Study On The Amine Oxidase Activity From Douchi Fermentation Microorganisms

Douchi is a kind of condiment used in medicine and food,which is fermented by microorganism.It not only can be used for cooking and flavoring,but also has high active health function of urokinase.The laboratory was detected in the early stage of existence of biogenic amines(BA)in Douchi,which the highest content of BA was tyramine and ethambutol in strengthening pure fermentation Douchi,and the content of tyramine and ethambutol were 63.25 mg/kg and 13.82 mg/kg,respectively;and most of the BA in natural fermentation Douchi were tyramine and spermidine,which were 27.94 mg/kg and 11.50 mg/kg,respectively.The results showed that when the content of tyramine is more than 100 m,and it will cause migraine and hypertension.Therefore,fermentation Douchi products have potential safety.At present,the most effective way to reduce BA in food is to increase the activity of AMO(Amine,oxidase)in fermented microorganisms and decompose biogenic amines.AMO consists of monoamine oxidase(MAO)and polyamine oxidase(PAO),because the previous experiment detected that the most content of biogenic amines was monoamine.Therefore,it is practical significance to improve food safety by clarifying the changes of AMO activity and influencing factors.Extract patent strains of Bacillus subtilis BJ3-2,the crude AMO was extracted from BJ3-2 strengthening pure fermentation Douchi and traditional nature fermentation Douchi,and the dynamic changes of AMO activity during the fermentation were detected.The results showed that AMO activity is the highest at the pre-fermentation 42 h and at the post-fermentation stage 7 d under the pure intensive fermentation conditions,and their activities are 4.74 U/mg and 2.73 U/mg respectively.AMO activity is the highest at the pre-fermentation 36 h and post-fermentation 3 d under the natural fermentation conditions,and their activities are 2.39 U/mg and 1.76 U/mg respectively.The recombinant plasmid pET28a-YobN was transferred to Escherichia coli BL21,the 1% recombination strain of pET28a-YobN/BL21 was inoculated to LB liquid medium containing kanamycin under condition of 100 r/min,16?.After overnight,1.0 mM IPTG was added for inducible expression.Ultrasonic broken cracking 30 min(working 5 s,intermittent 5 s)to obtain the fusion protein.Then the production was assayed by 15% SDS-PAGE.The results indicated that the protein expression molecular weight is about 53.4 kDa which is consistent with the expected molecular weight.Most of the proteins is expressed with the form of inclusion.Choose the inclusion part,get pure AMO after being purified by His target column and desalted through dialysis.Imidazole concentration was 300 mM.Stirring dialysis on protein samples using magnetic stirrer at 4?,and every 2 h change 1 dialysis solution(20 mmol/L Tris-HCl,pH=8.0),which changing 6 times.Then collecting the protein in dialysis bag.The analysis results showed solution was made up of 1.63 mg/m L protein by measuring with BCA.That illustrated that the sample of AMO is in high purity.Furthermore,the AMO activity was 4.8×106 U/g.The optimal enzyme activity of AMO was determined by adjusting the reaction temperature,pH and excipients concentration.The optimum conditions for the activity of amine oxidase were obtained:40?,pH =8,salt concentration of 4%,concentration of monosodium glutamate was as high as 12.5%,concentration of pepper was as high as 3%,concentration of tea was as high as 35%,concentration of ginger was as high as 3%,and the concentration of Zanthoxylum bungeanum was as high as 2%.MAO is alkaline enzyme,but weak acid is beneficial to prolong the shelf life of fermented food.Therefore,post-fermentation douchi,pH control in 7.0-7.5 can effectively improve its safety.In the add accessories,monosodium glutamate has almost no effect on monoamine oxidase activity.The addition of salt and pepper could inhibit the activity of amine oxidase.Ginger can promote the activity of amine oxidase.The effect of tea and pepper on amine oxidase is that low concentration promotes activity of oxidase and high concentration inhibits activity of oxidase.According to the demand of production,the content of monosodium glutamate is adjusted according to different plant formula,and the concentration of salt? pepper?ginger and tea are controlled at 5%?2? 3% and 35% respectively.This experiment not only has important guiding significance to the production practice but also provides reference for the production and processing of fermented bean products.