A method for preparing acid-hydrolyzed molecules of Poria cocos acidic polysaccharide for promoting the proliferation of lactic acid bacteria and its application

Abstract

The present invention provides a method for preparing acid-hydrolyzed molecules of Poria cocos acid polysaccharide that promotes the proliferation of lactic acid bacteria and its application, which belongs to the technical field of pharmaceutical raw material preparation. Among them, the method includes the step of treating water-insoluble Poria cocos acid polysaccharide with gluconic acid. The method provided by the present invention has mild preparation process conditions and does not use organic solvents and traditional industrial acids. The treated Poria cocos acid polysaccharide has significantly improved water solubility and shows good biological activity in promoting the proliferation of intestinal flora, which is conducive to the further development and utilization of Poria cocos acid polysaccharide and improves the application prospects of the products made therefrom.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical raw material preparation, and particularly relates to a method for preparing acid-hydrolyzed molecules of Poria cocos acidic polysaccharide for promoting the proliferation of lactic acid bacteria and an application thereof. Background Art

[0002] Poria cocos, the dried sclerotium of the fungus Poria cocos, belongs to the Polyporaceae family. First recorded as a medicinal substance in Shennong's Herbal Classic, it is listed as a top-grade medicinal product. It has the effects of promoting diuresis and dispelling dampness, strengthening the spleen, and calming the mind. It is used to treat edema, oliguria, phlegm and fluid retention, dizziness and palpitations, spleen deficiency, poor appetite, and loose stools and diarrhea. Polysaccharides are one of Poria cocos' main chemical components. Depending on the extraction method, Poria cocos polysaccharides can be divided into water-soluble polysaccharides and acidic polysaccharides. The water-soluble polysaccharides account for 0.7% to 2.6%, while the water-insoluble acidic polysaccharides account for as much as 70% to 90%. In clinical practice, these polysaccharides cannot be fully extracted due to their insolubility and are wasted when used in water decoction.

[0003] Acidic polysaccharides are an important biological substance containing acidic groups such as carboxyl or sulfate groups. They are widely available and have great development potential. Although existing studies have shown that Poria cocos polysaccharides have anti-tumor, antioxidant, and immune-regulating effects, for example, studies have confirmed that Poria cocos polysaccharides have the effect of regulating intestinal flora and immune function in rats with spleen deficiency (Reference: Zhang Yue. Study on the extraction and purification of Poria cocos polysaccharides and their regulatory effects on immune function and intestinal flora in rats with spleen deficiency [D]. Anhui University of Traditional Chinese Medicine, 2020.), however, current research on the efficacy of Poria cocos polysaccharides is mostly based on the biological activity of its water-soluble polysaccharides, and the biological activity of water-insoluble acidic polysaccharides remains to be explored. Moreover, based on the characteristics that acidic polysaccharides are poorly soluble in water or can only be dissolved in a slightly acidic environment, on the one hand, their bioavailability is seriously affected, which in turn greatly increases the difficulty of studying the biological activity of large molecular weight acidic polysaccharides themselves in living animals. The current method is to degrade them into sugars with a molecular weight of less than 10,000, or even monosaccharides to dozens of sugars for research; on the other hand, it can also lead to their inability to be fully utilized and developed into food or drug products that can be directly applied to the human body, such as beverages, injections, etc., which seriously limits their application prospects and industrial value.

[0004] Currently, the main degradation methods for Poria cocos acid polysaccharides include microbial degradation, enzymatic degradation, and acid degradation. However, microbial degradation takes a long time and is inefficient; enzymatic degradation requires large amounts of commercial enzymes and is not suitable for industrial production; traditional acid hydrolysis methods often use strong acids such as sulfuric acid for degradation. However, strong acids such as sulfuric acid are highly corrosive and oxidizing, posing significant safety risks, and directly degrade into sugars with very low molecular weight. In addition, whether the Poria cocos acid polysaccharides produced by the above-mentioned degradation methods are edible requires further verification. Summary of the Invention

[0005] In order to solve the above problems, the present application aims to use food-grade treatment agents to degrade water-insoluble Poria acid polysaccharides, develop a method with low cost, short time, high safety, and the ability to directly obtain highly hydrophilic, edible and highly biologically active Poria acid polysaccharide molecules, and enhance the industrial application value of Poria polysaccharides.

[0006] On the one hand, the present application provides a method for preparing acid-hydrolyzed molecules of Poria cocos acid polysaccharide that promote the proliferation of lactic acid bacteria, the method comprising the step of treating water-insoluble Poria cocos acid polysaccharide with gluconic acid.

[0007] In one embodiment, the molecular weight of the water-insoluble Poria acid polysaccharide is not less than 10,000 Da, such as 10,000-200,000 Da, such as 10,000-150,000 Da, and such as 10,000-100,000 Da.

[0008] In one embodiment, the reaction ratio of the water-insoluble Poria acid polysaccharide and gluconic acid is (0.01-10) g: (0.01-10) mol, for example, (0.01-5) g: (0.01-5) mol, for example, (0.01-2) g: (0.01-2) mol, for example, (0.01-1) g: (0.01-1) mol, for example, (0.1-0.5) g: (0.04-0.05) mol.

[0009] In one embodiment, the treatment comprises placing water-insoluble Pachycory acid polysaccharide and gluconic acid in water for static reaction.

[0010] Optionally, the water can be pure water, ultrapure water, deionized water or distilled water.

[0011] In one embodiment, the mass ratio of the water-insoluble Poria acid polysaccharide to water is 1:(80-120) (w / v), preferably 1:100 (w / v).

[0012] In one embodiment, the static reaction conditions include: reacting at 50-70° C. for 0.5-6 hours.

[0013] Preferably, the static reaction condition is: reacting at 60° C. for 2 hours.

[0014] In one embodiment, the water-insoluble Poria acid polysaccharide is prepared by the following method:

[0015] The water-soluble polysaccharide is removed from the Poria cocos by water extraction, the insoluble acidic polysaccharide precipitate is extracted with an alkaline solution, and the supernatant is collected, the pH value is adjusted to 6.5-7.5, purified, and freeze-dried to obtain the water-insoluble Poria cocos acidic polysaccharide.

[0016] In one embodiment, the Poria cocos in the above steps needs to be pre-treated before extraction, for example, the Poria cocos is crushed, then soaked in ethanol for decolorization and defatting, and then filtered and dried.

[0017] In one embodiment, the step of extracting Poria cocos with water specifically includes: extracting Poria cocos with water at a ratio of 1:(15-30) (w / v) at 80° C.-120° C. for 1-5 times, each time for 60-180 minutes.

[0018] In one embodiment, after water extraction of Poria cocos, the precipitate is collected by centrifugation and extracted with 1 mol / L sodium hydroxide solution at a ratio of 1:(40-80) for 30-120 min.

[0019] In one embodiment, after extraction, the supernatant is collected by centrifugation, the pH is adjusted to 7 with 1 mol / L hydrochloric acid solution, and dialyzed using a dialysis membrane with a molecular weight cutoff of 10,000 Daltons at 1-5° C. for 40-60 hours.

[0020] In one embodiment, the method further comprises the following steps: after the treatment is completed, adjusting the pH to 6.5-7.5, purifying, and freeze-drying.

[0021] In one embodiment, after the treatment of Poria acidic polysaccharide with gluconic acid is completed, the pH value of the solution is adjusted to 7 with an alkaline solution.

[0022] In one embodiment, the alkaline solution is a 1 mol / L sodium hydroxide solution.

[0023] In one embodiment, the purification step is dialysis.

[0024] In one embodiment, the dialysis is performed using a dialysis membrane with a molecular weight cut-off of 300 Daltons at 1-5° C. for 40-60 hours.

[0025] In one embodiment, the freeze-drying conditions are: freeze-drying for 48 hours at a cold trap temperature of -47.3°C and a vacuum degree of 1.0 Pa.

[0026] In one embodiment, the dialyzed product can be concentrated and then freeze-dried directly, or the dialyzed product can be centrifuged and then freeze-dried separately.

[0027] In one embodiment, the centrifugation is to centrifuge the dialyzed solution at 7000-10000 rpm for 20-40 min.

[0028] In a preferred embodiment, the method for preparing acid-hydrolyzed molecules of Poria cocos acidic polysaccharide provided in the present application comprises the following steps:

[0029] Step 1: extracting Poria cocos with water to remove water-soluble polysaccharides, extracting insoluble acidic polysaccharide precipitates with an alkaline solution, collecting the supernatant, adjusting the pH to 7, purifying, and freeze-drying to obtain water-insoluble Poria cocos acidic polysaccharide;

[0030] Step 2: mixing the water-insoluble Poria acid polysaccharide, gluconic acid and water, and allowing to react at 50-70° C. for 0.5-6 hours;

[0031] Step 3: Adjust the pH of the solution to 7 and dialyze using a dialysis membrane with a molecular weight cutoff of 300 Daltons at 4°C for 48 hours;

[0032] Step 4: centrifuge and freeze-dry the precipitate in vacuum.

[0033] On the other hand, the present application provides an acid-hydrolyzed molecule of Poria acidic polysaccharide, which is prepared using the above method.

[0034] In one embodiment, the molecular weight of the acid-hydrolyzed Poria acidic polysaccharide molecule is not higher than 55,000 Da, such as 300 to 52,000 Da.

[0035] On the other hand, the present application provides the use of acid-hydrolyzed molecules of Poria cocos acidic polysaccharide in the preparation of food and beverages, medicines, health products and / or daily chemical products containing Poria cocos active ingredients.

[0036] In one embodiment, the dosage form of the product includes but is not limited to oral solution, injection, and suspension.

[0037] Preferably, the acid-hydrolyzed molecules of Poria acidic polysaccharide are used to prepare medicines and / or health foods with anti-tumor, anti-fatigue, anti-inflammatory, antioxidant, diuretic, immunomodulatory and / or intestinal flora regulating functions.

[0038] Preferably, the acid-hydrolyzed molecules of Poria acidic polysaccharide are used to promote the proliferation of intestinal probiotics in animals.

[0039] Preferably, the probiotics include lactic acid bacteria.

[0040] On the other hand, the present application provides the use of gluconic acid as a water-insoluble acidic polysaccharide treating agent.

[0041] Preferably, the gluconic acid is used to increase the hydrophilicity of the water-insoluble acidic polysaccharide.

[0042] On the other hand, the present application provides a method for increasing the hydrophilicity of a water-insoluble acidic polysaccharide, comprising the step of treating the water-insoluble acidic polysaccharide with gluconic acid.

[0043] In one embodiment, the water-insoluble acidic polysaccharide may be derived from plants, fungi, etc., including but not limited to bupleurum, gum, pectin, Poria cocos, shiitake mushrooms, and Tremella fuciformis.

[0044] It can be understood that the hydrophilicity of the acidic polysaccharide described in the present application refers to the affinity of the acidic polysaccharide with water under a neutral environment of pH 7.

[0045] The present invention has the following advantages and effects compared to the prior art:

[0046] 1. The present application uses a variety of low-corrosive and edible organic acids to acid-hydrolyze homemade water-insoluble Poria cocos acid polysaccharides, and unexpectedly finds that the acid-hydrolyzed molecules of Poria cocos acid polysaccharides treated with gluconic acid, which has a relatively milder acidity, have significantly improved solubility in water under a neutral environment of pH 7; and the acid-hydrolyzed molecules obtained by treatment with gluconic acid still have good water solubility when they are redissolved after freeze-drying; at the same time, the molecular weight of the acid-hydrolyzed molecules can be controlled within a lower range, and low-molecular-weight polysaccharides account for more than 70% of the total sugars, with a higher yield; therefore, the method provided by the present application can improve the water solubility of water-insoluble Poria cocos acid polysaccharides, which is beneficial for its later preparation into a variety of aqueous preparations, improves bioavailability, and can also broaden its application fields and enhance its industrial value.

[0047] 2. The acid-hydrolyzed tuckahoe acidic polysaccharide molecules prepared by the preparation method of the present application, when concentrated, freeze-dried, and then reconstituted and used in animal experiments, selectively promoted the proliferation of intestinal flora, especially the abundance of lactic acid bacteria in the intestine.

[0048] 3. This application simplifies the degradation process of Poria acid polysaccharide, which is simple to operate and low in cost. No hazardous organic solvents are used in the preparation process, which is hygienic and safe. The drying process does not require spray drying and the conditions are mild. In addition, the gluconic acid used is biodegradable, highly safe, easily available, and can be produced by microorganisms, making it easy to realize the industrial production of Poria acid polysaccharide treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0050] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. For those skilled in the art, other drawings can be obtained based on these drawings without inventive work. In the drawings:

[0051] Figure 1This is a graph showing the hydrolysis results of crude water-insoluble Poria acidic polysaccharide in different food-grade acids, where A is acetic acid, B is citric acid, C is D / L-malic acid, and D is gluconic acid;

[0052] Figure 2 This is a graph showing the degradation effects of different concentrations of gluconic acid on crude water-insoluble Poria acid polysaccharide;

[0053] Figure 3 This is a graph showing the degradation effects of different concentrations of crude water-insoluble Poria acid polysaccharide in 4 mol / L gluconic acid;

[0054] Figure 4 This is a solution diagram of crude water-insoluble Poria acid polysaccharide in Example 4 after being degraded with gluconic acid and adjusted to pH 7;

[0055] Figure 5 This is a diagram showing the results of re-dissolving the polysaccharide powder of the first acid-hydrolyzed molecule (supernatant) (left) and the polysaccharide powder of the second acid-hydrolyzed molecule (precipitate) (right) in Example 4 with water;

[0056] Figure 6 This is a comparison chart of the molecular weights of the crude water-insoluble Poria acidic polysaccharide (left) and the first acid-hydrolyzed molecule (supernatant) polysaccharide (right) in Example 4;

[0057] Figure 7 This is a comparison chart of the molecular weights of the crude water-insoluble Poria acidic polysaccharide (top) and the second acid-hydrolyzed molecule (precipitate) polysaccharide (bottom) in Example 4;

[0058] Figure 8 This is a graph showing the abundance of intestinal flora genera in each group of mice in the D-galactose-induced aging mouse experiment. DETAILED DESCRIPTION

[0059] In order to more clearly illustrate the overall concept of the application, the following is described in detail in the form of embodiments. In the following description, a large amount of specific details are provided so that a more thorough understanding of the application is provided. However, it will be apparent to those skilled in the art that the application can be implemented without the need for one or more of these details. In other examples, in order to avoid confusion with the application, some technical features well known in the art are not described.

[0060] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0061] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0062] The main materials and instruments involved in the following examples are shown in Table 1:

[0063] Table 1

[0064] Materials or instruments factory Poria cocos China Beijing Tong Ren Tang (Group) Co., Ltd. Gluconic acid (food grade) Jiangxi Hongqi Biotechnology Co., Ltd. Glacial acetic acid (food grade) Luquan County Yiyeji Food Store (individual business owner) Citric acid (food grade) Weifang Yingxuan Industrial Co., Ltd. D / L Malic Acid (Food Grade) Henan Gaobao Industrial Co., Ltd. Laboratory-grade ultrapure water analyzer Dow Water Treatment Equipment Engineering Co., Ltd. Molecular weight tester Japan Shimadzu Corporation D-Galactose SALMART Balb / c mice Guangzhou Qingle Life Sciences Co., Ltd.

[0065] Example 1

[0066] This example provides a method for preparing acid-hydrolyzed molecules of Poria cocos acidic polysaccharide, and the specific steps are as follows:

[0067] (1) Preparation of water-insoluble Poria acid polysaccharide:

[0068] After the dried Poria cocos was ground into powder, it was soaked in anhydrous ethanol overnight for decolorization and defatting;

[0069] The decolorized and defatted Poria cocos powder was extracted twice with ultrapure water at a ratio of 1:20 (w / v) at 100°C for 120 min each time to remove the water-soluble polysaccharides of Poria cocos.

[0070] The precipitate was collected by centrifugation and extracted with 1 mol / L sodium hydroxide solution at a ratio of 1:60 for 60 min;

[0071] The supernatant was collected by centrifugation, adjusted to pH 7 with 1 mol / L hydrochloric acid solution, and dialyzed using a dialysis membrane with a molecular weight cutoff of 10,000 Daltons at 4°C for 48 h;

[0072] The crude water-insoluble Poria acid polysaccharide was obtained by freeze-drying at a cold trap temperature of -47.3°C and a vacuum degree of 1.0 Pa for 48 hours.

[0073] (2) Acid hydrolysis treatment of the crude water-insoluble Poria acid polysaccharide obtained in step (1) is performed using different food-grade organic acids, and the specific steps are as follows:

[0074] Weigh 0.1 g of crude water-insoluble Poria acid polysaccharide into a 15 ml centrifuge tube, then add 2.4 g (0.04 mol) of glacial acetic acid, and then make up to 10 ml with pure water. Incubate at 60°C for 2 hours.

[0075] Weigh 0.1 g of crude water-insoluble Poria acid polysaccharide into a 15 ml centrifuge tube, then add 7.68 g (0.04 mol) of citric acid, and then make up to 10 ml with pure water. Incubate at 60°C for 2 hours.

[0076] Weigh 0.1 g of crude water-insoluble Poria acid polysaccharide into a 15 ml centrifuge tube, then add 5.36 g (0.04 mol) of D / L malic acid, and then make up to 10 ml with pure water. Incubate at 60°C for 2 hours.

[0077] Weigh 0.1 g of crude water-insoluble Poria acid polysaccharide and put it into a 15 ml centrifuge tube, then add 7.85 g (0.04 mol) of gluconic acid, and then make up to 10 ml with pure water. Let it react at 60°C for 2 hours.

[0078] Among them, in order to exclude the influence of other physical reaction conditions, such as magnetic stirring, ultrasonic vibration, etc. on improving the solubility of crude Poria acid polysaccharide in water, this experiment adopts static reaction conditions. After the reaction is completed, the solution after the above acid treatment is as follows Figure 1 shown.

[0079] Depend on Figure 1 The results show that when the homemade crude Poria acid polysaccharide is treated with a variety of organic acids, there is no solid visible to the naked eye in the solution after gluconic acid degradation, and the Poria acid polysaccharide is completely dissolved in water. That is, compared with the relatively stronger acidic citric acid and malic acid and the relatively weaker acidic acetic acid, gluconic acid has the best effect on improving the hydrophilicity of Poria acid polysaccharide, and can be used to prepare highly hydrophilic Poria acid polysaccharide acid-hydrolyzed molecules.

[0080] Example 2

[0081] This embodiment optimizes the amount of gluconic acid in Example 1, and the specific steps are:

[0082] (1) Crude water-insoluble Poria acid polysaccharide was prepared using the method shown in Example 1.

[0083] (2) The crude water-insoluble Poria acid polysaccharide obtained in step (1) is subjected to acid hydrolysis treatment using different amounts of gluconic acid, and the specific steps are as follows:

[0084] Weigh 0.1 g of crude water-insoluble Poria acid polysaccharide into a 15 ml centrifuge tube, then add 3.92 g (0.02 mol) of gluconic acid, and then make up to 10 ml with pure water. Incubate at 60°C for 2 hours.

[0085] Weigh 0.1 g of crude water-insoluble Poria acid polysaccharide into a 15 ml centrifuge tube, then add 5.88 g (0.03 mol) of gluconic acid, and then make up to 10 ml with pure water. Incubate at 60°C for 2 hours.

[0086] Weigh 0.1 g of crude water-insoluble Poria acid polysaccharide and put it into a 15 ml centrifuge tube, then add 7.85 g (0.04 mol) of gluconic acid, and then make up to 10 ml with pure water. Let it react at 60°C for 2 hours.

[0087] That is, the final concentrations of gluconic acid were 2mmol / mL, 3mmol / mL and 4mmol / mL respectively. After the reaction was completed, the solution treated with the above acids was as follows Figure 2shown.

[0088] Depend on Figure 2 The results show that when the final concentration of gluconic acid is 4 mmol / mL, there is no solid visible to the naked eye in the solution after degradation, and the Poria acid polysaccharide is completely dissolved in water, indicating that the crude water-insoluble Poria acid polysaccharide precipitation is best degraded under this condition.

[0089] Example 3

[0090] This example optimizes the amount of water-insoluble Poria acid polysaccharide in Example 1, and the specific steps are as follows:

[0091] (1) Crude water-insoluble Poria acid polysaccharide was prepared using the method shown in Example 1.

[0092] (2) The crude water-insoluble Poria acid polysaccharide obtained in step (1) with different amounts was subjected to acid hydrolysis treatment with gluconic acid, and the specific steps were as follows:

[0093] Weigh 0.1 g of crude water-insoluble Poria acid polysaccharide into a 15 ml centrifuge tube, then add 7.85 g of gluconic acid, and then make up to 10 ml with pure water. Incubate at 60°C for 2 hours.

[0094] Weigh 0.2 g of crude water-insoluble Poria acid polysaccharide into a 15 ml centrifuge tube, then add 7.85 g of gluconic acid, and then make up to 10 ml with pure water. Incubate at 60°C for 2 hours.

[0095] Take 0.3 g of crude water-insoluble Poria acid polysaccharide and put it into a 15 ml centrifuge tube, then add 7.85 g of gluconic acid, and then make up to 10 ml with pure water. Let it react at 60 ° C for 2 hours;

[0096] Weigh 0.4 g of crude water-insoluble Poria acid polysaccharide into a 15 ml centrifuge tube, then add 7.85 g of gluconic acid, and then make up to 10 ml with pure water. Incubate at 60°C for 2 hours.

[0097] Weigh 0.5 g of crude water-insoluble Poria acid polysaccharide and put it into a 15 ml centrifuge tube, then add 7.85 g of gluconic acid, and then make up to 10 ml with pure water. Let it react at 60°C for 2 hours.

[0098] After the reaction is completed, the solution after the above acid treatment is as follows Figure 3 shown.

[0099] Depend on Figure 3The results show that as the concentration of crude water-insoluble Poria acid polysaccharide increases, the viscosity of the degraded solution increases accordingly. When the crude water-insoluble Poria acid polysaccharide increases to 0.5 grams, there is still no solid visible to the naked eye in the solution after acid hydrolysis, and Poria acid polysaccharide is completely dissolved in water, indicating that the acid-hydrolyzed Poria acid polysaccharide molecules treated by this method still have significantly improved hydrophilicity.

[0100] Example 4

[0101] This example detects the acid-hydrolyzed molecules obtained after degradation of water-insoluble Poria acidic polysaccharide, and the specific steps are as follows:

[0102] (1) Crude water-insoluble Poria acid polysaccharide was prepared using the method shown in Example 1.

[0103] (2) Weigh 6.3 g of the crude water-insoluble Poria acid polysaccharide obtained in step (1) into a 250 ml bottle, then add 164.85 g of gluconic acid and make up to 210 ml with pure water. Let it react at 60°C for 2 hours.

[0104] (3) The degradation solution obtained in step (2) was adjusted to pH 7 and dialyzed using a dialysis membrane with a molecular weight cutoff of 300 Daltons at 4°C for 48 hours.

[0105] The dialyzed solution can be directly concentrated and then freeze-dried to form a solid powdered polysaccharide, which is the acid-hydrolyzed molecule of Poria acid polysaccharide provided in the present application.

[0106] In this embodiment, 6.3 g of Poria acidic polysaccharide raw material can be subjected to acid hydrolysis to obtain about 4.8 g of acid-hydrolyzed molecules, with a yield of 76%.

[0107] (4) To detect the molecular weight composition of the acid-hydrolyzed molecules after degradation and to perform a biological activity test, the dialyzed solution obtained in step (3) was centrifuged at 8000 rpm for 30 min to separate the supernatant and the aggregated precipitate.

[0108] (5) The supernatant and precipitate obtained in step (4) are concentrated and vacuum freeze-dried respectively to obtain the first acid-hydrolyzed molecule (supernatant) polysaccharide powder and the second acid-hydrolyzed molecule (precipitate) polysaccharide powder.

[0109] (6) Take 1 gram of the first acid-hydrolyzed molecule (supernatant) polysaccharide powder and the second acid-hydrolyzed molecule (precipitate) polysaccharide powder obtained in step (5), and re-dissolve them in 5 ml of water at room temperature.

[0110] Wherein, the result of step (3) adjusting the pH of the degradation solution to 7 is as follows Figure 4 As shown. Figure 4The results show that after the crude water-insoluble Poria acid polysaccharide is degraded by gluconic acid and the pH is adjusted to 7, no visible precipitation is generated, indicating that the acid-hydrolyzed molecules obtained still maintain good hydrophilicity under a neutral environment.

[0111] In step (6), the first acid-hydrolyzed molecule (supernatant) polysaccharide powder and the second acid-hydrolyzed molecule (precipitate) polysaccharide powder are respectively redissolved, and the results are as follows: Figure 5 As shown. Figure 5 As can be seen from the results in , both can still be completely dissolved in water after redissolution, indicating that the method of this example improves the hydrophilicity of water-insoluble Poria acid polysaccharide.

[0112] Among them, 10 mg of the crude water-insoluble Poria acid polysaccharide obtained in step (1) was added to 1 ml of water, centrifuged at 10000 rpm for 10 minutes, and filtered through a 0.22 μm filter to obtain a test solution 1; 10 mg of the first acid-hydrolyzed molecule (supernatant) polysaccharide powder was added to 1 ml of water, and filtered through a 0.22 μm filter to obtain a test solution 2. The molecular weights of the test solutions 1 and 2 were tested, and the results were as follows: Figure 6 As shown in the left and right pictures.

[0113] The molecular weights of the crude water-insoluble Poria acid polysaccharide obtained in step (1) and the second acid-hydrolyzed molecule (precipitated) polysaccharide powder were tested respectively. The results were as follows: Figure 7 As shown in the upper and lower figures.

[0114] Depend on Figure 6 and Figure 7 The results show that the molecular weight of the crude water-insoluble Poria acid polysaccharide prepared in the examples of the present application is about 100,000. After degradation by gluconic acid, the molecular weight is reduced to 50,000-60,000, indicating that the substance before and after acid hydrolysis has changed.

[0115] Example 5

[0116] In this example, the acid-hydrolyzed molecules of Poria cocos acidic polysaccharide prepared in the above example were fed with D-galactose to induce aging in mice. The specific steps are as follows:

[0117] (1) The first acid-hydrolyzed molecular (supernatant) polysaccharide powder and the second acid-hydrolyzed molecular (precipitate) polysaccharide powder prepared in Example 4 were used.

[0118] (2) Sixty 6-8 week old Balb / c mice were randomly divided into five groups: blank group (CON), model group (MOD), supernatant group (SUP), precipitate group (PRE), and mixed group (MIX; SUP:PRE = 1:1). The model group, supernatant group, precipitate group, and mixed group were injected subcutaneously with D-galactose (200 mg / kg / day) in the morning to induce senescence, and polysaccharide (200 mg / kg) was gavaged in the afternoon. The blank group was injected subcutaneously with an equal amount of normal saline in the morning and gavaged with an equal amount of pure water in the afternoon. The experiment lasted for a total of 8 weeks.

[0119] (3) After 8 weeks of the experiment, the mice were euthanized and the intestinal contents were removed. The total genomic DNA of the microbial community was extracted according to the instructions of the soil DNA kit (Omega Bio-tek, Norcross, GA, US). The quality of the extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using NanoDrop2000 (Thermo Scientific, USA).

[0120] (4) Using the DNA extracted above as a template, PCR amplification of the V3-V4 variable region of the 16S rRNA gene was performed using the upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the downstream primer 806R (5'-GGACTACHV GGGTWTCTAAT-3') carrying the barcode sequence, wherein:

[0121] The PCR reaction system was as follows: 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 μM), 0.8 μL of downstream primer (5 μM), 0.4 μL of TransStart FastPfu DNA polymerase, and 10 ng of template DNA, made up to 20 μL.

[0122] The amplification procedure was as follows: 95°C pre-denaturation for 3 min, 27 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s), followed by 72°C stable extension for 10 min, and finally storage at 4°C (PCR instrument: ABI Model 9700).

[0123] The PCR products were recovered using 2% agarose gel, purified using a DNA gel recovery and purification kit (PCR Clean-Up Kit, Yuhua, China), and detected and quantified using Qubit 4.0 (Thermo Fisher Scientific, USA).

[0124] (5) Use The purified PCR products were constructed using a Rapid DNA-Seq Kit. The following steps were followed: adapter ligation; magnetic bead screening was used to remove adapter-ligated fragments; library template was enriched by PCR amplification; and magnetic bead-based PCR product recovery was performed to obtain the final library. Sequencing was performed using the Illumina Nextseq 2000 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). Raw data were uploaded to the NCBI SRA database.

[0125] (6) Fastp (https: / / github.com / OpenGene / fastp, version 0.19.6) software was used to perform quality control on the double-end original sequencing sequences, and FLASH (http: / / www.cbcb.umd.edu / softwa re / flash, version 1.2.11) software was used for splicing: the bases with a quality value of less than 20 at the end of the reads were filtered, and a 50 bp window was set. If the average quality value in the window was less than 20, the bases at the end were cut off from the window, and reads less than 50 bp after quality control were filtered, and reads containing N bases were removed; based on the overlap relationship between PE reads, paired reads were spliced ​​(merged) into a sequence with a minimum overlap length of 10 bp; the maximum mismatch ratio allowed in the overlap region of the spliced ​​sequence was 0.2, and non-compliant sequences were screened; samples were distinguished based on the barcodes and primers at both ends of the sequence, and the sequence direction was adjusted. The number of mismatches allowed for the barcode was 0, and the maximum number of primer mismatches was 2.

[0126] (7) Using UPARSE v7.1 software (http: / / drive5.com / uparse / ), the quality control spliced ​​sequences were clustered into operational taxonomic units (OTUs) at a similarity of 97% and chimeras were removed. To minimize the impact of sequencing depth on subsequent Alpha diversity and Beta diversity data analysis, the number of sequences in all samples was leveled to 20,000 (sequence leveling is recommended). After leveling, the average sequence coverage (Good's coverage) of each sample was still 99.09%. The OTU species taxonomic annotation was performed using RDP classifier (http: / / rdp.cme.msu.edu / , version 2.11) against the Silva 16S rRNA gene database (v138) with a confidence threshold of 70%. The community composition of each sample was statistically analyzed at different species classification levels. 16S functional prediction analysis was performed using PICRUSt2 (version 2.2.0) software.

[0127] (8) All data analyses were performed on the Majorbio Cloud Platform (https: / / cloud.majorbio.com), as follows: Mothur software (http: / / www.mothur.org / wiki / Calculators) was used to calculate the Alpha diversity knowledge Chao 1, Shannon index, etc., and the Wilcoxon rank sum test was used to analyze the inter-group differences in Alpha diversity; PCoA analysis (principal coordinate analysis) based on the Bray-Curtis distance algorithm was used to test the similarity of the microbial community structure between samples, and the PERMANOV A non-parametric test was combined to analyze whether the differences in microbial community structure between sample groups were significant; LEfSe analysis (Li near discriminant analysis Effect Size) (http: / / huttenhower.sph.harvard.edu / LEfSe e) (LDA>2, P<0.05) was used to determine the bacterial groups with significant differences in abundance from the phylum to the genus level between different groups. Distance-based redundancy analysis (db-RDA) was used to investigate the impact of soil physicochemical and clinical indicators on soil and intestinal bacterial community structure. Linear regression analysis was used to assess the impact of key soil physicochemical and clinical indicators identified in the db-RDA analysis on the microbial alpha diversity index. Species were selected for correlation network analysis based on Spearman correlations (r) > 0.6 and p < 0.05.

[0128] Among them, the bacterial species abundance results at the genus level in the mouse intestine are as follows: Figure 8 shown.

[0129] Depend on Figure 8 It can be seen that compared with the blank group and the model group, the acid-hydrolyzed molecules of Poria acid polysaccharide prepared in the examples of the present application can significantly increase the abundance of Lactobacillus and Bifidobacterium, while the abundance of Faecalibaculum and Lachnospiraceae-UCG-006 is lower than that of the control group. The abundance of the eubacterium-ventriosum-group in the PRE group is better than that in other groups. This shows that the acid-hydrolyzed molecules have a significant promoting effect on the proliferation of some probiotics in the intestinal tract of animals, especially lactobacilli and bifidobacteria.

[0130] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A method for preparing acid-hydrolyzed molecules of Poria cocos acidic polysaccharide for promoting the proliferation of lactic acid bacteria, characterized in that: The method comprises the steps of treating water-insoluble Poria acid polysaccharide with gluconic acid; the treatment comprises placing the water-insoluble Poria acid polysaccharide and gluconic acid in water for a static degradation reaction; the conditions of the static degradation reaction comprise: reacting at 50-70°C for 0.5-6 hours; and the ratio of the water-insoluble Poria acid polysaccharide to the gluconic acid is (0.1-0.5) g: (0.04-0.05) mol.

2. The method according to claim 1, characterized in that The molecular weight of the water-insoluble Poria acid polysaccharide is 100,000 to 150,000 Da.

3. The method according to claim 1, characterized in that The water-insoluble Poria acid polysaccharide is prepared by the following method: extracting Poria with water to remove water-soluble polysaccharides, extracting insoluble acid polysaccharide precipitates with alkaline solution and collecting the supernatant, adjusting the pH to 6.5-7.5, purifying, and freeze-drying to obtain the water-insoluble Poria acid polysaccharide.

4. The method according to any one of claims 1 to 3, characterized in that: After the degradation reaction is allowed to stand, the pH is adjusted to 6.5-7.5, dialyzed, and freeze-dried.

5. The method according to claim 4, characterized in that The dialysis is performed using a dialysis membrane with a molecular weight cut-off of 300 Daltons at 1-5° C. for 40-60 hours.

6. An acid-hydrolyzed molecule of Poria acidic polysaccharide, characterized in that: The method is prepared by any one of claims 1 to 5.

7. Use of the acid-hydrolyzed tuckahoe acidic polysaccharide molecules according to claim 6 in the preparation of medicines and / or health foods for promoting the proliferation of intestinal probiotics in animals, wherein the probiotics are lactobacilli and / or bifidobacteria.

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