Method for producing high-activity soybean isoflavone by fermenting soybean milk with lactobacillus pentosus and solid beverage prepared by method

Abstract

The invention discloses a method for producing high-activity soybean isoflavone by fermenting soybean milk through lactobacillus pentosus and a solid beverage prepared through the method, and relates to the technical field of microbial fermention.The method comprises the following steps that the soybean milk is inoculated with the lactobacillus pentosus 068-1, fermentation is conducted for 36-60 h at the temperature of 32-40 DEG C, and the high-activity soybean isoflavone is obtained. After fermentation with lactobacillus pentosus, the soybean isoflavone is hydrolyzed from combined soybean isoflavone to free soybean isoflavone, the fermentation process is simple and easy to control, and the method has the advantage of high repeatability of microbial fermentation, is not influenced by territory and environmental factors, and has a good hydrolysis effect.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a method for producing highly active soy isoflavones from soy milk using Lactobacillus pentosus and the resulting solid beverage. Background Technology

[0002] Soy isoflavones are bioactive substances extracted from the natural plant soybean. They are mainly distributed in the soybean seed coat, hypocotyl, and cotyledons. They are a class of non-steroidal substances with nutritional and therapeutic value. Soy isoflavones have a variety of physiological functions, not only participating in the regulation of plant growth activities, but also playing a beneficial physiological regulatory role in the human body.

[0003] Recent studies have found that soy isoflavones play an important role in the prevention and treatment of various diseases, including tumors, cardiovascular diseases, osteoporosis, and menopausal syndrome. Soy isoflavones exist in soybeans primarily in two forms: free aglycones, including genistein and daidzein, and bound glycosides. Glycosides mainly exist as malonyl genistein, malonyl daidzein, genistein, and daidzein. Soybeans are predominantly composed of bound glycosides, with free aglycones accounting for a relatively small proportion, approximately 2-3% of the total. However, the activity of aglycones is much higher than that of glycosides. Bound glycosides require hydrolysis into aglycones to exert their effects. Therefore, hydrolyzing glycosides into aglycones will yield higher physiological activity.

[0004] However, there are currently few technologies for preparing free aglycones through microbial fermentation, and the results are not good. Therefore, providing a microbial fermentation method with good results and high conversion rate is an urgent technical problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for producing highly active soy isoflavones from soy milk using Lactobacillus pentosus fermentation, and the resulting solid beverage.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for producing highly active soy isoflavones by fermenting soy milk with Lactobacillus pentosus, wherein the Lactobacillus pentosus is Lactobacillus pentosus 068-1, with accession number CGMCC No. 24424.

[0008] It was deposited on February 23, 2022, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Lactobacillus pentosus 068-1.

[0009] Fermentation by the strain of this invention can convert bound glycosides into free aglycones, increasing the amount of free aglycones in the product, and the conversion rate is significantly higher than that of related strains in the prior art.

[0010] Preferably, the method includes the following steps:

[0011] The Lactobacillus pentosus 068-1 was inoculated into soy milk and fermented at 32-40℃ for 36-60 hours to obtain soy isoflavones.

[0012] This invention provides Lactobacillus pentosus 068-1, which has high conversion activity against daidzein, genistein, and genistein in soy milk. Therefore, the soy isoflavones obtained by this method have high biological activity.

[0013] Preferably, the inoculum size of Lactobacillus pentosus 068-1 is 1% of the volume of the soy milk.

[0014] Preferably, the soy milk is black bean soy milk, and the black beans are brown black beans.

[0015] Preferably, the method for preparing the black soybean milk is as follows:

[0016] Grind dried beans and water into a paste, adjust the pH to 6.0-6.5, sterilize, and then add sterilized glucose solution to achieve a final concentration of 5-10 g / L.

[0017] Preferably, the mass-to-volume ratio of the dried beans to the water is 1g:10-20ml.

[0018] A solid beverage made from Lactobacillus pentosus is prepared using the method described above.

[0019] The Lactobacillus pentosus solid beverage prepared by this invention serves as an innovative carrier combining plant protein and probiotics, demonstrating significant potential in improving lipid metabolism, lowering cholesterol, protecting the liver, and aiding weight loss. Lactobacillus pentosus possesses excellent probiotic potential, and the product of this invention has a high content of live Lactobacillus pentosus, which can alleviate obesity and liver damage caused by a high-fat diet. The preparation method is simple, the product is easy to preserve, and the number of live bacteria remains high after long-term storage, indicating good commercial prospects.

[0020] Preferably, the fermented liquid after fermentation is completed is freeze-dried to obtain a powder, which is a solid beverage.

[0021] Preferably, the freeze-drying method is as follows: the fermentation broth is pre-cooled at -20℃ for 12-36 hours, placed in a vacuum freeze dryer for 24-60 hours, and then pulverized and sieved.

[0022] The viable count of Lactobacillus pentosaccharide solid beverage of this invention is not less than 1.0 × 10⁻⁶. 10 CFU / g, viable bacterial count not less than 2.0 × 10⁻⁶ after storage at 4-10℃ for 3 months. 9 cfu / g.

[0023] The application of Lactobacillus pentosus solid beverage in the preparation of antioxidant health food, as described above.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention, through fermentation with Lactobacillus pentosus, hydrolyzes soy isoflavones from bound soy isoflavones to free soy isoflavones. The fermentation process is simple and easy to control, with the advantage of strong reproducibility of microbial fermentation, unaffected by geographical and environmental factors, and with good hydrolysis effect. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. The drawings in this description are merely embodiments of the present invention.

[0027] Figure 1 Images of different soybean varieties;

[0028] Figure 2 Figure showing the results of antioxidant activity assays for products prepared from different soybean varieties;

[0029] Figure 3 HPLC chromatogram of changes in soybean isoflavones in fermented soybean milk broth prepared from different soybean varieties;

[0030] Figure 4 Bar chart showing the concentrations of three types of soy isoflavones in fermented soy milk broth prepared from different soybean varieties;

[0031] Figure 5 Image of the Lactobacillus pentosus solid beverage product prepared in Example 2;

[0032] Figure 6 Images of four groups of mice;

[0033] Figure 7 Bar chart showing the serum concentrations of total cholesterol, triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in four groups of mice.

[0034] Figure 8 Images of liver tissue sections from four groups of mice. Detailed Implementation

[0035] Embodiments of the present invention are described below, examples of which are shown in the accompanying drawings. The embodiments described with reference to the drawings are exemplary and intended to explain the present invention, but are not to be construed as limiting the present invention.

[0036] Example 1

[0037] This invention provides a method for producing highly active soy isoflavones from soy milk by fermenting with Lactobacillus pentosus, specifically including the following steps:

[0038] Lactobacillus pentosus 68-1 (CGMCC No. 24424, which has good probiotic properties) stored at -80℃ was continuously activated for two generations, inoculated into MRS liquid medium, and statically cultured at 37℃ for 12h. Then, 1% (v / v) of Lactobacillus pentosus 68-1 seed liquid was added to the soy milk fermentation medium and statically cultured at 37℃ for 24h to obtain fermented soy milk.

[0039] The soybean milk fermentation medium was prepared as follows: Selected Shaanxi specialty soybeans with good sensory quality and free from mold, including brown black soybeans (Shenmu), chicken-kidney green soybeans, chicken-kidney white soybeans, flail-shaped soybeans, black soybeans, and double-green soybeans (product details available). Figure 1 Wash with drinking water, soak overnight, add drinking water at a ratio of 1:14 by weight of material and liquid, blend with a household soymilk maker, adjust pH to 6.3 with sodium bicarbonate, dispense into 100ml / bottle, sterilize at 121℃ for 15min, cool to room temperature, add separately sterilized 500g / L glucose solution to make the final concentration reach 5g / L.

[0040] Antioxidant activity analysis of fermented soy milk

[0041] like Figure 2 The results of antioxidant activity determination of products made from different soybean varieties are shown in the figure. A represents the ABTS+ free radical scavenging rate, B represents the DPPH free radical scavenging rate, C represents the superoxide anion removal rate, and D represents the hydroxyl free radical scavenging rate. As can be seen from the antioxidant activity analysis results in the figure, the antioxidant activity of all six soybean milks was improved after fermentation with Lactobacillus pentosus. Among them, the fermentation liquid of brown and black soybeans ranked among the top three in the four antioxidant tests, with ABTS+, DPPH, hydroxyl free radical and superoxide anion removal rates of 60.80%, 71.92%, 43.75% and 22.67%, respectively.

[0042] Figure 3 HPLC chromatograms showing the changes in soy isoflavones in fermented soy milk broth prepared from different soybean varieties. In this chromatogram, A represents six soy isoflavone standards at 0.03 g / L; B–G represent the HPLC results of the six soy milk products before and after fermentation, with the black line representing before fermentation and the colored line representing after fermentation. Figure 3 It can be seen that after fermentation with 068-1, all three types of soybean isoflavones can be converted into their corresponding glycoside forms.

[0043] Table 1 shows the conversion rates of three types of soybean isoflavones in fermented soybean milk products made from different soybean varieties. As can be seen from the table, the conversion rates of the three types of soybean isoflavones in the 068-1 / brown-black soybean combination are the highest. The conversion rates of daidzein / daidzein and genistein / genistein in the 068-1 / black soybean combination are the second highest. The conversion rates of daidzein / daidzein in the 068-1 / chicken kidney white soybean combination are the second highest.

[0044] Table 1 shows the conversion rates of three types of soybean isoflavones in fermented soybean milk products made from different soybean varieties.

[0045] Conversion rate (%) daidzein / daidzein daidzein / daidzein Genistein / Gyroside Brown and black beans 78.55 55.33 90.85 flail 45.16 13.90 54.99 Double Green Peas 38.27 12.10 60.03 Chicken kidney white 51.05 16.38 58.09 Chicken kidney green 53.32 15.80 55.93 Black beans 58.01 15.04 70.33

[0046] Figure 4 The bar chart shows the concentrations of three types of soybean isoflavones in the fermentation broth of soybean milk prepared from different soybean varieties. As can be seen from the figure, after conversion, the concentrations of the three aglycone forms in the fermentation broth of 068-1 / brown black soybean combination are the highest, while the concentrations of the three aglycone forms in the 068-1 / chicken kidney white combination are the second highest.

[0047] Comparative Example 1

[0048] The comparative examples are comparisons between the fermentation products of the strains of this invention and other strains in the prior art. Except for the difference in raw materials and strains, the remaining steps are the same as in Example 1. The relevant conclusions are shown in Table 2. As can be seen from the table, compared with the two lactobacilli with the highest conversion rates mentioned in the article "Analysis of Fermentation Characteristics of Lactic Acid Bacteria with Different β-glucosidase Activities in Soy Milk", LPL043 has a conversion rate of 25.61% and 22.48% for daidzein and genistein, respectively, while 068-1 has a corresponding conversion rate of 78.55% and 90.85%, which are significantly higher than the comparative examples. The corresponding aglycone product concentrations are similar to or higher than those of the comparative examples. The conversion rate of 068-1 for daidzein is lower than that of the comparative examples, but the product concentration of daidzein is 8.4 times that of the comparative examples.

[0049] Compared with the patent "A type of Lactobacillus acidophilus, a method for fermenting soy milk with it, and the fermented soy milk prepared therefrom and its application", the daidzein content in soy milk after conversion by Lactobacillus acidophilus HAU-FR7 decreased from 0.184 mmol / L to 0.102 mmol / L, while the corresponding data in this experiment was 0.052 mmol / L, decreasing to 0; the genistein content after conversion by HAU-FR7 decreased from 0.222 mmol / L to 0.123 mmol / L, while the corresponding data in this experiment was 0.084 mmol / L, decreasing to 0.0016 mmol / L. Therefore, this invention is superior in terms of both conversion rate and final concentration.

[0050] Table 2. Comparison of fermentation products of the present invention strain with those of other existing strains.

[0051]

[0052] Example 2

[0053] This invention provides a method for preparing a Lactobacillus pentosaccharide solid beverage, specifically including the following steps:

[0054] Following the method in Example 1, fermented soy milk containing *Lactobacillus pentosus* 68-1 was obtained. This was pre-cooled at -20°C for 12 hours, freeze-dried in a vacuum freeze dryer for 24 hours, pulverized, and sieved to obtain *Lactobacillus pentosus* solid beverage. (See product image). Figure 5 ;

[0055] The Lactobacillus pentosus solid beverage was dissolved in sterile water and serially diluted. The viable count was 6.27 × 10⁻⁶. 9 CFU / g, viable bacterial count after 6 months of storage at 4-10℃ was 2.22×10⁻⁶. 9 CFU / g;

[0056] HPLC determination of soy isoflavone content:

[0057] Sample preparation: 1g of lyophilized Lactobacillus pentosaccharide-fermented soy milk and 1g of unfermented soy milk were dissolved in 20mL of 50% MeOH at 25℃. After 12h, the mixture was centrifuged at 3000g for 5min. The supernatant was aspirated with a syringe and filtered through a filter (0.45μm, Whatman, Inc., Maidstone, UK). The sample injection volume was 10μL.

[0058] Detection conditions: Mobile phase A: methanol solution, mobile phase B: water, flow rate at 260 nm: 0.8 mL / min, column temperature maintained at 25℃. Relevant detection conditions are shown in Table 3.

[0059] Table 3 Detection conditions

[0060] Time / min Mobile phase A% Mobile phase B% 0 40 60 6 40 60 10 58 42 25 58 42 26 40 60 27 40 60

[0061] The test results showed that after fermentation with *Lactobacillus pentosus* 68-1, the concentrations of three low-activity glycoside flavonoids (daidzein, genistein, and genistein) in six types of soy milk significantly decreased, while the concentrations of highly active aglycone flavonoids (daidzein, genistein, and genistein) increased. Among these, the conversion rates of the three flavonoids in the fermentation broth of brown and black soybeans were the highest, at 78.55%, 55.33%, and 90.85%, respectively. The highest concentrations of genistein and genistein were 2.71 mg / L and 33.11 mg / L, respectively, and the highest total concentration of the three aglycones was 52.87 mg / L. Therefore, *Lactobacillus pentosus* was selected to ferment brown and black soybeans for the production of solid beverages.

[0062] The Lactobacillus pentosus solid beverage obtained from fermenting brown and black soybean milk prepared in Example 2 was subjected to a high-fat mouse experiment to determine its weight loss, lipid-lowering, and liver-protecting effects, as detailed below:

[0063] Husbandry conditions: 40 male SPF grade C57BL / 6N mice were housed in a 12-hour light-dark cycle. The indoor temperature was controlled at 20-22℃ and the humidity at 60-65%. The animal room was kept well-ventilated, well-lit, and clean at all times. The mice were divided into a normal control group and a high-fat model group. The experimental conditions met the Ministry of Health's "Standards for Experimental Animal Environment and Facilities".

[0064] High-fat model establishment: Mice were fed with D12450B with 10% calories and D12492 with 60% calories, respectively. After 8 weeks of feeding, three mice were randomly selected from the control group and the high-fat group, respectively. The levels of triglycerides and total cholesterol in the fasting serum of the mice were detected. The differences were significant (p<0.05). The next step was to treat the mice with fermented soybean milk by gavage.

[0065] Grouping and feeding amount: Mice were divided into 4 groups according to the fact that there was no significant difference in average body weight among the groups:

[0066] The normal control group was fed D12450B + sterile water.

[0067] The high-fat model group (HFD) was fed D12492 + sterile water;

[0068] The low-fat unfermented soy milk solid beverage group (SM+HFD) was fed with D12492+low-fat unfermented Lactobacillus pentosaccharide 068-1 fermented soy milk solid beverage;

[0069] The low-fat fermented soy milk solid beverage experimental group (FSM+HFD) was fed with D12492+low-fat Lactobacillus pentosaccharide 068-1 fermented soy milk solid beverage.

[0070] The intervention group was given daily gavage treatment to mice at a dose of 50 g / kg BW / d. Mice had free access to food and water. Mouse weight and food intake were recorded every Monday for 8 weeks.

[0071] Sample Collection: Three days before euthanasia, fresh feces were collected from mice and placed in cryovials for storage at -80°C. Mice were fasted for 12 hours, and their final weight was recorded. Blood was collected from the eyeballs after euthanasia. Liver tissue blocks of equal size were removed from the abdominal cavity, fixed in 10% formaldehyde solution for 24 hours, stained with hematoxylin and eosin (HE), and analyzed for histopathological morphology. The remaining liver tissue was rapidly frozen in liquid nitrogen and stored at -80°C within 2 hours for liver lipid level detection. Epididymal fat was collected, weighed, and recorded. Relevant conclusions are shown in Tables 4-5 and below. Figure 6-8 ;

[0072] Table 4. Changes in mouse body weight after gavage.

[0073]

[0074]

[0075] Table 5 Comparison of epididymal fat and liver weight

[0076]

[0077] As shown in the table, after 8 weeks of gavage, compared with the high-fat model group, the mice in the low-fat fermented soy milk solid beverage experimental group showed significantly lower body weight, epididymal fat, and liver weight, which were superior to those in the low-fat unfermented soy milk solid beverage group and close to those in the normal group; the epididymal fat-to-body weight ratio in the low-fat fermented soy milk solid beverage experimental group was even lower than that in the normal group. This indicates that the low-fat fermented soy milk solid beverage of this invention has a significant effect on controlling body weight and visceral fat.

[0078] Figure 6 Images of four groups of mice. Figure 7 The bar chart shows the results of total cholesterol, triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) concentrations in the serum of four groups of mice. As can be seen from the chart, compared with the high-fat model group and the unfermented soy milk solid beverage control group, the mice in the Lactobacillus pentosus fermented soy milk solid beverage experimental group had significantly lower body weight, significantly lower levels of TC, TG, and LDL-C (P < 0.01), and significantly higher HDL-C levels than the high-fat model group (P < 0.01). This indicates that it can effectively reduce serum total cholesterol, triglycerides, and LDL concentrations, and effectively increase HDL concentration.

[0079] Figure 8 The images show liver tissue sections from four groups of mice. The results indicate that severe vacuolation was observed in the liver cytoplasm of rats in the high-fat model group and the unfermented soy milk solid beverage control group. Gavage administration of Lactobacillus pentosus 68-1 fermented soy milk solid beverage reduced the degree of lipid deposition and vacuolation in the liver tissue, essentially restoring it to the level of the normal control. The test results suggest that Lactobacillus pentosus 68-1 fermented brown and black soy milk solid beverage can alleviate obesity and liver damage caused by a high-fat diet.

[0080] The Lactobacillus pentosus solid beverage of the present invention has good antioxidant capacity and high conversion rate of soy isoflavones in soy milk. The solid beverage has been verified by mouse experiments to improve the blood lipid level in the serum of mice fed high-fat diets, reduce body weight and alleviate liver damage. The Lactobacillus pentosus solid beverage of the present invention has a simple preparation method, the product is easy to preserve, and the number of viable bacteria is high after long-term storage, which has good commercial prospects.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for producing highly active soy isoflavones from soy milk by fermentation with Lactobacillus pentosus, characterized in that, The Lactobacillus pentosus mentioned is Lactobacillus pentosus 068-1, with accession number CGMCC No. 24424.

2. The method for producing highly active soy isoflavones from soy milk by fermentation with Lactobacillus pentosus according to claim 1, characterized in that, The method includes the following steps: The Lactobacillus pentosus 068-1 was inoculated into soy milk and fermented at 32-40℃ for 36-60 hours to obtain highly active soy isoflavones.

3. The method for producing highly active soy isoflavones from soy milk by fermentation with Lactobacillus pentosus according to claim 2, characterized in that, The inoculation amount of the Lactobacillus pentosus 068-1 seed culture is 1% of the volume of the soy milk, wherein the viable count of the seed culture is 2*10⁻⁶. 8 cfu / ml.

4. The method for producing highly active soy isoflavones from soy milk by fermentation with Lactobacillus pentosus according to claim 2, characterized in that, The soy milk is black bean soy milk, and the black beans are brown black beans.

5. The method for producing highly active soy isoflavones from soy milk by fermentation with Lactobacillus pentosus according to claim 4, characterized in that, The preparation method of the black bean soy milk is as follows: Grind dried beans and water into a paste, adjust the pH to 6.0-6.5, sterilize, and then add sterilized glucose solution to achieve a final concentration of 5-10 g / L.

6. The method for producing highly active soy isoflavones from soy milk by fermentation with Lactobacillus pentosus according to claim 5, characterized in that, The mass-to-volume ratio of the dried beans to the water is 1g:10-20ml.

7. A solid beverage made from Lactobacillus pentosus, characterized in that, It is prepared by the method described in any one of claims 1-6.

8. A Lactobacillus pentosus solid beverage according to claim 7, characterized in that, After the fermentation process is complete, the fermentation liquid is freeze-dried to obtain a powder, which is a solid beverage.

9. A Lactobacillus pentosus solid beverage according to claim 8, characterized in that, The freeze-drying method is as follows: the fermentation broth is pre-cooled at -20℃ for 12-36 hours, placed in a vacuum freeze dryer for 24-60 hours, and then pulverized and sieved.

10. The use of the Lactobacillus pentosus solid beverage as described in any one of claims 7-9 in the preparation of antioxidant health food.

Images