Do nomadic lactobacilli fit as potential vaginal probiotics? The answer lies in a successful selective multi-step and scoring approach.

BACKGROUND: The goal of this study was to create a multi-strain probiotic gel that would foster a lactobacilli-dominated vaginal microbiota in pregnant women and ensure appropriate eubiosis for the newborn. Nomadic lactobacilli (95 strains), mostly isolated from food sources, were preliminarily screened for functional traits before being characterized for their capability to inhibit the two vaginal pathogens Streptococcus agalactiae and Candida albicans, which may lead to adverse pregnancy-related outcomes. Eight best-performing strains were chosen and furtherly investigated for their ability to produce biofilm. Lastly, the two selected potential probiotic candidates were analyzed in vitro for their ability to reduce the inflammation caused by C. albicans infection on the reconstituted human vaginal epithelium (HVE). RESULTS: Lactiplantibacillus plantarum produced both isomers of lactic acid, while Lacticaseibacillus paracasei produced only L-isomer. The production of hydrogen peroxide was strain-dependent, with the highest concentrations found within Lact. paracasei strains. The auto-aggregation capacity and hydrophobicity traits were species-independent. S. agalactiae 88II3 was strongly inhibited both at pH 7.0 and 4.0, whereas the inhibition of C. albicans UNIBZ54 was less frequent. Overall, L. plantarum strains had the highest pathogen inhibition and functional scoring. L. plantarum C5 and POM1, which were selected as potential probiotic candidates also based on their ability to form biofilms, were able to counteract the inflammation process caused by C. albicans infection in the HVE model. CONCLUSIONS: Our multi-step and cumulative scoring-based approach was proven successful in mining and highlighting the probiotic potential of two nomadic lactobacilli strains (L. plantarum C5 and POM1), being applicable to preserve and improve human vaginal health.

How multiple farming conditions correlate with the composition of the raw cow's milk lactic microbiome.

Questionnaires on farming conditions were retrieved from 2129 dairy farms and clustered, resulting in 106 representative raw cow's milk samples analysed in winter and summer. Substantiating the efficiency of our survey, some farming conditions affected the milk physicochemical composition. Culturing identified several species of lactic acid bacteria (LAB) per milk, whose number increased through 16S ribosomal RNA (rRNA) gene sequencing and shotgun metagenome analyses. Season, indoor versus outdoor housing, cow numbers, milk substitutes, ratio cattle/rest area, house care system during lactation, and urea and medium-chain fatty acids correlated with the overall microbiome composition and the LAB diversity within it. Shotgun metagenome detected variations in gene numbers and uniqueness per milk. LAB functional pathways differed among milk samples. Focusing on amino acid metabolisms and matching the retrieved annotated genes versus non-starter lactic acid bacteria (NSLAB) references from KEGG and corresponding to those identified, all samples had the same gene spectrum for each pathway. Conversely, gene redundancy varied among samples and agreed with NSLAB diversity. Milk samples with higher numbers of NSLAB species harboured higher number of copies per pathway, which would enable steady-state towards perturbations. Some farming conditions, which affected the microbiome richness, also correlated with the NSLAB composition and functionality.

Genetic Determinants of Hydroxycinnamic Acid Metabolism in Heterofermentative Lactobacilli.

Phenolic acids are among the most abundant phenolic compounds in edible parts of plants. Lactic acid bacteria (LAB) metabolize phenolic acids, but the enzyme responsible for reducing hydroxycinnamic acids to phenylpropionic acids (HcrB) was only recently characterized in Lactobacillus plantarum In this study, heterofermentative LAB species were screened for their hydroxycinnamic acid metabolism. Data on strain-specific metabolism in combination with comparative genomic analyses identified homologs of HcrB as putative phenolic acid reductases. Par1 and HcrF both encode putative multidomain proteins with 25% and 63% amino acid identity to HcrB, respectively. Of these genes, par1 in L. rossiae and hcrF in L. fermentum were overexpressed in response to hydroxycinnamic acids. The deletion of par1 in L. rossiae led to the loss of phenolic acid metabolism. The strain-specific metabolism of phenolic acids was congruent with the genotype of lactobacilli; however, phenolic acid reductases were not identified in strains of Weissella cibaria that reduced hydroxycinnamic acids to phenylpropionic acids. Phylogenetic analysis of major genes involved in hydroxycinnamic acid metabolism in strains of the genus Lactobacillus revealed that Par1 was found to be the most widely distributed phenolic acid reductase, while HcrB was the least abundant, present in less than 9% of Lactobacillus spp. In conclusion, this study increased the knowledge on the genetic determinants of hydroxycinnamic acid metabolism, explaining the species- and strain-specific metabolic variations in lactobacilli and providing evidence of additional enzymes involved in hydroxycinnamic acid metabolism of lactobacilli.IMPORTANCE The metabolism of secondary plant metabolites, including phenolic compounds, by food-fermenting lactobacilli is a significant contributor to the safety, quality, and nutritional quality of fermented foods. The enzymes mediating hydrolysis, reduction, and decarboxylation of phenolic acid esters and phenolic acids in lactobacilli, however, are not fully characterized. The genomic analyses presented here provide evidence for three novel putative phenolic acid reductases. Matching comparative genomic analyses with phenotypic analysis and quantification of gene expression indicates that two of the three putative phenolic acid reductases, Par1 and HcrF, are involved in reduction of hydroxycinnamic acids to phenylpropionic acids; however, the activity of Par2 may be unrelated to phenolic acids and recognizes other secondary plant metabolites. These findings expand our knowledge on the metabolic potential of lactobacilli and facilitate future studies on activity and substrate specificity of enzymes involved in metabolism of phenolic compounds.

Attenuated Lactococcus lactis and Surface Bacteria as Tools for Conditioning the Microbiota and Driving the Ripening of Semisoft Caciotta Cheese.

This study aimed at establishing the effects of attenuated starters and surface bacteria on various features of caciotta cheese. The cheese undergoes a ripening period during which the house microbiota contaminates the surface. Conventional cheese (the control cheese [CC]) is made using only primary starters. Primary starters and attenuated (i.e., unable to grow and synthesize lactic acid) Lactococcus lactis (Lc. lactis) subsp. lactis were used to produce caciotta cheese without (ATT cheese) or with an inoculum of surface bacteria: (i) Leuconostoc lactis (Le. lactis) (LL cheese), (ii) Vibrio casei (VC cheese), (iii) Staphylococcus equorum (SE cheese), (iv) Brochothrix thermosphacta (BX cheese), and (v) a mixture of all four (MIX cheese). Attenuated Lc. lactis increased microbial diversity during cheese ripening. At the core, attenuated starter mainly increased indigenous lactococci and Lactobacillus delbrueckii group bacteria. At the surface, the main effect was on Macrococcus caseolyticus Autochthonous Le. lactis strains took advantage of the attenuated starter, becoming dominant. Adjunct Le. lactis positively affected Lactobacillus sakei group bacteria on the LL cheese surface. Adjunct V. casei, S. equorum, and B. thermosphacta did not become dominant. Surfaces of VC, SE, and BX cheeses mainly harbored Staphylococcus succinus Peptidase activities were higher in cheeses made with attenuated starter than in CC, which had the lowest concentration of free amino acids. Based on the enzymatic activities of adjunct Le. lactis, LL and MIX cheeses exhibited the highest glutamate dehydrogenase, cystathionine-γ-lyase, and esterase activities. As shown by multivariate statistical analyses, LL and MIX cheeses showed the highest similarity for microbiological and biochemical features. LL and MIX cheeses received the highest scores for overall sensory acceptability.IMPORTANCE This study provides in-depth knowledge of the effects of attenuated starters and surface bacterial strains on the microbiota and related metabolic activities during cheese ripening. The use of attenuated Lc. lactis strongly impacted the microbiota assembly of caciotta cheese. This led to improved biochemical and sensory features compared to conventional cheese. Among surface bacterial strains, Le. lactis played a key role in the metabolic activities involved in cheese ripening. This resulted in an improvement of the sensory quality of caciotta cheese. The use of attenuated lactic acid bacteria and the surface adjunct Le. lactis could be a useful biotechnology to improve the flavor formation of caciotta cheese.

Effects of Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001 in IBS patients.

BACKGROUND: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder, which still lacks effective therapy. We aimed to investigate the effects of a novel formulation of Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001 with vitamin B6 (LBB) on symptoms, intestinal permeability, cultivable bacteria and metabolome in IBS subjects. MATERIALS AND METHODS: Twenty-five IBS patients (Rome IV criteria) (M:F = 8:17; age 48 years ± 11 SD) were randomized to treatment (LBB) or placebo (one month each) in a crossover randomized double-blind controlled trial. Symptoms, intestinal habits, disease severity, intestinal permeability and intestinal microbiota were analysed at 0, 30, 45 and 60 days. RESULTS: Percentage decrease from baseline of abdominal pain (-48.8% vs -3.5%), bloating (-36.35% vs +7.35%) and severity of disease (-30.1% vs -0.4%) was significantly (P < .0001) greater with LBB than placebo, respectively. In IBS-D patients, the improvement from baseline of Bristol score was more consistent with LBB (from 6 ± 0.4 to 4.3 ± 1.1, P < .00001) than placebo (from 6.2 ± 0.7 to 5.3 ± 1.1, P = .04). In IBS-C patients, Bristol score tended to improve from baseline after LBB (2.6 ± 1.1 vs 3.2 ± 0.5, P = .06). LBB significantly improved the percentage of sucralose recovery (colonic permeability) (1.86 ± 0.1 vs 1.1 ± 0.2, P = .01). During treatment, presumptive lactic acid bacteria and bifidobacteria, relative abundance of propanoic, butanoic, pentanoic acids and hydrocarbons increased, while phenol decreased. CONCLUSIONS: The novel formulation of B. longum BB536 and L. rhamnosus HN001 with B6 vitamin improves symptoms and severity of disease, restores intestinal permeability and gut microbiota in IBS patients.

Healthy dietary patterns to reduce obesity-related metabolic disease: polyphenol-microbiome interactions unifying health effects across geography.

PURPOSE OF REVIEW: The spread of the Western lifestyle across the globe has led to a pandemic in obesity-related metabolic disease. The Mediterranean diet (MedDiet), Okinawa diet (OkD) and Nordic diet, derived from very different regions of the world and culinary traditions, have a large whole plant food component and are associated with reduced disease risk. This review focuses on polyphenol : microbiome interactions as one possible common mechanistic driver linking the protective effects whole plant foods against metabolic disease across healthy dietary patterns irrespective of geography. RECENT FINDINGS: Although mechanistic evidence in humans is still scarce, animal studies suggest that polyphenol or polyphenol rich foods induce changes within the gut microbiota and its metabolic output of trimethylamine N-oxide, short-chain fatty acids, bile acids and small phenolic acids. These cross-kingdom signaling molecules regulate mammalian lipid and glucose homeostasis, inflammation and energy storage or thermogenesis, physiological processes determining obesity-related metabolic and cardiovascular disease risk. However, it appears that where in the intestine metabolites are produced, the microbiota communities involved, and interactions between the metabolites themselves, can all influence physiological responses, highlighting the need for a greater understanding of the kinetics and site of production of microbial metabolites within the gut. SUMMARY: Interactions between polyphenols and metabolites produced by the gut microbiota are emerging as a possible unifying protective mechanism underpinning diverse healthy dietary patterns signaling across culinary traditions, across geography and across domains of life.

The sourdough fermentation is the powerful process to exploit the potential of legumes, pseudo-cereals and milling by-products in baking industry.

In the era of fighting wastes and paying close attention to sustainability and new protein sources, legumes, pseudo-cereals and milling by-products deserve all the efforts for increasing their consumption. Even with obvious peculiarities, a common trait characterizes these heterogeneous matrixes: unquestionable nutritional and functional value combined with some technological, sensory and/or anti-nutritional weaknesses, which unfortunately limit the exploitation and consumption. With the perspective of their use to fortify staple baked goods, we reviewed the main technological, nutritional and functional features of various legumes and pseudo-cereals, and milling by-products. Notwithstanding the potential of other technological solutions, we reported numerous evidences that qualified the sourdough fermentation as the most sustainable and powerful process to exploit the technological, nutritional and functional features of these matrixes and to limit or eliminate weak attributes. Sourdough fermentations tailored for specific matrixes allowed the fortification of staple baked goods with abundant levels of legumes, pseudo-cereals or milling by-products while keeping high consumer acceptance.

How fructophilic lactic acid bacteria may reduce the FODMAPs content in wheat-derived baked goods: a proof of concept.

BACKGROUND: FODMAPs (Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) intake is associated with the onset of irritable bowel syndrome symptoms. FODMAPs in wheat-derived baked goods may be reduced via bioprocessing by endogenous enzymes and/or microbial fermentation. Because of the inherent enzyme activities, bread made by baker's yeast and sourdough may result in decreased levels of FODMAPs, whose values are, however, not enough low for people sensitive to FODMAPs. RESULTS: Our study investigated the complementary capability of targeted commercial enzymes and metabolically strictly fructophilic lactic acid bacteria (FLAB) to hydrolyze fructans and deplete fructose during wheat dough fermentation. FLAB strains displayed higher fructose consumption rate compared to conventional sourdough lactic acid bacteria. Fructose metabolism by FLAB was faster than glucose. The catabolism of mannitol with the goal of its reuse by FLAB was also investigated. Under sourdough conditions, higher fructans breakdown occurred in FLAB inoculated doughs compared to conventional sourdough bacteria. Preliminary trials allowed selecting Apilactobacillus kunkeei B23I and Fructobacillus fructosus MBIII5 as starter candidates, which were successfully applied in synergy with commercial invertase for low FODMAPs baking. CONCLUSIONS: Results of this study clearly demonstrated the potential of selected strictly FLAB to strongly reduce FODMAPs in wheat dough, especially under liquid-dough and high oxygenation conditions.

Selection of non-Lactobacillus strains to be used as starters for sourdough fermentation.

The suitability of forty-one non-Lactobacillus strains to be used as selected starters for sourdough fermentation was evaluated. According to the data collected, Pediococcus pentosaceus OA1 and S3N3 and Leuconostoc citreum PRO17 were selected based on the optimal acidification and growth performances and the intense proteolytic activity (increase of TFFA up to 80%) on whole wheat flour doughs. A relevant degradation of phytic acid (up to 58%) and the increase of phenols content and scavenging activity (4- and 2-folds, respectively) were also observed. The technological performances were compared to two representative Lactobacillus strains (Lactobacillus plantarum and Lactobacillus sanfranciscensis). The investigation of the robustness of the selected strains during the propagation (back-slopping procedure) showed their long-term dominance only when singly-inoculated; while Leuc. citreum PRO17 dominated the fermentation when the strains were co-inoculated. The sourdoughs obtained by the non-Lactobacillus selected strains (singly or pooled) were used for breadmaking. Selected sourdoughs allowed the production of breads characterized by in-vitro protein digestibility (IVPD) higher than that of breads obtained with Lactobacillus strains or baker's yeast. The aroma profile, estimated by GC/MS, was complex and characterized by high concentration of the typical compounds (hexanol, 3-methylbutanol and 2-pentylfuran) of sourdough bread.

Fructose-rich niches traced the evolution of lactic acid bacteria toward fructophilic species.

Fructophilic lactic acid bacteria (FLAB) are found in fructose-rich habitats associated with flowers, fruits, fermented foods, and the gastrointestinal tract of several insects having a fructose-based diet. FLAB are heterofermentative lactobacilli that prefer fructose instead of glucose as carbon source, although additional electron acceptor substrates (e.g. oxygen) remarkably enhance their growth on glucose. As a newly discovered bacterial group, FLAB are gaining increasing interest. In this review, the ecological context in which these bacteria exist and evolve was resumed. The wide frequency of isolation of FLAB from fructose feeding insects has been deepened to reveal their ecological significance. Genomic, metabolic data, reductive evolution, and niche specialization of the main FLAB species have been discussed. Findings to date acquired are consistent with a metabolic model in which FLAB display a reliance on environmental niches and the degree of host specificity. In light of FLAB proximity to lactic acid bacteria generally considered to be safe, and due to their peculiar metabolic traits, FLAB may be successfully exploited in food and pharmaceutical applications.

Clinical and Microbiological Effect of a Multispecies Probiotic Supplementation in Celiac Patients With Persistent IBS-type Symptoms: A Randomized, Double-Blind, Placebo-controlled, Multicenter Trial.

GOALS: The goals of this study were to evaluate the efficacy and safety of a probiotic mixture in patients with celiac disease (CD) with irritable bowel syndrome (IBS)-type symptoms despite a strict gluten-free diet (GFD). BACKGROUND: About 30% of patients with CD adherent to a GFD suffer from IBS-type symptoms; a possible cause resides in the imbalances of the intestinal microbiota in CD. Probiotics may represent a potential treatment. STUDY: CD patients with IBS-type symptoms entered a prospective, double-blind, randomized placebo-controlled study. A 6-week treatment period was preceded by a 2-week run-in and followed by a 6-week follow-up phase. Clinical data were monitored throughout the study by validated questionnaires: IBS Severity Scoring System (IBS-SSS); Gastrointestinal Symptom Rating Scale (GSRS); Bristol Stool Form Scale (BSFS); and IBS Quality of Life Questionnaire (IBS-QOL). The fecal microbiota were assayed using plate counts and 16S rRNA gene-based analysis. RESULTS: In total, 109 patients were randomized to probiotics (n=54) or placebo (n=55). IBS-SSS and GSRS decreased significantly in probiotics, as compared with placebo [(-15.9%±14.8% vs. 8.2%±25.9%; P<0.001) and (-19.8%±16.6% vs. 12.9%±31.6%; P<0.001)], respectively. Treatment success was significantly higher in patients receiving probiotics, as compared with placebo (15.3% vs. 3.8%; P<0.04). Presumptive lactic acid bacteria, Staphylococcus and Bifidobacterium, increased in patients receiving probiotic treatment. No adverse events were reported. CONCLUSIONS: A 6-week probiotic treatment is effective in improving the severity of IBS-type symptoms, in CD patients on strict GFD, and is associated with a modification of gut microbiota, characterized by an increase of bifidobacteria.

Novel solid-state fermentation of bee-collected pollen emulating the natural fermentation process of bee bread.

Structure of lactic acid bacteria biota in ivy flowers, fresh bee-collected pollen (BCP), hive-stored bee bread, and honeybee gastrointestinal tract was investigated. Although a large microbial diversity characterized flowers and fresh BCP, most of lactic acid bacteria species disappeared throughout the bee bread maturation, giving way to Lactobacillus kunkeei and Fructobacillus fructosus to dominate long stored bee bread and honeybee crop. Adaptation of lactic acid bacteria was mainly related to species-specific, and, more in deep, to strain-specific features. Bee bread preservation seemed related to bacteria metabolites, produced especially by some L. kunkeei strains, which likely gave to lactic acid bacteria the capacity to outcompete other microbial groups. A protocol to ferment BCP was successfully set up, which included the mixed inoculum of selected L. kunkeei strains and Hanseniaspora uvarum AN8Y27B, almost emulating the spontaneous fermentation of bee bread. The strict relationship between lactic acid bacteria and yeasts during bee bread maturation was highlighted. The use of the selected starters increased the digestibility and bioavailability of nutrients and bioactive compounds naturally occurring in BCP. Our biotechnological protocol ensured a product microbiologically stable and safe. Conversely, raw BCP was more exposed to the uncontrolled growth of yeasts, moulds, and other bacterial groups.

How Lactobacillus plantarum shapes its transcriptome in response to contrasting habitats.

Triplets of Lactobacullus plantarum strains were isolated from nine contrasting habitats. Without any passage through other culture media, isolation and cultivation were on model media that strictly reproduced the chemical and physical conditions and stressors of the habitats of origin. Here, we demonstrated how L. plantarum regulates and shapes its transcriptome in response to contrasting habitats. Firstly, multivariate clustering analysis of transcriptional data (RNA-Seq), complemented with metabolomics and phenomics, grouped the strains according to the habitats of origin. Subsequently, selected strains from each habitat switched to repeated cultivation on MRS medium and transcriptomes homogenized into a unique cluster. Adaptation to this common medium mainly relied on activation of genes for phage- and prophage-related proteins and transposases. Finally, the comparison of growth across model media and with respect to MRS medium showed that 44% of the overall 3112 gene transcripts changed depending on the specific habitat. Regulation and shaping of transcriptomes mainly concerned carbohydrate acquisition, pyruvate catabolism, proteolytic system and amino acid, lipid and inorganic ion transport and metabolism, with contrasting responses for contrasting habitats. Pathways reconstruction demonstrated how the large genome size of L. plantarum imparts transcriptome and metabolic flexibility as the basic mechanism for a nomadic lifestyle.

Joint Data Analysis in Nutritional Epidemiology: Identification of Observational Studies and Minimal Requirements.

Background: Joint data analysis from multiple nutrition studies may improve the ability to answer complex questions regarding the role of nutritional status and diet in health and disease. Objective: The objective was to identify nutritional observational studies from partners participating in the European Nutritional Phenotype Assessment and Data Sharing Initiative (ENPADASI) Consortium, as well as minimal requirements for joint data analysis. Methods: A predefined template containing information on study design, exposure measurements (dietary intake, alcohol and tobacco consumption, physical activity, sedentary behavior, anthropometric measures, and sociodemographic and health status), main health-related outcomes, and laboratory measurements (traditional and omics biomarkers) was developed and circulated to those European research groups participating in the ENPADASI under the strategic research area of "diet-related chronic diseases." Information about raw data disposition and metadata sharing was requested. A set of minimal requirements was abstracted from the gathered information. Results: Studies (12 cohort, 12 cross-sectional, and 2 case-control) were identified. Two studies recruited children only and the rest recruited adults. All studies included dietary intake data. Twenty studies collected blood samples. Data on traditional biomarkers were available for 20 studies, of which 17 measured lipoproteins, glucose, and insulin and 13 measured inflammatory biomarkers. Metabolomics, proteomics, and genomics or transcriptomics data were available in 5, 3, and 12 studies, respectively. Although the study authors were willing to share metadata, most refused, were hesitant, or had legal or ethical issues related to sharing raw data. Forty-one descriptors of minimal requirements for the study data were identified to facilitate data integration. Conclusions: Combining study data sets will enable sufficiently powered, refined investigations to increase the knowledge and understanding of the relation between food, nutrition, and human health. Furthermore, the minimal requirements for study data may encourage more efficient secondary usage of existing data and provide sufficient information for researchers to draft future multicenter research proposals in nutrition.

Wheat endophytic lactobacilli drive the microbial and biochemical features of sourdoughs.

The aim of this study was to assess whether wheat endophytic lactic acid bacteria (LAB) are able to dominate in sourdough ecosystem. To do that, a first experimental phase considered doughs produced under semi-sterile conditions and singly inoculated with different strains of endophytic LAB and Lactobacillus sanfranciscensis A4 isolated from sourdough. Notwithstanding the high frequency of Lactobacillus plantarum in the sourdoughs prepared in laboratory, only one of the starter strains, L. plantarum LB2, was detected after five days of back-slopping. Subsequently, the ability of this strain to dominate traditional sourdoughs was evaluated at bakery and laboratory level. Contamination of sourdoughs with L. plantarum LB2 caused an increased number of LAB and, accordingly, higher acidification, compared to the sourdoughs before this event. After six days of propagation, the wheat endophytic strain L. plantarum LB2 was retrieved as a component of the bacterial population, in all the sourdoughs and regardless of the place of propagation. In addition, the contamination event caused a modification of the lactic acid bacterium biota, which in turn influenced some sourdoughs biochemical features. In conclusion, this study showed that wheat endophytic LAB could represent a potential reservoir for selecting robust strains to be used as sourdough starters.

Dietary Fibers and Protective Lactobacilli Drive Burrata Cheese Microbiome.

This study was aimed at improving the functional attributes and shelf life of burrata cheese by using protective lactobacilli (Lactobacillus plantarum LPAL and Lactobacillus rhamnosus LRB), fructooligosaccharides, and inulin. Six burrata cheeses were made using (i) the traditional protocol (control), (ii) the addition of 0.5% fructooligosaccharides and inulin (DF cheese), (iii) protective lactobacilli in milk alone (PL cheese), (iv) protective lactobacilli in milk and governing liquid (2PL cheese), (v) protective lactobacilli in milk and dietary fibers (DF_PL cheese), and (vi) protective lactobacilli in milk and governing liquid and dietary fibers (DF_2PL cheese). As expected, DF, DF_PL, and DF_2PL cheeses showed 1.5% of total fibers. Burrata cheeses produced by adding protective lactobacilli only in milk (PL and DF_PL cheeses) showed the lowest acidification during cheese making and storage. Lactic and acetic acids and ethanol were found at the lowest concentrations in these samples. Analyses of cultivable microbiota and the microbiome showed that protective lactobacilli reduced the house microbiota components (e.g., Streptococcus thermophilus, Lactococcus lactis, and Leuconostoc lactis) during cheese making and storage. Protective lactobacilli slowed the growth of staphylococci, coliforms, and Pseudomonas spp., especially in early storage. According to the different microbiome assemblies, burrata samples differed in peptide profiles and the levels of free amino acids. As shown by a sensory analysis, the addition of protective lactobacilli in milk improved the flavor and increased the shelf life of burrata cheese. In comparison to cheeses made using protective cultures only in milk, the shelf lives of those containing cultures also in the governing liquid were not further prolonged and they received lower acceptability scores by the panelists.IMPORTANCE This study provides more in-depth knowledge of the microbiome of burrata cheese and the set-up for a novel biotechnology using prebiotic dietary fibers and protective probiotic Lactobacillus plantarum LPAL and Lactobacillus rhamnosus LRB in milk. The biotechnology proposed in this study should be considered a useful tool to improve the functional value of burrata cheese. The use of protective lactobacilli in milk enhanced the flavor formation and shelf life of burrata cheese.

Selected Probiotic Lactobacilli Have the Capacity To Hydrolyze Gluten Peptides during Simulated Gastrointestinal Digestion.

The aim of this study was to demonstrate the capacity of probiotic lactobacilli to hydrolyze immunogenic gluten peptides. Eighteen commercial strains of probiotic lactobacilli with highly variable peptidase activity (i.e., aminopeptidase N, iminopeptidase, prolyl endopeptidyl peptidase, tripeptidase, prolidase, prolinase, and dipeptidase), including toward Pro-rich peptides, were tested in this study. Ten probiotic strains were selected on the basis of their specific enzyme activity. When pooled, these 10 strains provided the peptidase portfolio that is required to completely degrade the immunogenic gluten peptides involved in celiac disease (CD). The selected probiotic mixture was able to completely hydrolyze well-known immunogenic epitopes, including the gliadin 33-mer peptide, the peptide spanning residues 57 to 68 of the α9-gliadin (α9-gliadin peptide 57-68), A-gliadin peptide 62-75, and γ-gliadin peptide 62-75. During digestion under simulated gastrointestinal conditions, the pool of 10 selected probiotic lactobacilli strongly hydrolyzed the wheat bread gluten (ca. 18,000 ppm) to less than 10 ppm after 360 min of treatment. As determined by multidimensional chromatography (MDLC) coupled to nanoelectrospray ionization (nano-ESI)-tandem mass spectrometry (MS/MS), no known immunogenic peptides were detected in wheat bread that was digested in the presence of the probiotics. Accordingly, the level of cytokines (interleukin 2 [IL-2], IL-10, and interferon gamma [IFN-γ]) produced by duodenal biopsy specimens from CD patients who consumed wheat bread digested by probiotics was similar to the baseline value (negative control). Probiotics that specifically hydrolyze gluten polypeptides could also be used to hydrolyze immunogenic peptides that contaminate gluten-free products. This could provide a new and safe adjunctive therapy alternative to the gluten-free diet (GFD).IMPORTANCE This study confirmed that probiotic Lactobacillus strains have different enzymatic abilities for hydrolyzing polypeptides, including the Pro-rich epitopes involved in the pathology of CD. Ten lactobacilli with complementary peptidase activities that hydrolyze gluten peptides during simulated gastrointestinal digestion were selected and tested. The results collected showed the potential of probiotic formulas as novel dietary treatments for CD patients.

Hydrolysate from a mixture of legume flours with antifungal activity as an ingredient for prolonging the shelf-life of wheat bread.

Aiming at identifying antifungal compounds from plant matrices to be used as ingredients in the bakery industry, a water/salt-soluble extract (WSE) was produced from a legume enzyme hydrolysate, consisting of a mixture of pea, lentil, and faba bean flours, and assayed towards Penicillium roqueforti DPPMAF1. Agar diffusion assays allowed the selection of the optimal processing conditions for hydrolysis. As shown by hyphal radial growth rate, the inhibition was observed towards several fungi, including Aspergillus parasiticus CBS971.97, Penicillium carneum CBS 112297, Penicillium paneum CBS 101032, Penicillium polonicum 112490. A multi-step purification was carried out to identify the active compounds. The antifungal activity was attributed to native proteins (nsLTP, ubiquitin, lectin alpha-1 chain, wound-induced basic protein, defensin-1, defensin-2) and a mixture of peptides, which were released during hydrolysis. Nine peptides were purified and identified as sequences encrypted in legume vicilins, lectins and chitinases. WSE was used as ingredient for making bread under pilot plant conditions. Chemical, structural and sensory characterization of bread showed the lack of significant changes compared to control. The bread made with the legume hydrolysate had a longer shelf-life than that of the control.

Multiple microbial cell-free extracts improve the microbiological, biochemical and sensory features of ewes' milk cheese.

This study used cell-free enzyme (CFE) extracts from Lactobacillus casei, Hafnia alvei, Debaryomyces hansenii and Saccharomyces cerevisiae to condition or accelerate Pecorino-type cheese ripening. Compositional, microbiological, and biochemical analyses were performed, and volatile and sensory profiles were obtained. Lactobacilli and cocci increased during ripening, especially in cheeses containing CFE from L. casei, H. alvei and D. hansenii (LHD-C) and L. casei, H. alvei and S. cerevisiae (LHS-C). Compared to control cheese (CC), several enzymatic activities were higher (P < 0.05) in CFE-supplemented cheeses. Compared to the CC (1907 mg kg-1 of cheese), the free amino acid level increased (P < 0.05) in CFE-supplemented cheeses, ranging from approximately 2575 (LHS-C) to 5720 (LHD-C) mg kg-1 of cheese after 60 days of CFE-supplemented ripening. As shown by GC/MS analysis, the levels of several volatile organic compounds were significantly (P < 0.05) lower in CC than in CFE-supplemented cheeses. All cheeses manufactured by adding multiple CFEs exhibited higher scores (P < 0.05) for internal structure, acid taste and juiciness than CC samples. This study shows the possibility of producing ewes' milk cheese with standardized characteristics and improved flavor intensity in a relatively short time.

Exploitation of grape marc as functional substrate for lactic acid bacteria and bifidobacteria growth and enhanced antioxidant activity.

This study aimed at using grape marc for the growth of lactic acid bacteria and bifidobacteria with the perspective of producing a functional ingredient having antioxidant activity. Lactobacillus plantarum 12A and PU1, Lactobacillus paracasei 14A, and Bifidobacterium breve 15A showed the ability to grow on grape marc (GM) based media. The highest bacterial cell density (>9.0 CFU/g) was found in GM added of 1% of glucose (GMG). Compared to un-inoculated and incubated control fermented GMG showed a decrease of carbohydrates and citric acid together with an increase of lactic acid. The content of several free amino acids and phenol compounds differed between samples. Based on the survival under simulated gastro-intestinal conditions, GMG was a suitable carrier of lactic acid bacteria and bifidobacteria strains. Compared to the control, cell-free supernatant (CFS) of fermented GMG exhibited a marked antioxidant activity in vitro. The increased antioxidant activity was confirmed using Caco-2 cell line after inducing oxidative stress, and determining cell viability and radical scavenging activity through MTT and DCFH-DA assays, respectively. Supporting these founding, the SOD-2 gene expression of Caco-2 cells also showed a lowest pro-oxidant effect induced by the four CFS of GMG fermented by lactic acid bacteria and bifidobacteria.