| Along with the improvement of people's living standard and food safety consciousness, the people pay more attention to the food safety and their own status of health, which suggests that the natural food without chemistry antiseptic and the organic foods is easier to be approved and accepted by the consumers. So, it is the main trend to research about searching a better antibacterial substance with broad-spectrum antibacterial activity, highly effective, stable, security, high production rate and low cost as a natural food antiseptic in the world.The followed are the reasons that using the antibacterial material produced by the microorganism as the natural antiseptic, it can be obtained widely and produced by fermentation, so it get more and more researchers'interest.Chigee is a famous traditional food in the Inner Mongolia Autonomic Region, and it will be a safty and novel way to screen lactic acid bacteria producted antibacterial substance from it.In this paper, lactic acid bacteria screened from chigee was selected as the object of study because the strains had a broad-spectrum antibacterial activity and their identification was followed.The purpose of this study were, (1) to isolate, identify lactic acid bacteria with high antibacterial activity, broad spectrum antibacterial substances from chigee. (2) to identify the antimicrobial substances structure with HPLC, ESI-MS/CID and MALDI-TOF/TOF-MS. (3) to study the physical and chemical properties of antibacterial substances (4) to investigate the Bacteriostasis mechanism to Escherichia coli. The result were described as foolowed:1.82 trains of lactic acid bacteria were isolated from six samples of fermented milk, collected from the nomadic families in the Inner Mongolia Autonomic Region, China. The lactic acid bacteria were identified as:14 strains of Lactobacillus acidophilus,11 strains of Lactobacillus casei,7 stains of Lactobacillus planatum,2 strains of Lactobacillus curvatus and 4 strains of Lactobacillus jensenii in 38 strains of Lactobacillus; 10 strains of Enterococcus faecalis,22 strains of Lactococcus lactic subsp. lactis,3 strains of Lactococcus mesenteriodes subsp. dextranium and 9 strains of Streptococcus thermophilus in 44 stains of Lactococcus, according to Lactobacillus acidophilus ATCC 4356, Lactobacilus delbrueckii subsp. Bulgaricus JCM 1002, Lactococcus lactis subsp. lactis IFO 12007 and Enterococcus faecalis IFO 3427 as the reference strains.2.There are two main kinds of strains in the chigee of Xilinhot in Inner Mongolia Autonomic Region which are Lactobacillus and Lactococcus. Lactobacillus acidophilus and Lactobacillus casei are two main kinds of Lactobacillus, which accounted for 17.1% and 13.4% respectively. Lactococcus lactic subsp. lactis, Enterococcus faecalis and Streptococcus thermophilus are the main kinds of Lactococcus. which accounted for 26.8%, 12.2% and 11.0% respectively. The proportion of Lactobacillus and Lactococcus in chigee is similar, which may be ralated to the sampling region and timing, the separation conditions (temperature, acidity), which can be a direct impect on the isolating rate of and the kinds of lactic acid bacteria.3.31 of 82 strains of lactic acid bacteria showed antibacterial activity, which acounted for 37.8%; 27 strains of them show antibacterial activity to Escherichia coli and Bacillus cereus, which accounted for 32.9%. There were 15 strains of Lactobacillus and 12 strains of Lactococcus, which accounted for 39.5% among Lactobacillus and 27.3% among Lactococcus separately. Lactobacillus acidophilus NX2-6 showed strong antibacterial activity to Escherichia coli, Bacillus cereus and Aspergillus oryzae after duplicate sieves, and be selected as the strain for reseach to study the antibacteria substance. Through physiological-biochemical and 16S rDNA sequence identification, it was identified as Lactobacillus acidophilus. The 16S rDNA is a 1480 bp DNA fragment, which was accessed in GenBank, and the number is EU878007.4. The antimicrobial substances produced by Lactobacillus acidophilus NX2-6 was stable with treatment of heat. It was stable at pH 2.0-5.0, which activity reduce gradually following the pH increasing and would lose activity at pH 6.0. It was sensitive to trypsin, pepsin and proteinase K but stable to a-chymotrypsin. Although the inhibition zone diameter significantly reduces when processed by trypsin, pepsin and proteinase K, it still has a strong antimicrobial effect, and antimicrobial substances has good stability, on the ultraviolet radiation. Therefore, the bacteriocins was a proteins or non-single antimicrobial substances. The result indicated that it had a high antibacterial activity to gram-positive, gram-negative bacteria and fungus which means it was a broad-spectrum antibacterial substance.5.The molecular weight of antimicrobial substances producted by Lactobacillus acidophilus NX2-6 is less than 50 kDa with largest UV absorption peak at 219.00 nm. Two component with antibacterial activity were separated and purified by HPLC, one of which molecular weight is 118.05 analyzed by ESI-MS/CID, and be determined as succinic acid. The other component was identified as a peptide with molecular weight of 825.149 kDa by MALDI-TOF/TOF-MS, which amino acid composition and structure was Asn-Thr-Arg-Tyr-Lys-Ser-Gly, suggested that it was a new type of antimicrobial peptide, and was named acidocin NX2-6.6.The submerged fermentation conditions for antimicrobial peptides produced by strain NX2-6 were optimized. On the base of single factor experment, the methodology of Plackett-Burman design was used to screen the key factors rapidly from the related 14 factors, such as ten compositions of the medium, the culture temperature, the culture time, the initial inoculum and the initial pH etc. The resluts showed that glucose, sodium acetate and culture time had a positive effect to producing bacteriocins but triammonium citrate dihydrate had a negative effect, and other tested factors had no significant effect.7.Some crucial factors, such as glucose, sodium acetate anhydrous, triammonium citrate dihydrate and incubation time affecting the yield of bacteriocins of Lactobacillus acidophilus NX2-6 in MRS medium, were further investigated and optimized by response surface methodology (RSM). The optimum concentration of culture medium were determined by second-order polynomial equation. Solving the model equations, we can get the largest diameter of inhibition zone with 21.37 mm forecasted when the factors are glucose 60.0 g/L, sodium acetate anhydrous 8.0 g/L, triammonium citrate dihydrate 5.0 g/L and incubation time 36 h. The predictive value has already been confirmed by the results of described in chapter 5 with the correlation coefficient R2 being 0.9918.8.The optimal fermentation conditions produced by Lactobacillus acidophilus NX2-6 is peptone 20.0 g/L, beef extract 20.0 g/L, yeast extract 10.0 g/L, glucose 60.0 g/L, Tween-80 3.0 g/L, K2HPO4·3H2O 4.0 g/L, sodium acetate 8.0 g/L, MgSO4·7H2O 400.0 mg/L, MnSO4·H2O 76.0 mg/L, sodium citrate hydrate 6.0 g/L, initial pH 6.0, culture temperature 40℃, inoculum 2%(V/V) and incubation time 36 h was obtained. Under these conditions, the antibacterial activity increased 80.0% than that of original.9.Sensibility and inhibition effect of antibacterial substance produced by strain NX2-6 to Escherichia coli were determined. The result showed that the minimal inhibitory concentration was 895.8μg/ml. And it had a high antibacterial activity to the Escherichia coli both on the breeding and non-breeding season. The antibacterial substance destroied integrity of cell membrance, and led to increasing of membrane permeability, leakied out of macromolecule in the cell, then cells breaked.
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