Cocoa Pulp as Alternative Food Matrix for Probiotic Delivery.

BACKGROUND: For screening probiotic strains with viability and stability in non-dairy foods for health benefits, we revised all patents relating to probiotics in food. OBJECTIVE: Screening of potential probiotics from Brazilian Minas artisanal cheese and verify their survival in frozen Brazilian cocoa pulp. METHODS: Isolation and identification of the strains. The potential probiotic characterization involved gastric juice and bile resistance, antibiotic and antimicrobial activity, hydrophobicity, autoaggregation, coaggregation and adhesion assay in HT-29 cells. Organoleptic, viability and stability of probiotic strain in frozen cocoa pulp were evaluated. RESULTS: Fourteen strains of Lactobacillus plantarum (9), Weissella paramesenteroides (3), Lactobacillus fermentum (1), and Leuconostoc mesenteroides (1) were obtained. Most of the strains were resistant to simulated gastric acidity and bile salts. Almost all strains were sensitive to the antibiotics tested, except to ciprofloxacin and vancomycin. About 47% of the strains are potential producers of bacteriocins. High hydrophobicity was observed for four strains. Autoaggregation ranged from 8.3-72.6% and the coaggregation capacity from 5.2-60.2%. All of the assessed strains presented more than 90% of adhesion to HT-29 intestinal cells. The percentage of Salmonella inhibition in HT-29 cells ranged from 4.7-31.1%. No changes in color, aroma, and pH were observed in cocoa pulps after storage at -20 °C for 90 days. CONCLUSION: Wild strains of acid lactic bacteria from cheese proved to be viable and stable in frozen Brazilian cocoa pulp. This work showed a promising application of L. plantarum isolated strains to be used with frozen cocoa pulp matrix in probiotics food industry.

Expression and Biochemical Characterization of a Yersinia intermedia Phytase Expressed in Escherichia coli.

BACKGROUND: Phytases are enzymes capable of degrading phytic acid and used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus. OBJECTIVE: The main goal of this study was to express and characterize a Yersinia intermedia phytase expressed in Escherichia coli cells. METHODS: The Y. intermedia phytase gene was synthesized and overexpressed in Escherichia coli cells. The phytase recombinante (rPHY) was purified to homogeneity using a Ni-NTA column. The biochemical and biophysical properties of the rPHY were measured in order to fully characterize the recombinant enzyme. The following patents database were consulted: Espacenet, USPTO, LATIPAT, Patent Scope, WIPO and Google Patents. RESULTS: The results showed that the rPHY is active at 37-40ºC and presented an optimal pH and temperature of 8.0 and 40°C, respectively. The phytase rPHY was activated by Cu2+ ion and showed resistance to trypsin and pepsin, retaining 55% of the activity at the ratio of 0.02. Furthermore, the dissociation constant (Kd = 1.1150 ± 0.0087 mM), as estimated by a fluorescence binding assay, suggests a medium affinity of the enzyme with the substrate. CONCLUSION: The results of this article can be considered as innovative and for this reason, they were protected by Intellectual Property Law in Brazil. Take together, the biochemical properties of the rPHY could be useful in future for its industrial application of this enzyme as an additive in the monogastric feed.

Biofactories for the Production of Recombinant Phytases and their Application in the Animal Feed Industry.

BACKGROUND: Phytases are enzymes capable of degrading phytic acid and are used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus. Recent inventions show interest in production and optimization of recombinant phytases with biochemical and physicochemical characteristics promising for animal feed industry. METHODS: This review article is focused on relevant patents of promising phytases, together with the commonly used expression systems for their production and tools currently employed to generate new phytases. We revised all patents related to recombinant phytases and their application in the animal feed industry. The following patents databases were consulted: European Patent Office (Espacenet), the United States Patent and Trademark Office (USPTO), the United States Latin America Patents (LATIPAT), Patent scope -Search International and National Patent Collections (WIPO) and Google Patents. RESULTS: In this review, information was collected from recent publications, including 38 patents related production systems for different recombinant phytases and their application in the animal feed industry. CONCLUSION: We showed that important recombinant phytases were successfully produced in different expression systems. In addition, this work highlights certain biotechnological tools such as mutagenesis for generation of novel enzymes with biochemical properties of use in the animal feed industry.

Gel mobility shift scanning of pectin-inducible promoter from Penicillium griseoroseum reveals the involvement of a CCAAT element in the expression of a polygalacturonase gene.

Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.

Gel mobility shift scanning of pectin-inducible promoter from Penicillium griseoroseum reveals the involvement of a CCAAT element in the expression of a polygalacturonase gene

Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.