Strains Breeding And Application Of A Bacillus Amyloliquefaciens For Camphor Tree Seed Kernel Oil Extracted By Aqueous Enzymatic Extraction

The strain Z37 was isolated from residue waste produced in camphor tree seed kernel oil production by using camphor tree seed kernel meal plate for enrichment culture,skim milk plate and oil culture medium plate for primary screening,Folin phenol method and camphor tree seed kernel oil medium shaking culture for re-screening,which was confirmed suitable for aqueous enzymatic extraction technology to extract camphor tree seed kernel oil,and its neutral protease production capability was 4536.5 U/mL,lipase production capability was 0.088 U/mL.The strain Z37 was identified as Bacillus amyloliquefaciens by morphological,physiological and biochemical characteristics and 16s rDNA molecular biological identification,and named Bacillus amyloliquefaciens Z37.Optimal fermentation medium and fermentation conditions were obtained through single factor experiments and orthogonal experiment.The experiment results showed that the optimum fermentation medium and enzyme producing condition were:corn meal 4.5%,wheat barn 2.5%,soybean meal 4%,CaCl2 0.2%,KH2PO40.03%,Na2HPO4�12H2O 0.4%,pH 7.5,inoculation amount 2%,37?,220 rpm,44 h.Under optimal fermentation condition,Bacillus amyloliquefaciens Z37's neutral proteinase producing capacity was 6984.3 U/mL.A strain named Bacillus amyloliquefaciens Z37-6 which have high production ability of neutral protease and good genetic stability was screened through Ultraviolet and nitrosoguanidine compound mutation.Its neutral protease production ability is 9116.1 U/mL,31.9%higher than the starting strain.After five consecutive culture,the neutral protease enzyme activity of fermentation liquid can be up to 96.2%of the starting strainNational standard method and CMC saccharification method were used to analysis Bacllus amyloliquefaciens Z37-6's enzymes.The experiment results showed that,extracellular enzymes producted by Bacillus amyloliquefaciens Z37-6 were:neutral protease,pectinase,cold-adapted amylase,saccharifying enzyme and cellulose enzyme.Using fermentation medium 37?,220rpm shake flask fermented 44 h,Under this fermentation condition,Bacillus amyloliquefaciens Z37-6's neutral protease activity is 9027 U/mL,pectic enzyme activity is 1537.0 U/mL,cold-adapted amylase activity is 28.2 U/mL,saccharifying enzyme activity is 8535.0 U/mL,cellulose enzyme activity of 5.6 U/mL.Optimal fermentation time and fermentation liquid enzyme adding proportion were obtained through single factor experiments.The experiment results showed that,the Optimal fermentation time and fermentation enzyme dosage used in aqueous enzymatic extraction of camphor tree seed kernel oil were:enzymolysis time 4 h,fermentation liquid enzyme adding proportion 20%,and under optimal condition the camphor tree seed kernel oil extraction rate was 95.1%.