP may be 10 times more abundant than MP derived from HM. (2) Raw cow's milk samples of Holstein and Jersey breeds, commercially available pasteurized milk, and milk from three human donors in the late lactation phase were subjected to chemical and microbiological analyzes. Microorganisms from the milk material were recovered, cultured, and their activities were tested. MPs were extracted and their immunoreactivity was tested with human high IgE pooled sera. The milk types were subjected to simulated digestion. Milk and microbial proteins were identified with LCMS and subjected to an in silico analysis of their activities. Their antioxidant potential was analysed with the DPPH method. (3) The MP of HM shows a stronger IgE and IgG immunoreactivity in the tests with human sera compared to the MP of CM (p = 0.001; p = 0.02, respectively). There were no significant differences between the microbes in the MP of different cattle breeds. The MS-identification and in silico tests of milk and microbial proteins confirmed the presence of MP with immunoreactivity and antioxidant potential. (4) MPs possess a broad bioactive effect, which was determined by an in silico tools. The balance between an MP's individual properties probably determines the raw material's safety, which undoubtedly requires further research.

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.

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.

The effect of lactic acid fermentation with different bacterial strains on the chemical composition, immunoreactive properties, and sensory quality of sweet buttermilk.

This paper describes the successful development of new low-immunoreactive buttermilk (BM)-based formulations which were fermented with 31 lactic acid bacteria (LAB) and Bifidobacterium strains. The aim of this study was to create a new formula, which can serve as potential candidates for the immunotherapy of allergy. Preparations were tested for their content of biologically active compounds, such as proteins, peptides, phospholipids, and short-chain fatty acids (SCFA), as well as for the survivability of LAB and sensory quality. The results showed that the BM was a matrix rich in nutritional components and displayed higher than expected susceptibility to the reduction of protein IgE-immunoreactivity (to 98%) and high bacterial-protecting capacity. The overall sensory quality of examined products was influenced by the profile of SCFA and free peptides, but two formulations fermented with Lactobacillus delbrueckii ssp. bulgaricus-151 and Lactobacillus casei-LcY were the most advantageous with desirable sensory, immunoreactive, and biochemical properties.

Two Faces of Milk Proteins Peptides with Both Allergenic and Multidimensional Health Beneficial Impact- Integrated In Vitro/In Silico Approach.

The main food-origin antigens that the infant's body is in contact with are cow's milk proteins (CMP). Still, CMP are one of the main sources of beneficial biologically active peptides that play a role in treatment of non-communicable diseases. Safe methods to quickly predict the sensitizing potential of food proteins among their range of health-promoting properties are essential. The aim of study was to adapt an integrated approach combining several in silico (IS) studies and in vitro (IV) assays to screen the multifunctionality of CMP-derived peptides. Major histocompatability complex type II MHC II-binders, interleukin-4 and -10 inducers, interferon γ -inducers and immunobioactivity tools were used to predict the peptide-power of inducing allergies or tolerance. A comparison of the peptide profiless revealed the presence of one identical and one overlapping sequence in IS and IV hydrolysate. By IS analysis, four of 24 peptides were found to have high affinity and stimulate IL-4 expression, and by IV, one of seven peptides had this potential (Bos d9 peptide DIPNPIGSENSEK (195-208)). Three IV peptides may induce IL-10 expression. The IV/IS assessment seems promising agents for peptides' potential determination dedicated only to preliminary screening of peptides. The IV verification is still crucial in further steps of studies.

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.

Gestational Dysfunction-Driven Diets and Probiotic Supplementation Correlate with the Profile of Allergen-Specific Antibodies in the Serum of Allergy Sufferers.

BACKGROUND: Maternal diet has significant effects on development of childhood atopic disease and hypersensitivity development. However, the gestational dysfunctions demanding special diets are becoming a widespread phenomenon, their immunological implications can be manifested in the profile of antibodies in the offspring's serum. METHODS: 153 allergic and 150 healthy individuals were diagnosed for allergy using specific antibody and cytokine immunoassay tests. The medical history of subjects along with mothers' course of pregnancy was completed by allergologist's anamnesis. A self-organizing neural network and multivariate analyses to complex data and pick basic interactions were used. RESULTS: Two significant explanatory modules were determined. The first was formed by gestational diabetic and cholestatic diet, infant formula feeding type, probiotic supplementation and its BMI index, moderate IgE, increased IgG levels of antibodies and single or poly-food allergy type (7 clusters). The second was formed by gestational vegan/vegetarian and elimination diet, maternal probiotic supplementation, sex, high IgE total antibodies and food and mixed poly-allergy to aero- and food-origin allergens (19 clusters). CONCLUSIONS: Significant associations were observed between special gestational diet intake underlying foetal programming and the mechanisms of childhood allergy. The novelty is the positive association between diabetic and cholestatic diet intake and IgE/IgG-mediated food hypersensitivity.