Isolation And Identification Of A Bacillus With Antibacterial Activity And Optimization Of Fermentation Conditions

The proliferation of microorganisms is one of the main causes of food spoilage,and the food safety problem caused by the abuse of chemical preservatives is becoming more and more obvious,so it is urgent for people to find a broad spectrum,safe and effective natural preservatives to make up for the shortage of chemical preservatives.Studies have shown that bacillus has a wide distribution range and antibacterial activity,so the separation of antibacterial substances from food source bacillus is a safe and effective way to obtain natural biological preservative materials.In this paper,a strain of bacillus with antibacterial activity was isolated from fermented douche.The strain was identified as bacillusamyloliquefaciens and named JY-1 by morphological,physiological and biochemical identification and 16 S r RNA gene sequence analysis.In order to preliminarily understand the safety of strain JY-1,20 antibiotics were used to conduct drug sensitivity test on bacillusamyloliquefaciens JY-1.The results showed that JY-1 was sensitive to all the 20 antibiotics tested,indicating that strain JY-1 had no drug resistance.The relationship between the growth curve of strain JY-1 and the generation of antibacterial substances was studied.It was found that the synthesis of antibacterial substances started when the growth of bacteria entered the logarithmic growth stage,maintained high antibacterial activity after entering the stable period,and almost did not experience the decay period.With listeria monocytogenes as the indicator bacteria,the stability of antibacterial substances produced by JY-1 was evaluated by the oxford cup agar diffusion method from four aspects,and the bacteriostatic spectrum of this antibacterial substance was tested: temperature,p H,protease and ultraviolet light.In p H2-10,it has good bacteriostatic effect.Protease K and neutral protease have little effect on the antagonism of bacterial substance,and trypsin and pepsin are partially sensitive.Ultraviolet irradiation has no effect on the stability of antibacterial substances.The antibacterial substances produced by strain JY-1 had a broad spectrum antibacterial effect,and had a strong inhibitory effect on most of the gram-positive bacteria and gram-negative bacteria,and also had a significant inhibitory effect on some molds.Fermentation conditions of bacillus amyloliquefaciens JY-1 were optimized by One-factor-at-a-time experiment,Plackett-burman test design and box-behnken response surface method.The results were as follows: soluble starch 29 g/L,beef paste 5 g/L,yeast 8 g/L,Mg SO4·7H2O 1g/L,inoculation quantity 0.9%,culture time49.5h,liquid loading quantity 30 m L/250 m L,culture temperature 33?,initial p H6.0,rotation speed 210r/min.Under this condition,the antibacterial effect was increased by48.6% compared with that before optimization.The above experimental results show that bacillus amyloliquefaciens JY-1 has a relatively stable broad-spectrum antibacterial effect,after optimized fermentation conditions,it had better bacteriostatic ability.This experiment has laid a theoretical foundation for the development of natural preservatives and also provided technical support for industrial production.