| Lactic acid bacteria(LAB) are gram positive bacteria that can ferment carbohydrate into lactic acid,they have different shapes, metabolism and physiological features. They are industrial fermentation microorganisms which are used widely in food and feed fermentation, biopharmaceuticals and other areas. Lactic acid bacteria were used in food and feed fermentation long ago, however, people didn’t begin to understand how the metabolic processes of lactic acid bacteria effect the food storage and worked as biopreservative since last century. lactic acid bacteria have their special advantages as probiotics, they are traditional fermentation stains and GRAS(Generally Recognized as Safe,GRAS) strains, they produce many flavor substances which can improve food flavor, in addition, bacteriocin and bacteriocinlike can prevent and cure some communicable diseases. In addition, lactic acid bacteria are normal bacteria of intestinal tract of human and animals, they can adjust the intestinal flora balance and enhance the body immunity. Another special property of Lactic acid bacteria is that they can wipe off toxins like aflatoxin from liquid culture medium by physical adsorption.We can learn from the facts above that lactic acid bacteria and its metabolic product have a huge potential in the area of biopreservative.in this article, three strains with broad antibacterial spectrum and great antibacterial ability have been screened and identified. We studied the basic properties of the strains and their fermentation broth and tested their adsorptive capacity towards aflatoxin by means of ELISA. Application potential of lactic acid bacteria fermentation broth in the area of grape common disease control and the extension of the bread preservation period. Peptides of fermentation broth of a lactic acid bacteria strain have been separated and tested. The specific content and results are as follows:1.Lactic acid bacteria screening and study of basic property of stains and their fermentation broth. In the process of lactic acid bacteria fermentation, some metabolic products which have the ability to inhibit the growth of other microorganisms so they can work as biopreservative, therefore, screening of stains which produce metabolic product with high antimicrobial activity is the primary task. In recent years there are many researchs on biopreservative but researchs on the inhibition of lactic acid bacteria towards fungal is relatively rare, inhibition of lactic acid bacteria towards fungal is studied in this article. Aspergillus niger was used as indicator in screening process,we got three lactic acid bacteria strains(2025b,2026b1,6-1-1) with broad antifungal spectrum and antifungal activity and their16s rDNA sequencing is performed, Blast comparison found that2025b and6-1-1are Lactobacillus fermentium strains,2026b1is a Lactobacillus plantarum strain. Basic properties of them was studied, growth curve in48hours and pH curve in36hours of the stains are drawned, we can learn from the growth curve that cell form of these lactic acid bacteria is best at their15hours growth in early stationary phase, cells in early stationary phase are used in aflatoxin adsorption experiment. There are most antifungal components in the fermentation broth in their36hours growth in late stationary phase, fermentation broth in this period is used in antifungal activity test, food preservation test and the separation of antifungal components. Antifungal spectrum towards6common fungal was tested. We found that the antifungal activity of the cell free fermentation broth can’t be effected by100℃water bath for5mins. The antifungal activity of fermentation broth of the strain6-1-1was found sensitive to protease and became lower in storage process.2. The inhibition of Lactic acid bacteria towards Aspergillus flavus and aflatoxin physical adsorptionAflatoxin is a series of powerful carcinogen with mutagenicity, carcinogenicity, hepatotoxicity and immunorepressive, its toxicity is10times greater than KCN, it is one of the strongest known carcinogens and polluted widely in food. Aflatoxin is the secondary metabolites of some fungi (Aspergillus flavus, Aspergillus parasiticus), aflatoxin B1, B2, G1, G2are the most commonly polluted aflatoxin in food and feed. Aflatoxin M1, M2is usually found in milk, aflatoxin B1is the most toxic compound among them and polluted most widely in food. Some lactic acid bacteria inhibit Aspergillus flavus growth and some lactic acid bacteria cells can remove aflatoxin by physical adsorption. This article tested the the antifungal ability of13strains of lactic acid bacteria towards Aspergillus flavus. Enzyme-linked immunosorbent assay tested the adsorption ability of3lactic acid bacteria towards aflatoxin B1. The result shows that lactic acid bacteria D52,D62,6-2-3have strong inhibitory effect on Aspergillus flavus.6-1-1,2025b,2026b1have only weak inhibitory effect on Aspergillus flavus. The experiment strains have strong adsorption effect compare with control bacteria Bacillus NattO,E.coli DH5a and Lactococcus lactis MG1614, lactic acid bacteria6-1-1adsorpt most aflatoxin B1among all these stains,60.69%of aflatoxin B1in the solution was adsorpted in experimental conditions by lactic acid bacteria6-1-1.3. Application potential research of lactic acid bacteria fermentation broth in the area of shelf-time prolongate of bread and grape common disease controlIn recent years, lactic acid bacteria were used as biopreservative in many areas such as bread baking, fruit and vegetable preservation, meat preservation and ensilage. In this article, shelf-time prolongate potential of cell free fermentation broth of3lactic acid bacteria was tested, it is found that all of the cell free fermentation broth can prolong shelf-time of bread, lactic acid bacteria2026b1is the best one among them.Grape downy mildew control experiment shows that fermentation broth of all concentrations have downy mildew control efficiency, disease leaf number and the degree of disease is realized effectively. When the the ratio of the fermentation broth and water is1:1, the fermentation broth performs better than common pesticides. Three Pathogenic molds were separated from affected grape, inhabitation activity of lactic acid bacteria towards pathogenic molds are tested,2strains of molds are sensitive towards the fermentation broth, another mold have a certain sensitivity to the inhibition of lactic acid bacteria.Merlot grape anthracnose control experiment shows that different concentrations of viable fermentation broth has a control effect towards the merlot grape anthracnose compare with control group. When the fermentation broth and water is sprayed at the ratio of1:1, the ratio of affected grape was2.91%which is much lower than the normal drug control group whose ratio of affected grape was3.82%, the amount of affected grape is only76.17%of the normal treatment group.4. Separation and purification of antifungal peptide substanceMetabolites produced by the lactic acid bacteria including many substances with mold inhibition ability, for instance organic acids, protein complexes,3-hydroxy propionaldehyde, fatty acids, benzene, lactic acid, cyclic peptides and other small molecular substances, etc. Among them, proteins and peptides with the mold inhibition ability are rarely reported. C18HPLC Columns are used in this article, analysis and preparation operations were done in order to separate peptide and protein substances with antifungal activity from lactic acid bacteria2025b,6-1-1fermentation broth and antifungal bioassay was done, several antifungal substances were found. However, electrophoretic bands can’t be found in polyacrylamide gel electrophoresis and the tris-tricine SDS-PAGE maybe due to the low sample concentration. |
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