Fermented soybean meal and preparation method thereof

Abstract

The invention discloses fermented soybean meal and a preparation method thereof, and belongs to the technical field of fermented food. After soybean meal and soybean slurry are mixed, bacillus natto and kluyveromyces marxianus are inoculated for fermentation, and the fermented soybean meal can be obtained. According to the method, bacillus natto and kluyveromyces marxianus are used as fermentation strains, a soybean meal processing technology is optimized through a single-bacterium and mixed-bacterium fermentation technology, the influence of fermentation on physicochemical properties, functional activity and flavor quality of soybean meal is systematically explored, a high-value fermented soybean meal product is prepared, and the nattokinase (NK) activity of the mixed-bacterium fermented soybean meal reaches 6562.49 IU / g. High-activity nattokinase can be produced by fermenting soybean meal with bacillus natto, and the flavor of the fermented soybean meal can be improved and the nutritional value of the soybean meal can be improved by mixed fermentation with kluyveromyces cerevisiae. According to the technical scheme, high-value utilization of soybean meal resources can be promoted, the cost of terminal products can be reduced, and dependence on soybean import can be reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of fermented foods, in particular to fermented soybean meal and a preparation method thereof. Background Art

[0002] Soybean meal (SBM) is a byproduct of soybean oil extraction. The nutritional composition of different soybean meals varies significantly depending on the soybean variety, growing region, and processing methods. As a cost-effective protein source, soybean meal has important applications in the food industry and animal feed.

[0003] Fermentation significantly enhances the functional components and nutritional properties of soybean meal. Natto, a popular superfood, is typically fermented from soybeans. Soybean meal significantly reduces production costs compared to soybeans. Currently, soybean meal is primarily used as a feed protein source, which hinders its high-quality applications. Therefore, maximizing the value of soybean meal and producing high-value natto-based products is crucial. Summary of the Invention

[0004] The purpose of the present invention is to provide a fermented soybean meal and a preparation method thereof, so as to solve the problems existing in the above-mentioned prior art.

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

[0006] One of the technical solutions of the present invention is a method for preparing fermented soybean meal. The fermented soybean meal is obtained by mixing soybean meal with soybean slurry, inoculating Bacillus natto and Kluyveromyces marxianus to ferment the mixture.

[0007] The second technical solution of the present invention is the fermented soybean meal prepared by the preparation method.

[0008] Based on the above technical solution, the present invention has the following technical effects:

[0009] The present invention uses Bacillus subtilis GUTU09 (B9) and Kluyveromyces marxianus (Klu) as fermentation strains, optimizes soybean meal processing technology through single-bacteria and mixed-bacteria fermentation technology, systematically explores the effects of fermentation on the physical and chemical properties, functional activity and flavor quality of soybean meal, and produces a high-value fermented soybean meal product. The nattokinase (NK) activity of the mixed-bacteria fermented soybean meal reaches 6562.49 IU / g. Using Bacillus subtilis GUTU09 (B9) to ferment soybean meal can produce highly active nattokinase, and using Kluyveromyces mixed fermentation can improve the flavor of the fermented soybean meal and increase its nutritional value. The technical solution of the present invention can not only promote the high-value utilization of soybean meal resources, but also reduce the cost of terminal products and reduce dependence on soybean imports. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Effects of inoculation ratio on amino nitrogen content (A), NK activity (A) and sensory score (B) of natto soybean meal.

[0011] Figure 2 Effects of fermentation time on the amino nitrogen content (A), NK activity (A) and sensory score (B) of natto meal.

[0012] Figure 3 Effects of fermentation temperature on amino nitrogen content (A), NK activity (A) and sensory score (B) of natto meal.

[0013] Figure 4 The changes in the number of viable bacteria during the fermentation of soybean meal (A), B9+Klu soybean meal (B) and Klu soybean meal (C).

[0014] Figure 5 The pH changes of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal during fermentation.

[0015] Figure 6 The changes in NK activity of B9 soybean meal and B9+Klu soybean meal during the fermentation process.

[0016] Figure 7 The changes in β-glucosidase activity during the fermentation of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal.

[0017] Figure 8 The changes in protease activity of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal during fermentation.

[0018] Figure 9 The changes in SOD activity during the fermentation of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal.

[0019] Figure 10 The changes in cellulase activity during the fermentation of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal.

[0020] Figure 11 The figure shows the changes in amino nitrogen of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal during fermentation.

[0021] Figure 12 The changes in soluble peptides of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal during fermentation.

[0022] Figure 13The changes in the content of soy isoflavones during the fermentation of B9 soybean meal, B9+Klu soybean meal, and Klu soybean meal, including genistein (A), genistin (B), daidzein (C), and daidzein (D).

[0023] Figure 14 The changes in antioxidant activity of B9 soybean meal, B9+Klu soybean meal, and Klu soybean meal during fermentation, including DPPH (A), FRAP (C), and ABTS (D).

[0024] Figure 15 Fourier transform infrared spectra of B9 soybean meal, B9+Klu soybean meal, Klu soybean meal and unfermented soybean meal (CK). DETAILED DESCRIPTION

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0027] The embodiment of the present invention provides a method for preparing fermented soybean meal. The fermented soybean meal is obtained by mixing soybean meal with soybean slurry, inoculating Bacillus natto and Kluyveromyces marxianus to perform fermentation.

[0028] In some specific embodiments, the weight ratio of the soybean meal to the soybean slurry is 1:2; the soybean slurry is obtained by mixing soybeans and water in a weight ratio of 1:8 and adding 4% (g / mL) sucrose.

[0029] In some specific embodiments, the inoculation amount of the Bacillus natto is 4%, and the inoculation amount of the Kluyveromyces marxianus is 1.3%, calculated by weight percentage.

[0030] In some specific embodiments, the fermentation conditions include: a fermentation time of 25 hours and a fermentation temperature of 37°C.

[0031] In some specific embodiments, after mixing the soybean meal and the soybean slurry, the method further comprises adding NaCl, wherein the amount of NaCl added is 1% (g / mL).

[0032] The embodiment of the present invention also provides fermented soybean meal prepared by the preparation method.

[0033] In this embodiment, soybeans were produced in Guiyang, Guizhou, and soybean meal was purchased from the Guizhou farmers' market.

[0034] In this example, Bacillus natto GUTU09 (B9 for short) was deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M2021641. This strain has been disclosed in patent CN118743447A.

[0035] In this example, Kluyveromyces marxianus (Klu for short) was purchased from Guangdong Provincial Microbiological Culture Collection Center, GDMCC 2.119.

[0036] Example 1

[0037] 1 Experimental methods

[0038] 1.1 Preparation of bacterial strains

[0039] After the B9 strain was inoculated into LB liquid medium, it was placed in a 37°C constant temperature shaking incubator (180 rpm) and cultured for 16-18 h. Then, the viable bacteria were counted by gradient dilution method and a concentration of 10 8 CFU / mL seed solution; Kluyveromyces marxianus strains were cultured in malt extract medium at 28°C for 24-48 hours, and bacterial suspensions of equal concentration were prepared using the same method. The above seed solution was centrifuged for 10 minutes using a high-speed refrigerated centrifuge (8000×g, 4°C), and finally resuspended in sterile saline to a standard concentration (1.0×10 8 CFU / mL) for later use.

[0040] 1.2 Sample preparation

[0041] Undamaged soybeans were screened, washed three times, and soaked at 20°C for 12 h. The soybeans were then pulped at a material-water ratio of 1:8. 4% (g / mL) sucrose was added to the pulped soybean slurry to obtain a soybean slurry, which was then refrigerated for later use.

[0042] After washing the soybean meal with pure water three times and filtering out the water, accurately weigh 20.0g and place it in a 250mL conical flask, then add 40.0g of soybean slurry and 1% (g / mL) NaCl. Sterilize the sample at 121°C for 20 minutes. Use an inoculum size of 4% (w / w) for B9 single strain fermentation, and an inoculum size of 1.3% (w / w) for Klu single strain fermentation. B9 and Klu are mixed and inoculated into the sample at a ratio of 3:1 (4%:1.3%) and fermented at 37°C for 24 hours. The soybean meal fermented from the mixed fermentation of B9+Klu is referred to as B9+Klu natto soybean meal.

[0043] Sample pretreatment: The fermented soybean meal sample was diluted 10 times with deionized water, crushed with a colloid mill, placed in a 4°C refrigerator for 24 h, and centrifuged (8000×g) for 10 min to obtain the supernatant for later use.

[0044] 2. Biochemical index detection

[0045] Amino acid nitrogen was analyzed according to the national standard GB5009.235-2016 "Determination of amino acid nitrogen in foods" (2016). Nattokinase activity was determined according to the standard DB 44 (2019).

[0046] The natto soybean meal samples to be tested were numbered and randomly arranged. Subsequently, a sensory evaluation team consisting of 20 teachers and graduate students from food-related majors with rich experience in sensory evaluation conducted a sensory evaluation in a dedicated sensory analysis laboratory with sufficient light and suitable space. The sensory characteristics of soybean meal were mainly evaluated from five dimensions: appearance, smell, viscosity, taste and chewiness. For these five sensory indicators, a scoring standard of 1.0-5.0 points (5 = very like, 4 = relatively like, 3 = average, 2 = not too like, 1 = very dislike) was used for independent evaluation. Finally, the average and standard deviation of the 20 scores were calculated. The higher the score, the better the quality of the soybean meal. The specific scoring standard refers to the literature (Zhang Qifeng, 2024), and the total score of the sensory evaluation was converted to a percentage system.

[0047] The initial fermentation process for natto soybean meal involved inoculating B9 and Klu at a ratio of 3:1 (4%:1.3%) and fermenting at 37°C for 24 hours. The effects of inoculation ratios (4:1, 3:1, 2:1, 1:1, and 1:2) (the B9 inoculum was always maintained at 4%), fermentation time (18, 21, 24, 27, and 30 hours), and fermentation temperature (25, 28, 31, 34, and 37°C) on the natto soybean meal were investigated, using amino nitrogen content, NK activity, and sensory evaluation as evaluation indicators. A response surface analysis was also conducted.

[0048] 3 Statistics and Analysis

[0049] Experimental data were processed and analyzed using statistical analysis software such as SPSS 22.0 and Microsoft Excel. Data are expressed as mean ± standard deviation (mean ± SD). One-way analysis of variance (ANOVA) was used to test for statistical significance (P < 0.05), and significant differences were indicated by letters. Data visualization was performed using Origin 21.0 software, and experimental design and response surface analysis were performed using Design Expert 8.0 software. All experimental results were based on data analysis from three independent replicates.

[0050] 4 Experimental results

[0051] 4.1 Single-factor experiment

[0052] In this example, Bacillus natto B9 and Kluyveromyces were combined to ferment soybean meal and the optimal inoculation ratio was explored. Figure 1 As shown, when the inoculation ratio of B9 to Kluyveromyces was 4:1, the aminopeptide nitrogen content and NK activity in the soybean meal were the highest, reaching 0.59 g / 100 g and 6257.86 IU / g, respectively. At this inoculation ratio, the soybean meal tasted slightly bitter and had a high viscosity. The characteristic natto odor was overpowering, and the chewiness score was low. As the inoculation ratio of Bacillus natto decreased, the aminopeptide nitrogen content and NK activity in the soybean meal also showed a decreasing trend. When the inoculation ratio of Bacillus natto to Kluyveromyces was 3:1, the aminopeptide nitrogen content was no significantly different from the 4:1 ratio, reaching 0.58 g / 100 g, and the NK activity was 5253.46 IU / g. At this ratio, the soybean meal had moderate viscosity, a reduced or barely perceptible bitterness, and a less pronounced natto odor. When the inoculation ratio of Bacillus natto to Kluyveromyces decreased to 2:1, 1:1, and 1:2, aminopeptide nitrogen content and NK activity decreased significantly (P < 0.05), ultimately dropping to 0.26 g / 100 g and 1000.74 IU / g. The soybean meal exhibited poor viscosity, a strong alcoholic flavor, and a loose appearance. Therefore, a B9 to Kluyveromyces inoculation ratio of 3:1 was selected for subsequent optimization experiments.

[0053] like Figure 2 As shown in Figure A, with the increase of fermentation time, the aminopeptide nitrogen content and NK activity in the fermented soybean meal showed a trend of first increasing and then decreasing. When the fermentation time was 24 hours, the aminopeptide nitrogen content was the highest, which was 0.61g / 100g; when the fermentation time was 27 hours, the NK activity was the highest, which was 6835.61IU / g. Figure 2 As shown in Figure B, fermentation time ranges from 18 to 24 hours. The soybean meal exhibits moderate viscosity, lacks a noticeable amine odor, and exhibits good sensory properties. At 18 hours, both the aminopeptide nitrogen content (0.43 g / 100 g) and NK activity (1564.11 IU / g) are low. As fermentation time increases, the aminopeptide nitrogen content decreases (from 0.61 g / 100 g to 0.43 g / 100 g) and the NK activity decreases (from 6835.61 IU / g to 4147.41 IU / g). Therefore, 24 hours represents the optimal fermentation condition.

[0054] like Figure 3As shown, as fermentation temperature increases, amino nitrogen content (0.21g / 100g to 0.57g / 100g) and NK activity (422.90 IU / g to 5674 IU / g) initially increase and then stabilize. Sensory scores initially increase and then decrease. As fermentation temperature increases, soybean meal darkens in color. Excessive fermentation causes the meal to become soft and mushy, with excessive stickiness and a reduced taste. Therefore, NK activity essentially ceases to increase above 37°C. When temperatures exceed 37°C, the amine flavor of mixed-bacteria fermented natto is overly pronounced. High temperatures may inhibit the growth of Kluyveromyces yeast, leading to a prominent amine flavor in soybean meal. Excessive protease degradation of soy protein is the primary source of amino nitrogen. Given the poor sensory properties of soybean meal above 37°C, 37°C was selected as the optimal fermentation temperature.

[0055] Response surface analysis revealed the optimal parameter combination to be an inoculation ratio of 2.8:1, a fermentation time of 24.83 hours, and a fermentation temperature of 36.99°C. Three replicates were conducted under these conditions. The results showed an amino nitrogen content of 0.70 g / 100 g, a nattokinase activity of 6562.49 IU / g, and a sensory score of 83.54. Based on the feasibility of industrial production, the process parameters were adjusted to integers: an inoculation ratio of 3:1, a fermentation time of 25 hours, and a fermentation temperature of 37°C. The actual values under these modified parameters were verified to be close to the predicted values, confirming the engineering application value of the optimization model and providing a reliable parameter benchmark for large-scale production.

[0056] Example 2

[0057] 1 Each sample prepared in Example 1 was taken and subjected to the following measurements: viable cell count determination, pH determination, nattokinase activity determination, β-glucosidase activity determination, protease activity determination, SOD activity in the sample was determined using a SOD activity detection kit (WST-1 method), cellulase activity was determined according to the agricultural industry standard of the People's Republic of China (NY / T 912-2004) (2005), amino nitrogen content was determined, soluble peptide content was determined, soy isoflavone content was determined based on the GBT26625-2011 standard, phenolic substance content, antioxidant activity, DPPH and ABTS free radical scavenging ability, and iron ion reducing ability (FRAP) determination.

[0058] 2 Experimental results

[0059] 2.1 Changes in viable bacterial count

[0060] Figure 4 A in the figure shows the changing trend of B9 live bacteria count during soybean meal fermentation with B9 single bacteria. Figure 4 B in the figure is the change of the number of live bacteria of B9 and Klu during the mixed fermentation of B9 and Klu. Figure 4Figure C shows the changes in Klu viable cell counts during Klu fermentation with a single strain. The results show that the B9 viable cell count showed a steady growth trend during both B9 fermentation and B9 + Klu mixed fermentation, reaching peak values of 13.44 lg CFU / g and 13.47 lg CFU / g at 25 h and 30 h, respectively. However, in the mixed fermentation, the B9 viable cell count decreased slightly in the later stages of fermentation. Furthermore, the Klu viable cell count continued to increase during single-strain fermentation, reaching 11.33 lg CFU / g at 30 h, but decreased significantly in the mixed fermentation due to changes in environmental conditions.

[0061] 2.2 pH changes

[0062] Figure 5 The pH of soybean meal changes during fermentation. The pH of B9 soybean meal gradually increases. Kluyveromyces Klu produces acid during the fermentation stage. Therefore, the pH of soybean meal fermented with mixed bacteria decreases in the early stage of fermentation and decreases in the later stage due to the decrease in the number of viable bacteria ( Figure 4 ), acid metabolites decreased, and the number of viable B9 bacteria continued to rise, which in turn led to an increase in pH. After 30 hours of fermentation, the pH of B9 soybean meal, B9+Klu soybean meal, and Klu soybean meal were 6.65, 6.56, and 5.79, respectively, representing increases of 0.2, 0.11, and 0.65, respectively (p < 0.05). Dynamic changes in pH significantly regulate microbial metabolic activity, thereby inducing the biotransformation of its nutritional components. This study found that Bacillus natto exhibits pH-regulating properties, causing the pH of the fermentation system to trend upward.

[0063] 2.3 Nattokinase activity analysis

[0064] like Figure 6 As shown, the changes in nattokinase activity showed significant differences (P<0.05). The nattokinase activity of B9 soybean meal gradually increased with the extension of fermentation time, tended to equilibrium after 25 hours, and the nattokinase activity was always higher than that of B9+Klu soybean meal. At 30 hours of fermentation, the nattokinase activity of B9 soybean meal reached its highest level, 5944.12 IU / g, which was not significantly different from the nattokinase activity at 25 hours (5648.44 IU / g). In the later stage of fermentation, the metabolites of Klu affected the growth metabolism of B9. Therefore, in the later stage of fermentation, the nattokinase activity remained basically unchanged. The nattokinase activity of Klu+B9 soybean meal continued to increase in the early stage of fermentation, reaching a maximum of 5252.86 IU / g at 25 hours of fermentation, and showed a downward trend after 25 hours (P<0.05), finally reaching 5163.15 IU / g. In the early stage of fermentation, due to the rich nutrients in the fermentation substrate, the nattokinase activity in B9 soybean meal and KLU+B9 soybean meal increased significantly (P<0.05). In the later stage, due to the joint consumption of nutrients by Kluyveromyces marxianus and B9 in B9+Klu soybean meal, resulting in insufficient nutrients, the nattokinase activity in the mixed soybean meal decreased.

[0065] 2.4 β-glucosidase activity analysis

[0066] Changes in β-glucosidase activity of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal during fermentation Figure 7 As shown in the results, fermentation significantly increased β-glucosidase activity in soybean meal. During fermentation, the β-glucosidase activity of B9 and B9+Klu soybean meals initially increased and then decreased. B9 soybean meal reached its maximum at 144.87 U / g at 10 hours of fermentation, while B9+Klu soybean meal reached its maximum at 151.28 U / g at 15 hours, representing 12.56 and 13.78 times the activity at 0 hours, respectively. In the later stages of fermentation, the β-glucosidase activity of both B9 and B9+Klu soybean meals decreased to varying degrees (P < 0.05), reaching 78.21 U / g and 110.25 U / g, respectively, by the end of fermentation. This suggests that mixed fermentation is more conducive to increasing and maintaining β-glucosidase activity. Klu soybean meal, on the other hand, showed an increasing trend, reaching a final content of 114.10 U / g, significantly higher than that of unfermented soybean meal (8.35 times) (P < 0.05). The results showed that fermentation of soybean meal with Kluyveromyces can increase the β-glucosidase activity of the product, and mixed fermentation with Bacillus natto can also increase the β-glucosidase activity.

[0067] 2.5 Protease activity analysis

[0068] Changes in protease activity of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal during fermentation Figure 8 As shown. Fermentation significantly increased the protease activity in soybean meal (P < 0.05), especially in B9 single-strain fermentation and B9 + Klu mixed-strain fermentation. During the fermentation process, the protease activity of B9 and Klu soybean meals showed a continuous upward trend, reaching maximum values of 555.93 U / g and 283.58 U / g, respectively, at 30 hours of fermentation, representing 8.87 and 4.49 times that of unfermented soybean meal, respectively. The protease activity in B9 + Klu soybean meal increased significantly and then decreased slightly, reaching a maximum of 565.39 U / g at 20 hours, representing 8.97 times that of unfermented soybean meal. The protease content of Klu soybean meal during fermentation was higher than that of unfermented soybean meal (P < 0.05), indicating that Kluyveromyces can produce proteases. The protease activity in soybean meal fermented with mixed bacteria increased.

[0069] 2.6 SOD activity analysis

[0070] Changes in SOD activity of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal during fermentation Figure 9As shown in the figure, fermentation significantly increased SOD activity in soybean meal (P < 0.05), especially in B9 + Klu mixed fermentation. During the fermentation process, the SOD activities of B9 soybean meal, B9 + Klu soybean meal, and Klu soybean meal continued to increase, reaching maximum values of 3796.44 U / g, 3985.17 U / g, and 2827.67 U / g, respectively, at 30 hours of fermentation. These activities were 2.29 times, 2.40 times, and 1.71 times those of unfermented soybean meal, respectively.

[0071] 2.7 Cellulase activity analysis

[0072] Changes in cellulase activity during fermentation of B9 soybean meal, B9+Klu soybean meal, and Klu soybean meal Figure 10 As shown. During the fermentation process, the cellulase activity in Klu soybean meal increased slowly, reaching a maximum of 2.32 U / g at 15 h, and then tended to balance. B9+Klu soybean meal showed a trend of first increasing and then decreasing, reaching a maximum of 3.70 U / g at 15 h, and then a significant decrease (P < 0.05). B9 soybean meal showed an upward trend throughout the fermentation process, with a maximum of 3.51 U / g. The highest value of cellulase activity in soybean meal fermented with mixed bacteria was greater than that of the other two single bacteria, indicating that fermentation with the two bacteria had a positive effect on the accumulation of cellulase activity. Overall, the cellulase activity in soybean meal fermented with mixed bacteria and B9 soybean meal was higher than that in Klu soybean meal, indicating that Bacillus natto has a higher cellulase production capacity than Kluyveromyces.

[0073] 2.8 Changes in amino nitrogen content

[0074] like Figure 11 The amino acid nitrogen content of B9, B9+Klu, and Klu soybean meals during fermentation was analyzed. The amino acid nitrogen content of B9 and B9+Klu soybean meals showed consistent changes during fermentation, initially increasing and then declining significantly, reaching maximum values of 0.65g / 100g and 0.66g / 100g, respectively, at 25 hours. After 30 hours, it decreased to 0.47g / 100g and 0.46g / 100g, respectively. The amino acid nitrogen content of Klu soybean meal slowly increased during fermentation before leveling off and remaining significantly lower than that of B9 and B9+Klu soybean meals, indicating that Bacillus natto has a greater ability to degrade protein than Kluyveromyces.

[0075] 2.9 Changes in soluble peptide content

[0076] Figure 12The changes in soluble peptides in B9 soybean meal, B9+Klu soybean meal and Klu soybean meal during the fermentation process. During the fermentation process, the soluble peptide content in different fermented soybean meals increased significantly (P < 0.05). At 0 hours of fermentation, the soluble peptide content in B9 soybean meal, B9+Klu soybean meal and Klu soybean meal was 17.56 mg / g, 17.86 mg / g and 17.26 mg / g, respectively. During the fermentation process of 0-30 hours, the soluble peptide content of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal showed a continuous upward trend. From 0 to 15 hours, the soluble peptide content of B9 soybean meal was higher than that of B9+Klu soybean meal. After 15 hours, the soluble peptide content of mixed fermentation soybean meal was higher than that of B9 soybean meal. At 20 hours. At 30 hours of fermentation, the soluble peptide contents in the three samples increased to 154.71 mg / g, 155.79 mg / g, and 26.16 mg / g, respectively, representing increases of 8.81-fold, 8.72-fold, and 1.52-fold compared to fermentation time 0. This indicates that Bacillus natto fermentation can significantly increase the soluble peptide content in soybean meal (P < 0.05).

[0077] 3.10 Analysis of Soy Isoflavone Content

[0078] like Figure 13 As shown in Figure 2, a total of four isoflavones were identified, namely genistein ( Figure 13 A), genistin ( Figure 13 B), daidzein ( Figure 13 C) and daidzein ( Figure 13 During the fermentation process, the contents of the four isoflavones in the three soybean meals were significantly increased compared with those in the unfermented meal (0 h) (P<0.05).

[0079] Changes in genistein such as Figure 13 As shown in Figure A, during the fermentation process, the genistein content of B9 soybean meal first increased, reaching a maximum of 353.36 μg / g at 5 h and then gradually decreased, with the final content being 279.86 μg / g; the content changes of B9+Klu soybean meal and Klu soybean meal were similar, showing a trend of first increasing, then decreasing, and then increasing. At 25 h of fermentation time, the genistein content of B9+Klu soybean meal and Klu soybean meal reached maximum values of 404.68 μg / g and 414.20 μg / g, respectively, which increased to 2.12 times and 2.18 times that of 0 h, respectively; the final contents were 377.24 μg / g and 397.04 μg / g, respectively. In the later stage of fermentation, the genistein content of the two was always higher than that of B9 soybean meal (P<0.05). This indicates that Kluyveromyces fermentation has a positive and significant effect on the accumulation of genistein in soybean meal. Figure 13Where B represents the change of genistin. B9 soybean meal and B9+Klu soybean meal showed a trend of first increasing and then decreasing. The genistin content of Klu soybean meal first increased and then decreased during the fermentation process, and then increased in the later stage of fermentation. In the early stage of fermentation (0-10h), the genistin content of B9 soybean meal was higher than that of other soybean meals. After 10h, the genistin content of B9+Klu soybean meal and Klu soybean meal did not change significantly and was higher than that of B9 soybean meal, while B9 soybean meal showed a significant decrease. When the fermentation was finally completed, the genistin content of B9 soybean meal, B9+Klu soybean meal and Klu soybean meal were 564.03μg / g, 763.50μg / g and 846.06μg / g, respectively. In the entire fermentation system, due to the decrease in the pH content of the system caused by Kluyveromyces fermentation and the interaction between microorganisms, the presence of Kluyveromyces in the later stage of fermentation caused the genistin content in the mixed fermentation soybean meal to be better accumulated. Figure 13 C in the figure represents the change of daidzein. The content of B9 soybean meal increased significantly after 5 hours and then maintained a balance. The final content was 768.12μg / g, which was 1.74 times that of unfermented soybean meal. The content of B9+Klu soybean meal first increased and then decreased. After 15 hours of fermentation, it increased and then decreased slightly. Klu soybean meal showed an overall upward trend and tended to be stable in the later period. Figure 13 The D in the figure represents the change in daidzein. The daidzein content in all three soybean meals showed an initial increase followed by a decrease. B9 soybean meal had the highest daidzein content at 5 hours of fermentation, reaching 1797.61 μg / g. The highest daidzein content in B9+Klu soybean meal and Klu soybean meal reached 1676.67 μg / g and 1114.94 μg / g, respectively, at 10 hours. The daidzein content decreased significantly in the later stages of fermentation.

[0080] This example shows that Kluyveromycin fermentation can increase the content of soybean isoflavones, and mixed fermentation of Kluyveromycin and B9 is conducive to the accumulation and retention of genistein and genistin.

[0081] 3.11 Analysis of phenolic content

[0082] High-performance liquid chromatography (HPLC) was used to qualitatively and quantitatively analyze several major phenolic compounds in fermented soybean meal. Six phenolic acid compounds, including ferulic acid, caffeic acid, chlorogenic acid, gallic acid, quercetin, and epicatechin, were detected. The results showed that fermentation significantly increased the phenolic content of soybean meal. Ferulic acid was relatively high in soybean meal. The ferulic acid content in B9 and B9+Klu soybean meal initially increased and then decreased during fermentation, with a significant decrease in both groups (P < 0.05) in the later stages of fermentation. The ferulic acid content in B9 and B9+Klu soybean meal reached its maximum at 5 h (233.30 μg / g and 252.56 μg / g, respectively). Klu soybean meal showed a continuous upward trend during fermentation, reaching a final content of 257.32 μg / g at 30 h of fermentation. The ferulic acid content in B9, B9+Klu, and Klu soybean meal increased by 85.73%, 101.07%, and 104.86%, respectively, compared to the 0 h content. But the decline in the later stage of fermentation may be due to the consumption and transformation of microorganisms.

[0083] During fermentation, the caffeic acid content in Klu soybean meal did not change significantly (P>0.05). The caffeic acid content in B9 soybean meal and B9+Klu soybean meal initially decreased and then increased, with B9+Klu soybean meal showing a downward trend after 25 hours. The caffeic acid content in B9 soybean meal increased significantly after 25 hours of fermentation, reaching a final value of 47.39 μg / g. The caffeic acid content in B9+Klu soybean meal reached a maximum of 50.34 μg / g at 25 hours. This suggests that B9 fermentation facilitates the accumulation and production of caffeic acid, and that the caffeic acid content in mixed fermentation was higher than that in single fermentation.

[0084] During the fermentation process, chlorogenic acid in B9 soybean meal initially increased and then decreased, reaching a maximum of 243.78 μg / g at 20 hours, a 33.40% increase compared to unfermented soybean meal (P < 0.05). A significant decrease occurred in the later stages of fermentation (P < 0.05). This may be due to an increase in metabolites in B9 soybean meal during the latter stages of fermentation, leading to changes in environmental pH and decreased activity of related metabolic enzymes, resulting in a decrease in chlorogenic acid content. Chlorogenic acid content in B9+Klu soybean meal and Klu soybean meal showed an overall upward trend, reaching maximum values at 20 hours (192.98 μg / g) and 30 hours (187.85 μg / g), respectively, significantly exceeding unfermented soybean meal.

[0085] Gallic acid content in B9 and B9+Klu soybean meal initially increased and then decreased during fermentation, while gallic acid content in Klu soybean meal increased slowly. The gallic acid content in B9 and B9+Klu soybean meal reached its maximum values at 124.42 μg / g and 85.29 μg / g, respectively, after 15 hours of fermentation. The gallic acid content in soybean meal fermented with B9 alone was significantly higher than that in B9+Klu soybean meal. Klu soybean meal reached its maximum value of 83.91 μg / g at 30 hours.

[0086] The fermentation of B9 and Klu significantly increased the quercetin content in fermented soybean meal. During the fermentation process, the quercetin content in B9 soybean meal, B9+Klu soybean meal, and Klu soybean meal first increased and then decreased. The maximum values were reached at 10h (41.56μg / g), 5h (43.25μg / g), and 15h (80.54μg / g), respectively. Compared with fermentation 0h, the content increased by 4.70 times, 5.56 times, and 8.42 times, respectively (P < 0.05). In the later stage of fermentation, the three decreased. The results showed that Klu had significantly higher accumulation and metabolic capacity for quercetin than B9. Compared with B9 single-bacteria fermentation, mixed fermentation was more conducive to the accumulation and retention of quercetin.

[0087] The epicatechin content in B9 soybean meal, B9+Klu soybean meal, and Klu soybean meal showed similar changes to quercetin, initially increasing and then decreasing. Maximum epicatechin content was reached at 5 h (82.29 μg / g) for B9 soybean meal, 20 h (93.95 μg / g) for B9 soybean meal, and 10 h (87.60 μg / g) for Klu soybean meal. Mixed fermentation resulted in higher epicatechin content than both B9 and Klu soybean meal, indicating a synergistic effect between B9 and Klu fermentation.

[0088] 3.12 Antioxidant Analysis

[0089] During the fermentation process, the antioxidant capacity of natto soybean meal increased significantly (P<0.05). Figure 14 In Figure A, in the DPPH system, the DPPH antioxidant activity of different fermented soybean meals increased differently. The highest DPPH antioxidant activity in B9 soybean meal, B9+Klu soybean meal, and Klu soybean meal were 83.63% (20h), 83.07% (30h), and 74.95% (30h), respectively. Figure 14 In the whole fermentation process, the ABTS free radical scavenging rate of B9 soybean meal and B9+Klu soybean meal was significantly higher than that of Klu soybean meal. When the fermentation time was 0-15h, the ABTS free radical scavenging rate of B9 soybean meal was higher than that of B9+Klu soybean meal. When the fermentation time was 15-30h, the ABTS free radical scavenging rate of B9+Klu soybean meal was higher than that of B9 soybean meal. At 30h, the ABTS free radical scavenging rate of B9+Klu soybean meal was 89.96%, and that of B9 soybean meal was 89.41%. In the ABTS system, the mixed fermentation samples had a better effect on scavenging ABTS free radicals. Figure 14 In C, the iron reducing capacity of natto soybean meal increased significantly during the fermentation process. The iron reducing capacity of B9 soybean meal, B9+Klu soybean meal, and Klu soybean meal reached the highest level at 30 h of fermentation, which were 0.40, 0.45, and 0.37, respectively.

[0090] Example 3

[0091] The sample fermented in Example 1 was freeze-dried and ground into powder, which was then packed in sealed bags and stored at 4°C for testing.

[0092] 2 Detection indicators

[0093] Perform Fourier transform infrared spectroscopy scan.

[0094] Peak Fit software was used for data analysis. -1 The protein secondary structure was calculated based on the absorbance peak position and area. The Gauss-Lorentz second derivative was used to resolve the overlap of individual bands. The content (%) of each secondary structure was determined by the peak located between 1600 and 1700 cm cm. -1 The ratio of the corresponding area to the total area is determined.

[0095] Microstructure observation, free amino acid determination and volatile flavor substance determination were carried out.

[0096] 3 Experimental results

[0097] 3.1 Fourier transform infrared spectroscopy analysis

[0098] like Figure 15 As shown, the four soybean meals were -1 ~400cm -1 The characteristics of the wavenumber range are generally consistent, but the intensity of the absorption peaks is significantly different. The absorption peaks in different bands represent various functional groups. B9 soybean meal and B9+Klu soybean meal have absorption peaks at about 3382cm- 1 There is a broad absorption peak at 2952cm, which is mainly attributed to the stretching vibration peak υ(O-H) of the intermolecular hydrogen bond and the functional group -O-H. -1 The characteristic absorption bands near the 1737cm-1 can be attributed to the CH asymmetric stretching vibrations of the methyl (-CH3) and methylene (-CH2-) groups in the polysaccharide molecules. The difference in their peak shapes reflects the conformational differences in the ratio of -CH / CH2 groups and their spatial arrangement in the molecular chain. In addition, the 1737cm-1 -1 and 1710cm -1 The peaks near 1646 cm-1 may be attributed to the stretching vibration of -C=O in the free carboxyl group and correspond to the protonated carboxylic acid (COOH) and the peaks at about 1646 cm-1. -1 The deprotonated COO- group is located at 1394 cm -1 and 1067cm -1 The characteristic absorption bands near the δ(CO) band can be attributed to the in-plane bending vibration mode of the CO bond in carbohydrates (glycosides, terpenes, and polysaccharides). -1The band corresponding to CO (COH and COC) stretching vibration. 886cm -1 The weak band at 3382 cm is the deformation vibration of β-CH, which is the characteristic band of β-glycosidic bond. -1 2952cm -1 The absorption peak intensity at is weak, indicating that fermentation has changed the chemical composition of soybean meal.

[0099] 3.2 Protein secondary structure

[0100] Table 1 Effect of fermentation on the secondary structure of proteins in soybean meal

[0101]

[0102] Peak fit v4 software was used to obtain the secondary structure of proteins after baseline correction and characteristic peak fitting. As shown in Table 1, fermentation significantly affected the four secondary structures of proteins in soybean meal (P < 0.05). β-sheet (1600-1640 cm -1 ) and β-angle (1650~1700cm -1 ) is the main secondary structure in soybean meal, accounting for 62.81% to 66.64%, β-sheet and β-turn account for 27.07% to 33.91% and 32.99% to 35.74% respectively. Compared with unfermented soybean meal, the content of β-sheet decreased after fermentation (P < 0.05), and the content of β-turn increased (P < 0.05). The results showed that the stability of the secondary structure of fermented soybean meal decreased (β-sheet decreased). Compared with unfermented soybean meal, B9+Klu soybean meal and Klu soybean meal had irregular curls (1640-1650cm -1 ) and α-helix (1650~1660cm -1 ) content in B9 soybean meal increased (P < 0.05), the α-helix content in B9 soybean meal increased (P < 0.05), and the random coil content decreased slightly, but the results were not significant. α-Helical conformation corresponds to a more ordered structure. As shown in Table 1, the β-sheet content in Klu soybean meal decreased from 33.91% to 27.07%, and the random coil and α-helix contents were significantly higher than those in B9 soybean meal and B9 + Klu soybean meal. Therefore, Kluyveromyces fermentation can further reduce the stability of the secondary structure of proteins in soybean meal. Microbial fermentation significantly affects the secondary structure of soybean meal proteins.

[0103] 3.3 Microstructure analysis

[0104] B9 and B9+Klu soybean meals have a dense structure, strong interparticle adhesion, and no obvious porosity. Kluyveromyces marxianus fermented soybean meal and unfermented soybean meal have a loose structure and more pores, which is significantly different from B9 fermented soybean meal. This suggests that fermentation enhances the viscous structure of B9 and B9+Klu soybean meals, helping to improve their water-holding capacity.

[0105] 3.4 Free amino acid analysis

[0106] A total of 17 amino acids were detected in the four soybean meals, including eight essential amino acids. The total free amino acid content of Klu soybean meal did not change significantly compared to unfermented soybean meal (P>0.05). The free amino acid content of B9 soybean meal and B9+Klu soybean meal increased significantly (P<0.05). The total free amino acid content of B9 soybean meal and B9+Klu soybean meal increased from 214.43 mg / 100g (unfermented soybean meal) to 2200.71 mg / 100g and 2147.48 mg / 100g, respectively, representing a 9.26-fold and 9.01-fold increase, respectively. Essential amino acid content also increased from 36.21 mg / 100g to 1288.53 mg / 100g and 1275.60 mg / 100g, respectively, significantly exceeding that of unfermented soybean meal. The amino acid with the highest concentration detected in B9 and B9+Klu soybean meals was phenylalanine, at 360.99 mg / 100g and 360.79 mg / 100g, respectively, followed by glutamic acid at 350.72 mg / 100g and 326.97 mg / 100g, respectively. Glutamic acid was also the highest in Klu soybean meal, at 112.08 mg / 100g. Furthermore, the concentrations of tyrosine, lysine, and leucine were significantly increased (P < 0.05). Threonine was newly detected in B9 and B9+Klu soybean meals. Fermentation significantly altered the content and composition of free amino acids in soybean meal.

[0107] The content of umami, sweet, and bitter amino acids in fermented soybean meal (B9, B9+Klu, and Klu) significantly increased. The TAV of umami amino acids (aspartic acid and glutamic acid) increased in B9, B9+Klu, and Klu soybean meal, contributing positively to the umami flavor of the soybean meal. The TAV of sweet amino acids increased to varying degrees in all three fermented soybean meals. With the exception of methionine, the TAV of the other sweet amino acids was ≤1, meaning they did not contribute significantly to the overall flavor of the soybean meal and were not noticeable. The TAV of bitter amino acids was significantly increased in soybean meal fermented with Bacillus natto (B9+Klu and B9), with the TAVs of histidine, isoleucine, leucine, phenylalanine, lysine, and valine all greater than 1, indicating that soybean meal fermented with Bacillus natto possessed a distinct bitter taste. With the exception of lysine, which was slightly higher in B9+Klu than in B9, the TAV of the remaining bitter amino acids was lower than in B9. This indicates that co-fermentation of Klu and B9 can reduce the bitterness in soybean meal.

[0108] 3.5 Volatile compound content

[0109] Esters, acids, alcohols, and terpenes were the primary flavor compounds in soybean meal samples, with Klu samples showing the highest ester content. Unfermented soybean meal contained relatively low levels of all flavor compounds, indicating that fermentation enriched the sensory qualities of soybean meal samples and the variety and content of flavor compounds.

[0110] A total of 731 volatile components (excluding tetramethylpyrazine) were identified, including aldehydes (63), acids (36), esters (151), alcohols (89), and ketones (86). Hydrocarbons (including 9 aromatic hydrocarbons), phenols (24), amines (19), terpenes (114), ethers (21), and heterocyclic rings (70) were also detected. Ten substances, including sulfur and nitrogen compounds, were also detected. 525 of these components were shared by the four soybean meal samples; 10 were shared by Klu soybean meal and B9+Klu soybean meal, and 89 by B9 soybean meal and B9+Klu soybean meal. The flavor compounds common to both Klu and B9+Klu soybean meal are metabolites produced by fermentation of Kluyveromyces marxianus.

[0111] The volatile component spectrum of sample B9 showed that a total of 687 flavor compounds were identified (total concentration 117,758.3 μg / kg), covering 13 categories, including esters (131 species / 21,418.39 μg / kg), terpenes (112 species / 14,705.63 μg / kg), heterocyclic compounds (66 species / 14,630.99 μg / kg), acids (34 species / 15,279.89 μg / kg), alcohols (85 species / 13,614.37 μg / kg), ketones (84 species / 11,892.33 μg / kg), aldehydes (54 species / 5,291.66 μg / kg), and amines (19 species / 4,055.41 μg / kg).

[0112] The B9+Klu sample contains 686 volatile organic components (total concentration 131748.3μg / kg), including esters (134 types / 24943.84μg / kg), terpenes (110 types / 15295.40μg / kg), heterocyclics (65 types / 15172.61μg / kg), acids (35 types / 19022.67μg / kg), alcohols (86 types / 14675.34μg / kg), ketones (84 types / 12612.28μg / kg), aldehydes (53 types / 7160.167μg / kg), etc.

[0113] A total of 617 volatile flavor compounds were detected in Klu soybean meal samples, with a total content of 94536.23 μg / kg, including 54 aldehydes (6672.41 μg / kg), 30 acids (16876.84 μg / kg), 126 esters (26064.23 μg / kg), 71 alcohols (6174.74 μg / kg), 71 ketones (5049.91 μg / kg), 38 hydrocarbons (623 9.07μg / kg), 7 aromatic hydrocarbons (3813.35μg / kg), 21 phenols (2072.54μg / kg), 13 amines (1875.97μg / kg), 104 terpenes (10174.38μg / kg), 17 ethers (1215.44μg / kg), 56 heterocyclics (7594.48μg / kg) and 8 others (712.83μg / kg).

[0114] A total of 588 volatile flavor substances were detected in CK soybean meal samples, with a total content of 71959.70 μg / kg, consisting of aldehydes (56 kinds / 6942.69 μg / kg), acids (27 kinds / 16078.82 μg / kg), esters (115 kinds / 11824.21 μg / kg), alcohols (68 kinds / 5034.88 μg / kg), ketones (64 kinds / 3901.94 μg / kg), hydrocarbons (37 kinds / 6214.71 μg / kg), and aromatic hydrocarbons (7 kinds / 700.14 μg / kg).

[0115] The types and total content of aldehyde volatile compounds were similar among the four fermented soybean meal varieties, but the concentrations of individual compounds varied significantly. For example, the levels of 2-isopropyl-5-methyl-2-hexenal and benzaldehyde were significantly lower in fermented soybean meal than in unfermented soybean meal. This decrease in their concentrations after fermentation suggests that fermented soybean meal significantly improves the inherent bitterness, grassiness, and beany flavors of soybean meal. 8-Nonenal, a volatile compound with a distinctive vanilla and floral aroma, was found at much higher levels in B9 and B9+Klu soybean meal than in CK and Klu soybean meal. 2,6,6-Trimethyl-2-cyclohexene-1-carboxaldehyde was also higher in B9+Klu and Klu soybean meal. This suggests that fermentation with Bacillus natto promotes the accumulation of 8-nonenal, while fermentation with Kluyveromyces yeast promotes the accumulation of 2,6,6-trimethyl-2-cyclohexene-1-carboxaldehyde.

[0116] The propionic acid content in soybean meal fermented by Bacillus natto and soybean meal fermented by mixed bacteria was significantly higher than that of other acid compounds, indicating that Bacillus natto is closely related to the production of propionic acid.

[0117] Ester content increased in fermented soybean meal, with significant variations in the content of individual ester compounds between different fermentation methods. The levels of 2-propenyl benzoate, 1-methyloctyl butyrate, phenylethyl isobutyrate, ethyl phenylpropionate, and methyl carbamate were significantly higher in soybean meal fermented with mixed bacteria and Kluyveromyces than in unfermented soybean meal and B9, indicating that the production of these esters is closely related to Kluyveromyces. Furthermore, the levels of 2-methacrylic anhydride and 2-hydroxy-2-methylpropyl and propionate were significantly higher in soybean meal fermented with Bacillus natto (B9 and B9+Klu) than in unfermented soybean meal and Kluyveromyces, indicating that the production of these two esters is primarily dependent on Bacillus natto.

[0118] Alcohols are one of the important flavor substances produced by Bacillus natto fermentation. Among the 89 alcohols identified, the contents of 1,4-butanediol, 2-heptanol (fruity, musty, mushroom), 2-pyridinemethanol (caramel, pistachio, and chocolate), 2,3-dimethyl-2-butanol (bitter, waxy), and 2-[(2-aminoethyl)amino]ethanol (bitter, astringent) in B9 soybean meal and B9+Klu soybean meal were significantly higher than those in unfermented soybean meal and Klu soybean meal, indicating that they mainly originated from Bacillus natto fermentation. Among the 86 ketones identified, 2-heptanone, 3-methylthio-2-butanone, 1-(5-methyl-2-pyrazinyl)-1-ethanone, and 2-methylcyclopentanone were mainly found in B9 soybean meal and B9+Klu soybean meal, while 3-ethyl-2-hydroxy-2-cyclopentenone (caramel) was mainly found in Klu soybean meal and B9+Klu soybean meal, indicating that mixed fermentation helps to coordinate the aroma of soybean meal.

[0119] Heterocyclic compounds detected in the samples include pyrazines, pyrimidines, and furans. Pyrimidines are relatively rare, but they contribute to the flavor of soybean meal, imparting a distinctive aroma and taste. Among phenols, ethers, and other compounds (sulfur- and nitrogen-containing compounds), benzyl methyl sulfide, guaiacol, and maltol (sweet, caramel-like) are naturally present in soybean meal, though their levels decrease after fermentation.

[0120] 3.6 Soybean meal aroma components OAVs

[0121] A total of 181 substances with OAV ≥ 1 were detected in the four soybean meal samples, including 36 aldehydes, 32 esters, 25 alcohols, 26 ketones, 4 hydrocarbons and aromatics, 12 phenols, 4 ethers, 1 sulfur-containing, 2 nitrogen-containing, 23 heterocyclic, and 16 terpenes.

[0122] Among the 36 aldehydes detected with an OAV ≥ 1, 8-nonenal had the highest OAV, reaching 53053.59 in B9 soybean meal. It is often associated with smoky and plastic notes. Next in line were (E)-2-nonenal (fatty, green, cucumber, aldehydes, citrus), (E,Z)-2,6-nonadienal (cucumber, green), (E)-6-nonenal, and 2-octenal (fatty, green, vanilla), which imparted grassy, cucumber, and citrus notes to the fermented soybean meal, enriching its flavor. Fermentation increased the OAVs of (E,Z)-2,6-nonadienal and 8-nonenal, while also decreasing those of nonanal and (E,E)-2,4-decadienal, harmonizing the flavor profile of the fermented soybean meal.

[0123] Esters can impart fruity and floral aromas to fermented soybean meal. Most have pleasant fruity and floral notes, contributing to a pleasant flavor profile. Among the 32 esters detected with an OAV ≥ 1, 2-phenylethyl 3-methylbutyrate had the highest OAV (2989.32-17694.47). It exhibits floral, fruity, sweet, rose, peach, and apricot aromas, contributing to a pleasant flavor profile in fermented soybean meal. Overall, fermentation contributes to the flavor profile of esters in soybean meal, contributing to a harmonious flavor profile.

[0124] Among the alcohol compounds, 25 have an OAV ≥ 1. The highest OAV is 2-furfurylthiol (sulfur, roasted, coffee, oil, fat, smoke), followed by 5-methyl-2-furylthiol (roasted, coffee), 1-nonen-3-ol (fat, creamy, green, earthy, mushroom), and 1-octen-3-ol (fat, fruity, grassy, mushroom, perfume, sweet). Compared to unfermented soybean meal, fermentation with Bacillus natto and Kluyveromyces significantly increases the OAV content of alcohols in soybean meal, enriching its flavor.

[0125] Among the 26 alcohols detected with OAV ≥ 1, 1-nonen-3-one (pungent, mushroom) and (5Z)-octane-1,5-dien-3-one (metallic, geranium) have lower thresholds and thus larger OAVs, making a significant contribution to the flavor of soybean meal.

[0126] Among the phenolic compounds, there are 12 phenolic substances with OAV ≥ 1, among which 2-methoxy-4-vinylphenol (spicy, raisin), p-cresol (narcissus, animal, mimosa) and guaiacol (nuts) have larger OAVs. In addition, the OAVs are larger in soybean meal fermented by Bacillus natto (B9 soybean meal and B9+Klu soybean meal), indicating that B9 fermentation increases the level of phenolic compounds.

[0127] Among the 22 heterocyclic compounds with OAV ≥ 1, tetramethylpyrazine (nutty, musty, chocolate, coffee, lard, burnt), 2-acetyl-3-methylpyrazine (nuts, roasted hazelnuts, roasted grains, corn, potato chips, vegetables, nut skins, caramel) and cis-3,5-diethyl-1,2,4-trithiolane have higher OAVs and make significant contributions to the flavor of fermented soybean meal.

[0128] Among other categories such as phenols and ethers, dodecanonitrile (citrus, orange, peel, metallic, spicy) and octanonitrile (fatty, green) were also found to have higher OAVs. Both are nitrogen-containing compounds, and their OAVs are larger in B9 soybean meal and B9+Klu soybean meal, significantly affecting their flavor.

[0129] 3.7 Dynamic distribution of metabolite content differences

[0130] Using unfermented soybean meal (CK) as a control, the most highly upregulated substances in B9 and B9+Klu soybean meal were similar, primarily ketones (D276, XMW1137), alcohols (YZMW0475, QWMW1034), and pyrazines (D1276, D351). Furthermore, due to the fermentation characteristics of Bacillus natto, amines (NMW0066034 and NMW0067034) were produced in B9 and B9+Klu soybean meals, while the most upregulated substances in Klu soybean meal were esters (XMW3510, XMW2230, KMW0105*008, KMW0106*008, KMW0437, and KMW0514). Kluyveromyces fermentation produces various esters, which contribute to the flavor of the sample. In metabolite analysis, upregulation indicates a significantly higher concentration of the metabolite compared to the control, while downregulation indicates a significantly lower concentration than the control. By analyzing up- and down-regulated metabolites, changes in metabolic pathways under specific conditions can be revealed.

[0131] Example 4

[0132] The shelf life of B9+Klu natto soybean meal was predicted. The results showed that the storage time of B9+Klu soybean meal at 4°C was significantly longer than that at high temperatures of 25°C and 37°C, which were 92 days (4°C), 72 days (25°C) and 62 days (37°C), respectively.

[0133] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing fermented soybean meal, characterized in that: The fermented soybean meal is obtained by mixing soybean meal with soybean slurry, inoculating Bacillus natto and Kluyveromyces marxianus to carry out fermentation.

2. The preparation method according to claim 1, characterized in that The weight ratio of the soybean meal to the soybean slurry is 1:2; the soybean slurry is obtained by mixing soybeans and water in a weight ratio of 1:8 and adding 4% (g / mL) sucrose.

3. The preparation method according to claim 1, characterized in that In terms of weight percentage, the inoculation amount of the Bacillus natto is 4%, and the inoculation amount of the Kluyveromyces marxianus is 1.3%.

4. The preparation method according to claim 1, characterized in that The fermentation conditions include: fermentation time of 25 hours and fermentation temperature of 37°C.

5. The preparation method according to claim 1, characterized in that After the soybean meal and the soybean slurry are mixed, the method further comprises the step of adding NaCl, wherein the amount of NaCl added is 1% (g / mL).

6. The fermented soybean meal prepared by the preparation method according to any one of claims 1 to 5.

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