Antibacterial Activity Of CP7 Fermentation Broth And Screening Of Optimization Solid Fermentation Conditions Of CP7 Strain

Paenibacillus polymyxa is a type of biocontrol bacteria that has been widely studied,and can produce a variety of biologically active substances such as antibacterial proteins.It has broad development prospects and application value in many fields such as industry,medicine,agriculture,and food.A strain of Bacillus polymyxa CP7 with high antibacterial protein production was screened by our group.Existing studies have shown that it has a good inhibitory and killing effect on fungi and bacteria,and has a broad antibacterial spectrum.In order to better develop and utilize CP7 bacteria,this paper studied the antibacterial activity of the fermentation broth of CP7 strain against eight bacteria,such as Klebsiella pneumoniae and other bateria,screened and optimized the conditions of CP7 solid-state fermentation.The main results are as follows:1.Using growth rate,filter paper method and other detection methods,the antibacterial activity of CP7 fermentation broth against Klebsiella pneumoniae,Salmonella typhimurium,Propionibacterium acnes,Staphylococcus epidermidis,Microsporum gypseum,Trichophyton rubrum,etc.,was tested.（1）The antibacterial effect of CP7 fermentation broth on Propionibacterium acnes ATCC 6919,Staphylococcus epidermidis ATCC 12228,Klebsiella pneumoniae and Salmonella typhimurium were determined by filter paper method and the results showed that CP7 fermentation broth had a strong antibacterial effect against Staphylococcus epidermidis ATCC 12228 with the inhibition zone diameter of 18.41mm and some antibacterial effect against Propionibacterium acnes ATCC 6919,Klebsiella pneumoniae and Salmonella typhimurium with the inhibition zone diameter of 7.98mm,8.15mm,8.41mm,respectively.（2）The antibacterial effects of CP7 fermentation broth against Microsporum gypseum ATCC 14683 and Trichophyton rubrum ATCC MYA-4438were tested through growth rate determination method.The results showed that the CP7fermentation broth had antibacterial effects on Microsporum gypseum and Trichophyton rubrum with the inhibition rates of 90.38%and 72%respectively.（3）The antibacterial potency of Staphylococcus aureus and Escherichia coli were measured by the agar hole diffusion method.The results showed that CP7 fermentation broth has strong antibacterial effect on Staphylococcus aureus and Escherichia coli,and the diameter of the inhibition zone can reach 15.81mm and 13.99mm,respectively.2.The culture medium formula and fermintion conditions of solid-state fermentation for CP7 were optimized with two indicators,the number of CP7 colonies and the size of the inhibitory zone of CP7 fermentation extract on Staphylococcus aureus,being used to evaluate the effect of solid-state fermentation.（1）Soybean meal was used as the nitrogen source while rice husk,bran and corn meal were used as the carbon source,respectly.Carbon source（g）and nitrogen source（g）were added in a ratio of 4:1.After fermentation at 31℃for 48 h,the results showed that the viable counts and diameter of the inhibition zone of bran and corn meal were 2.0×10⁸cfu/g and 1.55×10⁸cfu/g,as well as 14.60mm and 12.29mm,respectively while rice hulls had no antibacterial effect,therefore,the best carbon source was bran;（2）To select the best nitrogen source,yeast powder,soybean meal,and soybean meal were used as the nitrogen source with bran as the carbon source whilst the others being kept constant,the mass ratio of carbon source（g）to nitrogen source（g）were 4:1,the results showed that the number of viable cells feremented extract of soybean meal and soybean meal were2.53×10⁸cfu/g and 3.67×10⁸cfu/g,respectively,as well as their diameters of the inhibition zone o were 10.39mm and 12.14mm,respectively,while the one from yeast powder had no antibacterial effect and its number of viable cells was 0;Therefore,soybean meal was selected as the best carbon source;（3）The best mass ratio of bran to soybean meal was screened.The results showed that when the ratio of bran to soybean meal was 4:1,the fermentation effect was the best.At this time,the number of viable bacteria was 2.37×10⁸cfu/g,and the diameter of inhibition zone was 12.27mm;（4）K₂HPO₄·2H₂O with mass fractions of 2%,0.2%,0.02%,0.002%（w/w）were added to the substrate to produce antipeptied（eath treatment was applied whilst the others were kept constant）,the results showed that fermented extract with 0.2%（w/w）of K₂HPO₄·2H₂O was the best one,the number of viable bacteria was 3.33×10⁹cfu/g,and the diameter of the inhibition zone was19.05mm;On this basis,the optimal concentration of Mg SO₄ was screened with 4 kinds of concentrations of 1%,0.1%,0.01%,and 0.001%（w/w）,fermentation results showed that fermtation extract adding 0.01%（w/w）magnesium sulfate had the best effect with6.33×10⁹cfu/g of viable bacteria,and 17.01mm of inhibition zone diameter.Mn SO₄ was studied too,it had no efficiency on producing antisubstance.（5）Screening the best ratio of material to water,the results show that when the ratio of material to water was 3 g:4 m L,the fermentation effect was the best,the number of viable bacteria was 5.50×10⁹cfu/g,and the diameter of the inhibition zone was 17.01mm;（6）1～4 m L（about 10⁶cfu/m L）of CP7 seed solution was inoculated in the culture medium and the results showed that the fermentation effect was best when the inoculation amount was 3m L,the number of viable bacteria was2.43×10⁸cfu/g,and the diameter of the inhibition zone was 11.72mm.After optimization and screening,the best formula and fermentation conditions for CP7bacteria solid fermentation were established as follows:bran 12g,soybean meal 3g,K₂HPO₄·2H₂O 30mg,Mg SO₄·7H₂O 1.5mg,water 20m L,inoculation volume 3m L（about10⁶cfu/m L）,solid fermentation temperature 31℃,fermentation time 48h.3.Evaluation of CP7 solid state fermentation product.The fermented product was yellow,had a unique aroma of soybean meal,a slightly sour taste.The fermentation product extract has a good antibacterial effect,and the diameter of the inhibition zone against Staphylococcus aureus is 18.86mm.Compared with before fermentation,there was little change in nitrogen,calcium,phosphorus and p H while the available phosphorus content increased by 48.44%,the nitrogen content of protein solubility increased by 11.24%,the nitrogen content of acid-soluble protein increased by 35.85%.It shows that the fermented bran soybean meal is rich in antibacterial activity and other substances,and the nutrients are easier to use.