Microcin V Production in Lactobacillus plantarum LB-B1 Using Heterologous Leader Peptide from Pediocin PA-1.

Lactobacillus strains producing bacteriocins have attracted highly attention as probiotic cultures in animal nutrition since the use of antibiotics was forbidden in the livestock industry. Lactobacillus plantarum LB-B1 isolated from the fermented dairy product can produce pediocin PA-1, which has a strong inhibition of Listeria but hardly any influence on Gram-negative spoilage agents. In this work, L. plantarum LB-B1 was selected as the host to express microcin V using the leader peptide of pediocin PA-1. Well-diffusion assay combined with Tricine-SDS-polyacrylamide gel showed that microcin V could be successfully expressed and secreted in L. plantarum LB-B1. Meanwhile, the production of microcin V did not affect the secretion of pediocin PA-1. It is worthwhile noted that the supernatant from L. plantarum 8148-ColV had a more effective inhibition of Listeria than that from the control strain L. plantarum 8148. Furthermore, this supernatant also unexpectedly produced antibacterial activity against Staphylococcus aureus. Taken altogether, these results suggested that pediocin PA-1 and microcin V in the supernatant could generate synergistic effect, which not only enhanced the antibacterial ability but also expanded the antibacterial spectrum. Therefore, the recombinant strain has a great potential application as a probiotic to reduce the level of enteric pathogens in livestock industry.

Functional role of oppA encoding an oligopeptide-binding protein from Lactobacillus salivarius Ren in bile tolerance.

Lactobacillus salivarius is a member of the indigenous microbiota of the human gastrointestinal tract (GIT), and some L. salivarius strains are considered as probiotics. Bile tolerance is a crucial property for probiotic bacteria to survive the transit through the GIT and exert their beneficial effects. In this work, the functional role of oppA encoding an oligopeptide transporter substrate-binding protein from L. salivarius Ren in bile salt tolerance was investigated. In silico analysis revealed that the oppA gene encodes a 61.7-kDa cell surface-anchored hydrophilic protein with a canonical lipoprotein signal peptide. Homologous overexpression of OppA was shown to confer 20-fold higher tolerance to 0.5 % oxgall in L. salivarius Ren. Furthermore, the recombinant strain exhibited 1.8-fold and 3.6-fold higher survival when exposed to the sublethal concentration of sodium taurocholate and sodium taurodeoxycholate, respectively, while no significant change was observed when exposed to sodium glycocholate and sodium glycodeoxycholate (GDCA). Our results indicate that OppA confers specific resistance to taurine-conjugated bile salts in L. salivarius Ren. In addition, the OppA overexpression strain also showed significant increased resistance to heat and salt stresses, suggesting the protective role of OppA against multiple stresses in L. salivarius Ren.

Application and Effect of Pediococcus pentosaceus and Lactiplantibacillus plantarum as Starter Cultures on Bacterial Communities and Volatile Flavor Compounds of Zhayu, a Chinese Traditional Fermented Fish Product.

Zhayu is a type of traditional fermented fish product in China that is made through the fermentation of salted fish with a mixture of cereals and spices. Inoculation fermentation was performed using Pediococcus pentosaceus P1, Lactiplantibacillus plantarum L1, and a mixture of two strains, which were isolated from cured fish in Hunan Province. Compared with the natural fermentation, inoculation with lactic acid bacteria (LAB) accelerated the degradation of myosin and actin in Zhayu, increased the trichloroacetic acid (TCA)-soluble peptide content by about 1.3-fold, reduced the colony counts of Enterobacteriaceae and Staphylococcus aureus by about 40%, and inhibited their lipid oxidation. In the texture profile analysis performed, higher levels of hardness and chewiness were observed in the inoculation groups. In this study, the bacterial community and volatile flavor compounds were detected through 16S high-throughput sequencing and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Inoculation with L. plantarum L1 reduced around 75% abundance of Klebsiella compared with the natural fermentation group, which was positively correlated with 2,3-Butanediol, resulting in a less pungent alcohol odor in Zhayu products. The abundances of 2-pentylfuran and 2-butyl-3-methylpyrazine were increased over threefold in the L1 group, which may give Zhayu its unique flavor and aroma.

L-Malic Acid Protects Lacticaseibacillus paracasei L9 from Glycodeoxycholic Acid Stress via the Malolactic Enzyme Pathway.

Bile stress tolerance is a crucial characteristic of probiotics for surviving in the human gastrointestinal tract. The mechanism underlying the effect of l-malic acid on enhancing the glycodeoxycholic acid (GDCA) tolerance of Lacticaseibacillus paracasei L9 was investigated herein. Bile tolerance specificity assays revealed that Lc. paracasei L9 was more sensitive to GDCA than to taurocholic acid, glycocholic acid, and taurodeoxycholic acid. Notably, l-malic acid significantly enhanced the GDCA tolerance of Lc. paracasei L9 by increasing the pH of the medium. The role of the malolactic enzyme pathway in enhancing GDCA resistance was investigated using molecular techniques. Confocal laser scanning and scanning electron microscopy revealed that l-malic acid preserved membrane permeability and cellular morphology, thereby protecting bacterial cells from GDCA stress-induced damage. The study also demonstrated that l-malic acid enhanced bile tolerance in different species of lactobacilli. These findings provide a novel protective mechanism for coping with bile stress in lactobacilli.

Structural evolution, digestibility and inhibition on starch digestion of rice glutelin fibril aggregates as affected by incubation.

Structural evolution, digestibility and inhibition on starch digestion of rice glutelin fibril aggregates (RGFAs) as affected by incubation were investigated. Thioflavin T fluorescence intensity of the RGFAs, incubated for 4-day, reached the maximum values, which ranged from 845.00 ± 23.52 to 873.67 ± 50.30. Transmission electron microscopy (TEM) observed that the samples heated for 2 h (2 h fibril) were self-assembled from small glutelin aggregates and a few protofibrils into mature fibrils, the samples heated for 4-10 h (4-10 h fibril) were elongated into long, branched fibrils, and the longer fibrils of 15 h fibril sample dissociated into short fibrils after 4-day of incubation. Compared to rice glutelin, the RGFAs showed thermal stability and resistance to proteolysis. The fluorescence retention rate of 6 h fibril, after incubation for 4-day, was 8.62 ± 0.61 % after in vitro stomach and pancreas digestion, which was the highest among all of the samples. The RGFAs incubated for 1-day displayed much better inhibition effects on starch digestion. This was the first study to clarify the relationship between incubation and physicochemical/functional properties of protein fibrils, which could help understand the preservation of food protein fibrils and their application.

Corrigendum: Global Transcriptomic Analysis and Function Identification of Malolactic Enzyme Pathway of Lactobacillus paracasei L9 in Response to Bile Stress.

[This corrects the article DOI: 10.3389/fmicb.2018.01978.].

Global Transcriptomic Analysis and Function Identification of Malolactic Enzyme Pathway of Lactobacillus paracasei L9 in Response to Bile Stress.

Tolerance to bile stress is crucial for Lactobacillus paracasei to survive in the intestinal tract and exert beneficial actions. In this work, global transcriptomic analysis revealed that 104 genes were significantly changed (log2FoldChange > 1.5, P < 0.05) in detected transcripts of L. paracasei L9 when exposed to 0.13% Ox-bile. The different expressed genes involved in various biological processes, including carbon source utilization, amino acids and peptide metabolism processes, transmembrane transport, transcription factors, and membrane proteins. It is noteworthy that gene mleS encoding malolactic enzyme (MLE) was 2.60-fold up-regulated. Meanwhile, L-malic acid was proved to enhance bile tolerance, which could be attributed to the intracellular alkalinization caused by MLE pathway. In addition, membrane vesicles were observed under bile stress, suggesting a disturbance in membrane charge without L-malic acid. Then, genetic and physiological experiments revealed that MLE pathway enhanced the bile tolerance by maintaining a membrane balance in L. paracasei L9, which will provide new insight into the molecular basis of MLE pathway involved in bile stress response in Lactic acid bacteria.