Natto paste with anticoagulation and anti-inflammatory effects as well as preparation method and application of natto paste

Abstract

The invention relates to the technical field of health-care food, and particularly discloses natto paste with anticoagulation and anti-inflammatory effects as well as a preparation method and application of the natto paste. The natto sauce provided by the invention is prepared by fermenting natto through fungi. The fungus is preferably mucor racemosus HNmd, and the preservation number of the fungus is CGMCC (China General Microbiological Culture Collection Center) NO.41646. The invention further discloses a preparation method of the fungus. When the mucor racemosus HNmd is used for fermentation production of the natto paste, the prepared natto paste has nattokinase enzyme activity and anti-inflammatory activity, and has the effects of remarkably prolonging thrombin time and effectively scavenging free radicals, so that the natto paste is endowed with new anticoagulation, anti-inflammatory and anti-oxidation effects, the requirement trend of consumers on healthy food is met, and the natto paste has a good application prospect. The market application field of the soybean paste can be expanded, the added value and market competitiveness of the product can be improved, the traditional seasoning industry can be promoted to develop towards the healthy and high-end direction, and the soybean paste has remarkable economic benefits and social benefits and wide market prospects.

Description

Technical Field

[0001] The invention relates to the technical field of health food, in particular to natto paste with anticoagulant and anti-inflammatory effects and a preparation method and application thereof. Background Art

[0002] Soybean paste, also known as bean paste, is a traditional condiment made from beans through cooking, koji making, and fermentation. Soybean paste has a history of thousands of years in China. As early as the Zhou Dynasty, salty fermented sauces made from meat appeared. Later, the ancestors used grains and beans for brewing, and the word "sauce" came into being. The brewing of sauces has been recorded in the Western Han Dynasty. Shi You, who lived in the era of Emperor Yuan of the Western Han Dynasty, recorded "Wu Di Salt, Soybean, Vinegar, and Sauce" in "Jijiupian". As a traditional condiment with a long history and widespread consumption, soybean paste occupies an important position in the food culture of Asia and around the world. Its unique flavor and rich taste make it a common condiment for cooking various dishes, pickled foods, and side dishes. It is deeply loved by consumers and the market demand continues to grow steadily.

[0003] The koji making and fermentation process of traditional soybean paste is naturally fermented in an open-air environment, which specifically includes three stages: raw material processing, koji making and fermentation. Traditional koji making is to grind the cooked soybeans into square koji blocks, and then place the koji blocks in a cool and ventilated environment for natural fermentation. The making of soybean paste koji is the most important stage in the entire soybean paste making process, and its success or failure directly affects the quality of the soybean paste. After the koji making is completed, the mold spores and hyphae on the surface of the koji blocks are washed off, and the koji blocks are crushed into small pieces, mixed with salt water in a certain proportion and fermented, stirred every day, and soybean paste is made after 3 to 6 months. In order to shorten the koji making time and improve the consistency of different batches of soybean paste products, most companies currently use industrial koji making. The difference between industrial koji making and traditional koji making is the addition of bacterial powder, that is, flour and bacterial powder suitable for soybean paste fermentation are mixed into the steamed soybeans after cooling to make soybean paste, and the soybean paste is fermented into soybean paste by semi-natural fermentation or industrial equipment. Aspergillus is a common fungus powder used in soybean paste fermentation. Among them, Aspergillus oryzae is the most commonly used in soybean paste fermentation and koji making because of its high secretion capacity of protease, amylase, saccharifying enzyme, glutaminase, pectinase and cellulase. During the fermentation process of soybean paste, the fungus powder mixed in during koji making forms a rich microbial community on the surface of the paste together with the microorganisms in the air, produces various microbial enzyme systems, and synergistically promotes the release of volatile flavor substances such as aldehydes, acids, and phenols, ultimately giving soybean paste a sauce aroma, umami and sweetness.

[0004] Nattokinase (NK) is an alkaline serine protease with efficient thrombolytic effect. It can directly or indirectly dissolve fibrin in thrombi and shows great potential in the prevention and treatment of cardiovascular diseases. With the increasing incidence of cardiovascular diseases worldwide, the demand for natural, safe and effective thrombolytic drugs or functional food ingredients is becoming increasingly urgent. As a bioactive substance derived from traditional fermented foods, nattokinase has received extensive attention and in-depth research. The preparation of traditional natto sauce mainly focuses on the formation of flavor substances and the hydrolysis of proteins during the fermentation process to improve its taste and nutritional value. However, with the improvement of living standards, modern consumers' demand for food has gone beyond the simple enjoyment of taste, and they are increasingly concerned about the health attributes and nutritional value of food. Therefore, it is of great market value and health significance to develop a method for preparing natto sauce that has nattokinase enzyme activity and effectively maintains its stability throughout the preparation process. Summary of the invention

[0005] In view of this, the present invention provides a natto paste with anticoagulant and anti-inflammatory effects, and a preparation method and application thereof.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] The invention discloses a natto paste with anticoagulant and anti-inflammatory effects, which is prepared by fermenting natto with fungi.

[0008] Compared with the prior art, the present invention uses fungi to ferment natto to prepare natto paste. The prepared natto paste has nattokinase activity and anti-inflammatory activity, as well as the effects of prolonging thrombin time and scavenging free radicals. Therefore, new anti-coagulant, anti-inflammatory and antioxidant effects are endowed to natto paste, which not only conforms to the trend of consumer demand for healthy food, but also can expand the market application field of bean paste, enhance product added value and market competitiveness, and promote the development of the traditional seasoning industry in a healthy and high-end direction. It has significant economic and social benefits and broad market prospects.

[0009] Furthermore, the fungus includes at least one of Aspergillus, Rhizopus or Mucor.

[0010] Furthermore, the Aspergillus includes at least one of Aspergillus oryzae, Aspergillus niger or Aspergillus niger; and the Rhizopus includes Rhizopus oryzae.

[0011] Preferably, the fungus is Mucor racemosus HNmd, and its deposit number is CGMCCNO.41646.

[0012] The Mucor racemosus HNmd was isolated from Hunan moldy tofu, and the strain was isolated and identified. The strain was classified and named Mucor racemosus, and its 18S rRNA gene sequence is as shown in SEQ ID NO.3. The strain was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, abbreviated as CGMCC, on November 29, 2024, with the strain collection number CGMCC NO.41646, and the storage address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] When the Mucor racemosus HNmd provided by the present invention is used for fermentation to produce natto paste, the prepared natto paste can have higher nattokinase activity and anti-inflammatory activity, as well as significantly prolong thrombin time and effectively scavenge free radicals.

[0014] The present invention also provides a method for preparing natto paste, comprising the following steps:

[0015] S1, mixing the fungus and flour evenly to obtain koji flour;

[0016] S2, washing the natto to remove the mucus on the surface, obtaining the natto bean body; mixing the natto bean body with koji flour, and fermenting naturally to obtain koji;

[0017] S3, mixing the koji and the salt water evenly, and fermenting to obtain natto paste.

[0018] Compared with the prior art, the present invention creatively uses natto instead of traditional soybeans as raw material, and selects fungi to make natto sauce through traditional processes such as koji making, pre-fermentation and post-fermentation, and makes functional natto sauce containing nattokinase for the first time, which not only inherits the traditional production process of soybean sauce in my country, but also expands the development and utilization of natto food. The finished natto sauce prepared by the present invention is similar to traditional soybean sauce in my country in terms of color, tissue morphology and flavor, and is more easily accepted and liked by consumers. At the same time, it also has anti-coagulation, anti-inflammatory and antioxidant effects that traditional soybean sauce does not have, providing consumers with additional health care effects, helping to maintain the health of the body and the normal operation of physiological functions, and further enhancing the market competitiveness of the product.

[0019] Furthermore, in S1, the number of viable bacteria in the koji flour is 6.0×10 6 cfu / g~2.0×10 8 cfu / g.

[0020] Furthermore, in S1, the mass ratio of the fungus to the flour is 1:115 to 1:125.

[0021] Furthermore, in S2, the mass ratio of the natto beans to the koji flour is 1:0.45 to 1:0.65.

[0022] Furthermore, in S2, the natural fermentation time is 42h to 48h, and the relative humidity of the fermentation environment is 50% to 70%.

[0023] In the process of preparing soybean paste, koji making is a crucial link, which plays a vital role in the quality, flavor and nutritional value of soybean paste. In the present invention, koji is made with Mucor racemosus HNmd, and secondary fermentation can be used to degrade macromolecular proteins in natto, enrich the types of nutritional or functional components such as active peptides, and enhance its health care function. At the same time, more flavor substances can be produced during the fermentation process, so that the prepared natto paste has the color, structure and flavor of traditional soybean paste, and improves the acceptance of consumers.

[0024] Furthermore, in S2, the method for preparing natto bean bodies specifically comprises the following steps:

[0025] Place the fermented natto in a sterilized shallow container, pour in warm water that has been boiled and cooled to 50℃~60℃, covering the surface of the natto, soak for 25min~35min, stir gently to remove the natto mucus, drain the water, and get the natto bean body.

[0026] Furthermore, in S2, the natto beans are mixed with koji flour so that the natto beans are fully coated with koji flour, and then spread on a bamboo plate, the surface is covered with sterilized gauze, and placed under natural temperature and relative humidity of 50% to 70% for fermentation for 42h to 48h to obtain koji.

[0027] Furthermore, in S2, the method for preparing natto comprises the following steps:

[0028] The bacterial liquid of Bacillus subtilis HAU-SDZ6 with the preservation number of CGMCC NO.24151 is inoculated into steamed soybeans, and fermented at 37°C to 44°C for 18h to 22h to obtain natto.

[0029] It should be noted that Bacillus subtilis HAU-SDZ6 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 20, 2021, and disclosed in patent CN114292790B.

[0030] Furthermore, the soybean is Handan 13.

[0031] Handou 13 is a soybean variety suitable for summer sowing bred by Handan Academy of Agricultural Sciences, with the approval number: Jishendou 20180001. This variety has limited pod-bearing habits, a growth period of about 105 days, and a plant height of 82cm. There are 45 effective pods per plant, and the weight of 100 grains is 23.8g. The seeds are round, with yellow seed coat, brown hilum, faint light, and resistant to mosaic virus disease. The crude protein (dry basis) of the seeds is 39.09%, and the crude fat is 21.14%.

[0032] The enzyme activity of nattokinase in the prepared natto sauce can be improved by using Handan bean 13 as a raw material to prepare natto and using the prepared natto as a raw material to prepare natto sauce.

[0033] The bacterial solution of Bacillus subtilis HAU-SDZ6 is a bacterial solution of Bacillus subtilis HAU-SDZ6 cultured to a logarithmic growth phase. The OD value of the bacterial solution of Bacillus subtilis HAU-SDZ6 in a logarithmic growth phase is 4.0 or more.

[0034] Exemplarily, the mass volume ratio of the inoculation amount of the bacterial solution of Bacillus subtilis HAU-SDZ6 to the steamed soybeans is (8-12) mL:100 g.

[0035] Exemplarily, the steamed soybeans are obtained by soaking soybeans and water at a weight-to-volume ratio of 1:2.5-1:3.5 at 18°C-20°C for 12h-14h, and then steaming at 115°C-125°C for 30min-35min; wherein the unit of weight is gram and the unit of volume is milliliter.

[0036] Exemplarily, the method for preparing the bacterial solution of Bacillus subtilis HAU-SDZ6 includes the following steps: inoculating the activated bacterial solution of Bacillus subtilis HAU-SDZ6 into LB liquid culture medium at an inoculation rate of 1% to 2% (v / v), and culturing at 37° C. to 40° C. for 12 h to 15 h to obtain the culture solution of Bacillus subtilis HAU-SDZ6; resuspending the bacterial sludge obtained by centrifuging the culture solution in sterilized distilled water to obtain the bacterial solution of Bacillus subtilis HAU-SDZ6.

[0037] The activated bacterial solution of Bacillus subtilis HAU-SDZ6 is obtained by thawing the frozen bacterial strain of Bacillus subtilis HAU-SDZ6 in a glycerol tube, inoculating it into LB liquid culture medium at an inoculation rate of 1% to 2% (v / v), and culturing it at 37° C. for 24 to 36 hours.

[0038] Furthermore, S3 specifically includes the following steps:

[0039] The koji and the salt water are mixed evenly and put into a fermentation container, fermented at 30-40°C and 50-70% relative humidity for 6-8 days, poured out once, then continued to ferment for 14-16 days, poured out twice, and finally fermented for 13-15 days to obtain natto paste.

[0040] Furthermore, in S3, the mass volume ratio of the finished koji and the brine is 1 g:0.5 mL to 1 g:1.5 mL.

[0041] Furthermore, in S3, the concentration of the brine is 14°Bé~16°Bé.

[0042] Exemplarily, the brine used in the present invention is prepared by adding salt to water that has been boiled and cooled to 35° C. to 45° C.

[0043] The present invention also provides the use of the natto paste in preparing a product with the effects of prolonging thrombin time and anti-inflammatory effects.

[0044] The present invention uses fungi, especially Mucor racemosus HNmd, as a fermentation agent, and conducts secondary fermentation of natto using a traditional process. The prepared natto sauce retains the flavor characteristics of traditional soybean sauce and adds unique health care functions, and has high safety and no toxic side effects. It fills the gap in the health care functions of traditional soybean sauce, broadens the consumer group and market application scope, and provides strong support for the upgrading and development of the soybean sauce industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The nattokinase activity of the natto sauce made from the commercial fungus powder purchased in Example 2 of the present invention; wherein different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05);

[0046] Figure 2 The nattokinase enzyme activity of natto sauce made from fungal strains isolated from bean seeds or fermented bean products in Example 2 of the present invention; wherein different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05); wherein Chp is isolated from chickpea seeds, CDmd is isolated from soybean seeds, HM is isolated from Northeast sauce, and JXmd and HNmd are isolated from southern moldy tofu;

[0047] Figure 3 is the total phenol content of natto paste made from fungal strains isolated from bean seeds or fermented bean products in Example 2 of the present invention; wherein different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05);

[0048] Figure 4The DPPH free radical and ABTS free radical scavenging ability of natto paste made from fungal strains isolated from bean seeds or fermented bean products in Example 2 of the present invention; different letters for the same indicator indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05);

[0049] Figure 5 The phylogenetic tree of Mucor racemosus HNmd based on the 18S rRNA gene sequence in Example 3 of the present invention;

[0050] Figure 6 This is a graph showing the black tail ratio of tail thrombus in the normal control group, the intragastric saline group, and the intragastric natto paste group of mice in Example 5 of the present invention; wherein NC is the normal control group, Sal is the intragastric saline group, and NP is the intragastric natto paste group;

[0051] Figure 7 This is a comparison chart of the thrombin time (TT) in the plasma of mice in different test groups (normal control group, intragastric saline group, intragastric natto paste group) in Example 5 of the present invention, wherein ns: no significant difference (p>0.05); **: extremely significant difference (p<0.01); wherein NC is the normal control group, Sal is the intragastric saline group, and NP is the intragastric natto paste group;

[0052] Figure 8 This is a comparison chart of the tumor necrosis factor α (TNF-α) content in the serum of mice in the normal control group, the intragastric administration saline group, and the intragastric administration natto paste group in Example 5 of the present invention; wherein, ns: no significant difference (p>0.05); *: significant difference (p<0.05); wherein NC is the normal control group, Sal is the intragastric administration saline group, and NP is the intragastric administration natto paste group;

[0053] Fig. 9 This is a comparison chart of the interleukin 10 (IL-10) content in the serum of mice in the normal control group, the intragastric administration saline group, and the intragastric administration natto paste group in Example 5 of the present invention; wherein, ns: no significant difference (p>0.05); **: extremely significant difference (p<0.01); wherein NC is the normal control group, Sal is the intragastric administration saline group, and NP is the intragastric administration natto paste group;

[0054] Fig.10 This is a comparison chart of the interleukin 6 (IL-6) content in the serum of mice in the normal control group, intragastric saline group, and intragastric natto paste group in Example 5 of the present invention; wherein, ns: no significant difference (p>0.05); ***: extremely significant difference (p<0.001); wherein NC is the normal control group, Sal is the intragastric saline group, and NP is the intragastric natto paste group. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0056] The natural temperature mentioned in the following embodiments refers to an ambient temperature of 30°C to 40°C.

[0057] PDA solid culture medium: weigh 35 g of PDA powder and 16 g of agar, add 1 L of distilled water, sterilize at 121°C for 20 min, and set aside.

[0058] Example 1

[0059] Prepare Natto:

[0060] The frozen stock of Bacillus subtilis HAU-SDZ6 in a glycerol tube was thawed and inoculated into LB liquid culture medium at a 1% (v / v) inoculation amount, and cultured at 37° C. for 36 hours to obtain an activated bacterial solution of Bacillus subtilis HAU-SDZ6;

[0061] The activated bacterial solution of Bacillus subtilis HAU-SDZ6 is inoculated into LB liquid culture medium at an inoculum amount of 1% (v / v), and cultured at 37° C. for 12 hours to obtain a culture solution of Bacillus subtilis HAU-SDZ6; the bacterial sludge obtained by centrifuging the culture solution is resuspended in sterilized distilled water to obtain the bacterial solution of Bacillus subtilis HAU-SDZ6;

[0062] Select 20 g of full-grained Handan 13 soybean seeds, wash them clean, put Handan 13 in a glass bottle, add water 3 times the volume of the soybeans, soak them at 18°C-20°C for 12 hours, steam the soaked soybeans at 121°C in a high-temperature steam sterilizer for 30 minutes, cool the soybeans to 50°C after the sterilization, inoculate the above-mentioned Bacillus subtilis HAU-SDZ6 bacterial solution (OD value above 4.0) at an inoculation rate of 10 mL of bacterial solution per 100 g of dry soybeans, stir evenly, use light force when stirring to avoid puncturing the soybeans, ferment the inoculated soybeans in a 42°C incubator for 18 hours, and after the fermentation is completed, put the fermented soybeans into a 4°C refrigerator for 24 hours to obtain natto.

[0063] Example 2

[0064] 1. Screening of Mucor racemosus HNmd

[0065] 1.1 Isolation of a single bacterial species

[0066] The isolation sources include chickpeas, soybean seeds, ordinary white tofu sold in supermarkets, salted tofu from Ganzhou, Jiangxi, moldy tofu from the south, and farm tofu from the north.

[0067] ① Method for isolating fungi from chickpea and soybean seeds: Wash chickpea and soybean seeds with running water for 3 times, soak them for 12 hours, and then boil them with clean water for 15-20 minutes. After cooking, remove them and drain the water. Then sprinkle wheat flour on the surface of the cooked beans, stir them gently with wooden chopsticks disinfected with boiling water, put them in a porcelain bowl, and cover the surface with tin foil with small holes in advance to prevent dust. Place them at 28℃-35℃ for 48-72 hours to grow mycelium, pick the mycelium and inoculate it into PDA solid culture medium for cultivation and separation and purification, and finally obtain fungal mycelium of a single species.

[0068] ②The method of isolating fungi from tofu or fermented soy products: Use a sterile scalpel to take approximately 0.1g of ordinary white tofu sold in supermarkets, Jiangxi Ganzhou salted tofu, southern moldy tofu or northern bean paste, add 0.9mL of sterile water respectively, shake and mix well, take 50μL of the mixed solution respectively, apply it to PDA culture medium with sterile glass beads, culture for 48h to 72h, pick out the mycelium and inoculate it into a new PDA solid culture medium for separation and purification, and finally obtain the mycelium of a single species of fungus.

[0069] 1.2 Preparation of freeze-dried fungal powder

[0070] (1) Prepare 50 mL of PDA solid culture medium in a 250 mL conical flask, sterilize at 121°C for 20 min, and let cool and solidify for later use;

[0071] (2) picking an appropriate amount of fungal hyphae, streaking inoculated into the above-mentioned conical flask, and culturing in an incubator at 28° C. and a relative humidity of 60% for about 3 days until the hyphae completely cover the surface and a large number of spores appear;

[0072] (3) preparing bran culture medium: mixing bran and water in a ratio of bran: water = 2:3 (W / V), adding 1% glucose, 0.2% calcium chloride, 0.05% copper sulfate, and 0.05% ammonium sulfate by weight, stirring evenly and dispensing into 250 mL conical flasks, 50 g per bottle, and then sterilizing at 121° C. for 20 min for standby use;

[0073] (4) Preparation of spore suspension: Add 100 mL of sterile water and 5 to 7 sterile glass beads to the PDA medium where a large number of spores have grown, shake for 5 min until the mycelium falls off and the sterile water becomes turbid, and then filter through four layers of sterile gauze to obtain a mold spore suspension;

[0074] (5) Strain culture: 0.5 mL of the mold spore suspension was inoculated into a bottle (50 g) of bran culture medium and cultured at 28° C. and 60% relative humidity for 4 days;

[0075] (6) Spore concentration: add 50 mL of sterile saline to the bran medium after 4 days of culture, shake for 5 min, filter the sterile saline through four layers of sterile gauze after it becomes turbid, divide the filtrate into 2 mL centrifuge tubes, centrifuge at 3800 × g for 10 min, discard the supernatant, and obtain the spore precipitate;

[0076] (7) Preparation of protective agent: sterilize 10% skim milk powder solution at 113°C for 10 min and set aside. Use sterile water to prepare 5% sucrose solution, 5% maltodextrin solution, 7% sodium glutamate solution, and 7% sorbitol solution, respectively, shake and mix, and filter and sterilize with a 0.22 μm microporous filter membrane;

[0077] (8) Add 0.2 mL of skim milk solution, 0.4 mL of sucrose solution, 0.4 mL of maltodextrin solution, 0.4 mL of sodium glutamate solution and 0.4 mL of sorbitol solution to each 2 mL centrifuge tube containing spore pellet and mix well;

[0078] (9) Preparation of freeze-dried fungal powder: The mixed solution was placed in a -80°C refrigerator for pre-freezing, and then placed in a freeze dryer for freeze drying for 24 hours to obtain fungal powder.

[0079] 1.3 Preparation of Natto Sauce

[0080] Koji making: Place natto in a sterilized shallow container, pour in warm water that has been boiled and cooled to about 60°C, covering the surface of the natto, soak the natto for 30 minutes, stirring constantly during the period to dissolve the mucus on the surface of the natto in water, then drain the water and set aside. Mix 0.45g of fungus powder and 54g of flour to make koji flour, then sprinkle evenly on 100g of natto beans, gently stir to make each bean evenly covered with koji flour, then spread it flat on a bamboo plate, cover the surface with sterilized gauze to prevent dust and impurities from contamination, and place the bamboo plate in a ventilated place with a natural temperature and humidity of 65% to 75% for fermentation for 42h to 48h to make koji.

[0081] Pre-fermentation: Take a certain amount of edible salt and use boiled water cooled to 40℃ to prepare 15°Bé brine. Put the finished koji into the fermentation container, add brine according to the mass volume ratio of finished koji: brine = 1g:1mL, mix well, cover the container with dry sterilized gauze, and place it in an environment with natural temperature and humidity of 65% to 75% for fermentation. The fermentation cycle is 7 days, and the pool is emptied once after 7 days.

[0082] Mid-term fermentation: 15 days after pouring the pond is the mid-term fermentation, during which the sauce is stirred every 2 days. The number of stirrings can be appropriately increased or decreased according to the gas production during fermentation. At the end of the mid-term, the pond is poured for the second time. After the second pouring, add an appropriate amount of 10°Bé brine according to the wetness of the sauce.

[0083] Late fermentation: After the second pouring of the pool, it enters the late fermentation stage. During this period, the sauce mash is stirred once every 3 days. Stirring is stopped after about 14 days. Observe the surface of the sauce mash. If the surface is flat and no bubbles are generated, it means that the fermentation is over.

[0084] The commercially available fungus powder and the freeze-dried powder of the single fungus isolated under 1.1 were fermented under the same conditions according to the above method to prepare natto paste. The commercially available fungus powder was a product randomly selected from commercial products.

[0085] 1.4 Sensory evaluation of natto paste

[0086] Different levels were set for each fermented natto sauce in terms of color, state, smell, taste and comprehensive acceptance, and a sensory evaluation team of about 30 people was formed to score. The total sensory evaluation score of each product was calculated according to the formula, and the scoring results were averaged for analysis. The calculation formula was: sensory score = color × 5% + smell × 5% + tissue state × 30% + taste × 40% + comprehensive acceptance × 20%. The sensory scores of natto sauce fermented with commercially available fungal powder are shown in Table 1, and the sensory evaluation and scores of natto sauce fermented with freeze-dried powder of fungal strains isolated under 1.1 are shown in Table 2.

[0087] Table 1 Sensory evaluation of natto paste prepared from commercially available bacterial powder

[0088]

[0089]

[0090] Table 2 Sensory evaluation of natto paste prepared from freeze-dried powder of isolated fungi

[0091]

[0092]

[0093] Note: YD was isolated from salty tofu in Ganzhou, Jiangxi Province, SBS and SBL were isolated from ordinary white tofu in supermarkets, Chp was isolated from chickpea seeds, CDmd was isolated from ordinary soybeans in supermarkets, HM was isolated from bean paste of northern farmers, JXmd was isolated from moldy tofu in Jiangxi Province, and HNmd was isolated from moldy tofu in Hunan Province.

[0094] The results show that the type of fungal powder significantly affects the sensory quality of natto paste. Among them, the fermented mash of natto paste fermented by commercially available sweet potato Aspergillus, black Aspergillus, and Rhizopus oryzae is black, smells obviously moldy, tastes bitter when tasted, and is not easily accepted by people. The color of the fermented mash fermented by commercially available Mucor is obviously lighter, khaki, the mash is delicate, the taste of the sauce is special, with a slight natto flavor, and the taste is mixed. The color of the natto paste fermented by Aspergillus oryzae, composite Aspergillus oryzae, and composite black Aspergillus is brown-red or light brown-red, the smell of the sauce is relatively stronger, and the taste is closer to ordinary bean paste.

[0095] YD isolated from salty tofu in Ganzhou, Jiangxi, and SBS and SBL from ordinary white tofu in supermarkets have a lighter sauce flavor, and the color of the sauce is obviously lighter, light brown red or light brown yellow. YD tastes bitter and sour, and SBL tastes slightly sour, which is not easy to be accepted by people. The natto sauce prepared from the freeze-dried powder of other independently isolated fungi has a stronger sauce flavor.

[0096] 1.5 Determination of nattokinase activity in natto paste

[0097] The natto paste with good sensory evaluation is made into natto freeze-dried powder, specifically: put the natto paste into a -80°C refrigerator for pre-freezing for 2 hours, open the vacuum freeze dryer in advance for pre-cooling for 30 minutes, put the pre-frozen natto paste into a vacuum freeze dryer, freeze-dry for 48 hours at a temperature of -40°C and a vacuum degree of 30Pa, take out and grind it into powder with a pulverizer, and then transfer it into a vacuum bag for vacuuming to obtain natto paste freeze-dried powder.

[0098] Determination of nattokinase activity in natto paste:

[0099] 1.5.1 Preparation of urokinase standard curve:

[0100] 1.5.1.1 Solution preparation

[0101] Weigh 0.15 g of agarose powder and add 15 mL of normal saline to prepare a 1% agarose solution.

[0102] Weigh 20 mg of bovine fibrinogen, add 2 mL of normal saline, and stir continuously until completely dissolved to obtain a bovine fibrinogen solution.

[0103] Dissolve 1000U of thrombin powder in 1mL of sterile saline and dispense into small EP tubes, 10μL per tube. Add 90μL of sterile saline and mix by pipetting to make a thrombin solution with an enzyme activity of 10U per tube and a volume of 100μL.

[0104] 1.5.1.2 Preparation of fibrin plates

[0105] Heat 15 mL of agarose solution until completely dissolved, cool to about 50°C, add 2 mL of bovine fibrinogen solution and 100 μL of thrombin solution, shake gently to mix, avoid bubbles, and quickly pour into a 9 cm diameter dish. After solidification, obtain a fibrin plate and punch holes with a hole puncher.

[0106] 1.5.1.3 Preparation of standard curve

[0107] 50000U / g urokinase powder was prepared into a 100U / mL stock solution with sterile saline, and then the stock solution was diluted with sterile saline in proportion to 2.5U / mL, 5U / mL, 10U / mL, 20U / mL, 40U / mL, 60U / mL, and 80U / mL urokinase solutions. 10μL of each concentration of urokinase solution was spotted in the wells of the fibrin plate, and three parallel wells were set for each concentration. The wells were placed at room temperature for 15min, and then the fibrin plate was placed in a 37℃ incubator and inverted for reaction for 18h. After 18h, the diameter of the dissolution zone was measured and the area of ​​the dissolution zone was calculated to obtain the urokinase standard curve: y=2.706x+53.746, R 2 =0.9911.

[0108] 1.5.2 Determination of Nattokinase Activity in Natto Sauce:

[0109] The method for preparing agarose fibrin plates is the same as above.

[0110] Take 1 g of the natto paste freeze-dried powder sample prepared above, add 5 mL of physiological saline and mix well, put it in a 4°C refrigerator, let it stand for extraction for 6 hours, and then centrifuge it at 5066×g for 20 minutes at 4°C.

[0111] Take 10 μL of supernatant and spot it on the fibrin plate, invert and react at 37℃ for 18h, measure the diameter of the dissolution zone, calculate the area of ​​the dissolution zone, and use the standard curve to calculate the enzyme activity of the sample. Figure 1-2 As shown in Tables 3 and 4.

[0112] Table 3 Nattokinase activity of natto paste prepared from commercially available fungal powder

[0113]

[0114]

[0115] Note: Different letters indicate significant or extremely significant differences, and the same letters indicate no significant differences.

[0116] Table 4 Nattokinase activity of natto sauce prepared from freeze-dried powder of isolated fungi

[0117] Species name Nattokinase activity (U / mL) Chp <![CDATA[48.67±1.74 b <!-- 9 -->]]> CDmd <![CDATA[48.67±1.33 b ]]> HM <![CDATA[45.54±0.79 c ]]> JXj <![CDATA[38.67±0.76 d ]]> HkDJ <![CDATA[70.57±0.71 a ]]>

[0118] Note: Different letters indicate significant or extremely significant differences, and the same letters indicate no significant differences.

[0119] The results show that the nattokinase activity of natto sauce prepared by fermentation of commercially available fungal powder is the strongest in Mucor. The nattokinase activity of natto sauce prepared by fermentation of freeze-dried powder of fungi isolated independently is the strongest in HNmd. However, the color of the sauce fermented by commercial Mucor is obviously lighter, which is earthy yellow. The taste of the sauce is special, with a slight natto flavor. The taste is mixed, and the sensory evaluation score is not as high as that of HNmd.

[0120] 1.6 Determination of total phenol content

[0121] According to the activity of nattokinase in freeze-dried natto sauce powder under item 1.5, combined with the sensory evaluation results of natto sauce, 5 natto sauces with strong nattokinase activity and high sensory evaluation were selected to determine their total phenol content. The selected natto sauces include 1 natto sauce mAsp made by fermenting natto with purchased composite Aspergillus oryzae and 4 natto sauces fermented with isolated fungi (Chp, CDmd, HM, HNmd).

[0122] (1) Preparation of gallic acid standard curve

[0123] Prepare a 100μg / mL gallic acid mother solution and a 20% sodium carbonate solution. Dilute the gallic acid mother solution with distilled water to gallic acid solutions with concentrations of 0μg / mL, 10μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, 50μg / mL, 60μg / mL, and 70μg / mL. Take 1mL of gallic acid solutions of different concentrations, add 1mL of Folin phenol reagent, 2mL of 20% sodium carbonate solution, and 6mL of distilled water, mix well, and place in the dark for 30 minutes, then use a UV spectrophotometer to measure the absorbance at 760nm to draw a gallic acid standard curve.

[0124] (2) Determination of total phenol content of samples

[0125] After grinding evenly according to the ratio of natto paste freeze-dried powder: 70% ethanol solution = 1g: 20mL, place it in a 50mL centrifuge tube, extract it in a 37℃ shaker at 180rmp / min for 2h, then centrifuge it at 5066×g for 10min, keep the supernatant, dilute the supernatant 10 times, take 1mL of the diluted supernatant, react according to the same reaction system and method as above, measure the absorbance, bring the absorbance of the sample into the standard curve and calculate the total phenol content. The total phenol content of each natto paste is as follows Figure 3 And as shown in Table 5.

[0126] Table 5 Total phenol content of each natto paste

[0127] Natto paste name Total phenol content (mg / g) Aspergillus oryzae complex (mAsp) <![CDATA[5.68±0.47b c ]]> Chp <![CDATA[5.09±0.19 c ]]> CDmd <![CDATA[5.05±0.20 c ]]> HM <![CDATA[6.17±0.20 b ]]> HkDJ <![CDATA[7.55±0.38 a ]]>

[0128] Note: Different letters indicate significant or extremely significant differences, and the same letters indicate no significant differences.

[0129] Depend on Figure 3 As shown in Table 5, HNmd has the highest total phenol content among the five types of natto paste.

[0130] 1.7 In vitro antioxidant activity assay

[0131] The scavenging abilities of DPPH and ABTS radicals of natto paste prepared with purchased composite Aspergillus oryzae, Chp, CDmd, HM and HNmd isolated independently were tested.

[0132] (1) DPPH free radical scavenging ability

[0133] Take 10mg of natto paste freeze-dried powder in a 2mL centrifuge tube, add 2mL of distilled water, shake and mix, ultrasonic treatment for 20min, centrifuge the ultrasonically treated sample at 5066×g for 10min, take 300μL of supernatant, add 900μL of 0.1mmol / L DPPH anhydrous ethanol solution in the dark, shake and mix, react at room temperature in the dark for 30min, centrifuge at 5066×g after the reaction, measure the absorbance at 517nm with a UV spectrophotometer, set 3 parallels for each sample, and calculate the DPPH scavenging ability of natto paste freeze-dried powder with a formula. The UV spectrophotometer was zeroed with distilled water before the measurement; the blank control was replaced with an equal amount of distilled water instead of the supernatant, and the other experimental steps remained unchanged.

[0134] DPPH clearance rate (%) = (1-A1 / A0) × 100%.

[0135] Wherein, A1: absorbance of the sample; A0: absorbance of the blank control.

[0136] (2) ABTS free radical scavenging ability

[0137] In the dark, prepare 7mmol / L ABTS solution and 2.457mmol / L potassium persulfate solution with distilled water respectively; mix the ABTS solution and potassium persulfate solution in equal volumes to obtain ABTS mother solution. The ABTS mother solution can be used after being stable in the dark for 10h to 14h.

[0138] Take an appropriate amount of natto paste freeze-dried powder in a 2 mL centrifuge tube, add distilled water to prepare a 5 mg / mL sample solution, shake and mix, ultrasonically treat for 30 minutes, then centrifuge at 5066×g for 10 minutes, and keep the supernatant.

[0139] The ABTS stock solution was diluted with distilled water in dark conditions to an absorbance of 0.7±0.02 at 734 nm to obtain the ABTS working solution.

[0140] According to the volume ratio of supernatant: ABTS working solution of 1:4, the supernatant and ABTS working solution were mixed, and the reaction was protected from light for 5 minutes. After the reaction, it was centrifuged at 5066×g for 5 minutes, and the absorbance was measured at 734nm using a UV spectrophotometer. Three parallels were set for each sample, and the ABTS scavenging ability of natto paste freeze-dried powder was calculated using a formula. The UV spectrophotometer was zeroed with distilled water before the measurement; the blank control was replaced with an equal amount of distilled water instead of the supernatant, and the other experimental steps remained unchanged.

[0141] ABTS clearance rate (%) = (1-A1 / A0) x 100%.

[0142] Wherein, A1: absorbance of the sample; A0: absorbance of the blank control.

[0143] The scavenging abilities of DPPH free radical and ABTS free radical of each natto paste are as follows Figure 4 and as shown in Table 6.

[0144] Table 6 DPPH free radical and ABTS free radical scavenging rate of each natto paste

[0145]

[0146]

[0147] Note: Different letters after the data in the same column indicate significant or extremely significant differences, and the same letters indicate no significant differences.

[0148] Depend on Figure 4 As shown in Table 6, among the five kinds of natto paste, the scavenging rates of composite Aspergillus oryzae and HNmd on DPPH free radicals were higher; the scavenging rates of CDmd and HM on ABTS free radicals were higher.

[0149] Example 3

[0150] Based on the nattokinase activity of natto paste, the sensory quality of natto paste, combined with the results of total phenolic content and antioxidant capacity determination in natto paste, natto paste HNmd was selected for subsequent experiments.

[0151] The fungus HNmd isolated from Hunan moldy tofu was identified.

[0152] The total DNA of the above-mentioned HNmd strain was used as a template, and the universal primers ITS4 (5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO. 1)) and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3' (SEQ ID NO. 2)) were used as primers to amplify the 18SrRNA gene sequence by PCR. The PCR amplification product was sent to Shanghai Bioengineering Co., Ltd. for sequencing.

[0153] The 18S rRNA gene sequence of the fungus HNmd is:

[0154] (SEQ ID NO.3).

[0155] Sequence comparison revealed that the 18S rRNA gene of strain HNmd was most similar to that of Mucor racemosus isolate138a, at 99.68%. A phylogenetic tree was constructed using the 18S rRNA gene sequence of the fungus HNmd, as shown in Figure 1. Figure 5 shown.

[0156] Example 4

[0157] Safety evaluation of natto paste:

[0158] The safety of natto paste fermented by Mucor racemosus HNmd was evaluated. The natto paste was fermented by Mucor isolated from Hunan moldy tofu.

[0159] (1) In vitro safety testing

[0160] The plate counting method was used to detect the colony count of Escherichia coli, Salmonella and Staphylococcus aureus in natto paste prepared by fermentation of Mucor racemosus HNmd. The results are shown in Table 7.

[0161] Table 7 Pathogenic microorganism colony count

[0162] Pathogenic microorganisms Colony count (cfu / g) Escherichia coli Not detected salmonella Not detected Staphylococcus aureus 40

[0163] After culturing and counting, it was found that no Salmonella and Escherichia coli were detected in the natto paste. Staphylococcus aureus was counted, and the colony count of Staphylococcus aureus in the natto paste was 40 cfu / g, which is within the limit value of the acceptable level of microbial indicators specified in GB2718-2014 (100 cfu / g). Comprehensive plate counting results show that the content of pathogenic microorganisms in natto paste is within the range specified by national standards.

[0164] The content of aflatoxin B1 (AFB1) in the natto sauce was detected by liquid chromatography-tandem mass spectrometry. The results showed that the content of aflatoxin B1 in the natto sauce prepared by fermentation of Mucor racemosus HNmd was lower than the detection limit of 0.50 μg / kg. According to the national standard GB2718-2014, the content of AFB1 in soybean paste is limited to 5 μg / kg. Therefore, the content of aflatoxin B1 in the natto sauce is within the limit of the national standard.

[0165] The content of heavy metal lead and arsenic in natto paste prepared by fermentation of Mucor racemosus HNmd was tested. The results showed that the content of heavy metal lead and arsenic in the natto paste was lower than the detection limit of 0.05 mg / kg. According to the national standard GB2718-2014, the content of lead and arsenic in soybean paste is limited to 1.0 mg / kg and 0.5 mg / kg respectively. Therefore, the content of lead and arsenic in the natto paste is within the limit of the national standard.

[0166] (2) In vivo safety test

[0167] Male ICR mice weighing 35g to 40g were selected, and each was kept in a separate cage. The bedding and drinking water were changed once a week during the experiment. After the adaptive feeding, the mice were randomly divided into two groups, 10 in each group, a normal control group (NC) and a natto paste group (NP) for the experiment. The mice were fasted for 6h before the experiment and fasted for 4h after the gavage, with free drinking water during the period.

[0168] The normal control group was gavaged with distilled water, and the natto sauce group was gavaged with a natto sauce solution prepared from natto sauce freeze-dried powder prepared by fermentation of Mucor racemosus HNmd. The preparation method of the natto sauce solution is: add 1 mL of drinking water to every 0.33 g of natto sauce freeze-dried powder, mix well and ultrasonicate for 20 minutes. According to the weight of mice, the gavage dose of natto sauce solution for each mouse per day is 2.5 g / kg, and the gavage is continuous for 14 days. The physiological state of the mice was observed and recorded every day. The mice were weighed on the 0th, 7th and 14th days of the experiment, and the weight and food intake of the mice were recorded. The results are shown in Tables 8 and 9.

[0169] Table 8 Body weight of mice at different growth stages (g)

[0170] Group 0d 7d 14d Normal control group <![CDATA[36.84±1.78 a ]]> <![CDATA[38.46±1.97 a ]]> <![CDATA[38.67±2.54 a ]]> Natto paste set <![CDATA[36.86±1.42 a ]]> <![CDATA[38.17±2.11 a ]]> <![CDATA[38.18±1.45 a ]]>

[0171] Note: Different letters after the data in the same column indicate significant or extremely significant differences, and the same letters indicate no significant differences.

[0172] Table 9 Food intake of mice at different growth stages (g)

[0173] Group 0d 7d 14d Normal control group <![CDATA[4.20±0.54 a ]]> <![CDATA[4.68±0.61 a ]]> <![CDATA[4.31±0.69 a ]]> Natto paste set <![CDATA[4.12±0.56 a ]]> <![CDATA[4.42±0.60 a ]]> <![CDATA[4.38±0.57 a ]]>

[0174] Note: Different letters after the data in the same column indicate significant or extremely significant differences, and the same letters indicate no significant differences.

[0175] After the experiment, the mice were anesthetized, blood was collected from the eyeballs, and the mice were killed by dislocation of the neck and dissected. The internal organs of the mice were observed, and no edema or discoloration was found. Afterwards, the heart, liver, spleen, lung and kidney of the mice were weighed and recorded, and the organ index was calculated. The results are shown in Table 10.

[0176] Organ index = (organ weight / mouse body weight) × 100%.

[0177] Table 10 Effect of intragastric administration of natto paste solution on organ indexes of mice (%)

[0178]

[0179] Note: Different letters after the data in the same column indicate significant or extremely significant differences, and the same letters indicate no significant differences.

[0180] During the entire mouse test period, the fur of all mice in the normal control group (NC) and the oral natto paste group (NP) was white, smooth and shiny; the mouse eyeballs were pink, all mice ate and drank normally, had normal bowel movements, no abnormal symptoms were observed, and no deaths occurred.

[0181] As shown in Tables 8 to 10, the body weight, food intake and organ index of mice in the natto paste group (NP) gavaged for 2 weeks were not significantly different from those in the normal control group, indicating that the natto paste prepared by fermentation of Mucor racemosus HNmd was safe for mice and did not cause damage to their feeding and organs.

[0182] Example 5

[0183] Detection of anticoagulant and anti-inflammatory activities of natto paste fermented with Mucor racemosus HNmd in vivo:

[0184] Seven-week-old male ICR mice were purchased, weighing 35 g to 40 g. Each mouse was housed in a single cage, with an ambient temperature of 22°C to 25°C, a humidity of 50% to 60%, and a 12-hour alternating cycle of light and darkness. During the entire experiment, the mice were free to drink water and eat, and the bedding and drinking water were changed regularly.

[0185] Since the salt content in natto paste is relatively high, in order to understand the effect of salt itself on the anticoagulant activity and anti-inflammatory activity of mice, the present invention simultaneously determines the effect of edible salt with the same salinity as that in the natto paste freeze-dried powder for testing. After 10 days of adaptive feeding, the mice were randomly divided into three groups, with 20 mice in each group. The three groups were normal control group (NC), intragastric saline group (Sal) and intragastric natto paste group (NP). After grouping, intragastric gavage was performed at 9 am every day. The normal control group was gavaged with distilled water; the natto paste group was gavaged with a natto paste solution prepared by fermenting natto paste freeze-dried powder prepared by Mucor racemosus HNmd (1 mL of drinking water was added to every 0.33 g of natto paste freeze-dried powder, and ultrasonicated for 20 minutes after mixing); the saline group was gavaged with saline prepared by edible salt with the same salinity as the natto paste freeze-dried powder (1 mL of drinking water was added to every 0.0856 g of salt, and mixed). The intragastric dose of the natto paste group was 2.5 g / kg, and the intragastric volume of each mouse was 7.5 mL / kg; the intragastric dose of the saline group was 0.642 g / kg, and the intragastric volume of each mouse was 7.5 mL / kg.

[0186] The experimental period was 15 days in total, including 14 days of gavage and 1 day of modeling. The mice were weighed before gavage on days 0, 7, and 14, and the gavage volume was updated. 45 to 60 minutes after the end of gavage on day 14, each mouse was intraperitoneally injected with 50 mg / kg of carrageenan solution based on the weight of the mice, and the injection volume was 10 mL / kg. The mice injected with carrageenan were observed for 24 hours in an environment of 20±2°C and 50% humidity. After 24 hours, all mice were anesthetized, and blood was collected from the eyeballs. After blood collection, the mice were killed by dislocating the neck, the length of the black tail was measured, and the black tail ratio was calculated. The results are as follows Figure 6 As shown in Table 11.

[0187] Black tail ratio (%) = Black tail length (cm) / Tail length (cm) × 100%

[0188] Table 11 Black tail ratio of mice in each group

[0189] Group Black tail ratio (%) Normal control group (NC group) 76.63±3.9** Saline group (Sal group) <![CDATA[86.01±3.31 ## ]]> Natto paste group (NP group) <![CDATA[53.96±10.32 ## **]]>

[0190] Note: ## :Compared with the normal control group, there was a very significant difference p<0.01;

[0191] **: Very significant difference compared with the saline group, p<0.01.

[0192] Depend on Figure 6 As shown in Table 11, compared with the normal control NC group, the black tails of the Sal group mice gavaged with saline were longer and darker in color, indicating that the thrombosis was more serious; while the tail thrombosis of the NP group mice gavaged with natto sauce was improved compared with the Sal group, and the black tail length was even shorter than that of the NC group. Judging from the phenotype, gavage with natto sauce solution alleviated the formation of thrombus to a certain extent.

[0193] Eyeball blood was collected from 8 mice in each group, sodium citrate anticoagulant was added at a volume ratio of 9:1, the mixture was gently inverted and mixed, centrifuged at 1900×g for 10 min, the upper plasma was collected in a 1.5mL EP tube, and stored in a -80°C ultra-low temperature refrigerator for the detection of coagulation indicators thrombin time, prothrombin time, and activated partial thromboplastin time. Eyeball blood was collected from 6 mice in each group, centrifuged at 1900×g for 20 min at 4°C, the upper serum was collected in a 200μL EP tube, and stored in a -80°C ultra-low temperature refrigerator for the detection of coagulation indicators fibrinogen content and inflammatory indicators.

[0194] The commercially available kits for thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen content (Fig) were used to react and detect according to the methods described in the kit instructions. The results of the four coagulation tests are shown in Table 12 and Figure 7 shown.

[0195] Table 12 Results of four coagulation tests

[0196]

[0197] Note: ## :Compared with the normal control group, there was a very significant difference p<0.01;

[0198] **: Very significant difference compared with the saline group, p<0.01.

[0199] As can be seen from Table 12, compared with the normal control group, only the thrombin time of the gavage natto paste group increased significantly (p<0.05); there were no significant differences in the prothrombin time, activated partial thromboplastin time, and fibrinogen content between the normal control group, the gavage saline group, and the gavage natto paste group (p>0.05). The thrombin time of the mice in the gavage natto paste group was significantly longer than that of the mice in the normal control group. This result shows that the natto paste solution may contain substances that inhibit plasma coagulation. Gavage of the natto paste solution to mice for 2 weeks increased the substances that inhibit plasma coagulation in the blood of the mice. These anticoagulants may prevent thrombosis by prolonging the blood coagulation time.

[0200] Commercially available kits for the four inflammatory factors, tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 10 (IL-10), and interleukin 6 (IL-6), were used to react and detect according to the methods described in the kit instructions. The results of the inflammatory factor determination are shown in Tables 13 and Figure 8 to Figure 10 shown.

[0201] Table 13 Determination results of four inflammatory factors in the serum of mice in each group

[0202]

[0203] Note: # :Compared with the normal control group, there was a significant difference p<0.05;

[0204] ## :Compared with the normal control group, there was a very significant difference p<0.01;

[0205] ### :Compared with the normal control group, there was a very significant difference p<0.001;

[0206] *: significant difference compared with the saline group p < 0.05;

[0207] **: Very significant difference compared with the saline group p < 0.01;

[0208] ***: Very significant difference compared with the saline group, p < 0.001.

[0209] As shown in Table 13, compared with the normal control group, only the IL-6 content in the intragastric saline group was significantly reduced. Compared with the normal control group, the TNF-α, IL-10, and IL-6 contents in the intragastric natto paste group were significantly reduced; while there was no significant difference in the IL-1β content among the mice in each group.

[0210] Depend on Figure 8 It can be seen that compared with the normal control group, there was no significant difference in the TNF-α content in the intragastric saline group (p>0.05), and the TNF-α content in the intragastric natto paste group was significantly reduced (p<0.05); compared with the intragastric saline group, the TNF-α content in the intragastric natto paste group was also significantly reduced (p<0.05).

[0211] Depend on Fig. 9 It can be seen that compared with the normal control group, there was no significant difference in the IL-10 content of the intragastric saline group (p>0.05), and the IL-10 content of the intragastric natto paste group was extremely significantly reduced (p<0.01); compared with the intragastric saline group, the IL-10 content of the intragastric natto paste group was also extremely significantly reduced (p<0.01).

[0212] Depend on Fig.10 It can be seen that compared with the normal group, the IL-6 content in the intragastric saline group was significantly reduced (p<0.001), and the IL-6 content in the intragastric natto paste group was also significantly reduced (p<0.001); while there was no significant difference in IL-6 content between the intragastric saline group and the intragastric natto paste group (p>0.05).

[0213] These results all indicate that, except for IL-1β, natto paste can significantly reduce the expression of inflammatory factors TNF-α, IL-6 and IL-10 in mice.

[0214] In summary, the natto sauce prepared by secondary fermentation of Mucor racemosus HNmd provided by the present invention has nattokinase activity, and the nattokinase activity can reach 70.57±0.71U / mL. At the same time, it also has anti-inflammatory activity and antioxidant efficacy, and has no toxic side effects and high safety. It has good application prospects in the food field, fills the gap in the health care function of traditional soybean sauce, has significant economic and social benefits, and provides strong support for the upgrading and development of the soybean sauce industry.

[0215] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A natto paste having anticoagulant and anti-inflammatory effects, characterized in that: Prepared by fermenting natto with fungi.

2. The natto paste having anticoagulant and anti-inflammatory effects according to claim 1, characterized in that: The fungus includes at least one of Aspergillus, Rhizopus or Mucor.

3. The natto paste having anticoagulant and anti-inflammatory effects according to claim 2, characterized in that: The Aspergillus comprises at least one of Aspergillus oryzae, Aspergillus niger or Aspergillus niger; and / or The Rhizopus includes Rhizopus oryzae.

4. The natto paste having anticoagulant and anti-inflammatory effects according to claim 1, characterized in that: The fungus is Mucor racemosus HNmd, and its deposit number is CGMCC NO.41646.

5. The method for preparing the natto paste having anticoagulant and anti-inflammatory effects according to any one of claims 1 to 4, characterized in that: The steps include: S1, mixing fungus powder and flour evenly to obtain koji flour; S2, washing the natto to remove the mucus on the surface, obtaining the natto bean body; mixing the natto bean body with koji flour, and fermenting naturally to obtain koji; S3, mixing the koji and the salt water evenly, and fermenting to obtain natto paste with anticoagulant and anti-inflammatory effects.

6. The method for preparing natto paste having anticoagulant and anti-inflammatory effects according to claim 5, characterized in that: In S1, the number of viable bacteria in the koji flour is 6.0×10 6 cfu / g~2.0×10 8 cfu / g; and / or In S2, the mass ratio of the natto beans to the koji flour is 1:0.45 to 1:0.65; and / or In S2, the natural fermentation time is 42 hours to 48 hours, and the relative humidity of the fermentation environment is 50% to 70%.

7. The method for preparing natto paste having anticoagulant and anti-inflammatory effects according to claim 5, characterized in that: In S2, the method for preparing natto comprises the following steps: The bacterial liquid of Bacillus subtilis HAU-SDZ6 with the preservation number of CGMCC NO.24151 is inoculated into steamed soybeans, and fermented at 37°C to 44°C for 18h to 22h to obtain natto.

8. The method for preparing natto paste having anticoagulant and anti-inflammatory effects according to claim 7, characterized in that: The soybean is Handan 13.

9. The method for preparing natto paste having anticoagulant and anti-inflammatory effects according to claim 5, characterized in that: S3 specifically includes the following steps: The koji and the salt water are mixed evenly and put into a fermentation container, fermented at 30-40°C and 50-70% relative humidity for 6-8 days, poured out once, then continued to ferment for 14-16 days, poured out twice, and finally fermented for 13-15 days to obtain natto paste.

10. Use of the natto paste with anticoagulant and anti-inflammatory effects as claimed in claim 9 in the preparation of products with prolonged thrombin time and anti-inflammatory effects.

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