Preparation method of sophora flower bud powder containing quercetin and quercetin glucoside and application of sophora flower bud powder in preparation of respiratory tract health preparation

Abstract

The invention belongs to the technical field of plant extraction, and particularly relates to a preparation method of sophora flower bud powder containing quercetin and quercetin glucoside and application of the sophora flower bud powder in preparation of respiratory tract health preparations. The preparation method comprises the following steps: (1) crushing sophora flower buds to obtain sophora flower bud powder, (2) mixing the sophora flower bud powder with water, carrying out heating treatment, then cooling, and carrying out microwave treatment to obtain a pretreated sophora flower bud product; (3) adding a compound enzyme into the pretreated sophora flower bud product, wherein the compound enzyme comprises alpha-amylase, cellulase and hemicellulase; carrying out enzymolysis, and after the enzymolysis is finished, carrying out enzyme deactivation to obtain an enzymolysis product; and (4) adding compound bacteria into the enzymolysis product, carrying out anaerobic fermentation, sterilizing after fermentation, and carrying out spray drying to obtain the pagodatree flower bud powder. According to the preparation method disclosed by the invention, high contents of quercetin and quercetin glucoside in the sophora flower bud powder can be simultaneously kept, and the sophora flower bud powder is good in anti-inflammatory effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant extraction, and particularly relates to a preparation method of sophora japonica powder containing quercetin and quercetin glycoside and application thereof in preparation of respiratory health preparations. BACKGROUND

[0002] Sophora japonica is the dried flower bud of Sophora japonica L. of Leguminosae, which contains rich flavonoids, mainly quercetin and its glycoside derivatives. Quercetin is a natural flavonoid widely existing in plants, which has good antioxidant, anti-inflammatory, antiviral and immunomodulatory activities. Studies have shown that quercetin can protect respiratory tissues by clearing free radicals, inhibiting the release of inflammatory factors, regulating signal pathways, and help to relieve respiratory inflammation.

[0003] Chinese patent CN115997928B discloses an application of soluble sophora japonica powder in preparation of weight loss products. The soluble sophora japonica powder is obtained by enzymatic treatment of sophora japonica. The soluble sophora japonica powder is directly or indirectly used to prepare products for controlling body weight and reducing obesity. The preparation method comprises the following steps: heating extraction, enzymatic reaction, enzyme inactivation and drying to obtain the soluble sophora japonica powder.

[0004] Quercetin glycoside refers to a compound formed by the connection of quercetin and one or more sugar molecules through quercetin glycoside bond. Rutin is one of the main quercetin glycoside components in sophora japonica powder. Quercetin has low bioavailability as a glycoside form, but can directly play a role. Rutin needs to be converted into quercetin in the body to play a major role, which can improve the activity of endogenous antioxidant enzymes and has a lasting effect. Isoquercitrin is a single quercetin glycoside of quercetin, and has higher bioavailability than rutin and quercetin. Therefore, only the sophora japonica powder related extraction product with high content of quercetin and quercetin monoglucoside can ensure the effect and easy absorption. The sophora japonica powder obtained by microbial enzymatic treatment in the prior art has improved quercetin content, but has low bioavailability and cannot generate higher content of isoquercitrin, resulting in poor effect of sophora japonica product. SUMMARY

[0005] The application aims to provide a preparation method of sophora japonica powder containing quercetin and quercetin glycoside and application thereof in preparation of respiratory health preparations.

[0006] In order to achieve the above-mentioned purpose, the application provides the following technical solutions. A preparation method of sophora japonica powder containing quercetin and quercetin glycoside, comprising the following steps: (1) crushing sophora japonica to obtain sophora japonica powder, (2) Mix the Sophora japonica powder with water, heat it, then cool it down and microwave it to obtain the pretreated Sophora japonica product; (3) Add a complex enzyme to the pretreated Sophora japonica product. The complex enzyme includes α-amylase, cellulase and hemicellulase. Perform enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme to obtain the enzymatic hydrolysis product. (4) Add compound bacteria, including Lactobacillus branii and Lactobacillus gasseri, to the enzymatic hydrolysis product for anaerobic fermentation. After fermentation, sterilize and spray dry to obtain Sophora japonica powder.

[0007] Preferably, in step (1), the material is pulverized to a size of less than 60 mesh.

[0008] Preferably, the heating conditions in step (2) are: first heat to 80-90℃ and keep warm for 20-30 minutes.

[0009] Preferably, the microwave processing power in step (2) is 400W-500W and the processing time is 20min-30min.

[0010] Preferably, in step (3), the amount of the compound enzyme used in the pretreated Sophora japonica product is 110-120 U / mL; the enzyme activity ratio of α-amylase, cellulase and hemicellulase is 1:(0.2-0.4):(1.4-1.7).

[0011] Preferably, the enzymatic hydrolysis conditions in step (3) are: enzymatic hydrolysis at 40-45℃ and pH 6.2-6.5 for 3-5 hours.

[0012] Preferably, the compound bacteria in step (4) include Lactobacillus glutenosa and Lactobacillus gasseri.

[0013] Preferably, in step (4), the amount of the compound bacteria in the enzymatic hydrolysis product is 10. 7 -10 8 The CFU / mL ratio of viable Lactobacillus branii to Lactobacillus gasseri was (2-3):1.

[0014] Preferably, the fermentation conditions in step (4) are: anaerobic fermentation at 30-40℃ for 3-5 days.

[0015] This invention provides a method for preparing Sophora japonica powder containing quercetin and quercetin glycosides, and its application in the preparation of respiratory tract improvement formulations. The formulations are not limited to food, health products, or drugs.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This invention provides a method for preparing Sophora japonica bud powder containing quercetin and quercetin glycosides. The method utilizes a dual biotransformation technology of enzymatic hydrolysis and fermentation to deeply process Sophora japonica buds, significantly improving the extraction efficiency of the key active ingredient—quercetin—and its glycosides. Compared with traditional processing techniques, this method can effectively destroy the cell wall structure of Sophora japonica buds, releasing the encapsulated active substances. At the same time, through the action of enzymes and bacteria, some macromolecular flavonoids are converted into smaller molecules that are more easily absorbed, thereby greatly improving their bioavailability.

[0017] 2. In the enzymatic hydrolysis process, the present invention uses a specific complex enzyme system that can directionally cleave the polysaccharide structure in the plant cell wall, so as to fully expose the active ingredients. In the subsequent fermentation process, the complex bacteria are used for mild fermentation, which not only helps to further degrade complex components, but also generates a small amount of organic acids and metabolites, enhancing the antioxidant, anti-inflammatory and other functional properties of the product.

[0018] 3. This invention optimizes the extraction process to achieve a synergistic ratio of quercetin and its glycoside form (quercetin monosaccharide) in extracts related to Sophora japonica powder. Since quercetin, as an aglycone, can be rapidly absorbed and exert anti-inflammatory and antioxidant effects, while quercetin monosaccharide requires enzymatic hydrolysis in vivo to be converted into quercetin before slow release, thus prolonging the duration of action. The synergistic presence of both not only improves overall bioavailability but also ensures the immediacy and durability of the anti-inflammatory effect.

[0019] 4. This synergistic mechanism demonstrates significant advantages in respiratory health. Furthermore, both quercetin and rutin have the potential to regulate immune balance, and can help alleviate abnormal immune responses in respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Therefore, the Sophora japonica powder containing high levels of quercetin and quercetin glycosides described in this invention shows promising application prospects in the prevention and improvement of respiratory diseases, and is particularly suitable for daily nutritional support and health management of susceptible populations such as children, the elderly, and those exposed to air pollution. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] All raw materials used in the following embodiments of the present invention are commercially available products: *Lactobacillus fragrans* (strain number HZB231387), *Lactobacillus gasseri* (strain number HZB138217), *Aspergillus niger* (strain number HZB222717), *Bifidobacterium longum* (strain number HZB231326), and *Lactobacillus acidophilus* (strain number HZB359990) are all from Zhengzhou Fangjue Biotechnology Co., Ltd. *Lactobacillus plantarum* (strain number BNCC361473) is from Beina Chuanglian Biotechnology Co., Ltd. *Lactobacillus rhamnosus* (strain number HZB115392) is from the Hangzhou branch of Wuhan Gray Algae Biotechnology Co., Ltd. *Lactobacillus bulgaricus* (strain number HZB116558) is from the Hangzhou branch of Wuhan Gray Algae Biotechnology Co., Ltd. α-Amylase (strain number S10002) is from Shanghai Yuanye Biotechnology Co., Ltd. Cellulase is from Shanghai Yuanye Biotechnology Co., Ltd. Hemicellulase is from Shanghai Yuanye Biotechnology Co., Ltd.

[0022] Example 1 This embodiment provides a method for preparing Sophora japonica powder containing quercetin and quercetin glycosides, including the following steps: (1) Grind the Sophora japonica flowers to a finer mesh than 60 to obtain Sophora japonica flower powder. (2) Mix Sophora japonica powder and water at a mass ratio of 1:12, heat to 85°C, keep warm for 25 min, then cool to room temperature and microwave process. The microwave process has a power of 450W and a processing time of 25 min to obtain pretreated Sophora japonica product. (3) Add a complex enzyme to the pretreated Sophora japonica flower product. The complex enzyme includes α-amylase, cellulase and hemicellulase. The amount of complex enzyme used in the pretreated Sophora japonica flower product is 115 U / mL. The enzyme activity ratio of α-amylase, cellulase and hemicellulase is 1:0.3:1.6. The enzyme is inactivated by enzymatic hydrolysis at 42℃ and pH6.4 for 4 hours to obtain the enzymatic hydrolysis product. (4) Add a compound bacteria to the enzymatic hydrolysis product. The compound bacteria include Lactobacillus flavus and Lactobacillus gasseri. The amount of the compound bacteria in the enzymatic hydrolysis product is 5 × 10⁻⁶. 7 The live bacteria count ratio of Lactobacillus branii and Lactobacillus gasseri was 2.5:1. The mixture was anaerobic fermented at 37℃ for 4 days. After fermentation, the mixture was sterilized and spray-dried to obtain Sophora japonica powder.

[0023] Example 2 This embodiment provides a method for preparing Sophora japonica powder containing quercetin and quercetin glycosides, including the following steps: (1) Grind the Sophora japonica flowers to a finer mesh than 60 to obtain Sophora japonica flower powder. (2) Mix Sophora japonica powder and water at a mass ratio of 1:10, heat to 90°C, keep warm for 20 min, then cool down and microwave process. The microwave process has a power of 500W and a processing time of 20 min to obtain the pretreated Sophora japonica product. (3) Add a complex enzyme to the pretreated Sophora japonica flower product. The complex enzyme includes α-amylase, cellulase and hemicellulase. The amount of complex enzyme used in the pretreated Sophora japonica flower product is 120 U / mL. The enzyme activity ratio of α-amylase, cellulase and hemicellulase is 1:0.2:1.7. The enzyme is inactivated by enzymatic hydrolysis at 40℃ and pH6.5 for 3 h to obtain the enzymatic hydrolysis product. (4) Add a compound bacteria to the enzymatic hydrolysis product. The compound bacteria include Lactobacillus flavus and Lactobacillus gasseri. The amount of the compound bacteria in the enzymatic hydrolysis product is 10. 8 The live bacteria count ratio of Lactobacillus branii and Lactobacillus gasseri was 2:1. The mixture was anaerobic fermented at 37℃ for 3 days. After fermentation, the mixture was sterilized and spray-dried to obtain Sophora japonica powder.

[0024] Example 3 This embodiment provides a method for preparing Sophora japonica powder containing quercetin and quercetin glycosides, including the following steps: (1) Grind the Sophora japonica flowers to a finer mesh than 60 to obtain Sophora japonica flower powder. (2) Mix Sophora japonica powder and water at a mass ratio of 1:15, heat to 80°C, keep warm for 30 min, then cool down and microwave process. The microwave process has a power of 400W and a processing time of 30 min to obtain the pretreated Sophora japonica product. (3) Add a complex enzyme to the pretreated Sophora japonica flower product. The complex enzyme includes α-amylase, cellulase and hemicellulase. The amount of complex enzyme used in the pretreated Sophora japonica flower product is 110 U / mL. The enzyme activity ratio of α-amylase, cellulase and hemicellulase is 1:0.4:1.4. The enzyme is inactivated after 5 h of enzymatic hydrolysis at 45℃ and pH 6.2 to obtain the enzymatic hydrolysis product. (4) Add a compound bacteria to the enzymatic hydrolysis product. The compound bacteria include Lactobacillus flavus and Lactobacillus gasseri. The amount of the compound bacteria in the enzymatic hydrolysis product is 10. 7 The live bacteria count of Lactobacillus branii and Lactobacillus gasseri was 3:1 (CFU / mL). The mixture was anaerobic fermented at 37℃ for 5 days. After fermentation, the mixture was sterilized and spray-dried to obtain Sophora japonica powder.

[0025] Comparative Example 1 The difference between this comparative example and Example 1 is that the order of enzymatic hydrolysis and fermentation is different.

[0026] A method for preparing Sophora japonica flower powder containing quercetin and quercetin glycosides includes the following steps: (1) Grind the Sophora japonica flowers to a finer mesh than 60 to obtain Sophora japonica flower powder. (2) Mix Sophora japonica powder and water at a mass ratio of 1:12, heat to 85°C, keep warm for 25 min, then cool down and microwave process. The microwave process has a power of 450W and a processing time of 25 min to obtain the pretreated Sophora japonica product. (4) Add a compound bacteria to the pretreated Sophora japonica flower product. The compound bacteria include Lactobacillus flavus and Lactobacillus gasseri. The amount of the compound bacteria in the pretreated Sophora japonica flower product is 5 × 10⁻⁶. 7 The live bacteria count ratio of Lactobacillus branii and Lactobacillus gasseri was 2.5:1. The bacteria were anaerobic fermented at 37℃ for 4 days. After fermentation, the bacteria were sterilized to obtain the enzymatic hydrolysate. (3) Add a complex enzyme to the enzymatic hydrolysis product. The complex enzyme includes α-amylase, cellulase and hemicellulase. The amount of complex enzyme used in the pretreated Sophora japonica product is 115 U / mL. The enzyme activity ratio of α-amylase, cellulase and hemicellulase is 1:0.3:1.6. The product is enzymatically hydrolyzed at 42℃ and pH6.4 for 4 hours, the enzyme is inactivated, and the product is spray-dried to obtain Sophora japonica powder.

[0027] Comparative Example 2 The difference between this comparative example and Example 1 is that the compound bacteria includes Aspergillus niger and Bifidobacterium longum in a live bacteria ratio of 3:2.

[0028] Comparative Example 3 The difference between this comparative example and Example 1 is that the compound bacteria includes Lactobacillus plantarum and Lactobacillus rhamnosus with a live bacteria ratio of 2.5:1.

[0029] Comparative Example 4 The difference between this comparative example and Example 1 is that the compound bacteria includes Lactobacillus bulgaricus and Lactobacillus acidophilus with a live bacteria ratio of 2.5:1.

[0030] Comparative Example 5 The difference between this comparative example and Example 1 is that the complex enzyme includes cellulase, β-glucosidase and pectinase (all from Shanghai Yuanye Biotechnology Co., Ltd.), the amount of complex enzyme used in the pretreated Sophora japonica product is 115 U / mL, and the enzyme activity ratio of cellulase, β-glucosidase and pectinase is 1:0.5:1.

[0031] Comparative Example 6 The difference between this comparative example and Example 1 is that the enzyme activity ratio of α-amylase, cellulase and hemicellulase is 0.3:1.6:1.

[0032] Comparative Example 7 The difference between this comparative example and Example 1 is that the enzyme activity ratio of α-amylase, cellulase and hemicellulase is 1.6:1:0.3.

[0033] Comparative Example 8 This comparative example is the Sophora japonica powder prepared in Example 1 of Chinese Patent CN115997928B, which describes the application of soluble Sophora japonica powder in the preparation of weight loss products.

[0034] Performance testing The performance of the Sophora japonica powder prepared in Examples 1-3 and Comparative Examples 1-8 was tested.

[0035] (1) Referring to the method in "Establishment of Detection Method for Effective Components in Fermented Sophora japonica Flower Liquid" (Chen Caiyun et al.), the content of quercetin and quercetin glycoside in Sophora japonica flower powder was determined by HPLC.

[0036] (2) The anti-inflammatory effect of Sophora japonica powder was determined using the CCK method: After cell resuscitation and passage culture, RAW264.7 cells in the logarithmic growth phase were seeded in 96-well plates at a density of 0.8 × 10⁻⁶. 4 Cells were inoculated at 100 μL per well, with three replicates in the uninoculated group as a blank control. After overnight culture and cell adhesion, the supernatant was removed, and 100 μL of Sophora japonica powder (dissolved in cell culture medium) prepared in Examples 1-3 and Comparative Examples 1-8 (final concentration 100 μmol / L) was added to each well, with three replicates for each concentration. Three replicates were also added to each of the uninoculated group and the blank control group. Fresh cell culture medium was added to each well (100 μL per well), and the cells were incubated for 24 h. 10 μL of CCK-8 was added to each well, and the cells were incubated at 37°C with 5% CO2 for 2 h. The absorbance (OD value) was measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: Cell viability = (A with drug - A blank) / (A without drug - A blank) × 100%.

[0037] The results are shown in Table 1.

[0038] Table 1 Performance Test Results

[0039] As shown in Table 1, the Sophora japonica powder in Examples 1-3 has a high content of quercetin and quercetin monoglycoside, and has a good anti-inflammatory effect.

[0040] Comparative Example 1 shows that the order of enzymatic hydrolysis and fermentation has a significant impact on the content of quercetin monosaccharides. Enzymatic hydrolysis followed by fermentation can release more quercetin and quercetin monosaccharides from Sophora japonica powder into the solution. Although some rutin will be converted into quercetin during the microbial treatment, the content of quercetin monosaccharides in Sophora japonica powder can still be maintained at a high level, which helps Sophora japonica powder to have a long-lasting anti-inflammatory effect.

[0041] Comparative Examples 2-8 show that the use of specific enzymes in enzymatic hydrolysis and specific bacteria in fermentation affects the content of quercetin and quercetin monoglycosides in Sophora japonica flower powder. Comparative Example 3 produced Sophora japonica flower powder with a high quercetin content but a very low quercetin monoglycoside content. Analysis suggests that under these conditions, the quercetin naturally present in the Sophora japonica flower cannot be effectively released, and during fermentation, more rutin is converted to quercetin. Although the quercetin content is higher, the quercetin monoglycoside content is lower. Comparative Example 5, using different enzymes for enzymatic hydrolysis, showed a higher quercetin monoglycoside content but a lower quercetin content. Analysis suggests that the enzymatic hydrolysis system affects the activity of subsequent bacteria, thus affecting the efficiency of quercetin monoglycoside conversion to quercetin through fermentation. The specific mechanism requires further investigation.

[0042] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing Sophora japonica flower powder containing quercetin and quercetin glycoside, characterized in that, Includes the following steps: (1) Crush the Sophora japonica flowers to obtain Sophora japonica flower powder. (2) Mix the Sophora japonica powder with water, heat it, then cool it down and microwave it to obtain the pretreated Sophora japonica product; (3) Add a complex enzyme to the pretreated Sophora japonica product. The complex enzyme includes α-amylase, cellulase and hemicellulase. Enzymatic hydrolysis is performed, and after the enzymatic hydrolysis is completed, the enzyme is inactivated to obtain the enzymatic hydrolysis product. (4) Add compound bacteria, including Lactobacillus branii and Lactobacillus gasseri, to the enzymatic hydrolysis product for anaerobic fermentation. After fermentation, sterilize and spray dry to obtain Sophora japonica powder.

2. The method for preparing Sophora japonica powder containing quercetin and quercetin glycoside according to claim 1, characterized in that, In step (1), the material is pulverized to a size less than 60 mesh.

3. The method for preparing Sophora japonica powder containing quercetin and quercetin glycoside according to claim 1, characterized in that, The conditions for the heat treatment in step (2) are: first heat to 80-90℃ and keep warm for 20-30 minutes.

4. The method for preparing Sophora japonica powder containing quercetin and quercetin glycoside according to claim 1, characterized in that, The microwave processing power in step (2) is 400W-500W, and the processing time is 20min-30min.

5. The method for preparing Sophora japonica powder containing quercetin and quercetin glycoside according to claim 1, characterized in that, In step (3), the amount of compound enzyme used in the pretreated Sophora japonica product is 110-120 U / mL; the enzyme activity ratio of α-amylase, cellulase and hemicellulase is 1:(0.2-0.4):(1.4-1.7).

6. The method for preparing Sophora japonica powder containing quercetin and quercetin glycoside according to claim 1, characterized in that, The conditions for enzymatic hydrolysis in step (3) are: 40-45℃, pH 6.2-6.5 for 3-5 hours.

7. The method for preparing Sophora japonica powder containing quercetin and quercetin glycoside according to claim 1, characterized in that, The compound bacteria in step (4) include Lactobacillus glutenosa and Lactobacillus gasseri.

8. The method for preparing Sophora japonica powder containing quercetin and quercetin glycoside according to claim 1, characterized in that, In step (4), the amount of the compound bacteria in the enzymatic hydrolysis product is 10. 7 -10 8 The CFU / mL ratio of viable Lactobacillus branii to Lactobacillus gasseri was (2-3):

1.

9. The method for preparing Sophora japonica powder containing quercetin and quercetin glycoside according to claim 1, characterized in that, The fermentation conditions in step (4) are: anaerobic fermentation at 30-40℃ for 3-5 days.

10. The application of the Sophora japonica powder prepared by the method for preparing Sophora japonica powder containing quercetin and quercetin glycoside as described in any one of claims 1-9 in the preparation of a preparation for improving respiratory health.

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