Surface layer proteins from Lactobacillus helveticus ATCC® 15009™ affect the gut barrier morphology and function.

Paraprobiotics and postbiotics represent a valid alternative to probiotic strains for ameliorating and preserving a healthy intestinal epithelial barrier (IEB). The present study investigated the effects of surface layer proteins (S-layer) of the dairy strain Lactobacillus helveticus ATCC® 15009™ (Lb ATCC® 15009™), as paraprobiotic, on the morpho-functional modulation of IEB in comparison to live or heat-inactivated Lb ATCC® 15009™ in an in vitro co-culture of Caco-2/HT-29 70/30 cells. Live or heat-inactivated Lb ATCC® 15009™ negatively affected transepithelial electrical resistance (TEER) and paracellular permeability, and impaired the distribution of Claudin-1, a tight junction (TJ) transmembrane protein, as detected by immunofluorescence (IF). Conversely, the addition of the S-layer improved TEER and decreased permeability in physiological conditions in co-cultures with basal TEER lower than 50 ohmcm2, indicative of a more permeable physiological IEB known as leaky gut. Transmission electron microscopy (TEM) and IF analyses suggested that the S-layer induces a structural TJ rearrangement and desmosomes' formation. S-layer also restored TEER and permeability in the presence of LPS, but not of a mixture of pro-inflammatory cytokines (TNF-α plus IFN-γ). IF analyses showed an increase in Claudin-1 staining when LPS and S-layer were co-administered with respect to LPS alone; in addition, the S-layer counteracted the reduction of alkaline phosphatase detoxification activity and the enhancement of pro-inflammatory interleukin-8 release both induced by LPS. Altogether, these data corroborate a paraprobiotic role of S-layer from Lb ATCC® 15009™ as a possible candidate for therapeutic and prophylactic uses in conditions related to gastrointestinal health and correlated with extra-intestinal disorders.

Distinct Organotypic Platforms Modulate Rainbow Trout (Oncorhynchus mykiss) Intestinal Cell Differentiation In Vitro.

In vitro organotypic cell-based intestinal platforms, able to faithfully recapitulate the complex functions of the organ in vivo, would be a great support to search for more sustainable feed ingredients in aquaculture. We previously demonstrated that proliferation or differentiation of rainbow trout intestinal cell lines is dictated by the culture environment. The aim of the present work was to develop a culture platform that can efficiently promote cell differentiation into mature enterocytes. We compared four options, seeding the RTpiMI cell line derived from the proximal intestine on (1) polyethylene terephthalate (PET) culture inserts ThinCert™ (TC), (2) TC coated with the solubilized basement membrane matrix Matrigel® (MM), (3) TC with the rainbow trout fibroblast cell line RTskin01 embedded within the Matrigel® matrix (MMfb), or (4) the highly porous polystyrene scaffold Alvetex® populated with the abovementioned fibroblast cell line (AV). We evaluated the presence of columnar cells with a clear polarization of brush border enzymes, the formation of an efficient barrier with a significant increase in transepithelial electrical resistance (TEER), and its ability to prevent the paracellular flux of large molecules but allow the transit of small compounds (proline and glucose) from the apical to the basolateral compartment. All parameters improved moving from the simplest (TC) through the more complex platforms. The presence of fibroblasts was particularly effective in enhancing epithelial cell differentiation within the AV platform recreating more closely the complexity of the intestinal mucosa, including the presence of extracellular vesicles between fibroblasts and epithelial cells.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Current insights into non-thermal preservation technologies alternative to conventional high-temperature short-time pasteurization of drinking milk.

Milk is an important nutritional food source characterized by a perishable nature and conventionally thermally treated to guarantee its safety. In recent years, an increasing focus on competing non-thermal food processing technologies has been driven mainly by consumers' expectations for minimally processed products. Due to the heat sensitivity of milk, much research interest has been addressed to mild non-thermal pasteurization processing to keep safety, 'fresh-like' taste and to maintain the organoleptic qualities of raw milk. This review provides an overview of the current literature on non-thermal treatments as standalone alternative technologies to high-temperature short-time (HTST) pasteurization of drinking milk. Results of lab-scale experimentations suggest the feasibility of most emerging non-thermal processing technologies, including high hydrostatic pressure, pulsed electric field, cold plasma, cavitation and light-based technologies, as alternative to thermal treatment of drinking milk with premium in shelf life duration. Nevertheless, a series of regulatory, technological and economical hurdles hinder the industrial scaling-up for most of these substitutes. To date, only high hydrostatic pressure treatments are applied as alone alternative to HTSH pasteurization for processing of "cold pasteurized" drinking milk. Milk submitted to HTST treatment combined to ultraviolet light is currently accepted in EU countries as novel food.

Impact of in vitro static digestion method on the release of ß-casomorphin-7 from bovine milk and cheeses with A1 or A2 ß-casein phenotypes.

Beta-casomorphin-7 (BCM7) represents the fragment Val60-Ile66 of bovine ß-casein (ß-CN), and there is evidence that it is more easily released during gastrointestinal digestion (GID) of A1 ß-CN variant, in comparison to the A2 variant. This study aimed at investigating the effect of type of enzymes and the protease/protein (P/S) ratio on BCM7 release during the intestinal step of in vitro static GID of bovine milk and cheeses with A1 or A2 ß-CN phenotypes. BCM7 occurred in digests of both A1 and A2 samples, being the release more marked for A1 counterparts. Nonetheless, the BCM7 release depended on both the type of GID enzymes and the P/S ratio. These findings highlight the importance of GID conditions which may affect the outcomes for possible differences between A1 and A2 milk based on BCM7 release during in vitro GID.

Alzheimer's Disease Prevention through Natural Compounds: Cell-Free, In Vitro, and In Vivo Dissection of Hop (Humulus lupulus L.) Multitarget Activity.

The relevant social and economic costs associated with aging and neurodegenerative diseases, particularly Alzheimer's disease (AD), entail considerable efforts to develop effective preventive and therapeutic strategies. The search for natural compounds, whose intake through diet can help prevent the main biochemical mechanisms responsible for AD onset, led us to screen hops, one of the main ingredients of beer. To explore the chemical variability of hops, we characterized four hop varieties, i.e., Cascade, Saaz, Tettnang, and Summit. We investigated the potential multitarget hop activity, in particular its ability to hinder Aß1-42 peptide aggregation and cytotoxicity, its antioxidant properties, and its ability to enhance autophagy, promoting the clearance of misfolded and aggregated proteins in a human neuroblastoma SH-SY5Y cell line. Moreover, we provided evidence of in vivo hop efficacy using the transgenic CL2006Caenorhabditis elegans strain expressing the Aß3-42 peptide. By combining cell-free and in vitro assays with nuclear magnetic resonance (NMR) and MS-based metabolomics, NMR molecular recognition studies, and atomic force microscopy, we identified feruloyl and p-coumaroylquinic acids flavan-3-ol glycosides and procyanidins as the main anti-Aß components of hop.

Valorisation of Bovine Sweet Whey and Sunflower Press Cake Blend through Controlled Fermentation as Platform for Innovative Food Materials.

The current environmental challenge is pushing food systems towards more sustainable models of production that require reorganizing of processes by re-using side products still containing nutrients. This work aimed at valorising a mix of bovine sweet whey and sunflower press cake, through targeted fermentation. After preliminary screening based on growth rate, final pH, lactose/galactose assimilation, phytase activity, six Lactic Acid Bacteria strains (Lacticaseibacillus casei, L. paracasei (2), Lactococcus lactis, Lentilactobacillus parakefiri and Leuconostoc pseudomesenteroides) and three yeasts (Kluyveromyces lactis, K. marxianus and Torulaspora delbrueckii) were co-cultivated in pairs in microcosms (1-part ground press cake: 4-parts whey). All tested microorganisms were able to grow and acidify the blend: the LAB counts increased during the incubation (26 °C for 48 h) of +2.80 log CFU/g, whereas yeasts counts were of +1.98 log CFU/g, with significant differences among the different associations (p < 0.01). Mould counts were always <3 log CFU/g. Interestingly, the bacterial contaminants count significantly varied in samples with different pairs of strains (p < 0.001). Acidification level, acetic acid and ethanol contents were the limiting factors affecting the growth of spoilage micro-organisms. Best performances were attained in microcosms inoculated with L. lactis or L. paracasei and K. lactis or K. marxianus.

Transmission routes, preventive measures and control strategies of SARS-CoV-2 in the food factory.

SARS-CoV-2 virus represents a health threat in food factories. This infectious virus is transmitted by direct contact and indirectly via airborne route, whereas contamination through inanimate objects/surfaces/equipment is uncertain. To limit the potential spread of the pathogen in the food industry, close working between individuals should be avoided and both personal and respiratory hygiene activities should be enforced. Despite the high infectivity, SARS-CoV-2, being an enveloped virus with a fragile lipid envelop, is sensitive to biocidal products and sanitizers commonly used in the food factory. In the context of the building design, interventions that promote healthy air quality should be adopted, especially in food areas with high-occupancy rates for prolonged times, to help minimize the potential exposure to airborne SARS-CoV-2. Air ventilation and filtration provided by heating, ventilation and air conditioning systems, are effective and easy-to-organize tools to reduce the risk of transmission through the air. In addition to conventional sanitation protocols, aerosolization of hydrogen peroxide, UV-C irradiation or in-situ ozone generation are complementary techniques for an effective virucidal treatment of the air.

Model infant biscuits release the opioid-acting peptides milk ß-casomorphins and gluten exorphins after in vitro gastrointestinal digestion.

Infant biscuits (IBs) are commonly used during the complementary feeding of infants from the 6th month of life. They contain wheat flour and dairy ingredients, which can release the opioid-acting peptides ß-casomorphins (BCMs) and gluten exorphins (GEs) after gastrointestinal digestion. In the present study, five model IBs were prepared with or without gluten and different powdered milk derivatives in the formulations. IBs were digested simulating an in vitro static gastrointestinal digestion for infants aged 6-12 months. BCMs and GEs were identified and quantified by UPLC/HR-MS. The amounts of BCM7 and the GE A5 were related to the ß-CN and gluten content of the formulations. To date, levels of BCMs and GEs in digests of IBs have not been reported in literature. This work represents an in vitro investigation regarding the release of opioid-acting peptides in IBs. It could add additional knowledge on complementary foods for infant health.

Compartmentalization of bacterial and fungal microbiomes in the gut of adult honeybees.

The core gut microbiome of adult honeybee comprises a set of recurring bacterial phylotypes, accompanied by lineage-specific, variable, and less abundant environmental bacterial phylotypes. Several mutual interactions and functional services to the host, including the support provided for growth, hormonal signaling, and behavior, are attributed to the core and lineage-specific taxa. By contrast, the diversity and distribution of the minor environmental phylotypes and fungal members in the gut remain overlooked. In the present study, we hypothesized that the microbial components of forager honeybees (i.e., core bacteria, minor environmental phylotypes, and fungal members) are compartmentalized along the gut portions. The diversity and distribution of such three microbial components were investigated in the context of the physico-chemical conditions of different gut compartments. We observed that changes in the distribution and abundance of microbial components in the gut are consistently compartment-specific for all the three microbial components, indicating that the ecological and physiological interactions among the host and microbiome vary with changing physico-chemical and metabolic conditions of the gut.

Assessment of Possible Application of an Atmospheric Pressure Plasma Jet for Shelf Life Extension of Fresh-Cut Salad.

Ready-to-eat salads are very perishable with quality losses within 6-7 days, and the extension of their shelf life is still a challenge. In this work, an atmospheric pressure plasma jet (APPJ) was applied for the surface decontamination of fresh-cut lettuce baby leaves. The APPJ antimicrobial efficiency on the natural microbiota and its impact on some physicochemical attributes of lettuce were evaluated as a function of the treatment duration (0-30 s). Then, the influence of plasma treatment on the salad shelf life was studied, following the growth of aerobic mesophilic bacteria in both untreated and plasma-treated samples during 9 days of storage at 4 °C, together with the plasma-induced changes in physicochemical parameters of lettuce leaves. The APPJ induced a fast (15 s) microbial decontamination (1.3 log10 CFU/g) of the salad surface. Exposure time and salad-plasma plume distance were the parameters that substantially affected the microbial inactivation. APPJ treatment retarded bacterial growth during the refrigerated storage, as plasma-treated samples were noticeably less contaminated than the non-treated ones in the first 3-4 days. No significant effect were observed on electrolyte leakage, pH, and dry matter content in both the set up phase and the shelf life study.

Proteolytic Traits of Psychrotrophic Bacteria Potentially Causative of Sterilized Milk Instability: Genotypic, Phenotypic and Peptidomic Insight.

The proteolytic traits of the psychrotrophic strains Pseudomonas poae LP5, Pseudomonas fluorescens LPF3, Chryseobacterium joostei LPR1, Pseudomonas fulva PS1, Citrobacter freundii PS37, Hafnia alvei PS46, and Serratia marcescens PS92 were initially investigated by phenotypic and genotypic approaches. Six strains elicited extracellular proteolytic activity, and five expressed the thermostable AprX or (likely) Ser1 enzymes. Then, the strains were inoculated (104 CFU/mL) in microfiltered pasteurized milk and kept at 4 °C for five days. All of the strains reached 108 CFU/mL at the end of storage and five produced thermostable extracellular proteolytic enzymes. The freshly inoculated samples and the corresponding samples at 108 CFU/mL were batch-sterilized (131 °C, 30 s) and kept at 45 °C up to 100 days. The former samples did not gel until the end of incubation, whereas the latter, containing P. poae, P. fluorescens, C. joostei, C. freundii, and S. marcescens, gelled within a few days of incubation. The thermostable proteolytic activity of strains affected the peptidomic profile, and specific proteolyzed zones of ß-CN were recognized in the gelled samples. Overall, the results confirm some proteolytic traits of psychrotrophic Pseudomonas spp. strains and provide additional insights on the proteolytic activity of psychrotrophic bacteria potentially responsible for sterilized milk destabilization.

Correction to: Different phytotoxic effect of Lolium multiflorum Lam. leaves against Echinochloa oryzoides (Ard.) Fritsch and Oryza sativa L.

In the title, it should be Oryza instead of Oriza.

Different phytotoxic effect of Lolium multiflorum Lam. leaves against Echinochloa oryzoides (Ard.) Fritsch and Oryza sativa L.

Rice cultivation, particularly prone to weed issues, requires practices able to effectively control them, however reducing the use of herbicides, responsible for damage to human health and ecosystem sustainability. Alternative strategies for weed management can be based on plant-plant interaction phenomena. In this context, a group of organic farmers has developed a pragmatic approach for weed containment using Lolium multiflorum Lam. as a cover crop before rice. The present study aimed to confirm the farmer field observations reporting a preferential inhibitory effect of L. multiflorum on Echinochloa oryzoides (Ard.) Fritsch, one of the most yield-damaging rice weed, compared with Oryza sativa L. The study showed that L. multiflorum was able to significantly reduce the seed germination of E. oryzoides. It was found to be more susceptible than O. sativa both to the effect of the aqueous extract and powder of L. multiflorum leaves (23-79% vs. 3-57% and 26-100% vs. 23-31%, respectively). In addition, the leaf extract was able to affect E. oryzoides growth starting from 20% concentration both in relation to the root and shoot length while O. sativa exhibited differences compared with the control only under the influence of extract 50%. The L. multiflorum leaf characterization by NMR and UPLC-HR-MS analyses led to the identification of 35 compounds including several polyphenols, glycosyl flavonoids and glycosyl terpenoids, as well as different amino acids and organic acids. Some of them (e.g. protocatechuic and gallic acids) are already known as allelochemicals confirming that L. multiflorum is a source of plant growth inhibitors.

Occurrence, biological properties and potential effects on human health of ß-casomorphin 7: Current knowledge and concerns.

The genetic variant A1 of bovine ß-casein (ß-Cn) presents a His residue at a position 67 of the mature protein. This feature makes the Ile66-His67 bond more vulnerable to enzymatic cleavage, determining the release of the peptide ß-Cn f(60-66), named ß-casomorphin 7 (BCM7). BCM7 is an opioid-agonist for µ receptors, and it has been hypothesized to be involved in the development of different non-transmissible diseases in humans. In the last decade, studies have provided additional results on the potential health impact of ß-Cn A1 and BCM7. These studies, here reviewed, highlighted a relation between the consumption of ß-Cn A1 (and its derivative BCM7) and the increase of inflammatory response as well as discomfort at the gastrointestinal level. Conversely, the role of BCM7 and the effects of ingestion of ß-Cn A1 on the onset or worsening of other non-transmissible diseases as caused or favored by still need proof of evidence. Overall, the reviewed literature demonstrates that the "ß-Cn A1/BCM7 issue" remains an intriguing but not exhaustively explained topic in human nutrition. On this basis, policies in favor of breeding for ß-Cn variants not releasing BCM7 and consumption of "A1-like" milk appear not yet sound for a healthier and safer nutrition.

Effect of protein fortification on heat damage and occurrence of ß-casomorphins in (un)digested donor human milk intended for nutrition of preterm infants.

Pasteurized donor human milk (PDHM) for preterm infant nutrition is fortified with hydrolyzates of cow's milk proteins, which have been poorly investigated in relation to heat-damage and occurrence of the bioactive peptides ß-casomorphins (BCMs). Therefore, thermal protein modifications of three commercial fortifiers were assessed by measuring well-recognized indexes of heat load. The fortifiers did not contain pyrraline, whereas furosine and lysinoalanine levels roughly overlapped the lowest values reported for liquid formulas addressed to term infant nutrition. Bovine BCMs 3 to 7 and human BCMs 3 to 9 were searched. Bovine BCMs 3, 4, 6 and 7 were found in the undigested fortifiers. Following in vitro digestion simulating the digestive conditions of premature infant, bovine BCMs still occurred in fortified PDHM; the human BCMs 3, 7, 8 and 9 formed. Overall, these results better address the nutritional features of protein fortifiers and fortified PDHM intended for nutrition of preterm infants.

Bovine whey peptides transit the intestinal barrier to reduce oxidative stress in muscle cells.

Health benefits are routinely attributed to whey proteins, their hydrolysates and peptides based on in vitro chemical and cellular assays. The objective of this study was to track the fate of whey proteins through the upper gastrointestinal tract, their uptake across the intestinal barrier and then assess the physiological impact to downstream target cells. Simulated gastrointestinal digestion (SGID) released a selection of whey peptides some of which were transported across a Caco-2/HT-29 intestinal barrier, inhibited free radical formation in muscle and liver cells. In addition, SGID of ß-lactoglobulin resulted in the highest concentration of free amino acids (176 nM) arriving on the basolateral side of the co-culture with notable levels of branched chain and sulphur-containing amino acids. In vitro results indicate that consumption of whey proteins will deliver bioactive peptides to target cells.

Monitoring molecular composition and digestibility of ripened bresaola through a combined foodomics approach.

In this work, the effects of maturation time and simulated gastrointestinal digestion on the molecular and peptide profiles of "Bresaola Valtellina" were assessed through the foodomics approach, in this case food proteomics and peptidomics combined to other analytical and biological assays, aiming at depicting a holistic food quality. Human digestion of this Italian cured meat product was simulated using an in vitro static protocol and the degree of proteolysis and the in vitro bioactivity of the soluble free compounds in the digestates were evaluated by biochemical assays, e.g. SDS-PAGE, size exclusion HPLC, HPLC/MS, 1H NMR, enzymatic and antioxidant activities. The obtained results demonstrated that in vitro gastrointestinal digestion contributed to a considerable release of myofibrillar proteins by the muscle tissue. Data from SDS-PAGE, peptidomic and size exclusion HPLC assays showed that the in vitro digestion largely degraded proteins of muscle tissue to peptides smaller than 250 Da. The released peptides were likely responsible for the inhibitory activity on amylolytic enzymes and for the antioxidant properties elicited by the gastric digestates of Bresaola. Overall, the results demonstrated the negligible role of ripening in making meat proteins more bioaccessible, whereas they confirmed the highly in vitro digestibility of meat proteins from Bresaola. This study represents a new approach merging proteomics and foodomics to evaluate the effect of ripening and in vitro digestion on the bioactivity and bioaccessibility of proteins and peptides of meat products.

bioNMR-based identification of natural anti-Aß compounds in Peucedanum ostruthium.

The growing interest in medicinal plants for the identification of new bioactive compounds and the formulation of new nutraceuticals and drugs prompted us to develop a powerful experimental approach allowing the detailed metabolic profiling of complex plant extracts, the identification of ligands of macromolecular targets of biomedical relevance and a preliminary characterization of their biological activity. To this end, we selected Peucedanum ostruthium, a plant traditionally employed in Austria and Italy for its several potential therapeutic applications, as case study. We combined the use of NMR and UPLC-HR-MS for the identification of the metabolites present in its leaves and rhizome extracts. Due to the significant content of polyphenols, particularly chlorogenic acids, recently identified as anti-amyloidogenic compounds, polyphenols-enriched fractions were prepared and tested for their ability to prevent Aß1-42 peptide aggregation and neurotoxicity in a neuronal human cell line. STD-NMR experiments allowed the detailed identification of Aß oligomers' ligands responsible for the anti-amyloidogenic activity. These data provide experimental protocols and structural information suitable for the development of innovative molecular tools for prevention, therapy and diagnosis of Alzheimer's disease.

Functional characterization of Lactobacillus plantarum ITEM 17215: A potential biocontrol agent of fungi with plant growth promoting traits, able to enhance the nutritional value of cereal products.

In this work, we explored the potential of 25 Lactobacillus plantarum strains isolated from cereals and milk-based products, testing characteristics related to antifungal activity and to nutritional quality. The tested strains demonstrated interesting beneficial traits, such as the ability to utilize fructo-oligosaccharides, prebiotic substances that help probiotic microorganisms to grow in the human gut, and to reduce phytate, an antinutrient present in cereal sector. Regarding mould inhibition, we highlighted the ability of the strains to inhibit Penicillium roqueforti, Mucor circinelloides and mycotoxinogenic moulds associated with cereal grains as Aspergillus flavus, A. niger, Fusarium verticillioides. Moreover, a moderate reduction of the bioavailability of aflatoxin AFB1 was detected. The selected L. plantarum strain ITEM 17215, showed a strong inhibitory ability towards fungal growth and was able to produce 1,2-dihydroxybenzene, benzoic acid, p-hydroxyphenyllactic acid and 3-phenyllactic acid. The latter compound, already described as efficient antifungal inhibitor, was the most abundant and its concentration was further increased by adding phenylalanine and phenylpyruvic acid in the growth medium. The metabolites produced by strain ITEM 17215 could also be related to the ability of the strain to induce cereal germination and promote plant growth. This aspect, not yet investigated in L. plantarum, could have interesting applications in the agro-food sector.