Shotgun Phage Display of Lactobacillus casei BL23 Against Collagen and Fibronectin

7 pages, 4 figures.-- First published online 23 November 2010Lactobacilli are normal constituents of the intestinal microbiota, and some strains show the capacity to bind to extracellular matrix proteins and components of the mucosal layer, which represents an adaptation to persist in this niche. A shotgun phage-display library of Lactobacillus casei BL23 was constructed and screened for peptides able to bind to fibronectin and collagen. Clones showing binding to these proteins were isolated, which encoded overlapping fragments of a putative transcriptional regulator (LCABL_29260), a hypothetical protein exclusively found in the L. casei/rhamnosus group (LCABL_01820), and a putative phage-related endolysin (LCABL_13470). The construction of different glutathione S-transferase (GST) fusions confirmed the binding activity and demonstrated that the three identified proteins could interact with fibronectin, fibrinogen, and collagen. The results illustrate the utility of phage display for the isolation of putative adhesins in lactobacilli. However, it remains to be determined whether the primary function of these proteins actually is adhesion to mucosal surfaces.This work was supported by projects AGL2004-00176/ALI and Consolider Fun-c-Food CSD2007-00063 from the Spanish Ministry of Science and Innovation. Diego Muñoz-Provencio was the recipient of a research fellowship from the Instituto Danone.Peer reviewe

Reduction of Phytate in Soy Drink by Fermentation with Lactobacillus casei Expressing Phytases From Bifidobacteria

Plant-based food products can be modified by fermentation to improve flavour and the concentration of some biologically active compounds, but also to increase the mineral availability by eliminating anti-nutrient substances such as phytates. The objective of this study was to develop a fermented soybean drink with improved nutritional quality and source of probiotic bacteria by including as starter for fermentation Lactobacillus casei strains modified to produce phytase enzymes from bifidobacteria. The L. casei strains showed a good adaptation to develop in the soy drink but they needed the addition of external carbohydrates to give rise to an efficient acidification. The strain expressing the Bifidobacterium pseudocatenulatum phytase was able to degrade more than 90 % phytate during product fermentation, whereas expression of Bifidobacterium longum spp. infantis phytase only led to 65 % hydrolysis. In both cases, accumulation of myo-inositol triphosphates was observed. In addition, the hydrolysis of phytate in soy drink fermented with the L. casei strain expressing the B. pseudocatenulatum phytase resulted in phytate/mineral ratios for Fe (0.35) and Zn (2.4), which were below the critical values for reduced mineral bioavailability in humans. This investigation showed the ability of modified L. casei to produce enzymes with technological relevance in the design of new functional foods.This work was financed by projects Consolider Fun-C-Food CSD2007-00063 from the Ministry of Economy and Competitiveness (MINECO) and PROMETEO/2012/064 from the Generalitat Valenciana, Spain.Peer Reviewe

Application of phytases from bifidobacteria in the development of cereal-based products with amaranth

The effects of the inclusion of purified phytases from Bifidobacterium longum spp. infantis ATCC15697 and Bifidobacterium pseudocatenulatum ATCC27919 on phytate (InsP6) levels were analyzed during breadmaking process. Two different levels of whole amaranth (Amaranthus cruentus) flour (25 and 50 %) were used in bread dough preparation, and they were compared to control doughs made with 100 % wheat flour and 100 % whole wheat flour. Bread samples made with 50 % of amaranth flour showed a significant decrease in technological quality parameters in comparison with control white breads. However, a 25 % of amaranth flour improved the nutritional value of the bread, with only a slight depreciation in the quality. Addition of bifidobacterial phytases resulted in higher InsP6 degradation compared with a commercial fungal phytase, without affecting the bread quality. InsP 6 reduction was especially efficient in breads with 25 % amaranth, leading to InsP6 levels below the threshold of mineral bioavailability inhibition for Fe and Zn in human nutrition.This work was financially supported by grants AGL2011-22669 and Consolider Fun-C-Food CSD2007-00063 from the Ministry of Economy and Competitiveness (MINECO) and PROMETEO/2012/064 from the Generalitat Valenciana, Spain. The contract of I. García-Mantrana from the Consolider Fun-C-Food Project is gratefully acknowledged.Peer Reviewe

Bread with whole quinoa flour and bifidobacterial phytases increases dietary mineral intake and bioavailability

The purpose of the present work was to provide further information on how replacing wheat flour by whole quinoa flour (at 25 and 50g/100g of flour) affects bread performance and to assess its potential as a nutritious ingredient. Bread with quinoa resulted in a depreciation in quality in terms of loaf specific volume (from 4.48 to 3.46/2.63 cm3/g), crumb firmness (from 0.77 to 1.55/2.64 N) and acceptability (from 7.94 to 7.58/5.94). Quinoa increased the bread nutritional value, raising fibre (from 5.5 to 7.2 g/100g) and minerals (Ca from 0.35 to 1.28 mg/g, Fe from 17 to 34 ¿g/g, and Zn from 23 to 48 ¿g/g). The phytates were controlled by bifidobacterial phytase treatment during breadmaking (from 4.7 ¿mol/g to below the detection limit), which decreased phytate/mineral molar ratios to values lower than the threshold for inhibition of Fe and Zn absorption. Quinoa could partially replace wheat flour in bread, increasing its nutritional value in terms of dietary fibre, minerals, proteins of high biological value and healthy fats, with only a small depreciation in bread quality at 25g/100g of flour substitution. The high phytate contents were efficiently removed by phytase treatment and the breads were accepted by consumers.This work was financially supported by grants Consolider Fun-C-Food CSD2007-00063 and AGL2011-22669 from the Ministry of Economy and Competitiveness (MINECO) and PROMETEO/2012/064 from the Generalitat Valenciana, Spain.Peer Reviewe

Myo-inositol hexakisphosphate degradation by Bifidobacterium pseudocatenulatum ATCC 27919 improves mineral availability of high fibre rye-wheat sour bread

The goal of this investigation was to develop baking products using Bifidobacterium pseudocatenulatum ATCC27919, a phytase producer, as a starter in sourdough for the production of whole rye-wheat mixed bread. This Bifidobacterium strain contributed to myo-inositol hexakisphosphate (phytate) hydrolysis, resulting in breads with higher mineral availability as was predicted by the phytate/mineral molar ratios, which remained below the inhibitory threshold values for Ca and Zn intestinal absorption. The products with sourdough showed similar technological quality as their homologous without sourdough, with levels of acetic and d/l lactic acids in dough and bread baking significantly higher with the use of sourdough. The overall acceptability scores showed that breads with 25% of whole rye flour were highly accepted regardless of the inclusion of sourdough. This work emphasises that the in situ production of phytase during fermentation by GRAS/QPS microorganisms constitutes a strategy which is particularly appropriate for reducing the phytate contents in products for human consumption.This work was financially supported by grants Consolider Fun-C-Food CSD2007-00063 and AGL2011-22669 from the Ministry of Economy and Competitiveness (MINECO) and PROMETEO/2012/064 from the Generalitat Valenciana, Spain.Peer Reviewe

Therapeutic opportunities in intestinal microbiota-virus interactions

The host microbiota has emerged a third player in interactions between hosts and viral pathogens. This opens new possibilities to use different tools to modulate the intestinal microbial composition, aimed at reducing the risk of or treating viral enteric infections

The Interactions between host glycobiology, bacterial microbiota, and viruses in the gut

Rotavirus (RV) and norovirus (NoV) are the major etiological agents of viral acute gastroenteritis worldwide. Host genetic factors, the histo-blood group antigens (HBGA), are associated with RV and NoV susceptibility and recent findings additionally point to HBGA as a factor modulating the intestinal microbial composition. In vitro and in vivo experiments in animal models established that the microbiota enhances RV and NoV infection, uncovering a triangular interplay between RV and NoV, host glycobiology, and the intestinal microbiota that ultimately influences viral infectivity. Studies on the microbiota composition in individuals displaying different RV and NoV susceptibilities allowed the identification of potential bacterial biomarkers, although mechanistic data on the virus-host-microbiota relation are still needed. The identification of the bacterial and HBGA interactions that are exploited by RV and NoV would place the intestinal microbiota as a new target for alternative therapies aimed at preventing and treating viral gastroenteritis

Expression of bifidobacterial phytases in Lactobacillus casei and their application in a food model of whole-grain sourdough bread

Phytases are enzymes capable of sequentially dephosphorylating phytic acid to products of lower chelating capacity and higher solubility, abolishing its inhibitory effect on intestinal mineral absorption. Genetic constructions were made for expressing two phytases from bifidobacteria in Lactobacillus casei under the control of a nisin-inducible promoter. L. casei was able of producing, exporting and anchoring to the cell wall the phytase of Bifidobacterium pseudocatenulatum. The phytase from Bifidobacterium longum spp. infantis was also produced, although at low levels. L. casei expressing any of these phytases completely degraded phytic acid (2 mM) to lower myo-inositol phosphates when grown in MRS medium. Owing to the general absence of phytase activity in lactobacilli and to the high phytate content of whole grains, the constructed L. casei strains were applied as starter in a bread making process using whole-grain flour. L. casei developed in sourdoughs by fermenting the existing carbohydrates giving place to an acidification. In this food model system the contribution of L. casei strains expressing phytases to phytate hydrolysis was low, and the phytate degradation was mainly produced by activation of the cereal endogenous phytase as a consequence of the drop in pH. This work shows the capacity of lactobacilli to be modified in order to produce enzymes with relevance in food technology processes. The ability of these strains in reducing the phytate content in fermented food products must be evaluated in further models.This work was financed by projects Consolider Fun-C-Food CSD2007-00063 from the Ministry of Economy and Competitiveness (MINECO) and PROMETEO/2012/064 from the Generalitat Valenciana, Spain.Peer reviewe

Peptide and amino acid metabolism is controlled by an OmpR-family response regulator in Lactobacillus casei

A Lactobacillus casei BL23 strain defective in an OmpR-family response regulator encoded by LCABL_18980 (PrcR, RR11), showed enhanced proteolytic activity caused by overexpression of the gene encoding the proteinase PrtP. Transcriptomic analysis revealed that, in addition to prtP expression, PrcR regulates genes encoding peptide and amino acid transporters, intracellular peptidases and amino acid biosynthetic pathways, among others. Binding of PrcR to twelve promoter regions of both upregulated and downregulated genes, including its own promoter, was demonstrated by electrophoretic mobility shift assays showing that PrcR can act as a transcriptional repressor or activator. Phosphorylation of PrcR increased its DNA binding activity and this effect was abolished after replacement of the phosphorylatable residue Asp-52 by alanine. Comparison of the transcript levels in cells grown in the presence or absence of tryptone in the growth medium revealed that PrcR activity responded to the presence of a complex amino acid source in the growth medium. We conclude that the PrcR plays a major role in the control of the peptide and amino acid metabolism in L. casei BL23. Orthologous prcR genes are present in most members of the Lactobacillaceae and Leuconostocaceae families. We hypothesize that they play a similar role in these bacterial groups.This work was financed by funds from the former Spanish Ministry of Science and Innovation (AGL2007-60975 and AGL2010-15679) and Generalitat Valenciana (ACOMP2012/137). A. Revilla-Guarinos was the recipient of a research fellowship from the Instituto Danone.Peer reviewe