A lactococcus mellis and its application in improving the flavor of soybean seed coat

Abstract

The present application relates to the technical field of microbial strains and the technical field of soybean seed coat fermentation, in particular to a Pediococcus sp. of Pteropus sp. and application thereof in improving the flavor of soybean seed coat, wherein the Pediococcus sp. of Pteropus sp. is Pediococcus sp. of Pteropus sp. BL-2 (Lactococcus petauri BL-2) , which is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20252253. The Pediococcus sp. of Pteropus sp. BL-2 of the present application not only has significant antioxidant activity, but also can significantly increase the content of 3-hydroxy-2-butanone (acetoin) with a pleasant buttery flavor in soybean seed coat and significantly reduce the content of isovaleric acid, 2-methyl-2-pentenal and dimethyl disulfide and other substances with strong irritating odor after fermentation of soybean seed coat, thereby effectively improving the flavor of soybean seed coat.

Description

Technical Field

[0001] This invention relates to the fields of microbial strain technology and soybean seed coat fermentation technology, specifically to a type of Lactococcus sugar gliderus and its application in improving the flavor of soybean seed coats. Background Technology

[0002] Lactococcus is a class of lactic acid bacteria, and some species possess beneficial properties, such as promoting growth, enhancing immunity, and improving or maintaining intestinal flora homeostasis. *Lactococcus sugar gliderus* is a species of *Lactococcus*, and studies at the genomic level and in animal experiments have confirmed that this bacterium is mainly distributed in the intestines of humans and fish, as well as in cheese. It can promote growth, enhance the body's antioxidant capacity, and improve immunity. Furthermore, its genome has been predicted to contain genes encoding probiotic functions such as acid tolerance, bile salt tolerance, adhesion, and antioxidant activity. These results indicate that *Lactococcus sugar gliderus* is a potential probiotic.

[0003] Soybean seed coats, as a soybean byproduct, are abundant, but their unpleasant beany and grassy odors severely limit their direct application in the food industry. Currently, extrusion puffing and microbial fermentation are commonly used to improve the flavor of soybean seed coats. Compared to traditional microwave and enzymatic hydrolysis methods, microbial fermentation technology offers advantages such as safety and health benefits, increases the value of the substrate used during fermentation, and improves the nutritional and health properties of the food. Furthermore, microbial fermentation technology can promote the formation of flavor compounds, thereby endowing fermented products with unique flavor characteristics and ultimately achieving high-value transformation of byproducts. Therefore, microbial fermentation technology plays a positive role in improving the flavor of fermented products. Existing technologies often use yeast and lactic acid bacteria to ferment soybean milk, soybean residue, and soybean meal to improve their flavor characteristics. However, these strains are difficult to improve the odor in soybean seed coats; therefore, microbial fermentation is rarely used in current technologies to improve the flavor of soybean seed coats. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a *Lactococcus sugar glider* strain and its application in improving the flavor of soybean seed coats. This strain can effectively reduce unpleasant odors such as beany and grassy smells carried in soybean seed coats, making them more suitable for the needs of modern consumers.

[0005] According to a first aspect of the present invention, a *Lactococcus sugargrass* species is provided, wherein the *Lactococcus sugargrass* species is *Lactococcus sugargrass* BL-2. (Lactococcus petauri BL-2) It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20252253.

[0006] According to an embodiment of the present invention, the *Lactococcus honey gliderus* BL-2 is non-hemolytic, highly sensitive to antibiotics, and is a safe strain.

[0007] According to an embodiment of the present invention, the pH value of the *Lactococcus sugarfossa* BL-2 strain stabilizes at 4.56-4.59 and the total acid value stabilizes at 5.84-5.86 g / L within 16-24 h of culture.

[0008] According to an embodiment of the present invention, the Lactococcus honey gliderii BL-2 has certain acid resistance, salt resistance and high bile salt resistance.

[0009] According to embodiments of the present invention, the *Lactococcus honey gliderus* BL-2 exhibits high DPPH free radical scavenging rate and ABTS... + It exhibits significant antioxidant activity, with high free radical scavenging rate and high hydroxyl radical scavenging rate.

[0010] According to embodiments of the present invention, the fermentation of soybean seed coat powder by Lactococcus sugarfoss BL-2 can significantly increase the formation of flavor substances such as 3-hydroxy-2-butanone, linalool oxide and butyl acetate, thereby effectively improving the flavor of soybean seed coat.

[0011] According to a second aspect of the present invention, there is an application of Lactococcus sugar gliderus in improving the flavor of soybean seed coat, wherein Lactococcus sugar gliderus is used to ferment soybean seed coat.

[0012] According to a third aspect of the present invention, a method for fermenting soybean seed coats using Lactococcus sugarfossa is provided, comprising the following steps:

[0013] The *Lactococcus suis* BL-2 was activated and resuspended to obtain a *Lactococcus suis* BL-2 suspension. The Lactococcus honey gliderus BL-2 suspension was inoculated into sterile water to obtain a fermentation base liquid; The fermentation base liquid is mixed with soybean seed coat powder and fermented to obtain soybean seed coat fermented product.

[0014] According to an embodiment of the present invention, the method further includes a pretreatment of the soybean seed coat powder, comprising: drying the soybean skin at 60-65°C, pulverizing the dried soybean skin in a mill, and passing it through a 60-mesh sieve to obtain the soybean seed coat powder.

[0015] According to an embodiment of the present invention, the activation of *Lactococcus pyogenes* BL-2 of claim 1, followed by resuspending it to obtain a *Lactococcus pyogenes* BL-2 suspension, comprises: The activated Lactococcus sugar gliderus BL-2 was resuspended in 100 mmol / L sodium phosphate buffer to obtain a resuspension. The pH of the sodium phosphate buffer was 7.0-7.5. The OD value of the resuspended solution was adjusted to 1.2-1.5 to obtain the Lactococcus honeybage BL-2 suspension.

[0016] According to an embodiment of the present invention, the *Lactococcus sugar gliderus* BL-2 suspension accounts for 2-4% of the fermentation base liquid; The soybean seed coat powder and the fermentation base liquid are mixed in a weight-to-volume ratio of 1:2.

[0017] According to an embodiment of the present invention, the fermentation temperature is 30-37 ℃ and the fermentation time is 24-48 h.

[0018] According to a fourth aspect of the present invention, a soybean seed coat ferment obtained by the above-described fermentation method is provided.

[0019] According to a fifth aspect of the present invention, a food or beverage containing the aforementioned soybean seed coat fermentation product is provided.

[0020] This invention relates to a strain of Lactococcus sugar gliderus ( Lactococcus petauri BL-2 exhibits significant antioxidant capacity, with a DPPH free radical scavenging rate of 95.9%-97.6%, and ABTS. + The free radical scavenging rate was 81.4%-84.0%, and the hydroxyl radical scavenging rate was 68.3%-74.3%.

[0021] The present invention relates to Lactococcus honey gliderus ( Lactococcus petauri BL-2 fermentation of soybean seed coat powder can promote the release of flavor substances such as 3-hydroxy-2-butanone, linalool oxide and butyl acetate, thereby significantly improving the flavor of soybean seed coat powder and showing high application potential.

[0022] The *Lactococcus pyogenes* BL-2 of this invention not only has significant antioxidant activity, but also, after fermenting soybean seed coat, can significantly increase the content of 3-hydroxy-2-butanone (acetoin), which has a pleasant creamy aroma, and significantly reduce the content of substances such as isovaleric acid, 2-methyl-2-pentenal, and dimethyl disulfide, which have a strong pungent odor, thereby effectively improving the flavor of soybean seed coat. Attached Figure Description

[0023] Figure 1 The image shown is a scanning electron microscope image of Lactococcus sugargrass BL-2 in an embodiment of the present invention. Figure 2 This is a control diagram of the hemolytic activity test of *Lactococcus sugargrass* BL-2 in an embodiment of the present invention; a) *Lactococcus sugargrass* BL-2, b) positive control group (*Staphylococcus aureus*); Figure 3 This is a statistical graph showing the pH changes and total acid content of Lactococcus sugarfossa BL-2 in an embodiment of the present invention; Figure 4 The graph shows the results of the salt tolerance (a), acid tolerance (b), and bile salt tolerance (c) tests of Lactococcus sugarfossa BL-2 in this embodiment of the invention. Figure 5 The DPPH scavenging rate and ABTS of Lactococcus sugargrass BL-2 in the embodiments of the present invention are shown. + Figure 1 shows the results of the scavenging rate and hydroxyl radical scavenging rate measurements. Figure 6 This is a diagram illustrating the effect of Lactococcus sugar blisterii BL-2 fermentation on the content of aroma substances in soybean seed coats in an embodiment of the present invention. Detailed Implementation

[0024] This application describes a type of Lactococcus sugar glider and its application in improving the flavor of soybean seed coat.

[0025] Example 1: Isolation and culture of Lactococcus sugarbags BL-2 The *Lactococcus pyogenes* BL-2 of the present invention was cultured and activated in MRS medium, and the specific steps are as follows: Preparation of MRS medium: 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 5 g / L yeast extract, 3.02 g / L sodium acetate, 1.16 g / L dipotassium hydrogen phosphate, 0.05 g / L magnesium sulfate, 2 g / L triammonium citrate, 0.03 g / L manganese sulfate, and 1 mL / L Tween 80 were mixed thoroughly, dissolved in deionized water, and sterilized at 121℃ for 15-20 min to obtain the MRS medium.

[0026] Samples were taken from a naturally fermented mixture of soybean hulls and soybean residue. 1 g of the fermentation product was placed in a conical flask containing 9 mL of sterile physiological saline and thoroughly shaken to mix. The resulting naturally fermented sample was then serially diluted to 10⁻⁶. -6 Take 0.5-1 mL of each of the 10... -4 10 -5 and 10 -6 The gradient dilutions were evenly spread on MRS agar medium containing 1% calcium carbonate using the spreader method. The plates were then incubated in a constant temperature incubator at 30℃-37℃ for 24-48 hours. White colonies with a clear zone around them were picked and repeatedly inoculated and screened until uniform single colonies, namely Lactococcus sugarbags BL-2, were obtained.

[0027] Culture the lactococcus bacterial suspension, prepare scanning electron microscope slides, and observe the morphological characteristics of the bacteria using scanning electron microscopes. All operations in the preparation of scanning electron microscope slides of microbial strains should be carried out under strict aseptic conditions.

[0028] Experimental Example 1 1. Strain morphology detection: Scanning electron microscopy Culture the lactococcus bacterial suspension, prepare scanning electron microscope slides, and observe the morphological characteristics of the bacteria using scanning electron microscopes. All operations in the preparation of scanning electron microscope slides of microbial strains should be carried out under strict aseptic conditions.

[0029] The slide is gently attached to the conductive adhesive, ion sputtered, and finally observed under a microscope at an appropriate magnification from a suitable position.

[0030] The preparation steps for scanning electron microscope slides are as follows: Inoculate the activated bacterial culture into MRS liquid medium at an inoculation rate of 1.5%-2% and incubate at 37℃ and 180 r / min for 12-16 h. Centrifuge the cultured bacterial culture at 4℃ and 8000 r / min for 10 min, discard the supernatant, wash the precipitate 2-3 times with PBS buffer, centrifuge again and discard the supernatant, resuspend the bacterial culture in 2.5% glutaraldehyde solution, incubate at 4℃ for 4 h, centrifuge again and discard the supernatant, wash 2-3 times with PBS buffer, then dehydrate in 50%, 70%, and 90% ethanol solutions for 30 min each, centrifuge again, soak in 100% ethanol for 1 h, then drop the soaked bacterial culture onto a sterile zinc plate and allow it to air dry.

[0031] like Figure 1 The image shown is a scanning electron microscope (SEM) image (7k, 18k, 60k, 60k) of *Lactococcus sugargrass* BL-2 from this invention. *Lactococcus sugargrass* BL-2 cells are oval-shaped with a concave ring in the center, and are distributed singly or in pairs. Seven cells were selected, and their major and minor axes were measured using ImageJ-win64. See Table 1 for details. Table 1 shows the statistics of the major, minor, and area of ​​*Lactococcus sugargrass* BL-2 cells. As can be seen from Table 1, the major, minor, and area of ​​*Lactococcus sugargrass* BL-2 cells are 6.782–8.899 μm, 5.730–6.570 μm, and 48.260–37.088 μm, respectively. 2 between.

[0032] Table 1 shows the statistical data of the major diameter, minor diameter, and area of ​​Lactococcus sugar gliderus BL-2 cells.

[0033] The strain was inoculated into MRS liquid medium and cultured at 30-37℃ for 24-48 h. 16S rDNA sequencing was then performed by Shanghai Sangon Biotech Co., Ltd., and the result was identified as *Lactococcus sugarbagi*. Lactococcus petauri ).

[0034] Strain preservation: Preservation information of the biological material samples involved in this invention: The reference microorganism (strain) is BL-2, classified and named *Lactococcus honeybage* (…). Lactococcus petauri This entry was deposited by the China Center for Type Culture Collection (CCTCC) on October 20, 2025; accession number: CCTCC NO: M 20252253. The address of CCTCC is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0035] 2. Safety evaluation of the strain a. Hemolytic Sterilized Columbia agar medium was mixed thoroughly with 5-8% (v / v) sterile sheep blood. The activated *Lactococcus lactis* and control strains were then incubated at 30-37°C for 24-48 hours. *Staphylococcus aureus* was used as the positive control. The presence of hemolysis was observed on the plates. A greenish-yellow ring around the colony indicated α-hemolysis; a clearly defined, completely transparent hemolytic ring indicated β-hemolysis; and no change in the medium around the colony indicated γ-hemolysis. (See attached...) Figure 2 The diagram shown is a control image of the hemolytic activity of *Lactococcus sugargrass* BL-2 according to the present invention. In the diagram, a represents *Lactococcus sugargrass* BL-2, and b represents the positive control group (*Staphylococcus aureus*). It can be seen that the positive control *Staphylococcus aureus* colonies show a clear halo around their colonies, while *Lactococcus sugargrass* BL-2 does not produce a clear area on Columbia agar, which is defined as γ-hemolysis. This indicates that the *Lactococcus sugargrass* BL-2 described in this invention is non-hemolytic.

[0036] b. Antibiotic sensitivity The susceptibility of *Lactococcus sugargrass* strains to antibiotics was evaluated according to the Clinical Laboratory Standards Institute (CLSI) Standards for Antimicrobial Susceptibility Testing (2019). Eight antibiotics (kanamycin, gentamicin, vancomycin, ampicillin, penicillin, erythromycin, chloramphenicol, and tetracycline) were selected for the disk susceptibility testing to determine the antibiotic susceptibility of *Lactococcus sugargrass*. *Lactococcus sugargrass* was spread on MRS solid medium plates, and susceptibility test discs were placed on the surface. After standing for 5 min, the plates were inverted and incubated at 30–37 °C for 24–48 h. The diameter of the inhibition zone was measured using a ruler. Table 2 shows the susceptibility of *Lactococcus sugargrass* BL-2 to the disk susceptibility testing. As can be seen from Table 2, strain *Lactococcus sugargrass* BL-2 showed susceptibility to vancomycin, ampicillin, erythromycin, penicillin, and chloramphenicol; moderate susceptibility to gentamicin; and no susceptibility to kanamycin and tetracycline.

[0037] Table 2. Antibiotic susceptibility of Lactococcus sugar BL-2

[0038] Note: S: Inhibition zone diameter > 20 mm, indicating sensitivity; I: Inhibition zone diameter 15-19 mm, indicating moderate sensitivity; R: Inhibition zone diameter ≤ 14 mm, indicating insensitivity and resistance. 3. Physical and chemical characteristics of strains a. Determination of acid production rate of strains Freshly activated test strains (two generations in total) were inoculated into MRS liquid medium and cultured at 37°C for 24 hours. Uninoculated MRS liquid medium served as a blank control. pH was measured every 4 hours using a pH meter. pH reflects the acid-producing capacity of the strain. For a detailed pH curve of *Lactococcus sugar gliderus* BL-2, please refer to [link to relevant documentation]. Figure 3 .Depend on Figure 3 It can be seen that the pH of Lactococcus sugar gliderii BL-2 changes slowly and the acid production rate is low during fermentation from 0 to 4 hours; the strain has vigorous metabolism from 4 to 16 hours, and the acid production rate is the highest during this period; after 16 hours, the pH of the bacterial suspension stabilizes at 4.56-4.59.

[0039] b. Determination of acid production by strain Freshly activated test strains (two generations in total) were inoculated into MRS liquid medium and cultured at 37°C for 24 hours. Uninoculated MRS liquid medium was used as a blank control. Total acid (TTA) of *Lactococcus lactis* was measured every 4 hours. The determination was based on the national standard GB12456-2021, "National Food Safety Standard - Determination of Total Acid in Food". TTA reflects the acid production of the strain. For details on the changes in total acid of *Lactococcus sugar gliderus* BL-2, please refer to [link to relevant documentation]. Figure 3 .Depend on Figure 3 It can be seen that the total acid content of Lactococcus sugar gliderus BL-2 changes slowly and the acid production is low during fermentation from 0 to 4 hours; the total acid content increases rapidly from 4 to 16 hours, which may be related to the strain entering the logarithmic growth phase; after 16 hours, the total acid content tends to stabilize and remains at 5.84-5.86 g / L.

[0040] c. Salt tolerance analysis of strains Freshly activated *Lactococcus lactis* culture was inoculated at 2-4% of the culture into MRS liquid medium containing 2%, 4%, 6%, 8%, and 10% NaCl, respectively. After incubation at 37℃ and 180 r / min for 24 h, the absorbance of the culture was measured at 600 nm. The salt tolerance curve of *Lactococcus lactis* BL-2 is shown below. Figure 4 As shown in a. From Figure 4 As can be seen, the optical density of Lactococcus sugar gliderus BL-2 decreases with increasing NaCl concentration. When the NaCl concentration is greater than 10%, the strain can hardly survive. The mortality rate is highest when the concentration is between 6% and 8%, and the salt tolerance concentration of the strain is around 4%.

[0041] d. Analysis of the acid resistance of the strain Freshly activated *Lactococcus lactis* culture was inoculated at 2-4% of the culture into MRS liquid medium at pH 3, 3.5, 4, 4.5, and 5, respectively. The cultures were incubated at 37°C and 180 r / min for 4 h. The cultured culture was then centrifuged at 8000 r / min for 10 min at 4°C, and resuspended in an equal volume of fresh MRS medium. The cultures were then incubated at 37°C and 180 r / min for 12 h. The absorbance of the culture was measured at 600 nm. The survival rate of *Lactococcus lactis* was calculated as shown in equation (1). The acid tolerance curve of *Lactococcus lactis* BL-2 is shown in the figure. Figure 4 As shown in b. From Figure 4 As can be seen from b, the survival rate of Lactococcus sugarfoss BL-2 decreased with decreasing pH. The survival rate decreased significantly between pH 3.5 and 4, and the acid resistance of the strain was around pH 4.0.

[0042]

[0043] In the formula: OD 处理组 —Absorbance value of Lactococcus lactis in pH-adjusted MRS medium; OD 对照组 —Absorbance value of Lactococcus fermentation broth; OD 空白组 —Absorbance value of MRS medium without pH adjustment e. Analysis of bile salt tolerance of strains Freshly activated *Lactococcus lactis* culture was inoculated at a 2% inoculum into MRS liquid medium containing 0.3%, 0.5%, and 1.0% bovine bile salts, respectively. The cultures were incubated at 37°C and 180 r / min for 4 h. The cultures were then centrifuged at 8000 r / min for 10 min at 4°C, resuspended in an equal volume of fresh MRS medium, and incubated at 37°C and 180 r / min for 12 h. The absorbance of the cultures was measured at 600 nm. The survival rate of *Lactococcus lactis* was calculated as shown in equation (2). The bile salt tolerance curve of *Lactococcus lactis* BL-2 is shown in the figure below. Figure 4 As shown in c. From Figure 4 c shows that the survival rate of the bacteria in 0.3% bovine bile salts is as high as 91.83%-92.3%, and its bile salt tolerance is strong. At a concentration of 0.5%, its survival rate is about 16.88%.

[0044]

[0045] In the formula: OD处理组 —Absorbance value of MRS medium with added ox bile salts; OD 对照组 —Absorbance value of Lactococcus fermentation broth; OD 空白组 —Absorbance value of MRS medium without added ox bile salts 4. Determination of the antioxidant activity of the strain Lactococcus sugargrass strain BL-2 was inoculated into MRS medium at an inoculum rate of 2-4% and cultured in a constant temperature shaking incubator at 37℃ and 180 r / min for 24 h. The cultured bacterial solution was then centrifuged at 4℃ and 8000 r / min for 10 min, and the supernatant was collected.

[0046] a. Determination of the strain's DPPH free radical scavenging ability Mix 2 mL of 0.2 mmol / L DPPH solution with 1 mL of bacterial culture supernatant, vortex for 30 s, and react at room temperature in the dark for 30 min. Measure the absorbance of the sample at 517 nm. The DPPH free radical scavenging rate is calculated as shown in equation (3):

[0047] In the formula: A i : Sample group, i.e., the absorbance value of the mixture of bacterial culture supernatant and DPPH solution; A j : Control group, i.e., the absorbance value of the mixture of bacterial culture supernatant and anhydrous ethanol; A c : Blank group, i.e., the group consisting of a mixture of DPPH solution and deionized water.

[0048] b. Strain ABTS ＋ Free radical scavenging rate determination 7 mmol / L ABTS ＋ ABTS was prepared by mixing the solution with a 2.45 mmol / L potassium persulfate solution. + The stock solution was placed in the dark for 12-16 hours, and then diluted with anhydrous ethanol to prepare ABTS. ＋ Working solution. Mix 1 mL of bacterial culture supernatant with 3 mL of ABTS. ＋ After mixing the working solutions, vortex for 30 s, react at room temperature in the dark for 6 min, and then measure the absorbance of the sample at 734 nm. ABTS ＋ The free radical scavenging rate is calculated as shown in equation (4):

[0049] In the formula: A1: Sample group, i.e., bacterial culture supernatant and ABTS ＋ Absorbance value of the working fluid mixture group; A2: Control group, i.e., the absorbance value of the mixture of bacterial culture supernatant and anhydrous ethanol; A0: Blank group, i.e., ABTS ＋ Working solution mixed with deionized water.

[0050] c. Determination of hydroxyl radical scavenging rate 1 mL of bacterial culture supernatant was thoroughly mixed with 1 mL of 2.5 mmol / L o-phenanthroline, 1 mL of PBS buffer, 1 mL of 2.5 mmol / L FeSO4 solution, and 1 mL of 20 mmol / L H2O2, and reacted at 37℃ for 90 min. The absorbance of the sample was measured at 536 nm. The hydroxyl radical scavenging rate was calculated as shown in equation (3): (A) k Distilled water was used instead of the sample and H2O2 as the control group (A). j ); Distilled water was used instead of the sample as the blank group (A) i After thoroughly mixing, react at 37℃ for 90 min, and measure the absorbance at 536 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Each group was repeated 3 times. The hydroxyl radical scavenging capacity was calculated according to formula (5).

[0051]

[0052] In the formula: A k The absorbance values ​​of the sample group, namely the bacterial culture supernatant mixed with 2.5 mmol / L o-phenanthroline, 1 mL PBS buffer, 1 mL 2.5 mmol / L FeSO4 solution and 1 mL 20 mmol / L H2O2; A i The control group, i.e., the absorbance value of the mixture of distilled water and 2.5 mmol / L o-phenanthroline, 1 mL PBS buffer, and 1 mL 2.5 mmol / L FeSO4 solution; A j : Blank group, i.e., the absorbance value of the mixture of distilled water and 2.5 mmol / L o-phenanthroline, 1 mL PBS buffer, 1 mL 2.5 mmol / L FeSO4 solution and 1 mL 20 mmol / L H2O2.

[0053] Experimental results are as follows Figure 5 As shown, Lactococcus sugargrass BL-2 has high antioxidant capacity, with a DPPH free radical scavenging rate of 95.9%-97.6%, an ABTS+ free radical scavenging rate of 81.4%-84.0%, and a hydroxyl free radical scavenging rate of 68.3%-74.3%.

[0054] Example 2: Fermented soybean seed coat powder with Lactococcus BL-2 Fresh *Lactococcus sugargrass* BL-2, activated in MRS medium, was collected and resuspended in 100 mmol / L sodium phosphate buffer (pH 7.0-7.5) and the OD value was adjusted to 1.2-1.5 to obtain a *Lactococcus sugargrass* BL-2 suspension. The *Lactococcus sugargrass* BL-2 suspension was inoculated into sterile water at an inoculum rate of 2-4% (v / v). Sterilized soybean seed coat powder (crushed to 60 mesh) was mixed thoroughly with sterile water at a ratio of 1:2 (w / v), and fermented at 30-37℃ for 24-48 h.

[0055] Experimental Example 2: Determination of Flavor Compound Content in Soybean Seed Coarse Powder Fermented with Lactococcus BL-2 a) GC-IMS determination of fermented soybean seed coat powder Sample pretreatment: Weigh 10g of soybean seed coat powder sample fermented with Lactococcus honey gliderus BL-2, add 20mL of purified water and mix well, take 5mL of sample suspension and add 5μL of 0.8mg / kg 2-methyl-3-heptanone as internal standard.

[0056] Chromatographic conditions: GC-IMS injection volume 500 μL, IMS temperature 45℃, injection needle temperature 85℃, column temperature 60℃. High-purity nitrogen was used as the carrier gas, and the carrier gas flow rate was: The detection rates were 2 mL / min (0–2 min), 2–10 mL / min (2–10 min), 10–100 mL / min (10–20 min), and 100–150 mL / min (20–25 min), with a detection time of 25 min. Qualitative analysis was performed using GC-IMS library search software with an IMS database, and the linear retention index was determined using C4–C9 series ketones.

[0057] The results are shown in Table 3. A total of 73 flavor compounds were identified in both unfermented soybean seed coats and soybean seed coats fermented with *Lactococcus sugargrass* BL-2, including 14 esters, 17 alcohols, 19 aldehydes, 12 ketones, 3 terpenes, 2 furans, and some other compounds. Figure 6The results showed that the content of aldehydes and alcohols in soybean seed coats fermented with *Lactococcus sugargrass* BL-2 was significantly increased compared to unfermented soybean seed coats. Among the 36 flavor compounds that showed relatively significant changes, 3-hydroxy-2-butanone (acetoin), with its pleasant creamy aroma, is an intermediate in the synthesis of many flavor compounds and drugs. The content of this substance significantly increased after fermentation with *Lactococcus sugargrass* BL-2, which also contributed to the improvement of the flavor compounds in fermented soybean seed coats. Isovaleric acid, 2-methyl-2-pentenal, and dimethyl disulfide, among other substances, have strong, pungent odors that can directly cause discomfort. The content of these unpleasant flavor compounds significantly decreased after fermentation with *Lactococcus sugargrass* BL-2, indicating that fermentation with *Lactococcus sugargrass* BL-2 can significantly reduce the unpleasant odor of soybean seed coats.

[0058] Table 3 Flavor compounds of soybean hull fermented with Lactococcus sugar BL-2

[0059] In summary, the fermentation with *Lactococcus sugar gliderus* BL-2 in this invention significantly increases the content of aldehydes and alcohols in soybean seed coats, and significantly reduces the content of substances with strong, pungent odors, such as isovaleric acid, 2-methyl-2-pentenal, and dimethyl disulfide. Therefore, this demonstrates that *Lactococcus sugar gliderus* BL-2 in this invention can effectively improve the flavor of soybean seed coats.

[0060] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0061] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A type of Lactococcus pyogenes, characterized in that, The *Lactococcus sugarfossa* is *Lactococcus sugarfossa* BL-2. (Lactococcus petauri BL-2) It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M20252253.

2. The application of *Lactococcus sugargrass* as described in claim 1 in improving the flavor of soybean seed coat, characterized in that... The soybean seed coat is fermented using Lactococcus honey glider provided in claim 1.

3. A method for fermenting soybean seed coats using *Lactococcus sugargrass* as described in claim 1, characterized in that: Includes the following steps: Activate the Lactococcus sugargrass BL-2 of claim 1, and resuspend it to obtain a Lactococcus sugargrass BL-2 suspension; The Lactococcus honey gliderus BL-2 suspension was inoculated into sterile water to obtain a fermentation base liquid; The fermentation base liquid is mixed with soybean seed coat powder and fermented to obtain soybean seed coat fermented product.

4. The method for fermenting soybean seed coats using *Lactococcus sugargrass* according to claim 3, characterized in that, It also includes pretreatment of the soybean seed coat powder, including: drying the soybean skin at 60-65°C, pulverizing the dried soybean skin in a mill, and passing it through a 60-mesh sieve to obtain the soybean seed coat powder.

5. The method for fermenting soybean seed coats using *Lactococcus honey gliderus* according to claim 3, characterized in that, The activation of Lactococcus sugargrass BL-2 according to claim 1, followed by resuspending it to obtain a Lactococcus sugargrass BL-2 suspension, comprises: The activated Lactococcus sugar gliderus BL-2 was resuspended in 100 mmol / L sodium phosphate buffer to obtain a resuspension. The pH of the sodium phosphate buffer was 7.0-7.

5. The OD value of the resuspended solution was adjusted to 1.2-1.5 to obtain the Lactococcus honeybage BL-2 suspension.

6. The method for fermenting soybean seed coats using *Lactococcus sugargrass* according to claim 3, characterized in that, The *Lactococcus sugargrass* BL-2 suspension constitutes 2-4% of the fermentation base liquid; The soybean seed coat powder and the fermentation base liquid are mixed in a weight-to-volume ratio of 1:

2.

7. The method for fermenting soybean seed coats using *Lactococcus sugargrass* according to claim 3, characterized in that, The fermentation temperature is 30-37 ℃, and the fermentation time is 24-48 h.

8. A fermented soybean seed coat obtained by any one of the fermentation methods described in claims 3-7.

9. A food or beverage containing the soybean seed coat fermentation product of claim 8.

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