Breeding Of High-yield L-lactic Acid Strains Based On High-Efficiency Heavy Ion Beam Mutagenesis

As one of the most potential organic acids in the 21st century,because of its wide application in many fields such as food,medicine and environmental protection,it has received increasing attention.In recent years,in the field of new biodegradable polymer materials,poly lactic acid?PLA?has many advantages,such as non-toxicity,high heat resistance,high strength,good biocompatibility,non-irritation,excellent biodegradability and low environmental pollution.Therefore,the study of PLA synthesized by L-lactic acid as a monomer has become a bright spot.It can be completely decomposed by microorganisms to produce water and carbon dioxide without causing environmental pollution.Therefore,most of the researchers think that this biodegradable polymer material has great prospects for development in the future,and it has invested a lot of manpower and material resources,which has attracted widespread attention at home and abroad.Lactobacillus thermophilus can ferment at high temperature above 50?,which reduces other microbial contamination and has great advantages in L-lactic acid industrial production.Although the production of L-lactic acid by Lactobacillus thermophilus is very attractive,its efficiency of L-lactic acid and production capacity need to be further improved to meet commercial needs.Therefore,how to select high-yield L-lactic acid strains with good traits from Lactobacillus thermophilus is our research direction.In recent years,heavy ion beam mutation breeding has played a promotive role in the field of microorganisms.In this paper,Lactobacillus thermophilus SRZ-50mutagenized by heavy ion beam irradiation is taken as the initial strain,and relevant experiments are carried out with the three objectives of screening excellent strains,increasing lactic acid production and reducing lactic acid production cost.?1?Firstly,after the establishment and improvement of 24-well plate screening method,the original strain SRZ-50 irradiated by heavy ion beam was screened to obtain16 strains A21,A31,A34,A37,A45,A48,A56,A57,A58,A59,A62,A68,A69,A85,A86,A87 with higher Lactic acid production.They were preserved for the second round of rescreening experiments,the strains A59 and A69 produced higher L-lactic acid.Then the shake flask validation experiments were carried out on high lactic acid producing strains A59 and A69.The results were consistent with the results of 24-well plate screening,and finally the mutant strains A59 and A69 were increased respectively by 16.2%and 15.8%compared with the original strain L-lactic acid accumulation.The lactic acid production of mutant strains A59 and A69 was stable for 7 generations.?2?In the study of L-lactic acid fermentation technology,a key influence on the control of lactic acid production and the increase in lactic acid production is how to reduce or eliminate the inhibitory effect of fermentation products on fermentation acid production and cell growth.At present,batch fed-batch fermentation has become the main way of fermentation at home and abroad.In this experiment,the optimum fermentation conditions were seed liquid culture for 8 hours,initial glucose concentration of fermentation broth for 100 g/L and initial pH of 7.The yield of L-lactic acid reached 104.87 g/L after 24 hours once feeding,the yield of L-lactic acid reached114.23 g/L after 16 hours once feeding,the yield of L-lactic acid reached 130.67 g/L after 16 h and 32 h second feeding;and the accumulation of L-lactic acid reached 154.8g/L after four times feeding,The yield of L-lactic acid fermented with Ca?OH?2 as neutralizer reached 148 g/L,which was lower than that of NaOH adjusted pH L-lactic acid.Therefore,NaOH used by Lactobacillus thermophilus A69 was more suitable for regulating pH than Ca?OH?₂.?3?In order to reduce the cost of L-lactic acid fermentation raw materials,cottonseed cake powder is used as a cheap nitrogen source instead of expensive yeast meal to produce L-lactic acid.The composition of cottonseed cake powder is similar to yeast meal,in which the protein content is higher,in addition to protein,nutritional components are mainly amino acids,vitamins and other substances.Therefore,cottonseed cake meal has the important characteristics of high quality and low price,Nitrogen source cottonseed cake meal is used instead of yeast powder to produce L-lactic acid.In this experiment,the yield of L-lactic acid reached 76.88 g/L when cottonseed cake powder was added 15 g/L,and the accumulation of L-lactic acid reached 131 g/L after four times feeding at the same time,the yield is lower compared with yeast powder as nitrogen source.And 110.5 g/L after second-time feeding at 60 h in 5 L fermentor.It is determined that the production of L-lactic acid from cottonseed cake powder lays the foundation for the large-scale industrial production of this bacteria.