Characterization of Cell-Envelope Proteinases from Two Lacticaseibacillus casei Strains Isolated from Parmigiano Reggiano Cheese

In the present work, two cell-envelope proteinases (CEPs) from Lacticaseibacillus casei strains PRA205 and 2006 were characterized at both the biochemical and genetic levels. The genomes of both L. casei strains included two putative CEPs genes prtP2 and prtR1, but only prtR1 was transcribed. The extracted PrtR1 proteinases were serine proteinases with optimal activity at 40 °C and pH 7.5, and were activated by Ca2+ ions. Interestingly, PrtR1 from L. casei PRA205 exhibited high residual activity at pH 4 and at 5 °C, suggesting its possible exploitation for fermented food production. The caseinolytic activity against αS1- and β-casein indicated that both PrtR1s belonged to the PI/PIII type. These PrtR1s cleaved β-casein peptide bonds preferentially when amino acid M or N was present at the P1 subsite and amino acids A and D were at the P1′ subsite. Several bioactive peptides were found to be released from PrtR1 after αs1- and β-casein hydrolysis.

Genome-wide high-throughput signal peptide screening via plasmid pUC256E improves protease secretion in Lactiplantibacillus plantarum and Pediococcus acidilactici

Background Proteases catalyze the hydrolysis of peptide bonds of proteins, thereby improving dietary protein digestibility, nutrient availability, as well as flavor and texture of fermented food and feed products. The lactobacilli Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) and Pediococcus acidilactici are widely used in food and feed fermentations due to their broad metabolic capabilities and safe use. However, extracellular protease activity in these two species is low. Here, we optimized protease expression and secretion in L. plantarum and P. acidilactici via a genetic engineering strategy. Results To this end, we first developed a versatile and stable plasmid, pUC256E, which can propagate in both L. plantarum and P. acidilactici. We then confirmed expression and secretion of protease PepG1 as a functional enzyme in both strains with the aid of the previously described L. plantarum-derived signal peptide LP_0373. To further increase secretion of PepG1, we carried out a genome-wide experimental screening of signal peptide functionality. A total of 155 predicted signal peptides originating from L. plantarum and 110 predicted signal peptides from P. acidilactici were expressed and screened for extracellular proteolytic activity in the two different strains, respectively. We identified 12 L. plantarum signal peptides and eight P. acidilactici signal peptides that resulted in improved yield of secreted PepG1. No significant correlation was found between signal peptide sequence properties and its performance with PepG1. Conclusion The vector developed here provides a powerful tool for rapid experimental screening of signal peptides in both L. plantarum and P. acidilactici. Moreover, the set of novel signal peptides identified was widely distributed across strains of the same species and even across some closely related species. This indicates their potential applicability also for the secretion of other proteins of interest in other L. plantarum or P. acidilactici host strains. Our findings demonstrate that screening a library of homologous signal peptides is an attractive strategy to identify the optimal signal peptide for the target protein, resulting in improved protein export.

Positive Interactions Between Lactic Acid Bacteria Could Be Mediated by Peptides Containing Branched-Chain Amino Acids

Lactic acid bacteria (LAB) are responsible for the sanitary, organoleptic, and health properties of most fermented products. Positive interactions between pairs of LAB strains, based on nitrogen dependencies, were previously demonstrated. In a chemically defined medium, using milk and lupin proteins as sole nitrogen source, two proteolytic strains were able to sustain the growth of non-proteolytic strains, but one did not. The objective of the present study was, thus, to determine which specific peptides were implicated in the positive interactions observed. Peptides produced and involved in the bacterial interactions were quantified using tandem mass spectrometry (LC-MS/MS). About 2,000 different oligopeptides ranging from 6 to more than 50 amino acids in length were identified during the time-course of the experiment. We performed a clustering approach to decipher the differences in peptide production during fermentation by the three proteolytic strains tested. We also performed sequence alignments on parental proteins and identified the cleavage site profiles of the three bacterial strains. Then, we characterized the peptides that were used by the non-proteolytic strains in monocultures. Hydrophobic and branched-chain amino acids within peptides were identified as essential in the interactions. Ultimately, better understanding how LAB can positively interact could be useful in multiple food-related fields, e.g., production of fermented food products with enhanced functional properties, or fermentation of new food matrices.

Fate of influent microbial populations during medium chain carboxylic acid recovery from brewery and pre-fermented food waste streams

Waste streams continuously introduce active and inactive microbial populations that can influence assembly of microbial communities in chain elongation systems.

Karakteristik Organoleptik dari tempe modifikasi dengan kacang kedelai hitam (Glycine max (L) Merrit)

Tempeh is a fermented food from soybean seeds using the fungus Rhizopus oligosporus. Tempeh is usually made from ordinary fermented soybeans. However, there are various types of beans besides ordinary soybeans, one of which is black soybeans. Food product innovation is closely related to consumer acceptance. Acceptance can be measured using organoleptic test. Organoleptic testing is an important component in the process of making food product innovations. In this study, tempeh was made with a mixture of soybeans and black soybeans in a ratio of 50:50. The resulting tempeh was then tested organoleptically including the parameters of color, texture, aroma, and overall and compared with commercial tempeh on 45 panelists. Based on this research, it is known that the overall parameters, namely color, texture, aroma, and overall in soybean tempeh and 50:50 black soybeans have lower acceptability than commercial tempeh so that further development of these products is needed so that consumer acceptance is higher increase.

SlpB Protein Enhances the Probiotic Potential of L. lactis NCDO 2118 in Colitis Mice Model

Bacteria used in the production of fermented food products have been investigated for their potential role as modulators of inflammation in gastrointestinal tract disorders such as inflammatory bowel diseases (IBD) that cause irreversible changes in the structure and function of gut tissues. Ulcerative colitis (UC) is the most prevalent IBD in the population of Western countries, and it is marked by symptoms such as weight loss, rectal bleeding, diarrhea, shortening of the colon, and destruction of the epithelial layer. The strain Propionibacterium freudenreichii CIRM-BIA 129 recently revealed promising immunomodulatory properties that greatly rely on surface-layer proteins (Slp), notably SlpB. We, thus, cloned the sequence encoding the SlpB protein into the pXIES-SEC expression and secretion vector, and expressed the propionibacterial protein in the lactic acid bacterium Lactococcus lactis NCDO 2118. The probiotic potential of L. lactis NCDO 2118 harboring pXIES-SEC:slpB (L. lactis-SlpB) was evaluated in a UC-mice model induced by Dextran Sulfate Sodium (DSS). During colitis induction, mice receiving L. lactis-SlpB exhibited reduced severity of colitis, with lower weight loss, lower disease activity index, limited shortening of the colon length, and reduced histopathological score, with significant differences, compared with the DSS group and the group treated with L. lactis NCDO 2118 wild-type strain. Moreover, L. lactis-SlpB administration increased the expression of genes encoding tight junction proteins zo-1, cln-1, cln-5, ocln, and muc-2 in the colon, increased IL-10 and TGF-β, and decreased IL-17, TNF-α, and IL-12 cytokines in the colon. Therefore, this work demonstrates that SlpB recombinant protein is able to increase the probiotic potential of the L. lactis strain to alleviate DSS-induced colitis in mice. This opens perspectives for the development of new approaches to enhance the probiotic potential of strains by the addition of SlpB protein.

BIOTECHNOLOGY OF SOYA MULTI-COMPONENT FUNCTIONAL INGREDIENTS

The materials of the article present analytical and scientific-practical results of research of soy multi-component functional ingredients. Soy extract (or soy milk) is a valuable and cheap source of protein and other nutritious and biologically active components, like lipids, vitamins, mineral elements, isoflavones, flavonoids, saponins etc. But the presence of indigestible oligosaccharides and a bean flavor limit the consumption of soy milk. The soy oligosaccharides are known to be prebiotic for intestinal microbiota, particularly for probiotic microorganisms that hydrolyze them with the enzyme α-galactosidase. Isoflavones are known as phytoestrogens since they are similar to human estrogen in structure and function, and are recommended to prevent many hormonedependent diseases. In soy beans and unfermented soy products, isoflavones are present mainly as biologically inactive glycoside conjugates. Biologically active aglycones of soy isoflavones are substances that are assimilated by human organism from soy products faster and in higher amounts than their corresponding glycosides. The members of intestinal microbiota are predicted to play an important role also in the metabolism and bioavailability of isoflavones since they, synthesizing β-glycosidase, cause hydrolysis of glycoside components, thus releasing the bioavailable and biologically active form of aglycone. In this work, we estimated the potential of some Lactobacillus and Bifidobacterium strains to synthesize α-galactosidase and β-glycosidase, and to reduce respectively the amount of galactooligosaccharides and to convert isoflavones into their active forms, when growing in soy milk. Simultaneously, the profile of soy milk fermentation by the chosen probiotics was studied, as well as the proteolytic activity and accumulation of lactic acid by those probiotics, i. e. the possibility to produce soy-based polyfunctional fermented food products was investigated.

Examination of traditional fermented food consumption and product awareness of university students in Istanbul, Turkey

Background: Traditional fermented foods (TFF), which display positive effects on health, constitute a part of the traditions of a region and have continuity over many years. Familiarity with a product and the naturalness of food are positively associated with general attitudes toward traditional food consumption. Aims: To determine university students’ awareness and consumption of TFF and related factors in Turkey. Subjects and Methods: In this descriptive cross-sectional study, the faculties of Marmara University were stratified between January and June 2019, and a questionnaire on TFF consumption and related knowledge and attitudes was administered to 1,233 volunteer students selected using the random sampling method. The normality of data distribution was checked with the Kolmogorov-Smirnov test, and the data were analyzed with the chi-square test. Results: The students were familiar with the majority of TFF, with the most common being yoghurt (91.8%) and cheese (88.7%), while hardaliye (27.1%) and olives (27.1%) were less known TFF products. The majority of the students (60.7-99.2%) consumed cheese, yoghurt, pickled olives, pickle, soudjouk, tarhana, vinegar, and butter. The TFF were habitually consumed as industrial products, except tarhana, pickle, and yoghurt. It was also determined that the TFF consumption did not change according to the season in 76.4% of the students, and it was affected by the consumption of parents, whether they lived with their family or alone (p < 0.05). Conclusions: The students showed high awareness of TTF consumption, which was influenced by parent’s consumption and lifestyles. To ensure the continuity of TFF consumption, positive attitudes and behaviors must be maintained. Keywords: Consumption, familiarity, fermented foods, industrial products, Turkey.