Screening Of Highly Nirs-producing Strain From Douchi And Study Of Enzymatic Characteristics

Nitrite can oxidize hemoglobin carrying oxygen in the blood to methemoglobin,which renders hemoglobin to lose its oxygen carrying capacity and causes tissue hypoxia.Excessive intake of nitrite can lead to poisoning and death if severity.Nitrite also is a carcinogen,according to the study,the amount of nitrite that patients ingest is positively correlated with esophageal cancer incidence rate.The carcinogenic mechanism of nitrite is considered that in the acid environment,nitrite reacts with secondary amines,tertiary amine and amide in the foods,and produces strong carcinogens N-nitrosamines.Douchi is a traditional fermented soybeans product in China,and because of its unique processing technology,there are a large number of beneficial microorganisms,so we tried to isolate bacterias which can degrade nitrite from Hunan douchi.12 sodium nitrite biodegradation strains were selected from douchi sample,and they were all gram-positive,catalase positive,lysozyme positive.According to 16 SrDNA molecular identification,Acinetobacter guillouiae,Bacillus were found.From the growth curve of strains in the liquid broth medium we can see that all of strains were in the lag phase in the first 4 h,at about 16 h,Acinetobacter guillouiae NDS2,Acinetobacter bereziniae NDS4,Bacillus subtilis subsp.spizizenii NDS6,Bacillus amyloliquefaciens NDS7,Bacillus licheniformis NDS8,Bacillus methylotrophicus NDS9,Bacillus aryabhattai NDS10 fanished logarithmic phase and entered into stationary.While Bacillus subtilis subsp.spizizenii NDS1,Bacillus tequilensis NDS3,Bacillus subtilis subsp.subtilis NDS5,Bacillus amyloliquefaciens subsp.plantarum NDS11 and Bacillus subtilis NDS12 were stable at 28 h,and the number of bacteria in the logarithmic phase were higher than other strains.From the growing situation of strains in two kinds of liquid medium with nitrite or not,it can be seen that almost strains were preferred to surviving in a nitrite-containing environment,but only the growth of Bacillus subtilis subsp.spizizenii NDS6 and Bacillus subtilis NDS12 were not affected by nitrite content.The result of nitrite reduction ability showed that all the strains could degrade nitrite.Bacillus subtilis subsp.spizizenii NDS1,Acinetobacter guillouiae NDS2,Bacillus tequilensis NDS3,Bacillus subtilis subsp.Subtilis NDS5,Bacillus amyloliquefaciens NDS7 and Bacillus subtilis NDS12 could degrade more than 93 % sodium nitrite within 48 h,and the degradation rate of Bacillus subtilis subsp.spizizenii NDS1 reached 99.41 % at 48 h.However,the degradation rate of Acinetobacter bereziniae NDS4,Bacillus licheniformis NDS8,Bacillus methylotrophicus NDS9,Bacillus aryabhattai NDS10 could not exceed 50 % within 48 h.The result of nitrite reductase activity of all bacterias showed that at 24 h,Bacillus subtilis subsp.Subtilis NDS5 had the lowest enzyme activity of 4.25 U/mL,while Bacillus tequilensis NDS3 had the highest activity of 67.27 U/mL.And at 48 h,Bacillus methylotrophicus NDS9 showed the minimum enzyme activity of 21.88 U/mL,at this time the highest activity of Bacillus subtilis subsp.spizizenii NDS1 was 111.75 U/mL.It is consistent with the result of their nitrite reduction capacity experiment.At the same time,according to nasD gene sequence of Bacillus subtilis subsp.168,primers were designed for amplifing nitrite reductase gene from all strains.The clear single band on the agarose gel of all the isolated bacterias was gel extracted,sequenced and compared on BLAST.The results showed that the sequences were 97 % homologous to the nitrite reductase.After that,12 strains were put into jiangshui celery which had just been fermented in order to observe the effects of these strains on the nitrite content in the traditional flavor food.The results indicated that compared with control group,the jiangshui celery which have been inoculated Bacillus subtilis subsp.spizizenii NDS1,Acinetobacter guillouiae NDS2,Bacillus tequilensis NDS3,Acinetobacter bereziniae NDS4,Bacillus subtilis subsp.Subtilis NDS5,Bacillus subtilis subsp.spizizenii NDS6,Bacillus amyloliquefaciens NDS7,Bacillus amyloliquefaciens subsp.plantarum NDS11 and Bacillus subtilis NDS12 can not be detected nitrite in five days.