Isolation And Identification Of Lab Strains With A Broad-spectrum Antibacterial Activity And Extraction Purification And Identification Of The Bacteriocin

Bacteriocin,isolated from Lactic acid bacteria(LAB),is a type of ribosomally synthesized small molecules peptides with antimicrobial activity.Since the lactic acid bacteria is known as GRAS(generally recognized as safe,GRAS)strain,the products of lactic acid bacteria are called GRAS substances,bacteriocins of lactic acid bacteria are considered with natural,non-toxic and green,and have been widely used in food preservation areas in many countries.Microbiological contamination of food is the main cause of corruption,at the same time,food-borne pathogens is one of the main causes of food safety incidents.Therefore,pollution prevention and control of microorganisms in food is a food safety research in the field direction.In present,the most widely used food additives are chemical preservatives,people have come to realize that chemical preservatives in foods may lead to harm to human health,so natural,efficient and safe bacteriocins display broad potential applications in food preservation.The aim of this research was to isolate the LAB strain from Suan-Tsai(traditional fermented vegetables from Guizhou,Jilin,Liaoning,Heilongjiang and Hunan province,China)which produces bacteriocins with a broad-spectrum antimicrobial activity.And the bacteria was identified based on morphological,physiological and biochemical characteristics and 16S rRNA sequence analysis.We obtained purified antibacterial substances by the organic solvent extraction,gel filtration chromatography,HPLC and so on.The structure of the bacteriocin was identified by MALDI-TOF-MS analyses and the high resolution mass spectrum,in addition,the characterizations of the bacteriocin was explored.The main results are as follows:1.Isolation and identification of LAB producing bacteriocin with a broad inhibitory spectrum from fermented vegetables.A total of 2750 Gram-positive acid-producing strains were isolated from fermented vegetables from Guizhou,Jilin,Liaoning,Heilongjiang and Hunan provinces of China.We obtained 212 strains of LAB which have a strong antibacterial activity against Escherichia coli and Micrococcus aureus by primary screening.After secondary screening,6 strains displayed stronger antibacterial activity against Escherichia coli,Pseudomonas fluorescens,Bacillus cereus,Micrococcus aureus and Fusarium,which were numbered A65,A488,C174,C221,D474,D599.Among them,strain A65 exhibited the highest antibacterial activity,which was identified as Lactobacillus plantarum by morphological,physiological and biochemical characteristics and 16S rRNA sequence analysis,and named Lactobacillus plantarum JL-A65.2.Isolation and Purification of bacteriocins produced by Lactobacillus plantarum JL-A65.Lactobacillus plantarum JL-A65 grow slower than other bacterias during fermentation,the growth curve of this strain showed "S" shaped curve,the acids were constant produced by L.plantarum JL-A65 and pH start to drop at the end of lag phase,and then remain stable during the stationary phase,the pH value decreased from 6.2 to 3.6.L.plantarum JL-A65 started to produce bacteriocin at the mid-term of logarithmic phase,the production accumulated faster at the stationary phase,and the production reached to its maximum at the late decline phase with a litter drop at the end.The bacteriocins was extracted by 8 commom organic solvent,as results,higher extraction efficiency was obtained by butanol and ethyl acetate,but no bacteriocin was extracted by other organic solvent.Furthermore,different volumes ratio(0.5,1,1.5,2,2.5,3 times)of butanol or ethyl acetate to the fermentation broth were applied to extract bacteriocin,the result showed that the best volume ratio of butanol to the fermentation broth was 1.5 times.So butanol as the extractant was chosen.After extraction,the bacteriocins were further purified by Sephadex G-25 and Sephadex LH-20 gel chromatography to get a single peak,then a peak appeared in 11.2 min was separated by semi-preparative HPLC,at last,the bacteriocins in a single peak appeared in 8.15 min was obtained by RP-HPLC.3.Structure identification and characterizations of bacteriocins produced by Lactobacillus plantarum JL-A65.Bacteriocinwas purified using butanol extraction,gel filtration,and high-performance liquid chromatography.The molecular mass of bacteriocin was obtainded as 1036.69 Da by MALDI-TOF-MS analyses and the high resolution mass spectrum(HR-ESI-MS).The primary structure of the bacteriocins is VVGNLLLVPL.The bacteriocin was identified as a new bacteriocin by BLAST comparison and named plantaricin JL-A65.This bacteriocin exhibited broad-spectrum anti-bacteria activity,highly thermal stability and pH stability and it was sentive to common protease,such as pepsin,trypsin,proteinase K and a-chymotrypsin.