Study On Bacteriocin Produced By Pediococcus Pentosaceus C-2-1

With the continuous development of economy, more and more attention has been paid to the problem of food safety, the abuse of chemical preservatives, and the accumulation of toxin in human body make people worried. Bacteriocins were widely researched because of the characteristics of easy by protease degradation, no residue, non-toxic side effects and no drug resistance, at present, nisin which is widely used to food industry has been recognized as a GRAS natural food preservative. But with the constant study found that nisin has some shortcomings in application, the antibacterial spectrum is narrow only to near the source of lactic acid bacteria, staphylococcus aureus and other gram positive bacteria have inhibitory effect and only under acidic conditions has good antibacterial activity, thermal stability and dissolved characteristics,this insufficient greatly limits its application in food preservation. Relative to the shortage of nisin, pediocin, produced by pediococcus, has a widely antibacterial spectrum, especially for Listeria monocytogenes has a strong inhibitory effect and has good thermal stability, while most stable at pH = 2 to 10 range, in the field of food preservation have wider application prospect.This article screening a strain with strong inhibitory effect on L.monocytogenes from the plurality of cheese samples collected from Mongolia home and identified the strain. At the same time,in terms of the bacteriocins,produced by the strain, conduct in-depth research,such as biological properties, strain fermentation conditions and the gene cloning, the specific studies are as follows:1、Screening and identification of Pediococcus pentosaceus C-2-1.Lactic acid bacteria strain C-2-1, isolated from Mongolia cheese, can produced antimicrobial substance was screened by punch hole method. After eliminating the effect of organic acids and hydrogen peroxide, the antimicrobial substance was found to be sensitive to proteolytic enzyme, which was proved to be bacteriocin.This strain C-2-1 was identified as Pediococcus pentosaceus by using 16 S rDNA gene sequence homology analysis and morphological analysis, named as Pediococcus pentosaceus C-2-1, the bacteriocin produced by this strain named as pediocin C-2-1.2、Study on the biological properties of Pediococcus pentosaceus C-2-1.The highest bacteriocin production（5120 AU/ml）was noted at stationary phase after 22²6 h of growth in MRS broth at 30°C against L. monocytogenes ATCC 19114. Bacteriocin C-2-1 could significantly inhibit against a part of the gram positive bacteria, but was inactive against gram negative bacteria and Yeast. The mode of activity against L. monocytogenes was bactericidal. No change in activity was recorded after treatment at temperature range 60°C to 90 °C, and after treatment at pH values between 2.0 and 9.0. Besides, the bacteriocin was resistant to treatments with SDS, Tween-20, Tween-80, urea, NaCl and did not adhere to the surface of the producer cells and the molecular weight was between 5.8 k Da and 7.8 kDa, as determined by Tricine-SDS-PAGE.3、Optimizing of the fermentation conditions of Pediococcus pentosaceus C-2-1. Firstly using single factor method to determine the optimal conditions were two times after the activation of the seed solution with 2% inoculation amount 30 degrees in the static culture and the initial pH of the medium was 6.5. Then by the response surface method to optimize the fermentation medium for producing bacteriocin, by PB design to find the significant factors which can influence the bacteriocin produced,there are manganese sulfate, anhydrous sodium acetate and yeast powder, and then through the steepest ascent experiment to approach the optimal region, besides using CCD design to find the best fementation culture medium formula of pediococcus pentosaceus C-2-1, consisting of lactose 15 g/L, fish meal peptone 3.75 g/L, ammonium citrate 1.5 g/L, magnesium sulfate, 0.435 g/L, tween-80 1.125 g/L, manganese sulfate, 0.24 g/L, anhydrous sodium acetate 3.38 g/L, yeast powder 12.17 g/L, under this condition,the predict antibacterial activity of pediocin C-2-1 was 10566.4 AU/mL, the verification results was 10752 AU/mL close to the predict results, the antibacterial activity increased 2.1 times than before optimization。4、Heterologous expression of the ped A gene of the pediocin C-2-1 in Escherichia coli.To design primers（Pedio Pedio R, F） and obtained ped A gene by means of PCR method, construct the cloning vector pEASY- ped A and verify the sequence was right, after then using the Escherichia coli expression vector pET-28 a and transformed into the expression host strain BL21（DE3）, under the condition of 30 ℃, 1 mmol/L IPTG induced for 4h, realized the soluble expression of recombinant proteins. Using Ni²⁺ affinity chromatography column, a single component of the recombinant protein was collected, and the purified recombinant protein has significant antibacterial activity on L.monocytogenes measured by punching method.Pediococcus pentosaceus usually separated from vegetables and meat, and poor growth in dairy products,there are very few related reports; At the same time due to the L.monocytogenes can growth in dairy products in low temperature and low pH, caused frequently dairy products safety problems occur. Therefore this paper screening pediococcus pentosaceus C-2-1 from Mongolia cheese and make an in-depth study on its product of pediocin C-2-1 to provide a theoretical basis on industrial production and application of dairy products.