Breeding Of Highly Resistant Strains Of Bacillus Coagulans Using ARTP And Adaptive Laboratory Evolution For Soybean Meal Fermentation

Bacillus coagulans is a probiotic with a combined advantage of Lactic acid bacteria and Bacillus.Compared to other bacteria,B.coagulans has become one of the advantaged bacteria for the preparation of micro-ecological in food and feed industries due to the significant stress resistance to heat,acid,alkali and salt.However,B.coagulans is often subjected to various environmental stresses such as high temperature,low pH and bile salts during actual application processing,which significantly hinders its probiotic effect.To screen for target strains with high resistance to acids and bile salts,the present study attempted to apply atmospheric and room temperature plasma(ARTP)coupled with adaptive laboratory evolution(ALE)strategy for improving the stress resistant of a B.coagulans strain WT-03 with certain probiotic properties,isolated and genetically identified from the intestines of piglet farces.Furthermore,the possible mechanism of stress resistance was elucidated based on changes of intracellular pH,cell morphologies and cell membrane at multiple levels.Finally,solid-state fermentation conditions of mutated B.coagulans strain were optimized to improve the nutritional quality of on the basis of acid-soluble protein content.The main obtained results are as follows,(1)B.coagulans mutant with significantly improved tolerance to acid and bile salt were selected using ARTP and ALE.The lethal rate reached to 93.84 % when the optimal exposure time of ARTP was 15 s.Thirty bile-salt-tolerant mutants(artp-BC1,…..artp-BC30)surviving on 0.3 %(w/v)bile salts-MRS agar plate with good growth and large colonies were randomly picked out,and transferred to 24-well-plates containing pH 3.0,3.5 and 4.0 MRS broths for evaluation of their acid tolerance.Among the ARTP-mutated strains,three mutant isolates artp-BC5,artp-BC28 and artp-BC15 had the significant high survival rates of 18.5 %,18.8 % and 19.3 % at pH 3.0,respectively,compared to B.coagulans WT-03.A 40-day ALE results showed that mutant isolate artp-aleBC15 had the highest survival of 19.8 % at pH of 2.5,whereas the parent strain of B.coagulans only had a final survival rate of 7.15 %.In vitro simulated gastrointestinal tract experiments demonstrated that the final survival rates of artp-aleBC15,artp-BC15,and WT-03 were 22.4 %,15.2 % and 7.98 %,respectively.Therefore,ARTP-ALE mutant artp-aleBC15 was selected as the ideal strain and deposited in China General Microbiological Culture Collection(CGMCC)with the Accession number CGMCC 1.17145.(2)The possible mechanisms of stress resistance of B.coagulans mutants were investigated.The external pH stress caused the intracellular pH(pHi)to decrease.At pH 6.2,the pHi of artp-aleBC15,artp-BC15 and WT-03 were all approximately 7.10;and at pH 2.5,the pHi of artp-aleBC15,artp-BC15 and WT-03 were 4.90,4.45 and 4.23,respectively.Scanning electron microscopy results indicated that ARTP-ALE mutant isolate artp-aleBC15 could maintain a better intact cell structure at pH 2.5.Further studies presented that artp-aleBC15 exhibited a higher-strength biofilm with enhanced hydrophobicity and lower permeability compared to the parent strain of B.coagulans.Additionally,acid stress could result in reduced saturated fatty acids(FA),increased unsaturated fatty acids(UFA),and increased unsaturation in cell membrane.Quantitative real-time PCR(qRT-PCR)analysis revealed that the expressions of plsX,acpP and fabD encoding fatty acid synthesis genes in ARTP-ALE mutant strain artp-aleBC15 were significantly up-regulated to 7.00,15.99 and 9.47 times respectively,compared to the parent strain,at pH 2.5.(3)Solid-state conditions of B.coagulans artp-aleBC15 to ferment soybean meal were optimized on the basis of acid-soluble protein content,and changes of the nutritional quality before and after fermented were analyzed.One-factor-at-a-time and orthogonal design results showed that acid soluble protein in soybean meal increased from 2.29 % to 20.44 %,the crude protein content increased from 46.77 % to 49.13 %,and the total acidity increased from 2.28 to 8.46,total amino acids increased from 53.0 % to 55.8 % under the optimized conditions of inoculation size 4 %,feed-water ratio 1.0: 1.2,fermentation temperature 37℃,and fermentation time 72 h.SDS-PAGE results also indicated that majority of proteins in soybean meal were effectively degraded to the molecular weight of less than 20 kDa after fermentation by B.coagulans artp-aleBC15.