A method for removing the fishy smell from fermented soybean milk using fermented soybean 11S globulin.

Abstract

This invention discloses a method for removing the beany odor from fermented soy milk using fermented soybean 11S globulin. The method first employs alkaline extraction, acid precipitation, and ultrafiltration membrane technology to obtain high-purity soybean 11S globulin. Then, it ferments the soybean 11S globulin using *Lactobacillus fermentum* 56 and *Lactobacillus fermentum* 57 to obtain a soybean 11S globulin lactic acid bacteria fermentation product. This product is then added to sterilized soy milk and subjected to secondary fermentation using a compound lactic acid bacteria culture to obtain low-beany probiotic fermented soy milk. This invention can significantly reduce the content of beany odor substances in fermented soy milk, especially significantly reducing the concentration of characteristic beany flavor substances such as n-hexanol and 1-octen-3-ol, while increasing the concentration of ketones and esters, which generally have a fragrant aroma, thus giving the fermented soy milk a pleasant flavor.

Description

Technical Field

[0001] This invention relates to a method for removing the fishy smell from fermented soybean milk using fermented soybean 11S globulin, belonging to the field of food processing. Background Technology

[0002] Soy protein is a high-quality plant protein, mainly composed of soy protein isolate (SPI), soy globulin (11S globulin, 11S) and β-conglycinin (7S globulin, 7S), which account for more than 70% of the total soybean storage protein. It also contains small amounts of 2S and 15S proteins.

[0003] Several intermediate structures exist between the native and denatured states of soybean protein. The molten globule state is one of the most studied intermediates. It represents an intermediate state in the gradual conformational transition between protein folding and unfolding, possessing a secondary structure and intrinsic viscosity similar to the native state. However, it lacks the specific tertiary structure generated by side chains, and the protein molecule's radius is 10%–30% larger than in the native state. The abundant loosely packed hydrophobic cores in its structure increase the hydrophobic surface area compared to the native state. Globules can form molten globule structures under certain denaturing conditions, such as extreme pH values, heat treatment, high-pressure treatment, high-concentration salts, and the addition of exogenous denaturing agents (guanidine hydrochloride, urea). Generally, the most common and effective method is acid induction because pH changes are a milder denaturing agent compared to chemical denaturants and heat treatment, and conformational changes during denaturation are easier to observe.

[0004] When soy protein is in a molten globule state, its hydrophobic groups are exposed, enhancing its hydrophobicity. Theoretically, this allows it to bind more flavor compounds through hydrophobic interactions. In the presence of multiple flavor compounds, these compounds compete with soy protein for binding. Studies have shown that the binding enhancement for beany flavor compounds is greater in this state than for non-beany flavor compounds. Previous research has used equilibrium dialysis with β-cyclodextrin to remove 2-nonanone bound to soy protein, finding that a large amount of 2-nonanone bound to soy protein can be removed in a concentration-dependent manner in the presence of β-cyclodextrin. Therefore, it can be inferred that after fermentation, when the hydrophobic groups of soy protein are exposed, it can bind more flavor compounds through hydrophobic interactions, thereby reducing or even eliminating beany flavor compounds bound to soy protein. Summary of the Invention

[0005] The purpose of this invention is to provide a method for removing the beany odor from fermented soy milk using fermented soybean 11S globulin. This method first obtains an intermediate structure between the denatured and native states by fermenting soybean 11S globulin, increasing its ability to remove beany odor compounds. Then, the soy milk is mixed with the fermentation product of soybean 11S globulin for fermentation, thereby obtaining low-beany-odor probiotic fermented soy milk.

[0006] The technical solution for achieving the objective of this invention is as follows:

[0007] A method for removing the fishy smell from fermented soy milk using fermented soybean 11S globulin includes the following steps:

[0008] (1) Preparation of soybean 11S globulin:

[0009] After defatting soybean flour with hexane and ethanol, the defatted soybean flour was dissolved in deionized water at a mass-to-volume ratio of 1:10-15. The pH of the solution was adjusted to 8.5±0.5 with NaOH. The alkali-soluble soybean protein was extracted by stirring thoroughly. The mixture was centrifuged at 9000g for 30-40 minutes at 4°C. Sodium bisulfite (SBS) was added to the collected supernatant. The pH of the supernatant was adjusted to 6.4±0.5 with HCl. The protein was precipitated by standing at 4°C. The precipitate was centrifuged at 6500g for 20-30 minutes at 4°C. The precipitate was washed 3-5 times with deionized water. The mixture was concentrated using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1kDa to remove impurities. The pH of the final protein solution was adjusted to 7. The mixture was then freeze-dried to obtain soybean 11S globulin.

[0010] (2) Preparation of fermentation products from 11S globulin-producing lactic acid bacteria:

[0011] Lactobacillus fermentans.56 and Lactobacillus fermentans.57 were used to ferment soybean 11S globulin and glucose. The obtained soybean 11S globulin fermentation broth was concentrated by ultrafiltration using a polyethersulfone membrane with a molecular weight cutoff of 1 kDa. The retentate was recovered and freeze-dried to obtain the soybean 11S globulin lactic acid bacteria fermentation product.

[0012] (3) Preparation of sterilized soy milk:

[0013] Soybeans are washed after the skin is removed by heat drying, and then soaked in a 0.3% sodium bicarbonate solution for 14-16 hours. Soy milk is prepared at a material-to-liquid ratio of 1:10. 3% (w:v) inulin and 3% (w:v) white sugar are added. After sterilization, the mixture is cooled to 4°C to obtain sterilized soy milk.

[0014] (4) Mixed fermentation:

[0015] The soybean 11S globulin lactic acid bacteria fermentation product from step (2) is mixed evenly with the sterilized soy milk from step (3), and then inoculated with lactic acid bacteria for fermentation to obtain low-soybean-flavored probiotic fermented soy milk. The lactic acid bacteria are selected from any two or more of the following: Lactobacillus fermentum 56, Lactobacillus fermentum 57, Lactobacillus plantarum grx402, Lactobacillus fermentans grx501, and Lactobacillus paracasei grx701 with accession number CGMCC No. 27785.

[0016] Furthermore, in step (2), the inoculation ratio of Lactobacillus fermentum 56 and Lactobacillus fermentum 57 in the fermentation system is equal.

[0017] Furthermore, in step (2), the concentration of soybean 11S globulin in the fermentation system is 30 g / L, and the concentration of glucose is 25 g / L.

[0018] Furthermore, in step (2), the fermentation conditions are: 37℃, 16-24h.

[0019] Furthermore, in step (2), the concentration conditions are: transmembrane pressure 0.15 MPa, concentration factor 2.

[0020] Furthermore, in step (3), the mass of the 0.3% sodium bicarbonate solution is three times the mass of the soybeans after the skin has been removed.

[0021] Furthermore, in step (2) or step (4), the total inoculation amount of the strain is 5% (v:v).

[0022] Further, in step (4), the fermentation product of soybean 11S globulin lactic acid bacteria is mixed with sterilized soy milk at a concentration of 2% (w:v).

[0023] Further, in step (4), the lactic acid bacteria are Lactobacillus fermentum grx501 and Lactobacillus paracasei grx701, or Lactobacillus fermentum 56 and Lactobacillus fermentum 57, or Lactobacillus plantarum grx402 and Lactobacillus paracasei grx701 with accession number CGMCC No. 27785, or Lactobacillus plantarum grx402, Lactobacillus paracasei grx701 with accession number CGMCC No. 27785, Lactobacillus fermentum 56 and Lactobacillus fermentum 57, with each strain mixed in equal proportions.

[0024] The present invention also provides a low-soybean-flavor probiotic fermented soy milk prepared by the above method.

[0025] Compared with existing methods for removing the fishy smell from fermented soy milk, the present invention has the following advantages:

[0026] (1) This invention uses natural soybeans to extract soybean 11S globulin, and combines the classic alkaline extraction and acid precipitation method with ultrafiltration concentration to remove impurities that may have other effects on the flavor of soy milk. The soybean 11S globulin is then fermented with probiotics, using Lactobacillus fermentum 56 and Lactobacillus fermentum 57 as fermentation strains. The fermented soybean 11S globulin has a larger hydrophobic surface area and different secondary and tertiary structures compared to the native protein structure, allowing it to bind more flavor substances and exhibiting stronger antioxidant properties, thus providing favorable conditions for its addition to soy milk for further fermentation.

[0027] (2) The fermented soy milk obtained by the present invention using compound lactic acid bacteria fermentation and fermentation products of soybean 11S globulin lactic acid bacteria has significantly lower beany flavor content compared with traditional fermented soy milk. Taking fermented soy milk obtained by fermenting soy milk with Lactobacillus grx501 and Lactobacillus paracasei grx701 and fermentation products of soybean 11S globulin lactic acid bacteria as an example, the concentration of the main beany flavor volatile substances is: n-hexanol 55.48 μg / L and 1-octen-3-ol 115.77 μg / L. The concentration of volatile flavor substances detected in traditional fermented soy milk is: n-hexanol 168.97 μg / L and 1-octen-3-ol 337.49 μg / L. Compared with traditional fermented soy milk, its beany flavor content is significantly reduced.

[0028] (3) The method of the present invention is simple to operate, has a good effect on removing fishy smell, and all the materials used are edible, which improves the utilization rate of soybean resources, makes the product have the characteristics of natural nutrition, and adopts secondary fermentation, which makes the improved fermented soy milk have higher nutritional value. Attached Figure Description

[0029] Figure 1 Endogenous spectra of soybean 11S globulin after fermentation with different lactic acid bacteria.

[0030] Figure 2 Near-ultraviolet circular dichroism chromatograms of soybean 11S globulin after fermentation with different lactic acid bacteria.

[0031] Figure 3 Far-ultraviolet circular dichroism chromatograms of soybean 11S globulin after fermentation with different lactic acid bacteria.

[0032] Figure 4 A diagram showing the proportions of secondary structures of soybean 11S globulin after fermentation with different lactic acid bacteria.

[0033] Figure 5 The mass concentration of beany odor substances extracted from soybean 11S globulin using different methods is shown in the figure.

[0034] Figure 6 A graph showing the mass concentration of beany-smelling substances in soybean 11S globulin after fermentation with different lactic acid bacteria.

[0035] Figure 7 A comparison chart of volatile flavor compounds in traditionally fermented soy milk and mixed-fermented soy milk.

[0036] Figure 8 Frequency sweep curves of the rheological properties of different fermented soy milks. Detailed Implementation

[0037] In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the present invention will be further described in detail below with reference to the embodiments.

[0038] The Lactobacillus paracasei grx701 of this invention was deposited on July 3, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27785, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0039] The *Lactobacillus plantarum* grx402 of this invention was deposited on November 18, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28585 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. It has been fully disclosed in Chinese patent application CN 117511796A.

[0040] The Lactobacillus fermentum grx501 of the present invention was deposited on December 12, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.25349 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. It has been fully disclosed in Chinese patent application CN 115948290A.

[0041] The Lactobacillus plantarum grx16 of this invention was deposited on September 1, 2015, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.10921 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. It has been fully disclosed in Chinese patent application CN 116158532A.

[0042] The Lactobacillus plantarum 11, Lactobacillus fermentum 56 and Lactobacillus fermentum 57 of this invention have been disclosed in our laboratory in a paper [ZHANG LF, QU HX, LIU XX, et al. Comparison and selection of probiotic Lactobacillus from human intestinal tract and traditional fermented food in vitro via PCA, unsupervised clustering algorithm, and heat-map analysis[J]. Food Science & Nutrition, 2022, 10(12): 4247-57.].

[0043] The applicant, Yangzhou University, declares that it guarantees to distribute Lactobacillus fermentum 56, Lactobacillus fermentum 57, and Lactobacillus plantarum 11 to the public within twenty years from the date of application.

[0044] The relevant culture media, fermentation broth, and test methods in the following examples are as follows.

[0045] 1. MRS medium: glucose 20.0g, peptone 10.0g, anhydrous sodium acetate 5.0g, ammonium citrate 2.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate 0.05g, Tween-80 1.0ml, beef extract 10.0g, yeast extract 5.0g, water 1.0L.

[0046] 2. Determination of the tertiary structure of soybean 11S globulin:

[0047] (1) Determination of endogenous fluorescence spectroscopy: The soybean 11S globulin solution was serially diluted and the endogenous fluorescence was measured at room temperature. The endogenous fluorescence was measured on an F-7000 fluorescence spectrophotometer using a 1cm cuvette. The slit width was 5nm, the excitation wavelength was 290nm, and the spectral changes from 300 to 400nm were recorded. The scan was performed three times.

[0048] (2) Far-ultraviolet circular dichroism spectroscopy determination: The changes in the tertiary structure of the protein were determined by near-ultraviolet circular dichroism spectroscopy (250-400nm). The protein concentration was 1mg / mL, the optical path was 1.0cm, the measurement was performed at room temperature, and the scan was performed three times to record the changes in the spectrum.

[0049] 3. Determination of the secondary structure of soybean 11S globulin: The changes in the secondary structure of the protein were determined by far-ultraviolet circular dichroism (190-250 nm). The protein concentration was 0.1 mg / mL, the optical path length was 0.2 cm, the measurement was performed at room temperature, and the scans were repeated three times. The changes in the spectrum were recorded.

[0050] 4. Determination of Volatile Flavor Compounds: The mass concentration changes of volatile flavor compounds were determined by gas chromatography-mass spectrometry (GC-MS). 1 mL of sample was added to a GC-MS vial and placed at 4°C for analysis. Before analysis, 10 μL of 0.01% (v / v) 1,2-dichlorobenzene solution (internal standard) was added, and the vial was then sealed with a metal screw cup. GC was performed using an HP-5 capillary column (30 mm × 0.25 mm, 0.25 μm). A programmed heating method was used: initial temperature 35°C, held for 5 min; temperature increased to 140°C at a rate of 5°C / min, held for 2 min; temperature increased to 250°C at a rate of 10°C / min, held for 3 min. The vaporization chamber temperature was 250°C. The carrier gas was He, with a flow rate of 1.0 mL / min. Split injection was not performed. Mass spectrometry used an electron ionization source. The electron energy was 70 EV. The ion source temperature was 230℃, and the mass scan range was m / z 35~500. The emission current was 100μA. The SPME conditions were: extraction head aging temperature 250℃, aging time 20min, equilibration temperature 50℃, equilibration time 45min, and desorption at 250℃ for 3min.

[0051] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0052] Example 1

[0053] (1) Preparation of soybean 11S globulin:

[0054] Soybean flour was defatted using hexane and ethanol. Following the principle of alkaline extraction and acid precipitation, the defatted soybean flour was dissolved in deionized water (1:15, w:v). The pH of the solution was adjusted to 8.5 using 2M NaOH, and the mixture was stirred at room temperature for 1 hour. The solution was centrifuged at 9000g and 4℃ for 30 min, and the supernatant was collected. SBS was added to the supernatant to a concentration of 0.98 g / L, and the pH of the supernatant was adjusted to 6.4 using 2M HCl. The mixture was then incubated overnight at 4℃. The precipitate was centrifuged at 6500g for 20 min at 4℃. The precipitate was washed three times with deionized water and concentrated using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa to remove impurities and improve the purity of soybean 11S globulin. The final protein solution pH was adjusted to 7, and the pre-freezing temperature was set to -80℃. The freeze-drying thickness was 0.1–0.5 cm, yielding soybean 11S globulin (11S).

[0055] (2) Preparation of fermentation products from 11S globulin-producing lactic acid bacteria:

[0056] Soybean 11S globulin (30 g / L) and glucose (25 g / L) were dissolved in deionized water and sterilized at 85°C for 10 min. Then, equal proportions of *Lactobacillus fermentum* 56 and *Lactobacillus fermentum* 57 strains were inoculated, with a total inoculation amount of 5% (v:v). Soybean 11S globulin was fermented at 37°C for 16 h to obtain the fermentation broth. The broth was then concentrated using polyethersulfone membrane ultrafiltration at 1 kDa, transmembrane pressure of 0.15 MPa, and a concentration factor of 2. The retentate was recovered and pre-frozen at -80°C to a thickness of 0.2 cm for 24 h. Finally, it was freeze-dried for 48 h to obtain the lyophilized powder, which was named F11S-Lf.56+Lf.57.

[0057] (3) Preparation of sterilized soy milk:

[0058] Soybeans are dried to remove the skins and then washed. They are then soaked in a 0.3% sodium bicarbonate solution for 14-16 hours. Soy milk is prepared at a material-to-liquid ratio of 1:10. 3% (w:v) inulin and 3% (w:v) white sugar are added. After sterilization, the mixture is cooled to 4°C to obtain sterilized soy milk.

[0059] (4) Mixed fermentation:

[0060] The fermentation product of soybean 11S globulin lactic acid bacteria from step (2) was mixed with the sterilized soy milk from step (3) at a concentration of 2% (w:v). Second-generation lactic acid bacteria, Lactobacillus fermentum grx501 and Lactobacillus paracasei grx701, cultured to the logarithmic growth phase, were centrifuged at 8000g for 5 minutes to obtain bacterial cells. After washing twice with physiological saline, the two bacterial cells were mixed in equal proportions and then added to the above mixture at a total inoculum concentration of 5% (v:v). Fermentation was carried out at 37℃ for 16 hours to obtain low-soybean-flavor probiotic fermented soy milk, named FS-grx.501+grx.701-F11S, and stored at 4℃.

[0061] Comparative Example 1

[0062] This comparative example is roughly the same as Example 1, except that the fermentation strains used in step (2) are Lactobacillus plantarum grx402 and Lactobacillus paracasei grx701, and the fermentation product of soybean 11S globulin lactic acid bacteria is obtained and named F11S-grx.402+grx.70l.

[0063] Comparative Example 2

[0064] This comparative example is roughly the same as Example 1, except that the fermentation strains used in step (2) are Lactobacillus plantarum 11 and Lactobacillus plantarum grx16, and the fermentation product of soybean 11S globulin lactic acid bacteria is named F11S-Lp.11+grx.16.

[0065] Comparative Example 3

[0066] This comparative example is roughly the same as Example 1, except that the fermentation strain used in step (4) is a traditional fermentation agent.

[0067] Comparative Example 4

[0068] This comparative example is roughly the same as Example 1, except that the extraction method of soybean 11S globulin in step (1) is the traditional alkali dissolution and acid precipitation method, without ultrafiltration membrane treatment.

[0069] Example 2

[0070] This embodiment is largely the same as Embodiment 1, except that the fermentation strains used in step (4) are Lactobacillus plantarum grx402 and Lactobacillus paracasei grx701 in equal proportions, and the resulting low-soybean-flavor probiotic fermented soy milk is named FS-grx.402+grx.701-F11S.

[0071] Example 3

[0072] This embodiment is largely the same as embodiment 1, except that the fermentation strains used in step (4) are Lactobacillus fermentum 56 and Lactobacillus fermentum 57 in equal proportions, and the resulting low-soybean-flavor probiotic fermented soy milk is named FS-Lf.56+Lf.57-F11S.

[0073] Example 4

[0074] This embodiment is largely the same as Embodiment 1, except that the fermentation strains used in step (4) are Lactobacillus plantarum grx402, Lactobacillus paracasei grx701, Lactobacillus fermentum 56 and Lactobacillus fermentum 57 in equal proportions. The resulting low-soybean-flavored probiotic fermented soy milk is named FS-grx.402+grx.701+Lf.56+Lf.57-F11S.

[0075] Figure 1 The images show the endogenous fluorescence spectra of soybean 11S globulin after fermentation with different lactic acid bacteria. Figure 1It can be seen that the native 11S globulin exhibits maximum fluorescence intensity (FI) at 321 nm. This is because tryptophan residues are buried in the core of the 11S globulin and are quenched by fluorescence from adjacent amino acid side chains. After fermentation with different lactic acid bacteria, the maximum absorption wavelength of 11S globulin shows a significant red shift. This is because the lactic acid and other acids produced during the metabolism of lactic acid bacteria lower the pH of the protein's environment, partially unfolding the protein's tertiary structure and exposing some buried tryptophan side chain groups to a polar environment, resulting in a red shift of the maximum emission wavelength. Among these, the fermented soybean 11S globulin obtained using *Lactobacillus plantarum* grx402 and *Lactobacillus paracasei* grx701, *Lactobacillus plantarum* 11 and *Lactobacillus plantarum* grx16 showed a higher red shift wavelength than that obtained using *Lactobacillus fermentum* 56 and *Lactobacillus fermentum* 57. Simultaneously with the redshift of the maximum absorption wavelength, the fluorescence intensity decreased to varying degrees, indicating that the molecular conformation of soybean 11S globulin was shrinking after fermentation. The folding and shrinkage of soybean 11S globulin fermented by *Lactobacillus fermentum* 56 and 57 were stronger than the other two groups. Endogenous fluorescence spectroscopy results showed that the tertiary structure of 11S globulin, deformed by lactic acid bacteria fermentation, stretched, causing aromatic amino acid residues to migrate from nonpolar to polar environments. Simultaneously, varying degrees of refolding occurred in the tertiary structure, demonstrating that fermentation by different lactic acid bacteria can induce dynamic changes in protein conformation. Based on this, near-ultraviolet circular dichroism spectroscopy was used to characterize the changes in its tertiary structure, obtaining further information about these structural variations.

[0076] Figure 2 The images show near-ultraviolet circular dichroisms (NUV) spectra of soybean 11S globulin fermented with different lactic acid bacteria. The graphs reveal that natural soybean 11S globulin exhibits numerous tertiary structures and strong signal values. Completely denatured protein shows near-zero signal values, while the fermented proteins produced by different lactic acid bacteria all show strong signal values, indicating no complete denaturation. Furthermore, these fermented proteins differ from natural soybean 11S globulin, suggesting a more ordered intermediate state between the native and completely denatured states. Specifically, soybean proteins fermented with *Lactobacillus plantarum* grx402 and *Lactobacillus paracasei* grx701, and *Lactobacillus plantarum* 11 and grx16 show similar signal values ​​to unfermented soybean proteins, indicating minimal conformational change and thus similar tertiary structures. However, soybean proteins fermented with *Lactobacillus fermentum* 56 and 57 show a different signal value compared to unfermented soybean 11S globulin, indicating conformational changes and a more ordered spatial structure.

[0077] Figure 3The images show the far-ultraviolet circular dichroism chromatograms of soybean 11S globulin after fermentation with different lactic acid bacteria. As can be seen from the figures, compared to the spectrum of native soybean 11S globulin, the lowest negative peak in the spectrum of soybean 11S globulin begins to shift to lower wavelengths after lactic acid bacteria fermentation. This indicates that lactic acid bacteria fermentation alters the secondary structure of soybean 11S globulin, increasing the proportion of random coils and decreasing hydrophilicity, thus causing a blue shift. However, soybean 11S globulin obtained by fermentation with *Lactobacillus fermentum* 56 and *Lactobacillus fermentum* 57 did not exhibit a blue shift; instead, its lowest negative peak showed a red shift compared to soybean 11S globulin obtained by fermentation with other lactic acid bacteria.

[0078] Figure 4 This is a diagram showing the secondary structure content of soybean 11S globulin after fermentation with different lactic acid bacteria, as shown below. Figure 4 As shown, soybean 11S globulin obtained by fermentation with Lactobacillus fermentum 56 and Lactobacillus fermentum 57 has the highest retention rates of α-sheet and β-turn content, with the same random coil ratio as before fermentation, while the β-sheet ratio increases, indicating that the protein conformation becomes more ordered and closer to the native state.

[0079] Figure 5 The graph shows the mass concentration of beany-smelling substances extracted from soybean 11S globulin using different methods. After concentration and impurity removal via polyethersulfone ultrafiltration membrane, compared with the traditional soybean 11S globulin extraction method, the concentration of the main beany-smelling substances in the extracted soybean 11S globulin decreased to some extent. The mass concentrations of the main beany-smelling substances in the soybean 11S globulin after membrane filtration are as follows: hexanal 11.16 μg / L, 1-octen-3-ol 0.71 μg / L. The mass concentrations of the main beany-smelling substances in the soybean 11S globulin extracted using the traditional method are as follows: hexanal 13.95 μg / L, 1-octen-3-ol 2.34 μg / L. This indicates that during the impurity removal process of soybean 11S globulin, some of the beany-smelling substances were also removed.

[0080] Figure 6 The graph shows the mass concentration of beany-smelling substances in soybean 11S globulin after fermentation with different lactic acid bacteria. After lactic acid bacteria fermentation, the mass concentration of beany-smelling substances carried by natural soybean 11S globulin decreased, with the concentrations of beany-smelling substances that significantly affect flavor, such as hexanal and 1-octen-3-ol, approaching zero. Among the soybean 11S globulins obtained by fermentation with three different strains, those fermented with *Lactobacillus fermentum* 56 and *Lactobacillus fermentum* 57 had the lowest variety and concentration of beany-smelling substances. In conclusion, the soybean 11S globulins fermented with *Lactobacillus fermentum* 56 and *Lactobacillus fermentum* 57 show a certain degree of structural alteration compared to their native state, and the beany-smelling substances produced after fermentation have almost no adverse effect on subsequent fermented soymilk.

[0081] Figure 7 This is a comparison chart of volatile flavor compounds in traditionally fermented soy milk and mixed-fermented soy milk. The chart shows that the mixed-fermented soy milk fermented with *Lactobacillus plantarum* GRX402, *Lactobacillus paracasei* GRX701, *Lactobacillus fermentum* 56, and *Lactobacillus fermentum* 57 has the following concentrations of volatile flavor compounds contributing to the main beany odor: n-hexanol 129.34 μg / L, 1-octen-3-ol 278.55 μg / L. The mixed-fermented soy milk fermented with *Lactobacillus fermentum* GRX501 and *Lactobacillus paracasei* GRX701... The concentrations of volatile flavor compounds that primarily contribute to the beany aroma of fermented soy milk are as follows: n-hexanol 55.48 μg / L, 1-octen-3-ol 115.77 μg / L. The concentrations of volatile flavor compounds detected in traditionally fermented soy milk are as follows: n-hexanol 168.97 μg / L, 1-octen-3-ol 337.49 μg / L. Compared with traditionally fermented soy milk, the improved fermented soy milk shows a significant reduction in beany flavor compounds, and a certain increase in the concentrations of ketones and esters, which generally have a fragrant aroma, resulting in a better flavor.

[0082] Figure 8 The rheological properties of different fermented soy milks are shown in the frequency sweep curves. It can be seen from the figure that the G' and G” values ​​of different fermented soy milks increase with the increase of frequency (0.1-10Hz), and G' is greater than G”, indicating that the elastic component is dominant and the sample exhibits solid characteristics. Traditional fermented soy milk has the highest G' and G” values, followed by FS-grx.402+grx.701+Lf.56+Lf.57-F11S and FS-grx.501+grx.701-F11S, with the lowest being FS-Lf.56+Lf.57-F11S and FS-grx.402+grx.701-F11S. This result may be due to the lactic acid and acetic acid produced by lactic acid bacteria during fermentation, which continuously lowers the pH value, thereby reducing the surface activity of soybean protein molecules, forming colloidal particles, and increasing the viscosity of fermented soy milk. Similarly, in the preparation of fermented soy milk, the proteins and fats in the soy milk are homogenized under high pressure, resulting in a more uniform dispersion of protein and fat particles in the fermented soy milk. This facilitates the formation of a more uniform and dense protein gel network, ultimately giving the fermented soy milk higher elasticity and lower viscosity.

[0083] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations can be made based on the above description. It is impossible to illustrate all embodiments here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for deodorizing fermented soy milk using fermented soybean 11S globulin, characterized in that, Includes the following steps: (1) Preparation of soybean 11S globulin: After defatting soybean flour with hexane and ethanol, the defatted soybean flour was dissolved in deionized water at a mass-to-volume ratio of 1:10~15. The pH of the solution was adjusted to 8.5±0.5 with NaOH. The alkali-soluble soybean protein was extracted by stirring thoroughly. The mixture was centrifuged at 9000 g for 30~40 min at 4 ℃. Sodium bisulfite was added to the collected supernatant, and the pH of the supernatant was adjusted to 6.4±0.5 with HCl. The protein was precipitated by standing at 4 ℃ and centrifuged at 6500 g for 20~30 min at 4 ℃. The precipitate was washed with deionized water 3~5 times and concentrated using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa to remove impurities. The pH of the final protein solution was adjusted to 7 and then freeze-dried to obtain soybean 11S globulin. (2) Preparation of fermentation products of 11S globulin-producing lactic acid bacteria: Lactobacillus fermentum 56 and Lactobacillus fermentum 57 were used to ferment soybean 11S globulin and glucose. The obtained soybean 11S globulin fermentation broth was concentrated by ultrafiltration using a polyethersulfone membrane with a molecular weight cutoff of 1 kDa. The retentate was recovered and freeze-dried to obtain the soybean 11S globulin lactic acid bacteria fermentation product. (3) Preparation of sterilized soy milk: Soybeans are washed after removing the skins by heat drying, and then soaked in a 0.3% sodium bicarbonate solution for 14-16 hours. Soy milk is prepared at a material-to-liquid ratio of 1:

10. 3% (w:v) inulin and 3% (w:v) white sugar are added. After sterilization, the mixture is cooled to 4 ℃ to obtain sterilized soy milk. (4) Mixed fermentation: The soybean 11S globulin lactic acid bacteria fermentation product from step (2) is mixed evenly with the sterilized soy milk from step (3), and then inoculated with lactic acid bacteria for fermentation to obtain low-soybean-flavored probiotic fermented soy milk. The lactic acid bacteria are selected from any two or more of the following: Lactobacillus fermentum 56, Lactobacillus fermentum 57, Lactobacillus plantarum grx402 (CGMCC No. 28585), Lactobacillus fermentum grx501 (CGMCC No. 25349), and Lactobacillus paracasei grx701 (CGMCC No. 27785).

2. The method for removing fishy odors according to claim 1, characterized in that, In step (2), the inoculation ratio of Lactobacillus fermentum 56 and Lactobacillus fermentum 57 in the fermentation system is equal.

3. The method for removing fishy odors according to claim 1, characterized in that, In step (2), the concentration of soybean 11S globulin in the fermentation system is 30 g / L and the concentration of glucose is 25 g / L.

4. The method for removing fishy odors according to claim 1, characterized in that, In step (2), the fermentation conditions are: 37 ℃, 16 ~ 24 h.

5. The method for removing fishy odors according to claim 1, characterized in that, In step (2), the concentration conditions are: transmembrane pressure 0.15 MPa, concentration factor 2.

6. The method for removing fishy odors according to claim 1, characterized in that, In step (3), the mass of the 0.3% sodium bicarbonate solution is 3 times the mass of the soybeans after the skin has been removed.

7. The method for removing fishy odors according to claim 1, characterized in that, In step (2) or step (4), the total inoculation amount of the strain is 5% (v:v).

8. The method for removing fishy odors according to claim 1, characterized in that, In step (4), the fermentation product of soybean 11S globulin lactic acid bacteria is mixed with sterilized soy milk at a concentration of 2% (w:v).

9. The method for removing fishy odors according to claim 1, characterized in that, In step (4), the lactic acid bacteria are Lactobacillus fermentum grx501 with accession number CGMCC NO.25349 and Lactobacillus paracasei grx701 with accession number CGMCC No.27785, or Lactobacillus fermentum 56 and Lactobacillus fermentum 57, or Lactobacillus plantarum grx402 with accession number CGMCC No. 28585 and Lactobacillus paracasei grx701 with accession number CGMCC No.27785, or Lactobacillus plantarum grx402 with accession number CGMCC No.28585, Lactobacillus paracasei grx701 with accession number CGMCC No.27785, Lactobacillus fermentum 56 and Lactobacillus fermentum 57, with each strain mixed in equal proportions.

10. Low-beany-smelling probiotic fermented soy milk prepared by any one of the deodorization methods according to claims 1 to 9.

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