| Consumer preference regarding fresh-cut products favors those without artificial additives. This market demand has stimulated research interest in biopreservation that utilizes bacteriocinogenic lactic acid bacteria (LAB) and their metabolic products to inhibit undesired pathogens and spoilage organisms. Among various metabolic products, bacteriocin is regarded as the most promising bioactive substance. Molecular studies on bacteriocin and its producing strain will help LAB's further development and utilization.In this study, synergistic effects between bacteriocin and other metabolites produced by selected food-grade LAB species were observed, indicating LABs' potential as protective cultures. Selected LAB species have showed effective inhibitory effects on Listeria inoculated in fresh-cut mixed salad and fresh-cut onions. To study LAB proteins' expression during the cold storage of fresh-cut products, the utilization of food model system was proposed to establish the Listeria challenge test. The impact of media composition, inoculum level, LAB species and storage temperature on the anti-listerial activity were compared. In the simulated fish (salmon) media, any of the selected LAB species co-inoculated with L. innocua demonstrated no antimicrobial activity against L. innocua though normal growth of LAB was observed. In simulated fruit (cantaloupe) and vegetable (iceberg lettuce) media, the highest LAB inoculum level could inhibit the growth of L. innocua most effectively for the entire storage duration. At 10?, for the sample inoculated with Lactococcus lactis, Enterococcus faecium and Streptococcus thermophilus, a 4.5,4.4 and 3.8 log CFU mL-1 reduction of L. innocua in the fruit system was observed on day 21 and a 4.7,4.7 and 3.1 log CFU mL-1 reduction of L. innocua was observed in the vegetable system respectively. The selected LAB in the simulated vegetable media provided greater inhibitory effects against Listeria over the storage period. At 25 ?, vegetable samples inoculated with the highest level LAB demonstrated an average 1.1 log CFU mL-1 reduction of L. innocua after 72 h, whereas the fruit ones only permitted a 0.2 log CFU mL-1 reduction compared to initial level. L. innocua inhibition of LAB strains became weaken when the storage temperature decreased, and this trend was most obvious for the Strept. thermophilus. Lact. Lactis provided most constant anti-listerial activity for the tested conditions. These findings warrant consideration in the biopreservation of salads and other fresh-cut produce products.To find out the characterstic and change of Lact. Lactis proteome in fruit and vegetable media, the protein extraction method was optimized and proteomics platform based on liquid separation technology was established. Glass beads beating and acetone direct precipitation method was chosen for the preparation of soluble proteins in Lact. Lactis based on the experiments of protein recovery and SDS-PAGE result. OFFGEL electrophoresis (OGE) fractionators and Q-TRAP mass spectrometer (MS) were then utilized to establish a high-throughput proteomic research platform and identify Lact. Lactis proteome. OGE fractionation results indicated:most Lact. Lactis peptides distributed among fractions 1-5 and 8-11 (pI range 3.4-6.2), corresponding to 39.4% and 29.3% protein detected in the whole proteome; among the identified peptides,72.5% and 14.0% peptides were found in one or two fractions. The distribution of 7 important amino acids, aspartate (D), glutamate (E), arginine (R), lysine (K), histidine (H), tyrosine (Y), cysteine (C) by peptide and by fraction suggested:acidic peptides among fractions 1-5 had average of 2 D and 1.3 E amino acids, and the number of these two acidic amino acids by peptide globally decreased from acidic to basic fractions. The number of the basic amino acids R and K was relatively higher than the theoretical value, attributed to miss-cleavage of trypsin in certain peptides; cysteine and tyrosine (Y) were globally constant in each fraction, while histidine mainly presented in two regions, fractions 6-9 and 15-18. The results indicated a good separation was obtained by OGE and the method was suitable for LAB protein sample. Related data could be used as an reference for the enrichment of target peptides.Based on the aforementioned methods, stable isotope labeling by peptide dimethylation and high resolution QTOF MS were used for quantitative proteomics studies of Lact. lactis following cold storage in 10 ? fruit and vegetable media on day 0,3 and 7. Total 914 to 1192 proteins were identified in Lact. Lactis, with 98 and 113 being significantly regulated in vegetable and fruit media respectively. According the hierarchical clustering analysis,62.63% and 34.17% proteins showed up-regulated expression over the vegetable and fruit storage period. Results of protein functional analysis indicated:1. total 46 pair LAB proteins presented the similar changing trends in both systems, and this could be attributed to the cold storage condition they shared. Among the proteins, the cold shock proteins were the dominant ones. Triggered defence and repair mechanisms, up-regulation of energy metabolism, increased need for substrates for protein synthesis, and adjustment for cell envelop were found in Lact. lactis.2. Media composition also was a major influence for the LAB metabolism. Proteins in redox system and detoxification metabolism exhibited complexity during the storage, with glutamate- cysteine ligase (GCL) and cysteine synthase (CS) significantly up- regulated in both systems, glutathione peroxidase (GPX), superoxide dismutase (SOD) and FeS assembly protein SufD only significantly up-regulated in fruit matrix. GCL and CS catalyzes the formation of the antioxidant glutathione (GSH) and its structural component. While GPX and SOD detoxified cellular peroxides, hydroperoxides and superoxides. These results indicated that Lact. lactis faced a much serious oxidative stress in furit media.3. For the carbohydrate utilization and metabolism, in the Lact. lactis cultured in vegetable media, the component of PTS system, mannose/fructose/sorbose family, key enzyme in glycolysis and pyrvate pathway, and the enzyme involved in initiation and the elongation cycle for the biosynthesis of fatty acids were all up-regulated, while in the Lact. lactis cultured in fruit media, the down and steady regulation for some of these proteins were noticed, indicating maximal utilization for the available and the response for expending source in vegetable and fruit media, respectively. Above results illustrates Lact. Lactis could adapt dynamically to the harsh temperature condition and diverse sources available in their habitats. The study also detected several essential proteins that involved in bacteriocin biosynthesis and secretion process: EntP immunity protein, EntA immunity protein, preprotein translocase subunit SecA and SecY. These proteins remained constant expression during the storage period, suggesting Lact. lactis at least had one productive system for class ? a bacteriocin, which could guarantee the listericidal activity for Lact. Lactis under adverse stress.This study have provided a theoretical foundation for LAB strain selection in biopreservation on protein level. The peptides' OGE prefractionation characteristic obtained and Lact. lactis proteome library constructed in the study also offered scientific supports for future targeted proteomic analysis, especially the enrichment and absolute quantification of the proteins related to the bacteriocin production. |
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