Study On Antimicrobial Substances Of Weissellahellenica D1501 And Its Use In Novel Tofu As Protective Cultures

During production, transportation, storage and sales, foods are vulnerable to contamination caused by spoilage and pathogenic microorganisms. Contamination and spoilage of food not only cause huge economic losses and environmental pollution, but also are deleterious to human health. With the improvement of living standards and the progress of science and technology, issues of food safety receive consumers’attentions and demands for green and healthy food, thus safe, efficient, natural food preservatives need to be applied into food industry urgently. Lactic acid bacteria (LAB) are generally recognized as safe and probiotics, which are widely applied to many fermented foods with a history of thousands of years. LAB can produce many antimicrobial compounds such as organic acids, hydrogen peroxide, CO2 and bacteriocins which inhibit the growth of other microorganisms in different mechanisms, in order to extend the shelf-life of food and guarantee the quality and safety of food. In addition, LAB and their active metabolites are highly beneficial to human health. Therefore, as food preservative preservative, LAB and their metabolites are intensively applied into food productions.This paper starts with screeing a strain with broad-range antimicrobial activities among 148 strains isolated from different kinds of traditional fermented foods; it was identified as Weissella hellenica D1501; antimicrobial substance produced by W. hellenica D1501 in netrual broth was protein in nature; bacteriocin was purified by conventional separation methods and chromatographic techniques; bacteriocin was identified by MALDI-TOF-MS, N-termianl sequencing and PMF; physical-chemical characteristics and antimicrobial spectrums of bacteriocin were also studied; phenyllactic acid and hydroxyphenyllactic acid were also produced by W. hellenica D1501, and the concentrations of these two compounds were increased significantly by Plackett-Burman design and steepest ascent; Duo to its broad-range antimicrobial activities, a novel LAB tofu with long shelf-life was made by using W. hellenica D1501 as bio-coagulant and protective cultures. All studies provide theoretical basis for the development of new natural preservative agents produced by LAB and protective cultures applied in food preservation. The results are described as follows:1. Screening of strains with broad-range antimicrobial activities and study on fermentation conditionsThe antimicrobial activities of 148 strains isolated from various traditional fermented foods were determined by agar diffusion method. After primiary screening and secondary screening excluding effects of acids, a strain with broad-range antimicrobial activities was identified as W. hellenica D1501. By expxeriments of one factor at a time, in MRS broth with intial pH of 6.0 with inoculum of 3%(v/v) at 37℃ for 24 h, the antimicrobial activity of W. hellenica D1501 in neutral broth reached highest.2. Purification and characterization of bacteriocin produced by W, hellenica D1501Antimicrobial substance produced by W. hellenica D1501 was sensitive to protease, and it was bacteriocin designated as weissellicin D. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation, gel permeation and mono-Q anion-exchange chromgraphy. Weissellicin D had a molecular weight of 62425 Da by Tricine-SDS-PAGE and mass spectrometry, which was the largest among all known bacteriocins from Weissella. The Edman degradation resultes implied that the amino acid sequences of weissellicin D were belonged to N-terminal blocking group. Peptide fingerprinting mass (PMF) analyses mdicated that weissellicin D had no homology with any known bacteriocins or antimicrobial proteins. A similarity was between weissellicin D and ABC superfamily ATP binding cassette transporter of Lactobacillus ultunensis DSM 16047. However, the protein coverage was only 19%. Weissellicin D was a new kind LAB bacteriocin with special structure.3. Physical-chemical properties, antimicrobial spectrum and antimicrobial characteristics of weissellicin DThe bacteriocin activity was stable in a wide pH range between 2 and 8. A high thermal stability was also observed, and the bacteriocin remained active even after 121℃ for 15 min. The antimicrobial activities did not change after UV radiations. Chemicals such as methanol, ethanol, tween-80, SDS, urea and EDTA had no effect on antimicrobial activities. Interestingly, an increased activity of weissellicin D was observed in combined with EDTA. Weissellicin D exhibited a broad range of inhibitory activities against Gram-positive and Gram-negative bacteria and some molds and yeasts. The mode of weissellicin D against Escherichia coli K-12 was bacteriostatic. W. hellenica D1501 could effectively co-aggregate with E. coli K-12 in vitro. Co-cultured W. hellenica D1501 with E. coli K-12, the growth of E. coli K-12 was suppressed. At the same time, bacteriocin was produced in advance.4. Non-protein antimicrobial substances produced by W. hellenica D1501The production of PLA and OH-PLA reached 128.31 mg/L (0.77 mM) and 48.94 mg/L (0.27 mM) respectively when W. hellenica D1501 was incubated in MRS broth at 37℃ for 24 h, which were the highest in Weissella genus reported so far. The crude extracts containing PLA and OH-PLA exhibited inhibitory activities agains E. coli, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus and Bacillus cereus. Plackett-Burman design and steepest ascent were used to optimize the amounts of PLA and OH-PLA. The optimized condition was the medium composed of glucose 46 g/L, peptone 23 g/L, yeast extracts 5 g/L, beef extracts 10 g/L, sodium acetate 5 g/L, ammonium citrate 2 g/L, K2HPO4·3H2O 2.62 g/L, MgSO4·7H2O 0.58 g/L, MnSO4·H2O 0.198 g/L, Tween-80 1.0 mL/L, phenylpyruvic acid 6.5 g/L, tyrosine 1g/L, pH 8.0, the incubation temperature and time were 30℃ and 108 h. As a result, the amounts of PLA and OH-PLA reached the maximum, 2.40 g/L (14.46 M) and 242.95 mg/L (1.34 mM) respectively, which were increased by 18.70 times and 4.96 times respectively than before.5. Application into novel LAB tofu as protective culturesTo investigate the antagonistic activities of W. hellenica D1501 in soymilk, it was co-cultured with spoilage and pathogenic bacteria (Kurthia gibsonii, S. aureus and E. coli) in soymilk. The proliferations of three undesired strains were suppressed and the bacterial populations decreased to undetectable level after 36 h in co-cultures. Spoilage and pathogenic strains were presumably inhibited by various organic acids, bacteriocins, antimicrobial volatiles or other antimicrobial compounds generated by W. hellenica D1501 during the growth in soymilk.The formula of LAB tofu was optimized by using single factor experiments combined with sensory evaluations:ration of soybean to water was 1 to 8, inoculums concentration was 4%(v/v), additive MTG amount was 200 U/L, and fermentation temperature was 37℃. LAB tofu was white, smooth, soft and flexible. Compared with tofu prepared with traditional coagulants, W. hellenica D1501 in LAB tofu could strongly delay the microbial spoilage and extend its shelf-life based on microbiological analyses. During storage at 4℃ for 21 d,25℃ for 14 d and 37℃ for 7 d, LAB tofu exhibited a stability of quality properties (pH, titratable acidity, water holding capacity and texture) and viable lactic acid bacteria. In addition, various antimicrobial volatiles and bacteriocin-like substances were produced to guarantee the safety of LAB tofu. Based on all the results, D1501 as protective cultures was applied into soybean products, ont only could inhibit or kill spoilage organisms and pathogens, extend shelf-life, ensure the security and stability of the product, but also could enrich the organoleptic properties and the diversity of products.