Preparation method of gastrointestinal fluid-resistant intestinal flora solid, solid, freeze-dried powder and preparation method of freeze-dried powder

Abstract

The invention relates to the technical field of flora transplantation, in particular to a preparation method of a gastrointestinal fluid-resistant intestinal flora solid, the solid, freeze-dried powder and a preparation method of the freeze-dried powder. The preparation method of the gastrointestinal fluid-resistant intestinal flora solid comprises the following steps: taking whey calcium powder, adding normal saline, and uniformly mixing to prepare a whey calcium suspension; the method comprises the following steps: taking a fresh excrement sample of a healthy donor, adding normal saline, uniformly stirring and mixing, filtering, and collecting filtrate to obtain a bacterial suspension; centrifuging the bacterial suspension, removing the supernatant, collecting bacterial sludge, adding the bacterial sludge into the whey calcium suspension, uniformly mixing, incubating on a shaking table, centrifuging, removing the supernatant, and collecting to obtain the solid of the whey calcium embedded intestinal flora. According to the invention, the intestinal flora is endowed with a better gastrointestinal fluid-resistant effect through a simple and feasible technical method, the edible biocompatible material is adopted to ensure that a patient can take the composition safely, the production cost is reduced, and the composition is suitable for production and application.

Description

Technical Field

[0001] The present invention relates to the technical field of flora transplantation, and in particular to a preparation method of a gastrointestinal fluid-resistant intestinal flora solid, a solid, a freeze-dried powder and a preparation method thereof. Background Art

[0002] Microbiota transplantation is a new technology that transplants the intestinal flora from the feces of healthy donors to patients, thereby adjusting or reconstructing the balance of the patient's intestinal flora. Since the exogenous intestinal flora taken by patients needs to pass through the stomach to reach the intestines to play a role, and H in the stomach + It will change the Zeta potential of the ingested flora, thereby changing the permeability of the flora surface, leading to the death of the flora, and the bile salts in the intestine will also dissolve lipids, thereby destroying the cell structure of the flora and inactivating the flora. Therefore, how to protect the transplanted exogenous intestinal flora to smoothly enter the patient's intestines and play a role is the current difficulty and focus in the field of flora transplantation.

[0003] Encapsulation technology is a scientific and technological means widely used in the food, medicine, cosmetics, agriculture and textile industries. It involves encapsulating one or more core substances (such as active ingredients, cells, enzymes, etc.) in one or more layers of protective wall materials to improve their stability, control release, improve physical properties or provide other functional protection. At present, in the field of probiotics, encapsulation technology mainly acts to improve the survival rate of probiotics in the human digestive tract, that is, by adding biocompatible materials, the outer layer of the single bacteria forms an "armor", thereby giving it a certain ability to resist damage by gastrointestinal fluid, improving the survival rate and number of live bacteria in the gastrointestinal tract, and ensuring efficacy.

[0004] At present, there are many methods reported for probiotics embedding technology, such as endogenous emulsification method, Pickering emulsification method, complex coacervation method and multi-layer embedding method, etc. The above methods protect probiotics from the destruction of gastrointestinal fluid through different mechanisms, but all of them have corresponding disadvantages. The endogenous emulsification method requires the addition of oil as the dispersed phase, and the preparation process is relatively cumbersome, which increases the process cost. Therefore, it is currently mainly in the laboratory stage; the Pickering emulsification method is prone to interaction between Pickering emulsion droplets stabilized by single particles, resulting in degradation of the emulsion system, and heating, compounding and hydrolysis methods are required to improve its particle size, surface roughness and wettability. In addition, the types of edible Pickering solid particles are limited, and there are certain limitations in biocompatibility and environmental compatibility. The preparation process is complicated, the production cost is high, and there are difficulties in industrial application; the preparation cost of the composite coacervation method is relatively high, the process is difficult, the solubility and wettability of the microcapsules are poor, which brings inconvenience to the application of the product; and the multi-layer encapsulation method has many preparation steps, high production cost, and the particle size of the microcapsules is generally large, which increases the difficulty of releasing the probiotic core material and affects the release rate.

[0005] In addition, compared with general probiotics, the composition of intestinal flora is more complex, which puts higher requirements on the embedding materials and embedding methods. Based on this, it is of great significance to propose an embedding process suitable for intestinal flora for intestinal flora transplantation. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a preparation method of a solid intestinal flora resistant to gastrointestinal fluid, a solid, a lyophilized powder and a preparation method thereof. A simple and easy technical method is used to give the intestinal flora a good gastrointestinal fluid resistance effect, and an edible biocompatible material is used to ensure that the patient can take it safely, while reducing the production cost and adapting to production applications.

[0007] In order to achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention is to provide a method for preparing a solid substance of intestinal flora resistant to gastrointestinal fluid, comprising the following steps: S1. Preparation of whey calcium suspension: Take whey calcium powder, add physiological saline and mix well to prepare a suspension with a whey calcium concentration of 1% to 20%; S2. Preparation of bacterial suspension: Take a fresh stool sample from a healthy donor, add physiological saline, stir and mix, filter, and collect the filtrate to obtain a bacterial suspension; S3. Whey calcium encapsulated intestinal flora: After centrifuging the S2 bacterial suspension, remove the supernatant, collect the bacterial mud, and add the bacterial mud to the S1 whey calcium suspension, wherein the weight of the bacterial mud and the addition ratio of the whey calcium suspension is 1:1 to 1:20 (w:v / g:ml) according to the mass volume ratio. After mixing, incubate on a shaker and then centrifuge, remove the supernatant, and collect the solid matter of the intestinal flora embedded in whey calcium.

[0008] The present invention uses whey calcium as the only biocompatible material for embedding, and through a dual physical and chemical protection mechanism, it gives the intestinal flora the ability to resist damage by gastrointestinal fluid. In terms of the physical protection mechanism, the calcium ions in the whey react with the cell wall components of the flora (such as acidic polysaccharides and proteins) to form a stable protective film, namely a gel network. This process constructs a "physical armor" for the intestinal flora, effectively isolating and reducing the erosion of the flora by gastrointestinal fluid; in addition, the gel structure formed by the whey protein under the action of calcium ions further enhances the mechanical stability of this "physical armor", providing additional protection for the flora. In terms of the chemical protection mechanism, the calcium ions in the whey calcium can react with the hydrochloric acid in the gastric acid to form a soluble calcium salt. This pH buffering ability helps to neutralize the gastric acid, providing a "chemical armor" for the intestinal flora to protect it from gastric fluid H +In addition, the adhesion-promoting effect of whey calcium helps the flora colonize the intestinal mucosa, and as a nutrient, it can support the growth and activity of the flora in the gastrointestinal tract. The protein and calcium in whey also improve the heat resistance of the intestinal flora and enhance its stability during processing and storage.

[0009] The use of an appropriate concentration of whey calcium suspension and an appropriate ratio of bacterial mud weight to whey calcium suspension are very important for improving the ability of intestinal flora to resist gastrointestinal fluid; according to a further technical solution, the concentration of the whey calcium suspension described in S1 is 1% to 10%; it should be noted that the unit of the concentration of the whey calcium suspension should be understood as mass (g) volume (ml) concentration.

[0010] Preferably, the concentration of the whey calcium suspension in S1 is 1% to 5%.

[0011] According to a further technical solution, the addition ratio of the bacterial mud weight to the whey calcium suspension in S3 is 1:9 to 1:20. Further, the addition ratio of the bacterial mud weight to the whey calcium suspension in S3 is preferably 1:9.

[0012] According to a further technical solution, the incubation time used in S3 is 1 min to 30 min; Preferably, the incubation time used in S3 is 2 min to 5 min.

[0013] According to a further technical solution, the shaking table speed used in S3 is 80 rpm to 600 rpm; Preferably, the shaking table rotation speed used in S3 is 200 rpm to 600 rpm.

[0014] The second aspect of the present invention is to provide a solid material resistant to gastrointestinal fluid and intestinal flora, which is prepared by the above method.

[0015] The third aspect of the present invention is to provide a freeze-dried powder of intestinal flora resistant to gastrointestinal fluid, wherein the raw materials for preparing the freeze-dried powder include the solid material of intestinal flora resistant to gastrointestinal fluid and a freeze-dried protective agent for intestinal flora. The sample made into freeze-dried powder is easier to transport and store, and can be derived and developed into a variety of dosage forms, such as tablets, pills, etc.

[0016] According to a further technical solution, the intestinal flora freeze-drying protective agent described in S4 can be selected from at least one of maltitol, xylitol, fish protein peptide, sea urchin extract, hydroxypropyl methylcellulose, xanthan gum, manno-oligosaccharide, high-fat pectin, sorbitol, red algae sulfate polysaccharide, carrageenan, chitosan, sea urchin glycoprotein, mannitol, and seaweed pectin; According to a further technical solution, the intestinal flora freeze-drying protective agent in S4 is selected from at least one of xylitol, sorbitol, mannitol, maltitol, fucoidan, fish protein peptide, N-acetylglucose, and ice structure protein; Furthermore, the present application has screened a combination of freeze-drying protectants suitable for the above-mentioned solid materials of intestinal flora resistant to gastrointestinal fluid. The intestinal flora freeze-drying protectant is composed of xylitol, sorbitol, mannitol and maltitol in a mass ratio of 2:2:5:1; or it is composed of fucoidan, fish protein peptide, N-acetylglucose and ice structure protein in a mass ratio of 1:4:2.5:2.5. The latter is particularly suitable for solid materials of intestinal flora resistant to gastrointestinal fluid. The use of the latter freeze-drying protectant can significantly improve the freeze-dried survival rate of intestinal flora.

[0017] The fourth aspect of the present invention is to provide a method for preparing the freeze-dried powder of intestinal flora resistant to gastrointestinal fluid, comprising the following steps: (1) Preparation of lyophilized protective agent solution: Take intestinal flora lyophilized protective agent, add physiological saline to dissolve and mix to prepare lyophilized protective agent solution with a mass volume concentration of 5% to 20%, and sterilize for later use; (2) Preparation of whey calcium-encapsulated intestinal flora freeze-dried powder: The gastrointestinal fluid-resistant intestinal flora solids are mixed with a lyophilization protectant solution in a mass volume ratio of 1:1 to 1:15 (w:v / g:ml), and freeze-dried to obtain whey calcium-encapsulated intestinal flora freeze-dried powder.

[0018] It should be noted that the unit of the concentration of the lyoprotectant solution should be understood as mass (g) volume (ml) concentration.

[0019] According to a further technical solution, the ratio of the weight of the solid matter to the lyophilization protective agent solution in step (2) is 1:1 to 1:6.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. The intestinal flora encapsulation method provided by the present invention can endow the intestinal flora in the form of bacterial mud with gastrointestinal fluid resistance. At the same time, the intestinal flora in the form of freeze-dried powder also has good gastrointestinal fluid resistance after being embedded with whey calcium. This result can diversify the form of the final product on the one hand, and on the other hand, it also proves the effect of whey calcium embedding.

[0021] 2. The technical method of the present invention is simple and easy to implement. The whey calcium embedding process requires a short time from centrifugation, incubation to re-centrifugation. The process operation is easy and has fewer steps. It is more suitable for future production links. It can be expected that the requirements for operators will be greatly reduced, the process cost and time cost will be reduced, and the heating, compounding and hydrolysis methods required by other technologies can be avoided.

[0022] 3. The embedding structure formed by the technical method of the present invention is a single-layer micron-level structure as seen under microscopic examination, which avoids the problems of large microcapsule particle size and increased difficulty in releasing bacteria, and is easier for patients to take, especially for the elderly and young children.

[0023] 4. In the preparation process of the present invention, only edible whey calcium is used as the embedding material, which reduces the potential safety hazards and risks of patients taking the drug, enhances biocompatibility, and can also supplement corresponding nutrients.

[0024] In summary, the above-mentioned technical method based on whey calcium "armor" encapsulation of intestinal flora has important application prospects in transplanting intestinal flora to resist gastrointestinal fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the microscopic examination picture of the control group of Example 1; Figure 2 This is the microscopic examination picture of the test group of Example 1; Figure 3 This is the microscopic examination picture of the control group of Example 4; Figure 4 This is the microscopic examination picture of the test group of Example 4. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The whey calcium used in the following examples of the present invention is food-grade whey calcium, the main components of which are milk calcium and whey inorganic salt concentrate, purchased from Leshengyuan Biotechnology (Nanjing) Co., Ltd., item number S-6.

[0028] The methods are conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0029] Example 1: Preparation of solid materials resistant to gastrointestinal fluid and intestinal flora (1) Preparation of artificial gastric juice: Accurately measure 16.4 mL of dilute hydrochloric acid, add 800 mL of water and 10 g of pepsin and mix thoroughly, add water to make up to 1000 mL and mix thoroughly, adjust the pH to 2.5 with 1 mol / L HCl, filter and sterilize with a 0.22 μm microporous filter membrane after sufficient dissolution, and place in a 4 ℃ refrigerator for later use.

[0030] (2) Preparation of artificial intestinal fluid: Take 6.8 g of potassium dihydrogen phosphate and add 500 ml of water to dissolve it. Use 0.1 mol / L sodium hydroxide solution to adjust the pH value to 6.8. Take another 10 g of pancreatic enzyme and add appropriate amount of water to dissolve it. Mix the two liquids and dilute with water to 1000 ml. Place in a 4°C refrigerator for later use.

[0031] (3) Preparation of whey calcium suspension: Take 50 g of food-grade whey calcium powder, add it to 1000 ml of normal saline, vortex and mix well to complete the preparation of 5% whey calcium suspension, and place it in a 4 ℃ refrigerator for later use.

[0032] (4) Preparation of bacterial suspension: 100 g of fresh feces from three different healthy donors were selected and added to 800 ml of normal saline respectively. The mixture was placed in a blender and stirred for 5 min at a speed of 900 rpm / min and a temperature of 4°C. The mixture was then filtered through 20-mesh, 40-mesh, and 60-mesh filters in sequence, and the filtrate was collected to obtain the bacterial suspension.

[0033] (5) Preparation of whey calcium-encapsulated intestinal flora solids: The bacterial suspension prepared from fresh feces of different healthy donors was divided into two equal portions by volume, each with 400 ml, one as the experimental group and the other as the control group. Both groups were centrifuged at 7000 rpm / min and 4°C for 5 min, the supernatant was removed and the bacterial sludge was collected. The control group was added with normal saline at a ratio of 1:9 (w:v / g:ml) by weight of the bacterial sludge, and the experimental group was added with 5% whey calcium suspension at a ratio of 1:9 (w:v / g:ml) by weight of the bacterial sludge. The two groups were vortexed and incubated on a shaker at 4°C for 5 min at a speed of 200 rpm / min. The two groups were then centrifuged again at 7000 rpm / min and 4°C for 5 min, the supernatant was removed and the bacterial sludge was collected. At this point, the experimental group completed the whey calcium encapsulation.

[0034] (6) Artificial gastrointestinal fluid test of embedded intestinal flora: Part of the bacterial sludge from the above-mentioned test group and the control group was respectively entered into the flow cytometer to detect its initial activity. The specific operation was as follows: the bacterial sludge was diluted 1000 times with physiological saline and then stained with Syto-9 and PI (taken from LIVE / DEAD™ BacLight™ Bacterial Viability Kit). After 15 minutes of dark reaction, it was entered into the flow cytometer for detection. The remaining bacterial sludge samples of the experimental group and the control group were added with artificial gastric juice at a ratio of 1:9 (w:v / g:ml), vortexed and mixed, and incubated in a shaker at 37°C for 2h, with a shaker speed of 120rpm / min. After incubation, they were taken out and centrifuged at 7000rpm / min and 4°C for 5min, the supernatant was removed, and the bacterial sludge was collected. The control group and the experimental group were again added with artificial intestinal juice at a ratio of 1:9 (w:v / g:ml) by bacterial sludge weight, incubated in a shaker at 37°C for 3h, with a shaker speed of 120rpm / min. After incubation, they were taken out and centrifuged at 7000rpm / min and 4°C for 5min, the supernatant was removed, and the bacterial sludge was collected. Finally, they were respectively entered into the flow cytometer to detect their activity after treatment with artificial gastrointestinal juice. The dilution, staining, and reaction steps were the same as above. The comprehensive test data of artificial gastrointestinal juice of intestinal flora solids embedded with whey calcium are shown in Table 1 below: Table 1 Comprehensive test data of whey calcium encapsulated intestinal flora solids in artificial gastrointestinal fluid

[0035] From the above, we can see that the average survival rate of the unencapsulated intestinal flora after treatment with artificial gastrointestinal fluid is only 8.16%, while the average survival rate of the intestinal flora after encapsulation with whey calcium can reach 94.04%, which is much higher than that of the unencapsulated group, and the average survival rate is increased by 1052.45%.

[0036] The results show that on the one hand, whey calcium embedding treatment does not affect the initial activity of intestinal flora, and on the other hand, it can also give intestinal flora a stronger ability to resist artificial gastrointestinal fluid. In addition, by microscopic examination of the test group after embedding and the control group without embedding, the typical microscopic examination of the control group samples before artificial gastrointestinal fluid treatment is shown in the figure below. Figure 1 The typical microscopic examination pictures of the test group samples before artificial gastrointestinal fluid treatment are shown in Figure 2 As shown in the figure, it can be seen that there are a lot of adhesive substances on the outer layer of the bacterial flora in the test group, which aggregate them into a mass. It is considered that the calcium ions in the whey cross-link with the cell wall components of the bacterial flora to form a gel network.

[0037] Example 2: Preparation of solid materials resistant to gastrointestinal fluid and intestinal flora (1) The preparation method of artificial gastric juice is the same as that in Example 1.

[0038] (2) The preparation method of artificial intestinal fluid is the same as that in Example 1.

[0039] (3) Preparation of whey calcium suspension: Take 10 g of food-grade whey calcium powder, add it to 1000 ml of normal saline and vortex to mix well to complete the preparation of 1% whey calcium suspension. Place it in a 4 °C refrigerator for later use.

[0040] (4) The method for preparing the bacterial suspension is the same as that in Example 1.

[0041] (5) The preparation method of whey calcium-encapsulated intestinal flora solids is the same as that in Example 1.

[0042] (6) The test method for artificial gastrointestinal fluid of intestinal flora encapsulation is the same as that in Example 1. The comprehensive test data of artificial gastrointestinal fluid of intestinal flora encapsulation solid of whey calcium are shown in Table 2 below: Table 2 Comprehensive data of artificial gastrointestinal fluid test of embedded intestinal flora

[0043] From the above, we can see that the average survival rate of the unencapsulated intestinal flora after treatment with artificial gastrointestinal fluid is only 8.12%, while the average survival rate of the intestinal flora after encapsulation with whey calcium can reach 92.02%, which is much higher than that of the unencapsulated group, and the average survival rate is increased by 1033.25%.

[0044] Example 3: Preparation of solid materials resistant to gastrointestinal fluid and intestinal flora (1) The preparation method of artificial gastric juice is the same as that in Example 1.

[0045] (2) The preparation method of artificial intestinal fluid is the same as that in Example 1.

[0046] (3) Preparation of whey calcium suspension: Take 10 g of food-grade whey calcium powder, add it to 1000 ml of normal saline and vortex to mix well to complete the preparation of 1% whey calcium suspension. Place it in a 4 °C refrigerator for later use.

[0047] (4) The method for preparing the bacterial suspension is the same as that in Example 1.

[0048] (5) Preparation of whey calcium-encapsulated intestinal flora solids: The bacterial suspension was divided into two equal portions by volume, each portion was 400 ml, one portion was used as the experimental group and the other as the control group. Both groups were centrifuged at 7000 rpm / min and 4°C for 5 min, the supernatant was removed and the bacterial sludge was collected. The control group was added with physiological saline at a ratio of 1:20 (w:v / g:ml) by weight of the bacterial sludge, and the experimental group was added with 1% whey calcium suspension at a ratio of 1:20 (w:v / g:ml) by weight of the bacterial sludge. The two groups were vortexed and mixed separately, and then incubated on a shaker at 4°C for 2 min with a shaker speed of 600 rpm / min. Subsequently, the two groups were centrifuged again at 7000 rpm / min and 4°C for 5 min, the supernatant was removed and the bacterial sludge was collected. At this point, the experimental group completed the whey calcium encapsulation.

[0049] (6) The test method for artificial gastrointestinal fluid of encapsulated intestinal flora is the same as that in Example 1. The comprehensive test data of artificial gastrointestinal fluid of whey calcium encapsulated intestinal flora solids are shown in Table 3 below: Table 3 Comprehensive data of artificial gastrointestinal fluid test of embedded intestinal flora

[0050] From the above, we can see that the average survival rate of the unencapsulated intestinal flora after treatment with artificial gastrointestinal fluid is only 8.64%, while the average survival rate of the intestinal flora after encapsulation with whey calcium can reach 88.45%, which is much higher than that of the unencapsulated group, and the average survival rate is increased by 923.73%.

[0051] Example 4: Preparation of freeze-dried powder of embedded intestinal flora (1) Preparation of intestinal flora freeze-drying protective agent: Take 20g of food-grade xylitol, 20g of food-grade sorbitol, 50g of food-grade mannitol, and 10g of food-grade maltitol, add 1000ml of normal saline to dissolve and mix, then sterilize at high temperature and high pressure, take out and let it stand at room temperature to cool, then put it in a 4℃ refrigerator for use.

[0052] (2) Preparation method of embedded intestinal flora freeze-dried powder: fresh feces of 3 different healthy donors were selected, and bacterial suspensions were prepared according to the method of Example 1. A test group of whey calcium embedded intestinal flora solids and a control group of unembedded intestinal flora sludge were further prepared according to the method of Example 1. After obtaining the whey calcium embedded intestinal flora sludge (i.e., the test group) and the unembedded intestinal flora sludge (i.e., the control group), intestinal flora freeze-drying protective agents were added to the two groups at a ratio of 1:6 (w:v / g:ml) by weight of the sludge, and after vortex mixing, a portion of the sample was diluted, stained, reacted, and then entered into a flow cytometer to detect its pre-freeze-drying activity. The relevant detection steps were the same as above. The remaining samples were placed in a vacuum freeze dryer for freeze-drying. The freeze-drying was carried out according to the procedure in Table 3.

[0053] Table 4 Freeze-drying process formula of intestinal flora

[0054] After freeze-drying, the freeze-dried powder samples of the control group and the test group were collected, ground evenly and then respectively placed into a flow cytometer to detect their post-freeze-drying activities. The relevant detection steps were the same as those in Example 1, and the freeze-dried survival rate was calculated.

[0055] Freeze-dried survival rate (%) = sample activity after freeze-drying (%) / sample activity before freeze-drying (%) (3) Artificial gastrointestinal fluid test of freeze-dried powder for embedding intestinal flora: Then, the remaining freeze-dried powder samples of the control group and the experimental group were added with artificial gastric fluid at a ratio of 1:9 (w:v / g:ml) by weight of freeze-dried powder, vortexed and incubated in a shaker at 37°C for 2 h, with a shaker speed of 120 rpm / min. After incubation, the samples were taken out and centrifuged at 7000 rpm / min and 4°C for 5 min. The supernatant was removed and the bacterial sludge was collected. The control group and the experimental group were again added with artificial intestinal fluid at a ratio of 1:9 (w:v / g:ml) by weight of bacterial sludge, incubated in a shaker at 37°C for 3 h, with a shaker speed of 120 rpm / min. After incubation, the samples were taken out and centrifuged at 7000 rpm / min and 4°C for 5 min. The supernatant was removed and the bacterial sludge was collected. Finally, the samples were entered into a flow cytometer to detect their activity after treatment with artificial gastrointestinal fluid. The dilution, staining and reaction steps were the same as above. The results are shown in Table 5: Table 5 Comprehensive data of test on artificial gastrointestinal fluid of freeze-dried powder of embedded intestinal flora

[0056] From the above, we can see that the average survival rate of the unencapsulated intestinal flora freeze-dried powder after treatment with artificial gastrointestinal fluid is only 5.85%, while the average survival rate of the intestinal flora freeze-dried powder after whey calcium embedding can reach 95.78%, which is much higher than that of the unencapsulated group, and the survival rate is increased by an average of 1537.26%.

[0057] This result shows that the freeze-dried intestinal flora after being embedded with whey calcium still has a strong ability to resist gastrointestinal fluid. Therefore, more new dosage forms such as tablets and pills can be derived based on this method in the future. The freeze-dried powder products will be able to get rid of the capsule form and even be prepared into strip-shaped freeze-dried powder for consumption. In addition, the smell of the test group is better than that of the control group. It has a certain milky flavor of dairy products and is easier for patients to take. The freeze-dried powder samples of the test group and the control group were examined under a microscope. The typical microscopic examination of the freeze-dried powder of the control group before artificial gastrointestinal fluid treatment is shown in the figure below. Figure 3 The typical microscopic examination of the freeze-dried powder in the test group before artificial gastrointestinal fluid treatment is shown in Figure 4 It can be seen that after freeze-drying, the gel "physical armor" on the outer layer of the experimental group's flora still has a certain shape and has not been broken up, which may also be one of the reasons that give the intestinal flora the ability to resist gastrointestinal fluid.

[0058] (4) Tracking the activity of the freeze-dried powder of embedded intestinal flora after storage: In order to investigate the activity of the freeze-dried powder of embedded intestinal flora after storage at -20°C, the freeze-dried powder samples of the test group (the embedding and freeze-drying steps were the same as above) were stored at -20°C, and the samples were taken out on the 14th day for flow cytometry detection using the same detection method as above. The results showed that the average activity of the test group was 19.42±6.02% (the activity before storage was 20.55±5.61%), indicating that the activity of the freeze-dried powder of embedded intestinal flora was relatively stable after storage at -20°C for 14 days.

[0059] Example 5: Preparation of freeze-dried powder of embedded intestinal flora (1) The preparation method of the intestinal flora freeze-drying protective agent is the same as that of Example 4; (2) Preparation method of embedded intestinal flora freeze-dried powder: fresh feces of three different healthy donors were selected, and bacterial suspensions were prepared according to the method of Example 2. A test group of whey calcium embedded intestinal flora solids and a control group of unencapsulated intestinal bacteria mud were prepared according to the method of Example 2. The embedded intestinal flora freeze-dried powder was prepared according to the method of Example 4; (3) The test method for artificial gastrointestinal fluid of the embedded intestinal flora freeze-dried powder is the same as that in Example 4.

[0060] The comprehensive data of the test on artificial gastrointestinal fluid of the embedded intestinal flora freeze-dried powder are shown in Table 6: Table 6 Comprehensive data of test on artificial gastrointestinal fluid of freeze-dried powder of embedded intestinal flora

[0061] From the above, we can see that the average survival rate of the unencapsulated intestinal flora freeze-dried powder after treatment with artificial gastrointestinal fluid is only 8.02%, while the average survival rate of the intestinal flora freeze-dried powder after whey calcium embedding can reach 87.15%, which is much higher than that of the unencapsulated group, and the survival rate is increased by an average of 986.66%.

[0062] Example 6: Preparation of freeze-dried powder of embedded intestinal flora (1) Preparation of intestinal flora freeze-drying protective agent: same as Example 4 (2) Preparation method of freeze-dried powder of embedded intestinal flora: fresh feces of three different healthy donors were selected, and bacterial suspension was prepared according to the method of Example 3. A test group of whey calcium embedded intestinal flora solids and a control group of unencapsulated intestinal flora sludge were further prepared according to the method of Example 3. The embedded intestinal flora freeze-dried powder was prepared according to the method of Example 4; (3) Artificial gastrointestinal fluid test of freeze-dried powder of embedded intestinal flora: the method is the same as that of Example 4.

[0063] The comprehensive data of the test on artificial gastrointestinal fluid of the embedded intestinal flora freeze-dried powder are shown in Table 7: Table 7 Comprehensive data of test on artificial gastrointestinal fluid of freeze-dried powder of embedded intestinal flora

[0064] From the above, we can see that the average survival rate of the unencapsulated intestinal flora freeze-dried powder after treatment with artificial gastrointestinal fluid is only 10.38%, while the average survival rate of the intestinal flora freeze-dried powder after whey calcium embedding can reach 85.63%, which is much higher than that of the unencapsulated group, and the survival rate is increased by an average of 724.95%.

[0065] Example 7: Preparation of freeze-dried powder of embedded intestinal flora (1) Preparation of intestinal flora freeze-drying protective agent: Take 10g of food-grade fucoidan, 40g of food-grade fish protein peptide, 25g of food-grade N-acetylglucose, and 25g of food-grade ice structure protein, add 1000ml of normal saline to dissolve and mix, and the subsequent steps are the same as above.

[0066] (2) Preparation method of freeze-dried powder of embedded intestinal flora: the method is the same as that of Example 4.

[0067] (3) Artificial gastrointestinal fluid test of freeze-dried powder of embedded intestinal flora: the method is the same as that of Example 4.

[0068] The comprehensive data of the test on artificial gastrointestinal fluid of the embedded intestinal flora freeze-dried powder are shown in Table 8: Table 8 Comprehensive data of test on artificial gastrointestinal fluid of freeze-dried powder of embedded intestinal flora

[0069] From the above, we can see that the average survival rate of the unencapsulated intestinal flora freeze-dried powder after treatment with artificial gastrointestinal fluid is only 8.54%, while the average survival rate of the intestinal flora freeze-dried powder after whey calcium embedding can reach 94.42%, which is much higher than that of the unencapsulated group, and the survival rate is increased by an average of 1005.62%.

[0070] Example 8: Preparation of freeze-dried powder of embedded intestinal flora (1) The preparation method of the intestinal flora freeze-drying protective agent is the same as that of Example 4.

[0071] (2) Preparation method of freeze-dried powder of embedded intestinal flora: the method of step (2) of Example 4 is followed, except that a freeze-dried protective agent for intestinal flora is added at a ratio of 1:1 (w:v / g:ml) by weight of bacterial mud; (3) Artificial gastrointestinal fluid test of freeze-dried powder of embedded intestinal flora: the method is the same as that of Example 4.

[0072] The comprehensive data of the test on artificial gastrointestinal fluid of the embedded intestinal flora freeze-dried powder are shown in Table 9: Table 9 Comprehensive data of test on artificial gastrointestinal fluid of freeze-dried powder of embedded intestinal flora

[0073] From the above, we can see that the average survival rate of the unencapsulated intestinal flora freeze-dried powder after treatment with artificial gastrointestinal fluid is only 7.62%, while the average survival rate of the intestinal flora freeze-dried powder after whey calcium embedding can reach 92.13%, which is much higher than that of the unencapsulated group, and the survival rate is increased by an average of 1109.06%.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present application can still be modified or some technical features can be replaced by equivalents, which should all be included in the scope of the technical solution requested for protection in this application.

Claims

1. A method for preparing a solid substance resistant to gastrointestinal fluid and intestinal flora, characterized in that: The following steps are involved: S1. Preparation of whey calcium suspension: Take whey calcium powder, add physiological saline and mix well to prepare a suspension with a whey calcium concentration of 1% to 20%; S2. Preparation of bacterial suspension: Take a fresh stool sample from a healthy donor, add physiological saline, stir and mix, filter, and collect the filtrate to obtain a bacterial suspension; S3. Whey calcium encapsulated intestinal flora: After centrifuging the S2 bacterial suspension, remove the supernatant, collect the bacterial mud, and add the bacterial mud to the S1 whey calcium suspension, wherein the weight of the bacterial mud and the addition ratio of the whey calcium suspension is 1:1 to 1:20 according to the mass volume ratio. After mixing, incubate on a shaker and then centrifuge, remove the supernatant, and collect the solid matter of the intestinal flora embedded in whey calcium.

2. The method for preparing a gastrointestinal fluid-resistant intestinal flora solid according to claim 1, characterized in that: The concentration of the whey calcium suspension in S1 is 1% to 10%; Preferably, the concentration of the whey calcium suspension in S1 is 1% to 5%.

3. The method for preparing a gastrointestinal fluid-resistant intestinal flora solid material according to claim 1 or 2, characterized in that: S3: The addition ratio of the bacterial mud weight to the whey calcium suspension is 1:9 to 1:

20.

4. The method for preparing a solid substance resistant to gastrointestinal fluid and intestinal flora according to any one of claims 1 to 3, characterized in that: The incubation time used in S3 was 1 min to 30 min; Preferably, the incubation time used in S3 is 2 min to 5 min.

5. The method for preparing a solid substance resistant to gastrointestinal fluid and intestinal flora according to any one of claims 1 to 4, characterized in that: The shaking table speed used in S3 is 80 rpm to 600 rpm; Preferably, the shaking table rotation speed used in S3 is 200 rpm to 600 rpm.

6. A solid material resistant to gastrointestinal fluid and intestinal flora, characterized in that: It is prepared by the method described in any one of claims 1 to 5.

7. A freeze-dried powder of intestinal flora resistant to gastrointestinal fluid, characterized in that: The raw materials for preparing the lyophilized agent include the gastrointestinal fluid-resistant intestinal flora solid material and the intestinal flora lyophilization protective agent as claimed in claim 6.

8. The freeze-dried powder of intestinal flora resistant to gastrointestinal fluid according to claim 7, characterized in that: The intestinal flora freeze-drying protective agent is selected from at least one of xylitol, sorbitol, mannitol, maltitol, fucoidan, fish protein peptide, N-acetylglucose, and ice structure protein; Preferably, the intestinal flora freeze-dried protective agent is prepared by compounding xylitol, sorbitol, mannitol and maltitol in a mass ratio of 2:2:5:1; or is prepared by compounding fucoidan, fish protein peptide, N-acetylglucose and ice structure protein in a mass ratio of 1:4:2.5:2.

5.

9. The method for preparing a freeze-dried powder of intestinal flora resistant to gastrointestinal fluid according to claim 7 or 8, characterized in that: The following steps are involved: (1) Preparation of lyophilized protective agent solution: Take intestinal flora lyophilized protective agent, add physiological saline to dissolve and mix to prepare lyophilized protective agent solution with a mass volume concentration of 5% to 20%, and sterilize for later use; (2) Preparation of whey calcium-encapsulated intestinal flora freeze-dried powder: The gastrointestinal fluid-resistant intestinal flora solids are mixed with a lyophilization protectant solution in a mass-to-volume ratio of 1:1 to 1:15, and freeze-dried to obtain the whey calcium-encapsulated intestinal flora freeze-dried powder.

10. The method for preparing a freeze-dried powder of intestinal flora resistant to gastrointestinal fluid according to claim 9, characterized in that: The ratio of the weight of the solid matter to the lyophilization protective agent solution in step (2) is 1:1 to 1:6.

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