BIOACTIVES IN MILK-DERIVED PRODUCTS: BACTERIAL PRODUCTION OF IMMUNOMODULATORY CASEIN HYDROLYSATES AND TOOLS FOR IDENTIFICATION OF IMMUNOGENIC BACTERIAL PROTEIN

During hydrolysis of bovine milk caseins, cell envelope-associated proteinases (CEPs) of lactic acid bacteria (LAB) may be able to produce hydrolysates that possess in vitro immunomodulatory activity. We studied the proteolytic activity against bovine milk caseins exerted by specific strains of LAB selected on the basis of a preliminary genomic screening for the presence of CEPs. The tested strains demonstrated diverse hydrolytic ability against different casein fractions, alphaS1- and beta-casein in particular. The 3 kDa-ultrafiltered casein hydrolysates (CHs), produced after digestion with proteinases of Lactobacillus acidophilus ATCC 4356 and Lactococcus lactis subsp. lactis GR5 demonstrated immunomodulatory activity in vitro by significantly decreasing the basal NF-kB activity in recombinant Caco-2 cell layers. Both the strains digested beta-casein mostly. However, in the case of Lactobacillus helveticus MIMLh5, the investigation proved that the immunomodulatory activity of obtained CH was comparable to the one exerted by bacteria themselves (in the absence of caseinate) and therefore, was not due to the presence of casein-derived peptides. Indeed, the surface layer (S-layer) protein of L. helveticus MIMLh5 was identified as a molecule responsible for the immunomodulation. Overall, these results emphasize the importance of this bacterial cell-derived protein while evaluating the immunological activity of CHs, and it underlines the necessity to remove such molecule in order to properly assess the bioactivity of CHs deriving from the proteolytic activity of LAB. The second part of the research was focussed on the selection of S-layer-specific single-chain variable fragment antibodies (scFvs), a new powerful tool for the study of the S-layer of L. helveticus MIMLh5, the immunologically active protein, and for its identification in milk-derived products containing L. helveticus as a starter or non starter LAB. In this study, a mix of two human synthetic phage displayed libraries (protein-directed and hapten-directed) was used to select scFvs against L. helveticus MIMLh5 S-layer protein. After three rounds of panning, four monoclonal scFv binders capable of binding to the L. helveticus MIMLh5 S-layer protein and one capable of binding not only to the mentioned protein, but also to the S-layer protein of L. helveticus ATCC 15009, which is different only in five amino acids, were obtained. All five identified novel anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their basic characterisation was performed. Anti-S-layer-scFv PolyH4 was used to develop an assay, based on Western blot analysis, for detection of the S-layer protein in Grana Padano samples. These results showed promising applications of the method for the detection of the S-layer protein in food matrices

An analytical approach to reveal the addition of heat-denatured whey proteins in lab-scale cheese making

A simple analytical procedure for the detection of self-aggregated heat-denatured whey proteins (HDWP) in model cheeses was developed. The principle of the approach lies in the solubilization of the cheese matrix by a sodium citrate solution (0.2 M, pH 7.0) resulting in the dissociation of the casein micelles and the insolubilization of HDWP aggregates, which are collected in the pellet after a centrifugation step. The reliability of the procedure was tested in lab-scale cheeses from peroxidase-positive pasteurized milk with different protein-based ingredients (microparticulated whey protein concentrate, milk protein concentrate, whey protein isolate and Ricotta cheese) at concentrations ranging from 0.2 to 1.2% protein (w/v on cheese milk). A linear relationship between the amount of the HDWP added to cheese milk and that recovered from model cheeses was observed. Heat-damage indicators, furosine and lysinoalanine, showed levels in the experimental cheese samples not related with added HDWP, but represented a source of information on the ingredients other than liquid milk. Overall, in the model cheeses, the proposed method was an easy-to-apply and reliable tool for the evaluation of the presence of HDWP-based products. Further investigation is required for the application to real cheeses and for the evaluation of possible interferences from proteolysis during ripening

Role of extraction procedures on the concentration of varietal thiol precursors in Grillo white grape must

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Effect of pre-fermentative steps on thiol precursors in Grillo must

The varietal thiols, namely 3-mercaptohexan-1-ol (3MH), 3-mercaptohexylacetate (3MHA), and 4-mercapto-4-methylpentan-2-one (4MMP), are sulphur-containing aromas associated with the typical flavour of several white wines, such as Sauvignon blanc wine, conferring guava, citrus and passion fruit notes. These compounds occur as non-volatile sulphide precursors in grape berry, where they share their sulphur atom with a cysteine residue. 3MH bound with cysteine (Cys-3MH), glutathione (GSH-3MH) and also cysteine-glycine (CysGly-3MH) has been described, while 4MMP occurs as cysteine (Cys-4MMP) and glutathione (GSH-4MMP) conjugates. S-3-(hexan-1-al)-glutathione (GSH-3MHAl) was also identified, and it can be considered as a thiol precursor. Recently, the presence of thiol precursors was reported in the Italian autochthonous variety Grillo, and their concentrations strongly decreased when must was produced under commercial conditions. This study investigated the influence of pre-fermentative operations on thiol precursor concentrations. Grillo grape was pressed under industrial conditions; must samples were collected after crashing, at draining, at pressing yield of 20%, 40% and 60%, at the end of pressing, during transfer in clarification tank, in a clarification tank and after clarification. The must was either air-exposed or air-free during the pre-fermentative steps. Thiol precursors were determined in SPE-purified must samples by UPLC-HRMS. Cys-3MH, GSH-3MH and GSH-3MHAl strongly decreased after crashing, and small concentrations were found in drained must samples independently of the presence of air. In particular, the crashing played a major role on GSH-3MHAl content; a further decrease of both Cys-3MH and GSH-3MH was found due to the must transfer in clarification tank. In general, precursor amounts were lower in must samples produced in air-free condition, except for clarified musts where the precursor contents were comparable in both air-exposed and air-free conditions. For the must production at industrial conditions particular attention should be given to the grape pressing for limiting the loss of thiol precursors. The air-exposure of must has a limited positive influence on thiol precursors, since the removal of solid parts with the clarification is responsible for their further loss. The proper management of these winemaking steps could allow to preserve major levels of thiol precursors

In vitro antioxidant properties of digests of hydrolyzed casein and caseinophosphopeptide preparations in cell models of human intestine and osteoblasts

Three commercial samples consisting of enriched calcium-free caseinophosphopeptides (CPP), enriched calcium-bound caseinophosphopeptides (Ca-CPP) and an enzymatically hydrolyzed casein (hCN) were in vitro digested according to COST-Infogest protocol. As assessed by UPLC-HR-MS/MS, the digests contained 207\u2013235 unique caseinophosphopeptides, and the species presenting the cluster sssEE were more abundant in CPP digest. The antioxidant activity at three different doses of each digest was firstly evaluated on human intestinal Caco-2/HT-29 70/30 co-culture. In presence of AAPH, hCN and CPP digests displayed a dose-dependent antioxidant activity equal or even greater than Vitamin C. In presence of Fe2+, the digests exerted an antioxidant activity mainly at the highest dose. Antioxidant activities of the intestinal metabolized digests was then evaluated on human osteoblast (Saos-2) cells. The digests exerted an antioxidant activity in presence of AAPH, but not in presence of Fe2+. These results highlight milk-derived peptides as potential dietary supplements for gut and bone health

Purification and partial characterization of a novel bacteriocin produced by a thermophilic endospore-forming strain Geobacillus stearothermophilus 32A

The murB gene encodes UDP-N-acetylenolpyruvylglucosamine reductase and functions in bacterial peptidoglycan biosynthesis. A plasmid carrying the murB gene restored the temperature-sensitive growth of six Staphylococcus aureus mutants, in which peptidoglycan biosynthesis stopped at a restrictive temperature. Specific activity of UDP-N-acetylenolpyruvylglucosamine reductase in extracts from the mutants was lower than that from wild-type cells. Nucleotide sequence determination revealed that each mutant had a single amino acid substitution in the murB gene and five of six mutations were located within domain 3, where the proposed substrate binding site is located. These results suggest that the murB gene is essential for growth of S. aureus and that domain 3 is important for the MurB activity

Thiol precursors in Catarratto Bianco Comune and Grillo grapes and effect of clarification conditions on the release of varietal thiols in wine

Background and aims: Varietal thiols characterize the typical aroma of several white wines, as Sauvignon blanc. Their presence was suggested in two Sicilian grape cultivars, Catarratto Bianco Comune (CBC) and Grillo, thought it was not analytically proved to date. Methods and Results: Varietal thiol precursors and free varietal thiols were assessed in CBC and Grillo grapes, musts and wines by UPLC/high resolution mass spectrometry (HRMS). The isobaric compounds S-3-(hexanal)-glutathione (GSH-3MHAl) and S-3-(4-mercapto-4-methylpentan-2-one)-glutathione (GSH-4MMP) were discriminated by comparing their accurate masses and HR-MS/MS spectra with those of their synthetic standards. GSH-3MHAl, S-3-(hexan-1-ol)-glutathione (GSH-3MH) and S-3-(hexan-1-ol)-cysteine occurred in grape, must and wine, while GSH-4MMP and its hydrolysed forms did not. Their amounts decreased during the industrial winemaking processes, mostly following the grape pressing. We compared clarification conditions exposing must to either air or CO2 in terms of thiol precursors\u2019 and free thiols\u2019 content in wine. However, negligible differences were observed. Concentrations of free thiols in the range 400\u20131100 ng/L were found in the wines and they were not affected to the two clarification conditions adopted. Conclusion: The isobaric GSH-3MHAl and GSH-4MMP were clearly distinguished for the first time by UPLC-HRMS through their retention times and MS spectra. The varietal thiols were firstly revealed CBC and Grillo wines. The air-free and air- exposed clarification poorly affected the levels of varietal thiols. Significance of the study: This research highlights the major impact of the varietal thiols (mainly 3\u2013mercapto-hexan-1-ol and its acetate form) on the sensory properties of CBC and Grillo wines

A dairy bacterium displays in vitro probiotic properties for the pharyngeal mucosa by antagonizing group A streptococci and modulating the immune response

The probiotic approach represents an alternative strategy in the prevention and treatment of infectious diseases, not only at the intestinal level but also at other sites of the body where the microbiota plays a role in the maintenance of physiological homeostasis. In this context, we evaluated in vitro the potential abilities of probiotic and dairy bacteria in controlling Streptococcus pyogenes infections at the pharyngeal level. Initially, we analyzed bacterial adhesion to FaDu hypopharyngeal carcinoma cells and the ability to antagonize S. pyogenes on FaDu cell layers and HaCat keratinocytes. Due to its promising adhesive and antagonistic features, we studied the dairy strain Lactobacillus helveticus MIMLh5, also through in vitro immunological experiments. First, we performed quantification of several cytokines and measurement of NF-\u3baB activation in FaDu cells. MIMLh5 efficiently reduced the induction of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNF-\u3b1), in a dose-dependent manner. After stimulation of cells with IL-1\u3b2, active NF-\u3baB was still markedly lowered. Nevertheless, we observed an increased secretion of IL-6, gamma interferon (IFN-\u3b3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) under these conditions. These effects were associated with the ability of MIMLh5 to enhance the expression of the heat shock protein coding gene hsp70. In addition, MIMLh5 increased the GM-CSF/G-CSF ratio. This is compatible with a switch of the immune response toward a TH1 pathway, as supported by our observation that MIMLh5, once in contact with bone marrow-derived dendritic cells, triggered the secretion of TNF-\u3b1 and IL-2. In conclusion, we propose MIMLh5 as a potential probiotic bacterium for the human pharynx, with promising antagonistic and immunomodulatory properties

Modulation of fecal clostridiales bacteria and butyrate by probiotic intervention with Lactobacillus paracasei DG varies among healthy adults

Background: The modulation of gut microbiota is considered to be the first target to establish probiotic efficacy in a healthy population. Objective: This study was conducted to determine the impact of a probiotic on the intestinal microbial ecology of healthy volunteers. Methods: High-throughput 16S ribosomal RNA gene sequencing was used to characterize the fecal microbiota in healthy adults (23-55 y old) of both sexes, before and after 4 wk of daily consumption of a capsule containing at least 24 billion viable Lactobacillus paracasei DG cells, according to a randomized, double-blind, crossover placebo-controlled design. Results: Probiotic intake induced an increase in Proteobacteria (P = 0.006) and in the Clostridiales genus Coprococcus (P = 0.009), whereas the Clostridiales genus Blautia (P = 0.036) was decreased; a trend of reduction was also observed for Anaerostipes (P = 0.05) and Clostridium (P = 0.06). We also found that the probiotic effect depended on the initial butyrate concentration. In fact, participants with butyrate >100 mmol/kg of wet feces had a mean butyrate reduction of 49 ± 21% and a concomitant decrease in the sum of 6 Clostridiales genera, namely Faecalibacterium, Blautia, Anaerostipes, Pseudobutyrivibrio, Clostridium, and Butyrivibrio (P = 0.021), after the probiotic intervention. In contrast, in participants with initial butyrate concentrations <25 mmol/kg of wet feces, the probiotic contributed to a 329 ± 255% (mean ± SD) increment in butyrate concomitantly with an ~55% decrease in Ruminococcus (P = 0.016) and a 150% increase in an abundantly represented unclassified Bacteroidales genus (P = 0.05). Conclusions: The intake of L. paracasei DG increased the Blautia:Coprococcus ratio, which, according to the literature, can potentially confer a health benefit on the host. The probiotic impact on themicrobiota and on short-chain fatty acids, however, seems to strictly depend on the initial characteristics of the intestinal microbial ecosystem. In particular, fecal butyrate concentrations could represent an important biomarker for identifying subjects who may benefit from probiotic treatment