Composition for controlling postprandial blood sugar and preparation method thereof

Abstract

The invention discloses a composition for controlling postprandial blood sugar and a preparation method thereof, and relates to the technical field of fermentation processing of functional foods and natural products. The preparation method comprises the following steps: (1) preparing materials: mixing mulberry leaf powder and soybean powder in proportion, adding water to prepare a material with the water content of 55-60%, adding 0.3-0.5% of an inducer into the material, and uniformly mixing to obtain a mixture; (2) primary fermentation: inoculating a mixed bacteria solution of eurotium cristatum and bacillus subtilis into the mixture, and performing solid-state fermentation at 30-35 DEG C to obtain a first fermented material; (3) secondary fermentation: adding 1-4% of an enzyme production accelerant into the first fermentation material, and continuing fermentation at 30-35 DEG C to obtain a second fermentation material; and (4) enzyme deactivation and drying treatment: performing enzyme deactivation, cooling, homogenizing, drying and crushing on the second fermentation material to obtain the composition. The composition prepared by the invention contains a large amount of hypoglycemic active ingredients, has high inhibition rate on alpha-glucosidase, and can be used for controlling postprandial blood sugar.

Description

Technical Field

[0001] This invention relates to the field of functional food and natural product fermentation processing technology, and in particular to a composition for controlling postprandial blood glucose and its preparation method. Background Technology

[0002] Alpha-glucosidase is a key enzyme on the cell membrane of human small intestinal epithelial cells, which catalyzes the hydrolysis of disaccharides into monosaccharides. Its activity directly affects postprandial blood glucose levels. The stronger the alpha-glucosidase activity, the more starch and oligosaccharides are hydrolyzed, leading to faster absorption of glucose on the small intestinal mucosa and a faster rise in postprandial blood glucose. Therefore, it is of great significance to develop alpha-glucosidase inhibitors that are naturally derived, have mild effects, and are highly safe to control postprandial blood glucose elevation.

[0003] While commercially available chemically synthesized inhibitors such as acarbose are effective, they have significant gastrointestinal side effects. Therefore, finding highly effective, natural hypoglycemic drugs and / or foods has become a common desire, especially for patients with type 2 diabetes (who do not require insulin treatment and can effectively control their blood sugar through diet alone). The demand for natural hypoglycemic foods is even more urgent for them. Mulberry leaves and soybeans are traditional medicinal and edible plants, rich in various active ingredients such as 1-deoxynojirimycin (DNJ), flavonoids, polyphenols, and phytoisoflavones, which have potential hypoglycemic and antioxidant activities. *Aspergillus cristatus* (… Aspergillus crested Bacillus subtilis is widely used in traditional fermented food products, and it has multiple functions such as converting phenols and improving flavor. Bacillus subtilis It is a widely used strain that can produce hypoglycemic substances and various enzymes.

[0004] Existing technologies utilize the fermentation and extraction of active ingredients from the aforementioned medicinal and edible plants for hypoglycemic purposes, as illustrated by patents CN 116369462 A and CN 113144000 A. However, the extraction methods employed by these patents yield low levels of hypoglycemic active ingredients. Furthermore, the content of hypoglycemic active ingredients in mulberry leaves is inherently low; for example, 1-deoxynojirimycin (DNJ) is present in only 0.11% of mulberry leaves. Consequently, the resulting product exhibits a low α-glucosidase inhibition rate, typically only 40-70%, leading to high production costs. Therefore, improving the extraction yield of hypoglycemic active ingredients, increasing the α-glucosidase inhibition rate, and reducing production costs have become urgent issues requiring resolution. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a composition for controlling postprandial blood glucose and its preparation method. This fermentation method can significantly increase the content of hypoglycemic active ingredients and improve the α-glucosidase inhibition rate. The specific technical solution is as follows: A method for preparing a composition for controlling postprandial blood glucose includes the following steps: (1) Raw material preparation and processing: Mulberry leaf powder and soybean powder are mixed according to the ratio, and water is added to moisten the material to obtain a material with a moisture content of 55%-60%. An inducer of 0.3-0.5% by weight of the material is added to the material and mixed evenly to obtain a mixture. (2) Primary fermentation: A mixed bacterial solution of *Aspergillus cristatus* and *Bacillus subtilis* is inoculated into the mixture, wherein the mass ratio of *Aspergillus cristatus* to *Bacillus subtilis* in the mixed bacterial solution is (3-7):1, and the inoculation amount of the mixed bacterial solution is 2%-5% (v / w). Solid fermentation is carried out at 30-35℃ for 15-20 hours to obtain the first fermented material. (3) Secondary fermentation: Add 1-4% of the enzyme production promoter by weight of the first fermentation material to the first fermentation material, and adjust the pH of the fermentation system to 6.5-7.5, and continue fermentation at 30-35℃ for 20-30 hours to obtain the second fermentation material; (4) Enzyme inactivation and drying: The second fermentation material is inactivated at 80-85°C for 10-20 minutes, cooled, homogenized, dried and pulverized to obtain the composition.

[0006] Further, in step (1), the mass ratio of mulberry leaf powder to soybean powder is 1:(1-4).

[0007] Further, in step (1), the inducer comprises the following components in parts by weight: 6-9 parts of ornithine, 1-4 parts of tyrosine and 0.5-1.5 parts of quercetin.

[0008] Further, in step (3), the enzyme production promoter comprises the following components in parts by weight: 10-18 parts wheat bran, 15-22 parts orange peel powder, and 4-8 parts casein.

[0009] The present invention also provides a composition for controlling postprandial blood glucose, which is prepared by the above-described preparation method.

[0010] The present invention also provides the application of the above composition in health products and / or foods for controlling postprandial blood glucose.

[0011] Furthermore, the health products and / or foods are used to inhibit α-glucosidase activity.

[0012] The present invention also provides a health product and / or food for controlling postprandial blood glucose, wherein the health product and / or food contains the above-mentioned composition. Compared with the prior art, the beneficial effects of the present invention are: This invention uses mulberry leaf powder and soybean powder as fermentation substrates, and *Aspergillus cristatus* and *Bacillus subtilis* as fermentation strains. During the fermentation process, an inducer and an enzyme production promoter are added to assist fermentation. This not only improves the extraction rate of hypoglycemic active ingredients from mulberry leaves and soybeans, but also promotes the production of large amounts of hypoglycemic active ingredients by *Aspergillus cristatus* and *Bacillus subtilis*, thereby increasing the total content of active ingredients in the fermentation product. During the primary fermentation, *Aurorus cristatus* and *Bacillus subtilis* used ornithine, tyrosine, and quercetin as inducers to ferment within a set temperature range, producing various hypoglycemic active ingredients such as alkaloids, polysaccharides, and flavonoids. During the secondary fermentation, *Aurorus cristatus* and *Bacillus subtilis* were induced to produce large amounts of cellulase, pectinase, and protease under the action of enzyme promoters wheat bran, orange peel powder, and complexin. Cellulase and pectinase disrupted cell wall components, promoting the dissolution of more active ingredients from mulberry leaves and soybeans. Simultaneously, enzymes also promoted the transformation and structural modification of substances in the fermentation broth, such as breaking down proteins into polypeptides or small peptides and demethoxylating some active substances, thereby producing new active ingredients, enriching the variety of active ingredients, and increasing the types and content of hypoglycemic active ingredients such as alkaloids, polysaccharides, and flavonoids, thus improving the hypoglycemic effect. Detailed Implementation

[0013] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0014] The *Aspergillus cristatus* species mentioned below was deposited on July 3, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No: 66629. The *Bacillus subtilis* species mentioned below is *Bacillus subtilis* ATCC6633, purchased from Shanghai Jihe Biotechnology Co., Ltd.

[0015] Example 1 A method for preparing a composition for controlling postprandial blood glucose includes the following steps: (1) Raw material preparation and processing: Mulberry leaf powder and soybean powder are mixed in a mass ratio of 1:1, and water is added to moisten the material to obtain a material with a moisture content of 55%. 0.3% of the weight of the material is added to the material and mixed well to obtain a mixture. The inducing agent includes the following components in parts by weight: 6 parts of ornithine, 1 part of tyrosine and 0.5 parts of quercetin.

[0016] (2) Primary fermentation: A mixed bacterial solution of *Aspergillus cristatus* and *Bacillus subtilis* is inoculated into the mixture, wherein the mass ratio of *Aspergillus cristatus* to *Bacillus subtilis* in the mixed bacterial solution is 3:1, and the inoculation amount of the mixed bacterial solution is 2% (v / w). Solid fermentation is carried out at 30°C for 15 hours to obtain the first fermented material.

[0017] (3) Secondary fermentation: Add 1% of the enzyme production promoter of the first fermentation material to the first fermentation material, and adjust the pH of the fermentation system to 6.5, and continue fermentation at 30°C for 20 hours to obtain the second fermentation material; the enzyme production promoter includes the following components in parts by weight: 10 parts wheat bran, 15 parts orange peel powder and 4 parts fibroin.

[0018] (4) Enzyme inactivation and drying: The second fermentation material is inactivated at 80°C for 10 minutes, cooled, homogenized, dried and pulverized to obtain the composition.

[0019] Example 2 A method for preparing a composition for controlling postprandial blood glucose includes the following steps: (1) Raw material preparation and processing: Mulberry leaf powder and soybean powder are mixed in a mass ratio of 1:2, and water is added to moisten the material to obtain a material with a moisture content of 60%. 0.5% of the weight of the material is added to the material and mixed well to obtain a mixture. The inducing agent includes the following components in parts by weight: 9 parts of ornithine, 4 parts of tyrosine and 1.5 parts of quercetin.

[0020] (2) Primary fermentation: A mixed bacterial solution of *Aspergillus cristatus* and *Bacillus subtilis* is inoculated into the mixture, wherein the mass ratio of *Aspergillus cristatus* to *Bacillus subtilis* in the mixed bacterial solution is 7:1, and the inoculation amount of the mixed bacterial solution is 5% (v / w). Solid fermentation is carried out at 35°C for 20 hours to obtain the first fermented material.

[0021] (3) Secondary fermentation: Add 4% of the enzyme production promoter of the first fermentation material to the first fermentation material, and adjust the pH of the fermentation system to 7.5, and continue fermentation at 35°C for 30 hours to obtain the second fermentation material; the enzyme production promoter includes the following components in parts by weight: 18 parts wheat bran, 22 parts orange peel powder and 8 parts fibroin.

[0022] (4) Enzyme inactivation and drying: The second fermentation material was inactivated at 85°C for 20 minutes, cooled, homogenized, dried and pulverized to obtain the composition.

[0023] Example 3 A method for preparing a composition for controlling postprandial blood glucose includes the following steps: (1) Raw material preparation and processing: Mulberry leaf powder and soybean powder are mixed in a mass ratio of 1:3, and water is added to moisten the material to obtain a material with a moisture content of 58%. An inducer of 0.35% by weight of the material is added to the material and mixed evenly to obtain a mixture. The inducer includes the following components in parts by weight: 7 parts of ornithine, 2 parts of tyrosine and 0.6 parts of quercetin.

[0024] (2) Primary fermentation: A mixed bacterial solution of *Aspergillus cristatus* and *Bacillus subtilis* is inoculated into the mixture, wherein the mass ratio of *Aspergillus cristatus* to *Bacillus subtilis* in the mixed bacterial solution is 5:1, and the inoculation amount of the mixed bacterial solution is 3% (v / w). Solid fermentation is carried out at 32°C for 16 hours to obtain the first fermented material.

[0025] (3) Secondary fermentation: Add 2% of the enzyme production promoter of the first fermentation material to the first fermentation material, and adjust the pH of the fermentation system to 7, and continue fermentation at 32°C for 23 hours to obtain the second fermentation material; the enzyme production promoter includes the following components in parts by weight: 12 parts wheat bran, 16 parts orange peel powder and 5 parts fibroin.

[0026] (4) Enzyme inactivation and drying: The second fermentation material was inactivated at 82°C for 15 minutes, cooled, homogenized, dried and pulverized to obtain the composition.

[0027] Example 4 A method for preparing a composition for controlling postprandial blood glucose includes the following steps: (1) Raw material preparation and processing: Mulberry leaf powder and soybean powder are mixed in a mass ratio of 1:4, and water is added to moisten the material to obtain a material with a moisture content of 58%. 0.4% of the weight of the material is added to the material and mixed well to obtain a mixture. The inducing agent includes the following components in parts by weight: 8 parts of ornithine, 3 parts of tyrosine and 1 part of quercetin.

[0028] (2) Primary fermentation: A mixed bacterial solution of *Aspergillus cristatus* and *Bacillus subtilis* is inoculated into the mixture, wherein the mass ratio of *Aspergillus cristatus* to *Bacillus subtilis* in the mixed bacterial solution is 6:1, and the inoculation amount of the mixed bacterial solution is 4% (v / w). Solid fermentation is carried out at 35°C for 18 hours to obtain the first fermented material.

[0029] (3) Secondary fermentation: Add 3% of the enzyme production promoter of the first fermentation material to the first fermentation material, and adjust the pH of the fermentation system to 7.5, and continue fermentation at 35°C for 28 hours to obtain the second fermentation material; the enzyme production promoter includes the following components in parts by weight: 17 parts wheat bran, 20 parts orange peel powder and 7 parts casein.

[0030] (4) Enzyme inactivation and drying: The second fermentation material was inactivated at 82°C for 15 minutes, cooled, homogenized, dried and pulverized to obtain the composition.

[0031] Comparative Example 1: Step (1) without adding an inducing agent, and other steps are the same as in Example 1.

[0032] Comparative Example 2: The inducer in step (1) does not contain ornithine and quercetin, and the other steps are the same as in Example 1.

[0033] Comparative Example 3: Step (3) does not contain an enzyme production promoter, and the other steps are the same as in Example 1.

[0034] Comparative Example 4: The enzyme production promoter in step (3) does not contain wheat bran and complex protein, and the other steps are the same as in Example 1.

[0035] Active ingredient content detection: The contents of total alkaloids, total flavonoids, total polysaccharides, and 1-deoxynojirimycin in the compositions prepared in each example and comparative example were determined. Specifically, the Reichelk salt colorimetric method was used to determine the content of total alkaloids, the aluminum nitrate-sodium nitrite method was used to determine the content of total flavonoids, the phenol-sulfuric acid method was used to determine the content of total polysaccharides, and the high-performance liquid chromatography (HPLC) method was used to determine the content of 1-deoxynojirimycin. The results are shown in Table 1. Table 1. Content of hypoglycemic active ingredients in each group Table 1 shows that the compositions prepared using the method of the present invention have higher contents of hypoglycemic active ingredients such as alkaloids, polysaccharides, and flavonoids, especially 1-deoxynojirimycin, which can reach as high as 11 mg / g. A comparison of the data from Example 1 and the comparative examples shows that the use of inducing agents and enzyme-producing promoters during preparation in the present invention can increase the content of hypoglycemic active ingredients in the compositions.

[0036] Blood sugar lowering effect test: The inhibition rate of α-glucosidase by the compositions prepared in each example and comparative example was detected using the PNPG method. The results are shown in Table 2: Table 2. Inhibition rate of α-glucosidase in each group As shown in Table 2, the composition prepared by the method of the present invention has a higher inhibition rate of α-glucosidase.

[0037] Mouse experiment to lower postprandial blood glucose: Experimental subjects: Thirty healthy ICR mice aged 8-10 weeks and weighing 20-25g were selected and fed with standard diet for 1 week in an SPF-grade environment with a 12-hour light / dark cycle, free access to water.

[0038] Experimental Design: Mice were randomly divided into three groups of 10 mice each. Group 1: control group, administered physiological saline (0.1 mL / 10 g body weight) by gavage; Group 2: positive control group, administered acarbose (10 mg / kg body weight) by gavage; Group 3: experimental group, administered the composition prepared in Example 4 by gavage (2.5 g / kg body weight). Mice were fasted for 12 hours before the experiment (water was not restricted). Blood was collected from the tail tip, and fasting blood glucose (FBG) was measured using a glucometer. After the measurement, the prescribed dosage was administered, followed by gavage administration of glucose (2 g / kg body weight) 15 minutes later. Blood glucose was measured again after 120 minutes. The area under the blood glucose curve (AUC) from 0 to 120 minutes was calculated to assess overall glycemic control. A smaller AUC indicates a smaller range of blood glucose fluctuations and a lower overall blood glucose level, meaning the body can more effectively maintain stable blood glucose within a near-normal range.

[0039] The results are shown in Table 3: Table 3. Blood glucose changes in each group Table 3 shows that the composition of the present invention can reduce postprandial blood glucose.

[0040] In summary, this invention uses mulberry leaf powder and soybean powder as fermentation substrates, and *Aspergillus cristatus* and *Bacillus subtilis* as fermentation strains. Furthermore, it adds inducing agents and enzyme-producing promoters during fermentation to assist in the process. This not only improves the extraction rate of hypoglycemic active ingredients from mulberry leaves and soybeans but also promotes the production of large amounts of hypoglycemic active ingredients by *Aspergillus cristatus* and *Bacillus subtilis*, thereby increasing the total content of active ingredients in the fermentation product. In the primary fermentation, *Aurorus cristatus* and *Bacillus subtilis* used mulberry leaf powder as a carbon source, soybean powder as a nitrogen source, and ornithine, tyrosine, and quercetin as inducers. Fermentation was carried out within a set temperature range, producing various hypoglycemic active ingredients such as alkaloids, polysaccharides, and flavonoids. In the secondary fermentation, under the action of enzyme promoters wheat bran, orange peel powder, and chelate, *Aurorus cristatus* and *Bacillus subtilis* were induced to produce large amounts of cellulase, pectinase, and protease. Cellulase and pectinase disrupted cell wall components, promoting the dissolution of more active ingredients from mulberry leaves and soybeans. Simultaneously, enzymes also promoted the transformation and structural modification of substances in the fermentation broth, such as breaking down proteins into polypeptides or small peptides and demethoxylating some active substances, thereby producing new active ingredients, enriching the variety of active ingredients, and increasing the types and content of hypoglycemic active ingredients such as alkaloids, polysaccharides, and flavonoids, thus improving the hypoglycemic effect.

[0041] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for preparing a composition for controlling postprandial blood glucose, characterized in that, Includes the following steps: (1) Raw material preparation and processing: Mulberry leaf powder and soybean powder are mixed according to the ratio, and water is added to moisten the material to obtain a material with a moisture content of 55%-60%. An inducer of 0.3-0.5% by weight of the material is added to the material and mixed evenly to obtain a mixture. (2) Primary fermentation: Inoculate the mixture with a mixed bacterial solution of *Aspergillus cristatus* and *Bacillus subtilis* at an inoculation rate of 2%-5% (v / w), and ferment in a solid state at 30-35°C for 15-20 hours to obtain the first fermented material; (3) Secondary fermentation: Add 1-4% of the enzyme production promoter by weight of the first fermentation material to the first fermentation material, and adjust the pH of the fermentation system to 6.5-7.5, and continue fermentation at 30-35℃ for 20-30 hours to obtain the second fermentation material; (4) Enzyme inactivation and drying: The second fermentation material is inactivated at 80-85°C for 10-20 minutes, cooled, homogenized, dried and pulverized to obtain the composition.

2. The method for preparing a composition for controlling postprandial blood glucose according to claim 1, characterized in that, In step (1), the mass ratio of mulberry leaf powder to soybean powder is 1:(1-4).

3. The method for preparing a composition for controlling postprandial blood glucose according to claim 1, characterized in that, In step (1), the inducer comprises the following components in parts by weight: 6-9 parts of ornithine, 1-4 parts of tyrosine and 0.5-1.5 parts of quercetin.

4. The method for preparing a composition for controlling postprandial blood glucose according to claim 1, characterized in that, In step (3), the enzyme production promoter comprises the following components in parts by weight: 10-18 parts wheat bran, 15-22 parts orange peel powder, and 4-8 parts casein.

5. A composition for controlling postprandial blood glucose, characterized in that, The composition is prepared by the preparation method according to any one of claims 1 to 4.

6. The use of the composition according to any one of claims 1 to 5 in a health supplement and / or food for controlling postprandial blood glucose.

7. The application according to claim 6, characterized in that, The health products and / or foods are used to inhibit α-glucosidase activity.

8. A health product and / or food for controlling postprandial blood glucose, characterized in that, The health products and / or food products contain the composition as described in any one of claims 1 to 5.

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