The Construction Of Flavour Compound Acetoin-producing Strain And Research On Its Preparation Technology

Acetoin(also known as 3-hydroxy butanone), having a pleasant aroma of butter, naturally presents in some plants such as corn, grapes, cocoa, apples, bananas, and strawberries as well as in certain animal tissues. Acetoin is a favorite food flavor and is also an international widely used perfume. Acetoin is also an important chemical which was assigned as one of the 30 top platform chemicals by U.S. Department of Energy in 2004. Acetoin is produced by chemical synthesis, enzyme conversation and microbial fermentation. Currently, it is mainly produced by chemical synthesis with disadvantages such as complex process, high production cost, and serious environmental pollution. The application of chemical synthesized acetoin is restricted in food and pharmaceutical industries because the manufacture method does not meet with the natural, green and safe consumption concept. The industrial application of acetoin using enzyme conversation is also restricted because of the difficulty of obtaining enzymes with high activity. However, microbial fermentation method could use renewable biomass as substance with the widely distributed microbes as the producer. The advantages such as rich substance, moderate reaction, and eco-friendly and green safety, could reduce the pressure of resources and environment problem during the process of acetoin production. Hence, the microbial method has becoming a hotspot with the increasing of food safety and the lack of petroleum resources. An acetoin-producing strain, Bacillus subtilis SF3-102, was obtained according to traditional mutation method in this study. The work of molecular design, fermentation process and optimization of extraction process was also carried out. Here the main research results were presented as below:(1) In the previous study, B. subtilis SF4-3 with producing acetoin was isolated from wild samples. B. subtilis SF3-102 with high yield of acetoin was obtained through modifying B. subtilis SF4-3 by the traditional mutagenic methods including ultraviolet, diethyl sulfate and nitrosoguanidine. A further study on the culture conditions of strain SF3-102 for acetoin production found that the optimal inoculation size was 5%(v/v) and the optimal culture temperature was 36-38 ?. The effect of components on the fermentation medium was investigated by statistical analysis. The optimum culture components were as follows: glucose 160 g/L, yeast extract 6.5 g/L, corn steep liquor 13 g/L, urea 3 g/L, Mn SO4 0.05 g/L, and Fe SO4 0.05 g/L, respectively. The yield of acetoin achieved 60.64 g/L in the shake flask fermentation under the above optimized fermentation conditions.(2) To in-depth analyze the metabolic mechanism of acetoin accumulation in B. subtilis SF3-102, de novo whole genome shotgun sequencing was applied in B. subtilis SF3-102 by using Illumina Solexa technology. 4,153,882 bps of genomic data including 10 scaffolds and 4545 open reading frames(ORFs) were obtained by assembly. The average length of ORFs was 798 bp. The coding regions were 3,625,437 bps containing 87.28 percent of the whole genome. Protein encoding genes were analyzed by GO(Gene Ontology) annotation, COG(Cluster of Orthologous Groups of proteins) annotation, and KO pathway annotation. Based on the above analysis, comparative genomics analysis was also performed among strains SF3-102, 168, and BEST195. The key genes associated with excessive accumulation of acetoin and the mutation sites of individual amino acid on acetoin operon were confirmed eventually in B. subtilis.(3) Molecular modification was carried out in strain SF3-102 according to acetoin metabolic pathway and whole-genome sequencing. 2,3-butanediol as a by-product of acetoin fermentation was conversed by butanediol dehydrogenase(BDH), which could affect the yield of acetoin and extraction for downstream separation. Therefore, we knocked out BDH encoding gene bdh A by using Cre/lox system and obtained a strain, B. subtilis SFAP. The experiment of shake flask fermentation show that the yield of 2,3-butanediol decreased from 21.5 g/L to 6.1 g/L, and the yield of acetoin was increased by 23.6%(69.5 g/L with 0.97 g/L/h of production efficiency).(4) The fermentation conditions and control of strain SFAP for acetoin production were studied by 10-L and 50-L fermentors. The effects of dissolved oxygen(pressure, air rate and stirred rate) and p H were investigated for acetoin fermentation. The specific control strategy and parameters of acetoin production were determined: 0-15% of dissolved oxygen was controlled in the fermentation process, the stirred rate was 400-500 r/min(main control parameter), and the air rate was 1.0-1.2 m3/h. p H was controlled at 6.5-7.0 in the initial growth period of bacteria and 5.5-6.0 in the late period of product formation. The experiment was carried out in a 1000-L fermentor. The fermentation period was 70.2 h, the yield of acetoin was 64.84 g/L, and the conversion rate was 40.42%. The fermentation period of the 5000-L fermentor was 68.3 h and the highest yield was 64.90 g/L. Acetoin fed-batch fermentation was studied in the 1000-L fermentor, and the optimal feeding mode was determined. The highest yield of 78.9 g/L acetoin was obtained after 78 h.(5) The process of the downstream extraction of acetoin was studied systematically. The pretreatment and purification process of fermentation broth was determined. The ceramic membrane was used to remove bacteria cells. The temperature of the broth was controlled at less than 80?. The operation pressure was set at less than 0.2 MPa. The flow rate on the membrane was controlled at less than 5 m/s. The decolorization process of the supernatant liquid and the decolorization effect of activated carbon(model ZX-303) were determined. The decolorization temperature was 50-55?, the adding amount of activated carbon was 0.8-1.0%, and the decolorization time was 20-25 min. The extraction and purification of 3-hydroxy butanone was mainly studied. The separation route of 3-hydroxy butanone by distillation, salt distillation, and extraction was studied and compared. The purity of crystal 3-hydroxy butanone product was above 99.0% by our developed purification process.In the work, we have constructed acetoin high-producing strain and provided a route of fermentation process and extraction process, which lay the foundation of acetoin production.