Fermented whole soybean milk rich in polyphenols and preparation method thereof

Abstract

The present invention belongs to the technical field of fermented foods, and discloses a method for preparing fermented soy milk rich in polyphenols. Lactobacillus delbrueckii MT772183 is inoculated into whole bean soy milk for fermentation to obtain fermented soy milk rich in polyphenols. The present invention utilizes Lactobacillus delbrueckii MT772183 with β-glucosidase activity to ferment whole bean soy milk, and the presence of bean dregs effectively stimulates the ability of Lactobacillus delbrueckii MT772183 to produce β-glucosidase. During the whole bean fermentation process, a large amount of insoluble polyphenols in the bean dregs are dissolved, and the free phenols, bound phenols, total phenols, free flavonoids, bound flavonoids, total flavonoids and soy isoflavone aglycones therein are significantly higher than those in de-slag soy milk and semi-de-slag soy milk. At the same time, whole bean fermentation increases the thickness (viscosity) of the fermented soy milk; provides a rich mouthfeel; the fermented product contains prebiotics such as dietary fiber, oligosaccharides and polyphenols, and long-term use can regulate intestinal flora, thereby improving the bioabsorption rate and bioavailability of polyphenols.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermented foods, and more specifically relates to a polyphenol-rich fermented whole soybean milk and a preparation method thereof. Background Art

[0002] Soy milk has long been loved by Asians. It is a good source of protein for ordinary consumers or vegetarians, and a milk substitute for people with lactose intolerance. Soy milk contains a variety of bioactive substances, including isoflavones, polyphenols, phytates, saponins, lecithin, plant steroids and tocopherols. Among them, polyphenols and isoflavones mostly exist in natural soybeans in a bound form that is not easily absorbed by the human body, and their bioavailability is limited. If the bound polyphenols are converted into free form, the bioavailability of soy polyphenols can be further improved. At present, researchers have optimized the proportion of free polyphenols in de-slag soy milk from the perspectives of pulping process, fermentation process, enzymatic hydrolysis, etc., and have increased the content of free phenols in de-slag soy milk to a relatively good level, but there is limited room for breakthroughs.

[0003] It's worth noting that most soy product processing companies use a peeling and dregs removal process to prepare de-dreg soymilk, resulting in a significant amount of dregs being discarded or disposed of at low value. Dregs are rich in nutrients, with dietary fiber comprising approximately 55% of soybean dry matter and soy isoflavones accounting for 12-30% of the dry matter. Dregs contain a significant amount of phenolic compounds, which are often covalently or non-covalently bound together. When environmental conditions change or fermentation occurs, polyphenols may be released from the matrix of dietary fiber. Releasing insoluble phenolic compounds in dregs could increase the phenolic content of soymilk.

[0004] Fermented soy milk refers to a fermented protein beverage made by inoculating lactic acid bacteria into soy milk and culturing it at a suitable temperature for a period of time. Soy milk is a good culture medium for lactic acid bacteria, and its rich oligosaccharides can promote the growth of lactic acid bacteria. Lactic acid bacteria fermentation causes the fermented soy milk to contain microbial hydrolases (such as α-galactosidase, β-glucosidase, feruloyl esterase and protease, etc.), which in turn increases the nutritional value of soy milk, improves the taste of soy milk and optimizes the physical and chemical properties of soy milk. Studies have shown that α-galactosidase produced by lactic acid bacteria can reduce intestinal flatulence, and β-glucosidase can convert soy isoflavone glycosides into more biologically active aglycone forms. Therefore, lactic acid bacteria fermentation is an effective method to improve the nutritional value and sensory properties of soy milk.

[0005] Whole-soybean soymilk is made by beating soybeans without removing the slag. It retains nearly all of the soybean's nutrients, and contains higher levels of prebiotics such as polyphenols, soy isoflavones, and dietary fiber than de-slag soymilk. With the increasing advantages of whole-soybean and fermented soymilk, some researchers have combined the two to explore the nutritional and sensory properties of fermented whole-soybean soymilk. However, current research on fermented whole-soybean soymilk has largely focused on sensory, texture, and functional properties, with fewer reports on bioactive substances (such as polyphenols, polysaccharides, and bioactive peptides).

[0006] Polyphenols are hydroxyphenolic compounds with diverse biological activities, including antioxidant, anticancer, and cardiovascular disease prevention. Polyphenols have diverse structures, including flavonoids, phenolic acids, lignans, coumarins, and other low-molecular-weight components. Based on their solubility in organic solvents, polyphenols can be divided into non-extractable and extractable polyphenols. Non-extractable phenols are also called bound phenolic compounds (BPCs). Extractable polyphenols can be divided into free phenolic compounds (FPCs) and conjugated phenolic compounds (CPCs) based on their partitioning in ethyl acetate-water or ether-water systems. Soy isoflavones are the most important phenolic compounds in soybeans, with 12 isomers, which can be divided into bound glycosides and free aglycones. Free aglycones include genistein, daidzein, and glycitein. Bound glycosides include genistin, daidzin, glycitein, and their corresponding acetyl and malonyl derivatives.

[0007] Soybeans contain a large amount of polyphenol compounds, but most of them exist in a bound form that is not easily absorbed by the human body. Increasing the content of free phenols in food can improve the bioavailability of soy polyphenols. Studies have shown that lactic acid bacteria are widely used in the production of fermented soy products. Lactic acid bacteria that produce β-glucosidase can convert glycoside polyphenols into free forms. Whole soybean milk is rich in "prebiotics" such as polyphenols, dietary fiber, and oligosaccharides, but soybean dregs contain a large amount of insoluble phenolic substances. Using β-glucosidase-producing lactic acid bacteria to ferment whole soybean milk may promote the dissolution of soybean dregs polyphenols, thereby achieving the conversion and utilization of high-polyphenol soybean milk. Chinese patent CN112574905B discloses a strain of Lactobacillus delbrueckii MT772183 that has β-glucosidase activity and can convert soybean isoflavone glycosides. Its taxonomic name is Lactobacillus delbrueckii. Lactobacillus delbrueckii MT772183 was deposited with the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, under the accession number M 2020332, on July 20, 2020. Lactobacillus delbrueckii MT772183 can be used in soymilk fermentation to remove the beany odor, increase the content of soy isoflavone aglycones in the product, and enhance the antioxidant capacity of the soymilk product.

[0008] In summary, by fermenting soy milk with Lactobacillus delbrueckii MT772183 that produces β-glucosidase, it is expected to obtain a fermented soy milk rich in polyphenols. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the above-mentioned problems existing in the prior art. Firstly, a new application of Lactobacillus delbrueckii MT772183 is provided.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] The invention discloses an application of Lactobacillus delbrueckii MT772183 in promoting the dissolution of insoluble polyphenols in soy milk and soy dregs and the release of free phenols from conjugated phenols, wherein the insoluble polyphenols are conjugated phenols and conjugated flavonoids, and the conjugated phenols include conjugated polyphenols, conjugated flavonoids and soy isoflavone glycosides.

[0012] Firstly, under the premise of considering the comprehensive utilization of bean dregs, the present invention finds that the phenolic substance content of whole bean soy milk is significantly higher than that of semi-de-dregs soy milk and de-dregs soy milk, and compared with whole bean soy milk, the total soy isoflavone recovery rate of semi-de-dregs soy milk and de-dregs soy milk is significantly reduced, and decreases with the increase of the de-dregs degree.

[0013] Studies have reported that β-glucosidase-producing lactic acid bacteria can convert isoflavone glycosides into isoflavone aglycones, but there has been no systematic study on whether they can release bound polyphenols and bound flavonoids, and there has been no report on their ability to release bound phenols from soybeans.

[0014] Therefore, the applicant considered the feasibility of fermenting whole soybean soy milk. How to convert a certain amount of insoluble polyphenols in the dregs into soluble polyphenols that can be absorbed and utilized by humans became a new problem. In the previous research, the applicant discovered a Lactobacillus delbrueckii MT772183 that can produce β-glucosidase, which can be used to ferment de-dregs soy milk, remove the beany smell of soy milk, increase the content of soy isoflavone aglycones in the product, and improve the antioxidant capacity of the soy milk product. However, there has been no systematic study on the fermentation performance of whole soybean soy milk containing dregs.

[0015] Based on the fact that okara still contains a large amount of polyphenols and isoflavones, particularly bound polyphenols, this invention proposes the hypothesis that fermenting whole soybean milk with β-glucosidase-producing lactic acid bacteria can release the bound polyphenols in the okara and produce soymilk with a high polyphenol content. The research confirmed this hypothesis and unexpectedly found that the presence of okara can activate the β-glucosidase production capacity of lactic acid bacteria and improve the tolerance of the produced β-glucosidase in fermented soymilk.

[0016] Therefore, preferably, in the above-mentioned protected application of the present invention, whole soybean milk is fermented using Lactobacillus delbrueckii MT772183.

[0017] More preferably, the fermentation process comprises the following steps:

[0018] S1. Beat the soybeans with a water ratio of 1:6-12, sterilize and cool, and then inoculate at an inoculum level of 2-8% v / v;

[0019] S2. Ferment at 37-42°C until pH = 4.5. After fermentation, homogenize.

[0020] The present invention also provides a method for preparing a fermented whole soybean milk product, which comprises inoculating whole soybean milk with Lactobacillus delbrueckii MT772183 for fermentation. The soy milk product comprises a fermented whole soybean milk beverage and fermented soybean powder.

[0021] Preferably, when the fermented whole soybean milk product is a fermented whole soybean milk beverage, the method comprises the following steps:

[0022] S1. Beat the soybeans with a water ratio of 1:6-12, sterilize and cool them for later use;

[0023] S2, pick a single colony of Lactobacillus delbrueckii MT772183 for culture activation, and adjust the colony density to 10 9 CFU / mL;

[0024] S3. Ferment at 37-42°C until pH = 4.5. After fermentation is complete, homogenize to obtain a fermented whole soybean milk beverage with both soluble polyphenol content and consistency.

[0025] Whole bean fermentation can increase the thickness (viscosity) of fermented soy milk. Studies have shown that after fermentation, the viscosity of whole bean milk increases by 402.40±0.49 mPa·s, while the viscosity increases of semi-cleaned soy milk and cleaned soy milk are only 310.28±1.94 mPa·s and 235±1.68 mPa·s respectively.

[0026] Preferably, when the fermented whole soybean milk product is fermented soybean powder, the method comprises the following steps:

[0027] S1. Beat the soybeans with a water ratio of 1:6-12, sterilize and cool them for later use;

[0028] S2, pick a single colony of Lactobacillus delbrueckii MT772183 for culture activation, and adjust the colony density to 10 9 CFU / mL;

[0029] S3. Ferment at 37-42° C. until pH reaches 4.5. After fermentation is complete, homogenize to obtain a fermented whole soybean milk beverage having both soluble polyphenol content and consistency;

[0030] S4. Drying the whole bean soy milk beverage to obtain fermented soy powder.

[0031] More preferably, in the above method, the drying is selected from one or more of air drying, microwave drying, vacuum freeze drying, and spray drying.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention discloses a method for preparing fermented soymilk rich in polyphenols. Soybeans are soaked and pulped, and then whole soymilk is prepared without filtering. Lactobacillus delbrueckii MT772183 is inoculated into the whole soymilk for fermentation to obtain fermented soymilk rich in polyphenols. The present invention utilizes Lactobacillus delbrueckii MT772183 having β-glucosidase activity to ferment whole soymilk, and the presence of soybean dregs effectively stimulates the ability of Lactobacillus delbrueckii MT772183 to produce β-glucosidase. During the whole soymilk fermentation process, a large amount of insoluble polyphenols in the soybean dregs are dissolved, and the contents of free phenols, bound phenols, total phenols, free flavonoids (Bound flavonoid compounds, BFC), bound flavonoids (Bound flavonoid compounds, BFC), total flavonoids (Total flavonoid compounds, TFC) and soybean isoflavone aglycones are significantly higher than those in de-slag soybean milk and semi-de-slag soybean milk. At the same time, whole bean fermentation increases the thickness (viscosity) of fermented soy milk and provides a rich taste. The fermented product contains prebiotics such as dietary fiber, oligosaccharides and polyphenols. Long-term use can regulate intestinal flora, thereby improving the bioabsorption rate and bioavailability of polyphenols. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The soymilk fermentation characteristics of lactic acid bacteria were analyzed, among which no microorganisms were detected in WSM-LA, and no β-glucosidase activity was detected in WSM-b5 and WSM-LA;

[0035] Figure 2 The polyphenol conversion of soymilk with different dregs content fermented by lactic acid bacteria.

[0036] Figure 3 The flavonoid conversion of soy milk with different dregs content fermented by lactic acid bacteria. DETAILED DESCRIPTION

[0037] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0038] The Lactobacillus delbrueckii MT772183 used in the examples was deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 2020332 and a deposit date of July 20, 2020.

[0039] Example 1 Effect of Deslagging Process on Soymilk Polyphenol Content

[0040] 1. Sample preparation process

[0041] Soybeans → soak for 12 hours → wash → pulp (soybean-water ratio is 1:8) → filter or not filter.

[0042] Among them, whole soybean milk (WSM) was directly prepared without filtering process, semi-slag soybean milk (MSM) was prepared by passing through a 40-mesh sieve, and slag soybean milk (LSM) was prepared by passing through a 300-mesh sieve.

[0043] 2. Extraction and determination of polyphenols

[0044] Extraction of free phenol: Accurately weigh 0.500 g of freeze-dried sample and add 20 mL of 70% acetone solution (acetone:water:glacial acetic acid = 70:29.5:0.5, v / v / v). Incubate at 4°C for 2 h with constant shaking. Centrifuge at 4,000 rpm for 15 min at 4°C and collect the supernatant. Repeat the extraction twice. Combine the three supernatants and evaporate the organic solvent in vacuo at 35°C. Mix the remaining aqueous solution with ethyl acetate in a 1:1 ratio (v / v). Collect the ethyl acetate phase and repeat the extraction three times. Combine the ethyl acetate extracts and spin-dry the organic solvent in vacuo at 35°C. Collect the residual solution and dilute to 5 mL with 50% methanol to obtain the free phenol extract. Store in the dark at -20°C until assayed.

[0045] Extraction of conjugated phenols: Add an equal volume of 2 M NaOH solution to the phenol-free extract. After sealing with nitrogen, hydrolyze at room temperature for 6 h. Adjust the pH to 6 with 6 mol / L HCl solution and mix with ethyl acetate in a 1:1 ratio (v / v). Repeat the extraction three times. Combine the ethyl acetate phases, spin-dry the ethyl acetate at 35°C, collect the residual solution, and dilute to 5 mL with 50% methanol to obtain the conjugated phenol extract. Store the extract at -20°C in the dark until assayed.

[0046] Extraction of bound phenol: Add 10mL NaOH (4M) to the residue after free phenol extraction, purge with nitrogen at 35°C for 20min, and store at 4°C for 2h with continuous shaking. Neutralize the alkali solution to pH = 2 with HCl (6M) and store at 4°C for 2h. Add 20mL 70% acetone solution and store at 4°C for 2h with continuous shaking. Centrifuge (4,000r / min, 15min, 4°C) and collect the supernatant. Repeat the extraction of the residue twice, combine the three extraction supernatants, spin dry the organic reagent at 35°C, collect the residual liquid and dilute to 50mL with 50% methanol to obtain the bound phenol extract. Store it at -20°C in the dark until tested.

[0047] Determination of Polyphenols: Before analysis, filter the polyphenol extract through a 0.45 μm organic filter. Take 400 μL of the extract, add 2 mL of 10% Folin's phenol reagent, shake well, and add 1.6 mL of 7.5% Na2CO3 solution within 3-8 minutes. Incubate in the dark at room temperature for 60 minutes. Measure absorbance at 765 nm. Construct a calibration curve using gallic acid as the standard. Express the results as milligrams of gallic acid equivalent per 100 mL of sample (mg / 100 mL).

[0048] The relationship between phenolic compounds in WSM, MSM, and LSM and soybean polyphenols was expressed using the total phenol recovery rate. The recovery rate was calculated using Equation 1:

[0049]

[0050] The ratio of non-extractable polyphenols to extractable polyphenols (N / E) is used to represent the distribution of different types of phenolic compounds in the sample. The N / E ratio is calculated as shown in Equation 2:

[0051]

[0052] The growth rate is used to represent the change in polyphenol content in soymilk during lactic acid bacteria fermentation. The growth rate is calculated as shown in Formula 3:

[0053]

[0054] Table 1 Phenolic content of soybean flour and soybean milk

[0055]

[0056]

[0057] Note: ① The unit of phenolic substance content of soybean flour is mg / 100g; ② The unit of phenolic substance content of soy milk is mg / 100mL.

[0058] Using a combination of acetone and ethyl acetate methods, we separated extractable polyphenols (free and conjugated phenols) from non-extractable polyphenols (bound phenols) in soy flour and soy milk. The results (Table 1) showed that the polyphenol content in soy flour was primarily composed of non-extractable polyphenols (853.35 ± 14.56 mg / 100 g), with a ratio of non-extractable polyphenols to extractable polyphenols (the sum of free and conjugated phenols, i.e., 74.82 ± 3.27 mg / 100 g) greater than 10. Whole soybean milk, semi-debonded soy milk, and debonded soy milk also contained primarily bound phenols, with similar ratios to extractable polyphenols (10.60 ± 0.31, 10.32 ± 0.28, and 10.49 ± 0.32, respectively). Furthermore, the phenolic content in whole soybean milk was significantly higher than that in semi-debonded and debonded soy milks (p < 0.05).

[0059] 3. Determination of soybean isoflavones

[0060] The quantitative method for soy isoflavones was based on the national standard GBT26625-2011. Accurately weigh 0.500 g of freeze-dried sample and dissolve it in approximately 25 mL of 80% methanol. After sonication at room temperature for 20 minutes, the volume was adjusted to 25 mL with 80% methanol and centrifuged (8,000 rpm, 15 minutes, 4°C). The supernatant was collected, filtered through a 0.45 μm organic filter, and analyzed by high-performance liquid chromatography (HPLC). The mobile phase consisted of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile, and the sample was gradient eluted using a C18 column. The concentration of 0.1% acetic acid in acetonitrile was varied as follows: 10% to 30% from 0 to 12.5 minutes; 30% to 40% from 12.5 to 17.5 minutes; 40% to 100% from 17.5 to 18.5 minutes; 100% from 18.5 to 21 minutes; 100% from 100% to 10% from 21 to 22.5 minutes; and 10% from 22.5 to 26 minutes. The column temperature was maintained at 40°C, the flow rate was 1.0 mL / min, the injection volume was 20 μL, and the detection wavelength was 260 nm. Standard curves were constructed using standards for daidzein, glycitein, genistin, daidzein, glycitein, and genistein. Results are expressed as milligrams of standard per 100 mL of sample (mg / 100 mL).

[0061] The ratio of glycosides to total soy isoflavones (G / T) is used to represent the distribution of different types of phenolic compounds in the sample. The calculation method of G / T is shown in Equation 4:

[0062]

[0063] The growth rate is used to represent the change in the content of soy isoflavones during the fermentation of soy milk by lactic acid bacteria. The calculation method of the growth rate is shown in

[0064] Formula 5:

[0065]

[0066] Table 2 Soy isoflavone content of soy flour and soy milk

[0067]

[0068]

[0069] Note: ① The unit of soy isoflavone content in soy flour is mg / 100g; ② The unit of soy isoflavone content in soy milk is mg / 100mL.

[0070] Soy isoflavones are known to consist of two forms: glycosides and aglycones. Glycosides predominate in soy flour isoflavones (Table 2). Although heat treatment can lead to different forms of soy isoflavone conversion, reducing the proportion of isoflavone glycosides in total soy isoflavones, glycosides remain the predominant isoflavone content in the three soymilk types. As the okara content increases, the proportion of glycosides in the soymilk decreases and the composition deviates further from that of the original soy flour. Compared with whole soymilk, the recovery of total soy isoflavones in both semi-de ...

[0071] It can be seen from this that using whole soybean milk for fermentation can retain the nutrients in the soy milk to the greatest extent, thereby providing more nutrients for the human body to absorb and utilize.

[0072] Example 2 Fermentation characteristics of soy milk

[0073] 1. Sample preparation

[0074] Process flow: Soybeans → Soak for 12 hours → Wash → Beat (soybean to water ratio 1:8) → Filter or not filter → Sterilize (121°C, 20 minutes) → Cool → Inoculate (4%, v / v) → Ferment (37°C, 24 hours) → Homogenize → Assay. Whole soybean milk (WSM) is prepared directly without filtration, semi-sludged soymilk (MSM) is prepared by sieving through a 40-mesh sieve, and sludged soymilk (LSM) is prepared by sieving through a 300-mesh sieve.

[0075] Strain activation: After the strain is fully activated, a single colony is picked and inoculated into MRS liquid medium, cultured anaerobically at 37°C for 6-8 hours, centrifuged (8,000 rpm, 4°C, 5 minutes), washed twice with sterile water, and resuspended in sterile water, adjusting OD600≈0.9 (colony density is about 10 9 CFU / mL).

[0076] Inoculation: Lactobacillus delbrueckii MT772183 was inoculated into WSM, MSM, and LSM to obtain WSM-b8, MSM-b8, and LSM-b8. The cultures were fermented at 37°C for 24 hours, and samples were taken every 3 hours for measurement.

[0077] 2. Determination of pH, acidity, and viable lactic acid bacteria count

[0078] Measure the pH of the sample using a pH meter. Determine the acidity of the sample using sodium hydroxide titration, expressed as lactic acid (°T). Dissolve each sample (1 mL) in 9 mL of sterile water. Spread the diluted suspension onto MRS solid medium and incubate anaerobically at 37°C for 48 hours before counting the colonies.

[0079] 3. Determination of β-glucosidase activity

[0080] To 10 mL of fermented soy milk, 10 mL of 0.5 M sodium phosphate buffer (0.5 M, pH 6.5) was added. The mixture was shaken at room temperature (200 rpm for 30 min) and centrifuged (8,000 g for 10 min at 4°C). The precipitate was washed twice with sodium phosphate buffer (0.5 M, pH 6.5) and resuspended in an equal volume of sodium phosphate buffer (0.5 M, pH 6.5). The mixture was mixed with 1 mL of lysozyme (3 mg / mL, 20,000 U / mg) and incubated in a 37°C water bath for 30 min with continuous shaking (200 rpm). The cells were then disrupted by low-temperature sonication in a cell disruptor (2 s, 3 s rest, 45 W for 15 min) to obtain a disrupted solution. The supernatant was then centrifuged (8,000 g at 4°C for 10 min). The crude enzyme solution was used for further analysis.

[0081] To 100 μL of cell-free extract, 900 μL of sodium phosphate buffer (0.5 M, pH 6.5) was added. The solution was preheated in a 37°C waterbath for 10 minutes. Then, 1 mL of 5 mM p-NPG (preheated) was added and incubated at 37°C for 30 minutes. The reaction was immediately terminated by the addition of 1 mL of 1 M sodium carbonate (4°C). The solution was incubated at room temperature for 5 minutes, and the absorbance was measured at 400 nm. A crude enzyme solution inactivated at 100°C for 30 minutes and sodium phosphate buffer were used as controls. A standard curve was constructed using p-NP as the standard. Results were expressed as the enzymatic capacity to hydrolyze p-NPG to produce 1 μmol of p-NPG per minute (U / mL) in 1 mL of fermented soy milk at 37°C, pH 6.5.

[0082] Depend on Figure 1 It can be seen that when fermented with Lactobacillus delbrueckii MT772183, there was no significant difference in the pH value of WSM-b8, MSM-b8 and LSM-b8 ( Figure 1 A). However, under high acidity, the viable bacterial count of WSM-b8 decreased more slowly than that of MSM-b8 and LSM-b8, with LSM-b8 showing the fastest decline. This may be related to the fiber content, viscosity, and rheological properties of soymilk. WSM, with its higher okara fiber content and viscosity, provides a favorable environment for the survival of Lactobacillus delbrueckii MT772183.

[0083] During the fermentation of WSM-b8, β-glucosidase activity showed a trend of first increasing and then decreasing, which was related to the changing trend of the viable cell count of Lactobacillus delbrueckii MT772183. It is worth noting that after 6 hours of fermentation, the β-glucosidase activity of WSM-b8 (59.07±1.48U / mL) was 1.18 times that of MSM-b8 (49.95±1.90U / mL) and 1.62 times that of LSM-b8 (36.43±1.38U / mL). Furthermore, the β-glucosidase activity of WSM-b8 was retained the longest, showing a downward trend only after 18 hours of fermentation, followed by MSM-b8, and the shortest for LSM-b8. Therefore, the presence of okara is more conducive to the production and maintenance of β-glucosidase activity.

[0084] Example 3 Analysis of the conversion of phenolic substances in soymilk with different okara contents fermented by β-glucosidase-producing lactic acid bacteria

[0085] 1. Sample preparation

[0086] Process flow: Soybeans → Soak for 12 hours → Wash → Beat (soybean to water ratio 1:8) → Filter or not filter → Sterilize (121°C, 20 minutes) → Cool → Inoculate (4%, v / v) → Ferment (37°C, 24 hours) → Homogenize → Assay. Whole soybean milk (WSM) is prepared directly without filtration, semi-sludged soymilk (MSM) is prepared by sieving through a 40-mesh sieve, and sludged soymilk (LSM) is prepared by sieving through a 300-mesh sieve.

[0087] Strain activation: After the strain is fully activated, a single colony is picked and inoculated into MRS liquid medium, cultured anaerobically at 37°C for 6-8 hours, centrifuged (8,000 rpm, 4°C, 5 minutes), washed twice with sterile water, and resuspended in sterile water, adjusting OD600≈0.9 (colony density is about 10 9 CFU / mL).

[0088] Inoculation: Lactobacillus delbrueckii MT772183 was inoculated into WSM, MSM, and LSM to obtain WSM-b8, MSM-b8, and LSM-b8. The cultures were fermented at 37°C for 24 hours, and samples were taken every 3 hours for measurement.

[0089] 2. Extraction and determination of polyphenols

[0090] Extraction of free phenol: Accurately weigh 0.500 g of freeze-dried sample and add 20 mL of 70% acetone solution (acetone:water:glacial acetic acid = 70:29.5:0.5, v / v / v). Incubate at 4°C for 2 h with constant shaking. Centrifuge at 4,000 rpm for 15 min at 4°C and collect the supernatant. Repeat the extraction twice. Combine the three supernatants and evaporate the organic solvent in vacuo at 35°C. Mix the remaining aqueous solution with ethyl acetate in a 1:1 ratio (v / v). Collect the ethyl acetate phase and repeat the extraction three times. Combine the ethyl acetate extracts and spin-dry the organic solvent in vacuo at 35°C. Collect the residual solution and dilute to 5 mL with 50% methanol to obtain the free phenol extract. Store in the dark at -20°C until assayed.

[0091] Extraction of conjugated phenols: Add an equal volume of 2 M NaOH solution to the phenol-free extract. After sealing with nitrogen, hydrolyze at room temperature for 6 h. Adjust the pH to 6 with 6 mol / L HCl solution and mix with ethyl acetate in a 1:1 ratio (v / v). Repeat the extraction three times. Combine the ethyl acetate phases, spin-dry the ethyl acetate at 35°C, collect the residual solution, and dilute to 5 mL with 50% methanol to obtain the conjugated phenol extract. Store the extract at -20°C in the dark until assayed.

[0092] Extraction of bound phenol: Add 10mL NaOH (4M) to the residue after free phenol extraction, purge with nitrogen at 35°C for 20min, and store at 4°C for 2h with continuous shaking. Neutralize the alkali solution to pH = 2 with HCl (6M) and store at 4°C for 2h. Add 20mL 70% acetone solution and store at 4°C for 2h with continuous shaking. Centrifuge (4,000r / min, 15min, 4°C) and collect the supernatant. Repeat the extraction of the residue twice, combine the three extraction supernatants, spin dry the organic reagent at 35°C, collect the residual liquid and dilute to 50mL with 50% methanol to obtain the bound phenol extract. Store it at -20°C in the dark until tested.

[0093] Determination of Polyphenols: Before analysis, filter the polyphenol extract through a 0.45 μm organic filter. Take 400 μL of the extract, add 2 mL of 10% Folin-phenol reagent, shake well, and add 1.6 mL of 7.5% Na₂CO₃ solution within 3-8 minutes. Incubate in the dark at room temperature for 60 minutes. Measure the absorbance at 765 nm. Construct a standard curve using gallic acid as the standard. Express the results as milligrams of gallic acid equivalent per 100 mL of sample (mg / 100 mL).

[0094] Mix 1 mL of the polyphenol extract with 0.30 mL of a 5% (w / v) NaNO₂ solution. After reacting for 5 minutes, add 0.50 mL of a 2% (w / v) AlCl₃·6H₂O solution. After reacting at room temperature for 6 minutes, add 0.50 mL of a 1 mol / L sodium hydroxide solution to the test tube and react at room temperature for another 10 minutes. Measure the absorbance at 510 nm. A standard curve was constructed using rutin as the standard. Results are expressed as milligrams of rutin equivalent per 100 mL of soy milk (mg / 100 mL).

[0095] The growth rate is used to represent the change in polyphenol or flavonoid content in soy milk during lactic acid bacteria fermentation. The growth rate is calculated as shown in Equation 3.

[0096] Depend on Figure 2 A shows that the free phenol content of soy milk began to show significant differences around 6h of fermentation (p<0.05). The FPC growth rate of WSM-b8 was significantly higher than that of the other two groups, while that of LSM-b8 was the lowest. This trend continued until the peak of free phenol growth. Correspondingly, CPC ( Figure 2 B) showed the same downward trend, combined with phenol ( Figure 2 C) also showed a corresponding upward trend. Similarly, the flavonoid growth rate of WSM-b8 showed a similar trend ( Figure 3 Therefore, fermentation of soy milk with high dregs content by β-glucosidase-producing lactic acid bacteria is more conducive to the conversion and accumulation of soybean polyphenols.

[0097] Depend on Figure 1 C shows that the presence of okara enhances the β-glucosidase production capacity of Lactobacillus delbrueckii MT772183 and prolongs the retention of β-glucosidase activity. β-glucosidase activity is significantly correlated with changes in soy polyphenols. This suggests that higher okara content is associated with higher β-glucosidase activity, which is more conducive to the conversion and accumulation of soy polyphenols.

[0098] 3. Determination of soybean isoflavones

[0099] The quantitative method for soy isoflavones was based on the national standard GBT26625-2011. Accurately weigh 0.500 g of freeze-dried sample and dissolve it in approximately 25 mL of 80% methanol. After sonication at room temperature for 20 minutes, the volume was adjusted to 25 mL with 80% methanol and centrifuged (8,000 rpm, 15 minutes, 4°C). The supernatant was collected, filtered through a 0.45 μm organic filter, and analyzed by high-performance liquid chromatography (HPLC). The mobile phase consisted of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile, and the sample was gradient eluted using a C18 column. The content of 0.1% acetic acid in acetonitrile solution varied as follows: 10% to 30% from 0 to 12.5 minutes; 30% to 40% from 12.5 to 17.5 minutes; 40% to 100% from 17.5 to 18.5 minutes; 100% from 18.5 to 21 minutes; 100% from 100% to 10% from 21 to 22.5 minutes; and 10% from 22.5 to 26 minutes. The column temperature was maintained at 40°C, the flow rate was 1.0 mL / min, the injection volume was 20 μL, and the detection wavelength was 260 nm. Standard curves were drawn using daidzein, glycitein, genistin, daidzein, glycitein, and genistein standards. Results are expressed as milligrams of standard per 100 mL of sample (mg / 100 mL). The change in soy isoflavone content during lactic acid bacteria fermentation of soy milk was expressed as a growth rate. The growth rate was calculated as shown in Equation 5.

[0100] Table 3 Growth rate of soy isoflavones in soy milk with different dregs content fermented by lactic acid bacteria (%)

[0101]

[0102]

[0103] Note: Different superscript letters in the same column of each fermentation group represent significant differences (p<0.05).

[0104] The conversion of isoflavone glycosides in the three fermentation groups was similar, with all groups achieving complete hydrolysis of isoflavone glycosides after 18 hours of fermentation (Table 3). Lactobacillus delbrueckii MT772183 fermentation resulted in an increase in the aglycone content and a decrease in the total soy isoflavone content in the system. Furthermore, as fermentation progressed, the total soy isoflavone content in each fermentation group decreased in the order: whole soybean < semi-removed soybean < removed soybean. As previously mentioned, the hydrophobic effect of aglycones and the bonding of isoflavones with plant cell walls result in a large amount of unextractable soy isoflavones in soybean dregs. Lactic acid bacteria fermentation can promote the conversion of unextractable phenols in soybeans to extractable phenols. Figure 1A shows no significant difference in pH changes during fermentation (p>0.05), and β-glucosidase activity follows a trend from whole soybean to semi-removed soybean to removed soybean, indicating that the enzyme continuously hydrolyzes the non-extractable isoflavones in the okara, leading to an increase in aglycone content. In summary, β-glucosidase-producing Lactobacillus delbrueckii MT772183 can release non-extractable isoflavones from okara fiber, and whole soybean fermentation can produce more aglycone-type soy isoflavones.

[0105] Example 4 Viscosity of Fermented Soymilk

[0106] 1. Preparation of fermented soy milk

[0107] Process flow: Soybeans → Soak for 12 hours → Wash → Beat (soybean:water ratio 1:8) → Filter or not filter → Sterilize (121°C, 20 minutes) → Cool → Inoculate (4%, v / v) → Ferment to pH 4.5 → Homogenize → Assay. Whole soybean milk (WSM) is prepared directly without filtration, semi-sludged soymilk (MSM) is prepared by sieving through a 40-mesh sieve, and sludged soymilk (LSM) is prepared by sieving through a 300-mesh sieve.

[0108] Strain activation: After the strain is fully activated, pick a single colony and inoculate it into MRS liquid medium, culture it anaerobically at 37°C for 6-8h, centrifuge it (8,000r / min, 4°C, 5min), wash it twice with sterile water, and resuspend it in sterile water to adjust the OD 600 ≈0.9 (colony density is about 10 9 CFU / mL).

[0109] Inoculation: Lactobacillus delbrueckii MT772183 was inoculated into WSM, MSM, and LSM to obtain WSM-b8, MSM-b8, and LSM-b8, and the mixture was fermented continuously at 35-42°C for 10-15 hours.

[0110] 2. Viscosity

[0111] Take a sample from the homogenized sample, ensuring that there are no air bubbles. Place the sample in a circular container. Place the SP-3 coaxial measuring cylinder in the container and adjust the speed to 100 rpm / min. Measure the viscosity (mPa·s). The viscosity increase is expressed as the difference between the viscosity before and after fermentation.

[0112] Viscosity can reflect the viscosity production of the strain. The viscosity growth rate of fermented whole soybean milk (402.40±0.49 mPa·s) is significantly better than that of deboned soy milk (310.28±1.94 mPa·s) and semi-deboned soy milk (235±1.68 mPa·s), which may be related to the better growth state of the strain in whole soybean milk.

[0113] Example 5: Preparation method of fermented soybean powder rich in polyphenols

[0114] The following steps are involved:

[0115] (1) Process flow: soybeans → soaking for 12 hours → washing → beating (soybean-water ratio is 1:8) → sterilization (121°C, 20 minutes) → cooling → inoculation (4%, v / v) → fermentation (37-42°C, 10-15 hours) → homogenization → vacuum freeze drying (freezing chamber temperature 40°C, vacuum pressure 70Pa, heating plate temperature 55°C, freeze drying 48 hours) → fermented soybean powder rich in polyphenols.

[0116] (2) Strain activation: After Lactobacillus delbrueckii MT772183 was fully activated, a single colony was picked and inoculated into MRS liquid medium, cultured anaerobically at 37°C for 6-8 h, centrifuged (8,000 rpm, 4°C, 5 min), washed twice with sterile water, and resuspended in sterile water to adjust the OD 600 ≈0.9 (colony density is about 10 9 CFU / mL).

[0117] The contents of free phenol, bound phenol, conjugated phenol, total phenol, total aglycone and total soy isoflavones of the obtained fermented soy powder were 1.61±0.05, 14.82±0.26, 0.22±0.01, 16.65±0.17, 96.08±3.82 and 34.87±2.60 mg / 100 g, respectively.

[0118] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. Application of Lactobacillus delbrueckii MT772183 in promoting the dissolution of insoluble polyphenols in fermented whole soybean milk and the release of free phenols from conjugated phenols, characterized in that: The insoluble polyphenols are conjugated phenols and conjugated flavonoids, and the conjugated phenols include conjugated polyphenols, conjugated flavonoids and soy isoflavone glycosides; The Lactobacillus delbrueckii MT772183 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M2020332 and a deposit date of July 20, 2020.

2. The use according to claim 1, characterized in that Whole soybean milk was fermented using Lactobacillus delbrueckii MT772183.

3. The use according to claim 2, characterized in that The fermentation process includes the following steps: S1, beat the soybeans with a water ratio of 1:6-12, sterilize and cool, and then inoculate at an inoculum volume of 2-8% v / v; S2. Ferment at 37-42℃ until pH = 4.

5. Homogenize after fermentation.

4. The use according to claim 1, characterized in that Whole bean milk is inoculated with Lactobacillus delbrueckii MT772183 for fermentation to obtain a soy milk product, which includes a fermented whole bean milk drink and fermented soy powder; the whole bean milk is soy milk obtained by beating soybeans without performing a slag removal process.

5. The use according to claim 4, characterized in that When the fermented whole soybean milk product is a fermented whole soybean milk beverage, the method comprises the following steps: S1. Beat the soybeans with a ratio of 1:6 to 12, sterilize, cool and set aside; S2, pick a single colony of Lactobacillus delbrueckii MT772183 for culture activation, and adjust the colony density to 10 9 CFU / mL; S3. Inoculate and ferment at 37-42°C until pH = 4.

5. After fermentation is complete, homogenize to obtain a fermented whole soybean milk beverage with both soluble polyphenol content and consistency.

6. The use according to claim 4, characterized in that When the fermented whole soybean milk product is fermented soybean powder, the fermented soybean powder is obtained by drying the whole soybean milk beverage obtained in S3 of claim 5.

7. The use according to claim 6, characterized in that The drying is selected from one or more of air drying, microwave drying, vacuum freeze drying and spray drying.

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