Ginkgo functional edible gel product for relieving airway inflammation and preparation method thereof

Abstract

The invention discloses a ginkgo functional edible gel product for relieving airway inflammation and a preparation method of the ginkgo functional edible gel product. The ginkgo functional edible gel product is prepared from the following components in percentage by mass: 5.15% of ginkgo ethyl acetate extract, 8% of ginkgo RS3 type resistant starch, 2% of ginkgo polypeptide, 10% of gelatin, 15% of malt syrup, 0.19% of acidity adjusting solution and 0.3% of momordica grosvenori glucoside. According to the invention, an edible gel product is used as a carrier, and ginkgo seed ethyl acetate extract and ginkgo seed RS3 type resistant starch with functional activity of relieving airway inflammation, ginkgo seed polypeptide, low-calorie and decayed tooth-resistant momordica grosvenori glucoside and malt syrup are added as sweetening agents; according to the invention, gingko is used as a raw material to prepare an edible gel product containing gingko micromolecular active substances, resistant starch and other multi-component functional components, so that the application range of gingko resources is expanded, the utilization rate of each component of gingko is improved, and the variety of functional leisure food is enriched at the same time.

Description

Technical Field

[0001] This invention relates to the field of food processing technology for gummy candies containing plant extracts, and more particularly to a functional edible gel product made from ginkgo seeds that relieves airway inflammation and its preparation method. Background Technology

[0002] Chronic airway inflammation is a major pathological feature of chronic respiratory diseases. Currently, the focus of chronic respiratory disease treatment has shifted from solely treating acute exacerbations to preventing and treating chronic airway inflammation. Many traditional medications are used to treat chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD), including anticholinergics, glucocorticoids, β2-receptor agonists, sustained-release theophylline, and leukotriene modifiers. However, these medications have certain toxic side effects, such as palpitations, tremors, headaches, elevated blood pressure, cataracts, and osteoporosis. In contrast, extracts from traditional Chinese medicine and natural active ingredients are relatively safe for the body and show great potential for improving bodily functions. Therefore, developing a dietary supplement based on the active ingredients of food and medicine homologous substances to alleviate airway inflammation is of great significance for the adjunctive treatment of chronic respiratory diseases.

[0003] Ginkgo seeds, also known as silver apricot kernels or duck's foot seeds, are the mature, dried seeds of the ginkgo biloba L., a plant in the Ginkgoaceae family. They consist of an outer seed coat, a middle seed coat, an inner seed coat, and a kernel. In 1992, they were listed as a "dual-use" resource (medicinal and edible) by the Ministry of Health of my country. Ginkgo seeds have been used in my country for nearly a thousand years as both a traditional Chinese medicine and a food resource. As early as the Song Dynasty, they were listed as a royal tribute and could be made into snacks, candied fruits, and other foods. The earliest record of the pharmacological activity of ginkgo seeds is found in the Yuan Dynasty's *Daily Materia Medica*. Li Shizhen's *Compendium of Materia Medica* records that "cooked ginkgo seeds warm the lungs, benefit qi, relieve asthma and cough, reduce urination, and stop leukorrhea; raw ginkgo seeds reduce phlegm, relieve alcohol intoxication, disinfect, and kill parasites." The 2015 edition of the *Chinese Pharmacopoeia* also records that ginkgo seeds "have the effects of astringing the lungs, relieving asthma, stopping leukorrhea, and reducing urination." The Dingchuan Decoction, with ginkgo seeds as the principal ingredient, from *Prescriptions for Nourishing Life*, is a classic formula in modern TCM clinical treatment of asthma. Modern research indicates that ginkgo seeds not only contain nutrients such as protein, fat, and starch, but also active substances such as ginkgo flavonoids, ginkgo lactones, and ginkgo polysaccharides, which have various pharmacological activities such as anti-inflammatory, antioxidant, antibacterial, insecticidal, lipid-regulating, and neuroprotective effects.

[0004] China is the main producing area of ​​ginkgo and also the world's largest ginkgo planting area, accounting for 85% of the world's ginkgo resources. The main planting areas are in Jiangsu, Shandong, and Henan provinces. my country's ginkgo planting area is approximately 400,000 hectares. 2The cultivation area exceeds 2.5 billion ginkgo plants, with an annual ginkgo yield of 60,000 tons (incomplete statistics). Aside from a small amount used in medicine, the majority of ginkgo nuts are processed into low-value-added products such as ginkgo flour, pistachios, and canned ginkgo. The development of high-value-added products, such as dietary supplements with health benefits, is relatively lacking. The existing ginkgo processing industry cannot effectively promote the development of the ginkgo industry, resulting in ginkgo consumption accounting for less than 1% of total production, leading to a serious waste of ginkgo resources. Therefore, developing a ginkgo dietary supplement with airway inflammation-relieving effects is of great significance for the development and utilization of ginkgo resources. Summary of the Invention

[0005] Purpose of the invention: In view of the existing problems in the ginkgo market, such as the limited variety of products, low added value, and serious waste of ginkgo resources, this invention provides a high-value-added functional edible gel product made from ginkgo. The development of this functional edible gel product from ginkgo enriches the variety of ginkgo products, provides a new way for the development and utilization of ginkgo resources, and also enriches the category of functional edible gel products.

[0006] The present invention also provides a method for preparing ginkgo RS3 type resistant starch and a method for preparing ginkgo polypeptide.

[0007] Technical solution: In order to achieve the above objectives, the present invention provides a functional edible gel product of ginkgo that relieves airway inflammation, comprising ginkgo ethyl acetate extract, ginkgo RS3 resistant starch, and ginkgo polypeptide.

[0008] The ingredients include ginkgo ethyl acetate extract, ginkgo RS3 resistant starch, ginkgo polypeptide, gelatin, malt syrup, acidity regulator, and sweetener.

[0009] The ingredients, by mass percentage, include: 5-6% ginkgo ethyl acetate extract; 5-10% ginkgo RS3 resistant starch; 1-3% ginkgo polypeptide; 5-15% gelatin; 10-20% malt syrup; 0.1-0.3% acidity regulator; 0.1-0.5% sweetener; and the balance being water.

[0010] As a preferred embodiment, by weight percentage, it comprises: 5.15% ginkgo ethyl acetate extract; 8% ginkgo RS3 resistant starch; 2% ginkgo polypeptide; 10% gelatin; 15% malt syrup; 0.19% acidity regulator; 0.3% sweetener; and the balance being water.

[0011] As a preferred embodiment, the functional edible gel product for relieving airway inflammation contains ginkgo ethyl acetate extract, ginkgo RS3 resistant starch, ginkgo polypeptide, gelatin, malt syrup, acidity regulator and mogroside, with a mass percentage of 5.15:8:2:10:15:0.19:0.3.

[0012] This invention uses specific raw materials such as gelatin or malt syrup, while using other gelling agents will result in poor effects, and the sensory properties and textural characteristics of the product will be changed after using other syrups.

[0013] The preparation method of the ginkgo functional edible gel product for relieving airway inflammation according to the present invention includes the following steps:

[0014] (1) Sol: Mix gelatin with pure water, soak until the gelatin is fully swollen, stir until completely dissolved, and prepare a gelatin solution;

[0015] (2) Sweetness adjustment solution: Dissolve the sweetener in purified water to prepare a sweetness adjustment solution, and let it stand at room temperature for later use;

[0016] (3) Acidity adjustment solution: Dissolve the acidity regulator (tartaric acid: citric acid: sodium citrate = 4:8:3) in purified water to prepare an acidity adjustment solution, and store it at room temperature for later use;

[0017] (4) Gum base: Add Ginkgo RS3 resistant starch to gelatin solution, stir to dissolve, add Ginkgo polypeptide after complete dissolution, and continue stirring until completely dissolved to prepare a gel base solution for later use;

[0018] (5) Cooking sugar: Add malt syrup to purified water, heat and stir. After the syrup and water are evenly mixed, continue to cook until the sugar solution is slightly viscous and can be stretched into threads.

[0019] (6) Boiling: Add the prepared gum base solution in step (4) to the syrup prepared in step (5), stir evenly, and boil to make mixture A. Cool mixture A.

[0020] (7) Mixing and standing: Add the ginkgo ethyl acetate extract to the mixed solution A prepared in step (6), stir evenly, then add the sweetness adjustment solution prepared in step (2) and the acidity adjustment solution prepared in step (3), and continue stirring to make soft candy material;

[0021] (8) Molding: Pour the prepared soft candy material from step (7) into the mold while it is still hot, and demold to obtain the ginkgo functional edible gel product that relieves airway inflammation.

[0022] Preferably, the preparation method of the ginkgo functional gummies for relieving airway inflammation according to the present invention includes the following steps:

[0023] (1) Sol: Mix 10% of the gelatin in the formula with an equal amount of pure water, soak for 30 min, and stir continuously in a 45°C water bath until completely dissolved to prepare a gelatin solution and keep it at 45°C for later use.

[0024] (2) Sweetness adjustment solution: Dissolve 0.3% of the sweetener mogroside in the formula with an appropriate amount of water to make a sweetness adjustment solution, and leave it at room temperature for later use.

[0025] (3) Acidity adjustment solution: Dissolve 0.19% of the acidity regulator (tartaric acid: citric acid: sodium citrate = 4:8:3) in an appropriate amount of water to make an acidity adjustment solution, and store it at room temperature for later use.

[0026] (4) Gum base: Add 8% by mass of Ginkgo RS3 resistant starch in the formula to the gelatin solution, stir and dissolve in a water bath at 45°C. After complete dissolution, add 2% by mass of Ginkgo polypeptide in the formula, and continue stirring until completely dissolved to prepare a gel base solution and keep it at 45°C for later use.

[0027] (5) Cooking sugar: Put 15% of the maltose syrup in the recipe into a beaker, add 2 times the amount of pure water, heat and stir at the same time. After the syrup and pure water are evenly mixed, continue to cook at 85°C for 15 minutes until the sugar solution is slightly viscous and can be stretched into threads.

[0028] (6) Boiling: Add the gel-based solution prepared in step (4) to the syrup prepared in step (5), stir evenly, and continue to boil over low heat for 10 minutes to make mixture A. Cool mixture A to 45°C.

[0029] (7) Mixing and standing: Add 5.15% by mass of the ginkgo ethyl acetate extract in the formula to the mixed solution A prepared in step 4S6, stir at 45°C for 5 min, and after stirring evenly, add the sweetness adjustment solution prepared in step 2 and the acidity adjustment agent solution prepared in step 3, and continue stirring for 5 min to make soft candy material.

[0030] (8) Molding: Pour the gummy material prepared in step (7) into the mold while it is still hot, gently tap the mold to remove air bubbles and distribute the material evenly in the mold, and demold after refrigeration to obtain ginkgo functional gummy that relieves airway inflammation.

[0031] The ethyl acetate extract of ginkgo biloba in step (7) is prepared according to the following method:

[0032] Ginkgo kernel powder was mixed with ethanol solution and then heated and shaken for extraction. After extraction, the mixture was filtered and the residue was dried to obtain ginkgo residue. The filtrate was concentrated under reduced pressure to obtain ginkgo alcohol extract. The ginkgo alcohol extract was resuspended and extracted successively with petroleum ether and ethyl acetate. The ethyl acetate extracts were collected and combined and concentrated under reduced pressure to obtain ginkgo ethyl acetate extract.

[0033] Preferably, the extraction process of the ginkgo ethyl acetate extract in step (7) is as follows: dried ginkgo powder and 60-85% ethanol are added to a beaker at a ratio of 1:5 to 1:25 (m / v), mixed well, and then the beaker is placed in a water bath shaker at 50-70℃ for 0.5-2.5 h. After extraction, the mixture is filtered, and the residue is dried in an oven at 45℃ to obtain ginkgo residue. The filtrate is concentrated under reduced pressure to obtain ginkgo ethanol extract. The ginkgo ethanol extract is resuspended in an appropriate amount of distilled water and extracted successively with petroleum ether (boiling range: 30-60℃) and ethyl acetate, repeated 4 times each. The ethyl acetate extracts are collected and combined, and concentrated to obtain ginkgo ethyl acetate extract.

[0034] Further, dried ginkgo kernel powder and 80% ethanol solution were placed in a beaker at a ratio of 1:15 (w / v), mixed well, and then the beaker was placed in a 60℃ water bath shaker and extracted at 180 r / min for 2 h. After extraction, the mixture was filtered, and the residue was dried in a 45℃ oven to obtain ginkgo residue. The filtrate was concentrated under reduced pressure to obtain ginkgo ethanol extract. The ginkgo ethanol extract was resuspended in an appropriate amount of distilled water and extracted successively with petroleum ether (boiling range: 30~60 ℃) and ethyl acetate, repeated 4 times. The ethyl acetate extracts were collected and combined, and concentrated under reduced pressure to obtain ginkgo ethyl acetate extract.

[0035] The preparation process of ginkgo polypeptide in step (4) is as follows: Ginkgo pomace after alcohol extraction is defatted, the defatted pomace is mixed with water, and the pH of the suspension is adjusted to 10-11, preferably 10.5. Extraction is carried out at 45℃ for 4 h with stirring. The suspension is then centrifuged at room temperature. After centrifugation, the precipitate washing liquid is nearly neutral. The precipitate is dried to obtain ginkgo crude starch. The pH of the collected supernatant is adjusted to 4-5, preferably 4.5, and then allowed to settle at room temperature. The precipitate is collected by centrifugation, washed until the washing liquid is neutral, and then freeze-dried to obtain ginkgo protein. The ginkgo protein is prepared into a ginkgo protein solution with buffer solution, heated, and then enzymatically hydrolyzed with alkaline protease. After enzyme inactivation, the solution is centrifuged at room temperature, the supernatant is collected, dialyzed, and then freeze-dried to obtain ginkgo polypeptide.

[0036] Preferably, the preparation process of ginkgo peptides in step (4) is as follows: Ginkgo pomace after alcohol extraction is mixed with petroleum ether (boiling range: 30~60 ℃) at a ratio of 1:15 (m / v) for defatting for 12 h, repeated 3 times. The defatted ginkgo pomace is then added to a beaker with distilled water at a ratio of 1:25 (m / v). The pH of the suspension is adjusted to 10.5 using 1 M sodium hydroxide solution and 1 M hydrochloric acid solution. The beaker is then placed in a constant temperature stirring oil bath and stirred at 45 ℃ for 4 h. After extraction, the suspension is allowed to stand at room temperature for 2 h. After returning to room temperature, it is centrifuged at 12000 rpm for 15 min. The precipitate is washed with distilled water until the washing solution is nearly neutral. The precipitate is then dried at 45 ℃ to obtain crude ginkgo starch. The pH of the collected supernatant is adjusted to 4.5 using 1 M sodium hydroxide solution and 1 M hydrochloric acid solution, and then allowed to stand at room temperature for 2 h to allow protein precipitation. Centrifuge at 12000 rpm for 15 min. Collect the precipitate after centrifugation, wash the precipitate with distilled water until the washing solution is neutral, and then freeze-dry the precipitate to obtain ginkgo protein. Mix the ginkgo protein with phosphate buffer with a pH of 10 to prepare a 2% ginkgo protein solution. Heat to 40℃, add alkaline protease (5000 U / g) to the ginkgo protein solution for enzymatic hydrolysis for 4 h, inactivate the enzyme with boiling water for 10 min, let the enzymatic hydrolysate stand at room temperature for 1 h, then centrifuge at 12000 rpm for 15 min, collect the supernatant, dialyze at 4℃ for 48 h to desalt, and then freeze-dry to obtain ginkgo polypeptide.

[0037] Preferably, the pH value of the alkaline protease hydrolysis of ginkgo protein is 10.0, the enzyme addition amount is 5000 U / g, the hydrolysis temperature is 40℃, the hydrolysis time is 4 h, and the substrate concentration of ginkgo protein is 2%.

[0038] The RS3 resistant starch of ginkgo in step (4) is prepared by the following process: the crude ginkgo starch remaining after removing protein is mixed with buffer solution and stirred at room temperature to make a starch suspension. The suspension is subjected to pressure heating and then cooled. After stirring evenly, the pH value is adjusted, pullulanase is added for enzymatic hydrolysis, the enzyme is inactivated and cooled to room temperature and then refrigerated. The precipitate is obtained by centrifugation, the precipitate is washed three times, the precipitate is collected and freeze-dried to obtain RS3 resistant starch.

[0039] As a preferred method, the preparation process of the Ginkgo RS3 resistant starch in step (4) is as follows: the crude Ginkgo starch remaining after removing protein is mixed with phosphate buffer (pH 6.0), stirred at room temperature for 30 min to prepare a 20% (m / m) starch suspension, the suspension is subjected to pressure heat treatment and then cooled to 40-60℃, stirred evenly and the pH value is adjusted to 3.0-7.0, then pullulanase (0-5000 U / g) is added for enzymatic hydrolysis for 4-32 h, the enzyme is inactivated by boiling water for 10 min, cooled to room temperature and then refrigerated at 4℃ for 24 h, the precipitate is obtained by centrifugation, the precipitate is washed three times with distilled water, and the precipitate is collected and freeze-dried to obtain RS3 resistant starch.

[0040] Further, the Ginkgo RS3 resistant starch in step (4) is prepared according to the following process: the crude Ginkgo starch remaining after removing protein is mixed with phosphate buffer (pH 6.0), stirred at room temperature for 30 min to prepare a 20% (m / m) starch suspension. The suspension is subjected to pressure heat treatment and then cooled to 55°C. After stirring evenly, the pH value is adjusted to 4.0. Pullulanase (250 U / g) is added for enzymatic hydrolysis for 16 h. The enzyme is inactivated by boiling water for 10 min. After cooling to room temperature, it is refrigerated at 4°C for 24 h. The precipitate is obtained by centrifugation. The precipitate is washed three times with distilled water. The precipitate is collected and freeze-dried to obtain RS3 resistant starch.

[0041] In step (1), the food glue used for the sol is gelatin, and the preferred gelatin mass percentage is 10%.

[0042] In step (4), the mass percentage of Ginkgo RS3 resistant starch used to prepare the gel base is 2%~10%, and the mass percentage of Ginkgo polypeptide is 1%~5%.

[0043] Preferably, the mass percentage of Ginkgo RS3 resistant starch is 8%, and the mass percentage of Ginkgo polypeptide is 2%.

[0044] In step (5), the malt syrup has a mass percentage of 5-25%, and preferably 15%.

[0045] In step (6), the temperature for boiling the ginkgo edible gel product material is 70-90℃ and the time is 0-40min. Preferably, the temperature for boiling the ginkgo functional edible gel product material is 85℃ and the time is 10min.

[0046] In step (2), the sweetener is mogroside; in step S3, the acidity regulator solution is composed of tartaric acid, citric acid, and sodium citrate in a mass ratio of 4:8:3. In step (4), the gum base is prepared from gelatin, ginkgo RS3 resistant starch, and ginkgo polypeptide; the mass ratio of gelatin, ginkgo RS3 resistant starch, and ginkgo polypeptide is 10:8:2.

[0047] In step (7), the mass percentage of the ginkgo functional extract is 2.58%-12.88%, the mass percentage of the sweetness regulator mogroside is 0.1%-0.5%, the mass percentage of the acidity regulator (tartaric acid: citric acid: sodium citrate = 4:8:3) is 0.09%-1.5%, and preferably the mass percentage of the ginkgo functional extract is 5.15%, the mass percentage of the sweetness regulator is 0.3%, and the mass percentage of the acidity regulator (tartaric acid: citric acid: sodium citrate = 4:8:3) is 0.19%.

[0048] The process includes the following steps after step S8: after placing the desiccator in the packaging container before packaging, take out the packaging container, put in the gummies, and immediately seal it with plastic seal.

[0049] To comprehensively promote the development of the ginkgo processing industry, this invention focuses on developing a widely applicable health food product that can alleviate airway inflammation: an edible ginkgo gel product. To better leverage the airway inflammation-relieving effects of ginkgo, this invention employs multi-stage stepwise extraction of the ginkgo's active ingredients, obtaining a concentrated extract (ethyl acetate extract of ginkgo). This is the first time this component has been used to develop an edible ginkgo health food. This invention further explores the resources of the ginkgo residue remaining after preparing the active ingredients. We found that the main components of the residue are ginkgo protein (13.65%) and ginkgo starch (82.07%). Therefore, we used an alkaline dissolution and acid precipitation method to separate ginkgo protein and ginkgo starch. Ginkgo protein has a certain degree of allergenicity and low activity. Ginkgo starch consumption leads to elevated blood sugar. Therefore, to meet the attributes of a health food, we converted ginkgo starch into resistant ginkgo starch, which can slow down the rise in blood sugar, promote intestinal health, and alleviate inflammation; and we converted ginkgo protein into ginkgo polypeptides, which can regulate the body's immunity. Furthermore, resistant starch and peptides from ginkgo are added to edible gel products made from ginkgo. Taking the comprehensive utilization of all components of ginkgo and its tiered high-end processing as the starting point, we will carry out key technology innovation research and development and integration maturation for the tiered high-value utilization of all components of ginkgo. This will provide technical support for breaking the stagnation of the ginkgo processing industry, promoting more high-end ginkgo products to enter the market, and ensuring the healthy development of the ginkgo processing industry.

[0050] This invention is the first to utilize the concentrated active components of ginkgo to alleviate airway inflammation in the development of an edible ginkgo gel product. This invention is also the first to utilize ginkgo residue to prepare ginkgo resistant starch and ginkgo polypeptides, achieving comprehensive utilization of all ginkgo components. Both the ginkgo ethyl acetate extract and the ginkgo resistant starch in the product prepared in this invention have the effect of alleviating airway inflammation. This invention is also the first to propose converting the remaining ginkgo residue from the preparation of active ginkgo components into ginkgo resistant starch and ginkgo polypeptides with certain activity, and adding these components to the edible ginkgo gel product.

[0051] This invention, through research, discovered that both ginkgo ethyl acetate extract and ginkgo resistant starch have good effects in relieving airway inflammation, and ginkgo polypeptides also have good immunomodulatory effects. Therefore, this invention is the first to combine these components to prepare ginkgo edible gel gummies. Animal experiments were conducted on the final ginkgo edible gel product to study its efficacy in relieving airway inflammation. Simultaneously, this invention is the first to combine ginkgo ethyl acetate extract, ginkgo resistant starch, and ginkgo polypeptides to prepare ginkgo edible gel products, and optimized the preparation process parameters to achieve optimal sensory evaluation while ensuring the product's efficacy. The optimal ratio of raw materials for sensory evaluation was sought through a smaller proportion.

[0052] This invention fully utilizes various effective components of ginkgo and prepares them into by-products, ultimately forming the specific ginkgo edible gel product of this invention. Specifically, an ethyl acetate extract of ginkgo was prepared for the first time, exhibiting significantly superior efficacy in relieving airway inflammation compared to conventional ethanol extracts. To fully utilize the remaining ginkgo residue from the ethyl acetate extract, ginkgo polypeptides were obtained, which possess good immunomodulatory effects. Furthermore, ginkgo RS3 resistant starch was prepared using the remaining crude ginkgo starch from the preparation of the ginkgo polypeptides. Further research revealed that it also has a certain effect in relieving airway inflammation, and when combined with the ethyl acetate extract, it further enhances the effect of relieving airway inflammation. Therefore, this invention not only fully utilizes various components of ginkgo, avoiding waste of ginkgo resources, but also develops novel ginkgo products using these resources, showing significant effects in relieving airway inflammation.

[0053] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0054] 1. The ginkgo edible gel product prepared by this invention contains ginkgo ethyl acetate extract, which has a significant effect in relieving airway inflammation in asthma.

[0055] 2. The ginkgo edible gel product prepared by this invention contains ginkgo RS3 type resistant starch, which has significant effects on regulating blood sugar and improving intestinal health.

[0056] 3. This invention uses mogrosides to replace the sweetness provided by sucrose, white sugar, etc. in ordinary soft candies, and does not cause an increase in blood sugar in the human body.

[0057] 4. This invention uses ginkgo ethyl acetate extract and ginkgo RS3 resistant starch as the main functional ingredients, and adds ginkgo polypeptides to prepare a functional edible gel product containing multiple active ingredients to relieve airway inflammation.

[0058] 5. The preparation process of the ginkgo edible gel product of the present invention is simple, easy to operate and control, and has high production efficiency, which is conducive to industrial production. The ginkgo functional edible gel product for relieving airway inflammation has good product stability, food safety and taste.

[0059] 6. The functional edible gel product of ginkgo that relieves airway inflammation prepared by this invention not only contributes to the development of ginkgo-related industries, but also improves the utilization rate of ginkgo and provides consumers with more diverse choices. Attached Figure Description

[0060] Figure 1 A schematic diagram of the standard curves for NO, IL-1β, IL-6, and TNF-α;

[0061] Figure 2 The effect of different polarity extracts of Ginkgo biloba on LPS-induced RAW264.7 cell viability (GBS, ethanol extract of Ginkgo biloba; GBSP, petroleum ether extract of Ginkgo biloba; GBSE, ethyl acetate extract of Ginkgo biloba; GBSB, n-butanol extract of Ginkgo biloba; GBSW, aqueous extract of Ginkgo biloba).

[0062] Figure 3 Schematic diagram of the anti-inflammatory activity of Ginkgo biloba extracts of different polarities (GBS, ethanol extract of Ginkgo biloba; GBSP, petroleum ether extract of Ginkgo biloba; GBSE, ethyl acetate extract of Ginkgo biloba; GBSB, n-butanol extract of Ginkgo biloba; GBSW, aqueous extract of Ginkgo biloba). Compared with the model group, p < 0.001 compared with the blank group;

[0063] Figure 4 A schematic diagram illustrating the anti-inflammatory activity of ginkgo ethyl acetate extract; Compared with the model group, p < 0.001 compared with the blank group;

[0064] Figure 5 The effects of ethanol concentration, extraction temperature, extraction time, and solid-liquid ratio on the anti-inflammatory activity of ginkgo ethyl acetate extract;

[0065] Figure 6 A schematic diagram illustrating the effect of ginkgo ethyl acetate extract (GBSE) on the number of inflammatory cells in the airways of asthmatic mice;

[0066] Figure 7 This is a schematic diagram illustrating the effect of ginkgo ethyl acetate extract (GBSE) on inflammatory cell infiltration in the lung tissue of asthmatic mice.

[0067] Figure 8 A schematic diagram showing the effect of ginkgo ethyl acetate extract (GBSE) on goblet cell proliferation and mucus secretion in the lung tissue of asthmatic mice;

[0068] Figure 9 The effects of enzyme dosage, substrate concentration, pH, temperature, and time on the yield of ginkgo peptides were investigated.

[0069] Figure 10 The effects of temperature, enzyme dosage, hydrolysis time, and pH on resistant starch content were investigated.

[0070] Figure 11 A schematic diagram illustrating the effect of ginkgo resistant starch (RS) on the number of inflammatory cells in the airways of asthmatic mice; Compared with the model group, p < 0.001 compared with the blank group;

[0071] Figure 12 A schematic diagram illustrating the effect of ginkgo resistant starch (RS) on inflammatory cell infiltration in the lung tissue of asthmatic mice; Compared with the model group, p < 0.001 compared with the blank group;

[0072] Figure 13 A schematic diagram showing the effect of ginkgo resistant starch (RS) on goblet cell proliferation and mucus secretion in the lung tissue of asthmatic mice; Compared with the model group, p < 0.001 compared with the blank group;

[0073] Figure 14 A schematic diagram of the standard curve for ginkgo flavonoids;

[0074] Figure 15 This is a schematic diagram of the standard curve for ginkgolides.

[0075] Figure 16 This is a product illustration of ginkgo edible gel products;

[0076] Figure 17 Schematic diagram showing the effects of ginkgo edible gel products on body weight and organ index in asthmatic mice.

[0077] Figure 18 A schematic diagram illustrating the effect of ginkgo edible gel products on the number of inflammatory cells in the airways of asthmatic mice. Compared with the model group, p < 0.001 compared with the blank group;

[0078] Figure 19 Schematic diagram of the effect of ginkgo edible gel products on inflammatory cell infiltration in the lung tissue of asthmatic mice; Compared with the model group, p < 0.001 compared with the blank group;

[0079] Figure 20 Schematic diagram showing the effects of ginkgo edible gel products on goblet cell proliferation and mucus secretion in the lung tissue of asthmatic mice. Compared with the model group, p < 0.001 compared with the blank group;

[0080] Figure 21 A schematic diagram illustrating the effect of ginkgo edible gel products on the levels of inflammatory factors in the BALF of asthmatic mice; different lowercase letters represent significant differences in the levels of inflammatory factors. Detailed Implementation

[0081] The present invention will be further described below with reference to the accompanying drawings and embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0082] Materials: Ginkgo nuts were harvested on October 1, 2023, in Taizhou City, Jiangsu Province, of the variety Dafozhi. Alkaline protease (FDG-2202, 200,000 U / g) and pullulanase (FDY-2224, 2,000 U / g) were purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd.

[0083] Food additives citric acid, sodium citrate, tartaric acid, gelatin, malt syrup, and mogrosides were purchased from Zhongchen Biotechnology.

[0084] Example 1

[0085] Preparation of Ginkgo Biloba Ethyl Acetate Extract

[0086] (1) Peel the fresh ginkgo seeds, remove the red inner seed coat, freeze them in an ultra-low temperature freezer at -80℃ for 24 h, then transfer them to a vacuum freeze dryer and freeze for 72 h. After pulverizing them in a high-speed grinder, pass them through a 40-mesh sieve to obtain ginkgo kernel powder.

[0087] (2) Add the dried powder of ginkgo kernels and 80% ethanol (v / v) to a beaker at a ratio of 1:15 (g / mL), mix well, place the beaker in a 60℃ water bath shaker, and extract by shaking at 180 rpm for 2 h. After extraction, filter, concentrate the filtrate under reduced pressure to obtain ginkgo alcohol extract (GBS), and dry the filter residue at 45℃ to obtain ginkgo residue.

[0088] (3) After suspending GBS thoroughly in an appropriate amount of distilled water, extract it four times in sequence with equal volumes of petroleum ether (boiling range: 30~60℃), ethyl acetate, and n-butanol, respectively, until the color of the organic solvent layer no longer lightens. The remaining aqueous extract is the water-soluble substance. Collect the petroleum ether extract, ethyl acetate extract, n-butanol extract, and the remaining water-soluble substance obtained above, and then concentrate them under vacuum to obtain petroleum ether extract (GBSP), ethyl acetate extract (GBSE), n-butanol extract (GBSB), and water-soluble substance (GBSW), and store them at -80℃ for later use. The present invention has been verified through subsequent experiments to preferably use the ethyl acetate extract (GBSE) obtained by sequential extraction with equal volumes of petroleum ether (boiling range: 30~60℃) and ethyl acetate.

[0089] Example 2

[0090] Effects of different extracts of ginkgo on LPS-induced viability of RAW264.7 cells

[0091] RAW264.7 cells in logarithmic growth phase were fed at a rate of 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μL per well in 96-well plates and incubated at 37 °C with 5% CO2. Once cell confluence reached 80%-90%, the old complete culture medium was discarded, and the cells were washed twice with PBS. Different polar extracts of Ginkgo biloba were then added to achieve final concentrations of 0, 25, 50, 100, 200, and 400 μg / mL. Next, 100 μg / mL of LPS was added to each well to achieve a final concentration of 1 μg / mL. The plates were gently shaken to mix, and then incubated at 37 °C with 5% CO2 for another 24 h. The culture medium was then discarded, and the plates were preheated to 37 °C and washed twice with PBS. Finally, 100 μL of complete culture medium containing 10% CCK-8 was added to each well. After 1 h of incubation, the absorbance was measured at 450 nm. Five replicates were set up for each drug concentration, and the experiment was repeated three times.

[0092] Example 3

[0093] Evaluation of the anti-inflammatory activity of different extracts from ginkgo

[0094] RAW264.7 cells in logarithmic growth phase were seeded into 12-well plates (1×10⁻⁶ cells per well). 5RAW264.7 cells were cultured in wells at 37°C in a 5% CO2 incubator. After 12 h, the cells were divided into four groups: a control group, a model group, a Dex positive control group, and low, medium, and high dose groups of GBS, GBSP, GBSE, GBSB, and GBSW. The control and model groups received no drug treatment. The experimental groups were treated with GBS, GBSP, GBSE, GBSB, and GBSW at final concentrations of 50, 100, and 200 μg / mL, respectively. The positive control group was treated with Dex solution at a final concentration of 10 μg / mL. The cells were gently shaken to mix the culture medium and incubated for 4 h. Then, 100 μg / mL LPS solution was added to the model, control, and experimental groups to bring the final LPS concentration to 1 μg / mL, and the cells were cultured for 20 h. At the end of the intervention, the cell supernatant was collected from each group, centrifuged at 12000 rpm / min for 10 min at 4°C, and 1 mL of the supernatant was aliquoted and stored at -80°C for later use.

[0095] The NO content in each group of culture media was determined using the Griess method. Following the kit manufacturer's instructions, a certain amount of 1 M NaNO2 standard solution was taken and prepared into 0, 1, 2, 5, 10, 20, 40, 60, and 100 µM standard solutions using complete culture medium as the solvent to establish NO standard curves (e.g., ...). Figure 1 Add 50 μL of supernatant and a series of standard solutions to each well of a 96-well plate. Then, add 50 μL of Griess Reagent I and Griess Reagent II to each well at room temperature, and incubate with shaking for 15 min at room temperature. - The dye undergoes a diazotization coupling reaction with Griess Reagent to generate a pinkish-purple azo dye with strong absorption at 540 nm. The absorbance value is read using an ELISA reader, and the NO content is calculated based on the NO standard curve.

[0096] Example 4

[0097] Anti-inflammatory activity assay of ginkgo ethyl acetate extract.

[0098] (1) RAW264.7 cells in logarithmic growth phase were seeded in 24-well plates (IL-6 and TNF-α, 5×10⁻⁶ cells). 4 / well) and 12-well plate (IL-1β, 1×10) 5RAW264.7 cells were cultured in wells at 37°C in a 5% CO2 incubator. After 12 h, the cells were divided into control, model, Dex positive control, and low, medium, and high dose GBSE groups. The control and model groups received no drug treatment; GBSE was added to the experimental groups at final concentrations of 50, 100, and 200 μg / mL; Dex solution was added to the positive control group at a final concentration of 10 μg / mL. The cells were gently shaken to mix the culture medium and incubated for 4 h. Then, 100 μg / mL LPS solution was added to the model, control, and experimental groups to bring the final LPS concentration to 1 μg / mL, and the cells were cultured for 20 h. At the end of the intervention, the cell supernatant was collected from each group, centrifuged at 12000 rpm / min for 10 min at 4°C, and 1 mL of the supernatant was aliquoted and stored at -80°C for later use.

[0099] (2) The secretion levels of inflammatory factors IL-1β, IL-6, and TNF-α in the culture medium of each group were detected according to the ELISA kit instructions. The concentrations of IL-1β, IL-6, and TNF-α in the cell supernatant of each group were calculated based on the standard curves of IL-1β, IL-6, and TNF-α. The standard curves of IL-1β, IL-6, and TNF-α are shown below. Figure 1 As shown.

[0100] Example 5

[0101] In vivo experiments were conducted to evaluate the effect of ginkgo ethyl acetate extract in relieving airway inflammation in asthma.

[0102] Thirty SPF-grade BABL / c female mice were randomly divided into five groups of six each: a control group, an asthma group (Model group), a dexamethasone group (Dex, 1 mg / kg bw), a high-dose group of ginkgo ethyl acetate extract (GBSEH, 200 mg / kg bw), and a low-dose group of ginkgo ethyl acetate (GBSEL, 100 mg / kg bw). In the modeling group, mice were intraperitoneally injected with 250 μL of OVA sensitization solution on days 0 and 7, respectively. Starting on day 14, mice were challenged by ultrasonic nebulization with 5% OVA solution three times a week for 45 minutes each time, for five consecutive weeks. Thirty minutes before nebulization challenge, mice were administered gavage with ginkgo ethyl acetate extract solution and intraperitoneally injected with dexamethasone solution. The control group was treated with physiological saline in the same manner as the modeling group.

[0103] Example 6

[0104] In Example 5, bronchoalveolar lavage fluid (BALF) was collected from mice in each group 24 hours after the last challenge. The specific steps were as follows: Mice were placed supine on an operating table, and their limbs and teeth were fixed. PBS was slowly injected three times via endotracheal intubation, 0.4 mL each time, followed by slow aspiration, for a total of 1 mL of BALF. The collected BALF was centrifuged at 1500 rpm / min for 10 min at 4°C. The supernatant was aliquoted and stored at -80°C for subsequent cytokine assays. The cell pellet was used for cell classification and counting in the BALF.

[0105] Example 7

[0106] Mouse lung tissue pathological section staining

[0107] Following bronchoalveolar lavage in mice, the thoracic cavity was opened, and a needle connected to a 20 mL syringe was inserted into the right ventricle, with 10 mL of PBS slowly injected. Right lower lobe lung tissue was collected and fixed in 4% paraformaldehyde solution. After 24 h, it was embedded in paraffin for pathological sectioning. H&E staining was used to observe peritracheal and perivascular inflammatory cell infiltration in the lung tissue, and PAS staining was used to observe goblet cell proliferation and mucus secretion.

[0108] Example 8

[0109] Ginkgo polypeptide preparation

[0110] (1) The ginkgo pomace obtained after alcohol extraction in Example 1 was mixed with petroleum ether (boiling range: 30~60℃) at a ratio of 1:15 (g / mL) for defatting for 12 h, and repeated 3 times. The defatted ginkgo pomace and distilled water were added to a beaker at a ratio of 1:25 (g / mL), and the pH of the suspension was adjusted to 10.5 with 1 M sodium hydroxide solution and 1 M hydrochloric acid solution. The suspension was then placed in a constant temperature stirred oil bath and stirred for 4 h at 45℃. After extraction, the suspension was allowed to stand at room temperature for 2 h. After returning to room temperature, it was centrifuged at 12000 rpm for 15 min. The supernatant after centrifugation was placed in a new beaker for acid precipitation. The precipitate was washed with distilled water until the washing liquid was neutral, and then the precipitate was dried at 45℃ to obtain crude ginkgo starch.

[0111] (2) Adjust the pH of the collected supernatant to 4.5 with 1 M sodium hydroxide solution and 1 M hydrochloric acid solution, and then let it stand at room temperature for 2 h to allow the protein to precipitate. Centrifuge at 12000 rpm for 15 min. After centrifugation, collect the precipitate, wash the precipitate with distilled water until the washing solution is neutral, and then freeze-dry the precipitate to obtain ginkgo protein.

[0112] (3) Prepare a 2% ginkgo protein solution using a phosphate buffer solution with a pH of 10. Heat the ginkgo protein solution to 40°C and add alkaline protease (5000 U / g) to the ginkgo protein solution for 4 h.

[0113] (4) After the enzymatic hydrolysis is completed, the enzyme is inactivated by boiling water for 10 min. The hydrolysate is left to stand at room temperature for 1 h, then centrifuged at 12000 rpm for 15 min. The supernatant is collected and desalted by dialyzing at 4℃ for 48 h using a 0.5 kDa dialysis bag. The resulting product is then freeze-dried to obtain ginkgo polypeptide.

[0114] Example 9

[0115] Preparation of RS3 type resistant starch from ginkgo.

[0116] (1) The crude ginkgo starch prepared in Example 8 was mixed with a phosphate buffer solution at pH 6.0 and stirred at room temperature for 30 min to prepare a 20% (m / m) ginkgo starch suspension.

[0117] (2) After the suspension was subjected to high pressure treatment at 121℃ for 20 min, it was cooled to 55℃, stirred evenly, and the pH value was adjusted to 4.0. Pullulanase (250 U / g) was added for enzymatic hydrolysis for 16 h, the enzyme was inactivated by boiling water for 10 min, cooled to room temperature, and then stored at 4℃ for 24 h. The precipitate was obtained by centrifugation, the precipitate was washed three times with distilled water, the precipitate was collected and freeze-dried to obtain Ginkgo RS3 type resistant starch.

[0118] Example 10

[0119] In vivo experiments were conducted to evaluate the effect of ginkgo resistant starch in relieving airway inflammation in asthma.

[0120] Thirty SPF-grade BABL / c female mice were randomly divided into 5 groups, with 6 mice in each group: a control group, an asthma group (Model), a dexamethasone group (Dex, 1 mg / kg·bw), a high-dose ginkgo resistant starch group (RSH, 200 mg / kg·bw) prepared in Example 9, and a low-dose ginkgo resistant starch group (RSL, 100 mg / kg·bw). In the modeling group, mice were intraperitoneally injected with 250 μL of OVA sensitization solution on days 0 and 7, respectively. Starting on day 14, mice were challenged by ultrasonic nebulization with 5% OVA solution three times a week for 45 minutes each time, for 5 consecutive weeks. Thirty minutes before nebulization challenge, mice were administered gavage with ginkgo resistant solution and intraperitoneally injected with dexamethasone solution. The control group was treated with physiological saline in the same manner as the modeling group.

[0121] Example 11

[0122] Determination of the textural properties of 9 commercially available edible gel products.

[0123] The hardness, elasticity, viscosity, chewiness, and resilience of nine commercially available gummies were determined using a TA.XT.plus texture analyzer. The TPA mode was selected, a P / 0.5 probe was used, and the instrument's initial and return speeds were both set to 1 mm / s, with a testing speed of 2 mm / s; target value: 3 mm; trigger force: 5.0 g; residence time: 5 s. Three replicates were performed for each group of commercially available gummies.

[0124] Example 12

[0125] Sensory evaluation of edible gel products.

[0126] The sensory evaluation was conducted by 15 sensory evaluators based on the sensory scoring criteria (Table 1).

[0127] Table 1 Sensory Evaluation Standards for Edible Gel Products

[0128]

[0129] Example 13

[0130] Preparation of acidity-adjusting solution for ginkgo edible gel products

[0131] Weigh out the three components of the acidity regulator—tartaric acid, citric acid, and sodium citrate—in a mass ratio of 4:8:3. Dissolve them in an appropriate amount of distilled water at room temperature to prepare a 1 g / mL solution. Stir well and set aside at room temperature.

[0132] Example 14

[0133] Preparation of sweetness-adjusting solution for ginkgo edible gel products

[0134] Weigh out 0.3% of the sweetener mogroside by weight of the gummies, dissolve it in an appropriate amount of distilled water at room temperature to prepare a 1 g / mL solution, stir well and set aside at room temperature.

[0135] Example 15

[0136] Preparation of Ginkgo Biloba Edible Gel Base

[0137] (1) Weigh 10% of the gelatin according to the mass percentage of the gummies, mix the gelatin with an equal amount of pure water and soak for 30 min. When the gelatin is fully swollen, stir continuously in a 45℃ water bath until completely dissolved to prepare a gelatin solution and keep it at 45℃ for later use.

[0138] (2) Weigh out 8% and 2% of ginkgo RS3 resistant starch and ginkgo polypeptide according to the mass percentage of the gummies. Add ginkgo RS3 resistant starch to gelatin solution and stir in a water bath at 45°C until dissolved. After complete dissolution, add ginkgo polypeptide and continue stirring until completely dissolved to prepare a gel-based solution and keep it at 45°C for later use.

[0139] Example 16

[0140] Preparation of edible ginkgo gel products.

[0141] Formula for functional edible gel products containing ginkgo (by weight percentage of gummies): 5.15% ginkgo ethyl acetate extract; 8% ginkgo RS3 resistant starch; 2% ginkgo polypeptide; 10% gelatin; 15% malt syrup; 0.19% acidity regulator (tartaric acid:citric acid:sodium citrate = 4:8:3); 0.3% mogroside sweetener.

[0142] Specific preparation steps:

[0143] (1) Sol: Mix 10% of the gelatin in the formula with twice the amount of pure water, soak for 30 min, and stir continuously in a 45°C water bath until completely dissolved to prepare a gelatin solution and keep it at 45°C for later use.

[0144] (2) Sweetness adjustment solution: Dissolve 0.3% of the sweetener mogroside in the formula with an appropriate amount of water to prepare a sweetness adjustment solution of 1 g / mL, and store at room temperature for later use.

[0145] (3) Acidity adjustment solution: Dissolve the acidity regulator (0.19% by mass) in the formula with an appropriate amount of water to prepare an acidity adjustment solution of 1 g / mL, and store at room temperature for later use.

[0146] (4) Gum base: Add 8% by mass of Ginkgo RS3 resistant starch in the formula to the gelatin solution, stir and dissolve in a water bath at 45°C. After complete dissolution, add 2% by mass of Ginkgo polypeptide in the formula, and continue stirring until completely dissolved to prepare a gel base solution and keep it at 45°C for later use.

[0147] (5) Cooking sugar: Put 15% of the maltose syrup in the recipe into a beaker, add 2 times the amount of pure water, heat and stir at the same time. After the syrup and pure water are evenly mixed, continue to cook at 85°C for 15 minutes until the sugar solution is slightly viscous and can be stretched into threads.

[0148] (6) Boiling: Add the gel-based solution prepared in step (4) to the syrup prepared in step (5), stir evenly, and continue to boil over low heat for 10 minutes to make mixture A. Cool mixture A to 45°C.

[0149] (7) Mixing and standing: Add 5.15% by mass of the ginkgo ethyl acetate extract in the formula to the mixed solution A prepared in step 6, stir at 45°C for 5 min, stir evenly, add the sweetness adjustment solution prepared in step 2 and the acidity adjustment agent solution prepared in step 3, and make up the remainder with water to make the total amount 100%, and continue stirring for 5 min to make soft candy material.

[0150] (8) Molding: Pour the gummy material prepared in step (7) into the mold while it is still hot, gently tap the mold to remove air bubbles and distribute the material evenly in the mold, and demold after refrigeration to obtain ginkgo functional gummy that relieves airway inflammation.

[0151] After step (8), the following steps are also included: after placing the desiccator in the packaging box in advance, before packaging, take out the packaging box, put in the gummy candies, and immediately seal it with plastic.

[0152] Example 17

[0153] Determination of textural properties and sensory evaluation of ginkgo edible gel products

[0154] The method for determining the textural properties in Example 17 is the same as that for determining the textural properties of 10 commercially available edible gel products in Example 11. The method for sensory evaluation in Example 17 is the same as that for sensory evaluation of edible gel products in Example 12. The difference is that the sample in Example 17 is the ginkgo edible gel product prepared in Example 16.

[0155] Example 18

[0156] Detection of ginkgo flavonoid content in the ginkgo edible gel product prepared in Example 16.

[0157] Flavonoids were detected using a colorimetric method with rutin as the standard. The specific detection method was as follows: Take 0.5 mL of the sample to be tested, add 0.3 mL of 5% NaNO2 solution, let stand for 6 min, add 0.3 mL of 10% Al(NO3)3 solution, let stand for 6 min, add 4 mL of 4% NaOH solution and 4.5 mL of 70% ethanol solution, mix well and let stand for 20 min, and then detect the absorbance at 510 nm.

[0158] Prepare standard solutions using rutin standards and dilute them to the appropriate concentration gradients of 0.000, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.5, and 1 mg / mL. Detect the solution after color development and plot a standard curve.

[0159] The standard curve for flavonoids detected using the rutin method is Y = 0.04904 + 0.35462x, R0 2 =0.9999. The standard curve is as follows: Figure 14 As shown.

[0160] Example 19

[0161] Detection of ginkgolide content in the ginkgo edible gel product prepared in Example 16.

[0162] Terpene lactones were detected using the absorbance method. The specific detection method was as follows: Take 0.5 mL of the sample to be tested, add 0.4 mL of alkaline hydroxylamine mixed solution (13.9% hydroxylamine hydrochloride aqueous solution - 12.3% sodium hydroxide solution (1:2) mixed, freshly prepared before use), let stand for 5 min, add 0.4 mL of 3 mol / L HCl solution and 0.2 mL of 6% FeCl3 solution, mix well, add 3 mL of 70% ethanol solution, mix well, and detect the absorbance value at 517 nm.

[0163] Ginkgolide A standard solution was prepared and diluted to the corresponding concentration gradients of 0.000, 0.0625, 0.125, 0.250, 0.500, and 1 mg / mL. After color development, the solution was detected and a standard curve was plotted.

[0164] The standard curve for terpene lactones is y = 0.1507x + 0.05514, R0 2 =0.99931. The standard curve is as follows: Figure 15 As shown.

[0165] Example 20

[0166] Determination of reducing sugar content in ginkgo edible gel products

[0167] The reducing sugar content of the ginkgo edible gel product prepared in Example 16 was determined according to GB.5009.7.

[0168] Example 21

[0169] Determination of Moisture Content in Ginkgo Biloba Edible Gel Products

[0170] The moisture content of the ginkgo edible gel product prepared in Example 16 was determined according to the direct drying method in GB 5009.3-2016 National Food Safety Standard - Determination of Moisture in Food. The moisture content of the ginkgo edible gel product should meet the requirements of SB / T 10021-2017 Confectionery and Gel Confectionery Standard.

[0171] Example 22

[0172] The total bacterial count and coliform count in the ginkgo edible gel product prepared in Example 16 were determined according to the standards GB 4789.2-2022 (National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count) and GB 4789.3-2016 (National Food Safety Standard - Microbiological Examination of Food - Coliform Count). The total bacterial count and coliform count in the ginkgo edible gel product should meet the requirements of GB 17399-2016 (National Food Safety Standard - Candy).

[0173] Example 23

[0174] Animal experiments were conducted to verify the potential effects of ginkgo edible gel products on airway inflammation.

[0175] Thirty SPF-grade BABL / c female mice were randomly divided into 5 groups, with 6 mice in each group: a control group, an asthma group (Model), a dexamethasone group (Dex, 1 mg / kg·bw), a high-dose group (RTH, 2000 mg / kg·bw) of the ginkgo edible gel product prepared in Example 16, and a low-dose group (RTL, 1000 mg / kg·bw). In the modeling group, mice were intraperitoneally injected with 250 μL of OVA sensitization solution on days 0 and 7, respectively. Starting on day 14, mice were challenged by ultrasonic nebulization with 5% OVA solution three times a week for 45 minutes each time, for 5 consecutive weeks. Thirty minutes before nebulization challenge, mice were administered the ginkgo edible gel product solution by gavage and intraperitoneally injected with dexamethasone solution. The control group was treated with physiological saline in the same manner as the modeling group. Mouse weight was measured daily during the experiment.

[0176] Example 24

[0177] Collection of mouse lung and spleen tissue samples.

[0178] After bronchoalveolar lavage in mice, the thoracic cavity was opened, and a needle connected to a 20 mL syringe was inserted into the right ventricle, followed by slow injection of 10 mL of PBS. The left lung and spleen tissues were weighed, their weight recorded, and immediately placed in liquid nitrogen for cryopreservation.

[0179] Experimental Example 1

[0180] Effects of different extracts of Ginkgo biloba on LPS-induced RAW264.7 cell viability.

[0181] Different extracts of ginkgo were prepared using the method in Example 1, and the effects of these extracts on LPS-induced RAW264.7 cell viability were determined using the method in Example 2. The results are as follows: Figure 2 As shown.

[0182] from Figure 2 It can be seen that the cell survival rate of the blank group without any added samples was 100%. With the increase of sample concentration, the cell survival rate of each sample group did not change significantly, and the cell activity of all sample groups remained at around 100%, indicating that the experimental samples had no significant effect on the LPS-induced RAW264.7 cell viability. This shows that different extracts of Ginkgo biloba have no toxic side effects on LPS-induced RAW264.7 cells, and subsequent cell experiments can be conducted within this concentration range.

[0183] Experimental Example 2

[0184] Evaluation of the anti-inflammatory activity of different extracts of ginkgo.

[0185] NO is a key mediator in inflammatory signaling pathways. In anti-inflammatory activity studies, detecting the effect of a sample on LPS-induced NO production in RAW264.7 cells is a common method for evaluating the anti-inflammatory activity of a sample. This invention uses the method of Example 1 to prepare different extracts of Ginkgo biloba, and uses the method of Example 3 to evaluate the anti-inflammatory activity of these extracts. The results are as follows: Figure 3 As shown.

[0186] from Figure 3 As can be seen, compared with the Control group, stimulation with 1 μg / mL LPS for 20 h significantly increased the NO concentration in RAW264.7 cells (by approximately 15-fold), indicating that LPS stimulation successfully activated RAW264.7 cells and induced a significant inflammatory response. Pretreatment with unfractionated GBS extract inhibited LPS-induced excessive NO production in RAW264.7 cells. This inhibition was dose-dependent, with statistically significant inhibitory effects observed only at higher concentrations (100 and 200 μg / mL). This suggests that unfractionated GBS extract contains too many chemical components, with relatively low levels of anti-inflammatory components, requiring accumulation at higher concentrations to exert its effect. Therefore, unfractionated GBS extract only exerts its inhibitory effect on NO production at a certain concentration. Pretreatment with GBSP, GBSE, GBSB, and GBSW extracts obtained from the GBS extract of Ginkgo biloba significantly inhibited LPS-induced excessive NO production in RAW264.7 cells, and this inhibitory effect was dose-dependent (p<0.05). At the same concentration, GBSE showed a higher inhibitory effect on NO production than the other extracts. This indicates that the ethyl acetate extract of Ginkgo biloba (GBSE), obtained from the systematic fractional extraction of the ethanol extract of Ginkgo biloba (GBS), has significantly stronger anti-inflammatory activity than the ethanol extract of Ginkgo biloba (GBS) and the other three extracts.

[0187] Experimental Example 3

[0188] Further evaluation of the anti-inflammatory activity of ginkgo ethyl acetate extract (GBSE).

[0189] LPS-stimulated RAW264.7 cells participate in the body's defense and immune regulation by releasing pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α, etc.). The amount of pro-inflammatory cytokines secreted directly reflects the degree of inflammatory damage in the body. Therefore, the method of Example 4 was used to evaluate the effect of the ethyl acetate extract of Ginkgo biloba (GBSE) prepared by the method of Example 1 on the production of IL-1β, IL-6, and TNF-α in LPS-induced RAW264.7 cells, and the anti-inflammatory activity of the ethyl acetate extract of Ginkgo biloba was further evaluated. The results are as follows: Figure 4 As shown.

[0190] from Figure 4 As can be seen, compared with the control group, stimulation with 1 μg / mL LPS significantly increased the concentrations of inflammatory factors IL-1β, IL-6, and TNF-α in RAW264.7 cells (p<0.001), indicating the successful establishment of the cellular inflammation model. GBSE pretreatment significantly reduced the secretion of IL-1β, IL-6, and TNF-α (p<0.05) in a good dose-dependent manner. This further demonstrates that the GBSE extract of Ginkgo biloba has good anti-inflammatory activity.

[0191] Test Example 4

[0192] Ginkgo biloba ethyl acetate extract was prepared using the method described in Example 1. One parameter was adjusted using a single-factor controlled variable method (see details of the adjustment). Figure 5 With other preparation parameters unchanged, the effects of different extraction methods, ethanol concentration, solid-liquid ratio, temperature, time, and number of extractions on the anti-inflammatory activity of the ginkgo ethyl acetate extract were investigated. The anti-inflammatory activity of the ginkgo ethyl acetate extract was assessed by detecting NO secretion using the method described in Example 3, where the final concentration of the ginkgo ethyl acetate extract was 200 μg / mL, the pretreatment time was 4 h, and the LPS stimulation time was 20 h. The results are as follows: Figure 5 As shown.

[0193] from Figure 5 As shown in Figure A, there was no significant difference in the NO inhibition rate between the ethyl acetate extracts prepared by ultrasound-assisted extraction and water bath extraction, but the yield of the ethyl acetate extract prepared by water bath extraction was significantly higher than that prepared by ultrasound-assisted extraction. Figure 5 BF analysis shows that the NO inhibition rate of the ethyl acetate extract of ginkgo biloba initially increases and then decreases with increasing material-to-liquid ratio, ethanol concentration, extraction temperature, and extraction time. However, the NO inhibition rate of the ethyl acetate extract of ginkgo biloba decreases with increasing extraction cycles. Considering all factors, water bath extraction is recommended, with the optimal material-to-liquid ratio, ethanol concentration, water bath temperature, water bath time, and extraction cycles being 1:15, 80%, 60℃, 2 h, and 1 extraction.

[0194] Experimental Example 5

[0195] Ethyl acetate extract of ginkgo biloba was prepared using the method described in Example 1. In vitro experiments were conducted using the method described in Example 5 to investigate the alleviating effect of the ethyl acetate extract of ginkgo biloba on airway inflammation in asthmatic mice. Bronchoalveolar lavage fluid (BALF) from each group of mice was collected using the method described in Example 6. The number of inflammatory cells in BALF was detected using a blood cell counter, and the results are as follows: Figure 6 As shown.

[0196] In the asthma model group, a large number of inflammatory cells infiltrated into the BALF. Figure 6 As can be seen, compared with the control group, the total cell count and differential count (eosinophils, neutrophils, and lymphocytes) in the BALF of mice in the model group were significantly increased (p<0.05). Compared with the model, the total cell count, eosinophil count, neutrophil count, and lymphocyte count in the BALF of mice treated with the positive control drug dexamethasone were all significantly decreased. Similar to the effect of the positive control drug dexamethasone, the total cell count, eosinophil count, neutrophil count, and lymphocyte count in the BALF of mice treated with low-dose (GBSEL) and high-dose (GBSEH) ginkgo biloba ethyl acetate extract were significantly reduced compared with the model group (p<0.05), and this reduction showed a certain dose-dependent effect.

[0197] Experimental Example 6

[0198] Ethyl ginkgo biloba extract was prepared using the method described in Example 1. In vivo experiments were conducted using the method described in Example 5 to investigate the alleviating effect of the ethyl ginkgo biloba extract on airway inflammation in asthmatic mice. Mouse lung tissue pathological sections were stained using the method described in Example 7. The results are as follows: Figure 7 and Figure 8 As shown.

[0199] from Figure 7 It can be seen that the airway epithelium of mice in the Control group was smooth, with no obvious edema of the airway mucosa, smooth lumen, and no obvious inflammatory cell infiltration around the airway and blood vessels, and no abnormalities were observed in the alveolar spaces. Mice in the Model group showed obvious pathological manifestations of airway inflammation, with incomplete damage to airway epithelial cells, airway mucosal edema, and a large number of inflammatory cells infiltrating around the airway and blood vessels. After treatment with low-dose (GBSEL), high-dose (GBSEH) ginkgo ethyl acetate extract and the positive control drug dexamethasone, the infiltration of inflammatory cells was significantly reduced compared with the Model group, and the inflammation score was significantly reduced (p<0.05).

[0200] from Figure 8It can be seen that there were very few PAS-positive cells in the Control group mice, while the model group mice had obvious dark PAS-positive cells, namely goblet cells, in the tracheal wall, and the mucus secretion in the lumen was significantly increased (p<0.05). Compared with the model group, the increase of goblet cells in the airway wall of mice was reduced and the mucus secretion was reduced after administration of Dex group and ethyl acetate (GBSEL group and GBSEH group) (p<0.05).

[0201] Experimental Example 7

[0202] Preparation of ginkgo polypeptide.

[0203] Ginkgo polypeptide was prepared using the method described in Example 8. One parameter was adjusted using a single-factor controlled variable method (see details of the adjustment). Figure 9 With other preparation parameters unchanged, the effects of alkaline protease hydrolysis pH, enzyme dosage, hydrolysis temperature, hydrolysis time, and ginkgo protein substrate concentration on the yield of ginkgo polypeptide in Example 7 were investigated. The results are as follows: Figure 9 As shown.

[0204] from Figure 9 B. Figure 9 C and Figure 9 E shows that with the increase of alkaline protease, hydrolysis pH, hydrolysis temperature, and ginkgo biloba protein substrate concentration, the yield of crude polypeptide first increases and then decreases. From Figure 9 As shown in Figure A, the amount of ginkgo polypeptide initially increases and then stabilizes with increasing enzyme dosage. When the enzyme dosage is too high, the enzyme in the system becomes supersaturated and cannot bind to the substrate to react. From... Figure 9 As shown in Figure D, the yield of ginkgo biloba polypeptide initially increases and then stabilizes with increasing enzymatic hydrolysis time. At the beginning of the hydrolysis reaction, both enzyme and substrate concentrations are high, resulting in a faster reaction rate and a more significant increase in polypeptide yield. However, as the hydrolysis time increases, the concentrations of enzyme and substrate in the reaction system decrease, thus slowing the increase in polypeptide yield. Considering all factors, the optimal parameters for alkaline protease addition, hydrolysis pH, hydrolysis temperature, hydrolysis time, and ginkgo biloba protein substrate concentration are: 5000 U / g, 10.0 μg / g, 40℃, 4 h, and 2%.

[0205] Experimental Example 8

[0206] Preparation of RS3 type resistant starch from ginkgo.

[0207] Ginkgo RS3 resistant starch was prepared using the method described in Example 9. One parameter was adjusted using a single-factor controlled variable method (see details of the adjustment). Figure 10 With other preparation parameters unchanged, the effects of pullulanase hydrolysis temperature, enzyme dosage, hydrolysis time, and hydrolysis pH on the yield of RS3 resistant starch were investigated. The results are as follows: Figure 10 As shown.

[0208] from Figure 10 The AD analysis shows that the content of RS3 resistant starch in ginkgo increases with increasing pullulanase hydrolysis temperature, enzyme dosage, hydrolysis time, and hydrolysis pH. With increasing hydrolysis temperature and pH, pullulanase activity is activated, the hydrolysis rate increases, and the RS3 resistant starch content increases. However, once the temperature and pH reach a certain value, further increases in temperature and pH destroy pullulanase activity, decrease the hydrolysis rate, and reduce the RS3 resistant starch content. Considering all factors, the optimal pullulanase hydrolysis temperature, enzyme dosage, hydrolysis time, and hydrolysis pH are 55℃, 250 U / g, 16 h, and 4.0.

[0209] Experimental Example 9

[0210] Ginkgo biloba resistant starch was prepared using the method described in Example 9, and in vivo experiments were conducted using the method described in Example 10 to investigate its alleviating effect on airway inflammation in asthmatic mice. Bronchoalveolar lavage fluid (BALF) was collected from each group of mice using the method described in Example 4. The number of inflammatory cells in BALF was detected using a blood cell counter, and the results are as follows: Figure 11 As shown.

[0211] from Figure 11 As can be seen, compared with the control group, the total cell count and differential count (eosinophils, neutrophils, and lymphocytes) in the BALF of mice in the model group were significantly increased (p<0.05). Compared with the model, the total cell count, eosinophil count, neutrophil count, and lymphocyte count in the BALF of mice treated with the positive control drug dexamethasone were all significantly decreased. Similar to the effect of the positive control drug dexamethasone, the total cell count, eosinophil count, neutrophil count, and lymphocyte count in the BALF of mice treated with low-dose (RSL) and high-dose (RSH) ginkgo resistant starch were all significantly reduced compared with the model group (p<0.05), and this reduction showed a certain dose-dependent effect.

[0212] Experimental Example 10

[0213] Ginkgo biloba resistant starch was prepared using the method described in Example 9, and in vivo experiments were conducted using the method described in Example 10 to investigate its alleviating effect on airway inflammation in asthmatic mice. Mouse lung tissue pathological sections were stained using the method described in Example 7. The results are as follows... Figure 12 and Figure 13 As shown.

[0214] from Figure 12It can be seen that the airway epithelium of mice in the Control group was smooth, with no obvious edema of the airway mucosa, smooth lumen, and no obvious inflammatory cell infiltration around the airway and blood vessels, and no abnormalities were observed in the alveolar spaces. Mice in the Model group showed obvious pathological manifestations of airway inflammation, with incomplete damage to airway epithelial cells, airway mucosal edema, and a large number of inflammatory cells infiltrating around the airway and blood vessels. After treatment with low-dose (RSL), high-dose (RSH) ginkgo resistant starch and the positive control drug dexamethasone, the infiltration of inflammatory cells was significantly reduced compared with the Model group, and the inflammation score was significantly reduced (p<0.05).

[0215] from Figure 13 It can be seen that there were very few PAS-positive cells in the Control group mice, while the tracheal wall of the model group mice showed obvious dark PAS-positive cells, namely goblet cells, and the mucus secretion in the lumen was significantly increased (p<0.05). Compared with the model group, the increase of goblet cells in the airway wall of mice was reduced and the mucus secretion was reduced after administration of Ginkgo biloba resistant starch (RSL combined with RSH group) in the Dex group and Ginkgo biloba resistant starch group (RSL combined with RSH group) (p<0.05).

[0216] Experimental Example 11

[0217] Determination of the textural properties of nine commercially available gummies.

[0218] The textural properties of nine commercially available gummies were determined using the method described in Example 11. The results are shown in Table 2.

[0219] Table 2. Texture characteristics of 9 commercially available functional edible gel products

[0220]

[0221] As shown in Table 2, there are significant differences in hardness, elasticity, viscosity, chewiness, and resilience among the nine commercially available functional edible gel products, which has a significant impact on the sensory indicators of commercially available edible gel products.

[0222] Experimental Example 12

[0223] The method of Example 12 was used to evaluate the mouthfeel, taste, color, stickiness, and texture of nine commercially available gel products, attempting to assess the intensity (weight) of sensory indicators. ) and sensory evaluation index discrimination (weight) The weights of each sensory evaluation index for the gummy candy are determined using two dimensions. The scores given by each sensory evaluator for each index are normalized, and the average of the normalized values ​​is the weight. The weight is the value obtained after normalizing the coefficient of variation of sensory evaluators' scores for a certain indicator of the gel product. The sensory evaluation scores of the nine commercially available gel products, as well as the mean and standard deviation of each score, are shown in Table 3.

[0224] Table 3. Sensory evaluation and statistical analysis of nine commercially available functional edible gel products.

[0225]

[0226] The coefficient of variation for each indicator is calculated by dividing the standard deviation by the mean, and the mean of each indicator is... ,variance and coefficient of variation The calculation formula is as follows:

[0227]

[0228] in, Let m be the score of the nth evaluated gel product on the mth indicator (m=1, 2, 3, 4, 5). Normalization is performed to obtain the sensory evaluation indicators of the gel product. Weight, The weighting results are shown in Table 3, and the specific formulas are as follows:

[0229]

[0230] The scores given by each sensory evaluator for a specific indicator were normalized. The results are shown in Table 4, and the specific formula is as follows:

[0231]

[0232] in This represents the normalized value of gel sample n on index m; Let be the score given by the j-th sensory evaluator to the m-th indicator of sample n. Based on this, the sensory evaluation indicators for the gel product can be calculated. Weight, The weighting results are shown in Table 4.

[0233] Table 4 Normalized data and mean and standard deviation

[0234]

[0235] The combination weights of the gel products were calculated according to formula (1-4), and the results are shown in Table 5. The specific calculation formula is as follows:

[0236]

[0237] This invention, for the first time, utilizes the sensory evaluation results of commercially available gel products to determine the weights of five sensory evaluation indicators: color, texture, taste, stickiness, and texture. As shown in Table 5, the combined weights of these five sensory evaluation indicators are 0.194, 0.205, 0.190, 0.212, and 0.198, respectively. Subsequently, the sensory scores of the ginkgo nut edible gel products are calculated according to the sensory evaluation standards in Example 10 and the sensory indicator weights in Table 5. It should be noted that, in this invention, to facilitate subsequent data statistics and result interpretation, the sensory evaluation of the ginkgo nut gel products is quantified using a percentage system. The total percentage score is obtained by summing the scores of each evaluation indicator and multiplying by a conversion factor of 2.

[0238] Table 5. Weights of Sensory Evaluation Indicators for Gummy Candies

[0239]

[0240] Experimental Example 13

[0241] Ginkgo biloba edible gel base was prepared according to Example 15, wherein the mass percentage of gelatin and ginkgo biloba polypeptide was 0, and the effect of different amounts of resistant starch added from ginkgo biloba on the matrix properties of the ginkgo biloba edible gel base was investigated.

[0242] The results are shown in Table 6. The gelling properties of ginkgo nut resistant starch were poor; 2% and 4% by mass of RS3-type resistant starch failed to form a gel. 6%-10% by mass of RS3-type resistant starch could form a gel, and with increasing RS3-type resistant starch content, the gel's hardness and chewiness increased, but elasticity, viscosity, and resilience decreased. Furthermore, the higher the RS3-type resistant starch content, the faster the gel structure collapsed. Therefore, considering all factors, the optimal mass percentage of RS3-type resistant starch was 8%.

[0243] Table 6. Concentration screening of RS3 resistant starch from ginkgo biloba.

[0244]

[0245] Test Example 14

[0246] Ginkgo biloba edible gel base was prepared according to Example 15, wherein the mass percentage of gelatin was 0% and the mass percentage of RS3 resistant starch was 8%. The effect of different amounts of ginkgo biloba peptides added on the structural properties of the ginkgo biloba edible gel base was investigated. The results are shown in Table 7.

[0247] Table 7. Concentration screening of ginkgo peptides

[0248]

[0249] As shown in Table 7, with the increase of ginkgo peptide addition, the hardness, elasticity, and chewiness of the ginkgo edible gel base initially increased and then decreased. Ginkgo peptide has certain gelling properties, and adding it can increase the gelling properties of the gel base. However, with further increases in ginkgo peptide concentration, a large number of bubbles are generated during stirring, leading to a decrease in the hardness, elasticity, chewiness, and resilience of the ginkgo gum base. Therefore, considering all factors, the optimal mass percentage of ginkgo peptide is 2%, with the data for no peptide addition being 8% RS3 in Table 5.

[0250] Experimental Example 15

[0251] Ginkgo biloba edible gelatin base was prepared according to Example 15, wherein the mass percentage of RS3 resistant starch was 8% and the mass percentage of ginkgo biloba polypeptide was 2%. The effect of different gelatin addition amounts on the matrix properties of the ginkgo biloba edible gelatin base was investigated. The results are shown in Table 8.

[0252] Table 8. Concentration Screening of Gelatin

[0253]

[0254] As shown in Table 8, with the increase of gelatin content, the hardness, viscosity, and resilience of the ginkgo edible gel base first increased sharply and then increased steadily at a small rate. However, with the increase of gelatin content, the elasticity of the ginkgo edible gel base first increased and then decreased. Gelatin is a high-molecular-weight protein. Within a certain content range, the higher the gelatin content, the more peptide chains participate in building the network structure, the more sufficient the intermolecular interactions, and the better the hardness and elasticity of the resulting gel. Considering the acceptance of the hardness and elasticity of the gummies by the elderly and children, a gelatin mass percentage of 10% was selected.

[0255] Experimental Example 16

[0256] The effect of the amount of mogroside added on the texture and sensory score of ginkgo edible gel.

[0257] Ginkgo biloba edible gel products were prepared according to Example 16, wherein the gelatin, RS3 resistant starch, gelatin peptides, acidity regulator, malt syrup, ginkgo biloba ethyl acetate extract, cooking time, and cooking temperature in the formulation were 10%, 8%, 2%, 0.38%, 5%, 5.15%, 0 min, and 85℃, respectively. The effect of the amount of sweetener dissolved and added according to Example 12 on the textural properties and sensory evaluation of the ginkgo biloba edible gel products was investigated. The textural properties of the samples were tested using the method of Example 11, and the sensory evaluation of the samples was performed according to the method of Example 12. The sensory scores were calculated according to the weights in Table 5 of Example 12, and the results are shown in Table 9.

[0258] Table 9. Effects of mogroside addition on textural properties and sensory scores of ginkgo functional edible gel products.

[0259]

[0260] As shown in Table 9, with the increase of mogroside addition, the hardness, viscosity, and chewiness of the ginkgo edible gel product all showed a trend of first increasing and then decreasing. Within the range of 0.1%-0.5% mogroside, the elasticity and resilience of the ginkgo edible gel product basically stabilized. The hardness, viscosity, and chewiness of the ginkgo edible gel product reached their maximum when the mogroside addition reached 0.4%. With the increase of mogroside addition, the sensory evaluation score of the ginkgo edible gel product also showed a trend of first increasing and then decreasing, reaching its maximum when the mogroside addition reached 0.3%. Therefore, considering all factors, the optimal mass percentage of mogroside is 0.3%.

[0261] Experimental Example 17

[0262] The effect of acidity regulator addition on the texture properties and sensory scores of ginkgo edible gel products.

[0263] Ginkgo biloba edible gel products were prepared according to Example 16, wherein the gelatin, RS3 resistant starch, gelatin peptides, mogrosides, malt syrup, ginkgo biloba ethyl acetate extract, cooking time, and cooking temperature in the gummy candy formulation were 10%, 8%, 2%, 0.3%, 5%, 5.15%, 0 min, and 85℃, respectively. The effect of the acidity-adjusting dissolving addition amount prepared according to Example 13 on the textural properties and sensory evaluation of the ginkgo biloba edible gel products was investigated. The textural properties of the samples were tested using the method of Example 11, and the sensory evaluation of the samples was performed according to the method of Example 12. The sensory scores were calculated according to the weights in Table 5 of Example 12, and the results are shown in Table 10.

[0264] Table 10 Effects of Acidity Regulator Addition Amount on Texture Properties and Sensory Scores of Ginkgo Biloba Functional Edible Gel Products

[0265]

[0266] As shown in Table 10, with the increase of acidity regulator addition, the hardness and chewiness of the ginkgo edible gel product gradually decreased, while elasticity, viscosity, and resilience basically tended to be within the range of temperature. The sensory evaluation score of the ginkgo edible gel product showed a trend of first increasing and then decreasing, reaching its maximum when the acidity regulator addition reached 0.19%. Therefore, considering all factors, the optimal mass percentage of acidity regulator addition is 0.19%.

[0267] Experimental Example 18

[0268] The effect of maltose syrup addition on the texture and sensory score of ginkgo edible gel products.

[0269] Ginkgo biloba edible gel products were prepared according to Example 16, wherein the gelatin, RS3 resistant starch, gelatin peptides, mogrosides, acidity regulator, ginkgo biloba ethyl acetate extract, cooking time, and cooking temperature in the gummy candy formulation were 10%, 8%, 2%, 0.3%, 0.19%, 5.15%, 0 min, and 85℃, respectively. The effect of maltose syrup addition on the textural properties and sensory evaluation of the ginkgo biloba edible gel products was investigated. The textural properties of the samples were tested using the method of Example 11, and the sensory evaluation of the samples was performed according to the method of Example 12. The sensory scores were calculated according to the weights in Table 5 of Example 12, and the results are shown in Table 11.

[0270] As shown in Table 11, with the increase of maltose syrup addition, the hardness and chewiness of the ginkgo edible gel product showed a trend of first increasing and then decreasing, reaching their maximum when the maltose syrup addition reached 15%. The elasticity, viscosity, and resilience of the ginkgo edible gel product remained relatively stable within the range of 5%-25% maltose syrup addition. With the increase of maltose syrup addition, the sensory evaluation score of the ginkgo edible gel product showed a trend of first increasing and then decreasing, reaching its maximum when the maltose syrup addition reached 15%. Therefore, considering all factors, the optimal mass percentage of maltose syrup is 15%.

[0271] Table 11 Effects of maltose syrup addition on the textural properties and sensory scores of ginkgo functional edible gel products

[0272]

[0273] Experimental Example 19

[0274] The effect of the amount of ginkgo ethyl acetate extract added on the textural properties and sensory scores of ginkgo edible gel products.

[0275] Ginkgo biloba edible gel products were prepared according to Example 16, wherein the gelatin, RS3 resistant starch, gelatin peptides, mogrosides, acidity regulator, malt syrup, cooking time, and cooking temperature in the gummy candy formula were 10%, 8%, 2%, 0.3%, 0.19%, 15%, 0 min, and 85℃, respectively. The effect of the amount of ginkgo biloba ethyl acetate extract added on the textural properties and sensory evaluation of the ginkgo biloba edible gel products was investigated. The textural properties of the samples were tested using the method of Example 11, and the sensory evaluation of the samples was performed according to the method of Example 12. The sensory scores were calculated according to the weights in Table 5 of Example 12, and the results are shown in Table 12.

[0276] Table 12 Effects of extract addition amount on the textural properties and sensory scores of ginkgo functional edible gel products

[0277]

[0278] As shown in Table 12, with the increase of the amount of ethyl acetate extract of ginkgo biloba, the hardness, elasticity, viscosity, chewiness, and resilience of the ginkgo edible gel product gradually decreased. The sensory evaluation score of the ginkgo edible gel product showed a trend of first increasing and then decreasing with the increase of the amount of ethyl acetate extract, reaching its maximum when the amount of ethyl acetate extract reached 5.15%. Therefore, considering all factors, the optimal mass percentage of ethyl acetate extract of ginkgo biloba is 5.15%.

[0279] Test Example 20

[0280] The effect of cooking temperature on the textural properties and sensory scores of ginkgo edible gel products.

[0281] Ginkgo biloba edible gel products were prepared according to Example 16, wherein the gelatin, RS3 resistant starch, gelatin peptides, mogrosides, acidity regulator, malt syrup, cooking time, and ginkgo biloba ethyl acetate extract in the gummy candy formula were 10%, 8%, 2%, 0.3%, 0.19%, 15%, 0 min, and 5.15%, respectively. The effect of cooking temperature on the textural properties and sensory evaluation of the ginkgo biloba edible gel products was investigated. The textural properties of the samples were tested using the method of Example 11, and the sensory evaluation of the samples was performed according to the method of Example 12. The sensory scores were calculated according to the weights in Table 5 of Example 12, and the results are shown in Table 13.

[0282] Table 13 Effects of cooking temperature on the textural properties and sensory scores of ginkgo functional edible gel products

[0283]

[0284] As shown in Table 13, with increasing cooking temperature, the hardness and chewiness of the ginkgo edible gel product's texture properties initially increased and then decreased, reaching their maximum at 85℃. Within the 70-90℃ range, the elasticity and viscosity of the ginkgo edible gel product's texture properties remained relatively stable, while the resilience initially increased slightly, then decreased and stabilized. With increasing cooking temperature, the sensory evaluation score of the ginkgo edible gel product initially increased and then decreased, reaching its maximum at 85℃. Therefore, considering all factors, the optimal cooking temperature is 85℃.

[0285] Experimental Example 21

[0286] The effect of cooking time on the textural properties and sensory scores of ginkgo edible gel products.

[0287] Ginkgo biloba edible gel products were prepared according to Example 16, wherein the gelatin, RS3 resistant starch, gelatin peptides, mogrosides, acidity regulator, malt syrup, cooking temperature, and ginkgo biloba ethyl acetate extract in the gummy candy formula were 10%, 8%, 2%, 0.3%, 0.19%, 15%, 85℃, and 5.15%, respectively. The effect of cooking time on the textural properties and sensory evaluation of the ginkgo biloba edible gel products was investigated. The textural properties of the samples were tested using the method of Example 11, and the sensory evaluation of the samples was performed according to the method of Example 12. The sensory scores were calculated according to the weights in Table 5 of Example 12, and the results are shown in Table 14.

[0288] Table 14 Effects of cooking time on the textural properties and sensory scores of ginkgo functional edible gel products

[0289]

[0290] As shown in Table 14, with increasing cooking time, the hardness and chewiness of the ginkgo edible gel product's texture properties initially increased and then decreased, reaching their maximum at 20 min. With increasing cooking time, the viscosity of the ginkgo edible gel product's texture properties generally stabilized, while elasticity and resilience initially increased slightly and then decreased before stabilizing, reaching their maximum at 10 min. With prolonged cooking time, the sensory evaluation score of the ginkgo edible gel product initially increased and then decreased, reaching its maximum at 10 min. Therefore, considering all factors, the optimal cooking time is 10 min.

[0291] Test Example 22

[0292] Determination of flavonoid and lactone content in ginkgo gel gummies.

[0293] Ginkgo biloba edible gel product was prepared according to Example 16. The prepared edible gel product is as follows: Figure 16 As shown, the gummy candy formula is as follows: 10% gelatin, 8% RS3 resistant starch, 2% gelatin polypeptide, 0.3% mogroside, 0.19% acidity regulator (tartaric acid:citric acid:sodium citrate = 4:8:3), 15% maltose syrup, 5.15% ginkgo ethyl acetate extract, cooking temperature 85℃, cooking time 10 min. The ginkgo flavonoid content in the ginkgo edible gel product was determined according to the method in Example 18, and the ginkgo lactone content in the ginkgo edible gel product was determined according to the method in Example 19. The results are shown in Table 15.

[0294] As shown in Table 15, the contents of ginkgo flavonoids and ginkgo lactones in the ginkgo edible gel products are 4.55 mg / g and 7.735 mg / g, respectively.

[0295] Experimental Example 23

[0296] Determination of reducing sugar and moisture content in ginkgo jelly gummies.

[0297] Ginkgo biloba edible gel product was prepared according to Example 16. The prepared edible gel product is as follows: Figure 16 As shown, the gummy candy formula is the same as in Example 22. The reducing sugar content of the ginkgo edible gel product was determined according to the method of Example 20, and the moisture content of the ginkgo edible gel product was determined according to the method of Example 21. The results are shown in Table 15.

[0298] As shown in Table 15, the reducing sugar content in ginkgo edible gel products is 10.19%. The moisture content in ginkgo edible gel products is 33%, which is less than 35%, meeting the moisture content requirements for starch-type edible gel products in the standard SB / T 10021-2017 Candy Gel Candy.

[0299] Test Example 24

[0300] Determination of total bacterial count and coliform count in ginkgo jelly gummies.

[0301] Ginkgo biloba edible gel product was prepared according to Example 16. The prepared edible gel product is as follows: Figure 16 As shown, the gummy candy formula is the same as in Example 22. The total bacterial count and coliform count in the ginkgo edible gel product were determined according to the method of Example 22. The results are shown in Table 15.

[0302] As shown in Table 15, the total bacterial count in the ginkgo edible gel product was 72 CFU / g, and coliform bacteria were not detected. Both the total bacterial count and coliform bacteria count in the ginkgo edible gel product were less than 100 CFU / g, meeting the requirements for total bacterial count and coliform bacteria count in the "GB 17399-2016 National Food Safety Standard for Candy".

[0303] Table 15 Physicochemical and Hygienic Indicators of Ginkgo Biloba Functional Edible Gel Products

[0304]

[0305] Test Example 25

[0306] Evaluation of the effect of ginkgo edible gel products on relieving airway inflammation and cell infiltration in asthma.

[0307] Ginkgo biloba edible gel product was prepared according to Example 16. The prepared edible gel product is as follows: Figure 16 As shown, the gummy formulation was the same as in Example 22. The intervention effect of ginkgo edible gel on asthmatic airway inflammation was evaluated according to the method in Example 23. BALF from mice in each group was collected using the method in Example 6, and the number of inflammatory cells and the level of inflammatory factors in BALF were analyzed. The results are shown below. Figure 18 and Figure 21 As shown in Example 6, the lower lobe of the right lung of mice was collected for histopathological staining of mouse lung tissue sections, and the results are as follows. Figure 19 and Figure 20 As shown; the weights of left lung tissue and spleen tissue of mice were collected using Example 22, and the results are as follows. Figure 17 As shown.

[0308] from Figure 17 As can be seen from A, the overall body weight of mice in all groups tended to remain stable, with almost no change in body weight between the experimental group and the control group. Figure 17 B shows that, compared with the Control group, the lung organ index of the Model group mice was significantly increased (p<0.05), indicating that the Th2 immune regulation in the lung tissue of asthmatic mice was significantly enhanced; intervention with different concentrations of ginkgo nut edible gel products could significantly reverse the excessive increase in lung tissue (p<0.05), indicating that ginkgo nut edible gel products have a certain inhibitory effect on Th2 immune system. From Figure 17 As can be seen from C, compared with the control group, the spleen organ index of the model group mice was significantly increased (p<0.05), indicating that the spleen of asthmatic mice has a negative immune regulatory response; intervention with different concentrations of ginkgo edible gel products can significantly reverse the excessive increase of spleen tissue (p<0.05), indicating that ginkgo edible gel products have a certain immunomodulatory effect.

[0309] from Figure 18 As can be seen, compared with the Control group, the total cell count and differential count (eosinophils, neutrophils, and lymphocytes) in the BALF of mice in the Model group were significantly increased (p<0.05). Compared with the Model group, the total cell count, eosinophil count, neutrophil count, and lymphocyte count in the BALF of mice in the positive control group (dexamethasone) were significantly decreased. Similar to the effect of the positive control group (dexamethasone), the total cell count, eosinophil count, neutrophil count, and lymphocyte count in the BALF of mice treated with low-dose (RTL) and high-dose (RTH) ginkgo edible gel products were all reduced compared with the model group (p<0.05), and this was dose-dependent, indicating that the ginkgo edible gel product has the effect of alleviating airway inflammatory cell infiltration in asthmatic mice.

[0310] from Figure 19It can be seen that the airway epithelium of mice in the Control group was smooth, with no obvious edema of the airway mucosa, smooth lumen, and no obvious inflammatory cell infiltration around the airway and blood vessels, and no abnormalities were observed in the alveolar spaces. Mice in the Model group showed obvious pathological manifestations of airway inflammation, with incomplete damage to airway epithelial cells, airway mucosal edema, and a large number of inflammatory cells infiltrating around the airway and blood vessels. After treatment with low-dose (RTL) and high-dose (RTH) ginkgo edible gel products and the positive control drug dexamethasone, the infiltration of inflammatory cells was significantly reduced compared with the Model group, and the inflammation score was significantly reduced (p<0.05), indicating that ginkgo edible gel products have the effect of relieving inflammatory cell infiltration in the lung tissue of asthmatic mice.

[0311] from Figure 20 It can be seen that the Control group mice had very few PAS-positive cells, while the model group mice had obvious dark PAS-positive cells, i.e. goblet cells, in the tracheal wall, and the mucus secretion in the lumen was significantly increased (p<0.05). Compared with the model group, the Dex group and mice treated with low-dose (RTL) and high-dose (RTH) ginkgo edible gel products showed a decrease in the increase of goblet cells in the airway wall, a decrease in mucus secretion, and a decrease in mucus secretion score (p<0.05), indicating that ginkgo edible gel products have the effect of relieving goblet cell hyperplasia and mucus secretion in the lung tissue of asthmatic mice.

[0312] from Figure 21 It can be seen that, compared with the control group, the levels of pro-inflammatory cytokines IL-6, TNF-α, IL-13, IL-4 and IL-1β in the BALF of the model group mice were significantly increased (p<0.05), while the level of anti-inflammatory cytokine IL-10 was significantly decreased (p<0.05). Intervention with different concentrations of ginkgo edible gel products can significantly reduce the levels of pro-inflammatory cytokines IL-6, TNF-α, IL-13, IL-4 and IL-1β (p<0.05) and significantly increase the level of anti-inflammatory cytokine IL-10 (p<0.05), and this effect is dose-dependent. This indicates that ginkgo edible gel products have the effect of relieving airway inflammation in asthmatic mice.

[0313] In summary, the formula for the ginkgo edible gel product prepared according to this invention is as follows: 10% gelatin, 8% RS3 resistant starch, 2% gelatin polypeptide, 0.3% mogroside, 0.19% acidity regulator, 15% malt syrup, 5.15% ginkgo ethyl acetate extract, with a cooking temperature of 85℃ and a cooking time of 10 min. The ginkgo edible gel product prepared according to this invention contains abundant ginkgo flavonoids and ginkgo lactones. The water content of the ginkgo edible gel product prepared according to this invention meets the requirements of the standard SB / T 10021-2017 Candy Gel Candy. The total bacterial count and coliform count of the ginkgo edible gel product prepared according to this invention meet the requirements of the standard GB 17399-2016 National Food Safety Standard for Candy. The ginkgo edible gel product prepared according to this invention has a certain alleviating effect on airway inflammatory cell infiltration in asthma.

Claims

1. A functional edible gel product made from ginkgo seeds that relieves airway inflammation, characterized in that, It includes ginkgo ethyl acetate extract, ginkgo RS3 resistant starch, and ginkgo polypeptide.

2. The ginkgo functional edible gel product for relieving airway inflammation according to claim 1, characterized in that, It includes ginkgo ethyl acetate extract, ginkgo RS3 resistant starch, ginkgo polypeptide, gelatin, malt syrup, acidity regulator, and sweetener.

3. The ginkgo functional edible gel product for relieving airway inflammation according to claim 1, characterized in that, The preferred composition by weight percentage includes: 5-6% ginkgo ethyl acetate extract; 5-10% ginkgo RS3 resistant starch; 1-3% ginkgo polypeptide; 5-15% gelatin; 10-20% malt syrup; 0.1-0.3% acidity regulator; 0.1-0.5% sweetener; and the balance being water.

4. A method for preparing a ginkgo functional edible gel product for relieving airway inflammation as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Sol: Mix gelatin with pure water, soak until the gelatin is fully swollen, stir until completely dissolved, and prepare a gelatin solution; (2) Sweetness adjustment solution: Dissolve the sweetener in purified water to prepare a sweetness adjustment solution, and let it stand at room temperature for later use; (3) Acidity adjustment solution: Dissolve the acidity regulator in pure water to prepare an acidity adjustment solution, and store it at room temperature for later use; (4) Gum base: Add Ginkgo RS3 resistant starch to gelatin solution, stir to dissolve, add Ginkgo polypeptide after complete dissolution, and continue stirring until completely dissolved to prepare a gel base solution for later use; (5) Cooking sugar: Add malt syrup to purified water, heat and stir. After the syrup and water are evenly mixed, continue to cook until the sugar solution is slightly viscous and can be stretched into threads. (6) Boiling: Add the prepared gum base solution in step (4) to the syrup prepared in step (5), stir evenly, and boil to make mixture A. Cool mixture A. (7) Mixing and standing: Add the ginkgo ethyl acetate extract to the mixed solution A prepared in step (6), stir evenly, then add the sweetness adjustment solution prepared in step (2) and the acidity adjustment solution prepared in step (3), and continue stirring to make soft candy material; (8) Molding: Pour the prepared soft candy material from step (7) into the mold while it is still hot, and demold to obtain the ginkgo functional edible gel product that relieves airway inflammation.

5. The method for preparing the ginkgo functional edible gel product for relieving airway inflammation according to claim 4, characterized in that, The ethyl acetate extract of ginkgo in step (7) is prepared by the following method: the dried powder of ginkgo kernels is mixed with ethanol solution and heated and shaken for extraction. After extraction, the mixture is filtered, the residue is dried to obtain ginkgo residue, and the filtrate is concentrated under reduced pressure to obtain ginkgo alcohol extract. The ginkgo alcohol extract is resuspended in distilled water and extracted with petroleum ether and ethyl acetate in sequence. The ethyl acetate extracts are collected and combined and concentrated under reduced pressure to obtain ginkgo ethyl acetate extract.

6. The method for preparing the ginkgo functional edible gel product for relieving airway inflammation according to claim 5, characterized in that, The preparation process of ginkgo polypeptide in step (4) is as follows: defatting the ginkgo residue after alcohol extraction, mixing the defatted ginkgo residue with water, adjusting the pH of the suspension to 10-11, stirring and extracting, centrifuging the suspension at room temperature, and washing the precipitate after centrifugation until the washing solution is nearly neutral, drying the precipitate to obtain ginkgo crude starch; adjusting the pH of the collected supernatant to 4-5, then allowing the protein to precipitate at room temperature, collecting the precipitate after centrifugation, washing the precipitate until the washing solution is neutral, and then freeze-drying the precipitate to obtain ginkgo protein; preparing ginkgo protein solution with buffer solution, heating and adding alkaline protease to digest, inactivating the enzyme, centrifuging at room temperature, collecting the supernatant, dialysis and freeze-drying to obtain ginkgo polypeptide.

7. The method for preparing the ginkgo functional edible gel product for relieving airway inflammation according to claim 6, characterized in that, The RS3 resistant starch of ginkgo in step (4) is prepared by the following process: the crude ginkgo starch remaining after removing protein is mixed with buffer solution and stirred at room temperature to make a starch suspension. The suspension is subjected to pressure heating and then cooled. After stirring evenly, the pH value is adjusted, pullulanase is added for enzymatic hydrolysis, the enzyme is inactivated, cooled to room temperature and then refrigerated. The precipitate is obtained by centrifugation, the precipitate is washed three times, the precipitate is collected and freeze-dried to obtain RS3 resistant starch.

8. The method for preparing the ginkgo functional edible gel product for relieving airway inflammation according to claim 4, characterized in that, The sweetener in step (2) is mogroside.

9. The method for preparing the ginkgo functional edible gel product for relieving airway inflammation according to claim 4, characterized in that, The acidity regulator solution in step S3 is composed of tartaric acid, citric acid and sodium citrate.

10. The method for preparing the ginkgo functional edible gel product for relieving airway inflammation according to claim 4, characterized in that, After step (8), the following steps are also included: after placing the desiccator in the packaging container in advance, before packaging, take out the packaging container, put in the soft candy, and immediately seal it with plastic seal.

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