A mixed fermentation coix seed natto and a preparation method thereof

Abstract

The application discloses a kind of mixed fermentation of coix seed rice natto and its preparation method, belong to natto fermentation technical field.The preparation method includes that clean soybean is soaked with coix seed saccharification liquid, NaCl is added after soaking and sterilized, Bacillus subtilis and bifidobacterium are inoculated in the material obtained by sterilization, and mixed fermentation is carried out.The application first proposes the method for preparing natto by soaking soybean with coix seed saccharification liquid and mixed fermentation of Bacillus subtilis and bifidobacterium, and the method for preparing natto by soaking soybean with coix seed saccharification liquid and mixed fermentation of Bacillus subtilis and bifidobacterium, coix seed saccharification liquid soaking and double bacteria fermentation promote the number of viable bacteria during natto fermentation and natto kinase enzyme activity, further affect the composition and content of active substance and flavor substance in subsequent fermentation process.The application improves the flavor of natto by process optimization, increases its nutrition, and obtains a novel unique, taste rich, flavor good high natto kinase activity flavor natto.

Description

Technical Field

[0001] The invention relates to the technical field of natto fermentation, in particular to coix seed natto fermented by mixed bacteria and a preparation method thereof. Background Art

[0002] Natto is a traditional fermented soy product made from soybeans fermented with Bacillus subtilis. During the fermentation process, the concentrations of various beneficial components, such as nattokinase, isoflavones, vitamin K2, and antioxidants, increase. Nattokinase, a key active substance that dissolves fibrin, is the most biologically active component in natto. It is a natural, safe, highly effective, and economical thrombolytic enzyme, and is safer and more effective than clinical thrombolytic drugs in treating various thrombotic vascular diseases. Isoflavones, a subclass of flavonoids, are among the most important plant-derived polyphenolic compounds, exhibiting antioxidant, anti-inflammatory, antiviral, and anticancer properties. Phenolic acids, produced during natto fermentation, are natural antioxidants widely recognized for their protective effects in health and disease. Therefore, natto is a functional food with high nutritional value that can be used to prevent a variety of diseases, making it a true super health food. Natto may also reduce the risk of cancer in women because soy contains isoflavone compounds (genistein, daidzein, and glycine). Therefore, fermented natto is considered to be more nutritious and healthier than raw soy.

[0003] Although natto contains a variety of active nutrients, its unique aroma and flavor limit its acceptability. The fermentation process significantly impacts natto quality. Therefore, improving the natto fermentation process and producing better natto products through scientific methods are crucial for expanding the natto food market and improving consumer acceptance. Summary of the Invention

[0004] The present invention aims to provide a mixed fermentation of coix seed and barley natto and a preparation method thereof to solve the problems existing in the above-mentioned prior art. The present invention uses coix seed and barley saccharification liquid to soak soybeans and ferments them with a dual-bacteria mixed fermentation method. Through process optimization, the flavor of natto is improved and its nutrition is increased.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for preparing coix seed natto fermented with mixed bacteria, comprising: soaking cleaned soybeans in coix seed saccharification liquid, adding NaCl after soaking and sterilizing, inoculating Bacillus subtilis and Bifidobacterium into the sterilized material, and fermenting the mixture;

[0007] Wherein, the Bacillus subtilis is Bacillus subtilis GUTU09, which was deposited in the China Center for Type Culture Collection on May 31, 2021, with the deposit number: CCTCC NO: M 2021641;

[0008] The bifidobacterium is Bifidobacteriμm animalis subsp. Lactis BLH1 or Bifidobacteriμm animalis subsp. Lactis BLH6; the Bifidobacterium animalis subsp. Lactis BLH1 was deposited in the China Center for Type Culture Collection on December 19, 2022, with a deposit number of CCTCC NO: M20221979; the Bifidobacterium animalis subsp. Lactis BLH6 was deposited in the China Center for Type Culture Collection on May 29, 2023, with a deposit number of CCTCC NO: M 2023843.

[0009] Furthermore, the bifidobacterium is Bifidobacterium animalis BLH6.

[0010] Furthermore, the inoculation ratio of the Bacillus subtilis and the Bifidobacterium is 1:1.

[0011] Furthermore, the inoculation amount of the Bacillus subtilis and Bifidobacterium is 8 wt % of the sterilized material. In the mixed fermentation, the fermentation temperature is 35° C. and the fermentation time is 27 h.

[0012] Furthermore, the amount of NaCl added is 0.8% of the mass of the soybeans after being soaked in the coix seed saccharification liquid.

[0013] Furthermore, the soaking time of the coix seed saccharification liquid is 12-14 hours.

[0014] Furthermore, the coix seed saccharification liquid is prepared by soaking clean coix seed, beating with water, heating and gelatinizing, and then adding enzymes for enzymatic hydrolysis; the enzymes include high-temperature α-amylase and glucoamylase.

[0015] Furthermore, the soaking is performed at 25° C. for 12 hours.

[0016] Furthermore, in the step of adding water and beating, the mass ratio of the soaked coix rice to water is 1:12.

[0017] The present invention also provides coix seed natto prepared by the preparation method.

[0018] The present invention discloses the following technical effects:

[0019] The present invention proposes for the first time a method for preparing natto by soaking soybeans in coix seed saccharification liquid and fermenting them with a mixed culture of Bacillus subtilis and Bifidobacterium. The soaking in coix seed saccharification liquid and the dual-bacteria fermentation both promote the number of viable bacteria and the activity of nattokinase during the fermentation process of natto, further affecting the composition and content of active substances and flavor substances in the subsequent fermentation process.

[0020] The present invention improves the flavor of natto and increases its nutrition through process optimization, thereby obtaining a novel and unique flavored natto with rich taste, good flavor and high nattokinase activity. The final product can not only add new color to the natto market, but also broaden the development market of natto and coix rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The effect of fermentation time on nattokinase activity (A), GABA content (A) and sensory score (B) of coix seed natto fermented by BLH1+B9 mixed bacteria;

[0023] Figure 2 The effect of fermentation temperature on nattokinase activity (A), GABA content (A) and sensory score (B) of coix seed natto fermented by BLH1+B9 mixed bacteria;

[0024] Figure 3 Effects of NaCl addition on sensory scores (A), nattokinase activity (B) and GABA content (B) of coix seed natto fermented by BLH1+B9 mixed bacteria;

[0025] Figure 4 The effect of inoculum size on nattokinase activity (A), GABA content (A) and sensory score (B) of coix seed natto fermented by BLH1+B9 mixed bacteria;

[0026] Figure 5 Counts of viable bacteria in BLH1+B9 Natto (A) and B9 Natto (B) samples;

[0027] Figure 6 The changes of nattokinase activity during the fermentation of BLH1+B9 natto, BLH6+B9 natto and B9 natto;

[0028] Figure 7Changes in pH during fermentation for B9 natto, BLH1+B9 natto and BLH6+B9 natto;

[0029] Figure 8 Changes in amino acid nitrogen content during fermentation for B9 natto, BLH6+B9 natto and BLH1+B9 natto;

[0030] Figure 9 Changes in soluble peptide content during fermentation for B9 natto, BLH6+B9 natto and BLH1+B9 natto;

[0031] Figure 10 Changes in soy isoflavone (daidzin (A), glycitein (B), genistein (D) and glycitinn (C)) content during fermentation for BLH1+B9 natto, BLH6+B9 natto and B9 natto;

[0032] Figure 11 Changes in phenolic substances (gallic acid (A), chlorogenic acid (B), ferulic acid (C) and caffeic acid (D)) content during fermentation for BLH1+B9 natto, BLH6+B9 natto and B9 natto;

[0033] Figure 12 Changes in organic acid substances (oxalic acid (A), tartaric acid (B), malic acid (D), formic acid (C), lactic acid (E), acetic acid (F), succinic acid (G)) content during fermentation for BLH1+B9 natto, BLH6+B9 natto and B9 natto;

[0034] Figure 13 Changes in antioxidant activity (DPPH (A), FRAP (B), ABTS (C)) during fermentation for BLH1+B9 natto, BLH6+B9 natto and B9 natto;

[0035] Figure 14 Principal component analysis biplot for unfermented soybean, B9 natto, BLH6+B9 natto and BLH1+B9 natto (OAV>1);

[0036] Figure 15 Electronic tongue response radar chart for unfermented soybean, B9 natto, BLH6+B9 natto and BLH1+B9 natto. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings, which are not to be construed as limiting the present application, but are to be understood as being illustrative of certain aspects, features and embodiments of the present application.

[0038] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, unless indicated otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0039] Various modifications and variations of the described methods and materials of the application will be apparent to those skilled in the art from the foregoing disclosure, and further will be apparent after examination of the drawings and detailed description. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the description contained herein does not limit the scope of the application. The specification and examples given herein are exemplary only. It is recognized that equivalents exist within the scope of the application and are covered by the disclosure.

[0040] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.

[0041] The main materials used in the present application are as follows:

[0042] 1. Strains

[0043] The three strains used in the present application are screened by the 542 laboratory of the Brewing College of Guizhou University and sent to the preservation agency for preservation. Among them, strain B9 is isolated from Guizhou douchi, its classification name is Bacillus subtilis GUTU09, the preservation date is May 31, 2021, and the preservation number is CCTCC NO: M 2021641; strain BLH1 is isolated from red sour soup, its classification name is Bifidobacterium animalis subsp. Lactis BLH1, the preservation date is December 19, 2022, and the preservation number is CCTCC NO: M 20221979; strain BLH6 is isolated from Guizhou douchi, its classification name is Bifidobacterium animalis subsp. Lactis BLH6, the preservation date is May 29, 2023, and the preservation number is CCTCC NO: M 2023843. The above three strains are preserved in the China Center for Type Culture Collection, the preservation address is Wuhan, Wuhan University, China.

[0044] 2. Culture medium

[0045] (1) B9 seed culture medium: 10 g / L glucose, 5 g / L yeast extract, 10 g / L beef extract, 5 g / L NaCl, pH 7.0-7.5, 121℃ sterilization for 20 min.

[0046] (2) PTYG medium: 5 g tryptone, 5 g soy peptone, 10 g yeast powder, 10 g glucose, 1 mL Tween 80, 15-20 g agar, 0.05 g L-cysteine ​​hydrochloride, 1000 mL distilled water, 4 mL salt solution (2% agar is required for solid culture medium), sterilized at 121°C for 20 min. Preparation of salt solution: 0.2 g anhydrous calcium chloride, 1 g dipotassium hydrogen phosphate, 1 g potassium hydrogen phosphate, 0.48 g magnesium sulfate heptahydrate, 10 g sodium carbonate, 2 g sodium chloride (dissolve calcium chloride and magnesium sulfate heptahydrate in 300 mL distilled water, add 500 mL water after dissolution and slowly add other salts. After all substances are dissolved, add 200 mL distilled water and store at 4°C until used).

[0047] (3) Casein medium: 5 g / L casein, 1 g / L glucose, 1 g / L yeast extract, 1 g / L K2HPO4, 0.5 g / L KH2PO4, 0.1 g / LMgSO4, 20 g / L agar, pH 7.0-7.5, sterilized at 121°C for 20 min.

[0048] (4) MRS medium: 10.0 g / L peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 5.0 g / L sodium acetate, 2.0 g / L ammonium citrate, 20.0 g / L glucose, 2.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 1.0 mL / L Tween-80, adjust pH to 6.2-6.4, 20.0 g / L agar (solid medium), sterilize at 121°C for 20 min.

[0049] (5) E9 medium: glucose 5.0 g / L, ammonium sulfate 1.3 g / L, potassium dihydrogen phosphate 0.5 g / L, disodium hydrogen phosphate 1.5 g / L, sodium chloride 1.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, biotin 0.5 g / L, and agar powder 20 g / L.

[0050] The bacterial strain used for natto fermentation and the coix seed saccharification liquid of the present invention are prepared as follows:

[0051] 1. Strain B9 was screened in medium E9 and confirmed to be Bacillus natto (the method is: Bacillus natto does not grow in E9 medium without biotin, but only grows in the presence of biotin. This method further identified Bacillus subtilis as Bacillus natto). Strain B9 was inoculated into B9 seed medium and cultured at 37°C for 18 hours. The cells were collected by centrifugation and resuspended in sterile saline (the concentration of the strain was 1.0×10 8 CFU / mL). Strains BLH1 and BLH6 were inoculated into PTYG liquid culture medium, respectively, and cultured at 37°C for 24-48 hours before use.

[0052] 2. Preparation of Coix Seed Rice Saccharification Solution Reference (Lin Ruimin. Study on the Preparation of Coix Seed Rice Extract for Cosmetics by Composite Enzyme Method [J]. Fujian Light Industry, 2018, No. 349(06): 34-39) with slight modifications. Screen the undamaged Coix Seed Rice and wash it three times with clean water. Then soak it at 25℃ for 12h. Then, beat the Coix Seed Rice with a material-water ratio of 1:12 (W / W). The Coix Seed Rice slurry was heated at 90℃ for gelatinization for 20min. After gelatinization was complete, enzymes were added for enzymatic hydrolysis. The liquefaction enzyme was high-temperature α-amylase, the dosage was 300U / g, the liquefaction temperature was 85℃, and the liquefaction time was 45min. The saccharification enzyme was glucoamylase, the dosage was 300U / g, the saccharification temperature was 65℃, and the saccharification time was 80min. After cooling, it was placed in the refrigerator for use.

[0053] Example 1 Optimization of soybean fermentation process by mixing strains BLH1 and B9

[0054] 1. Sample preparation

[0055] Wash the intact and uniform soybeans three times and soak them in coix seed saccharification liquid for 13 hours. After filtering, place 50 grams of coix seed saccharification liquid soybeans in a conical flask, add 1wt% NaCl, and sterilize (121°C, 20min). Mix the BLH1 and B9 bacterial liquids in a volume ratio of 1:1, and use an 8wt% inoculation amount for mixed fermentation. The mixed bacteria are inoculated into the coix seed soybeans and fermented for 24 hours at a fermentation temperature of 37°C and a ripening time of 24h. The natto made by sterilizing the soybeans soaked in coix seed saccharification liquid and inoculating BLH1+B9 mixed fermentation is referred to as BLH1+B9 natto.

[0056] Weigh 5 g of the fermented natto sample, add deionized water to dilute it 10 times, grind it with a grinder, place it in a 4°C refrigerator for 24 hours, and then centrifuge (7500×g) to collect the supernatant for later use.

[0057] 2. Methods

[0058] 1. Determination of Nattokinase

[0059] The specific steps are as follows: dilute the natto extract a certain number of times, using deionized water as a blank control, and set aside until the absorbance is between 0.04 and 0.08. Add 1.4 mL of Tris-HCl (50 mM, pH 7.8) buffer and 0.4 mL of fibrinogen solution (7.2 mg / mL) to a test tube. Incubate at 37°C for 5 minutes, then add 0.1 mL of thrombin (20 U / mL). Incubate at 37°C for another 10 minutes to form an artificial thrombus. Add 0.1 mL of the test sample, incubate at 37°C for 60 minutes, and add 2 mL of trichloroacetic acid (0.2 mol / L) to terminate the reaction. Centrifuge at 11,000 × g for 10 minutes, and measure the absorbance of the supernatant at 275 nm. Enzyme activity is defined as 1 unit of fibrin-degrading enzyme activity, which is defined as an increase of 0.01 in absorbance per minute at 275 nm.

[0060] 2. Determination of γ-aminobutyric acid (GABA) content

[0061] 2.4-Dinitrofluorobenzene (FDNB) was used for pre-column biochemistry to determine the GABA content in Job's tears and natto.

[0062] Sample extraction: Accurately weigh 5g of natto sample in a 50mL centrifuge tube, add 15mL of deionized water, vortex and shake thoroughly for 5min, centrifuge (6000×g, 15min) and collect the supernatant. Repeat the shaking extraction twice according to the above steps. Combine the extracts and dilute to 50mL with deionized water, and analyze by HPLC. Drawing of the standard curve: Prepare GABA standards of different concentrations, take 0.2mL of standard solution, add 0.2mL of 0.5mol / L sodium bicarbonate and 0.2mL of 1% FDNB solution (dissolved in acetonitrile and placed in this bottle), and place in a 60℃ water bath in the dark for 1h for derivatization reaction. After the reaction is completed, add 1.4mL of 0.12% phosphoric acid aqueous solution. Pass through the membrane and put on the machine. The standard curve equation is: Y=0.1172x-0.9054, R 2 =0.9996

[0063] Chromatographic conditions: An AclaimM 120CI8 column (inner diameter 4.6 x 250 mm particle size, Sum), detector wavelength 370 nm, column temperature 35°C, analysis time 10 min, flow rate 1 mL / min, injection volume 10 μL, mobile phase: acetonitrile: aqueous phosphoric acid (0.12% by volume) = 50:50.

[0064] 3. Sensory evaluation

[0065] The natto samples were randomly sorted and then evaluated by a sensory panel of 20 experienced sensory assessors in a dedicated sensory food laboratory with ample light and space. The sensory characteristics of natto are primarily determined by appearance, smell, stickiness, mouthfeel, and chewiness. These five sensory characteristics were independently evaluated using a 1.0-5.0 scale (5 = very like, 4 = somewhat like, 3 = average, 2 = not too like, 1 = very dislike). The average of the 20 scores was taken, and the standard deviation was calculated. A higher score indicates better quality natto. The natto sensory scoring criteria are shown in Table 1.

[0066] Table 1 Natto sensory scoring standards

[0067]

[0068] 4. Single-factor design of fermentation parameters

[0069] (1) Effect of NaCl addition on natto fermentation

[0070] Select small soybeans, wash and soak them for 14 hours, drain the water and maintain a certain water content, package them in 50g / bottle, add NaCl at 0.2%, 0.4%, 0.6%, 0.8% and 1%, high-pressure steaming at 121℃ and 0.1MPa for 20 minutes, after cooling, inoculate the seed liquid at 8% of the total inoculum amount (inoculation ratio 1:1), ferment at 37℃ for 24 hours, transfer to a 4℃ refrigerator, and ripen for 24 hours to obtain the natto product, and test various indicators.

[0071] (2) Fermentation temperature

[0072] Temperature is an important prerequisite for bacterial growth and enzyme activity. Temperature control is crucial in natto fermentation. Five temperature gradients were selected: 28°C, 31°C, 34°C, 37°C, and 40°C. Other conditions remained the same as the basic process.

[0073] (3) Inoculation volume

[0074] The appropriate starting bacterial concentration can shorten the growth period of the strain and quickly enter the fermentation stage. The cultured biological seed liquid was centrifuged to collect the bacteria and resuspended and diluted with sterile saline. The bacterial concentration was maintained at 1.0×10 8 CFU / mL, the two bacterial solutions were mixed at a 1:1 ratio for use. Five gradients of total inoculum volume were set: 2%, 4%, 6%, 8%, and 10%. Other conditions were the same as the basic process for natto fermentation.

[0075] (4) Fermentation time

[0076] The control of fermentation time is particularly important. A suitable fermentation time, on the basis of sufficient fermentation of the material, can avoid the waste of nutrients and prevent the accumulation of harmful metabolites. Therefore, according to previous experience, five gradients of fermentation time were selected: 18 h, 21 h, 24 h, 27 h, and 30 h, and the other conditions were the same as the basic process, so as to ferment the natto.

[0077] 5. Response surface test

[0078] The BBD response surface design method was used to optimize the significant factors by taking the natto kinase activity, GABA content, and sensory score as the response values. The factor levels and codes are shown in Table 2.

[0079] Table 2 Response surface experimental design

[0080]

[0081] III. Results

[0082] 1. Single factor experiment

[0083] (1) Effect of fermentation time on coix seed natto

[0084] As Figure 1 the fermentation time was 21-27 h, the natto had a good sensory score. With the extension of the fermentation time, the color of the natto became darker, the ammonia smell was rich, and excessive fermentation made the beans softer, and the sensory score gradually decreased. The natto kinase activity content (18-27 h) showed an upward trend. When the bacteria reached the logarithmic phase, the rapid growth of the bacteria would consume a large amount of nutrients, and continued fermentation would inhibit the growth of bacteria due to insufficient nutrients, while the biochemical reactions under the action of various enzyme systems did not stop, and the fermentation products were gradually accumulated. However, too long fermentation time would lead to aging of the bacterial cells and the production of various harmful metabolites.

[0085] (2) Effect of fermentation temperature on coix seed natto

[0086] As Figure 2 , the natto kinase activity was the highest at 37℃, but the sensory score was not the best at this temperature. The GABA content was the highest at 34℃. In terms of taste, flavor, texture, viscosity, and appearance, temperature had a greater impact on these sensory characteristics. When the temperature was between 28-34℃, the bacteria grew slowly and had an impact on various biochemical reactions throughout the fermentation process. When the temperature was higher than 37℃, the sensory score gradually decreased, and the natto kinase activity first increased and then decreased. In addition, with the increase of temperature, the GABA content first increased and then decreased, and the metabolites did not increase but decreased at the end of fermentation, which may be due to the decomposition rate of certain enzymes and other substances being greater than the metabolic rate of bacteria.

[0087] (3) The effect of salt addition on BLH1+B9 natto

[0088] The effect of NaCl addition on natto is shown in Figure 3 , the sensory quality of natto was significantly affected by different NaCl concentrations. The highest sensory score was obtained with 0.6% NaCl. On the other hand, there was a significant difference in sensory score between 0% and 1% (p>0.05). However, when the salt concentration increased above 0.6%, the sensory score decreased rapidly, with a highly significant difference (p<0.01). Figure 3 (B) of Table 1 shows that with the increase of NaCl level, the GABA content and sensory score showed a trend of first increasing and then decreasing, and the natto kinase activity level was basically unchanged, with the highest index at 0.8% addition.

[0089] Salt is often added during food fermentation, which not only affects the growth of fermentation microorganisms, but also affects the final sensory quality and shelf life of the product. Different salt contents significantly affect the sensory properties and GABA content of natto. The best sensory score is obtained when the addition is 0.6%, and the GABA content decreases rapidly when the addition exceeds 0.6%, with a highly significant difference. With the increase of NaCl content, the metabolic capacity of natto kinase is not affected, but the GABA content is greatly affected, which shows that the salt tolerance of the fermentation strain does not affect the production of natto kinase within a certain range, and natto bacteria need salt during growth and fermentation, and are suitable for growth and fermentation in an environment with osmotic pressure. Therefore, from the perspective of health and nutrition, it is appropriate to add a certain amount of salt during fermentation to promote the growth of natto bacteria. The salt content should not be too high, as it will affect the sensory score of natto, and too low will affect the metabolism of active substances. Such low-salt natto food can not only prevent and control hypertension but also reduce blood volume, which is very friendly to patients with coronary heart disease accompanied by hypertension, and meets the requirements of food in the era of health.

[0090] (4) The effect of inoculum size on natto with coix seed

[0091] As shown in Figure 4 , when the inoculum size is 8-10%, the NK activity of natto, GABA content and sensory score are all higher. When the inoculum size is small, the initial bacterial concentration is low, and it takes longer to multiply the bacterial population, the natto fermentation speed is slow, and the product accumulation is less; while the inoculum size is too large, the bacterial reproduction speed is too fast, the primary metabolism of cells is vigorous, and the nutrients are quickly consumed, which does not contribute to the accumulation of fermentation products. Therefore, when the inoculum size exceeds 8%, the natto kinase activity and sensory score are significantly decreased, and the GABA content level is basically unchanged. In addition, with the increase of inoculum size, the water content of the substrate also increases, and maintaining a certain water content is beneficial to cell metabolism, but too high water content makes the granular feeling of the beans weak, and there is a tendency to become paste-like, which greatly affects the stringiness and chewiness of natto.

[0092] 2. Response Surface Experiment

[0093] The response surface central composite experimental design and experimental results are shown in Table 3.

[0094] Table 3 Response surface design results

[0095]

[0096]

[0097] Design Expert 8 data processing software was used for analysis, and regression analysis was performed separately on the three response values ​​of sensory value (Y1), nattokinase activity content (Y2) and GABA content (Y3). The variance analysis results of the quadratic model of the response surface experiment are shown in Tables 4-6.

[0098] Table 4 Analysis of variance of sensory

[0099]

[0100] Table 5 Analysis of variance of GABA

[0101]

[0102]

[0103] Table 6 Analysis of variance of nattokinase activity

[0104]

[0105] The variance analysis with sensory response value as the response value is shown in Table 4. The p value of the model is <0.0001, indicating that the model is extremely significant. The lack of fit term p=0.9274>0.05 is not significant, indicating that the model has a good degree of fit. Sensory response value R 2 =92.74%, indicating that there is a high correlation between the actual and predicted sensory values. 2 、B 2 、C 2 、D 2 The corresponding p values ​​were < 0.05, indicating that these factors significantly affected the sensory content. The order of influence of the factors on the sensory content in the first-order terms was: fermentation temperature > fermentation time > inoculation amount > salt addition. Regression analysis of the experimental data yielded a quadratic regression model with sensory value as the objective function and the response value:

[0106] Y1=19.17+0.79×A-0.73×B-0.017×C+0.40×D-0.075×AB-0.55×AC-0.50×AD-0.075×BC+0.13×BD+0.72×CD.

[0107] Analyzing the interaction of relevant variables in Table 4, there are different degrees of interaction between factors. The p values ​​of the interaction between factors AB, AC, AD, BC, BD, and CD are all greater than 0.05, indicating that the interaction between factors has no significant effect on the sensory value. The order of the interaction of factors on the sensory value is AB>AC>CD>BD>AD=BC.

[0108] The variance analysis of the response value GABA is shown in Table 5. The p value of the model is <0.0001, indicating that the model is extremely significant. The lack of fit term p = 0.2369> 0.05 is not significant, indicating that the model fits well. The R 2 =90.83%, indicating that the correlation between the actual and predicted values ​​of GABA is high. 2 、B 2 、C 2 The corresponding p<0.05 indicates that these factors have a significant impact on GABA content. The order of influence of several factors on GABA content in the first term is: fermentation time > salt addition > inoculation amount > fermentation temperature. Based on the above analysis, the multiple regression equation for the response value GABA (Y3) and the independent variables fermentation time (A), fermentation temperature (B), inoculation amount (C), and salt addition (D) can be obtained:

[0109] Y3=156.11+11.83×A+0.24×B-7.03×C-37.36×D+7.05×AB-20.89×AC-1.73×AD-10.14×BC+43.85×BD+8.90×CD+2.80×A 2 -13.39×B 2 -1.94×C 2 +37.81×D 2 .

[0110] Analyzing the interaction of relevant variables in Table 5, there is an interaction between fermentation time and salt addition amount, p=0.0003<0.05, so the interaction of AD has a more significant effect on GABA content. With the increase of fermentation temperature and fermentation time, the GABA content first increases slowly and then tends to decrease. This is because fermentation time and fermentation temperature affect the growth and fermentation activity of microorganisms, thereby affecting the GABA content.

[0111] The variance analysis of the response value nattokinase activity is shown in Table 6. The model p = 0.0031, indicating that the model is extremely significant. The lack of fit term p = 0.0674> 0.05, the lack of fit term is not significant, indicating that the model fit is good. The R 2= 82.64, which indicates that the actual value and the predicted value of the nattokinase enzyme activity have a good fitting degree. A, C, AB, B 2 , C 2 The corresponding p < 0.05 indicates that the effects of these factors on the nattokinase content are significant. The order of the effects of the factors in the primary term on the nattokinase enzyme activity content from large to small is as follows: fermentation temperature > salt addition amount > inoculation amount > fermentation time. Regression analysis is performed on the experimental data, and the quadratic regression model of the target function with the nattokinase enzyme activity as the response value is as follows:

[0112] Y2 = 333.20 + 32.50 x A + 62.50 x B + 48.50 x C - 14.00 x D + 31.00 x AB + 71.25 x AC - 4.25 x AD - 55.25 x BC - 38.25 x BD + 54.50 x CD + 2.57 x A 2 - 9.18 x B 2 - 7.43 x C 2 - 39.18 x D 2 .

[0113] The interaction of the related variables in Table 6 is analyzed, and the factors have different degrees of interaction. The p of AB is 0.0533 < 0.05, the p of BC is 0.0613 > 0.05, the p of BD is 0.1067 > 0.05, the p of AC is 0.1502 > 0.05, and the p of AD is 0.4327 > 0.05, which indicates that the interaction of the fermentation temperature and the inoculation amount, the fermentation temperature and the NaCl addition amount, and the fermentation time and the inoculation amount has no significant effect on the nattokinase enzyme activity, while the interaction of the fermentation time and the fermentation temperature has a significant effect on the nattokinase content. With the increase of the fermentation temperature and the fermentation time, the nattokinase enzyme activity first increases and then decreases, which is because the fermentation time and the fermentation temperature have an effect on the growth of the microorganism and the fermentation activity, thus affecting the ability of the microorganism to produce metabolic nattokinase and GABA.

[0114] 3. Verification experiment

[0115] To verify the effectiveness and practicality of the model, three validation experiments were conducted under optimal culture conditions. Based on the three response values ​​of GABA content, enzyme activity, and sensory evaluation, the optimal response was obtained: fermentation time 27 hours, fermentation temperature 34.96°C, inoculum size 8.4%, salt addition 0.8%, and a sensory score of 20.87. Nattokinase activity was 379.885 FU / g, and GABA content was 135.764 μg / g. To verify the accuracy of the response surface optimization results and facilitate experimental operation, the optimized fermentation conditions were revised to: inoculum size 8%, fermentation temperature 35°C, fermentation time 27 hours, and salt addition 0.8 wt%. The revised optimized conditions were used for verification experiments. Under these conditions, the GABA content of BLH1+B9 natto was 125.749 μg / g, the nattokinase activity was 347.67 FU / g, and the sensory value was 22.07, which were close to the predicted values ​​of the response surface analysis. This shows that the fermentation industrial conditions obtained in this example are credible.

[0116] Example 2 Characterization of the physical and chemical properties of natto and changes in viable bacterial content during fermentation by different fermentation strains and coix seed saccharification liquid

[0117] 1. Sample preparation

[0118] According to the process finally determined in Example 1, soybeans soaked in coix seed saccharification liquid fermented by strain B9 and strain BLH1 or BLH6 (referred to as BLH1+B9 natto and BLH6+B9 natto) were prepared respectively. Steamed soybean samples (unfermented samples) were obtained by sterilizing the soybeans soaked in the saccharification liquid; natto samples of soybeans soaked in coix seed saccharification liquid fermented by a single strain B9 (referred to as B9 natto), and natto samples of soybeans not soaked in coix seed saccharification liquid fermented by a single strain B9 / BLH1 and a mixed strain B9 and BLH1 (referred to as B9 natto', BLH1 natto', and BLH1+B9 natto') were used as controls.

[0119] For natto fermentation without soaking soybeans in coix seed saccharification solution, whole and uniform soybeans were soaked in deionized water for 12-14 hours. After filtering the water, 50g of soybeans were placed in a conical flask, 1wt% sucrose was added, and sterilization was performed. The fermentation was then carried out by inoculation.

[0120] When mixed bacteria were fermented, the mixing ratio of all bacteria species was 1:1.

[0121] 2. Characterization of the physical and chemical properties of natto and changes in viable bacterial content during fermentation

[0122] 1. Changes in the content of total viable bacteria (TVC) during fermentation

[0123] One gram of natto was homogenized in 9 mL of sterile saline for 15 seconds, serially diluted, and poured onto MRS and casein plates (20 g agar per liter of liquid medium). Samples were taken every 3 hours to evaluate TVC. Strain BLH1 / BLH6 was cultured anaerobically at 37°C on MRS plates (anaerobic conditions were 80% H2, 10% N2, and 10% CO2) for 48 hours, and strain B9 was cultured aerobically at 37°C on casein plates for 24 hours. The colonies were counted and the viable colony count was expressed as colony forming units per gram of sample (CFU / g). The results are shown in Figure 2. Figure 5 .

[0124] Figure 5 The effect of fermentation time on the viable bacterial counts of B9 and BLH1 during the fermentation of BLH1+B9 natto and B9 natto. After 12 hours of fermentation, the viable bacterial count of BLH1 in BLH1+B9 was lower than that of B9. B9 grew steadily throughout the fermentation process. Fermentation was carried out in an aerobic environment, and the growth of BLH1 on the surface of natto was affected by oxygen. After 30 hours of fermentation with the two strains, the average maximum viable bacterial counts of B9 and BLH1 were 14.2 and 11.8 log CFU / g, respectively. The total viable bacteria in single-bacteria fermentation were 13.58 log CFU / g. The addition of BLH1 promoted the growth of B9. Mixed-bacteria fermentation was significantly better than samples fermented with single bacteria. Bacillus subtilis and Bifidobacterium produced a synergistic effect, promoting each other's coordinated growth.

[0125] The effects of fermentation time on the viable counts of B9 and BLH1 during the fermentation of B9 Natto', BLH1 Natto' and BLH1+B9 Natto' are shown in Table 7.

[0126] Table 7

[0127]

[0128] As shown in Table 7, the bacterial strain growth trend during fermentation of soybeans not soaked in the Job's tears saccharification solution was consistent with that during fermentation of soybeans soaked in the Job's tears saccharification solution. However, the viable bacterial count after 30 h of fermentation was lower than that of natto soaked in the Job's tears saccharification solution. This suggests that soaking soybeans in the Job's tears saccharification solution can help increase bacterial strain growth during fermentation.

[0129] 2. Nattokinase activity in different natto products

[0130] The nattokinase activity was the same as in Example 1. The changes in nattokinase activity during the fermentation of BLH1+B9 natto, BLH6+B9 natto and B9 natto were as follows: Figure 6 .

[0131] Depend on Figure 6Significant differences were observed in the changes in nattokinase activity (p < 0.05). Nattokinase activity gradually increased with fermentation time. Before 18 hours, nattokinase activity in BLH1+B9 natto was higher than in BLH6+B9 natto. At 25 hours, the nattokinase activity in the two groups was similar. After 15 hours of fermentation, BLH6+B9 natto exhibited higher nattokinase activity than BLH1+B9 natto. The viable count of B9 in BLH1+B9 natto was much higher than that in BLH6+B9 natto, and nattokinase is produced by B9. This suggests that it is not the greater the number of viable B9 cells that leads to higher nattokinase activity, but rather that other factors, such as bifidobacteria, regulate this activity. In the later stages of fermentation, BLH6 / BLH1 promoted nattokinase production in B9. After 30 hours of fermentation, nattokinase levels increased from 253.33 FU / g to 435.24 and 393.27 FU / g, respectively.

[0132] After 30 h of fermentation, the nattokinase activities of B9 Natto' and BLH1+B9 Natto' were 143.9 FU / g and 176.1 FU / g, respectively, which were significantly lower than those of B9 Natto and BLH1+B9 Natto.

[0133] Since the number of viable bacteria and nattokinase activity of soybean fermented natto soaked in coix seed saccharification liquid are better than those of soybean fermented products soaked in ordinary sucrose solution, it shows that soaking soybeans in coix seed saccharification liquid has a beneficial effect on fermented natto. Therefore, the following experiments only test the physical and chemical properties of soybean fermented natto products soaked in coix seed saccharification liquid.

[0134] 3. Changes in pH during fermentation

[0135] The pH value of the fermented soybean extract obtained according to "2. Determination of γ-aminobutyric acid (GABA) content" in "II. Method" of Example 1 was measured with a pH meter. Figure 7 .

[0136] Depend on Figure 7 It can be seen that during the entire fermentation process, the pH of the B9 natto sample gradually increased, and the pH of the mixed fermentation of coix seed natto gradually decreased, from the initial 6.3 to 4.9 ± 0.008, a significant decrease (p < 0.05), and Bifidobacterium BLH1 produced acid during the fermentation stage. The pH of BLH1 + B9 natto and BLH6 + B9 natto decreased during the fermentation process, falling to 6.29 ± 0.052 and 6.03 ± 0.09, respectively. The effect of pH on the acidity and alkalinity of natto affects the biological metabolism of the strain, thereby affecting the changes in nutrient content. pH also affects the shelf life of natto. pH can affect the activity of some enzymes in the cell, thereby affecting the storage period of food. The present invention found that Bacillus subtilis natto can regulate the rise of pH value, and the difference between the pH curves was significant (p < 0.05).

[0137] 4. Changes in amino nitrogen content during fermentation

[0138] The amino nitrogen content of three samples of B9 natto, BLH6+B9 natto, and BLH1+B9 natto during fermentation was tested. The results are as follows: Figure 8 shown.

[0139] Depend on Figure 8 It can be seen that the amino nitrogen content of the three samples increased during the fermentation process. The content in B9 natto and BLH1+B9 natto showed an upward trend, and the BLH6+B9 natto sample decreased slightly after 27h. From 0h to 30h, the amino nitrogen content of BLH1+B9 natto increased from 0.322±0.22g / 100g to 0.55±0.029g / 100g. The amino nitrogen content of natto samples fermented by a single bacteria was higher than that of samples fermented by mixed bacteria, among which the content of BLH1+B9 natto sample was the lowest. The test results of free amino acids showed that the free amino acid content of BLH1+B9 natto was the lowest, and the content of B9 natto was the highest, which was consistent with the research results of amino nitrogen.

[0140] 5. Changes in soluble peptide content during fermentation

[0141] The changes in the content of soluble peptides in B9 natto, BLH6+B9 natto, and BLH1+B9 natto during the fermentation process were detected. Figure 9 shown.

[0142] Depend on Figure 9 It can be seen that there are significant differences between different samples during the fermentation process (P < 0.05). The soluble polypeptide content in the unfermented sample was 45.25 ± 4.81 mg / g, and reached 249.34 ± 22.36 mg / g after 30 hours of single-bacteria fermentation, and its soluble polypeptide content increased by 5.5 times. As the fermentation time increases, the soluble polypeptide content in the mixed-bacteria fermentation sample shows an upward trend. During the fermentation process of 0-30 hours, the soluble polypeptide content of BLH1 + B9 natto continues to increase. When the content is the lowest at 45.25 ± 4.81 mg / g at 0 hours of fermentation, it rises to 132.146 ± 4.44 mg / g at 30 hours, which is 2.92 times that of the unfermented sample. This shows that Bacillus subtilis single-bacteria fermentation of soybeans can significantly increase the content of soluble polypeptides (P < 0.05). The soluble protein content of single-bacteria fermentation is the highest. In contrast, the soluble protein content of BLH1+B9 natto sample was significantly lower than that of the other two groups of samples.

[0143] 6. Changes in soybean isoflavone content during fermentation

[0144] High-performance liquid chromatography (HPLC) was used to quantitatively analyze the isoflavone content during soybean fermentation, and four isoflavones were identified: daidzein, daidzein, genistein, and genistin. Both soy isoflavones and genistein exhibit relatively high biological activity.

[0145] like Figure 10 As shown, the content of the four isoflavones in the three natto samples first increased and then decreased during fermentation. More importantly, the total isoflavone content in the crude natto extract was significantly increased compared to the unfermented soy sample (p < 0.05), especially daidzein. Changes in isoflavone content before and after fermentation are related to cellular metabolism and consumption. The daidzein content in the BLH6+B9 natto sample increased from 300.33±13.15μg / g to 746.65±12.97μg / g, and in the BLH1+B9 natto sample, it increased to 617.31±16.88μg / g. The final fermentation content of daidzein in the three sample groups (BLH1+B9 natto, BLH6+B9 natto, and B9 natto) was 190.29±18.07μg / g, 198.26±8.85μg / g, and 112.36±3.69μg / g, respectively. Soy isoflavones are phytoestrogens primarily found in soybeans and soy products. The genistin content in BLH1+B9 natto, BLH6+B9 natto, and B9 natto increased from 153.09±18.05μg / g to 795.69±23.16μg / g, 1133.72±35.964μg / g, and 235.40±18.42μg / g, respectively, before and after fermentation. The significant differences in genistin content among the three sample groups after fermentation suggest that BLHI itself can metabolize genistin, or that BHL1 promotes the metabolic conversion of B9 to genistin. Therefore, the above data analysis shows that mixed fermentation is beneficial for the accumulation of active substances. Of all the soy isoflavones detected in natto, daidzein has the highest content. Compared to pre-fermentation levels, daidzein content has increased significantly. Mixed fermentation increases the content of all soy isoflavones in natto, further enhancing its health benefits.

[0146] 7. Changes in phenolic content during fermentation

[0147] Phenolic compounds are important secondary metabolites in natto, playing a role in the detoxification of reactive oxygen species, disease response, and defense. High-performance liquid chromatography (HPLC) was used to qualitatively and quantitatively analyze the phenolic compounds in B9 natto, BLH1+B9 natto, and BLH6+B9 natto.

[0148] like Figure 11Significant differences were observed in the changes in phenolic compounds during fermentation (p < 0.05). Phenolic activity gradually increased with fermentation time. Ferulic acid was the primary phenolic compound in natto samples, with high levels in all three natto samples. Gallic acid content in B9 natto and BLH1+B9 natto varied similarly during fermentation. Gallic acid content in B9 initially decreased and then increased during fermentation, reaching a final level of 11.67 ± 6.03 μg / g. The late decrease in gallic acid content in BLH6+B9 natto was clearly due to the influence of Bifidobacterium BLH6. Ferulic acid content in the mixed fermentation natto samples initially increased, then decreased, and then increased again. After fermentation for over 20 hours, BLH1+B9 natto exhibited higher levels than BLH6+B9 natto. This suggests that phenolic compounds are not regulated solely by the B9 strain but are also influenced by other factors, such as Bifidobacterium BLH1 and BLH6. In many cases, co-fermentation with both strains promotes metabolite production. After 30 hours of fermentation, ferulic acid levels in BLH6+B9 natto and BLH1+B9 natto increased from 5.87±0.23μg / g to 33.61±1.68μg / g and 35.61±2.34μg / g, respectively. The addition of Bifidobacterium promoted the accumulation of ferulic acid in natto, increasing the content and quality of active substances. These results indicate that mixed fermentation can alter the composition and content of phenolic compounds. Fermentation methods using different bacterial strains also affected the total flavonoid content and the activity of enzymes involved in isoflavone metabolism.

[0149] 8. Changes in organic acid content during fermentation

[0150] The contents of oxalic acid, tartaric acid, succinic acid, malic acid, lactic acid, acetic acid and formic acid in B9 natto, BLH1+B9 natto and BLH6+B9 natto were determined. Figure 12 .

[0151] Depend on Figure 12The higher concentrations of organic acids in mixed-fermentation natto may be partially attributed to acid production by bifidobacteria, with a higher concentration of lactic acid, oxalic acid, and acetic acid. A one-way analysis of variance (ANOVA) was performed on each component to investigate the variation in organic acid content among the different samples. The oxalic acid content of B9 natto, BLH1+B9 natto, and BLH6+B9 natto was 24.88±1.99μg / g, 274.41±21.4μg / g, and 232.33±11.1μg / g, respectively. The tartaric acid content of BLH1+B9 natto, BLH6+B9 natto, and B9 natto was 57.07±3.39μg / g, 43.01±1.85μg / g, and 8.59±1.42μg / g, respectively. Lactic acid was detected in all samples. The lactic acid contents of BLH1+B9 natto, BLH6+B9 natto, and B9 natto were 290.36±12.89μg / g, 188.28±6.4μg / g, and 24.11±1.35μg / g, respectively. Lactic acid was the most abundant organic acid, followed by oxalic acid, acetic acid, and malic acid, all of which were detected in nearly all samples. During the fermentation process, the addition of bifidobacteria led to significant differences in the content of different organic acids. Organic acids in mixed fermentation are derived from the metabolic activity of bifidobacteria. The malic acid contents of BLH1+B9 natto, BLH6+B9 natto, and B9 natto were 82.45±2.163μg / g, 76±2.122μg / g, and 24.22±2.029μg / g, respectively. The acetic acid contents of B9 natto, BLH6+B9 natto, and BLH1+B9 natto were 13.36±0.4μg / g, 116.69±3.73μg / g, and 142.28±4.28μg / g, respectively. The above results indicate that the acid production capacity of Bifidobacterium BLH1 is greater than that of BLH6.

[0152] 9. Analysis of the antioxidant activity of natto during fermentation

[0153] The changes in antioxidant activity of B9 natto, BLH6+B9 natto, and BLH1+B9 natto samples during fermentation were evaluated.

[0154] like Figure 13As shown, in the DPPH system, mixed fermentation samples showed the best DPPH free radical scavenging activity, with BLH1+B9 natto achieving the highest scavenging activity. The DPPH free radical scavenging rate for all samples exceeded 85%. During natto fermentation, the antioxidant capacity of the three natto varieties increased significantly (p < 0.05). Comparison of the data with the significant increase in phenolic content revealed that samples with higher phenolic content also exhibited greater antioxidant activity. The composition and content of phenolic compounds change during soybean fermentation, and so does the antioxidant activity. The DPPH antioxidant capacities of BLH1+B9 natto, BLH6+B9 natto, and B9 natto were 97.4% ± 2.5, 94.6% ± 5.45, and 86.5% ± 4.26, respectively. The iron reducing capacities of BLH1+B9 natto, B9 natto, and BLH6+B9 natto samples were 95.95% ± 2.4, 92.45% ± 1.95, and 97.45% ± 3.35, respectively. The ABTS free radical scavenging rates of BLH1+B9 natto, B9 natto, and BLH6+B9 natto were 96.4% ± 1.39, 93.9% ± 1.41, and 85.2% ± 2.38, respectively. The composition and content of phenolic compounds and antioxidant peptides change during soybean fermentation, as does the antioxidant activity. The antioxidant activity of natto is correlated with the content of various active substances in natto.

[0155] Example 3 Effect of mixed fermentation on the flavor and taste of Job's tears natto

[0156] 1. Volatile substances in different natto products

[0157] Flavor is a complex characteristic, resulting from a combination of sugars, acids, and numerous volatile chemicals. This study used a combined SPME and GC–MS method to analyze and identify the aroma components of natto. Approximately 142 volatile ester compounds with a distinctive fruity aroma were detected in natto. Eight pyrazines were the most volatile compounds in B9 natto, BLH6+B9 natto, BLH1+B9 natto, and unfermented samples.

[0158] Generally, compounds with an OAV greater than 1 are considered to be key aroma-active compounds. The main flavor compounds produced by BLH6+B9 natto fermentation are 2,5-dimethyl-pyrazine (A2), trimethyl-pyrazine (A3), methyl nonanoate (B7), methyl hexadecanoate (B10), benzaldehyde (C2), octanal (C9), 2,3-butanedione (D9), 3-methyl-butyric acid (E6), 2-pentylamine (J5), and formamide (J2). The key flavor components in the mixed fermentation samples of BLH1 and B9 strains were trimethylpyrazine (A3), methyl hexadecanoate (B10), methyl anthranilate (B11), (Z)-2-decenal (C3), (E)-2-octenal (C5), hexanal (C7), octanal (C9), 3-octanone (D3), 3-methyl-butyric acid (E6), ethanol (F6), 2-acetylthiazole (I3), and naphthalene (I11). B9 natto produced mainly methyl octanoate (B9), benzaldehyde (C2), hexanal (C7), 3-octanone (D3), 3-methyl-butyric acid (E6), 2-hydroxypropionic acid (E16), and dimethyl disulfide (I10). The advantages of mixed fermentation of the two strains can be attributed to the synergistic effect between the different strains, which improves the flavor of natto. Compared to cooking saccharified soybeans, fermentation produces an increase in esters. It is speculated that the acids and alcohols produced by Bifidobacterium BLH1 / BLH6 can be esterified to form a large number of esters. Natto flavor is positively influenced by pyrazines. In particular, 2,5-dimethylpyrazine contributes significantly to natto flavor. Most amines have a pungent taste, which is the primary cause of natto's amine flavor. Twelve amines were detected, and fermentation with Bifidobacterium natto significantly reduced amine content compared to samples fermented with only one strain. Natto samples containing BLH1+B9 contained fewer volatile amines than samples containing BLH6+B9. This may be due to BLH1's greater acid production compared to BLH6, which neutralizes amines and improves natto flavor.

[0159] To show the overall distribution of the 94 odor-active compounds (OAV>1) in all samples, principal component analysis ( Figure 14). Principal component analysis showed the main differences between the volatile characteristics of the four samples. The distribution positions of the four samples were far apart, indicating that there were significant differences in the flavor substances between the samples. The characteristic components of B9 natto were 2-butanone (D10), 2-nonanone (D11), methyl tetradecanoate (B8), methyl octanoate (B9), 2-hydroxypropionic acid (E16), biphenyl (H9) and dimethyl disulfide (I10). Therefore, the characteristic flavor components of natto can be changed by mixed fermentation of two bacteria. Cluster analysis was performed on the content of volatile compounds in the four samples. CMSS included high levels of propionic anhydride (E11), nonanoic acid (E4), hexanoic acid (E8), n-hexadecanoic acid (E18) 3-octanol (F3), 1-undecene (G6), p-xylene (H3), estragole (I2), anethole (I5) and 2-pentyl-furan (I7). The characteristic volatile compounds that contribute most to the flavor of B9 natto include styrene (H1), 2-butanone (D10), methyl myristate (B8), methyl octanoate (B9), 2-nonanone (D11), 2-hexanone (D12), 2-hydroxypropionic acid (E16), biphenyl (H9), dimethyl disulfide (I10), cyclobutylamine (J4), and ethosuximide (J7). The characteristic volatile compounds that contribute most to the flavor of BLH1+B9 natto include esters (B), aldehydes (C), and ketones (D). The characteristic volatile compounds that contribute most to the flavor of BLH6+B9 natto include pyrazines (A), ketones (D), acids (E), and amines (J). The accumulation of volatile organic compounds (VOCs) in BLH6+B9 natto was greater than that in BLH1+B9 natto, and the flavor compounds that contributed to the overall flavor profile were significantly different from those in BLH6+B9 natto. Principal component analysis and hierarchical cluster analysis showed that mixed fermentation of BLH1+B9 natto significantly altered the VOC composition of natto, thereby influencing sensory scores and improving people's acceptance of natto.

[0160] 2. Electronic Tongue Data Analysis

[0161] Taste analysis of B9 natto, BLH6+B9 natto, BLH1+B9 natto and unfermented samples was performed using SA402B electronic tongue.

[0162] like Figure 15, there were no significant differences among the four samples in terms of richness, aftertaste A, and aftertaste B. However, there were significant differences in bitterness, sourness, saltiness, and umami. The sourness of the unfermented samples was significantly lower than that of the fermented samples, among which the BLH1+B9 natto sample tasted the sourest, once again proving that the acid production capacity of Bifidobacterium BLH1 was greater than that of BLH6. The astringency value of the unfermented samples was lower than that of the fermented samples, among which BLH1+B9 had the highest astringency value. Bitterness also increases after fermentation, which may be caused by bitter peptides and some bitter amino acids. Among them, BLH1+B9 natto had the lowest bitterness, and the single-bacteria fermented natto sample had the highest bitterness. The umami of all fermented natto samples was lower than that of the unfermented samples.

[0163] 3. Analysis of biogenic amine content

[0164] Table 8 lists the biogenic amine contents of three types of natto produced by fermentation with different strain combinations.

[0165] Table 8 Analysis of biogenic amines in natto

[0166]

[0167] Note: ND: Not detected (amine content is less than 0.1 mg / kg).

[0168] Only six biogenic amines—tryptamine, putrescine, cadaverine, tyramine, spermidine, and spermine—were detected in natto fermented with B9 alone and in the two mixed fermentation groups. 2-phenylethylamine and histamine were not detected. The highest total amine content in all three groups reached 359.53±13.03 mg / kg. The total biogenic amine content of all three natto samples tested was below 390.76±11.98 mg / kg, indicating that all products were within the safety range. The study found that the biogenic amine content of mixed fermentation samples was higher than that of single fermentation samples. The total amine content of B9 natto, BLH1+B9 natto, and BLH6+B9 natto was 359.53±13.03 mg / kg, 335.11±8.95 mg / kg, and 330.09±7.63 mg / kg, respectively. Tyramine, spermidine, and spermine were found at higher levels in B9 natto, BLH1+B9 natto, and BLH6+B9 natto. This indicates that they are mainly produced by B9 metabolism, and the addition of bifidobacteria will promote B9 metabolism to produce these biogenic amines.

[0169] 4. Free amino acid composition and analysis

[0170] An automatic amino acid analyzer was used to determine the content of hydrolyzed amino acids and free amino acids, and the ratio of hydrolyzed amino acids to free amino acids and the taste characteristics of these foods were systematically compared. The results showed that the total amount of hydrolyzed amino acids in the three types of natto ranged from 298.9 to 661.2 mg / 100 g. Comparative analysis of the mass fraction and composition of amino acids showed that different strains fermented the natto. The taste characteristics of the protein were evaluated by calculating the taste activity value (TAV) of the free amino acids that produced the taste. The TAV values ​​of the two types of natto, B9 fermented natto and BLH6+B9 fermented natto, were most significantly in Phe, His, and Lys, which are all bitter amino acids. Table 9 lists the amino acid content of raw unfermented soybeans, soybeans fermented with BLH6+B9, and soybeans fermented with BLH1+B9 natto.

[0171] Table 9 Comparison of free amino acid content of unfermented samples B9 Natto, BLH1+B9 Natto and BLH6+B9 Natto

[0172]

[0173] Note: RT: retention time; umami free amino acids (Asp and Glu), sweet free amino acids (Met, Ala, Gly, Ser, Pro and Thr), bitter free amino acids (Arg, His, Ile, Leu, Phe, Trp, Lys and Val) and tasteless free amino acid (Cys); values ​​are mean ± standard deviation (n = 3).

[0174] As shown in Table 9, the free amino acid content increased significantly in both the BLH6+B9 natto and B9 natto groups. In the case of BLH1+B9 fermentation, the free amino acid content increased less, suggesting that Bifidobacterium BLH1 has a low level of protein utilization or that the free amino acids produced during the fermentation process are consumed by the microorganisms. However, after being treated with Bacillus subtilis B9 alone, the amino acid content increased to 2.21 times that of the raw material, with significant increases in the Asp, Thr, Ser, Leu, Tyr, Phe, His, and Lys amino acid contents. The increase in free amino acid content in the BLH1+B9 mixed fermentation was lower than that in the BLH1+B9 natto group, likely due to the influence of BLH6. During mixed fermentation, the growth and metabolism of B9 produces proteases, which promote protein hydrolysis to produce various polypeptides, short peptides, and amino acids. These substances can be metabolized and oxidized by the bifidobacteria to provide energy, serving as a nitrogen source for bacterial growth, resulting in a decrease in free amino acid content.

[0175] The content of free amino acids affects food quality. Bitter amino acids are ranked as the primary factor contributing to poor taste. Fatty acids in food, such as lysine, threonine, and methionine, can affect protein quality and flavor. A total of eight bitter amino acids were detected in the four sample groups. As shown in Table 10, the taste thresholds of the bitter amino acids in the BLH6+B9 natto samples, with the exception of Phe, His, and Lys, were all below the human taste threshold. However, the bitter amino acids in the BLH1+B9 mixed fermentation samples were all below the taste threshold, meaning that no bitterness was detected. These data demonstrate that mixed fermentation with two bacteria can reduce the content of bitter amino acids.

[0176] Table 10 Taste activity values ​​of four groups of samples

[0177]

[0178] Note: “—” indicates not calculated.

[0179] 5. Peptide molecular weight distribution analysis

[0180] The present invention uses HPLC chromatography to determine the molecular weight of soybean polypeptides. Fermentation with different strains can cause significant changes in the molecular weight of polypeptides, as shown in Table 11.

[0181] Table 11 Distribution of molecular weight of polypeptides in natto

[0182]

[0183] As can be seen from Table 11, all samples are between 19-22 minutes, and the peak shape of each standard is uniform and symmetrical, all within the standard peak time range. The molecular weight range of the standards selected for this experiment is between 400Da and 12500Da, which generally includes the molecular weight of all protein peptides. The changes in the molecular weight of soluble proteins and polypeptides in natto were analyzed by liquid phase analysis, and the results are shown in the figure. The natto finished product has significantly less soluble proteins with a molecular weight greater than 6kD, while there are no soluble proteins with a molecular weight less than 2kD. The fermentation of Bacillus subtilis can accumulate molecular weights in the range of 2-6kD. Mixed fermentation of Bacillus subtilis and Bifidobacterium can produce a large amount of soluble polypeptides with a molecular weight of 2-6kD, and most of them are concentrated around 3kD.

[0184] Example 4 Prediction of Storage Stability and Shelf Life of BLH1+B9 Natto

[0185] 1. Construction of prediction model

[0186] This example uses the Arrenius model to predict the shelf life of BLH1+B9 natto. The initial pH value of this natto product was 6.17, the storage endpoint was 5.23, the initial sensory value was 23.6, the endpoint was 15, and the initial color difference value was 1.21, the storage endpoint was 3.5. t1 (pH), t2 (sensory), t3 (color difference). The pH and sensory prediction model can be determined as:

[0187]

[0188]

[0189] In all prediction models, T is the temperature.

[0190] 2. Validation of the Prediction Model

[0191] The temperature dependence of color difference, pH, and sensory parameters during storage of natto samples fermented with a mixed culture of BLH1 and B9 was statistically analyzed. Based on these temperature-dependent and statistical analyses, color difference, pH, and sensory parameters were selected as quality indicators for the development of a shelf life prediction model. Table 12 reports the predicted shelf lives of fermented natto samples stored at 4°C, 27°C, and 37°C, calculated using the pH and sensory degradation kinetics models: 12, 7, and 6 days; 12, 8, and 6 days; and 11, 9, and 8 days, respectively. As expected, low temperatures enhance the quality of BLH1 and B9 natto. The relative error between the predicted and actual shelf life values ​​also indicates that the shelf life prediction model at low temperatures is more accurate. Currently, the sensory-based prediction model demonstrates satisfactory results in predicting the shelf life of natto samples. Accuracy verification of the three constructed prediction models revealed that the error between the predicted pH value and the experimental value was 0, indicating high accuracy. This indicates that the zero-order reaction model combined with the Arrhenius model is feasible for predicting the shelf life and quality changes of natto. It can effectively predict the shelf life of BLH1+B9 natto within the 4-37°C temperature range. Shelf life prediction is crucial for distributors and consumers to determine storage time and optimal consumption time, and it also provides a reference for natto preparation and distribution.

[0192] Table 12 Predicted and actual values ​​of shelf life of BLH1+B9 natto at different temperatures

[0193]

[0194] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing coix seed natto fermented by mixed bacteria, characterized in that: include: Soaking cleaned soybeans in coix seed saccharification liquid, adding NaCl after soaking and sterilizing, inoculating Bacillus subtilis and Bifidobacterium into the sterilized material, and mixing and fermenting; Wherein, the Bacillus subtilis is Bacillus subtilis GUTU09, which was deposited in the China Center for Type Culture Collection on May 31, 2021, with the deposit number: CCTCC NO: M 2021641; The bifidobacterium is Bifidobacterium animalis subsp. Lactis BLH6; the Bifidobacterium animalis subsp. Lactis BLH6 was deposited in the China Center for Type Culture Collection on May 29, 2023, with a deposit number of CCTCC NO: M 2023843; The amount of NaCl added is 0.8% of the mass of soybeans after soaking in the coix seed saccharification liquid; The soaking time of the coix seed saccharification liquid is 12-14 hours; The coix seed saccharification liquid is prepared by soaking clean coix seed at 25° C. for 12 hours, adding water to pulp, heating to gelatinize, and then adding enzymes for enzymatic hydrolysis; the enzymes include high-temperature α-amylase and glucoamylase; In the step of adding water and beating, the mass ratio of the soaked Job's tears rice to water is 1:12; The inoculation ratio of the Bacillus subtilis and the Bifidobacterium is 1:1; The inoculation amount of the Bacillus subtilis and Bifidobacterium is 8 wt % of the sterilized material. During the mixed fermentation, the fermentation temperature is 35° C. and the fermentation time is 27 h.

2. Coix seed natto prepared by the preparation method according to claim 1.

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