Purification And Analysis Of Active Substances Produced By Lactobacillus Plantarum HS-R9

Currently,China is still a country that uses chemical preservatives on a large scale.As relevant research reveals more and more potential hazards from using chemical preservatives,we have started to look for a safe and natural preservative to replace chemical preservatives.Lactobacillus bacteriocins are a class of small molecule polypeptides synthesized by lactic acid bacteria ribosomes and exhibits bacteriostatic activities.Presently,it is known that lactic acid bacteria can produce a variety of bacteriocins.Because of its natural,safe and non-toxic characteristics,lactic acid bacteria bacteriocin has been widely used in food and other product preservation in many countries in the world.Nisin,produced by Streptococcus faecalis,is a safe natural food preservative that has been recognized worldwide,but Nisin only inhibits Gram-positive bacteria.Therefore,developmental studies on other types of bacteriocins has become very important,especially to identify a natural,efficient and safe bacteriocin with broad-spectrum resistance.The purpose of this experiment is to find a lactic acid bacteria bacteriocin that can produce a broad spectrum of antibacterial activity,isolate and identify the production strain,and conduct a series of studies on the bacteriocin produced by it.The main research contents are as follows:A lactic acid bacteria with bacteriostatic activity was isolated from the intestinal tract of sea cucumber,based on the sequencing analysis of the 16S rDNA gene,the strain was initially identified as Lactobacillus plantarum,and was named Lactobacillus plantarum HS-R9.The bacteriocin produced by this strain was named Plantaricin HS-R9.PCR technology was used to design primers based on the partial structural gene of Plantaricin,and the genome of the strain was amplified.After sequencing the amplified sequence,the results of NCBI alignment analysis confirmed that the strain HS-R9 genome contained Plantaricin’s structural gene plnEF.The cells free supernatant of the strain HS-R9 exhibits a broad-spectrum antibacterial activity.Following studies on its fermentation process,its logarithmic growth phase was identified to occur within 0¹6 hours.Its stable period is reached after 16⁴8 hours.After 48hours,it enters its decline phase.Acids are produced continuously during the process,reducing the pH gradually.The acidity trends to become stable in the late stage of the stable phase,and the acid yield is basically unchanged.In the late logarithmic growth phase,the antibacterial activity gradually increased,peaking during 40⁴8 hours.After entering the decay period,the total amount of antibacterial active substances remained basically unchanged.The antibacterial active substances in the fermentation supernatant of the strain HS-R9were retrieved by extraction method,ammonium sulfate method,and ultrafiltration method.The results showed that the extraction method showed the optimal results.It was found by Tricine-SDS-PAGE electrophoresis that the fermentation extract had a molecular weight of about 3.3 kDa.According to the analysis of its biological characteristics,the active substances produced by the HS-R9 strain can maintain high activity after treatment under different temperature and pH conditions.The crude extract was extracted from the fermentation supernatant of the HS-R9 strain with organic solvent ethyl acetate,and purified by Sephadex LH-20 and Sephadex G-50chromatography.The eluted fraction was collected.The light absorbance of each component was measured under OD₂₈₀,and their elution curves were established.Both curves have two absorption peaks.Oxford Cup Method was used to detect the antibacterial activity of the absorption peaks,and a single antibacterial activity peak was obtained from both activity curves.The above active components were collected and concentrated for HPLC analysis to obtain a single bacteriostatic activity peak.The strain HS-R9 produced plantaricin and Nisin solutions of the same concentration were prepared and the antibacterial activity was verified by the filter paper method.It was found that the antibacterial activity of the HS-R9 strain produced plantaricin was significantly higher than that of Nisin.Moreover,Nisin exhibits a significantly lower antibacterial activities against Gram-negative bacteria than against Gram-positive bacteria,and it has no inhibitory effects on Escherichia coli.Listeria monocytogenes is a pollutant in certain foods that can cause severe food poisoning.The presence of L.monocytogenes in food is dangerous to humans.This type of bacteria can grow and reproduce in a 4℃environment,and it is one of the main pathogens that cause frozen foods to threaten human health.Vibrio parahaemolyticus is a kind of Gram-negative bacteria and has become the leading food-borne pathogen in China.The proportion of food poisoning caused by poisoning from this kind of bacteria has reached more than 60%.The inhibitory effect of strain HS-R9 produced plantaricin on L.monocytogenes and V.parahaemolyticus was significantly higher than that of Nisin,supporting the potential application of Plantaricin HS-R9 in the food industry.