Abstract BACKGROUND: Oat and barley beta-glucans are prebiotic fibers known for their cholesterol-lowering activity, but their action on the human gut microbiota metabolism is still under research. Although the induction of short-chain fatty acids (SCFA) following their ingestion has previously been reported, no study has investigated their effects on proteolytic uremic toxins p-cresyl sulfate (pCS) and indoxyl sulfate (IS) levels, while others have failed to demonstrate an effect on the endothelial function measured through flow-mediated dilation (FMD). OBJECTIVE: The aim of our study was to evaluate whether a nutritional intervention with a functional pasta enriched with beta-glucans could promote a saccharolytic shift on the gut microbial metabolism and improve FMD. METHODS: We carried out a pilot study on 26 healthy volunteers who underwent a 2-month dietary treatment including a daily administration of Granoro "Cuore Mio" pasta enriched with barley beta-glucans (3g/100g). Blood and urine routine parameters, serum pCS/IS and FMD were evaluated before and after the dietary treatment. RESULTS: The nutritional treatment significantly reduced LDL and total cholesterol, as expected. Moreover, following beta-glucans supplementation we observed a reduction of serum pCS levels and an increase of FMD, while IS serum levels remained unchanged. CONCLUSIONS: We demonstrated that a beta-glucans dietary intervention in healthy volunteers correlates with a saccharolytic shift on the gut microbiota metabolism, as suggested by the decrease of pCS and the increase of SCFA, and associates with an improved endothelial reactivity. Our pilot study suggests, in addition to cholesterol, novel pCS-lowering properties of beta-glucans, worthy to be confirmed in large-scale trials and particularly in contexts where the reduction of the microbial-derived uremic toxin pCS is of critical importance, such as in chronic kidney disease.

Dietary polyphenols are associated with a wide range of health benefits, protecting against chronic diseases and promoting healthy aging. Dietary polyphenols offer a complementary approach to the treatment of inflammatory bowel diseases (IBDs), a group of common chronic intestinal inflammation syndromes for which there is no cure. Tomato is widely consumed but its content of polyphenols is low. We developed a tomato variety, Bronze, enriched in three distinct classes of polyphenols: flavonols, anthocyanins, and stilbenoids. Using Bronze tomatoes as a dietary supplement as well as Indigo (high anthocyanins and flavonols), ResTom (high stilbenoids) and wild-type tomatoes, we examined the effects of the different polyphenols on the host gut microbiota, inflammatory responses, and the symptoms of chronic IBD, in a mouse model. Bronze tomatoes significantly impacted the symptoms of IBD. A similar result was observed using diets supplemented with red grape skin containing flavonols, anthocyanins, and stilbenoids, suggesting that effective protection is provided by different classes of polyphenols acting synergistically.

The role played by glutamatedehydrogenase (GDH) in Lactobacillusplantarum was investigated by preparing a GDH-deficient mutant UC1001G. The growth rate and acidification displayed by this mutant were lower than those of the wild-type UC1001. Compared with UC1001G, UC1001 increased the synthesis of proteins that confer a competitive advantage of cell resistance. When both strains were used as adjunct starter for cheese-making, the highest cell survival was found for UC1001. Cheeses made with the adjunct of UC1001 showed the lowest level of free aminoacids and the highest content of volatile organic compounds. Both strains produced high level of γ-aminobutyric acid in cheese and under simulated gastrointestinal conditions. The findings of this study provide an evidence of the important role of GDH on growth and survival of Lb. plantarum under different environmental conditions that influences some important functional features of the strain.

Background: Epidemiology of celiac disease (CD) is increasing. CD mainly presents in early childhood with small intestinal villous atrophy and signs of malabsorption. Compared to healthy individuals, CD patients seemed to be characterized by higher numbers of Gram-negative bacteria and lower numbers Gram-positive bacteria. Results: This study aimed at investigating the microbiota and metabolome of 19 celiac disease children under gluten-free diet (treated celiac disease, T-CD) and 15 non-celiac children (HC). PCR-denaturing gradient gel electrophoresis (DGGE) analyses by universal and group-specific primers were carried out in duodenal biopsies and faecal samples. Based on the number of PCR-DGGE bands, the diversity of Eubacteria was the higher in duodenal biopsies of T-CD than HC children. Bifidobacteria were only found in faecal samples. With a few exceptions, PCRDGGE profiles of faecal samples for Lactobacillus and Bifidobacteria differed between T-CD and HC. As shown by culture-dependent methods, the levels of Lactobacillus, Enterococcus and Bifidobacteria were confirmed to be significantly higher (P = 0.028; P = 0.019; and P = 0.023, respectively) in fecal samples of HC than in T-CD children. On the contrary, cell counts (CFU/ml) of presumptive Bacteroides, Staphylococcus, Salmonella, Shighella and Klebsiella were significantly higher (P = 0.014) in T-CD compared to HC children. Enterococcus faecium and Lactobacillus plantarum were the species most diffusely identified. This latter species was also found in all duodenal biopsies of T-CD and HC children. Other bacterial species were identified only in T-CD or HC faecal samples. As shown by Randomly Amplified Polymorphic DNA-PCR analysis, the percentage of strains identified as lactobacilli significantly (P = 0.011) differed between T-CD (ca. 26.5%) and HC (ca. 34.6%) groups. The metabolome of T-CD and HC children was studied using faecal and urine samples which were analyzed by gas-chromatography mass spectrometry-solid-phase microextraction and 1H-Nuclear Magnetic Resonance. As shown by Canonical Discriminant Analysis of Principal Coordinates, the levels of volatile organic compounds and free amino acids in faecal and/or urine samples were markedly affected by CD. Conclusion: As shown by the parallel microbiology and metabolome approach, the gluten-free diet lasting at least two years did not completely restore the microbiota and, consequently, the metabolome of CD children. Some molecules (e.g., ethyl-acetate and octyl-acetate, some short chain fatty acids and free amino acids, and glutamine) seems to be metabolic signatures of CD.

Weissella cibaria, Lactobacillus plantarum, Lactobacillus sp. and Lactobacillus pentosus were variously identified from blackberries, prunes, kiwifruits, papaya and fennels by partial 16S rRNA gene sequence. Representative isolates from each plant species were screened based on the kinetics of growth on fruit juices. A protocol for processing and storage of red and greensmoothies (RS and GS) was set up, which included fermentation by selected lacticacid bacteria starters and exo-polysaccharide producing strains. Starters grew and remained viable at ca. 9.0 log cfu g−1 during 30 days of storage at 4 °C. No contaminating Enterobacteriaceae and yeast were found throughout storage. Values of soluble solids, total titratable acidity and viscosity distinguished started RS and GS compared to spontaneously (unstarted) fermented smoothies. Color difference dE∗ab and browning index were positively affected by lacticacidfermentation. Consumption of carbohydrates by lacticacid bacteria was limited as well as it was the lacticfermentation. Consumption of malic acid was evident throughout storage. Polyphenolic compounds and, especially, ascorbic acid were better preserved in started RS and GS compared to unstarted samples. This reflected on the free radical scavenging activity. A statistical correlation was only found between the level of ascorbic acid and free radical scavenging activity. As shown by a first-order equation, the rate of degradation of ascorbic acid through storage were found to be higher in the unstarted compared to started RS and GS. Fermentation by lacticacid bacteria clearly improved the sensory attributes of RS and GS.

Lactic acid bacteria strains were identified from wheatgerm by 16S rRNA partial sequencing, subjected to RAPD-PCR typing and screened. Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5 were used as starters to produce sourdough fermented wheatgerm (SFWG). The chemical and nutritionalcharacteristics of SFWG were compared to those of the raw wheatgerm (RWG). Lipase activity in SFWG was ca. 2.6-fold lower than that found in RWG. As shown by SPME/GC/MS analysis, most of the volatile compounds derived from lipid oxidation during storage (40 days) were at markedly lower levels in SFWG compared to RWG. Fermentation of wheatgerm increased of ca. 50% the concentration of free amino acids. Glu markedly decreased in SFWG, due to its conversion in GABA. The concentration of the anti-nutritional factor raffinose also decreased in SFWG. The in vitro protein digestibility, the concentration of total phenols, phytase and antioxidant activities were increased by fermentation.

In this study, we compared the fecal microbiota and metabolomes of 26 healthy subjects before (HS) and after (HSB) 2 months of diet intervention based on the administration of durum wheat flour and whole-grain barley pasta containing the minimum recommended daily intake (3 g) of barley β-glucans. Metabolically active bacteria were analyzed through pyrosequencing of the 16S rRNA gene and community-level catabolic profiles. Pyrosequencing data showed that levels of Clostridiaceae (Clostridium orbiscindens and Clostridium sp.), Roseburia hominis, and Ruminococcus sp. increased, while levels of other Firmicutes and Fusobacteria decreased, from the HSB samples to the HS fecal samples. Community-level catabolic profiles were lower in HSB samples. Compared to the results for HS samples, cultivable lactobacilli increased in HSB fecal samples, while the numbers of Enterobacteriaceae, total coliforms, and Bacteroides, Porphyromonas, Prevotella, Pseudomonas, Alcaligenes, and Aeromonas bacteria decreased. Metabolome analyses were performed using an amino acid analyzer and gas chromatography-mass spectrometry solid-phase microextraction. A marked increase in short-chain fatty acids (SCFA), such as 2-methyl-propanoic, acetic, butyric, and propionic acids, was found in HSB samples with respect to the HS fecal samples. Durum wheat flour and whole-grain barley pasta containing 3% barley β-glucans appeared to be effective in modulating the composition and metabolic pathways of the intestinal microbiota, leading to an increased level of SCFA in the HSB samples.

An experimental investigation evaluated the possibility of increasing the nutritional value of fermented table olives by adding olive leaf extract (OLE). OLE was added to table olives fermented using indigenous bacteria and yeasts, and a commercial starter (Lactobacillus plantarum strain). Microbiological, physico-chemical, and sensory analyses showed that OLE addition resulted in fermented olives with higher levels of antioxidant, anti-inflammatory, and antimicrobial substances, but did not adversely affect their qualities. Moreover, OLE and the commercial starter functioned synergically against spoilage microorganisms. In addition, fermented olives had higher values of hardness, total phenols, antioxidant activity, hydroxytyrosol, and verbascoside. Nonanal and ethanol contents were lower in fermented olives when Lactobacillus plantarum and OLE were used, indicating lower degrees of oxidation and fermentation. Finally, olives fermented with OLE had a less bitter taste.

Proceedings of the 1th International conference on "Microbial Diversity 2011 - Environmental Stress and Adaptation, October 26 – 28, Milan (Italy). Pages 102-105.

This study aimed at investigating the fecal microbiota and metabolome of children with Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) and autism (AD) in comparison to healthy children (HC). Bacterial tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) of the 16S rDNA and 16S rRNA analyses were carried out to determine total bacteria (16S rDNA) and metabolically active bacteria (16S rRNA), respectively. The main bacterial phyla (Firmicutes, Bacteroidetes, Fusobacteria and Verrucomicrobia) significantly (P<0.05) changed among the three groups of children. As estimated by rarefaction, Chao and Shannon diversity index, the highest microbial diversity was found in AD children. Based on 16S-rRNA and culture-dependent data, Faecalibacterium and Ruminococcus were present at the highest level in fecal samples of PDD-NOS and HC children. Caloramator, Sarcina and Clostridium genera were the highest in AD children. Compared to HC, the composition of Lachnospiraceae family also differed in PDD-NOS and, especially, AD children. Except for Eubacterium siraeum, the lowest level of Eubacteriaceae was found on fecal samples of AD children. The level of Bacteroidetes genera and some Alistipes and Akkermansia species were almost the highest in PDD-NOS or AD children as well as almost all the identified Sutterellaceae and Enterobacteriaceae were the highest in AD. Compared to HC children, Bifidobacterium species decreased in AD. As shown by Canonical Discriminant Analysis of Principal Coordinates, the levels of free amino acids and volatile organic compounds of fecal samples were markedly affected in PDD-NOS and, especially, AD children. If the gut microbiota differences among AD and PDD-NOS and HC children are one of the concomitant causes or the consequence of autism, they may have implications regarding specific diagnostic test, and/or for treatment and prevention.

Functional microorganisms and health benefits represent a binomial with great potential for fermented functional foods. The health benefits of fermented functional foods are expressed either directly through the interactions of ingested live microorganisms with the host (probiotic effect) or indirectly as the result of the ingestion of microbial metabolites synthesized during fermentation (biogenic effect). Since the importance of high viability for probiotic effect, two major options are currently pursued for improving it—to enhance bacterial stress response and to use alternative products for incorporating probiotics (e.g., ice cream, cheeses, cereals, fruit juices, vegetables, and soy beans). Further, it seems that quorum sensing signal molecules released by probiotics may interact with human epithelial cells from intestine thus modulating several physiological functions. Under optimal processing conditions, functional microorganisms contribute to food functionality through their enzyme portfolio and the release of metabolites. Overproduction of free amino acids and vitamins are two classical examples. Besides, bioactive compounds (e.g., peptides, γ-amino butyric acid, and conjugated linoleic acid) may be released during food processing above the physiological threshold and they may exert various in vivo health benefits. Functional microorganisms are even more used in novel strategies for decreasing phenomenon of food intolerance (e.g., gluten intolerance) and allergy. By a critical approach, this review will aim at showing the potential of functional microorganisms for the quality of functional foods.

The in vitro screening of several non starter lactic acid bacteria (NSLAB) strains from Lactobacillus plantarum, Lactobacillus casei ssp. casei, Lactobacillus curvatus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus parabuchnerii, Lactobacillus alimentarius and Lactobacillus paraplantarum revealed the presence of NADP-dependent glutamate dehydrogenase (GDH). NADP-GDH activity was markedly strain dependent and varied according to the interactions between temperature, pH and NaCl. The role played by GDH in Lb. plantarum was investigated by preparing a GDH-deficient mutant strain of Lb. plantarum UC1001 in which the gdh gene was disrupted by plasmid integration. The growth rate and acidification displayed by this mutant strain were lower than those of the wild-type strain, especially when cultivated in reconstituted skim milk powder (SMP). When both strains were used as adjunct starter for cheese-making, the highest cell survival was found for wild-type. In comparison with the GDH-deficient mutant, cheeses made with the adjunct of wild-type showed a lower level of free amino acids (FAA) (7181 ± 52 mg/kg for mutant vs. 4790 ± 40 mg/kg for wild-type). Accordingly, the highest level of volatile organic compounds (VOC) (alcohols, aldehydes, miscellaneous and carboxylic acids) was found in cheeses made by wild-type strain. The findings of this study provide evidence of the important role of NADP-GDH in cheese ripening and flavour development.

Objectives: This study was aimed at showing the safety, for young patients with celiac disease (CD), of sweet baked goods made of wheat flour, which was rendered gluten-free during sourdough fermentation. Methods and Results: As shown by R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay (ELISA), selected lactobacilli and fungal proteases, routinely used in bakeries, degraded gluten to &lt;10 ppm during sourdough fermentation. The resulting flour was mainly a mixture of water-/salt-soluble low-size peptides and free amino acids. Gliadin and glutenin fractions extracted from the pepsin-trypsin (PT) digest of the fermented wheat flour induced the expression of interferon (IFN)-γ at the level comparable with the negative control. After fermentation, the wheat flour was spray dried and used for making sweet baked goods. Eight patients with CD in remission were enrolled for the clinical challenge, and they daily consumed 200 g of sweet baked goods equivalent to 10 g of native gluten. Hematology, serology (total serum IgA, IgG and IgA antigluten, endomysial and tissue transglutaminase IgA antibodies), and intestinal permeability analyses were carried out over time. One patient interrupted the trial after 15 days and another after 30 days only due to difficulties in the compliance of the daily consumption. All of the other patients showed normal values of hematology, serology, and intestinal permeability during 60 days of challenge. Conclusions: This study showed that a wheat flour–fermented product, having gluten completely degraded, is not toxic for patients with CD. Nevertheless, these foods should not be recommended for patients with celiac disease until a formal trial has been done.

The aim of this study was to optimize the production of exopolysaccharides (EPS) by sourdough LactobacilluscurvatusDPPMA10 for industrial application. The effects of pH, temperature, planktonic or attached cells and of some food matrices as substrates were studied. Wheatflour hydrolysate (WFH), reconstituted skimmed milk (RSM) and whey milk were supplemented with fresh yeast extract, mineral salts, and/or molasses. Non-controlled pH, starting from 5.6 to 3.5, was the optimal condition for L. curvatusDPPMA10. Temperature of 30 °C was also found to be optimal. Solid surfaces (agar culture media) stimulated attached bacteria to synthesize EPS (≥ of two-fold, P < 0.05) with respect to planktonic cells (broth media). The highest production of EPS (ca. 46–50 g/kg of wet medium) was found during growth as attached cells in WFH agar supplemented with glucose, sucrose or molasses, mineral salts and fresh yeast extract at 30 °C for 48 h. As shown by high-performance liquid chromatography analysis, glucose was the only hydrolysis end-product for EPS synthesized during 48 h of incubation. The EPS synthesized by L. curvatusDPPMA10 improved the quality of bread and was utilized as carbon course by intestinal strains of lactobacilli and bifidobacteria. The synthesis of EPS by L. curvatusDPPMA10 under the conditions of this study may open new perspectives for their industrial applications.

Sourdough fermentation is one of the oldest food biotechnologies, which has been studied and recently rediscovered for its effect on the sensory, structural, nutritional and shelf life properties of leavened baked goods. Acidification, proteolysis and activation of a number of enzymes as well as the synthesis of microbial metabolites cause several changes during sourdough fermentation, which affect the dough and baked good matrix, and influence the nutritional/functional quality. Currently, the literature is particularly rich of results, which show how the sourdough fermentation may affect the functional features of leavened baked goods. In the form of pre-treating raw materials, fermentation through sourdough may stabilize or to increase the functional value of bran fractions and wheat germ. Sourdough fermentation may decrease the glycaemic response of baked goods, improve the properties and bioavailability of dietary fibre complex and phytochemicals, and may increase the uptake of minerals. Microbial metabolism during sourdough fermentation may also produce new nutritionally active compounds, such as peptides and amino acid derivatives (e.g., g-amino butyric acid) with various functionalities, and potentially prebiotic exo-polysaccharides. The wheat flour digested via fungal proteases and selected sourdough lactobacilli has been demonstrated to be probably safe for celiac patients.

Seven mature type I sourdoughs were comparatively back-slopped (80 days) at artisan bakery and laboratory levels under constant technology parameters. The cell density of presumptive lactic acid bacteria and related biochemical features were not affected by the environment of propagation. On the contrary, the number of yeasts markedly decreased from artisan bakery to laboratory propagation. During late laboratory propagation, denaturing gradient gel electrophoresis (DGGE) showed that the DNA band corresponding to Saccharomyces cerevisiae was no longer detectable in several sourdoughs. Twelve species of lactic acid bacteria were variously identified through a culture-dependent approach. All sourdoughs harbored a certain number of species and strains, which were dominant throughout time and, in several cases, varied depending on the environment of propagation. As shown by statistical permutation analysis, the lactic acid bacterium populations differed among sourdoughs propagated at artisan bakery and laboratory levels. Lactobacillus plantarum, Lactobacillus sakei, and Weissella cibaria dominated in only some sourdoughs back-slopped at artisan bakeries, and Leuconostoc citreum seemed to be more persistent under laboratory conditions. Strains of Lactobacillus sanfranciscensis were indifferently found in some sourdoughs. Together with the other stable species and strains, other lactic acid bacteria temporarily contaminated the sourdoughs and largely differed between artisan bakery and laboratory levels. The environment of propagation has an undoubted influence on the composition of sourdough yeast and lactic acid bacterium microbiotas.

This work aimed to select heat-resistant probiotic lactobacilli to be added to Fior di Latte (high-moisture cow milk Mozzarella) cheese. First, 18 probiotic strains belonging to Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus reuteri were screened. Resistance to heating (65 or 55°C for 10 min) varied markedly between strains. Adaptation at 42°C for 10 min increased the heat resistance at 55°C for 10 min of all probiotic lactobacilli. Heat-adapted L. delbrueckii ssp. bulgaricus SP5 (decimal reduction time at 55°C of 227.4 min) and L. paracasei BGP1 (decimal reduction time at 55°C of 40.8 min) showed the highest survival under heat conditions that mimicked the stretching of the curd and were used for the manufacture of Fior di Latte cheese. Two technology options were chosen: chemical (addition of lactic acid to milk) or biological (Streptococcus thermophilus as starter culture) acidification with or without addition of probiotics. As determined by random amplified polymorphic DNA-PCR and 16S rRNA gene analyses, the cell density of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 in chemically or biologically acidified Fior di Latte cheese was approximately 8.0 log10 cfu/g. Microbiological, compositional, biochemical, and sensory analyses (panel test by 30 untrained judges) showed that the use of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 enhanced flavor formation and shelf-life of Fior di Latte cheeses.

SelectedLactobacillus paracasei FC2-5, Lactobacillus casei LC01 and Lactobacillus curvatus 2770 were used for cheese making as adjunct cultures (AC) or attenuatedadjunct cultures (AAC). AAC were obtained by sonication treatment. ItalianCaciotta-typecheeses were manufactured on an industrial plant scale, including control cheese (CC) without AC, and ripening was lasted 60 days at 10 °C. AAC did not increase the production of lactic acid compared with the CC. AC acidified during manufacture and throughout ripening, affected the moisture and texture of the cheese. As shown by the plate count and confirmed by RAPD-PCR, the cell numbers of non-starterlactobacilli varied between cheeses manufactured with AC or AAC. The major differences between cheeses were the accumulation of free amino acids and the synthesis of some key volatile components. As shown by PT-GC/MS analysis, the levels of ketones, secondary alcohols and sulphur compounds were highest in the cheese manufactured with AAC.

This study aimed at investigating the fecal microbiota, and the fecal and urinary metabolome of non progressor (NP) and progressor (P) patients with immunoglobulin A nephropathy (IgAN). Three groups of volunteers were included in the study: (i) sixteen IgAN NP patients; (ii) sixteen IgAN P patients; and (iii) sixteen healthy control (HC) subjects, without known diseases. Selective media were used to determine the main cultivable bacterial groups. Bacterial tag-encoded FLX-titanium amplicon pyrosequencing of the 16S rDNA and 16S rRNA was carried out to determine total and metabolically active bacteria, respectively. Biochrom 30 series amino acid analyzer and gas-chromatography mass spectrometry/solid-phase microextraction (GC-MS/SPME) analyses were mainly carried out for metabolomic analyses. As estimated by rarefaction, Chao and Shannon diversity index, the lowest microbial diversity was found in P patients. Firmicutes increased in the fecal samples of NP and, especially, P patients due to the higher percentages of some genera/species of Ruminococcaceae, Lachnospiraceae, Eubacteriaceae and Streptococcaeae. With a few exceptions, species of Clostridium, Enterococcus and Lactobacillus genera were found at the highest levels in HC. Bacteroidaceae, Porphyromonadaceae, Prevotellaceae and Rikenellaceae families differed among NP, P and HC subjects. Sutterellaceae and Enterobacteriaceae species were almost the highest in the fecal samples of NP and/or P patients. Compared to HC subjects, Bifidobacterium species decreased in the fecal samples of NP and P. As shown by multivariate statistical analyses, the levels of metabolites (free amino acids and organic volatile compounds) from fecal and urinary samples markedly differentiated NP and, especially, P patients.

Aims:  To screen the glutamate dehydrogenase (GDH) activity of nonstarter lactic acid bacteria (NSLAB) and to determine the effects of temperature, pH and NaCl values used for cheese ripening on enzyme activity and expression of GDH gene. Methods and Results:  A subcellular fractionation protocol and specific enzyme assays were used. The effect of temperature, pH and NaCl on enzyme activity was evaluated. The expression of GDH gene was monitored by real-time PCR. One selected strain was also used as adjunct starter for cheese making to evaluate the catabolism of free amino acids and the production of volatile organic compounds (VOC) during cheese ripening. The cytoplasm fraction of all strains showed in vitro NADP-dependent GDH activity. NADP-GDH activity was markedly strain dependent and varied according to the interactions between temperature, pH and NaCl. Lactobacillus plantarum DPPMA49 showed the highest NADP-GDH activity under temperature, pH and NaCl values found during cheese ripening. RT-PCR analysis revealed that GDH expression of Lact. plantarum DPPMA49 was down-expressed by low temperature (&lt;13°C) and over-expressed by NaCl (1·87–5·62%). According to NADP-GDH activity, the highest level of VOC (alcohols, aldehydes, miscellaneous and carboxylic acids) was found in cheeses made with DPPMA49. Conclusions:  The results of this study may be considered as an example of the influence of temperature, pH and NaCl on enzyme activity and expression of functional genes, such as GDH, in cheese-related bacteria. Significance and Impact of the Study:  It focuses on the phenotypic and molecular characterization of the NADP-GDH in lactobacilli under cheese-ripening conditions. The findings of this study contribute to the knowledge about enzymes involved in the catabolism of amino acids, to be used as an important selection trait for cheese strains.

This work showed the effect of pheromone plantaricinA (PlnA) on the proliferation and migration of the humankeratinocytes NCTC 2544. PlnA was chemically synthesized and used as pure peptide or biologically synthesized during co-cultivation of Lactobacillusplantarum DC400 and Lactobacillus sanfranciscensis DPPMA174. The cell-free supernatant (CFS) was used as the crude preparation containing PlnA. The inductive effect of PlnA on the proliferation of NCTC 2544 cells was higher than that found for hyaluronic acid, a well known skin protective compound. As shown by scratch assay and image analyses, PlnA enhanced the migration of NCTC 2544 cells. Compared to the basal serum free medium (control), the highest inductive effect was found using 10 μg/ml of chemically synthesized PlnA. Similar results (P &gt; 0.05) were found for CFS. In agreement, the percentage of the starting scratch area was decreased after treatment (24 h) with PlnA. The expression of transforming growth factor-β1 (TGF-β1), keratinocyte growth factor 7 (FGF7), vascular endothelial growth factor (VEGF-A), and interleukin-8 (IL-8) genes was affected by PlnA. Compared to control, TGF-β1gene was under expressed in the first 4 h of treatments and up-regulated after 8–24 h. On the contrary, FGF7gene was strongly up-regulated in the first 4 h of treatments. Compared to control, VEGF-A and IL-8genes were always up-regulated during the 4–24 h from scratching. Since capable of promoting the proliferation and migration of the humankeratinocytes and of stimulating IL-8 cytokine, the use of PlnA for dermatological purposes should be considered.

This work aimed at showing the effect of pheromone plantaricin A (PlnA) by Lactobacillus plantarum DC400 towards other sourdough lactic acid bacteria and the potential of PlnA to protect the function of the human intestinal barrier. Growth and survival of sourdough lactic acid bacteria were differently affected by co-cultivation with L. plantarum DC400. Compared to mono-cultures, Lactobacillus sanfranciscensis DPPMA174 and Pediococcus pentosaceus 2XA3 showed growth inhibition and decreased viability when co-cultured with L. plantarum DC400. L. sanfranciscensis DPPMA174 induced the highest synthesis of PlnA. Survival of strain DPPMA174 only slightly varied by comparing the addition of PlnA to the culture medium and the co-cultivation with L. plantarum DC400. Compared to mono-culture, the proteome of L. sanfranciscensis DPPMA174 grown in co-culture with L. plantarum DC400 showed the variation of expression of 58 proteins (47 over expressed and 11 repressed). Thirty-four of them were also over expressed or repressed during growth of DPPMA174 with PlnA. Fifty-one of the above 58 proteins were identified. They had a central role in stress response, amino acid, energy and nucleotide metabolisms, membrane transport, regulation of transcription, and cell redox homeostasis. PlnA markedly increased the viability of human Caco-2/TC7 cells and the transepithelial electrical resistance.

This study aimed at investigating the robustness of selected sourdough strains of Lactobacillusplantarum. Seven strains were singly used as sourdoughtype I starters under daily back-slopping propagation (ten days) using wheatflour. Cell numbers of presumptive lactic acid bacteria varied slightly (median values of 9.13–9.46 log cfu g−1) between and within started sourdoughs, as well as the acidifying activity (median values of 1.24–1.33). After three days also the control sourdough (unstarted) had the same values. As shown by RAPD-PCR analysis, five (DB200, 3DM, G10C3, 12H1 and LP20) out of seven strains maintained elevated cell numbers (ca. 9 log cfu g−1) throughout ten days. The other two strains progressively decreased to less than 5 log cfu g−1. As identified by partial sequencing of 16S rRNA and recA genes, L. plantarum (11 isolates), pediococci (7), Lactobacillus casei (3) and Lactobacillus rossiae (2) dominated the flour microbiota. Monitoring of lactic acid bacteria during sourdoughpropagation was carried out by culture dependent approach and using PCR-DGGE (Denaturing Gradient Gel Electrophoresis). Except for the sourdough started with L. plantarum LP20, in all other sourdoughs at least one autochthonous strain of L. plantarum emerged. All emerging strains of L. plantarum showed different RAPD-PCR profiles. L. rossiae and Pediococcus pentosaceus were only found in the control and sourdough started with strain 12H1. The characterization of the catabolic profiles of sourdoughs (Biolog System) showed that sourdoughs containing persistent starters behaved similarly and their profiles were clearly differentiated from the others. One persistent strain (DB200) of L. plantarum and Lactobacillus sanfranciscensis LS44, previously shown to be persistent (Siragusa et al., 2009), were used as the mixed starter to produce a wheatfloursourdough. Both strains cohabited and dominated during ten days of propagation.

The use of sourdough, even in combination with cryoprotectant (skim milk, sucrose and trehalose), conventional additives (guar gum, diacetyl tartaric acid esters of monoglycerides, ascorbic acid), honey or fructose and glucose, in frozen dough technology was investigated. After frozen storage, the leavening performance of doughs, and the hardness and texture of breads were compared to those of an unfrozen dough, and to those of a conventional frozen dough. All frozen doughs showed a longer fermentation time and a lower volume increase, with respect to unfrozen dough. When sourdough was combined with cryoprotectant, honey or both, the leavening performance improved compared to the use of sourdough alone. Compared to the conventional frozen dough, higher leavening performance was reached combining sourdough with cryoprotectant alone or together with honey. Sourdough combined with honey, fructose and glucose, honey and cryoprotectant, or conventional additives decreased bread hardness compared to the unfrozen dough bread and to the conventional frozen dough bread. Independently from the use of sourdough, conventional additives allowed to reach a specific volume not significantly different from that of unfrozen dough bread, and breads containing honey were characterized by low values of hardness and by high values of red index.

Aims:  This study aimed at characterizing the lactic acid bacteria microbiota and selecting mixed endogenous starters to be used for sourdough fermentation of spelt or emmer flours. Methods and Results:  Identification of lactic acid bacteria was carried out by partial sequencing of the 16S rRNA, recA, 16S/23S rRNA spacer region and pheS genes. Spelt flour showed the largest biodiversity, while Lactobacillus plantarum dominated in emmer flour. Isolates were subjected to RAPD-PCR analysis and screened based on the kinetics of growth and acidification, quotient of fermentation and liberation of free amino acids (FAA) during sourdough fermentation. After selection, mixed starters were used according to a two-step fermentation process. Wheat flour was fermented by the same starters. Spelt and emmer sourdoughs had slightly higher pH than wheat sourdoughs but titratable acidity, concentration of FAA and phytase activity were higher. Specific volume and crumb grain of emmer and, especially, spelt breads approached those of wheat breads. Sensory analysis confirmed the suitability of spelt and emmer for bread making. Conclusions:  The sourdough biotechnology was indispensable to completely exploit the potential of spelt and emmer flours. Significance and Impact of the Study:  Results filled up the lack of knowledge on the lactic acid bacteria microbiota and technological performances of spelt and emmer flours.

Agriculture surplus were used as substrates to synthesize !-aminobutyric acid (GABA) by Lactobacillus plantarum DSM19463 for the manufacture of a functional beverage or as a novel application for dermatological purposes. Dilution of the grape must to 1 or 4% (w/v) of total carbohydrates favored higher cell yield and synthesis of GABA with respect to whey milk. Optimal conditions for synthesizing GABA in grape must were: initial pH 6.0, initial cell density of Log 7.0 cfu/mL, and addition of 18.4 mM L-glutamate. L. plantarum DSM19463 synthesized 4.83 mM of GABA during fermentation at 30°C for 72 h. The fermented grape must also contain various levels of niacin, free minerals, and polyphenols, and Log 10.0 cfu/g of viable cells of L. plantarum DSM19463. Freeze dried preparation of grape must was applied to the SkinEthic® Reconstructed Human Epidermis or multi-layer human skin model (FT-skin tissue). The effect on transcriptional regulation of human beta-defensin-2 (HBD-2), hyaluronan synthase (HAS1), filaggrin (FGR), and involucrin genes was assayed through RT-PCR. Compared to GABA used as pure chemical compound, the up-regulation HBD-2 was similar while the effect on the expression of HAS1 and FGR genes was higher.

One hundred and three strains of lactic acid bacteria, isolated from various food ecosystems, were assayed for β-glucosidase activity toward p-nitrophenyl-β-d-glucopyranoside substrate. Lactobacillus plantarum DPPMA24W and DPPMASL33, Lactobacillus fermentum DPPMA114, and Lactobacillus rhamnosus DPPMAAZ1 showed the highest activities and were selected as the mixed starter to ferment various soy milk preparations, which mainly differed for chemical composition, protein dispersibility index, and size dimension. The soy milk made with organically farmed soybeans (OFS) was selected as the best preparation. All selected strains grew well in OFS soy milk, reaching almost the same values of cell density (ca. 8.5 log cfu/mL). After 96 h of fermentation with the selected mixed starter, OFS soy milk contained 57.0 μM daidzein, 140.3 μM genistein, 20.4 μM glycitein, and 37.3 μM equol. Fermented and nonfermented OFS soy milks were used for the in vitro assays on intestinal human Caco-2/TC7 cells. Fermented OFS soy milk markedly inhibited the inflammatory status of Caco-2/TC7 cells as induced by treatment with interferon-γ (IFN-γ) (1000 U/mL) and lipopolysaccharide (LPS) (100 ng/mL), maintained the integrity of the tight junctions, even if subjected to negative stimulation by IFN-γ, and markedly inhibited the synthesis of IL-8, after treatment with interleukin-1β (2 ng/mL). As shown by using chemical standards, these effects were due to the concomitant activities of isoflavone aglycones and, especially, equol, which were synthesized in the fermented OFS soy milk preparation.

Plantaricin A (PlnA) is a peptide with antimicrobial and pheromone activities. PlnA was synthesized chemically and used as a pure peptide or synthesized biologically using Lactobacillus plantarum DC400 co-cultured with Lactobacillus sanfranciscensis DPPMA174. Cell-free supernatant (CFS) was used as a crude PlnA preparation. As estimated using the 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide and the 2',7'-dichlorofluorescein diacetate assays, both PlnA preparations increased the antioxidant defenses of human NCTC 2544 keratinocytes. PlnA (10 ?g/ml) had a higher activity than hyaluronic acid or 125 ?g/ml ?-tocopherol. Effects on the transcriptional regulation of filaggrin (FLG), involucrin (IVL), hyaluronan synthase (HAS2), human ?-defensin-2 (HBD-2) and tumor necrosis factor-alpha (TNF-?) genes were assayed. Compared with the control, expression of the FLG gene in NCTC 2544 cells increased in cells treated with hyaluronic acid, 1 or 10 ?g/ml PlnA. Compared with the control, the level of IVL gene expression increased in NCTC 2544 cells treated with 10 ?g/ml PlnA. No significant difference was found between the level of the HAS2 gene expressed by control cells and cells treated with PlnA. Compared with chemically synthesized PlnA, the up-regulation of the HBD-2 gene by CFS was higher. Compared with the control, expression of TNF-? decreased in NCTC 2544 cells after treatment with 1 or 10 ?g/ml of chemically synthesized PlnA. In contrast, the level of TNF-? was highest in the presence of 10 ?g/ml CFS-PlnA. These findings suggest that the PlnA was positively sensed by human keratinocytes, promoting antioxidant defenses, barrier functions and antimicrobial activity of the skin.

Gut microbiota, the largest symbiont community hosted in human organism, is emerging as a pivotal player in the relationship between dietary habits and health. Oral and, especially, intestinal microbes metabolize dietary components, affecting human health by producing harmful or beneficial metabolites, which are involved in the incidence and progression of several intestinal related and non-related diseases. Habitual diet (Western, Agrarian and Mediterranean omnivore diets, vegetarian, vegan and gluten-free diets) drives the composition of the gut microbiota and metabolome. Within the dietary components, polymers (mainly fibers, proteins, fat and polyphenols) that are not hydrolyzed by human enzymes seem to be the main leads of the metabolic pathways of gut microbiota, which in turn directly influences the human metabolome. Specific relationships between diet and microbes, microbes and metabolites, microbes and immune functions and microbes and/or their metabolites and some human diseases are being established. Dietary treatments with fibers are the most effective to benefit the metabolome profile, by improving the synthesis of short chain fatty acids and decreasing the level of molecules, such as p-cresyl sulfate, indoxyl sulfate and trimethylamine N-oxide, involved in disease state. Based on the axis diet-microbiota-health, this review aims at describing the most recent knowledge oriented towards a profitable use of diet to provide benefits to human health, both directly and indirectly, through the activity of gut microbiota.

Cholesterol gallstone disease is a major health problem in Westernized countries and depends on a complex interplay between genetic factors, lifestyle and diet, acting on specific pathogenic mechanisms. Overweigh, obesity, dyslipidemia, insulin resistance and altered cholesterol homeostasis have been linked to increased gallstone occurrence, and several studies point to a number of specific nutrients as risk- or protective factors with respect to gallstone formation in humans, motivating a rising interest in the identification of common and modifiable dietetic factors that put the patients at risk of gallstones or that are able to prevent gallstone formation and growth. In particular, dietary models characterized by increased energy intake with highly refined sugars and sweet foods, high fructose intake, low fiber contents, high fat, consumption of fast food and low vitamin C intake increase the risk of gallstone formation. On the other hand, high intake of monounsaturated fats and fiber, olive oil and fish (ω-3 fatty acids) consumption, vegetable protein intake, fruit, coffee, moderate alcohol consumption and vitamin C supplementation exert a protective role. The effect of some confounding factors (e.g., physical activity) cannot be ruled out, but general recommendations about the multiple beneficial effects of diet on cholesterol gallstones must be kept in mind, in particular in groups at high risk of gallstone formation.

Abstract Abstract Objective: A supervised multivariate model to classify the metabolome alterations between autistic spectrum disorders (ASD) patients and controls, siblings of autistic patients, has been realized and used to realize a network model of the ASD patients' metabolome. METHODS: In our experiment we propose a quantification of urinary metabolites with the Mass Spectroscopy technique couple to Gas Chromatography. A multivariate model has been used to extrapolate the variables of importance for a network model of interaction between metabolites. In this way we are able to propose a network-based approach to ASD description. RESULTS: Children with autistic disease composing our studied population showed elevated concentration of several organic acids and sugars. Interactions among diet, intestinal flora and genes may explain such findings. Among them, the 3-(3-hydroxyphenyl)-3-hydroxypropanoic acid has been previously described as altered in autistic subjects. Other metabolites increased are 3,4-dihydroxybutyric acid, glycolic acid and glycine, cis-aconitic acid; phenylalanine, tyrosine, p-hydroxyphenylacetic acid, and homovanillic acid are all involved in the tyrosine pathway leading to neurotransmitter cathecolamine. CONCLUSION: GC-MS-based metabolomic analysis of the urinary metabolome suggests to have the required sensitivity and specificity to gain insight into ASD phenotypes and aid a personalized network-based medicine approach.

Food allergy is recognized as one of the major health concerns. It is estimated that ca. 4% of the population is affected by food allergenic disorders. Food allergies are defined as IgE-mediated hypersensitivity reactions. Foods such as peanuts, tree nuts, wheat, soy, cow's milk, egg, fish and shellfish are regarded as responsible for the majority of reactions. The ubiquitous presence of allergens in the human foods coupled with an increased awareness of food allergies warrants to undertake appropriate preventive measures for protecting sensitive consumers from unwanted exposure to offending food allergens. 2-DE followed by immunoblotting and identification of IgE-reactive proteins, as a proteomic approach to identify new allergens in foods, are reviewed. Specific examples of identification of allergens in foods and beverages by using 2-DE and IgE are described. Protein profiling using 2-DE and allergens detection by IgE has become a powerful method for analyzing changes of allergens content in complex matrix during food processing.

This study aimed at investigating the addition of legume (chickpea, lentil and bean) flours to wheat flour bread. Type I sourdough containing legumes or wheat–legume flours were prepared and propagated (back slopped) in laboratory, according to traditional protocols that are routinely used for making typical Italian breads. Based on kinetic of acidification and culture-dependent data, the wheat–legume sourdough was further characterized and selected for bread making. As determined by RAPD-PCR and partial sequencing of 16S rDNA gene analyses, lactic acid bacteria in wheat–legume sourdough included Lactobacillus plantarum, Lactobacillus sanfranciscensis, Leuconostoc mesenteroides, Lactobacillus fermentum, Weissella cibaria, Lactobacillus pentosus, Lactobacillus coryneformis, Lactobacillus rossiae, Lactobacillus brevis, Lactobacillus parabuchneri and Lactobacillus paraplantarum. Two breads containing 15% (w/w) of legume (chickpea, lentil and bean) flours were produced using selected wheat–legume sourdough (WLSB) and traditional wheat sourdough (WSB). Compared to wheat yeasted bread (WYB), the level of total free amino acids (FAA) was higher in WSB and WLSB. Phytase and antioxidant activities were the highest in WLSB. Compared to bread WYB, the addition of legume flours decreased the in vitro protein digestibility (IVPD) (WYB versus WSB). However, the dough fermentation with WSLB favored an increase of IVPD. According to the levels of carbohydrates, dietary fibers and resistant starch, WSB and WLSB showed lower values of hydrolysis index (HI) compared to WYB. As showed by texture and image analyses and sensory evaluation of breads, a good acceptability was found for WSB and, especially, WLSB breads.

In this review we elucidate the role of gut microbiota as the plausible missing link between food and health, focusing on chronic kidney disease (CKD). Microbiota, the microbial community harboured in the large intestine, is considered a symbiotic “supplementary organ”. It contributes to digestion, mainly through two catabolic pathways: saccharolytic (fermentation) or proteolytic (putrefaction). It also interacts with host influencing immunity, metabolism, and health status. It is believed that a balanced healthy microbiota is primarily saccharolytic and diet has a deep effect on its composition. Mediterranean Diet, UNESCO “Intangible Cultural Heritage of Humanity”, prevents cardiovascular and metabolic systemic diseases, thanks to the high supply of fibres and antioxidants. Mediterranean Diet also favours the prevalence of saccharolytic species, while Western Diet promotes the shift towards a proteolytic profile (dysbiosis). Emerging evidences highlight the association between a wide range of diseases and dysbiosis. In CKD a vicious circle exists, in which proteolytic-derived microbial metabolites (p-cresol and indoxyl sulphate), represent the main circulating uremic toxins: their accumulation worsens dysbiosis and promotes CKD progression. Gut microbiota shaping through nonpharmacologic nutritional treatments, based on Mediterranean Diet, represents an innovative approach in CKD, potentially restoring microbiota balance, ameliorating CKD conditions and slowing down disease progression.

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.

In this review we elucidate the role of gut microbiota as the plausible missing link between food and health, focusing on chronic kidney disease (CKD). Microbiota, the microbial community harboured in the large intestine, is considered a symbiotic "supplementary organ". It contributes to digestion, mainly through two catabolic pathways: saccharolytic (fermentation) or proteolytic (putrefaction). It also interacts with host influencing immunity, metabolism, and health status. It is believed that a balanced healthy microbiota is primarily saccharolytic and diet has a deep effect on its composition. Mediterranean Diet, UNESCO "Intangible Cultural Heritage of Humanity", prevents cardiovascular and metabolic systemic diseases, thanks to the high supply of fibres and antioxidants. Mediterranean Diet also favours the prevalence of saccharolytic species, while Western Diet promotes the shift towards a proteolytic profile (dysbiosis). Emerging evidences highlight the association between a wide range of diseases and dysbiosis. In CKD a vicious circle exists, in which proteolytic-derived microbial metabolites (p-cresol and indoxyl sulphate), represent the main circulating uremic toxins: their accumulation worsens dysbiosis and promotes CKD progression. Gut microbiota shaping through non-pharmacologic nutritional treatments, based on Mediterranean Diet, represents an innovative approach in CKD, potentially restoring microbiota balance, ameliorating CKD conditions and slowing down disease progression.

La presente invenzione ha per oggetto un metodo per l’autenticazione di pani tradizionali/tipici, cioè pani lievitati con lievito naturale (lievito madre, pasta madre o impasto acido), mediante un metodo molecolare che permette il rilevamento e la quantificazione del DNA di batteri lattici eventualmente presente nel pane. In particolare, l’invenzione concerne l’uso di primer che consentono di effettuare una reazione a catena della polimerasi quantitativa (qPCR) che permette di verificare la densità cellulare di batteri, vantaggiosamente di batteri lattici, contenuta nei prodotti alimentari lievitati e determinare se tale densità sia compatibile con la lievitazione naturale. L’invenzione ha altresì per oggetto i suddetti primer e un kit che li contiene.

The present invention concerns a mixture of lactic bacteria for yeasting of gluten-free baked products. Particularly, the invention concerns the use of “natural yeast” based on selected lactic bacteria as yeasting agent for the production of gluten-free bread, with improved sensory and nutritional property, designed for celiac patients feeding.

This invention relates to a process for the production of gamma- aminobutyric acid (GABA) from Lactococcus lactis ssp. DSM 19464 and from Lactobacillus plantarum DSM 19463 or from their associations on must. In particular, this invention contemplates the selection and use of Lactococcus lactis ssp. DSM 19464 and of Lactobacillus plantarum DSM 19463 or their associations on must whose composition has been suitably optimized as to its composition, for the production of a preparation based on GABA, containing also vitamins, minerals, polyphenols and alive vital lactic bacteria for its potential use in the dermatological field.

PROCESS OF MICROBIC BIOTECHNOLOGY FOR COMPLETELY DEGRADING GLUTEN IN FLOURS The present invention concerns the use of lactic acid bacteria selected and fungal enzymes for the gluten complete degradation from both bread and durum wheat, barley, rye and oat flour. In particular, the invention concerns the use of lactic acid bacteria selected and fungal enzymes for the gluten complete degradation (residual gluten concentration lower than 20 ppm) of cereal flours, which after detoxification can be used according to a standardized biotechnological protocol for the production of various gluten- free foods.