CN111040958A

CN111040958A - Preparation method of fermentation product with multiple physiological function activities and special bacillus amyloliquefaciens thereof

Info

Publication number: CN111040958A
Application number: CN201811195573.9A
Authority: CN
Inventors: 闫巧娟; 吴珊; 杨绍青; 江正强; 卢帅; 骆珅
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-10-15
Filing date: 2018-10-15
Publication date: 2020-04-21
Anticipated expiration: 2038-10-15
Also published as: CN111040958B

Abstract

The invention discloses a preparation method of a fermentation product with various physiological functional activities and a special Bacillus amyloliquefaciens thereof, wherein the preparation method comprises the steps of inoculating Bacillus amyloliquefaciens CAUNDJ118CGMCC No.16050 into sterile red beans or sterile bean curd, and fermenting to obtain the fermentation product, wherein compared with the sterile red beans or the sterile bean curd, the fibrinolytic activity and/or anticoagulant activity and/or α -glucosidase inhibition rate and/or total phenol content and/or polypeptide content of the fermentation product are obviously improved.

Description

Preparation method of fermentation product with multiple physiological function activities and special bacillus amyloliquefaciens thereof

Technical Field

The invention belongs to the field of fermentation, and particularly relates to a preparation method of a fermentation product with multiple physiological functional activities and a special bacillus amyloliquefaciens thereof.

Background

Thrombotic diseases are cardiovascular and cerebrovascular diseases that seriously threaten human health. It is statistically determined that thrombus blockage is an important factor in lethal disabling of thrombotic diseases (Lau et al Circulation Journal of the Japanese Circulation Society, 2017, 81: 920-. The current thrombolytic drugs for treating thrombotic diseases commonly used in clinic mainly comprise streptokinase, urokinase and tissue-type plasminogen activator (t-PA), however, most of these drugs have the disadvantages of high price, short half-life or easy bleeding (Wei et al. journal of agricultural and Food Chemistry, 2011, 59: 3957-. The exploration of safe and economic novel thrombolytic foods becomes one of the research hotspots at home and abroad in recent years.

Fermentation is a traditional processing technique that changes the physicochemical properties of food and enhances the functional activity of food. The application of fermentation technology is particularly extensive in asian regions. Asian people have low prevalence rate of thrombotic diseases, and the method is closely related to the frequent eating of fermented foods such as natto, fermented soybean, soybean paste and tempeh (Kotb. Springer briefs in Microbiology, 2012: 1-74). The use of microorganism fermentation of cereal beans not only eliminates anti-nutritional factors but also produces active ingredients and functional activities that cereal beans do not have originally (Chang et al food Chemistry, 2012, 133: 1611-. Bacillus is an important genus for producing plasmin activity. Sumi et al, Japan, isolated and extracted an active substance having an ability to efficiently dissolve fibrin from soybeans fermented with Bacillus subtilis (Sumi et al, Experientia, 1987, 43: 1110-1111). Jhan et al use Bacillus subtilis and Lactobacillus bulgaricus to ferment red beans in a mixed manner to obtain a fermented product with plasmin activity and enrich the probiotic species in the product (Jhan et al LWT-Food Science and Technology, 2015, 63: 1281) -1287.). Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a bacterium with high affinity with Bacillus subtilis, is generally present in fermented foods such as fermented soya beans or soybean paste and the like, and is widely applied as an antibacterial agent in the industries of food, feed and the like (Peng et al. comprehensive Biochemistry & Physiology B-Biochemistry & Molecular Biology, 2003, 134: 45-52; Chakraborty et al. food chemistry.2017, 218: 427-434; Liu Shi Zhe et al. A Bacillus amyloliquefaciens, an antibacterial agent prepared from the same and application thereof, Chinese patent application No. 201510330551.9). In recent years, the research on the preparation of plasmin by using bacillus amyloliquefaciens is gradually increased. Wang Xiao lan, etc. separates bacillus amyloliquefaciens producing plasmin from fermented soya beans, and the activity of the plasmin after liquid fermentation culture reaches 324.6-410.2IU/mL (Wang Xiao lan, etc., a fermented soya bean plasmin and its culture method, Chinese patent, application number: 201710320905.0). Although there are many reports on the utilization of the activity of Bacillus amyloliquefaciens to produce plasmin at home and abroad at present, the activity of the plasmin is mostly 100-3000IU/g (Gao Yang Xin et al, China brewing, 36: 58-62). The plasmin-producing activity level of bacillus utilized at home and abroad is still not high, and the plasmin-producing bacillus has less application in fermented foods.

Small red bean (Vigna umbellata), also known as small red bean, is sour and sour in taste, neutral in nature, non-toxic, and has the effects of inducing diuresis to alleviate edema, detoxifying and expelling pus, etc., and China is the world with the largest small red bean yield (Chenqiao, etc. Chinese patent medicine, 2017, 39: 1414 and 1422). The research finds that the small red beans are rich in essential amino acids, a plurality of unsaturated fatty acids, minerals and dietary fibers, and are functional food with high protein, low fat and multiple nutrients. The functional activity of small red beans in Vigna is higher than that of beans of other varieties (Bishtet al. genetic & Genomic Resources of gain Legend Improvement, 2013, 74: 237. 267.), which are rich in polyphenol, flavone and pigment, etc. (Yao et al. International Journal of molecular Sciences, 2012, 13: 2707. 2716). So far, small red bean processed products are few and are not fully developed and utilized (Pengxue et al. food industry science and technology, 2013, 34: 389-395). Research on the red bean peptide oral liquid with the effects of reducing blood pressure and blood sugar by fermenting red bean with aspergillus oryzae has been carried out, and no specific data are disclosed about the effects of reducing blood pressure and blood sugar (Zhangli et al. a method for preparing the red bean peptide oral liquid with the effects of reducing blood pressure and blood sugar by a fermentation method. Chinese patent application No. 201611146609.5). So far, no literature report and patent publication of the fibrinolytic enzyme activity produced by fermenting small red beans by utilizing bacillus amyloliquefaciens exist.

Bean curd is one of the traditional soybean products in China, and is a gel containing water, lipid and other components formed by extracting soybeans with water and coagulating protein with salt or acid. The bean curd contains abundant protein, and is a good source for obtaining high-quality protein. However, tofu is perishable and perishable due to its high moisture and high nutrition, and further has a short shelf life (Tian Qi & Hua Lian Fei. food and fat 2007, 137: 7-10). The fermentation can improve the storage property of food, and can also enhance the biological activities of raw materials such as oxidation resistance, obesity resistance, bacteria resistance and the like (the theory, university of south China, 2012, vector separation). Fermented bean curd with large consumption at home and abroad comprises Chinese fermented bean curd, stinky bean curd, Japanese Tofuyo, Tau ju in Malaya and the like, and the fermented food needs high salt content (4-14%) to inhibit the growth of harmful microorganisms in the fermentation process, and has a long fermentation period which is generally 3-6 months (Lioe et al food Chemistry, 2018, 252: 265. sup. 270.). So far, no research related to bean curd fermentation by using bacillus amyloliquefaciens exists.

Disclosure of Invention

The invention aims to provide a fermentation product with various physiological functional activities, and the fermentation product can be fermented small red beans or fermented bean curd.

In order to protect the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118, the strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 of Xilu No.1 of Beijing Kogyo-Yang district) in 2018, at the 03 th of 07 th and 03 th, and the preservation number is CGMCC No. 16050. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118CGMCC No.16050 is simply called Bacillus amyloliquefaciens CAUNDJ 118.

The invention also protects a microbial inoculum which contains the bacillus amyloliquefaciens CAUNDJ 118.

The microbial inoculum can be used for preparing at least one of A) small red beans through fermentation culture, A) products with plasmin activity, A) products with anticoagulant activity, A) products with blood sugar reducing function, A) products with antioxidant activity, A) products for eliminating DPPH free radicals, A) products for eliminating ABTS free radicals, A) products for improving FRAP reducing power, A) products for improving glucosidase inhibiting power, A) products for improving DPP-IV inhibiting power, A) products for improving angiotensin converting enzyme inhibiting power, A) products for improving acetylcholine esterase inhibiting power, A) products for improving total phenol content, A) products for improving flavone content, A) products for improving polypeptide content, A) products for improving SOD enzyme content, A) products for preventing and/or treating cardiovascular and cerebrovascular diseases, A) products for preparing products for preventing and/or treating thrombotic diseases, A) products for improving thrombocyte inhibiting power and/or treating hypertension, A) products for improving DPP-IV inhibiting power, A) products for improving DPP-A inhibiting power and/or treating hypertension, A) products for improving DPP-IV inhibiting power, A) products for improving free radical inhibiting power and/or treating diabetes mellitus, A) products for improving free radical inhibiting power, A) products for treating or preventing hypertension and/or treating diabetes mellitus, A) products for improving hypertension and/or treating hypertension, and/or treating senile dementia, A) products for improving free radical inhibiting power, and/or improving DPP-IV, and/or preventing hypertension, A) products for treating hypertension, and/or treating hypertension, and/or improving hypertension, and/or treating hypertension, or improving hypertension, and/or treating hypertension, or improving hypertension, and/or preventing hypertension, or treating hypertension, and/or treating hypertension, or improving hypertension, or treating hypertension, and/or treating hypertension.

The invention also provides a preparation method of the microbial inoculum, which comprises the following steps: inoculating the bacillus amyloliquefaciens CAUNDJ118 to a bacterial culture medium and culturing to obtain a bacterial liquid, namely the microbial inoculum.

The bacterial culture medium can be LB liquid culture medium. The preparation method of the LB liquid culture medium can be specifically as follows: dissolving 10g of peptone, 10g of sodium chloride and 5g of yeast powder in deionized water, and then using the deionized water to fix the volume to 1L; sterilizing at 121 deg.C for 15 min.

In the preparation method of the microbial inoculum, the specific culture conditions can be as follows: 35-39 deg.C (such as 35-37 deg.C, 37-39 deg.C, 35 deg.C, 37 deg.C or 39 deg.C), 100-300rpm (such as 100-200rpm, 200-300rpm, 100rpm, 200rpm or 300rpm), and culturing for 10-20h (such as 10-15h, 15-20h, 10h, 15h or 20h) with shaking.

The microbial inoculum may include a carrier in addition to the active ingredient. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymeric compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth. The plant material may be at least one of corn flour, bean flour and starch. The high molecular compound may be polyvinyl alcohol and/or polyglycol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water. The organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

The invention also discloses application of the bacillus amyloliquefaciens CAUNDJ118 or any microbial inoculum described above, which can be at least one of A1) -A43), A1) fermentation culture of small red beans, A2) preparation of products with fibrinolytic enzyme activity, A3) preparation of products with anticoagulant activity, A4) preparation of products with blood sugar reducing function, A5) preparation of products with antioxidant activity, A6) preparation of products for eliminating DPPH free radicals, A7) preparation of products for eliminating ABTS free radicals, A8) preparation of products for improving FRAP reducing power, A8) preparation of products for improving 8-glucosidase rate, A8) preparation of products for improving DPP-IV inhibiting rate, A8) preparation of products for improving angiotensin converting enzyme inhibiting rate, A8) preparation of products for improving angiotensin A inhibiting enzyme inhibiting rate or improving angiotensin A8) inhibiting rate, B-A activity, B-8) preparation of products for preparing products for improving, A8) preparation of products for improving, 8) for improving angiotensin-B-8) for improving, 8) preparation of products for improving angiotensin-B-8) for treating, or 8) for treating diabetes mellitus, or 8) for treating or improving blood glucose-B-8) for treating diabetes mellitus, or improving blood sugar-8) treating diabetes mellitus, or improving blood glucose-8) for treating diabetes mellitus, or improving blood glucose-8) treating diabetes mellitus, or improving blood sugar-8) treating diabetes mellitus, or improving total-8) treating diabetes mellitus, or improving blood sugar-8) treating diabetes mellitus, or improving blood-8.

In the application, the product can be functional food, health-care product and medicine.

In the above application, the product may be fermented red bean or fermented bean curd prepared by any one of the following methods.

The invention also provides a preparation method of the fermented red bean, which comprises the steps of inoculating bacillus amyloliquefaciens into the sterile red bean, and fermenting to obtain the fermented red bean, wherein compared with the sterile red bean, the fibrinolytic enzyme activity and/or the anticoagulant activity and/or the antioxidant activity and/or the DPPH free radical clearance rate and/or the ABTS free radical clearance rate and/or the FRAP reducing power and/or α -glucosidase inhibition rate and/or the DPP-IV inhibition rate and/or the total phenol content and/or the flavone content and/or the polypeptide content and/or the SOD enzyme content of the fermented red bean are improved (obviously improved).

In the method, the steps for preparing the sterile small red beans can be as follows: soaking semen Phaseoli in water, and steaming for 20-40min (such as 20-30min, 30-40min, 20min, 30min or 40 min).

The time for soaking the small red beans can be more than 9 hours.

The soaking time of semen Phaseoli is 9-12h (such as 9-11h, 11-12h, 9h, 11h or 12 h).

The temperature for soaking semen Phaseoli can be 22-30 deg.C (such as 22-25 deg.C, 25-30 deg.C, 22 deg.C, 25 deg.C or 30 deg.C).

The small red bean is soaked in water, and the soaking degree can be as follows: the wet weight of the soaked small red beans is 2.5 +/-0.5 times of that of the small red beans before soaking.

In the method, the inoculation amount of the bacillus amyloliquefaciens for inoculating the sterile small red beans can be 10⁵-10⁷CFU/100g (e.g., 10)⁵-10⁶CFU/100g、10⁶-10⁷CFU/100g、10⁵CFU/100g、10⁶CFU/100g or 10⁷CFU/100g) sterile red bean.

The invention also provides a preparation method of the fermented bean curd, which comprises the steps of inoculating the bacillus amyloliquefaciens into the sterile bean curd, and fermenting to obtain the fermented bean curd, wherein compared with the sterile bean curd, the fibrinolytic enzyme activity and/or the anticoagulation activity and/or the acetylcholinesterase inhibition rate and/or the angiotensin converting enzyme inhibition rate and/or the α -glucosidase inhibition rate and/or the total phenol content and/or the polypeptide content of the fermented bean curd are improved (obviously improved).

In the above method, the step of preparing the sterilized bean curd may be: oven drying bean curd, and steaming for 10-40min (such as 10-20min, 20-30min, 30-40min, 10min, 20min, 30min or 40 min).

The drying temperature can be 25-40 deg.C (such as 25-30 deg.C, 30-40 deg.C, 25 deg.C, 30 deg.C or 40 deg.C)

The degree of drying may be: the water content of the bean curd block is more than 55%.

The degree of drying may specifically be: the moisture content of the bean curd block is 55-85% (m/m) (such as 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%).

The protein content of the bean curd can be more than 5% (m/m). The water content of the bean curd is 70-90%.

The protein content of the bean curd may be 5-10% (m/m) (e.g. 5-8%, 8-10%, 5%, 8% or 10%).

The bean curd can be commercially available bean curd (raw materials can be common beans such as soybean, soybean and the like), and can also be prepared by using common beans (such as soybean, mung bean, small red bean and the like) as raw materials.

The bean curd can be cut into bean curd blocks firstly, and then the bean curd blocks are dried.

The length, width and height of the bean curd block can be more than 2 cm.

The length, width and height of the bean curd block can be 2-5cm (such as 2-3cm, 3-4cm, 4-5cm, 2cm, 3cm, 4cm or 5 cm).

In the above method, the inoculation amount of the "inoculating Bacillus amyloliquefaciens into sterile bean curd" may be 10⁷-10⁹CFU/100g (e.g., 10)⁷-10⁸CFU/100g、10⁸-10⁹CFU/100g、10⁷CFU/100g、10⁸CFU/100g or 10⁹CFU/100g) sterilized bean curd.

In any of the above methods, the cooking temperature may be 95-121 deg.C (e.g., 95-110 deg.C, 121 deg.C, 95 deg.C, 110 deg.C, or 121 deg.C).

In any of the above methods, the fermentation may specifically be performed at 35-40 deg.C (e.g., 35-37 deg.C, 37-40 deg.C, 35 deg.C, 37 deg.C or 40 deg.C) for 24-48h (e.g., 24-36h, 36-48h, 24h, 36h or 48 h).

In any of the above methods, the Bacillus amyloliquefaciens may specifically be Bacillus amyloliquefaciens CAUNDJ 118.

The invention also protects K1) or K2).

K1) The application of the fermented red bean prepared by any one of the methods can be at least one of A22), A22) for producing plasmin, A22) for anticoagulation, A22) for reducing blood sugar, A22) for antioxidation, A22) for eliminating DPPH free radicals, A22) for eliminating ABTS free radicals, A22) for improving the reducing power of FRAP, A22) for improving 22-glucosidase inhibition rate, A22) for improving DPP-IV inhibition rate, A22) for improving total phenol content, A22) for improving flavone content, A22) for improving polypeptide content, A22) for improving the enzyme content, A22) for treating a cardiovascular disease or preventing a diabetic disease, and/or treating a diabetic/diabetic disease.

K2) The fermented bean curd prepared by any one of the methods can be at least one of A22), A23), A24), A29), A31), A32), A33), A35), A37), A38), A39), 40), 41) and A43), wherein A22) produces plasmin, A23) anticoagulation, A24) reduces blood sugar, A29) improves α -glucosidase inhibition rate, A31) improves angiotensin converting enzyme inhibition rate, A32) improves acetylcholine esterase inhibition rate, A33) improves total phenol content, A35) improves polypeptide content, A37) prevents and/or treats cardiovascular and cerebrovascular diseases, A38) prevents and/or treats thrombotic diseases, A39) prevents and/or treats diabetes, 40) prevents and/or treats hypertension, 41) prevents and/or treats senile 43) and reduces senile dementia.

Experiments prove that the plasmin activity of the fermented red bean prepared by the method provided by the invention is as high as 78.0FU/g wet weight (4890.0IU/g wet weight) and 220FU/g dry weight (13814IU/g dry weight), compared with the unfermented red bean, the anticoagulant activity, the antioxidant activity, the α -glucosidase inhibition rate and the DPP-IV inhibition rate of the fermented red bean are all obviously improved, the total phenol content, the flavone content, the polypeptide content and the SOD enzyme content are also obviously improved, the fermented red bean has the effects of reducing blood sugar and resisting blood coagulation, and has great application value in developing products for preventing and/or treating cardiovascular and cerebrovascular diseases and/or diabetes.

Drawings

FIG. 1 shows the effect of cooking time on the viscosity (▲), firmness (●) and plasmin activity (■) of B.amyloliquefaciens fermented red beans.

FIG. 2 shows the effect of inoculum size on the viscosity (▲), firmness (●) and plasmin activity (■) of B.amyloliquefaciens fermented red beans.

FIG. 3 is a graph showing the effect of fermentation time on the viscosity (▲), firmness (●) and plasmin activity (■) of B.amyloliquefaciens fermented red beans.

FIG. 4 shows DPP-IV inhibitory effects of unfermented red bean (▲) and fermented red bean (●), in which (■) is a positive control, statin A.

FIG. 5 shows the α -glucosidase inhibitory effect of unfermented red bean (▲) and fermented red bean (●), in which (◆) is the positive control acarbose.

FIG. 6 shows the anticoagulant activity of unfermented red bean (▲) and fermented red bean (●), in which (×) is the positive control heparin sodium.

Deposit description

The strain name is as follows: bacillus amyloliquefaciens

Latin name: bacillus amyloliquefaciens

The strain number is as follows: CAUNDJ118

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: year 2018, month 07, 03

Registration number of the preservation center: CGMCC No.16050

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The following examples were carried out using the fibrin plate method for detecting plasmin activity as follows:

a clean culture dish is taken, and 5mL of 1% (w/v) agarose aqueous solution, 5mL of 0.6% (w/v) fibrinogen solution (solvent is pH8.0, 50mmol/L borax buffer solution) and 100 μ L of thrombin (concentration is 10U/mL) are added into each culture dish and mixed uniformly. After it is cooled (i.e. fibrin plate), the plate is perforated uniformly with a hole puncher, the hole diameter is 3mm, and the residual water in the hole is sucked dry. 10 μ L of the sample was added to the wells, incubated at 37 ℃ for 18h, and the diameter of the transparent circle was measured using an electronic ruler using a cross method.

And (3) drawing a standard curve by taking urokinase as a standard substance: with urokinase fibrinolytic enzyme activity (IU/mL): 20. 40, 60, 80, 100 and 200, 400, 600, 800 and 1000 are vertical coordinates, the area of a transparent ring is horizontal coordinates, two urokinase standard curves with different concentration series are respectively drawn, and the equivalent of the fibrinolytic enzyme activity of the sample is calculated according to a standard regression equation, wherein the unit IU/g wet bean.

The following examples were carried out using the fibrinolysis method to detect the fibrinolytic enzyme activity as follows:

adding 1.4mL of pH8.0 and 50mmol/L borax buffer solution and 0.4mL of 0.72% (w/v) fibrinogen solution (the solvent is pH8.0 and 50mmol/L borax buffer solution) into a test tube, uniformly mixing, preheating in a water bath kettle at 37 ℃ for 5min, adding 0.1mL of thrombin (the concentration is 20U/mL), uniformly mixing, and reacting at 37 ℃ for 10min to solidify. Adding 0.1mL of sample, mixing uniformly, reacting at 37 ℃ for 60min, and shaking and mixing uniformly every 20 min. Immediately after the reaction, 2.0mL of a 0.2M trichloroacetic acid solution (terminator) was added, the mixture was centrifuged at 10000rpm for 10min, and the supernatant was collected to determine the absorbance at 275 nm.

Definition of Activity: the amount of enzyme required for an increase in absorbance at 275nm per minute of 0.01 localizes the fibrin degrading enzyme activity (FU) of 1 unit.

The calculation formula is as follows: x is (Ar-Ac). times.N/(60X 0.01X 0.1), wherein X represents the plasmin activity (FU/mL), Ar represents the absorbance of the sample, Ac represents the absorbance of the control, 60 represents the reaction time 60min, and 0.1 represents 0.1mL of the enzyme solution participating in the reaction, measured in 1 min. N represents the dilution factor.

The procedure for determining hardness in the following examples is as follows:

the small red bean sample was taken out from the refrigerator at 4 ℃ and left to stand at room temperature, and the hardness was measured by a texture analyzer. The test method comprises the following steps: 25000g of load unit and TA 4/100038.1-mm D of probe, 3 small red bean samples are parallelly placed, the compression deformation amount is 60%, the trigger point load is 5g, the testing speed is 1mm/s, each sample is parallelly tested for 6 times, and the average value is taken.

The procedure for determining the viscosity in the following examples is as follows:

taking 10g of small red bean sample, adding 100mL of distilled water, shaking and extracting for 30min, centrifuging at 10000rpm for 20min, collecting supernatant, and measuring viscosity at 30 ℃.

LB liquid medium: dissolving 10g of peptone, 10g of sodium chloride and 5g of yeast powder in deionized water, and then using the deionized water to fix the volume to 1L; sterilizing at 121 deg.C for 15 min.

LB solid medium: dissolving 10g of peptone, 10g of sodium chloride, 5g of yeast powder and 7g of agar in deionized water, and then fixing the volume to 1L by using the deionized water; sterilizing at 121 deg.C for 15 min.

LB solid plate: and pouring the LB solid culture medium with the temperature of about 55 ℃ into a sterile culture dish, and cooling to obtain an LB solid plate.

Methods for determining total phenol content and flavone content are described in the following documents: yao, Y., Cheng, X.Z., Wang, L.X., Wang, S.H., & Ren, G. (2012). Major phenolic compounds, antioxidant capacity and antibiotic beta potential of a rice bean (Vigna umbellate L.). International Journal of Molecular Sciences, 13, 2707-.

Methods for determining the content of polypeptides are described in the following documents: zhang, b., Sun, q., Liu, h.j., Li, s.z., & Jiang, Z.Q. (2017.) the mutation of action from Chinese customers and matters applications in the medium termination and production of antibiotic i-converting enzyme (ACE) inhibition peptides lwt-Food Science and technology, 78, 1-7.

Methods for determining the SOD enzyme content are described in the following documents: dong, D, Yin, L, Qi, Y, Xu, L, & Peng, J. (2015). Protective effect of the total saponin from a rock strain of microxfrouit against carbon nanoparticles, 7(6), 4829.

Methods for determining the reducing sugar content are described in the following documents: sritongtae, B., Sangsukiam, T., Morgan, M.R., & Duangmal, K. (2017). Effect of acid prediction and simulation on the composition and antibiotic activity of edge bean (Vigna umbellata). Food Chemistry, 227, 280-.

Methods for determining DPPH radical clearance, ABTS radical clearance and FRAP reduction are described in the following documents: dudonn, S., Vitrac, X., Courtere, P., Wolinez, M., & M rillon, J.M. (2009).

Example 1 isolation, identification and preservation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118CGMCCNo.16050

Isolation of the bacterium CAUNDJ118

1. Adding 1g of commercially available Lentinus Edodes sauce into 10mL LB liquid culture medium, mixing, and culturing at 37 deg.C and 200rpm for 12 hr to obtain enrichment solution.

2. After the step 1 is completed, the enrichment solution is taken and added into a sterile test tube filled with 9mL of sterile physiological saline to be fully and uniformly mixed (the dilution is marked as 10-¹) Then sucking 1mL of the mixture from the test tube, adding the mixture into another sterile test tube containing 9mL of sterile physiological saline, and uniformly mixing, and so on to obtain 10^-2、10^-3、10^-4、10^-5Bacterial suspensions of various dilutions. 0.1mL of each dilution was uniformly spread on an LB solid plate, 37Standing at constant temperature for 24 h.

3. And (3) after the step 2 is completed, selecting the colonies, and streaking the colonies on an LB solid plate to obtain single colonies.

4. And (3) respectively inoculating the single colonies obtained in the step (3) to an LB liquid culture medium, and culturing at 37 ℃ and 200rpm for 12h to obtain a culture bacterial liquid.

5. Dongnong 690 (hereinafter abbreviated as "Xiao-jin") was collected and soaked in water for 12 hours.

6. Inoculating the small-particle soybeans obtained in the step 5 with the culture solution obtained in the step 4 (the inoculation amount is 10 percent)⁷CFU/100g), mixing, fermenting and culturing at 40 deg.C for 24 hr, and after-ripening at 4 deg.C for 24 hr to obtain fermented semen glycines. The degree of stringiness and odor of each fermented soybean was recorded and fibrinolytic activity was measured by the fibrin plate method.

7. And (6) after the step 6 is finished, taking fermented soybeans which have natto flavor and high wire drawing degree and generate larger transparent rings on a fibrin flat plate, and detecting the activity of fibrinolytic enzyme by adopting a fibrin degradation method.

The single colony corresponding to the fermented soybean with higher plasmin activity is repeatedly confirmed for 3-5 times and named as bacterium CAUNDJ 118.

Identification of the bacterium CAUNDJ118

The isolated and purified bacterium CAUNDJ118 was identified in several ways:

1. morphological identification

The single colony status of the bacteria CAUNDJ118 growing on the LB solid plate was observed, and mainly included the color, transparency, wettability, colony surface status (smooth, protruding, wrinkled, dented, etc.), and colony edge status (neat, irregular, radial, etc.) of the colonies.

The result shows that the colony surface of the bacterium CAUNDJ118 separated and purified in the first step is round, slightly convex, milky yellow, semitransparent and sticky after being picked up; the colony is smooth and moist when young, and the colony edge is irregular and has the fold after the maturity.

Microscopic observation revealed that the bacterial bodies of CAUNDJ118 were in the form of short rods.

2. Physiological and biochemical characteristic analysis

The physiological and biochemical characteristics of the above-mentioned bacterium CAUNDJ118 were determined with reference to "Manual of identification of common bacterium System" (Dongxu bead, Chuia Miaoying. Manual of identification of common bacterium System. Beijing: scientific Press, 2011.) and "microbiological experiments" (Shenping, Fangxiu capacity, Liguanwu. microbiological experiments (third edition); Beijing: advanced education Press, 1999.).

The physiological and biochemical characteristics of the bacterium CAUNDJ118 are as follows: gram-positive bacteria, positive starch hydrolysis, positive gelatin liquefaction test, positive lecithin enzyme, negative phenylalanine dehydrogenase, positive V-P test and negative M.R enzyme, and can utilize glucose, sucrose, lactose, D-xylose and L-arabinose.

3. 16S rDNA sequence analysis

The isolated and purified bacterium CAUNDJ118 obtained in one of the above steps was cultured by a conventional method, the total DNA of the strain was extracted and used as a template, and the DNA sequence of the bacterium 16S rDNA universal primer 27 f: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492 r: 5'-GGTTACCTTGTTACGACTT-3' PCR amplification is carried out to obtain PCR amplification product. Reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 51 ℃ for 45s, extension at 72 ℃ for 1.5min, and 30 cycles; extension at 72 ℃ for 10 min.

The PCR amplification product was subjected to sequencing by Biotechnology Ltd of Bomaide, Beijing. And carrying out gene comparison analysis on the sequencing result, and constructing a phylogenetic tree by using MEGA 5.0. Gene comparison was done online using the national center for Biotechnology information NCBI database (http:// www.ncbi.nlm.nih.gov).

The 16S rDNA sequence of the bacterium CAUNDJ118 is shown as a sequence 1 in a sequence table. Phylogenetic trees were constructed by MEGA5.0 software, showing that the bacterium CAUNDJ118 belongs to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

The bacterium CAUNDJ118 isolated and purified in step one was identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) in view of the above morphological characteristics, physiological and biochemical characteristics analysis and 16S rDNA sequence analysis. The bacterium CAUNDJ118 has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 3 Xilu No.1 Beijing, Chaoyang, China) in 2018, No. 07/03, and the deposition number is CGMCC No. 16050. The bacterium CAUNDJ118 is called Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118CGMCC No.16050, and is called Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118 for short.

Example 2 Effect of cooking time, inoculum size and fermentation time on the viscosity, firmness and plasmin Activity of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118 fermented Red Bean

Preparation of bacterial liquid

1. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118 was inoculated into LB solid medium and cultured at 37 ℃ for 12 hours (for activation).

2. And (3) inoculating the single colony obtained in the step (1) to a10 mLLB liquid culture medium, and culturing at 37 ℃ and 200rpm to obtain a bacterial liquid.

Second, red bean pretreatment

1. Soaking semen Phaseoli in water at 25 deg.C for 9-12 hr to obtain soaked semen Phaseoli.

The wet weight of the soaked red bean is 2.5 +/-0.5 times of that of the red bean before soaking.

2. And (3) after the step (1) is finished, taking the soaked beans, and removing the soaked beans which are incomplete and/or uneven in color and/or poor in water absorption to obtain selected soaked beans.

3. After the step 2 is finished, selecting the selected soaked beans, steaming at 121 ℃ under high pressure for 10, 20, 30, 35, 40, 45 or 50min, and then cooling to 30-40 ℃ to obtain the unfermented small red beans.

Thirdly, fermentation

Inoculating the unfermented red bean obtained in the step two with the bacterial liquid obtained in the step one (the inoculation amount is 10)⁵CFU/100g、10⁶CFU/100g、10⁷CFU/100g、10⁸CFU/100g or 10⁹CFU/100g), mixing well, fermenting and culturing at 40 ℃ for 6h, 12h, 18h, 24h, 30h or 36h to obtain the fermented red bean.

Fourthly, detecting the fibrinolytic enzyme activity, hardness and viscosity of the fermented small red beans

Taking fermented red bean, and detecting the fibrinolytic enzyme activity by adopting a fibrin degradation method and a fibrin plate method respectively.

Taking fermented small red beans, and measuring hardness and viscosity.

The results show that the hardness of the fermented red bean is decreased and the viscosity and the plasmin activity are increased along with the increase of the cooking time. When the inoculation amount is 10⁵When CFU/100g and fermentation time are 24h, the fibrinolytic enzyme activity, hardness and viscosity of the fermented small red beans are shown in figure 1; the cooking time is 40min, the fibrinolytic enzyme activity of the fermented red bean is highest and reaches 73.7FU/g wet weight. When the fermentation time is 24h and the cooking time is 40min, the fibrinolytic enzyme activity, hardness and viscosity of the fermented red bean are shown in figure 2; the inoculation amount is 10⁷CFU/100g (namely the inoculation amount is logCFU/100g and is 7), the fibrinolytic enzyme activity of the fermented small red beans is the highest and reaches 78.0FU/g wet weight. When the inoculation amount is 10⁷When CFU/100g and cooking time is 40min, the fibrinolytic enzyme activity, hardness and viscosity of the fermented red bean are shown in figure 3; the fermentation time is 24 hours, the plasmin activity of the fermented red bean is the highest, the plasmin activity detected by a fibrin degradation method is 78.0FU/g wet weight (220FU/g dry weight), and the plasmin activity detected by a fibrin plate method is 4890.0IU/g wet weight (13814IU/g dry weight).

Example 3 analysis of active ingredients and functional Activity of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118 fermented red Bean

Preparation of bacterial liquid

2. And (3) inoculating the single colony obtained in the step (1) to a10 mLLB liquid culture medium, and culturing at 37 ℃ and 200rpm for 12h to obtain a bacterial liquid.

3. Adding LB liquid culture medium into the bacterial liquid obtained in the step 2 for dilution to obtain the bacterial liquid with the concentration of 10⁷CFU/100g of bacterial liquid.

Second, red bean pretreatment

3. After the step 2 is finished, selecting the selected soaked beans, steaming at 121 ℃ for 40min under high pressure, and then cooling to 30-40 ℃ to obtain the unfermented small red beans.

Thirdly, fermentation

Inoculating the bacterial liquid obtained in the first step with the unfermented small red beans, wherein the inoculation amount is 10⁷Mixing with CFU/100g, fermenting and culturing at 40 deg.C for 24 hr to obtain fermented semen Phaseoli.

Analysis of active ingredients

1. Measuring total phenol content, flavone content, polypeptide content and SOD enzyme content

(1) Mixing 1 volume part of fermented red bean with 5 volume parts of water, performing ultrasonic extraction for 3 times (ultrasonic parameters: 100W, 20min), centrifuging at 4000rpm for 10min, and collecting supernatant 1. Mixing 1 volume part of fermented red bean with 5 volume parts of 80% (v/v) ethanol water solution, performing ultrasonic extraction for 3 times (ultrasonic parameters: 100W, 20min), centrifuging at 4000rpm for 10min, and collecting supernatant 2.

(2) And (3) taking the supernatant fluid 1, and performing rotary evaporation and concentration to obtain a water extract of the fermented small red beans. And (3) taking the supernatant 2, and performing rotary evaporation and concentration to obtain an alcohol extract of the fermented small red beans.

(3) And (3) taking the water extract of the fermented small red beans or the alcohol extract of the fermented small red beans, and measuring the total phenol content, the flavone content, the polypeptide content and the SOD enzyme content.

2. Taking fermented small red beans, and measuring hardness and viscosity.

3. Determination of reducing sugar content

Taking fermented small red beans, and measuring the content of reducing sugar.

4. Determination of the moisture content

Taking fermented small red beans, and determining the moisture content according to GB/T5009.3-2010.

Replacing the fermented red bean with unfermented red bean according to the above steps, and using the rest steps as a control.

The results are shown in Table 1.

The results show that compared with the unfermented red bean, the total phenol content, the flavone content, the polypeptide content and the SOD enzyme content in the fermented red bean are respectively improved by 3.3 times, 1.5 times, 5.1 times and 4.8 times, the hardness is obviously reduced, and the viscosity, the reducing sugar content and the water content are all obviously improved.

TABLE 1 physicochemical Properties of fermented and unfermented red beans

Note that a, b are different, which indicates that the difference between groups is significant, and p is less than 0.05; GAE, gallic acid equivalent.

Fifth, analysis of functional Activity

1. Preparation of fermented red bean freeze-dried powder and unfermented red bean freeze-dried powder

Placing the unfermented red bean in a refrigerator at-20 deg.C for freezing for 24h, transferring to a vacuum freeze drier (cold trap temperature is-48 deg.C-60 deg.C), and freeze drying for 35-40h to obtain unfermented red bean lyophilized powder.

Freezing the fermented red bean in a refrigerator at-20 deg.C for 24h, transferring to a vacuum freeze drier (cold trap temperature is-48 deg.C to-60 deg.C), and freeze drying for 35-40h to obtain fermented red bean lyophilized powder.

2. Antioxidant activity of fermented red bean

The antioxidant activity is mainly reflected in DPPH free radical clearance rate, ABTS free radical clearance rate and FRAP reducing power.

(1) Mixing 1 volume part of fermented red bean lyophilized powder and 5 volume parts of water, performing ultrasonic extraction for 3 times (ultrasonic parameters: 100W, 20min), centrifuging at 4000rpm for 10min, and collecting supernatant 1. Mixing 1 volume part of fermented red bean lyophilized powder and 5 volume parts of 80% (v/v) ethanol water solution, performing ultrasonic extraction for 3 times (ultrasonic parameters: 100W, 20min), centrifuging at 4000rpm for 10min, and collecting supernatant 2.

(3) And (3) taking the water extract of the fermented small red beans or the alcohol extract of the fermented small red beans, and measuring DPPH free radical clearance rate, ABTS free radical clearance rate and FRAP reducing power.

Replacing the fermented red bean with unfermented red bean according to the steps, and obtaining the DPPH free radical clearance, ABTS free radical clearance and FRAP reducing power of the water extract or the alcohol extract of the unfermented red bean without changing other steps.

The results are shown in Table 2.

The results show that compared with the unfermented red bean, the DPPH free radical clearance rate, ABTS free radical clearance rate and FRAP reducing power in the fermented red bean are obviously improved, which indicates that the fermented red bean has higher antioxidant activity.

TABLE 2 antioxidant action of unfermented and fermented red beans

Note that a, b, c, d show significant differences between groups, p < 0.05.

3. Hypoglycemic effect of fermented small red beans

(1) The DPP-IV inhibition ratio of samples to be tested (fermented red bean lyophilized powder of different concentrations, unfermented red bean lyophilized powder of different concentrations or dipeptide inhibin A) was determined according to the method described in the literature (Wang, T.Y., Hsieh, C.H., Hung, C.C., Jao, C.L., Lin, P.Y., & Hsieh, Y.L., et al (2017). A study to estimate the potential of peptide-IV inhibitor activity in vitamin A. food Chemistry, 234, 431 and 438). Aprotinin a was a positive control.

The results of the experiment are shown in FIG. 4. The result shows that the DPP-IV inhibition rate of the fermented small red bean freeze-dried powder is obviously higher than that of the unfermented small red bean. When the concentrations of the fermented red bean freeze-dried powder and the unfermented red bean freeze-dried powder are both 6.4mg/mL, the DPP-IV inhibition rate (65.6%) of the fermented red bean freeze-dried powder is 2.2 times of that of the unfermented red bean.

(2) α -glucosidase inhibition rate of red bean samples to be tested (fermented red bean lyophilized powder of different concentration, unfermented red bean lyophilized powder of different concentration or acarbose) was detected as positive Control according to the method described in the literature (Shukla, s., Park, j., Kim, d.h., Hong, s.y., Lee, j.s., & Kim, M. (2016) (Total phenolic content, antioxidant, tyrosinase and α -glucopyranoside extraction of soluble mutant consumer enzyme, food Control, 59, 854-one 861.).

The experimental result is shown in figure 5. the result shows that the α -glucosidase inhibition rate of the fermented red bean freeze-dried powder is obviously higher than that of the unfermented red bean, and when the concentrations of the fermented red bean freeze-dried powder and the unfermented red bean freeze-dried powder are both 11.2mg/mL, the α -glucosidase inhibition rate (88.9%) of the fermented red bean freeze-dried powder is 3.2 times of that of the unfermented red bean.

The results show that the fermented small red beans have the function of reducing blood sugar.

4. Anticoagulant activity of fermented small red beans

The anticoagulant activity of the samples to be tested (freeze-dried powder of fermented red bean at different concentrations, freeze-dried powder of unfermented red bean at different concentrations or heparin sodium) was tested according to the method described In the literature (Zhang, S.B, (2016). Heparin sodium was the positive control.

The results of the experiment are shown in FIG. 6. The result shows that the anticoagulant activity of the fermented small red bean freeze-dried powder is obviously higher than that of the unfermented small red bean. When the concentrations of the fermented red bean freeze-dried powder and the unfermented red bean freeze-dried powder are both 1.0mg/mL, the anticoagulant activity (98.2%) of the fermented red bean freeze-dried powder is 15.6 times of that of the unfermented red bean and is 1.7 times of that of heparin sodium. Therefore, the fermented small red beans have good anticoagulant activity.

Example 4 analysis of active ingredients and functional Activity of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118 fermented Bean curd

The tofu in this example is commercially available, and the raw material for processing tofu is soybean.

Preparation of bacterial liquid

2. And (3) inoculating the single colony obtained in the step (1) to 10mL of LB liquid culture medium, and culturing at 37 ℃ and 200rpm for 12h to obtain a bacterial liquid.

3. Adding LB liquid culture medium into the bacterial liquid obtained in the step 2 for dilution to obtain the bacterial liquid with the concentration of 10⁸CFU/100g of bacterial liquid.

Second, pretreatment of bean curd

1. Cutting bean curd into blocks with length, width and height of 2-5cm, and oven drying in oven at 25-40 deg.C until water content is 55-65% to obtain dried bean curd block.

2. And (3) after the step (1) is finished, taking the dried bean curd blocks, and removing the bean curd blocks which are incomplete and/or uneven in color and/or high in moisture to obtain the selected bean curd blocks.

3. And (3) after the step 2 is finished, taking the selected bean curd blocks, cooking for 10-40min at the temperature of 121 ℃, and then cooling to 30-40 ℃ to obtain the unfermented bean curd blocks.

Thirdly, fermentation

Taking the unfermented bean curd block, inoculating the bacterial liquid obtained in the step one, wherein the inoculation amount is 10⁸Mixing CFU/100g, fermenting and culturing at 40 deg.C for 48 hr to obtain fermented bean curd.

Analysis of active ingredients

1. Preparation of fermented bean curd lyophilized powder and unfermented bean curd lyophilized powder

Placing the unfermented bean curd in a refrigerator of-20 deg.C, freezing for 24 hr, transferring to a vacuum freeze drier (cold trap temperature is-48 deg.C to-60 deg.C), and freeze drying for 35-40 hr to obtain unfermented bean curd lyophilized powder.

Freezing the fermented bean curd in a refrigerator at-20 deg.C for 24 hr, transferring to a vacuum freeze drier (cold trap temperature is-48 deg.C to-60 deg.C), and freeze drying for 35-40 hr to obtain fermented bean curd lyophilized powder.

2. Mixing 1 volume part of fermented bean curd lyophilized powder and 5 volume parts of water, performing ultrasonic extraction for 3 times (ultrasonic parameters: 100W, 20min), centrifuging at 4000rpm for 10min, and collecting supernatant 1. Mixing 1 volume part of fermented bean curd lyophilized powder and 5 volume parts of 80% (v/v) methanol water solution, ultrasonic extracting for 3 times (ultrasonic parameters: 100W, 20min), centrifuging at 4000rpm for 10min, and collecting supernatant 2.

3. And (3) taking the supernatant 1, carrying out rotary steaming and concentration to obtain a fermented bean curd water extract, and further carrying out freeze drying to obtain the fermented bean curd water extract freeze-dried powder 1. And (3) taking the supernatant 2, carrying out rotary steaming and concentration to obtain a fermented bean curd alcohol extract, and further carrying out freeze drying to obtain the fermented bean curd alcohol extract freeze-dried powder 2.

And calculating the yield of the fermented bean curd water extract freeze-dried powder 1 and the fermented bean curd alcohol extract freeze-dried powder 2 according to a formula.

The yield (%) is the weight of the extract freeze-dried powder/the weight of the raw material freeze-dried powder x 100.

4. And (3) taking the freeze-dried powder of the water extract of the fermented bean curd or the freeze-dried powder of the alcohol extract of the fermented bean curd, and measuring the content of the polypeptide and the content of the total phenol.

According to the above steps, the fermented bean curd is replaced by unfermented bean curd, and other steps are not changed as a control.

The results are shown in Table 3.

TABLE 3 Total phenol content and polypeptide content of unfermented tofu and fermented tofu

Note: the difference of the shoulder mark letters (a, b, c) in the same row indicates that the difference is remarkable among groups, and p is less than 0.05; GAE, gallic acid equivalent.

The results show that compared with unfermented bean curd, the polypeptide content and the total phenol content in the aqueous extract of the fermented bean curd are respectively improved by 8.1 times and 2.7 times; the content of polypeptide and total phenol in the alcohol extract of fermented bean curd are respectively increased by 12.1 and 4.7 times.

Fifth, analysis of functional Activity

1. Preparation of aqueous extract and alcoholic extract of fermented bean curd

(1) Mixing 1 volume part of fermented bean curd lyophilized powder and 5 volume parts of water, performing ultrasonic extraction for 3 times (ultrasonic parameters: 100W, 20min), centrifuging at 4000rpm for 10min, and collecting supernatant 1. Mixing 1 volume part of fermented bean curd lyophilized powder and 5 volume parts of 80% (v/v) methanol water solution, ultrasonic extracting for 3 times (ultrasonic parameters: 100W, 20min), centrifuging at 4000rpm for 10min, and collecting supernatant 2.

(2) And (3) taking the supernatant 1, carrying out rotary steaming and concentration to obtain a fermented bean curd water extract, and further carrying out freeze drying to obtain the fermented bean curd water extract freeze-dried powder 1. And (3) taking the supernatant 2, carrying out rotary steaming and concentration to obtain a fermented bean curd alcohol extract, and further carrying out freeze drying to obtain the fermented bean curd alcohol extract freeze-dried powder 2.

According to the steps, the fermented bean curd is replaced by unfermented bean curd, and other steps are not changed, so that the unfermented bean curd water extract freeze-dried powder and the unfermented bean curd alcohol extract freeze-dried powder are obtained.

2. Antithrombotic activity of fermented bean curd

The antithrombotic activity is mainly reflected in fibrinolytic enzyme activity and anticoagulant activity.

And (3) detecting the plasmin activity and the anticoagulant activity of a sample to be detected (the freeze-dried powder of the fermented bean curd water extract, the freeze-dried powder of the fermented bean curd alcohol extract, the freeze-dried powder of the unfermented bean curd water extract or the freeze-dried powder of the unfermented bean curd alcohol extract).

The results are shown in Table 4. The result shows that compared with the non-fermented bean curd, the fibrinolytic enzyme activity and the anticoagulation activity in the fermented bean curd are both obviously improved, the fibrinolytic enzyme activity and the anticoagulation activity of the aqueous extract of the fermented bean curd are the strongest, which are 296.1FU/g and 0.04mg/mL respectively, and the anticoagulation effect of the aqueous extract of the fermented bean curd is superior to that of positive control heparin sodium, which indicates that the fermented bean curd has higher antithrombotic activity.

TABLE 4 functional Activity of unfermented and fermented tofu

Note: the difference of the shoulder mark letters (a, b, c) in the same row indicates that the difference is remarkable among groups, and p is less than 0.05; positive control: heparin sodium (0.55 +/-0.06), huperzine A (0.06 +/-0.01), captopril (0.000002 +/-0.000) and acarbose (0.18 +/-0.03); ACHE, acetylcholinesterase; ACE, angiotensin converting enzyme.

3. Acetylcholinesterase inhibitory activity of fermented bean curd

The acetylcholinesterase inhibitory activity of the sample to be tested (lyophilized powder of fermented soybean curd aqueous extract, lyophilized powder of unfermented soybean curd aqueous extract or huperzine A) was measured according to the method described in the literature (Liu, Y.Q., Wang, L.J., Cheng, Y.Q., Saito, M., Yamaki, K., & Qiao, Z.H., et al (2009). Huperzine A is used as positive control.

The results are shown in Table 4. The results show that the acetylcholinesterase inhibition activity of the aqueous extract and the alcohol extract of the fermented bean curd is obviously higher than that of the unfermented bean curd. The acetylcholinesterase inhibitory activity of the fermented bean curd alcohol extract is 2.14mg/mL most strongly.

The above results indicate that the fermented bean curd has a stronger acetylcholinesterase inhibitory effect.

4. Blood pressure lowering effect of fermented bean curd

Angiotensin converting enzyme inhibition of samples to be tested (lyophilized fermented bean curd water extract powder, lyophilized unfermented bean curd alcohol extract powder or captopril) was tested according to the method described in literature (Zhang, b., Sun, q., Liu, h.j., Li, s.z., & Jiang, Z.Q. (2017.) Characterization of action from Chinese chicken kit primers and inhibitors in medium termination and production of enzyme i-converting enzyme (ace). Captopril was the positive control.

The results are shown in Table 4. The result shows that the angiotensin converting enzyme inhibition rate of the aqueous extract and the alcohol extract of the fermented bean curd is obviously higher than that of the unfermented bean curd. The angiotensin converting enzyme inhibition rate of the fermented bean curd alcohol extract is 0.63mg/mL most strongly.

The above results indicate that the fermented bean curd has a stronger blood pressure lowering effect.

5. Hypoglycemic effect of fermented bean curd

The inhibition ratio of α -glucosidase in the sample to be tested (freeze-dried powder of fermented bean curd extract, freeze-dried powder of non-fermented bean curd extract or acarbose) was determined as a positive Control according to the method described in the literature (Shukla, s., Park, j., Kim, d.h., Hong, s.y., Lee, j.s., & Kim, M. (2016.). Total phenolic content, antioxidant, tyrosinase and α -glucosidase inhibitors in the publications of water soluble extracts of non-fermented starter culture doenjang, a korean transferred sol bean curd sample variation.food Control, 59, 854-kava 861.).

The experimental results are shown in Table 4, and the results show that the inhibition rates of α -glucosidase of the fermented bean curd aqueous extract and the fermented bean curd alcohol extract are obviously higher than that of unfermented bean curd, and the inhibition rate of α -glucosidase of the fermented bean curd alcohol extract is strongest and is 4.07 mg/mL.

The results show that the fermented bean curd has stronger blood sugar reducing effect.

<110> university of agriculture in China

<120> preparation method of fermentation product with multiple physiological function activities and special bacillus amyloliquefaciens thereof

<160>1

<170>PatentIn version 3.5

<210>1

<211>1352

<212>DNA

<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)

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gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga 660

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gtgctaagtg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg 780

cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg 840

gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc 900

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Claims

1. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118, the preservation number of which in China general microbiological culture Collection center is CGMCC No. 16050.

2. A microbial inoculum, which is characterized in that: the microbial inoculum contains the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118CGMCC No.16050 in claim 1.

3. The use of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118CGMCCNo.16050 or the microbial inoculum according to claim 2) in at least one of A) fermentation culture of red bean, A) preparation of a product with fibrinolytic enzyme activity, A) preparation of a product with anticoagulant activity, A) preparation of a product with hypoglycemic function, A) preparation of a product with antioxidant activity, A) preparation of a product for eliminating DPPH free radicals, A) preparation of a product for eliminating ABTS free radicals, A) preparation of a product for improving FRAP reducing power, A) preparation of a product for improving glucosidase inhibiting power, A) preparation of a product for improving DPP-IV inhibiting power, A) preparation of a product for improving angiotensin converting enzyme inhibiting power, A) preparation of a product for improving acetylcholine esterase inhibiting power, A) preparation of product for improving total phenol content, A) preparation of product for improving flavone content, A) preparation of product for improving angiotensin converting enzyme inhibiting power, A) preparation of product for improving angiotensin converting enzyme inhibiting enzyme content, A) preparation of product for improving angiotensin converting enzyme A) and/or improving content of SOD, A) and/or preventing hypertension, A) treating or treating diabetes mellitus, and/or treating hypertension, and/or preventing hypertension, A) and/or treating hypertension, A) or preventing hypertension, A) and/or treating hypertension, and/or treating or preventing hypertension, and/or treating a high content of a cardiovascular diseases.

4. Use according to claim 3, characterized in that: the product is fermented small red beans prepared by the method of any one of claims 5 to 9.

5. A process for preparing fermented red bean includes inoculating Bacillus amyloliquefaciens to aseptic red bean, fermenting to obtain fermented red bean, and increasing the fibrinolytic activity, anticoagulant activity, antioxidizing activity, DPPH free radical eliminating rate, ABTS free radical eliminating rate, FRAP reducing power, α -glucosidase inhibiting rate, DPP-IV inhibiting rate, total phenol content, flavone content and polypeptide content and SOD enzyme content.

6. The method of claim 5, wherein: the preparation method of the sterile small red beans comprises the following steps: soaking semen Phaseoli in water, and steaming for 20-40 min.

7. A method for preparing fermented bean curd comprises inoculating Bacillus amyloliquefaciens into sterile bean curd, and fermenting to obtain fermented bean curd, wherein compared with the sterile bean curd, the fermented bean curd has improved fibrinolytic enzyme activity and/or anticoagulant activity and/or acetylcholinesterase inhibition rate and/or angiotensin converting enzyme inhibition rate and/or α -glucosidase inhibition rate and/or total phenol content and/or polypeptide content.

8. The method of claim 7, wherein: the steps for preparing the sterile bean curd are as follows: oven drying bean curd, and steaming for 10-40 min.

9. The method of any of claims 5 to 8, wherein: the Bacillus amyloliquefaciens is the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CAUNDJ118CGMCC No.16050 in claim 1.

10, K1) or K2):

K1) use of fermented red bean prepared by the method according to claim 5 or 6, which is at least one of A22), A22) and A22), for producing plasmin A22), for anticoagulation A22), for lowering blood glucose, A22) for antioxidation, A22) for scavenging DPPH radicals, A22) for scavenging ABTS radicals, A22) for increasing FRAP reducing power, A22) for increasing 22-glucosidase inhibition, A22) for increasing DPP-IV inhibition, A22) for increasing total phenol content, A22) for increasing flavone content, A22) for increasing polypeptide content, A22) for increasing SOD content, A22) for preventing and/or treating cardiovascular diseases and/or treating diabetes mellitus;

K2) the fermented bean curd prepared by the method according to claim 7 or 8 is at least one of A22), A23), A24), A29), A31), A32), A33), A35), A37), A38), A39), 40), 41) and A43), wherein A22) produces plasmin, A23) anticoagulation, A24) reduces blood sugar, A29) increases α -glucosidase inhibition rate, A31) increases angiotensin converting enzyme inhibition rate, A32) increases acetylcholinesterase inhibition rate, A33) increases total phenol content, A35) increases polypeptide content, A37) prevents and/or treats cardiovascular and cerebrovascular diseases, A38) prevents and/or treats thrombotic diseases, A39) prevents and/or treats diabetes, 40) prevents and/or treats hypertension, 41) and/or treats senile dementia, and A43) reduces dementia.