| Natto, is the popular traditional fermented soybeans food in the Japan, which isfermented using Bacillus subtilis natto from soybeans. The sticky polyglutamic acid on thesurface of natto contains some physiological active substances, such as nattokinase,Bacillus natto, angiotensin converting enzyme inhibitor, vitamin K2, pyridine dicarboxylicacid, isoflavone, etc, which have a lot of health functions such as antitumor, thrombolytic,lowering blood pressure, antioxidant activity, prevention of osteoporosis, antibacterial.Bacillus subtilis (natto) is a strain isolated from Japanese traditional food and the typestrain is the same as Bacillus subtilis. which is a subspecies of Bacillus subtilis.Nattokinase is producted by Bacillus subtilis (natto) and a kind of extracellular enzyme,which has fibrinolytic function with a strong alkaline serine protease. Nattokinase hasstrong fibrinolytic activity and good ability to thrombolysis for the treatment andprevention of cardiovascular disease. Nattokinase comes from traditional fermented food,which is safe and persistent effect raw material for thrombosis treatment drugs andfunctional health food.This study contains experiments of Bacillus subtilis (natto) isolation, determination ofnattokinase activity, optimization of solid state fermentation conditions of natto nattokinase,safety evaluation of natto, active substances of natto, which are theoretical bases of furtherresearch and functional food development.According to the characteristics of the heat resistance of Bacillus subtilis (natto) andproduction of serine protease hydrolyzed casein-Nattokinase, which can hydrolyze caseinthen form clear zone. Two strains NB-7and NB-8were isloated from commerciallyavailable natto which were confirmed as Bacillus subtilis (natto) according to Gram stain,comparison with the known Bacillus subtilis (natto), the microscopic morphology andphysiological and biochemical identification using morphological characteristics as well asobservation plate and reference of Bergey’s Manual of Systemaic Bacteriology (EighthEdition).This work explored the determination of nattokinase activity assay using subtilisin tofind an inexpensive and efficient method to determine the activity of nattokinase. Theassociation fibrin plate method and Folin-phenol method was established to measureenzyme activity of Nattokinase. The result indicated that fibrin plate method and theFolin-phenol method had highly correlation and the correlation equation was follows: Y(NK activity)=0.9433X (protease)-30.891, correlation coefficient R2=0.9906. Solidstate fermentation conditions of six strains provided by the Microbiology Department ofSchool of Life Science and two strains screened out by us were investigated. Andnattokinase activities of these eight strains were studied individually. Based on the results,the strain NB-7was chosen as the objective strain for further study.According to the growth curve of Bacillus subtilis (natto), the optimal fermentationtime is20h. The effects of material thickness, carbon source, growth factor supplements,minerals, water content, inoculation amount, post-fermentation time on production ofnattokinase were investigated by single factor experiments. The effects of glucose, yeastextract, MgSO4, incubation time on nattokinase were further studied using softwareDesign Expert7.0Box-Behnken design experiments and the optimal levels of the4factors were archived. The optimal culture medium was glucose carbon source6%, growth factorsupplement yeast extract1.65%, MgSO40.3%. And the optimal culture conditions werematerial thickness2cm, moisture content of materials60%, inoculation amount6%,fermentation time38.85h, post-ferment time1d and under this condition the maximumtheoretical nattokinase activity could reach up to2797.17IU/g.Safety of natto was evaluated by gastric lavage (thick liquid of natto powder) intohealthy mice. All the mice were observed after treatment for7d. There was no death and noother obvious toxic symptoms of major organs as well. The results indicated that themaximum tolerated dose for mice was119.2758times. According to acute toxicity testrequirements of Toxicological Assessment Standard for Health Food, more than100timesof the dose indicated that there was natto is safe and no acute toxicity.The isoflavones of raw soybean powder, sterilized soybean powder and natto powderwere analyzed preliminary by high performance liquid chromatography-mass spectrometry,respectively. The effects of freeze-drying and hot-air drying on the isoflavones of sterilizedsoybean powder and natto powder were studied as well. The results showed two unknownpeaks appeared at retention times of8.9min and12.4min and those were much higherthan that of no fermented soybean powder, while the peak at retention time2.1min was low.There was a peak at15.83min of the freeze-drying natto powder, but no peak at15.83minof hot-air drying natto powder. The treatment of hot drying also affected the forms ofisoflavone. There were two low peaks of raw soybean powder at the retention time24.6min and26.3min, but the peaks of hot drying soybean powder and natto powder wereobvious higher than thoes of raw soybean powder the same time. The results suggested thatthe solid fermentation using Bacillus subtilis (natto) had an impact on the form isoflavonesof the soybean.Volatile compounds of raw soybean powder, sterilized soybean powder and nattopowder were analyzed by simultaneous distillation extraction and gas chromatography andgas chromatography–mass spectrometry. The results indicated that indole37.85%andbenzyl alcohol16.88%are main volatile compounds in Natto. By this method,40compounds including alcohols, acids, esters, aldehydes, ketones, phenols, pyrazines, furansand others were found in Natto powder. While only naphthalene and butylatedhydroxytoluene (BHT) which was up to99%in raw soybean. Soybeans were sterilized at121℃for20min, three new volatile compounds were found such as1-octene-3-alcohol,phenylethyl alcohol,2-methoxy-4-vinylphenol, but the BHT was still very rich as87.12%.The results showed that the major volatile compounds in natto soybeans were come fromfermentation of Bacillus subtilis. High concentration of indole has strong fecal odor,maybe that why natto has the special smell.Based on the above studies, KangTai natto capsules has been developed and produced.The production technology and enterprise standard: Q/NZFT0004S-2009wereestablished.The fortified health food2009No.640100SQ002has been approved byNingxia Hui Autonomous Region and it has conducted industrial production. |
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