| Natto is a healthy functional food with rich nutrition,which has physiological activities such as thrombosis,hypotension and antioxidation.However,in the process of natto fermentation,Bacillus subtilis will decompose soybean protein,thus producing a special ammonia odor.The"bad smell"limits the promotion and development of natto in China.Nattokinase（NK）is the main physiologically active component in natto.NK is an alkaline serine protease,which not only has strong fibrinolytic ability,but also can activate plasminogen in human body to convert it into plasmin.As an oral drug,NK has great development potential in the pharmaceutical market,but its stability is poor and it is easy to be inactivated.γ-polyglutamic acid（γ-PGA）is an adhesive substance in natto,which has excellent biological safety and environmental protection molecular materials,and is widely used in agriculture,cosmetics,medicine and materials.The main purpose of this study is to improve the flavor of soybean products and ensure the stability of NK.The research methods and results are as follows:（1）Lactic acid bacteria is a kind of probiotics widely used to improve food flavor,and can be used as a fermentation strain of innovative flavor natto.Using Bacillus subtilis and Lactobacillus bulgaricus to co-ferment natto can not only obtain mixed fermented natto with unique flavor,but also increase the nutritional value of natto.Taking NK enzyme activity as the index,the fermentation process of natto mixed bacteria was optimized by single factor and response surface method.The experimental conditions were optimized as follows:inoculation rate was 4%,strain ratio（Bacillus subtilis:Lactobacillus bulgaricus）was 1:1,fermentation time was 20 h,and fermentation temperature was 35℃.Under the optimized conditions,the fibrinolytic activity A574of natto fermented by mixed bacteria could reach 0.82±0.02,which was 18.84%higher than that of natto fermented by single bacteria,and the isoflavone content andγ-PGA yield were also at a high level.（2）The flavor of natto fermented by mixed bacteria was analyzed by GC-MS,electronic nose,biogenic amine relative content and sensory analysis,and the basic physical and chemical indexes,nutrition and active components of natto fermented by mixed bacteria were compared.Finally,the two fermentation methods were compared with the monitoring of fermentation process.Through the determination of volatile compounds,30compounds were detected,among which natto samples fermented by mixed bacteria contained more acids and esters;Natto fermented by single bacteria is rich in pyrazines and ketones.This is consistent with the characteristics measured by electronic nose,indicating that natto fermented by mixed bacteria has a unique lactic acid fruity flavor.According to the qualitative analysis of biogenic amines by LC-MS,a total of 4 biogenic amines were detected in natto samples,and the qualitative relative content of biogenic amines in mixed fermentation decreased.Finally,the ammonia odor of natto was improved and the innovative lactic acid flavor was obtained.The quality analysis of natto with mixed bacteria also shows that natto fermented with mixed bacteria has higher biological activity（fibrinolytic activity and antioxidant ability）,and lactic acid bacteria,an intestinal probiotic,is introduced.（3）The two main products of natto,NK andγ-PGA,were extracted continuously by salting-out and alcohol precipitation,and then NK was prepared and encapsulated by Chitosan（CS）andγ-PGA,through electrostatic interaction.The results are as follows:With encapsulation efficiency as the index,the single factor optimization was performed from six factors,including stirring time,NK concentration,suspension and CS/γ-PGA ratio,stirring speed,γ-PGA concentration,and CS concentration.The most significant influence factors were NK concentration,the ratio of suspension to CS/γ-PGA（wall material）,stirring speed andγ-PGA concentration.For each factor,three levels of orthogonal test methods are selected:the ratio of suspension to wall material was 1:1,the stirring speed was 350 r/min,theγ-PGA concentration was 1.5 mg/m L,the NK concentration was 0.3 g/m L,and the encapsulation efficiency was 96.08±2.99%.The number of embedding layers was analyzed according to the stability and slow-release benefit of microcapsules,and the optimal embedding layer number was determined to be 2 layers.（4）A series of characterization of microcapsules were performed,and the digestive stability of microcapsules was analyzed and applied through in vitro simulated digestion.Firstly,the NK was successfully embedded into the microcapsules by infrared and ultraviolet spectra.The particle size and potential of the microcapsules could be measured by electron microscopy and dynamic light scattering.Secondly,the stability of microcapsules was analyzed,and the temperature,acid base and storage stability of microcapsules were measured respectively.It is known that the stability of embedded NK microcapsules in various extreme environments and long-term storage is significantly improved.At 60℃,the activity of embedded NK remained 76.58%after 120 min.Higher than 52.68%of naked NK;When p H was 1,4 and 12,the relative activity of pure NK decreased by 35.48%,25.93%and 33.02%compared with microencapsulated NK,respectively.When microcapsules were stored for 60 days,more than 80%of their NK fibrinolytic activity was still maintained,while the unencapsulated drugs were deactivated in 30 about days.This indicated that NK maintained high activity and stability under the protection of microcapsule shell.Finally,the NK microcapsules were simulated digestion in the gastrointestinal tract in vitro,and the kinetic model was fitted.It could be seen that the activity of NK is stable in the gastrointestinal tract digestion,and has a good slow-release effect in intestinal digestion.In the process of gastric digestion,the release of NK on fast-state condition and fed state condition to the Korsmeyr-Peppas and Zero-order kinetics,respectively,while in the intestinal digestion,the NK is more consistent with the First-order and Higuchi kinetics.The main content of this project is the improvement and innovation of natto flavor and the application of natto products,which has certain guiding significance for the development of natto food and the application of NK in medicine. |
