Bacteriocin-producing lactic acid bacteria isolated from mangrove forests in southern Thailand as potential bio-control agents in food: Isolation, screening and optimization

International audienceA total of 386 isolates of lactic acid bacteria isolated from mangrove forests (soil, water, leaf, twig and fruit) in southern Thailand were screened for bacteriocin production. Only 4 strains that produced bacteriocin-like inhibitory substance (BUS) in MRS broth, named KT2W2G, KT2W2L, TS9S17 and TS9S19 showed an inhibition zone against Lactobacillus sakei subsp. sakei JCM 1157, Listeria monocytogenes DMST 17303 and Brochothrix thermosphacta DSM 20171 as indicators by using agar well diffusion assay. None of the inhibitory activities were related to the production of either organic acids or hydrogen peroxide. The BLISs produced by these strains were not affected by heating but were sensitive to proteolytic enzymes. The isolate KT2W2L was identified as Lactococcus lactis subsp. lactis while the isolates KT2W2G, T59S17 and TS9S19 were identified as Enterococcus faecalis. These BLISs showed a wide range of antibacterial activity against similar bacterial strains, food-spoilage and food-borne pathogens, but were inactive against the Gram-negative bacteria tested. Statistical experimental designs, based on the Plackett-Burman protocol, were applied to optimize the bacteriocin production by Ent faecalis KT2W2G in flask cultures. By using Plackett-Burman protocol, lactose and temperature were found to be the most important factors for bacteriocin production. The effects of the two main factors on bacteriocin activity were further investigated using a central composite design (CCD) and the optimum composition was found to be lactose 14.85 g/I and temperature 25.59 degrees C. Optimum conditions were validated by experiment in which bacteriocin activity (Arbitrary Unit/ml) was increased 8-fold (640 AU/ml) in 18 h fermentation

Inhibition of food-spoilage and foodborne pathogenic bacteria by a nisin Z-producing Lactococcus lactis subsp. lactis KT2W2L

The growth of food-spoilage and foodborne pathogenic bacteria was inhibited by a nisin Z-producing Lactococcus lactis subsp. lactis KT2W2L as determined by the agar spot test and agar well diffusion assay. The growth of Brochothrix thermosphacta DSMZ 20171T and Staphylococcus aureus CIP 76.25 was inhibited when co-cultivated with L. lactis subsp. lactis KT2W2L whereas the growth of Escherichia coli CIP 76.24 and Salmonella Enteritidis CIP 81.3 was not. However, the growth level of E. coli CIP 76.24 and S. Enteritidis CIP 81.3 co-cultivated with L. lactis subsp. lactis KT2W2L was diminished compared to cultivation without L. lactis subsp. lactis KT2W2L. The neutralized cell-free supernatant (NCFS) collected from the culture of L. lactis subsp. lactis KT2W2L, cultivated with and without indicator strain, exhibited inhibition zones against the indicator strains as determined by the agar well diffusion assay. The results reported in this study indicate that a nisin Z-producing L. lactis subsp. lactis KT2W2L may find application as a bio-preservative for reducing food-spoilage and foodborne pathogens in food products

Bacteriocin producing Enterococcus faecalis isolated from chicken gastrointestinal tract originating from Phitsanulok, Thailand: Isolation, screening, safety evaluation and probiotic properties

A total of 230 isolates of lactic acid bacteria were isolated from gastrointestinal tracts (GIT) of indigenous chickens purchased from local market in Bang Rakam district, Phitsanulok, Thailand. Among 230 isolates, only 7 isolates, named CM6CR07, CM6CR11, CF1GI15, CF1GI14, CF1GI17-1, CF1GI17-2 and CF1GI 19 showed an inhibition zone against indicator strains. Bacteriocin activity was completely inactivated when Neutralized Cell Free Supernatants (NCFS) of the isolates were treated with proteases, confirming the proteinaceous nature of the bacteriocin component. NCFS of all selected bacteriocin producing lactic acid bacteria (SBP-LAB) retained their bacteriocin activity after heating at 100 C-omicron for 30 min. All of SBP-Lwere identified as Enterococcus faecalis. These Ent. faecalis isolates harbored the same virulence genes. All strains were positive for gelE (gelatinase), efaA(fs) (cell wall adhesins), hyl (hyaluronidase), ace (adhesin of collagen protein), cylL(L) (cytolysin structural subunit) and cylL(s) (cytolysin structural subunit) and were negative for asal (aggregation substance), agg (aggregation protein involved in adherence to eukaryotic cells), and esp (enterococcal surface protein). These Ent. faecalis showed a wide range of inhibitory spectrum against food-spoilage and food-borne pathogens when studied by agar spot test. The CFS and NCFS also inhibited some of food-spoilage and food-borne pathogenic indicator strains when checked by agar well diffusion assay. All of these Ent. faecalis survived simulated stomach conditions, whereas all of them could survive in conditions of simulated intestinal juice

Effect of Bacillus toyonensis BCT-7112T supplementation on growth performance, intestinal morphology, immune-related gene expression, and gut microbiome in Barbary ducks

ABSTRACT: This study aimed to investigate the effect of Bacillus toyonensis BCT-7112T supplementation on growth performance, intestinal morphology, immune-related gene expression, and the cecal microbiota of meat ducks. A total of 150 one-day-old male Barbary ducks were divided into 3 groups with 5 replicates (n = 10 ducks per replicate) by completely randomized design and offered diets supplemented with the commercial product Toyocerin (containing 1 × 109 B. toyonensis BCT-7112T viable spores/g product) at the levels of 0, 500, or 1,000 mg/kg (0, 500, or 1,000 ppm), respectively, for 8 wk. The results showed that although ducks in the 500 ppm B. toyonensis BCT-7112T group displayed numerically better values (e.g., weight gain and feed conversion ratio) than those in the control group, the growth performance of ducks fed diets supplemented with B. toyonensis BCT-7112T did not differ significantly from that of the control group (P > 0.05). There were no significant differences in the intestinal mucosal morphology of ducks across the experimental groups (P > 0.05). However, ducks in the 500 ppm B. toyonensis BCT-7112T group showed a trend of greater values, for example, villus height per crypt depth of duodenum (P = 0.16) and ileum (P = 0.12) compared with those in the control group. The relative expression of immune-related genes, for example, interferon (IFN) and interleukin-6 (IL-6) in the meat duck spleen was significantly lower in both B. toyonensis BCT-7112T groups at 14 d and 35 d than in the control group (P < 0.05). Beta diversity analysis of the cecal microbiota of ducks in either the 500 ppm or the 1,000 ppm B. toyonensis BCT-7112T group showed to have higher diversity than that in the control group, where at the phylum level, Bacteroidetes was the most abundant, followed by Firmicutes, and at the genus level, Bacteroides, Fusobacterium, and Ruminococcaceae were the top 3 most abundant genera. In conclusion, our study demonstrates that 500 ppm supplementation with B. toyonensis BCT-7112T in duck diets can reduce proinflammatory cytokine gene expression, improve immunological function, and increase the variety of microbial communities in the ceca of meat-type ducks

Application of a nisin Z-producing <em>Lactococcus lactis</em> subsp. lactis KT2W2L isolated from brackish water for biopreservation in cooked, peeled and ionized tropical shrimps during storage at 8 °C under modified atmosphere packaging

International audienceLactococcus lactis subsp. lactis KT2W2L was used for biopreservation in cooked, peeled and ionized tropical shrimps (CPITS) during storage at 8 A degrees C under modified atmosphere packaging (MAP). The neutralized and filtered cell-free supernatant produced by this strain exhibited a broad spectrum of inhibition against 23 strains from 24 indicator strains of food-spoilage bacteria and food-borne pathogens by agar well diffusion assay. The growth of Brochothrix thermosphacta CD274, Carnobacterium maltaromaticum CD263 and Listeria innocua CIP 80.11T was inhibited by L. lactis subsp. lactis KT2W2L when co-cultivated at 25 A degrees C. However, only L. innocua CIP 80.11T was inhibited in a co-cultivation at 8 A degrees C. L. lactis subsp. lactis KT2W2L was then used for biopreservation in CPITS. Growth of bacteria group inoculated in CPITS was monitored at regular intervals during storage period under MAP at 8 A degrees C by microbial counts and the thermal temperature gradient gel electrophoresis (TTGE) technique. L. lactis subsp. lactis KT2W2L inhibited the growth of B. thermosphacta and L. innocua CIP 80.11T batches after 7 days of storage. However, it was inactive against C. maltaromaticum batch. The growth of L. lactis subsp. lactis KT2W2L alone or in the presence of indicators increased to reach about 8-9 log CFU/g within 7 days of storage and remained constant until the end of the experiment. In the batches without L. lactis subsp. lactis KT2W2L, all bacterial groups grew well on the cooked shrimp matrix, reaching their maximal levels after 2 weeks of storage. The pH of all homogenized suspensions of MAP-CPITS was quite stable at 6.0-6.7. The control batches were under the enumeration threshold (< 1.70 +/- A 0.00 log CFU/g) until 14 days of storage, and then, an increase in growth was detected on agar plates. In addition, TTGE revealed to be an excellent tool to monitor the change of the microbial ecosystem in this product

Bacteriocin-producing <em>Enterococcus faecalis</em> KT2W2G isolated from mangrove forests in southern Thailand: Purification, characterization and safety evaluation

International audienceThe aim of this work was to purify and characterize the bacteriocin produced by Enterococcus faecalis KT2W2G isolated from the mangrove forest in southern Thailand, in order to evaluate its potential as a new food protective agent. The active peptide from the cell-free supernatant of Ent. faecalis KT2W2G was purified in 4 steps: (i) precipitation with 70% saturated ammonium sulfate, (ii) elution on a reversed phase cartridge (Sep-Pak C8) using different concentrations of acetonitrile, (iii) cation-exchange chromatography and (iv) final purification by reversed phase-HPLC on a C8 column. Each purification step increased the specific activity and reduced the amount of contaminating non-bacteriocin proteins. The specific activity of purified bacteriocin was 13,470.53 AU/mg of protein, which corresponded to a 48.10- fold increase. TricineeSDS-PAGE of the purified bacteriocin gave molecular weight ranging between 3.5 and 6.5 kDa. The activity of the partially purified bacteriocin was unaffected by pH (2.0e12.0) and thermostable, but was sensitive to proteolytic enzymes. This bacteriocin maintained full stability after storage at 20, 4 and 37 C for 2 months. It was stable when incubated for 1 month at 4 C in 0e30% NaCl. Inhibitory spectrum of this bacteriocin showed a wide range of activities against other LAB, foodspoilage and food-borne pathogens. Ent. faecalis KT2W2G was sensitive to kanamycin, tetracycline and vancomycin but resistant to ampicillin, gentamicin and penicillin. PCR amplification demonstrated that Ent. faecalis KT2W2G does not harbor virulence genes cylA, cylB and esp but has virulence genes ace, asa1and efaAfs. The bacteriocin and its producing strain may find application as bio-preservatives for reduction of food-spoilage and food-borne pathogens in food products