Screening Of A Lactobacillus Rhamnosus Strain For Producing Antimicrobial Peptides And Purification Of Antimicrobial Peptides

With the improvement of consumers'living standards,the enhancement of people safety awareness,and the increase of reports which chemical preservatives are damaging human,manufacturers urgently need to develop a safety preservative instead of chemical preservatives.Lactic acid bacteria are recognized as safe strains.Antibacterial peptides produced by them are safe,non-toxic,and can effectively inhibit a variety of food-borne spoilage bacteria,so it has become a research hotspot at home and abroad.However,only few antibacterial peptides such as Nisin had been approved as food-grade antimicrobial peptides and achieved industrial production.But their own shortcomings are obvious,such as only inhibiting Gram-positive bacteria.Therefore,there is an urgent need to develop new antimicrobial peptides for food preservation.In this paper,a strain of L.rhamnosus SCB0119which has a good inhibitory effect on both Gram-positive bacteria and Gram-negative bacteria was screened from the laboratory strain library.We clarified the biological characteristics of the strain,optimized the fermentation conditions for producing antimicrobial peptides,and purified the antimicrobial peptide by various methods.Finally,the antibacterial spectrum and stability of the antibacterial peptides were studied.The main experimental results are as follows:(1)142 kinds of lactic acid bacteria were screened from the research group by agar diffusion method.It was found that 13 strains of the lactic acid bacteria had significant inhibitory on Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922.Among them,SCB0119 had the strongest activity with the inhibition zone diameter 12.02±0.18 mm.We confirmed that SCB 0119 was L.rhamnosus by the morphological observation,physiological and biochemical,16S r DNA sequencing and the blast alignment on the NCBI website.(2)When cultured in MRS liquid medium at 37°C,SCB0119 entered stable phase at 24 h and the p H of fermentation liquid also decreased to the minimum of 3.74.The optimal growth temperature and p H range of SCB0119 was 37?42°C and 5.0?7.0.The survival numbers of SCB0119 were(5.04±0.48)×10~6CFU/m L and(1.39±0.11)×10~6CFU/m L respectively(control number(4.2±0.24)×10~8CFU/m L)after treated for 4 h in MRS broth whose p H was3.0 or bovine bile salt concentration was 0.1%.(3)The medium composition and culture conditions of L.rhamnosus SCB0119 for producing antimicrobial peptide were optimized by single factor and response surface methodology.The results showed that the optimal basal medium was 14%skim milk medium containing1.8%soybean peptone(m/v),the fermentation temperature was 37°C,and the fermentation time was 60 h.Under the optimal conditions,the predicted inhibition zone diameter of the model was 17.68 mm.The verification test showed that the inhibition zone diameter under this condition was 16.22±0.26 mm,which was close to the predicted value of 17.68 mm and the inhibition zone diameter was 35%larger than before.(4)After ultrafiltration,reversed-phase chromatography and high-precision preparative liquid phase separation,a high-purity antibacterial peptide which had an inhibition zone diameter of 9.45 mm for S.aureus ATCC25923(paper diameter is 6.00 mm)was collected.It showed a single peak at 7.8 min whose purity was over 95%in HPLC.Five possible amino acid sequences of the antimicrobial peptide were identified,but the specific sequence needs further verification.(5)Antibacterial property test and antibacterial spectrum experiment showed that the antibacterial peptide of L.rhamnosus had a good inhibitory effect on Bacillus subtilis,B.cereus,and multiple foodborne pathogenic pathogens such as S.aureus,Vibrio parahaemolyticus,E.coli and Salmonella,but had no inhibition effect on Saccharomyces cerevisiae,Aspergillus Niger.The antibacterial peptide performance good thermal stability after treated at 121°C for 20 min and with different proteases,but it is sensitive to p H.