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Study of Isolation and Identification on Antimicrobial Substances Produced by Lactobacillus Acidophilus NX2-6

Author: ZhouZuoZuo
Tutor: LuZhaoXin
School: Nanjing Agricultural College
Course: Of Food Science
Keywords: Lactobacillus acidophilus NX2-6 Antibacterial substances Purification Identification
CLC: TS201.3
Type: Master's thesis
Year: 2011
Downloads: 18
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Abstract

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 conditions of health, which suggests that the natural food without chemistry antiseptic and the organic food are easier to be accepted by the consumers. So, it is the main trend of research fields to search better antibacterial substances with broad-spectrum antibacterial activity, highly effective, stable, security, high production 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 obtain more and more researchers’ interest.In this paper, Lactobacillus acidophilus NX2-6, selected from chigee in the Inner Mongoliaas, was a strain with high production. The subject of study focused on the fermentation conditions of broad-spectrum antibacterial substances, pursued the pathes of their purification and identification and examed sound research on food preservations. The main results are followed:1. Discussion on cultivated conditions and fermentation dynamics of Lactobacillus acidophilus NX2-6 in producing antibacterial substance was carried out. The microscopic examination of strain before fermentation were:G+, small white colonies and both tidy ends in single or chain; The optimum growth conditions were inoculated with 18 hours’ seed age, contained 90% culture volume and fermented 48 hours; Besides, the reducing sugar concentration, dry cells weight and pH over periods from fermentation dynamics were studied, showing 18.02 g/L,6.01 g/L and 4.05 respective when came to maximum.2. A broad-spectrum antibacteria substance was separated and purified from strain NX2-6’s fermentation broth, along with organic extraction, silica gel chromatography and analytical high performance liquid chromatography(HPLC) been applied. The results were significant that UV-spectrum of antibacterial substance was 220 nm; the optimum silica gel chromatography conditions was investingated thoroughly:200～300 mesh number, V/V(chloroform/methanol)=80/20,0.6 mL/min flow rate in eluent; the retention time of purified target in HPLC was 19.669 min with 98% purity.3. Structure identification on the antibacteria substance from Lactobacillus acidophilus NX2-6 have been studied preliminarily. The retention time of purified target, isolated by preparative HPLC, was 19.832 min. LC-MS showed that the antibacteria substance molecular was 566, and MS/MS analysis demonstrated that the antibacterial substance was composed of characteristic fragments of m/z 549.00,523.30,456.85 and 284.06. The results above, compared with similar reviews, suggested that it was a new type of antimicrobial substance in Lactobacillus acidophilus strains.4. Study on preservations of milk and fruits associate with the antibacteria substance from Lactobacillus acidophilus NX2-6 were carried on. The results demonstrated respectively:when preserved at 4℃, the raw milk added antibacteria substance had antimicrobial effect on Mesophiles partly although it would spoiled over 3 days; While the pasteurized milk added antibacteria substance could keep in good preservations without spoilage at 4℃and 20℃; The antibacteria substance had also antimicrobial effect on Penicillium notatum and Rhizopus stolonife in fresh after-cut apples, which could reduced rotting rate, diameter of disease spot and extended shelf life.

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