Preliminary Exploration On Metabolites Of Propionibacterium Shermanii Fermentation, Purification And Identification

Propionibacterium shermanii is a common fermentation strains in producing Swiss cheese,which has an important influence on its texture. We can obtain its crude extract by centrifugal,pH adjustment and spray drying its supernatant,which is called Propionibacteria Metabolites(PM). PM can be used as a food biological preservatives to prolong its shelf life,not only it can inhibit gram-gegative bacteria(G-) growth in food products,but also can play a role of inhibition to some fungi, actinomycetes and a handful of gram positive bacteria(G+). Compared to potassium sorbate and sodium benzoate et.al traditional chemical preservatives,It has broad application prospects in the industry of food preservation because it has so many advantages such as natural sources, trace the advantages of efficient and non-toxic side effects.But present technical conditions are faced up with many problems such as a long fermentation cycle, low unit of antimicrobial activity and a too deep colour with PM. Excessive content seriously affect the taste and flavor of food; Color too deep in acid milk beverage products will influence the color and finish of the products, which will become unacceptable to consumers. These technical problems limited its widely use in food industry. Antibacterial peptides can be used for the preparation of the burn wound infection drugs,which exists in PM. But unfortunately PM also contains macromolecular protein, propionic acid, acetic acid, and inorganic salts and other impurities besides antibacterial peptides. So the separation and purification of antimicrobial peptides become particularly urgent and important because only high purity of propionic acid bacillus antibacterial peptides can provide in the use of anti-infective drugs.In order to solve the related problems above, the research of this paper mainly include the following:By feeding glucose and sodium lactate so as to improve the unit activity of PM;Through four kinds of yeast extract and eight kinds of hydrolyzed vegetable protein (HVP) fermentation experiments to find out the best alternative nitrogen source; Tried the decolorization of propionibacteria supernatant by DOWEXTMPTIPORETMSD-2 decoloring resin; The protease sensitivity, thermal stability, antibacterial spectrum and the impact on spore germination of Alicyclobacillus acidoterrestris of PM were also studied on the basis of getting the PM by fermentation. The purification of antimicrobial peptides were preliminary explored in this paper, and the physicochemical properties were identified at the same time by TLC technique, Fourier transform infrared spectroscopy(FTIS),MALDI-TOF technology and the analysis of the amino acid composition.The feeding carbon source experiments indicated that:reducing sugar and sodium lactate of initial fermented medium were consumed within 72 hour and 24 hour respectively,while the metabolites were in an important stage of synthesis and accumulation.They can all improve the antimicrobial activity of fermentation products in different level by feeding glucose and sodium lactate, a single feeding glucose effect is better than that of single feeding sodium lactate. The best feeding batch model was feeding sodium lactate once at the 24hours and again at the 48hours,then give a glucose feeding at the 72 hours amd 96 hours respectively, meanwhile, the bacteriostatic activity of PM can get a 30.02% increase than that of control group.Four kinds of light colour yeast powder and eight kinds of HVP fermentation experiments of alternative source of nitrogen showed that:FM802 yeast powder and HVP-FSC hydrolyzed vegetable protein were the best alternatives of nitrogen source; The bacteriostatic activity of control and experimental group were respectively 22.91Au/mL and 23.27Au/mL when FM802 was used as the alternative, it can rech a fairly antimicrobial activity compared to the control,while increase is not obvious,and the price of FM802 was only about half of the control,so it can be used as an alternative of nitrogen source. Bacteriostatic activity of the experimental group and the control group were 31.1 Au/mL and 25.38 Au/mL when HVP-FSC was used to fermentation, bacteriostatic activity increased 22.54%,and only a quarter of the price of the control.The nature of crude extract from propionibacterium supernatant research results show that:PM was sensitive to pepsin, but not to trypsin and papain, which was preparation by CXY-2 strain. The temperature sensitivity experiments indicated that it has good thermal stability; Bacteriostatic spectrum experiment showed that PM had inhibition to organisms, not only for Pseudomonas putida et.al 5 kinds of G’also had a strong inhibitory effect on mould, yeasts and actinomyces.It had no inhibitory effect on Bacillus subtilis et.al 6 kinds of G+,but it played a role of inhibition to spores germination of Alicyclobacillus acidoterrestris.It was indicated that the unit antimicrobial activity of propionicin which was purified by Sephadex G-10 and Superdex peptide improved 6.8 fold,the rate of activity was 10.9%.This PM contains a very small molecular weight of 699Da bacteriostatic substance by the preliminary purification experiment. TLC analysis showed that the sample was a kind of strong polarity, containing free alpha amino or alpha carboxyl polypeptide materials. Fourier transform infrared spectral analysis indicated that the sample contained unsaturated bond such as peptide bond. The analysis results of amino acid composition showed that the propionicin mainly consisted of seven kinds of amino acids, which were glutamic, glycine, leucine, valine, threonine, proline and isoleucine respectively, its content was 82.4% of all the amino acids. Comprehensive the above experimental results, this propionicin was likely to be a strong polarity, a very small molecular antimicrobial peptide with bacteriostatic action.