Method for enhancing bean flavor of soybean milk and reducing beany flavor through combination of composite enzymolysis and bacterium fermentation

Abstract

The invention relates to the technical field of food processing, in particular to a method for enhancing the bean flavor of soybean milk and reducing the beany flavor through composite enzymolysis and bacterium fermentation. According to the method for enhancing the bean flavor of the soybean milk and reducing the beany flavor through the combination of the composite enzymolysis and the bacterium fermentation, the soybeans are firstly soaked, then the soaked soybeans are mixed with water, and the mixture is ground and cooked to obtain original soybean milk; adding porcine pancreas lipase into the original soybean milk to carry out enzymolysis, then adding alkaline protease to carry out enzymolysis, and then carrying out enzyme deactivation to obtain enzymolysis soybean milk; and performing sterilization treatment on the enzymolysis soybean milk, and then inoculating lactobacillus gasseri for fermentation treatment to obtain a fermented soybean milk finished product. According to the method disclosed by the invention, the beany flavor and the bean flavor of the soybean milk are taken as a comprehensive target, and the bean flavor can be effectively improved on the basis of removing the beany flavor through organic combination of enzyme treatment and microbial fermentation, so that a theoretical basis can be provided for accurate regulation and control of the flavor of the soybean milk, and a technical support can be provided for processing and production of high-quality soybean milk.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a method for enhancing the aroma of soybean milk and reducing its beany odor through compound enzymatic hydrolysis combined with bacterial fermentation. Background Technology

[0002] Soy milk, a traditional plant-based protein beverage, is favored by consumers worldwide for its high nutritional value. However, its distinctive beany odor severely impacts sensory acceptance and limits its further promotion in the global market. Research indicates that the beany odor primarily originates from hydroperoxides produced by the catalysis of lipoxygenase (LOX) from polyunsaturated fatty acids (such as linoleic acid and linolenic acid) in soybeans. These intermediate products further degrade into small-molecule volatile substances such as alcohols, aldehydes, and ketones, collectively contributing to the unpleasant smell. Eliminating the beany odor has become a key issue for increasing soy milk consumption and promoting industry development.

[0003] Currently, methods for controlling the beany odor mainly include physical, chemical, and biological methods. Physical methods primarily remove the beany odor through heat treatment; the content of several key beany odor components in raw soy milk prepared through heat treatment is significantly reduced. However, heat treatment easily leads to denaturation of soy protein, loss of nutritional value, and may produce a cooked taste, affecting product quality. Chemical methods block oxidation pathways by adding LOX inhibitors (such as dimercaptothreitol). Although these can completely inhibit enzyme activity under laboratory conditions, the potential risk of chemical residues and consumers' pursuit of clean labels limit their large-scale application and promotion in the food industry.

[0004] In comparison, biological methods have become a research hotspot due to their advantages such as safety and lack of pollution, with enzymatic hydrolysis and microbial fermentation showing the greatest potential. Enzymatic hydrolysis mainly utilizes enzymes such as proteases to specifically hydrolyze soybean protein or modify flavor precursors to remove the fishy smell. However, excessive enzymatic hydrolysis can easily produce unpleasant flavors. Microbial fermentation relies on the rich metabolic systems of microorganisms (such as lactic acid bacteria, yeast, and mold) to transform or mask the fishy smell. Unfortunately, while lactic acid bacteria fermentation can reduce the content of unpleasant flavor substances such as hexanal and hexanol, it also suffers from low yield, insufficient aroma production, and a long aroma production cycle. Furthermore, existing biological methods, while removing the fishy smell of soy milk, often also reduce the amount of soy aroma compounds, resulting in bland and tasteless soy milk that seriously affects the product's edible quality.

[0005] Therefore, developing a soy milk processing method that can effectively enhance the aroma of soybeans while removing the beany smell, in order to meet the production needs of high-quality soy milk, has become an urgent technical problem to be solved. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a method for enhancing the aroma of soy milk and reducing its beany odor through compound enzymatic hydrolysis combined with bacterial fermentation. This method effectively enhances the aroma of soy milk while removing its beany odor, making it suitable for the preparation of high-quality soy milk.

[0007] Another objective of this invention is to provide a finished fermented soybean milk product prepared using the above method.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for enhancing the aroma of soybean milk and reducing its beany odor through compound enzymatic hydrolysis and combined bacterial fermentation includes the following steps:

[0010] (1) Soak soybeans, then mix the soaked soybeans with water, grind and cook them to obtain raw soybean milk;

[0011] (2) Add porcine pancreatic lipase to the original soy milk for enzymatic hydrolysis, then add alkaline protease for enzymatic hydrolysis, and then inactivate the enzyme to obtain enzymatically hydrolyzed soy milk;

[0012] (3) The enzymatically hydrolyzed soy milk is sterilized and then inoculated with Lactobacillus gasseri for fermentation to obtain the fermented soy milk product.

[0013] As a preferred option, in step (1), the soaking temperature is 2~8℃, the time is 8~20h, and the mass-volume ratio of soybeans to water during the soaking process is 1:8~10.

[0014] As a preferred option, in step (1), the mass-to-volume ratio of the soaked soybeans to the water is 1:7~10.

[0015] As a preferred option, in step (1), the pulping and cooking time is 10-50 min, more preferably 30 min.

[0016] As a preferred embodiment, in step (2), the total amount of porcine pancreatic lipase and alkaline protease added is 0.3%~0.6% of the original soybean milk mass, more preferably 0.4%. The mass ratio of alkaline protease to porcine pancreatic lipase is 1:8~12, more preferably 1:10. The enzymatic hydrolysis temperature is 50~60℃, and the pH is 7.2~7.8; the enzymatic hydrolysis time with porcine pancreatic lipase is 3.5~4.5h, and the enzymatic hydrolysis time with alkaline protease is 0.4~0.6h. Through experimental research, this invention has found that when the mass ratio of alkaline protease to porcine pancreatic lipase is 1:10, the best effect of removing the beany smell and enhancing the beany aroma can be achieved.

[0017] As a preferred option, in step (2), the enzyme inactivation temperature is 90~100℃ and the time is 5~20min.

[0018] As a preferred option, in step (3), the sterilization process is carried out at a temperature of 110~120℃ for 10~20min.

[0019] As a preferred embodiment, in step (3), the Lactobacillus gasseri is Lactobacillus gasseri JM1; the inoculation amount of Lactobacillus gasseri is 3%~5% v / v; and the effective bacterial concentration in the enzymatically hydrolyzed soy milk after inoculation is 3×10⁻⁶. 6 ~8×10 6 CFU / g.

[0020] As a preferred option, in step (3), the fermentation treatment temperature is 35~39℃ and the time is 6~10h.

[0021] A fermented soybean milk product prepared using the method described above.

[0022] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0023] This invention provides a method for enhancing the aroma and reducing the beany smell of soy milk through combined enzymatic hydrolysis and microbial fermentation. With both beany and beany smells as comprehensive objectives, it organically combines enzymatic treatment with microbial fermentation, specifically selecting alkaline protease and porcine pancreatic lipase for combined enzymatic hydrolysis. This overcomes the shortcomings of single methods in terms of flavor formation efficiency and metabolic pathway integrity, constructing a more efficient and controllable flavor metabolism system to achieve synergistic enhancement of both beany smell and flavor in soy milk products. Furthermore, this invention systematically investigates the effects of enzymatic hydrolysis-fermentation synergistic treatment on the functional properties (LOX enzyme activity, trypsin inhibitors) and storage properties (titerable acidity, viable cell count) of soy milk. Using GC-IMS and LC-MS technologies, it comprehensively analyzes the dynamic changes of volatile flavor substances and non-volatile flavor precursors during fermentation, revealing the transformation pathways and metabolic relationships between key flavor substances and their precursors. This systematically elucidates the mechanism of action of enzymatic hydrolysis and fermentation in promoting the deodorization and aroma enhancement of fermented soy milk.

[0024] Therefore, the enzymatic hydrolysis-fermentation synergistic regulation of soy milk quality provided by this invention can not only remove the beany smell but also effectively enhance the soy aroma. It can provide a theoretical basis for the precise regulation of soy milk flavor and technical support for the processing and production of high-quality soy milk. Attached Figure Description

[0025] Figure 1 This invention illustrates the effects of different types of enzyme treatments on the soybean aroma and beany flavor compounds in soybean milk.

[0026] Figure 2 This invention illustrates the effect of different ratios of lipase and protease on the aroma and beany flavor compounds in soy milk.

[0027] Figure 3 This invention illustrates the effect of different protease hydrolysis times on the degree of hydrolysis and bitter amino acid content in soy milk.

[0028] Figure 4 This invention describes the effects of different treatment groups on the functional properties of soy milk (including lipoxygenase activity, trypsin inhibitory activity, phytic acid content, and total phenol content).

[0029] Figure 5 This invention illustrates the effects of different treatment groups on the storage characteristics of soy milk (including pH value, titratable acidity, and viable bacteria count).

[0030] Figure 6 This invention illustrates the effects of different treatment groups on the nutritional properties of soy milk (including the content of free amino acids and free fatty acids).

[0031] Figure 7 The results of electronic nose and electronic tongue measurements of soy milk in different treatment groups in this invention;

[0032] Figure 8 This invention illustrates the effect of different treatment groups on volatile flavor compounds in soy milk.

[0033] Figure 9 This invention illustrates the effects of different treatment groups on non-volatile flavor compounds in soy milk. Detailed Implementation

[0034] The technical solutions and effects of the present invention will be clearly and completely described below with reference to specific embodiments and experimental examples. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical solutions of the present invention and should not be regarded as limiting the scope of protection of the present invention.

[0035] In the following embodiments, the Lactobacillus gasseri JM1 involved was isolated from infant intestinal feces and has been disclosed in the following existing patents and existing literature: (1) CN120173854A A fermentation culture additive for Lactobacillus gasseri JM1 and its uses; (2) Sun Linlin. Study on the immunomodulatory effect and regulatory pathway of a strain of Lactobacillus gasseri [D]. Northeast Agricultural University. Therefore, this invention will not describe it in detail. In other embodiments, those skilled in the art may choose other strains of Lactobacillus gasseri, and this invention does not impose any special restrictions on them. Specifically, the Lactobacillus gasseri JM1 used below is stored in the Key Laboratory of Dairy Science of the Ministry of Education of Northeast Agricultural University and is stored at -80℃; before use, the strain is inoculated in MRS liquid medium at 4% (v / v) and then activated by culturing at 37℃ for 24h, and then passaged twice to obtain the seed culture of Lactobacillus gasseri JM1.

[0036] In the following embodiments, the soybeans used were non-GMO Fook brand soybeans, produced in Heilongjiang Province, China. The alkaline protease had an enzyme activity of 2.4 AU / g, product code Alcalase 2.4L, sourced from Novozymes, Denmark. The porcine pancreatic lipase had an enzyme activity of 20000 U / g. The commercially available starter culture was purchased from Inner Mongolia Yisige Biotechnology Co., Ltd. (specifically, Suangeyizu KKJ01 starter culture). The soymilk maker was model SUPOR DJ08B-P69E.

[0037] Example 1

[0038] This embodiment provides a method for enhancing the aroma of soybean milk and reducing its beany odor through compound enzymatic hydrolysis combined with bacterial fermentation, specifically including the following steps:

[0039] (1) Select plump soybeans, wash them three times with clean water, and soak them in water at 4℃ for 12 hours. During the soaking process, the ratio of soybeans to water is 1:9 (W / V). Then take out the soaked soybeans, put the soybeans and water into the soymilk maker at a ratio of 1:8 (W / V), turn on the soymilk making mode to cook and stir for 30 minutes, and let it cool naturally to obtain the original soymilk;

[0040] (2) Adjust the pH of the original soy milk obtained in step (1) to 7.5±0.1, then add porcine pancreatic lipase and hydrolyze it in a 55℃ water bath for 4 hours, then add alkaline protease and hydrolyze it at the same temperature for 0.5 hours; wherein, the mass ratio of alkaline protease to porcine pancreatic lipase is 1:10; the total added mass of alkaline protease and porcine pancreatic lipase is 0.4% of the original soy milk mass (of which porcine pancreatic lipase is 0.36% and alkaline protease is 0.04%). After the hydrolysis is completed, inactivate the enzyme at 95℃ for 10 minutes, and cool to room temperature to obtain hydrolyzed soy milk;

[0041] (3) The enzymatically hydrolyzed soy milk obtained in step (2) was sterilized at 115℃ for 15 min to obtain sterilized soy milk. After one subculture of the Lactobacillus gasseri JM1 seed culture, it was inoculated into the sterilized soy milk at 4% (v / v). The effective bacterial concentration in the enzymatically hydrolyzed soy milk after inoculation was 5 × 10⁻⁶. 6 The fermented soybean milk product of this embodiment (denoted as ENJMI group) was prepared by fermenting at 37°C for 8.5 hours with CFU / g.

[0042] Comparative Example 1

[0043] This comparative example provides a method for preparing unenzymatically hydrolyzed, unfermented soy milk, specifically including the following steps:

[0044] (1) Same as step (1) in Example 1;

[0045] (2) Adjust the pH of the original soy milk obtained in step (1) to 7.5±0.1, and then place the soy milk in a 55℃ water bath for 4.5h without adding enzymes, then treat it at 95℃ for 10min, and cool it to room temperature to obtain control soy milk.

[0046] (3) The control soy milk obtained in step (2) was sterilized at 115℃ for 15 min to obtain sterilized soy milk. The sterilized soy milk was then treated at 37℃ for 8.5 h to obtain the finished soy milk product of the comparative ratio (denoted as UN group).

[0047] Comparative Example 2

[0048] This comparative example provides a method for preparing soy milk using compound enzymatic hydrolysis, specifically including the following steps:

[0049] (1) Same as step (1) in Example 1;

[0050] (2) Adjust the pH of the original soy milk obtained in step (1) to 7.5±0.1, then add porcine pancreatic lipase and enzymatically hydrolyze it in a 55℃ water bath for 4 hours, and then add alkaline protease and enzymatically hydrolyze it at the same temperature for 0.5 hours; wherein, the mass ratio of alkaline protease to porcine pancreatic lipase is 1:10; the total added mass of alkaline protease and porcine pancreatic lipase is 0.4% of the mass of the original soy milk. After enzymatic hydrolysis, inactivate the enzyme at 95℃ for 10 minutes, and cool to room temperature to obtain enzymatically hydrolyzed soy milk;

[0051] (3) Sterilize the enzymatically hydrolyzed soy milk obtained in step (2) at 115°C for 15 min. The sterilized soy milk obtained is the soy milk product of this comparative example (denoted as EN group).

[0052] Comparative Example 3

[0053] This comparative example provides a method for preparing soy milk by fermentation with Lactobacillus gasseri, specifically including the following steps:

[0054] (1) Same as step (1) in Example 1;

[0055] (2) Adjust the pH of the original soy milk obtained in step (1) to 7.5±0.1, and then place the soy milk in a 55℃ water bath for 4.5h without adding enzymes. After the end, treat it at 95℃ for 10min and cool it to room temperature to obtain unhydrolyzed soy milk.

[0056] (3) The unenzymatically hydrolyzed soy milk obtained in step (2) was sterilized at 115℃ for 15 min to obtain sterilized soy milk. After one subculture of the Lactobacillus gasseri JM1 seed culture, it was inoculated into the sterilized soy milk at 4% (v / v). The effective bacterial concentration in the enzymatically hydrolyzed soy milk after inoculation was 5 × 10⁻⁶. 6 CFU / g, fermented at 37℃ for 8.5h, to prepare the soy milk of this comparative ratio (denoted as JMI group).

[0057] Comparative Example 4

[0058] This comparative example provides a method for preparing soy milk using alkaline protease alone followed by bacterial fermentation, specifically including the following steps:

[0059] (1) Same as step (1) in Example 1;

[0060] (2) Adjust the pH of the original soy milk obtained in step (1) to 7.5±0.1, then place it in a 55℃ water bath for 4 hours, and then add alkaline protease to the 55℃ water bath for 0.5 hours for enzymatic hydrolysis; the mass of alkaline protease added is 0.4% of the mass of the original soy milk. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95℃ for 10 minutes, and cool to room temperature to obtain enzymatically hydrolyzed soy milk;

[0061] (3) The enzymatically hydrolyzed soy milk obtained in step (2) was sterilized at 115℃ for 15 min to obtain sterilized soy milk. After one subculture of the Lactobacillus gasseri JM1 seed culture, it was inoculated into the sterilized soy milk at 4% (v / v). The effective bacterial concentration in the enzymatically hydrolyzed soy milk after inoculation was 5 × 10⁻⁶. 6 CFU / g, fermented at 37℃ for 8.5h, to prepare the soy milk of this comparative ratio (denoted as APJMI group).

[0062] Comparative Example 5

[0063] This comparative example provides a method for preparing soy milk by single enzymatic hydrolysis of porcine pancreatic lipase combined with bacterial fermentation, specifically including the following steps:

[0064] (1) Same as step (1) in Example 1;

[0065] (2) Adjust the pH of the original soy milk obtained in step (1) to 7.5±0.1, and then add porcine pancreatic lipase to a water bath at 55℃ for 4.5h for enzymatic hydrolysis; the mass of porcine pancreatic lipase added is 0.4% of the mass of the original soy milk. After enzymatic hydrolysis, inactivate the enzyme at 95℃ for 10min, and cool to room temperature to obtain enzymatically hydrolyzed soy milk;

[0066] (3) The enzymatically hydrolyzed soy milk obtained in step (2) was sterilized at 115℃ for 15 min to obtain sterilized soy milk. After one subculture of the Lactobacillus gasseri JM1 seed culture, it was inoculated into the sterilized soy milk at 4% (v / v). The effective bacterial concentration in the enzymatically hydrolyzed soy milk after inoculation was 5 × 10⁻⁶. 6 CFU / g, fermented at 37℃ for 8.5h, to prepare the soy milk of this comparative ratio (denoted as PLJMI group).

[0067] Comparative Example 6

[0068] This comparative example provides a method for preparing fermented soybean milk using a commercially available starter culture alone, specifically including the following steps:

[0069] (1) Same as step (1) in Example 1;

[0070] (2) Adjust the pH of the original soy milk obtained in step (1) to 7.5±0.1, then place it in a 55℃ water bath for 4.5h, and after the end, treat it at 95℃ for 10min, and cool it to room temperature to obtain unhydrolyzed soy milk.

[0071] (3) The unenzymatically hydrolyzed soy milk obtained in step (2) was sterilized at 115℃ for 15 minutes to obtain sterilized soy milk. A commercially available starter culture was inoculated into the sterilized soy milk at 5% (v / v). After inoculation, the effective bacterial concentration in the soy milk was 5 × 10⁻⁶. 6 CFU / g, fermented at 37℃ for 8.5h, to prepare the soy milk of this comparative ratio (denoted as CC group).

[0072] Experiment Example 1: Determination of Enzyme Type, Enzyme Ratio, and Enzymatic Hydrolysis Time

[0073] 1.1 Types of enzymes

[0074] This experiment used nine commercially available enzymes to enzymatically hydrolyze raw soy milk to investigate the effects of different enzymes on the beany and soy aroma flavor compounds in soy milk. The nine enzymes tested were: flavor protease (20 U / mg), alkaline protease (2.4 AU / g), papain (16 U / mg), neutral protease (50 U / mg), porcine pancreatic lipase (20000 U / g), Aspergillus niger lipase (12000 U / g), α-glucosidase (50 U / mg), β-glucosidase (20 U / mg), and α-galactosidase (1000 U / g). The specific testing method is as follows: Prepare raw soy milk according to step (1) of Example 1; divide the raw soy milk into 10 soy milk samples, adjust the pH value of each soy milk sample to 7.5±0.1, and then add the enzyme to be tested to each sample in a 55℃ water bath for enzymatic hydrolysis. The hydrolysis time is 4.5h, and the amount of enzyme used is 0.4% of the mass of soy milk. After the hydrolysis is completed, inactivate the enzyme at 95℃ for 10min, and cool to room temperature to obtain enzymatically hydrolyzed soy milk. At the same time, a blank control group was prepared under the same conditions without adding enzyme. After the hydrolysis is completed, inactivate the enzyme at 95℃ for 10min, and then perform GC-MS analysis on the soy milk samples obtained after each group of treatment (GC-MS measurement parameters can be referred to in Section 5.2 of this invention, the same below). The flavor substances in the sample group were detected by GC-MS technology. The effects of different enzyme treatments on the soy aroma flavor substances and beany flavor substances in soy milk are shown in Tables 1-2 and Figure 1 As shown. Figure 1 In the figure, Figure a represents the flavor compounds of bean aroma, and Figure b represents the flavor compounds of bean-like aroma.

[0075] Table 1. Effects of different enzyme treatments on soybean aroma and flavor compounds in soybean milk

[0076] Table 2. Effects of different enzyme treatments on beany flavor compounds in soy milk

[0077] Combining Table 1 and Table 2 Figure 1 The GC-MS analysis revealed 13 bean aroma compounds (including 3-methylbutanol, ethyl acetate, and 3-hydroxy-2-butanone) and 14 beany flavor compounds (including hexanal, 1-octen-3-ol, and 2-pentylfuran). Treatment with porcine pancreatic lipase resulted in the highest content of bean aroma compounds, reaching 1297.3 μg / kg. Treatment with alkaline lipase resulted in the lowest content of beany flavor compounds, at 595.39 μg / kg. Therefore, porcine pancreatic lipase treatment enhances the bean aroma of soy milk, while alkaline protease treatment reduces the beany odor. Based on this, the subsequent investigation in this invention focuses on the effects of combined fermentation by porcine pancreatic lipase and alkaline protease on the quality of soy milk.

[0078] 1.2 Enzyme ratio

[0079] To investigate the combined effects of different protease and lipase ratios on the beany and beany flavor of soy milk, the original soy milk prepared according to step (1) of Example 1 was divided into 10 portions. The pH was adjusted to 7.5 ± 0.1, and then different proportions of porcine pancreatic lipase and alkaline protease were added. The mixture was enzymatically hydrolyzed in a 55℃ water bath for 4.5 h. The ratios of alkaline protease and porcine pancreatic lipase were 1:1, 1:2, 1:3, 1:5, 1:10, 1:15, and 1:20, respectively, with the total amount of both enzymes added being 0.4% of the soy milk mass. Simultaneously, samples prepared under the same conditions—without enzymes, with only alkaline protease (0.4%), and with only porcine pancreatic lipase (0.4%)—were used as control groups. After enzymatic hydrolysis, the enzymes were inactivated at 95℃ for 10 min. The soy milk samples obtained after each treatment were analyzed by GC-MS, and the flavor compounds in the sample groups were detected using GC-MS technology. The effects of different enzyme ratios on the aroma and flavor compounds and beany flavor compounds in soy milk, such as Figure 2 As shown. Figure 2 In the figure, Figure a represents the flavor compounds of bean aroma, and Figure b represents the flavor compounds of bean-like aroma.

[0080] Depend on Figure 2 It was found that after treatment with alkaline protease: porcine pancreatic lipase at a ratio of 1:10, the content of bean-like flavor compounds was the highest, reaching 1400.74 μg / kg. Although the content of beany flavor was not the lowest at this point, it was significantly reduced compared to the content of porcine pancreatic lipase alone. Therefore, an alkaline protease: porcine pancreatic lipase ratio of 1:10 was chosen for subsequent experiments.

[0081] 1.3 Enzymatic hydrolysis time

[0082] Excessive protein hydrolysis may cause the protein in soy milk to be hydrolyzed into small peptides and free amino acids. At the same time, a large amount of hydrophobic amino acids are exposed, forming a large number of small hydrophobic peptides with bitter taste, thus significantly increasing the bitterness of soy milk. Therefore, in order to avoid this phenomenon, this invention investigated the effect of different alkaline protease hydrolysis times on the degree of hydrolysis and bitter amino acid content of soy milk. The specific experimental method is as follows: Take the original soy milk prepared according to step (1) of Example 1, adjust its pH value to 7.5±0.1, and then add 0.36% of the mass of soy milk porcine pancreatic lipase and hydrolyze it in a 55℃ water bath for 4.5h. At different time points during the hydrolysis process of porcine pancreatic lipase, add 0.04% of the mass of soy milk alkaline protease. Control the hydrolysis time of alkaline protease to be 4.5h, 3.5h, 2.5h, 1.5h, 0.5h and 0.1h respectively. At the same time, a sample prepared under the same conditions without enzyme is set as a blank control group. After the hydrolysis is completed, the enzyme is inactivated at 95℃ for 10min. Finally, the degree of hydrolysis and bitter amino acid content in the soy milk samples obtained after each treatment were determined. The effects of different alkaline protease hydrolysis times on the bitter amino acid content in soy milk are shown in Table 3. Figure 3 As shown. Figure 3 In the figure, Figure a represents the degree of hydrolysis, and Figure b represents the content of bitter amino acids.

[0083] Table 3. Effects of different protease hydrolysis times on the content of bitter amino acids in soy milk

[0084] Based on Table 3 Figure 3 It is evident that with increasing alkaline protease hydrolysis time, both the degree of hydrolysis and the content of bitter amino acids significantly increase. This is likely because alkaline protease, as an endopeptidase, can rapidly cleave peptide bonds in proteins, leading to a significant increase in the content of bitter amino acids and small peptides. When the hydrolysis time exceeds 1.5 hours, the degree of hydrolysis also exceeds 30%, and the content of bitter amino acids increases significantly. A 30% degree of hydrolysis falls within the range of deep hydrolysis, at which point the original protein structure is completely destroyed, resulting in a large accumulation of bitter amino acids and small peptides, which negatively impacts the flavor of soy milk. When the hydrolysis time is 0.5 hours, the degree of hydrolysis is 17.03%, falling within the range of moderate hydrolysis, and the content of bitter amino acids is also low. Hydrolysis at 0.1 hours shows no significant difference compared to unhydrolyzed soy milk. Therefore, this invention ultimately determines the alkaline protease hydrolysis time to be 0.5 hours.

[0085] Experiment Example 2: Functional Characteristic Determination

[0086] The soy milk products prepared in Example 1 and Comparative Examples 1-6 were used as soy milk samples to be tested. Their lipoxygenase activity, trypsin inhibitory activity, phytic acid content, and total phenol content were tested to investigate the functional characteristics of different soy milk samples.

[0087] 2.1 Assay of Lipoxygenase (LOX) Activity

[0088] Take 2g of the soybean milk sample to be tested and soak it in phosphate buffer (0.05mol / L, pH 6.0) for 1h, then centrifuge at 4000rpm for 30min. Take the supernatant, add 0.5g of anhydrous ammonium sulfate, let it stand for 3h, and then centrifuge at 12000rpm for 20min. Repeat twice. Dialyze the precipitate obtained by centrifugation in phosphate buffer and concentrate to obtain crude enzyme solution. Mix 140μL of linoleic acid and 140μL of Tween 20 and emulsify in 8mL of borate buffer (0.2mol / L, pH 9.0), then add 1.1mL of sodium hydroxide solution and make up to 50mL with borate buffer to obtain a mixture. Dilute this mixture 40 times before use to obtain a diluted solution. Add 50μL of crude enzyme solution to 3.5mL of the above diluted solution, mix quickly, start timing, and measure the increase in absorbance at a wavelength of 234nm. An increase in absorbance of 0.001 per minute is defined as one unit of enzyme activity.

[0089] 2.2 Assay for trypsin inhibitory activity

[0090] Take 1g of the soy milk sample to be tested into an Erlenmeyer flask, add 50mL of 0.006mol / L NaOH solution, shake at room temperature for 30min, adjust the pH to 9.5-9.8, shake for 2.5h, then remove the sample solution and let it stand for later use. Take two test tubes, one for the blank reaction and one for the enzyme reaction. Add 1mL of distilled water to the blank reaction tube and 1mL of the sample solution to the enzyme reaction tube. Then add 1mL of trypsin solution (0.15mg / mL) to each test tube and mix well. Then add 2.5mL of L-BAPA solution (i.e., NA-benzoyl-L-arginine yl-4-nitroaniline hydrochloride solution, concentration 0.06wt%, dissolved in 1wt% dimethyl sulfoxide), incubate at 37℃ for 10min, remove and add 0.5mL of acetic acid to terminate the reaction, and measure the absorbance at 410nm. Trypsin inhibitory activity is expressed as trypsin inhibition units per milligram of sample.

[0091] 2.3 Phytic acid

[0092] Take 0.5g of the soy milk sample to be tested into an Erlenmeyer flask, add 10mL of 2.4% dilute hydrochloric acid, and shake at room temperature for 2 hours. After removal, centrifuge at 3000rpm for 20 minutes and collect the supernatant. Add 3mL of 30wt% sodium chloride solution to the supernatant, let stand at 4℃ for 1 hour, centrifuge at 3000rpm for 20 minutes, and collect the supernatant for later use. Take 5mL of the supernatant into a test tube, add 4mL of Wade's reagent (FeCl3•H2O, 0.3% sulfosalicylic acid), mix well, centrifuge at 3000rpm for 20 minutes, let stand for 10 minutes, and measure the absorbance at 500nm. Plot a standard curve using the absorbance values ​​of different concentrations of phytic acid, and calculate the phytic acid content in the sample using the standard curve.

[0093] 2.4 Total phenols

[0094] Take 0.5g of the soy milk sample to be tested, add 4.5mL of 80% methanol, sonicate for 20min, centrifuge at 8000rpm for 15min, and collect the supernatant. Add Folin-Ciocalteu and 7.5wt% Na2CO3 solution to the supernatant, and react at room temperature in the dark for 1h. Measure the absorbance at 765nm. Plot a standard curve based on the absorbance values ​​of different concentrations of gallic acid, and calculate the total phenol content in the sample using the standard curve.

[0095] The effects of different treatment groups on the functional properties of soy milk (including lipoxygenase activity, trypsin inhibitory activity, phytic acid content, and total phenolic content) are as follows: Figure 4 As shown in the figure. Among them, Figure a shows the activity of lipoxygenase, Figure b shows the activity of trypsin inhibitory activity, Figure c shows the content of phytic acid, and Figure d shows the content of total phenols.

[0096] Lipoxygenases oxidize polyunsaturated fatty acids to produce hydroperoxides. These hydroperoxides are extremely unstable and can further degrade to form volatile compounds with different odor thresholds, among which aldehydes are the main source of the beany odor. Figure 4 As shown in Figure a, compared with the UN group, the activities of lipoxygenase in the JM1, APJM1, PLJM1, and ENJM1 groups were all significantly reduced. Furthermore, compared with the JM1 group, the LOX activities in the APJM1 and ENJM1 groups decreased by 8.17% and 14.83%, respectively, while there was no significant difference in the PLJM1 group. This indicates that alkaline proteases can directly attack and hydrolyze lipoxygenase, destroying its three-dimensional structure and activity, thus leading to its rapid inactivation.

[0097] Trypsin, an important anti-nutritional factor in soybeans and other legumes, hinders protein digestion and reduces its absorption and utilization. Figure 4As shown in Figure b, compared with the UN group, the trypsin inhibitory activity in the JM1 group decreased by 10.26%. This is mainly because the acidic environment generated during the fermentation of Lactobacillus gasseri JM1 led to the destruction of some trypsin inhibitory structures, thus inactivating them. Compared with the JMI group, the trypsin inhibitory activities in the APJM1 and ENJM1 groups were significantly reduced (30.08% and 36.91%, respectively), while there was no significant difference in the PLJM1 group. This indicates that trypsin inhibitors are proteins, which can be directly hydrolyzed and destroyed by proteases, while lipase hydrolysis has little effect on them.

[0098] Phytic acid can rapidly bind with mineral ions (such as calcium, iron, and zinc) to form insoluble complexes and can interact with dietary proteins, thereby significantly reducing the bioavailability of nutrients. Therefore, reducing the phytic acid content in soybean-derived products such as soy milk is crucial. Figure 4 As shown in Figure c, compared with the UN group, the phytic acid content in the JM1, APJM1, PLJM1, and ENJM1 groups was significantly reduced. Compared with the JM1 group, the phytic acid content in the PLJM1 and APJM1 groups decreased by 4.65% and 5.44%, respectively, while that in the ENJM1 group decreased by 14.14%. This may be due to the synergistic effect of lipase and protease hydrolysis, leading to a significant reduction in phytic acid content.

[0099] Soy milk contains abundant phenolic compounds, some of which exist in free form, while others are bound to cellulose, protein, and other substances, known as bound phenolic compounds. Therefore, breaking the bonds between phenolic compounds and other substances to release them helps improve their nutritional value. Figure 4 As shown in Figure d, the total phenol content of the JM1 group increased by 12.62% compared to the UN group. Compared to the JM1 group, the total phenol content of the APJM1, EN, and ENJM1 groups increased significantly. This is because the protease can effectively hydrolyze soybean protein, destroying the phenol-protein complex, thereby releasing a large amount of the bound phenolic substances. This also helps the lactic acid bacteria to further enhance the phenol content during subsequent fermentation, resulting in a secondary increase in phenol content and thus increasing the total phenol content of the soy milk.

[0100] Experimental Example 3: Storage Characteristics

[0101] The soy milk products prepared in Example 1 and Comparative Examples 1-6 were used as soy milk samples to be tested. Their pH value, titratable acidity, and viable bacteria count were tested to investigate the storage characteristics of different soy milk samples.

[0102] 3.1 pH value and titratable acidity

[0103] The prepared soy milk samples were stored at 4℃ for 28 days, with samples taken every 7 days. 10g of sample was placed in a 50mL centrifuge tube, and the pH value was measured using a pH meter. The titratable acidity of the soy milk samples was determined using acid-base titration. 10g of sample was weighed into an Erlenmeyer flask, diluted with 20mL of deionized water, and thoroughly mixed. Then, 2mL of 1% phenolphthalein indicator was added, and titration was performed with 0.1mol / L sodium hydroxide standard solution. The titration endpoint was reached when the reaction color changed from colorless to light pink and did not fade within 30 seconds. Each sample group was measured in triplicate.

[0104] 3.2 Viable Bacterial Count Determination

[0105] Soy milk samples were collected at storage times of 0, 7, 14, 21, and 28 days. 1 g of sample was accurately weighed and serially diluted with 9 mL of 0.85% sterile physiological saline. Based on the predicted concentration gradient of the target bacterial strain, 3-4 adjacent suitable dilution gradients were selected for the experiment. MRS solid medium was used, and the culture was carried out at 37°C. After a standard culture period of 48 hours, the colonies on the plates were counted. Plates with 30-300 colonies were considered valid data.

[0106] The effects of different treatment groups on the storage characteristics of soy milk (including pH, titratable acidity, and viable cell count) are as follows: Figure 5 As shown. Figure 5 In the figure, Figure a represents the pH value, Figure b represents the titration acidity, and Figure c represents the viable bacteria count.

[0107] pH is a key parameter for monitoring probiotic fermentation and an important indicator for determining the fermentation endpoint; pH 4.5 is typically used as the critical value for fermentation completion. Furthermore, suitable acidity contributes to excellent flavor and texture, while excessive acidification impairs taste and quality. Figure 5 As shown in Figures a and b, compared to the UN and EN groups, the pH values ​​of fermentation groups such as JM1 and PLJM1 slowly decreased and the acidity slowly increased during storage. This is because during fermentation, probiotics continue to convert lactose in the soy milk into lactic acid, and at lower storage temperatures, the lactic acid bacteria enter a dormant state, resulting in a slow decrease in pH and a slow increase in acidity. Compared to the JM1 group, the APJM1 and ENJM1 groups had even lower pH values ​​and higher acidity.

[0108] The number of live bacteria directly affects the sensory and functional properties of fermented soy milk and is a key indicator for evaluating its quality. Figure 5As shown in Figure c, the viable cell count of the fermentation samples (JM1, PLJM1, APJM1, ENJM1, and CC groups) generally decreased with increasing storage days. This can be attributed to two factors: firstly, the pH value decreased due to lactic acid accumulation, which inhibited bacterial growth; and secondly, the probiotics, even when stored in a low-temperature environment, still require nutrients to maintain their normal physiological activities. As the available nutrients gradually decreased, the lactic acid bacteria gradually died out.

[0109] Experimental Example 4: Nutritional Characteristics

[0110] The soy milk products prepared in Example 1 and Comparative Examples 1-6 were used as soy milk samples to be tested. The contents of free amino acids and fatty acids were measured to investigate the nutritional characteristics of different soy milk samples.

[0111] 4.1 Free amino acids

[0112] A 10% sulfosalicylic acid solution was added to the soy milk sample at a 1:1 (v / v) ratio and incubated at room temperature for 1 hour. The sample was then centrifuged at 12000 rpm for 15 minutes, and the supernatant was collected and refrigerated overnight at 4°C. The soy milk sample was then centrifuged again using the same parameters. The supernatant was filtered through a 0.22 μm aqueous filter membrane, and 1 mL of the filtrate was transferred to a sample vial to complete sample preparation. The free amino acids in the sample were then quantitatively analyzed using an automated amino acid analyzer (model LA8080, Hitachi, Japan).

[0113] 4.2 Determination of Free Fatty Acid Content

[0114] Lipids in the soy milk sample were extracted using chloroform-methanol. After drying the extract with nitrogen, the residue was redissolved in a 14 wt% boron trifluoride methanol solution and methylated in a 60°C water bath for 1 h. Hexane was then added for extraction, and the mixture was shaken and washed with distilled water. After standing and separating the layers, the upper organic phase was collected, and excess water was removed using anhydrous sodium sulfate powder. The sample was then filtered through a 0.22 μm filter before gas chromatography detection. The gas chromatography conditions were as follows: Thermo TG-FAME capillary column, injection port temperature 250°C, ion source temperature 300°C; initial temperature 80°C held for 1 min, increased to 200°C at 20°C / min and held for 10 min, and finally increased to 250°C at 5°C / min and held for 3 min. Qualitative analysis was performed using a mixed standard containing saturated and unsaturated fatty acids, and the content of various fatty acids was quantified using peak area external standard.

[0115] The effects of different treatment groups on the nutritional properties of soy milk (including free amino acid content and free fatty acid content) are as follows: Figure 6 As shown. Figure 6In the figure, Figure a shows the content of free amino acids, and Figure b shows the content of free fatty acids.

[0116] like Figure 6 As shown in Figure a, unfermented soy milk contained 256.29 μg / mL of total free amino acids. After fermentation with Lactobacillus gasseri JM1, the total free amino acid content in the JM1 group was 323.21 μg / mL, indicating that fermentation significantly increased the total concentration of free amino acids. This suggests that during fermentation, Lactobacillus gasseri secretes proteases to degrade proteins, producing peptides and free amino acids, ultimately improving the nutritional value of the soy milk. Compared to the EN group, the free amino acid content in the APJM1 group decreased. Compared to the JM1 group, the free amino acid content in the PLJM1 group increased by 24.95%, the APJM1 group by 106.89%, and the ENJM1 group by 160.83%. Therefore, the free amino acid content in soy milk increases after fermentation, making it a valuable nutritional supplement. Further statistical analysis of individual amino acid content revealed that, compared to the JM1 group, the ENJM1 group showed a significant increase not only in the content of sweet and umami free amino acids such as glutamic acid, glutamine, alanine, and glycine, but also in the content of bitter amino acids such as leucine, tyrosine, and phenylalanine. During fermentation, bitter amino acids can synergistically interact with umami and sweet amino acids and other flavor compounds to enhance the fullness and complexity of the flavor.

[0117] like Figure 6As shown in Figure b, the total free fatty acid content of unfermented soy milk (UN group) was 265.81 mg / L. After fermentation with Lactobacillus gasseri JM1 (495.96 mg / L), the free fatty acid content increased significantly. This indicates that probiotic fermentation has a significant impact on the free fatty acid composition of soy milk. Furthermore, compared with the JM1 group, the free fatty acid content increased by 20.8% in the APJM1 group, 64.7% in the PLJM1 group, and 115.4% in the ENJM1 group. Simultaneously, compared with JM1, the content of both saturated and unsaturated fatty acids in the PLJM1 group also increased significantly. The significant increase in fatty acid content in the ENJM1 group compared to the PLJM1 group can be attributed to the action of alkaline protease. Further analysis of individual fatty acid content revealed that compared with the UN group, the JM1 group showed a significant increase in the content of saturated fatty acids such as palmitic acid and stearic acid, and unsaturated fatty acids such as oleic acid, linoleic acid, and α-linolenic acid. Moreover, after complex enzymatic hydrolysis (ENJM1 group), the content of these substances further increased. Analysis of free fatty acids also revealed a large number of functional fatty acids, including alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), arachidonic acid (DPA), gamma-linolenic acid (DGLA), and myristic acid (MA). These functional amino acids help enhance the nutritional value of soy milk. All of the above studies collectively confirm that enzymatic hydrolysis combined with fermentation effectively improves the nutritional characteristics of soy milk.

[0118] Experimental Example 5: Flavor Characteristics

[0119] The soy milk products prepared in Example 1 and Comparative Examples 1-6 were used as soy milk samples to be tested. The electronic nose and electronic tongue, volatile flavor compounds, and non-volatile flavor compounds were measured to investigate the flavor characteristics of different soy milk samples.

[0120] 5.1 Electronic nose and electronic tongue

[0121] Electronic noses and electronic tongues are analytical systems that mimic human olfaction, enabling rapid identification, detection, and analysis of the odors and tastes of various foods. The testing method for an electronic nose is as follows: Weigh 10±0.01g of the soy milk sample to be tested into a headspace vial, incubate in an 85℃ water bath for 15 minutes, and then allow to cool to room temperature for measurement. Use a disposable sterile needle to aspirate volatile gases from the headspace vial. Air is used as the carrier gas at a flow rate of 230mL / min and a flow velocity of 200mL / min. The washing time is 60s, the sampling interval is 1s, and the total detection time is 120s. Each sample is measured three times for PCA plotting, and the average value is used for radar plotting. The electronic nose contains 10 metal oxide sensors: W1C (aromatic components), W3C (aromatic components, amines), W5C (short-chain alkane aromatic components), W1S (methyl compounds), W2S (alcohols, aldehydes, and ketones), W3S (long-chain alkanes), W5S (nitrogen oxides), W6S (hydrides), W1W (sulfides), and W2W (sulfur-containing organic compounds). The testing method for the electronic tongue is as follows: Weigh 30 ± 0.01 g of sample into the electronic tongue sampling cup. Before testing, seal the sample with sealing film and let it stand for half an hour. The sensors of the electronic tongue need to be soaked in buffer solution for 24 hours before use. Testing begins after instrument calibration. Each sample is measured three times for PCA plotting, and the average value is used for radar plotting. The taste sensors of an electronic tongue mainly include: Sourness, Bitterness, Astringency, Aftertaste-A (astringency aftertaste), Aftertaste-B (bitter aftertaste), Unami, Richness, and Saltiness.

[0122] The results of electronic nose and electronic tongue measurements of soy milk in different treatment groups are as follows: Figure 7 As shown. Figure 7 In the middle, the left image shows an electronic nose, and the right image shows an electronic tongue. Figure 7The changes in response values ​​of fermented soybean milk samples treated with different enzymes on the sensors are presented in radar chart form. The electronic nose detection results show that, compared with unfermented soybean milk (UN group), all fermented soybean milk samples showed lower response values ​​on sensors W1S, W3S, and W6S, and higher response values ​​on sensors W1C and W2S. This indicates that lactic acid bacteria fermentation significantly reduces the content of harmful substances such as alkanes and hydrides, and increases the content of aromatic components such as alcohols, aldehydes, ketones, terpenes, and aromatic substances. Compared with group JM1, group ENMJ1 showed higher response values ​​on sensors W1C, W3C, W5C, and W2S, indicating that enzymatic hydrolysis promotes the production of aromatic components such as alcohols, aldehydes, ketones, and short-chain alkanes in fermented soybean milk, thereby enhancing the aroma of the soybean milk. The electronic tongue detection results show that Lactobacillus gasseri JM1 fermentation also has a significant impact on the taste of soybean milk. Specifically, the response values ​​of the sourness and umami sensors increased significantly, while the response values ​​of the bitter aftertaste, astringent aftertaste, and salty taste sensors decreased significantly. These results indicate that the fermentation process of lactic acid bacteria can significantly improve the sourness of soy milk while reducing the associated bitterness, astringency, and saltiness. Compared with the JM1 group, the ENJM1 group showed higher response values ​​for umami and umami aftertaste. The increase in umami may be related to the hydrolysis of soy protein during enzymatic hydrolysis. Under the action of alkaline protease, the protein is hydrolyzed into free amino acids, resulting in an increase in the content of aspartic acid and glutamic acid, thereby enhancing the umami flavor of soy milk. Therefore, enzymatic hydrolysis combined with lactic acid bacteria fermentation can significantly improve the flavor characteristics of soy milk.

[0123] 5.2 Volatile flavor compounds

[0124] In this experiment, to investigate the effects of different treatments on volatile flavor compounds in soy milk, GC-IMS technology was used to identify the volatile flavor compounds in the samples, and they were classified and analyzed in conjunction with existing literature and odor descriptions. The testing procedure was as follows: 3 ± 0.01 g of the soy milk sample was accurately weighed and placed in a 20 mL headspace vial, incubated at 60℃ for 15 min, and then injected. The injection needle temperature was 85℃. The testing instrument was a FlavorSpec® gas ion mobility spectrometer, manufactured by GAS. The chromatographic column type was FS-SE-54-CB-1 (15 m × 0.53 mm, 1 μm), the column temperature was 60℃, and the carrier gas was N2. The pressure program was as follows: initial flow rate of 2.0 mL / min, held for 2 min, linearly increased to 10.0 mL / min within 8 min, linearly increased to 100.0 mL / min within 10 min, held for another 10 min, and then terminated. The chromatographic run time was 30 min; the injection port temperature was 80℃. IMS conditions: Tritium (3H) ionization source; migration tube length: 98 mm; electric field strength: 500 V / cm; migration tube temperature: 45 °C; drift gas: N2, flow rate: 150.0 mL / min. A mixed standard of six ketones was analyzed to establish calibration curves for retention time and retention index. The retention index of the target analyte was then calculated based on its retention time. Qualitative analysis of the target analyte was performed using the GC retention index database (NIST 2020) and the IMS migration time database built into the VOCal software for retrieval and comparison.

[0125] The effects of different treatments on volatile flavor compounds in soy milk are shown in Table 4. Figure 8 As shown. Figure 8 In the figure, Figure a is the distribution map of volatile flavor compounds; Figure b is the PLS-DA diagram; Figure c is the annular heat map; Figure d is the clustering characteristic map of beany flavor compounds; and Figure e is the clustering heat map of beany aroma compounds.

[0126] Table 4. Effects of different treatments on volatile flavor compounds in soy milk

[0127]

[0128]

[0129]

[0130] Depend on Figure 8As shown in Table 4, a total of 30 beany flavor compounds and 38 beany aroma compounds were detected in all soy milk samples, along with 12 other flavor components not listed. Aldehydes, ketones, alcohols, and esters are the main volatile flavor compounds in soy milk, playing a crucial role in the unique flavor of fermented soy milk (Figure a). The annular heatmap (Figure c) shows that the content of volatile flavor compounds in the JM1 group was significantly higher than that in the UN group. Furthermore, in the PLS-DA score (Figure b), the UN and JM1 groups were located in different quadrants, indicating a significant difference between the two sample groups. This suggests that JM1 fermentation significantly improved the flavor of the soy milk. In addition, the content of volatile flavor compounds in the ENJM1 group was significantly higher than that in the JM1 group, and the PLS-DA results showed a significant difference in their volatile flavor compound composition, indicating that complex enzymatic hydrolysis also has a certain impact on the overall flavor of fermented soy milk.

[0131] 5.2.1 Analysis of beany flavor in volatile flavor compounds

[0132] Lipoxygenase-catalyzed lipid oxidation is a major source of typical soybean off-flavors in fermented soy milk, and at high concentrations, it can negatively impact the quality of the fermented soy milk due to undesirable flavors. (Based on Table 4...) Figure 8 As shown in Figure d, among the 30 detected beany-smelling compounds, there were 17 aldehydes, 6 alcohols, 5 ketones, and 2 furans. Compared to unfermented soy milk (UN group), the contents of aldehydes such as hexanal, heptanal, pentanal, and benzaldehyde; alcohols such as 1-pentanol, 1-penten-3-ol, and 1-octen-3-ol; ketones such as 2-heptanone and 3-penten-2-one; and furans such as 2-pentylfuran and 2-ethylfuran were all significantly reduced. This indicates that fermentation can effectively reduce the beany smell of soy milk. Furthermore, compared to the JM1 group, the contents of beany-smelling substances in the APJM1 and ENJM1 groups were also significantly reduced. Compared with the JM1 group, the content of aldehydes such as E)-2-hexenal, hexanal-D, 1-penten-3-one-D, and propional in the PLJM1 group did not decrease, but instead showed an upward trend. This may be because lipase mainly converts triglycerides into free fatty acids, and free fatty acids are more easily oxidized than triglycerides, which leads to the beany smell increasing rather than decreasing.

[0133] 5.2.2 Analysis of Bean Flavor Compounds

[0134] The flavor formation of fermented soy milk is a dynamic process. Its unique flavor is not dominated by a single compound, but rather the result of a dynamic balance and synergistic effect among various volatile molecules such as acids, alcohols, esters, and ketones. (See Table 4 and...) Figure 8As shown in Figure e, a total of 38 aroma components were detected in the samples, including 9 esters, 12 alcohols, 6 aldehydes, 7 ketones, and 4 other aroma components. Furthermore, compared with unfermented soy milk (UN group), the concentration of aroma compounds was significantly increased in all soy milk samples (P < 0.05). Among them, 2,3-butanedione, 2,3-pentanedione, 2-butanone, ethanol, acetal, and ethyl acetate are key flavor compounds in fermented soy milk, possessing a pleasant aroma.

[0135] Esters typically possess pleasant floral or fruity aromas. Nine esters were detected in this invention, including methyl octanoate (fruity / orange aroma), ethyl heptanoate (pineapple aroma), and ethyl isovalerate (banana flavor), with significantly increased levels compared to unfermented soy milk (UN group). This may be related to the sugar fermentation and amino acid metabolism processes of lactic acid bacteria. Compared to the JM1 group, the PLJM1 and ENJM1 groups had higher concentrations of esters, which may be related to the enzymatic hydrolysis by lipases. These esters impart a fresh aroma to the fermented soy milk, similar to apples, bananas, oranges, and pineapples. They effectively mask the potentially pungent and irritating odors of aldehydes and acids, and significantly improve the smoothness and flavor complexity of the fermented soy milk.

[0136] Twelve alcohols were detected in this invention, among which ethanol, 3-methylbutanol, and 1-butanol showed a significant increase in content after fermentation, imparting characteristic flavors such as banana, floral, and alcoholic aromas to fermented soy milk. Furthermore, ketones are common and very important flavor compounds in fermented soy milk. Since most ketones have low odor thresholds, they have a significant impact on the flavor of food. In this invention, seven ketones were detected, including 3-hydroxy-2-butanone, 2,3-pentanedione, and 2,3-butanedione, all exhibiting buttery, creamy, and caramel flavors. 2,3-butanedione and 3-hydroxy-2-butanone have been identified as common flavoring substances in yogurt. The content of these ketones increased significantly after fermentation with Lactobacillus gasseri JM1. Compared to the JM1 group, the PLJM1, APJM1, and ENJM1 groups had higher ketone content, but the increase in the EN group was not significant. Aldehydes are also common substances in fermented soy milk. In this invention, six soy aroma compounds were identified as aldehydes, with 3-methylbutyraldehyde and acetal being the most abundant. 3-Methylbutyraldehyde has been reported in fermented yogurt, exhibiting a flavor similar to banana and grape, and is primarily produced by the degradation of leucine by leucine transferase and α-ketoacid decarboxylase in lactic acid bacteria. Acetal, on the other hand, emits a unique floral and fruity aroma, and its content was significantly higher in the ENJM1 group than in the JM1 group, suggesting it may play a key role in the aroma of fermented soy milk.

[0137] 5.3 Non-volatile flavor compounds

[0138] In this experiment, to investigate the effects of different treatments on non-volatile flavor compounds in soy milk, GC-IMS was used to identify non-volatile metabolites in the samples. The testing procedure was as follows: 0.2 ± 0.01 g of the soy milk sample was accurately weighed and placed in a centrifuge tube. Appropriate amounts of methanol and methyl tert-butyl ether solution were added, vortexed, and centrifuged at 12000 rpm for 15 min. The supernatant was filtered through a 0.22 μm filter membrane for LC-MS detection. An ACQUITY UPLC® HSS T3 (2.1 × 100 mm, 1.8 µm) Waters column was used at a flow rate of 0.3 mL / min, a column temperature of 40 °C, and an injection volume of 2 μL. The mobile phase consisted of 0.1% formic acid acetonitrile (B2) and 0.1% formic acid water (A2). The gradient elution program was: 0–1 min, 10% B2; 1–5 min, 10%–98% B2; 5–6.5 min, 98% B2; 6.5–6.6 min, 98%–10% B2; 6.6–8 min, 10% B2. A Thermo Q Exactive Focus mass spectrometer (ThermoFisher Scientific) was used, with an electrospray ionization (ESI) source at 3.50 kV, a sheath gas of 40 alb, and an auxiliary gas of 10 alb. The capillary temperature was 325 °C, and the resolution was 70,000. Unnecessary MS / MS information was removed using dynamic exclusion. Baseline filtering, peak identification, retention time correction, peak alignment, and characteristic peak identification were performed using Compound Discoverer 3.1.0 software. Relative quantification was based on normalized peak area. Subsequently, compounds were identified by analyzing retention time, isotope ratio, and MS / MS similarity against libraries from publicly available databases such as LipiDblast and MassBank. The effects of different treatments on non-volatile flavor compounds in soy milk are shown in Table 5. Figure 9 As shown. Figure 9 In the figure, Figure a is the PCA diagram; Figure b is the clustering heatmap; Figure c is the OPLS-DA diagram; and Figure d is the distribution map of non-volatile flavor compounds.

[0139] Table 5. Effects of different treatments on non-volatile flavor compounds in soy milk

[0140]

[0141]

[0142]

[0143] PCA is a multidimensional data analysis method for unsupervised pattern recognition, which can be used to simplify data and reveal the relationships between different samples. Figure 9 As shown in Figure a, PC1 and PC2 accounted for 60.7% and 23.7% respectively, with a cumulative contribution rate of 84.4%. The results indicate that the UN, JM1, and CC groups were clearly separated, suggesting significant differences in overall metabolites among the three groups. Furthermore, the JM1, PLJM1, APJM1, and ENJM1 groups were far apart and located in different quadrants, indicating that enzymatic hydrolysis significantly alters the metabolites in soy milk.

[0144] OPLS-DA is a regression-based supervised pattern recognition technique that identifies characteristic variables by distinguishing between predicted and orthogonal components. To more intuitively analyze the effects of different enzymatic treatments on non-volatile flavor compounds in fermented soybean milk, OPLS-DA analysis was performed on the identified non-volatile compounds. The results are as follows: Figure 9 As shown in Figure c, the identified non-volatile flavor compounds were screened using the OPLS-DA model based on VIP > 1 and P < 0.05, and their significantly different metabolites were identified. Table 5 shows the distribution of 76 significantly different metabolites identified. Figure 9 As shown in Figure d, there are 18 amino acids, peptides and analogs, 11 carbohydrates, 10 fatty acids and their derivatives, 9 carboxylic acids and their derivatives, 4 phosphate derivatives, 4 nucleotides and their derivatives, 3 vitamins, 3 soy isoflavones, 2 carbonyl compounds, 2 flavonoids, 2 benzoic acid and its derivatives, and 8 other compounds.

[0145] To more intuitively analyze the differences in non-volatile flavor compounds among different samples, cluster heatmap analysis was performed using the relative content of significantly different metabolites in different samples as indicators. Different color gradients were used to visualize the differences in the data. The results are as follows: Figure 9 As shown in Figure b, compared with the UN group, the content of most non-volatile flavor compounds showed an upward trend, with only 16 non-volatile flavor compounds, including L-histidine, genistein, 3-indoleacetic acid, D-glucose, mannoheptanose, and p-hydroxybenzaldehyde, showing a downward trend. The downward trend in glucosamine, D-glucose, and mannoheptanose may be due to two reasons: firstly, Lactobacillus gasseri JM1 produced metabolic enzymes that break down sugars in soy milk, thus breaking them down into monosaccharides; secondly, it may be related to the growth of the inoculated Lactobacillus itself.

[0146] Furthermore, the biosynthesis and metabolism of amino acids also have a significant impact on the flavor of soy milk. From... Figure 9As shown in Figure b, compared with the unfermented group (UN group), the JM1 group showed a slight increase in the contents of L-serine, 2-methylserine, alanine, glutamic acid, and aspartic acid, while the APJM1 and ENJM1 groups showed a significant increase in the contents of these amino acids. This indicates that Lactobacillus gasseri JM1 fermentation can effectively promote the generation of key flavor amino acids in soy milk, thereby affecting the flavor of soy milk. Furthermore, the combined enzymatic hydrolysis pretreatment with alkaline protease and lipase efficiently hydrolyzes the complex macromolecular proteins in soy milk into smaller peptides and amino acids. This increases the content of flavor amino acids in soy milk and provides a rich nitrogen source for the growth and metabolism of the strain. The synergistic effect of these two processes greatly promotes the accumulation of key sweet and umami amino acids, thus improving the flavor of soy milk.

[0147] Furthermore, carbohydrates also have a profound impact on the formation of soy milk flavor, serving not only as a direct source of sweetness but also as the foundation of the entire flavor system. Metabolites such as D-glucose, D-fructose, D-galactose, and D-glucose-6-phosphate were all identified in the samples. The dynamic changes of these substances directly drive the transformation of fermented soy milk from a simple soy flavor to a complex flavor system with a balanced sweet and sour taste and a rich milky aroma. Various saturated fatty acids (stearic acid, 2-oxostearic acid, 16-hydroxypalmitic acid, behenic acid) and unsaturated fatty acids (linoleic acid, arachidonic acid, erucic acid, γ-linolenic acid) were identified in the fermented samples, and the content of these substances was significantly higher in the PLJM1 and ENJM1 groups than in the JM1 group. After fermentation, the contents of vitamin B5, vitamin B2, and vitamin B1 also increased significantly, indicating that the fermentation of Lactobacillus gasseri JM1 not only significantly improved the nutritional components of soy milk but also converted proteins, lipids, and carbohydrates in soybeans into flavor precursors. The combined enzymatic hydrolysis of lipase and protease not only generates a large number of flavor precursors, but also provides the optimal nutritional substrate for the growth and metabolism of Lactobacillus gasseri JM1. This not only enhances the complexity of soy milk flavor, but also achieves nutritional fortification, thus improving the quality of soy milk in both flavor and nutrition.

[0148] In summary, the combined enzymatic hydrolysis and microbial fermentation strategy provided by this invention takes the beany and beany aroma of soy milk as a comprehensive goal. By organically combining enzyme treatment and microbial fermentation, it can not only remove the beany aroma but also effectively enhance the beany aroma. Therefore, it can provide a theoretical basis for the precise control of soy milk flavor and provide technical support for the preparation of high-quality soy milk.

Claims

1. A method for enhancing the aroma of soybean milk and reducing its beany odor through compound enzymatic hydrolysis combined with bacterial fermentation, characterized in that, Includes the following steps: (1) Soak soybeans, then mix the soaked soybeans with water, grind and cook them to obtain raw soybean milk; (2) Add porcine pancreatic lipase to the original soy milk for enzymatic hydrolysis, then add alkaline protease for enzymatic hydrolysis, and then inactivate the enzyme to obtain enzymatically hydrolyzed soy milk; (3) The enzymatically hydrolyzed soy milk is sterilized and then inoculated with Lactobacillus gasseri for fermentation to obtain the fermented soy milk product.

2. The method for enhancing the soybean aroma and reducing the beany odor of soybean milk through compound enzymatic hydrolysis combined with bacterial fermentation according to claim 1, characterized in that, In step (1), the soaking temperature is 2~8℃, the time is 8~20h, and the mass-volume ratio of soybeans to water during the soaking process is 1:8~10.

3. The method for enhancing the soybean aroma and reducing the beany odor of soybean milk through compound enzymatic hydrolysis combined with bacterial fermentation according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of the soaked soybeans to the water is 1:7~10.

4. The method for enhancing the soybean aroma and reducing the beany smell of soybean milk through compound enzymatic hydrolysis combined with bacterial fermentation according to claim 1, characterized in that, In step (1), the pulping and cooking time is 10~50 min.

5. The method for enhancing the soybean aroma and reducing the beany odor of soybean milk through compound enzymatic hydrolysis combined with bacterial fermentation according to any one of claims 1 to 4, characterized in that, In step (2), the total amount of porcine pancreatic lipase and alkaline protease added is 0.3% to 0.6% of the original soybean milk mass; the mass ratio of alkaline protease to porcine pancreatic lipase is 1:8 to 12; the enzymatic hydrolysis temperature is 50 to 60°C and the pH is 7.2 to 7.8; the enzymatic hydrolysis time with porcine pancreatic lipase is 3.5 to 4.5 hours and the enzymatic hydrolysis time with alkaline protease is 0.4 to 0.6 hours.

6. The method for enhancing the soybean aroma and reducing the beany odor of soybean milk through compound enzymatic hydrolysis combined with bacterial fermentation according to any one of claims 1 to 4, characterized in that, In step (2), the enzyme inactivation temperature is 90~100℃ and the time is 5~20min.

7. The method for enhancing the soybean aroma and reducing the beany odor of soybean milk through compound enzymatic hydrolysis combined with bacterial fermentation according to any one of claims 1 to 4, characterized in that, In step (3), the sterilization process is carried out at a temperature of 110-120°C for 10-20 minutes.

8. The method for enhancing the soybean aroma and reducing the beany odor of soybean milk through compound enzymatic hydrolysis combined with bacterial fermentation according to any one of claims 1 to 4, characterized in that, In step (3), the Lactobacillus gasseri is Lactobacillus gasseri JM1; the inoculation amount of Lactobacillus gasseri is 3%~5% v / v; and the effective bacterial concentration in the enzymatically hydrolyzed soy milk after inoculation is 3×10⁻⁶. 6 ~8×10 6 CFU / g.

9. The method for enhancing the soybean aroma and reducing the beany odor of soybean milk through compound enzymatic hydrolysis combined with bacterial fermentation according to any one of claims 1 to 4, characterized in that, In step (3), the fermentation treatment is carried out at a temperature of 35~39℃ for 6~10h.

10. A fermented soybean milk product prepared by the method described in any one of claims 1 to 9.

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