Study On Fermentation Preparation Of Antimicrobial Substance By Lactobacillus Reuteri And Its Characteristics

Lactobacillus reuteri was considered to be a safe microbial strain and has been approved for use in health foods,foods,and infant foods.It was a lactic acid bacterium that could produce a variety of antimicrobial substances.In this study,Lactobacillus reuteri LYS-1 was used as the research object,and the antimicrobial spectrum and tolerance to p H,heat treatment and enzyme treatment of LYS-1 fermentation liquid were studied.Based on the influence of carbon and nitrogen sources on the antimicrobial effect of LYS-1 fermentation liquid,the Lactobacillus reuteri medium formulation was optimized.What's more,the main antimicrobial components in the LYS-1 fermentation liquid was explored,and the peptides were isolated and extracted.The study could provide theoretical reference and basis for preparation of antimicrobial substances by Lactobacillus reuteri.The main results were as follows:(1)It was found that the inhibitory effect on the indicator bacteria of Lactobacillus reuteri LYS-1 fermentation liquid was:Escherichia coli>Pseudomonas aeruginosa>Staphylococcus aureus>Bacillus subtilis.LYS-1 fermentation liquid had the potential of broad-spectrum antimicrobial,and the effect of inhibiting gram-negative bacteria was stronger than that of gram-positive bacteria.(2)The antimicrobial effect of the fermentation liquid of Lactobacillus reuteri LYS-1was greatly affected by p H value.When p H=2.00?5.00,lowering the p H could significantly increase its antimicrobial effect,but would disappear when the p H?5.50.(3)Lactobacillus reuteri LYS-1 fermentation liquid(p H=3.80)had strong thermal stability and ability to resist protease.The antimicrobial activity could be maintained above99%after treated at 50�C,60�C,70�C,80�C,90�C,100�C and 121�C for 30 minutes.The influence of proteinase K,trypsin,pepsin,papain and bromelain on the antimicrobial effect of LYS-1 fermentation liquid were weak.(4)The carbon source had a great influence on the antimicrobial effect of the Lactobacillus reuteri LYS-1 fermentation liquid.When lactose was used as the carbon source,the number of viable bacteria and acidity of the fermentation liquid was low.When sucrose was used as the main carbon source,the production of acid was high and the fermentation liquid began to have antimicrobial effect when the number of viable bacteria reached 10~9CFU/m L.Then the maximum of the number of viable bacteria and antimicrobial effect was reached after 36 h of fermentation.The combination of glucose and sucrose or glucose and fructose significantly affected the antimicrobial activity of LYS-1 fermentation liquid,and the replacement of sucrose with glucose and fructose was beneficial to promote the antimicrobial effect of LYS-1 fermentation liquid.When the carbon source formula was 6.26%glucose+5.26%fructose(corresponding to 1.00%glucose+10.00%sucrose),LYS-1fermentation liquid had the strongest antimicrobial effect.(5)Nitrogen source was also a key factor affecting the antimicrobial effect of Lactobacillus reuteri LYS-1 fermentation liquid.LYS-1 fermentation liquid had antimicrobial activity only when yeast extract and soybean peptone was used as the nitrogen source,but the combination of them could not improve the antimicrobial effect.2.00%yeast extract as the nitrogen source could get better antimicrobial effect,and the appropriate addition of semicarbazide hydrochloride also could increase the antimicrobial effect,the effect was the best when added amount was 0.04%.(6)The carbon-to-nitrogen ratio of the Lactobacillus reuteri medium had a great influence on the antimicrobial effect of the Lactobacillus reuteri LYS-1 fermentation liquid.The number of viable bacteria and the diameter of inhibition zone of LYS-1 fermentation liquid had the best when the carbon-to-nitrogen ratio was 5:1,was 5.60?10~9CFU/m L and12.95 mm,respectively.At this time,the medium formula was 6.26%glucose+5.26%fructose as the carbon source and 2.30%yeast extract as the nitrogen source.(7)After peroxidase treatment,gas chromatography,strong acid hydrolysis,and HS-SPME-GC-MS measurement of volatile compounds,it was found that Lactobacillus reuteri LYS-1 fermentation liquid did not contain 3-hydroxypropanal.The main antimicrobial components were peroxide,volatile aldehydes,volatile carboxylic acids and small molecule peptides.The volatile aldehydes were mainly trans-2-nonanal,2-undecenal,trans-2-octenal,benzaldehyde and decanal,and the volatile carboxylic acids were mainly acetic acid,caprylic acid,lauric acid and hexanoic acid.Peptides of LYS-1 fermentation liquid were separated and purified by ammonium sulfate precipitation,dialysis,ultrafiltration and ion exchange chromatography.Finally,the molecular weight of the extract was measured,and found that it was a antimicrobial peptide of small molecule which mainly composed of dipeptide(242 Da,250 Da)and tripeptide(353 Da,368 Da).