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.

Effects of Dietary Supplementation with Prebiotics and Pediococcus Acidilactici on Gut Health, Transcriptome, Microbiome, and Metabolome in Atlantic Salmon (Salmo Salar L) after Seawater Transfer

BackgroundGiven the importance of gut microbiota for health, growth and performance of the host, the aquaculture industry has taken measures to develop functional fish feeds aiming at modulating gut microbiota and inducing the anticipated beneficial effects. However, present understanding of the impact of such functional feeds on the fish is limited. The study reported herein was conducted to gain knowledge on performance and gut health characteristics in post-smolt Atlantic salmon fed diets varying in content of functional ingredients. Three experimental diets, a diet containing fructo-oligosaccharides (FOS), a diet with a combination of FOS and Pediococcus acidilactici (BC) and a diet containing galacto-oligosaccharides (GOS) and BC, were used in a 10-weeks feeding trial. A commercial diet without functional ingredients was also included as a control/reference. Samples of blood plasma, mucosa and digesta were subjected to microbiome, transcriptome and metabolome profiling for evaluation of the diet effects. Results No significant growth differences were observed between fish fed the supplemented diets, but FOS-BC fed fish showed significantly higher growth than the control fed fish. The microbiome results showed that the BC was present in both the digesta and the mucosa samples of fish fed the FOS-BC and GOS-BC diets. Digesta-associated microbiota was altered, while mucosa-associated microbiota was relatively unaffected by diet. Replacing FOS with GOS increased the level of metabolites linked to phospholipid, fatty acid, carnitine and sphingolipid metabolism. Variation in metabolite levels between the treatments closely correlated with genera mainly belonging to Firmicutes and Actinobacteria phyla. The transcriptome analyses indicated diet effects of exchanging FOS with GOS on immune functions, oxidative defense and stress responses. No significant diet effect was observed on intestinal inflammation in the pyloric ceca or in the distal intestine, and no steatosis was observed in the pyloric ceaca. ConclusionsDietary supplementation with BC induced moderate effects on the microbiota of the digesta, while the effects of replacing FOS with GOS were more marked and was observed also for nutrient metabolism. Our data indicates therefore that the quality of a prebiotic may be of great importance for the effects of a probiotic on gut microbiota, function and health.

Citrulline Accumulation Mechanism of Pediococcus acidilactici and Weissella confusa in Soy Sauce and the Effects of Phenolic Compound on Citrulline Accumulation

Citrulline is one of the major precursors of ethyl carbamate in soy sauce, and the accumulation of citrulline is attributed to the metabolism of arginine by bacteria with the arginine deiminase (ADI) pathway. However, key strains and factors affecting citrulline accumulation are not yet clear. In this study, two key strains of Pediococcus acidilactici and Weissella confusa were isolated from soy sauce moromi, and the regularity of citrulline formation was studied. Results showed that the conversion rates from arginine to citrulline (A/C rate) and the citrulline accumulation ability of W. confusa and P. acidilactici significantly increased in the presence of different concentrations of NaCl, indicating that salt stress was the main factor for citrulline accumulation. The inconsistent expression of arc genes by salt stress was the reason for citrulline accumulation for P. acidilactici, but for W. confusa, it may be due to the influence of arginine/citrulline on the transportation system: the intracellular citrulline could neither transport to extracellular space nor convert into ornithine. Environmental factors greatly influenced citrulline accumulation of the two key bacteria; A/C rate and citrulline formation in both strains decreased at low temperature (15°C) under high salt stress, but opposite effects were observed for the two key strains under anaerobic light condition. Moreover, quercetin and gallic acid significantly decreased the A/C rate and citrulline accumulation ability of the two key strains. The optimal quercetin and gallic acid as suggested by simulation experiment were 100 and 10 mg/l, respectively, and the lowest A/C rate of 28.4% and citrulline level of 1326.7 mg/l were achieved in the simulation system. This study explored the main factors for citrulline formation by the two key strains and proposed a targeted way to control citrulline in soy sauce.

In vitro studies on anti-inflammatory, antioxidant and antihyperglycemic activities of potential probiotic Pediococcus acidilactici NCDC 252

Probiotics are live microbes which positively influence the health when consumed in adequate amount. Lactic acid bacteria (LAB) are commonly used probiotics and are generally found in yogurt and fermented foods. They provide barrier for pathogens by secreting peptides and other metabolites. Pediococcus acidilactici NCDC 252 is a LAB of dairy origin with probiotic attributes. NCDC 252 was studied for in vitro anti-inflammatory, antioxidant and antihyperglycemic activities. Anti-inflammatory activity was studied by human red blood cell (HRBC) membrane stabilization method, protein (albumin) denaturation inhibitory activity and heat induced haemolysis. Antioxidant activity was evaluated by α, α-diphenyl-β- picrylhydrazyl (DPPH) radical and hydrogen peroxide (H2O2) scavenging assays. Alpha amylase inhibition assay was performed to examine antihyperglycemic effect. NCDC 252 exhibited potent anti-inflammatory but moderate antioxidant and antihyperglycemic activities as compared to control. NCDC 252 exhibited 65%, 70% and 49% membrane stabilization, protein denaturation and heat induced activity respectively. Scavenging effect was 45 % and 60% in H2O2 and DPPH assays respectively. Alpha amylase inhibition was 48 %. These results suggest therapeutic potential of NCDC 252 and open new avenues to treat disorders related to free radical generation such as inflammation and diabetes mellitus after in vivo evaluation of NCDC 252 to confirm its efficacy in animals.