Enhancing Acid Tolerance Of Lactobacillus Acidophilus With Atmospheric And Room Temperature Plasma (ARTP) Coupled With Adaptive Laboratory Evolution(ALE)

Lactobacillus acidophilus is a genetically stable lactic acid bacteria with a wide range of industrial applications in food production,the agricultural industry,and as a probiotic.However,it is often exposed to acid stress during industrial processing which has a significant impact on its growth.Primarily,L.acidophilus can rapidly acidify the environment it grows in by converting carbohydrates into lactic acid.The accumulation of lactic acid has an inhibitory effect on the bacteria.It results in a lower intracellular p H,decreased metabolism and a reduction in growth,thus negatively affecting fermentation processes and the viability and concentration of probiotic cultures.Moreover,as a probiotic L.acidophilus is often exposed to acid stress as it travels down the gastrointestinal tract.It must survive the acidic gastric environment to reach the small intestines and colonize the host,thereby imparting its probiotic benefits.Considering the effect that acid stress has on L.acidophilus,this study aimed to screen a new probiotic strain of L.acidophilus with improved tolerance to lactic acid stress using atmospheric and room temperature plasma(ARTP)mutation in combination with adaptive laboratory evolution(ALE).The obtained results are as follows:(1)To improve the acid tolerance of L.acidophilus,atmospheric and room temperature plasma(ARTP)mutation in combination with adaptive laboratory evolution(ALE)was used.Sixty seconds was determined as the ideal exposure time for ARTP mutation of L.acidophilus with a survival rate of 5.91 %.The ARTP-ALE mutant strain LAartp-ale2 displayed increased lactic acid stress tolerance with survival rates of 75.67 % and 25.78 % when cultured in p H 3.0 and 2.5,respectively,for 3 h.The physiological analysis revealed that the ARTP-ALE mutant exhibited a lower inner membrane permeability than that of the parental strain during acid stress.Furthermore,the mutant LAartp-ale2 produced more biofilm in response to lactic acidinduced acid stress and showed an increased hydrophobicity(87.2 %)when compared to the parent strain(76.2 %)at p H 2.5.LAartp-ale2 exhibited a higher unsaturated fatty acid(UFA)to saturated fatty acids(SFA)ratio that affected the physical state of the cell membrane for increased survival in p H 3.0 and 2.5.ARTP,coupled with ALE,proved to be an effective method in enhancing the acid tolerance of L.acidophilus for potential industrial use.(2)The survival of L.acidophilus against lactic acid-induced stress depends on the expression of acid stress response genes.Acid tolerance response(ATR)mechanisms of L.acidophilus were investigated at a transcriptional level.Reverse transcription-quantitative PCR was used to determine the expression of several genes associated with ATR in L.acidophilus.The results indicated that the genes were regulated differently in ATR mechanisms that involved changes in the cell membrane,energy production,and cellular metabolism,Class I stress response pathways and cell signaling.The expression of genes pls X,slp X,atp C,gad C,lux S,and dna K were upregulated with exposure to acid stress in both the parent and mutant strain LAartp-ale2.Interestingly,a comparison made between the parent strain and mutant strain,revealed that the mutant strain had significantly higher expression of the six genes,which could account for its improved acid tolerance.(3)Finally,the application of solid-state fermentation to soybean meal using the acidtolerant probiotic L.acidophilus LAartp-ale2 was considered to be an effective method to improve the overall nutritional value of the soybean meal.The fermentation process was optimized using the Taguchi orthogonal design.The effect of three various levels for four factors,including inoculum size,moisture content,time and temperature,were investigated.The optimum conditions identified by the L9 Taguchi orthogonal design for increased trichloroacetic soluble proteins,crude protein and protease activity for L.acidophilus LAartpale2 from this experiment were an inoculum size of 6 %,moisture content of 80 %,5 d incubation time and a temperature of 37 oC.These optimum conditions produced 14.10 % trichloroacetic soluble proteins,61.77 % crude protein and protease activity of 155.87 ?/g.