Research On Breeding Of High Acetic Acid Producing Strain And Its Metabolism Controlling

Acetic acid,as the main component of vinegar,is used as acidifying agent,flavoring agent and spice in the food industry.It is widely used in anhydride acetic acid,vinyl acetate,acetate fiber,metal acetate,acetic ester and other chemical industry.There are many methods for the production of acetic acid,the acetic acid used in the food industry needs to be produced by biological method.Acetobacter pasteurianus has been the main strain of vinegar fermentation in China.However,there are too many specific strains and very few strong acid producing strains,and its characteristics are unstable.In order to improve the ability of acetic acid fermentation and obtain the dominant strains,in this study,a strain?Acetobacter pasteurianus JST-S?with good growth and high acid intensity was firstly selected from several strains of common acetic acid bacteria.The strain was mutated by UV.Finally,a fine strain with acetic acid bacteria YHL3 was selected.Combined with its fermentation characteristics and metabolic flux analysis,the mutagenic mechanism of Acetobacter pasteurianus YHL3 was discussed.The mechanism controlling of key emzymes activities with YHL3 was also performed.Its semi-continuous fermentation process parameters were optimized,and its high concentration acetic acid fermentation system was established.The main research results were as follows:?1?A fine acetic acid bacteria were selected.It was determined that Acetobacter pasteuribacillus JST-S was the dominant strain by comparing the fermentation characteristics of three strains.The yield of its total acid in fermented liquid was 60.15 g/L.It was mutated by UV and performed for high alcohol pressure culture.A strain was screened and named as Acetobacter pasteuribacillus YHL3.The highest total acid concentration reached 42.55 ± 0.86 g/L under high alcohol pressure culture.It was subcultured for 5 times and performed the fermentation experiment.When the fermentation time was 48 h,the total acid concentation with YHL3 was maintained at 58.10⁵9.68 g/L,and its fermentation intensity was maintained at 1.21¹.24 g/?L·h?.?2?Mutagenic mechanism of mutant strain with YHL3 were analyzed.The tolerance test results with YHL3 and BJST-S showed that under the high acohol content,specific emzymes acitivies of ADH and ALDH with YHL3 were respectively 5.96±0.26 U/mg and 4.82±0.22 U/mg,they were higher than those of BJST-S?respectively 4.78±0.28 U/mg and 4.12±0.24 U/mg?;under the initial acid,specific emzymes acitivies of ADH and ALDH with YHL3 were respectively 6.08±0.18 U/mg and 5.47±0.16 U/mg,and they were also higher than those of BJST-S?respectively 5.66±0.16 U/mg and 4.87±0.12 U/mg?.The expression results of ADH enzyme protein analyzed by SDS-PAGE gel electrophoresis was in line with determination results of emzyme acivities.Combined with the references,the metabolic network pathway of Acetobacter pasteuriens was constructed.47 metabolic flux equations were listed.Compared with the internal metabolic rate of BJST-S and YHL3,it was found that the enzyme system of YHL3 in ethanol oxidative respiratory chain pathway was better than that of BJST-S.?3?The metabolism controlling of YHL3 was performed.The seed medium composition of YHL3 strain was optimized by single factor and response surface method,the results were as follows: ethanol,glucose,yeast extract,MgSO₄ and K₂HPO₄ were respectively 2.4%?V/V?,10.7 g/L,10 g/L,1.2 g/L,and 3.4 g/L.The content of acetic acid in fermentation broth with acetic acid bacteria reached 54.75 ± 0.72 g/L under the optimal condition.The effects of 4 metal ions on key emzyme activities of metabolic process with acetic acid bacteria was studied.It was determined that Mg²⁺ could be used as a metal ion activator.For the medium adding Mg²⁺,the biomass was 2.12 + 0.16 g/L and the yield of acetic acid was 56.84 + 1.42 g/L when the fermentation time was 72 h.?4?A semi-continuous acetic acid fermentation system was established.The optimization results for culture medium of batch fermentation in shake flask were as follows: the glucose content was 10 g/L,the yeast extract dosage was 8 g/L,the ethanol volume fraction was 5%,and the metal Mg²⁺ ion addition amount was 10 mmol/L.Under the optimal conditions,the highest yield of acetic acid in fermentation broth reached 72.94 g/L after batch feeding in four batches.Combined with the results of metabolic network and flux analysis,the best aeration volume of the first batch process was determined to be 0.4 L/min/L.After semi-continuous acetic acid fermentation for four batches,the highest acetic acid yield of fermentation broth could reach 75.24 g/L.The above results provide a theoretical basis for the industial fermentation of high concentration acetic acid and the breeding of acetic acid bacteria.