Maria Calasso

Effect of lactose on gut microbiota and metabolome of infants with cow's milk allergy

To cite this article: Francavilla R, Calasso M, Calace L, Siragusa S, Ndagijimana M, Vernocchi P, Brunetti L, Mancino G, Tedeschi G, Guerzoni E, Indrio F, Laghi L, Miniello VL, Gobbetti M, De Angelis M. Effect of lactose on gut microbiota and metabolome of infants with cow’s milk allergy. Pediatric Allergy Immunology 2012: 23: 420–427.

Mechanism of degradation of immunogenic gluten epitopes from Triticum turgidum L. var. durum by sourdough lactobacilli and fungal proteases.

ABSTRACT As shown by R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay (ELISA), selected sourdough lactobacilli, in combination with fungal proteases, hydrolyzed gluten (72 h at 37°C) of various cultivars of Triticum turgidum L. var. durum to less than 20 ppm. Complementary electrophoretic, chromatography, and mass spectrometry techniques were used to characterize the gluten and epitope hydrolysis. Nine peptidases were partially purified from the pooled cytoplasmic extract of the sourdough lactobacilli and used to hydrolyze the 33-mer epitope, the most immunogenic peptide generated during digestion of Triticum species. At least three peptidases (general aminopeptidase type N [PepN], X-prolyl dipeptidyl aminopeptidase [PepX], and endopeptidase PepO) were necessary to detoxify the 33-mer without generation of related immunogenic epitopes. After 14 h of incubation, the combination of all or at least six different peptidases totally hydrolyzed the 33-mer (200 mM) into free amino acids. The same results were found for other immunogenic epitopes, such as fragments 57-68 of α9-gliadin, 62-75 of A-gliadin, and 134-153 of γ-gliadin. When peptidases were used for fermentation of durum wheat semolina, they caused the hydrolysis of gluten to ca. 2 ppm. The in vivo digestion was simulated, and proteins/peptides extracted from pepsin-trypsin (PT) digestion of durum wheat semolina fermented with selected sourdough lactobacilli induced the expression of gamma interferon and interleukin 2 at levels comparable to those of the negative control. Durum wheat semolina fermented with sourdough lactobacilli was freeze-dried and used for making Italian-type pasta. The scores for cooking and sensory properties for this pasta were higher that those of conventional gluten-free pasta.

Quorum sensing in sourdough Lactobacillus plantarum DC400: Induction of plantaricin A (PlnA) under co-cultivation with other lactic acid bacteria and effect of PlnA on bacterial and Caco-2 cells

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.

Selection and use of autochthonous multiple strain cultures for the manufacture of high-moisture traditional Mozzarella cheese.

Lactobacillus plantarum 18A, Lactobacillus helveticus 2B, Lactobacillus delbrueckii subsp. lactis 20F, Streptococcus thermophilus 22C, Enterococcus faecalis 32C and Enterococcus durans 16E were the most acidifying strains within 146 isolates for natural whey starters. The effect of media and temperature on 2 autochthonous multiple strain cultures (AMSI: 18A, 2B, 20F and 22C, 32C and 16E and AMSII: 18A, 2B, 20F and 22C) was studied. Genomic analysis showed a constant cell numbers for AMSII during 16 days of propagation in whey milk. Mozzarella cheese was made by using AMSII, commercial starter (CS) or citric acid (DA). Compared to other cheeses, the DA had a lower level of protein, ash, Ca, free amino acids and a higher level of moisture. Based on confocal laser scanning microscopy analysis, AMSII cheese showed the lowest microstructural variations during the period of storage compared to other cheeses. All the sensory attributes were scored highest for AMSII cheese. ASMII extend the shelf-life to ca. 12-15 days instead of the 5-7 days of traditional high-moisture Mozzarella cheese.

Use of sourdough fermentation and mixture of wheat, chickpea, lentil and bean flours for enhancing the nutritional, texture and sensory characteristics of white bread.

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.

Metabolic and proteomic adaptation of Lactobacillus rhamnosus strains during growth under cheese‐like environmental conditions compared to de Man, Rogosa, and Sharpe medium

The aim of this study was to demonstrate the metabolic and proteomic adaptation of Lactobacillus rhamnosus strains, which were isolated at different stages of Parmigiano Reggiano cheese ripening. Compared to de Man, Rogosa, and Sharpe (MRS) broth, cultivation under cheese‐like conditions (cheese broth, CB) increased the number of free amino acids used as carbon sources. Compared with growth on MRS or pasteurized and microfiltrated milk, all strains cultivated in CB showed a low synthesis of d,l‐lactic acid and elevated levels of acetic acid. The proteomic maps of the five representative strains, showing different metabolic traits, were comparatively determined after growth on MRS and CB media. The amount of intracellular and cell‐associated proteins was affected by culture conditions and diversity between strains, depending on their time of isolation. Protein spots showing decreased (62 spots) or increased (59 spot) amounts during growth on CB were identified using MALDI‐TOF‐MS/MS or LC‐nano‐ESI‐MS/MS. Compared with cultivation on MRS broth, the L. rhamnosus strains cultivated under cheese‐like conditions had modified amounts of some proteins responsible for protein biosynthesis, nucleotide, and carbohydrate metabolisms, the glycolysis pathway, proteolytic activity, cell wall, and exopolysaccharide biosynthesis, cell regulation, amino acid, and citrate metabolism, oxidation/reduction processes, and stress responses.

Fermentation and proteome profiles of Lactobacillus plantarum strains during growth under food-like conditions

UNLABELLED This study aimed at investigating the proteomic adaptation of Lactobacillus plantarum strains. Cultivation of L. plantarum strains under food-like conditions (wheat flour hydrolyzed, whey milk, tomato juice) affected some metabolic traits (e.g., consumption of carbohydrates and synthesis of organic acids) compared to de Man, Rogosa and Sharpe (MRS) broth. The analysis of the fermentation profile showed that the highest number of carbon sources metabolized by L. plantarum strains was found using cells cultivated in media containing low concentration of glucose or no glucose at all. The proteomic maps of the strains were comparatively determined after growth on MRS broth and under food-like conditions. The amount of proteins depended on strain and, especially, on culture conditions. Proteins showing decreased or increased amounts under food-like conditions were identified using MALDI-TOF-MS/MS or LC-nano-ESI-MS/MS. Changes of the proteome concerned proteins that are involved in carbohydrate transport and metabolism, energy metabolism, Sec-dependent secretion system, stress response, nucleotide metabolism, regulation of nitrogen metabolism, and protein biosynthesis. A catabolic repression by glucose on carbohydrate transport and metabolism was also found. The characterization of the proteomes in response to changing environmental conditions could be useful to get L. plantarum strains adapted for specific applications. BIOLOGICAL SIGNIFICANCE Microbial cell performance during food biotechnological processes has become one of the greatest concerns all over the world. L. plantarum is a lactic acid bacterium with a large industrial application for fermented foods or functional foods (e.g., probiotics). The present study compared the fermentation and proteomic profiling of L. plantarum strains during growth under food-like conditions and under optimal laboratory conditions (MRS broth). This study provides specific mechanisms of proteomic adaptation involved in the microbial performances (carbohydrates utilization, energy metabolism, stress resistance, etc.) affecting the main biotechnological tracts of L. plantarum strains. The finding of this study provides evidences that may be exploited to get strains adapted for specific applications in food biotechnology.

Diversity of the lactic acid bacterium and yeast microbiota in the switch from firm- to liquid-sourdough fermentation.

ABSTRACT Four traditional type I sourdoughs were comparatively propagated (28 days) under firm (dough yield, 160) and liquid (dough yield, 280) conditions to mimic the alternative technology options frequently used for making baked goods. After 28 days of propagation, liquid sourdoughs had the lowest pH and total titratable acidity (TTA), the lowest concentrations of lactic and acetic acids and free amino acids, and the most stable density of presumptive lactic acid bacteria. The cell density of yeasts was the highest in liquid sourdoughs. Liquid sourdoughs showed simplified microbial diversity and harbored a low number of strains, which were persistent. Lactobacillus plantarum dominated firm sourdoughs over time. Leuconostoc lactis and Lactobacillus brevis dominated only some firm sourdoughs, and Lactobacillus sanfranciscensis persisted for some time only in some firm sourdoughs. Leuconostoc citreum persisted in all firm and liquid sourdoughs, and it was the only species detected in liquid sourdoughs at all times; it was flanked by Leuconostoc mesenteroides in some sourdoughs. Saccharomyces cerevisiae, Candida humilis, Saccharomyces servazzii, Saccharomyces bayanus-Kazachstania sp., and Torulaspora delbrueckii were variously identified in firm and liquid sourdoughs. A total of 197 volatile components were identified through purge and trap–/solid-phase microextraction–gas chromatography-mass spectrometry (PT–/SPME–GC-MS). Aldehydes, several alcohols, and some esters were at the highest levels in liquid sourdoughs. Firm sourdoughs mainly contained ethyl acetate, acetic acid, some sulfur compounds, and terpenes. The use of liquid fermentation would change the main microbial and biochemical features of traditional baked goods, which have been manufactured under firm conditions for a long time.

Plantaricin A synthesized by Lactobacillus plantarum induces in vitro proliferation and migration of human keratinocytes and increases the expression of TGF-β1, FGF7, VEGF-A and IL-8 genes.

This work showed the effect of pheromone plantaricin A (PlnA) on the proliferation and migration of the human keratinocytes NCTC 2544. PlnA was chemically synthesized and used as pure peptide or biologically synthesized during co-cultivation of Lactobacillus plantarum 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 10μg/ml of chemically synthesized PlnA. Similar results (P>0.05) were found for CFS. In agreement, the percentage of the starting scratch area was decreased after treatment (24h) 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-β1 gene was under expressed in the first 4h of treatments and up-regulated after 8-24h. On the contrary, FGF7 gene was strongly up-regulated in the first 4h of treatments. Compared to control, VEGF-A and IL-8 genes were always up-regulated during the 4-24h from scratching. Since capable of promoting the proliferation and migration of the human keratinocytes and of stimulating IL-8 cytokine, the use of PlnA for dermatological purposes should be considered.

Lactobacillus rossiae, a vitamin B12 producer, represents a metabolically versatile species within the Genus Lactobacillus.

Lactobacillus rossiae is an obligately hetero-fermentative lactic acid bacterium, which can be isolated from a broad range of environments including sourdoughs, vegetables, fermented meat and flour, as well as the gastrointestinal tract of both humans and animals. In order to unravel distinctive genomic features of this particular species and investigate the phylogenetic positioning within the genus Lactobacillus, comparative genomics and phylogenomic approaches, followed by functional analyses were performed on L. rossiae DSM 15814T, showing how this type strain not only occupies an independent phylogenetic branch, but also possesses genomic features underscoring its biotechnological potential. This strain in fact represents one of a small number of bacteria known to encode a complete de novo biosynthetic pathway of vitamin B12 (in addition to other B vitamins such as folate and riboflavin). In addition, it possesses the capacity to utilize an extensive set of carbon sources, a characteristic that may contribute to environmental adaptation, perhaps enabling the strains ability to populate different niches.