Process for preparing intestinal conditioning prebiotics from clostridium butyricum and radix astragali

Abstract

The invention relates to the technical field of probiotic preparations, in particular to a process for preparing intestinal conditioning prebiotics from clostridium butyricum and radix astragali seu hedysari, which comprises the following steps: (1) multi-stage synergistic activation of clostridium butyricum; (2) carrying out composite enzymolysis on the astragalus extract; (3) symbiotic fermentation; (4) performing multi-layer embedding; the prepared prebiotics have a multi-dimensional intestine regulating mechanism, the bacteriostasis and mucous membrane repairing functions of the clostridium butyricum cooperate with the prebiotics effect and antioxidant activity of the astragalus polysaccharide to form a multi-protection system, and the intestinal flora imbalance is more effectively improved compared with single probiotics or prebiotics. The multi-layer embedding technology solves the problems of gastric acid inactivation and difficulty in intestinal colonization of probiotics, and spore induction of three-stage fermentation is matched, so that the viable count maintenance rate of the product is kept at a relatively high level after the product is stored for 6 months at normal temperature, and the shelf life is remarkably prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic preparations, and in particular to a process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus. Background Art

[0002] Clostridium butyricum is an anaerobic, Gram-positive bacillus. As a key member of intestinal probiotics, it metabolizes and produces short-chain fatty acids (such as butyrate and acetate), regulating intestinal pH, inhibiting the proliferation of harmful bacteria, and promoting repair of the intestinal mucosal barrier. Furthermore, Clostridium butyricum can stimulate the activity of intestinal immune cells and enhance the body's immune function.

[0003] Due to factors such as an unbalanced diet (high in fat and sugar) and the overuse of antibiotics, modern people generally suffer from intestinal flora imbalance, which manifests as constipation, diarrhea, and inflammatory bowel disease. An imbalance in the intestinal flora can further lead to systemic health problems such as metabolic syndrome and decreased immunity.

[0004] Regulating the balance of intestinal flora by supplementing with probiotics and prebiotics is an effective means of improving intestinal health. However, the practical application of single probiotics or prebiotics faces many problems: probiotics are easily destroyed by gastric acid and bile acid, and have a low survival rate.

[0005] Traditional prebiotics are mostly derived from plant-based dietary fiber (such as oligofructose and inulin). Their core function is to provide nutritional substrates for probiotics and improve the intestinal microbiome. Astragalus, a traditional Chinese medicinal herb, is rich in astragalus polysaccharides and flavonoids, which have natural prebiotic properties and can form a synergistic effect with probiotics. This provides new ideas for the development of new prebiotics.

[0006] In order to solve the above problems, the present invention provides a process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus. Summary of the Invention

[0007] The invention relates to a process for preparing intestinal regulating prebiotics by using Clostridium butyricum and astragalus.

[0008] The present invention provides the following technical solutions:

[0009] A process for preparing intestinal regulating prebiotics from Clostridium butyricum and Astragalus membranaceus comprises the following steps:

[0010] (1) Multi-stage synergistic activation of Clostridium butyricum: The original strain is treated through three stages: temperature gradient control, nutritional enhancement, and stress adaptation;

[0011] (2) Complex enzymatic hydrolysis of Astragalus extract: using a cellulase-xylanase-pectinase system for step-by-step enzymatic hydrolysis;

[0012] (3) Symbiotic fermentation: inoculating the activated strain from step (1) into a culture medium containing the astragalus extract from step (2) to perform a three-stage oxygen-controlled fermentation;

[0013] (4) Multi-layer encapsulation: The bacteria are encapsulated in a sodium alginate-chitosan matrix, and the outer layer of the encapsulated bacteria is coated with a pH-sensitive enteric coating.

[0014] As a further technical solution, the activation treatment in step (1) includes:

[0015] (11) Temperature gradient control: The original strain was prepared into a bacterial solution with a concentration of OD 600 The bacterial suspension was prepared by refrigerating at 4°C ± 0.5°C for 12-14 hours and then inoculating into RCM medium at an inoculum size of 5-5.2% (v / v) and incubating at 37°C under anaerobic conditions for 18 hours. Finally, the cultured bacterial suspension was transferred to fresh RCM medium at an inoculum size of 5-5.2% (v / v) and heat-shocked at 42°C ± 0.5°C, shaking at 150 rpm, and aeration at 1.5-1.8 L / min for 2 hours; the aeration was nitrogen.

[0016] (12) Nutritional fortification: RCM medium and fortified Clostridium medium were used alternately for culturing, with the inoculum volume of each culture being 5-5.2% (v / v); the RCM medium was cultured at 37°C under anaerobic conditions for 12-14 hours; the fortified Clostridium medium was cultured at 37°C under anaerobic conditions for 12-14 hours;

[0017] (13) Stress adaptation: During the culture phase in the enhanced Clostridium medium, sodium butyrate was added every 2 hours from the start of the culture. The first addition of sodium butyrate was sufficient to make the concentration of sodium butyrate in the culture medium reach 10 g / L. The concentration of sodium butyrate was increased by 5 g / L each time thereafter until the concentration reached 40-45 g / L. The culture was continued for two alternating cycles.

[0018] (14) During the entire activation process, the volume of the culture system at each stage remained consistent, all at 1 L.

[0019] As a further technical solution, in step (12), the R3M culture medium and the enhanced Clostridium culture medium are alternately used every 12 hours.

[0020] As a further technical solution, the specific preparation method of the composite enzymatic hydrolysis Astragalus extract in step (2) includes:

[0021] (21) Pretreatment: After the Astragalus root is sliced, it is defatted using supercritical carbon dioxide at a pressure of 30-35 MPa and a temperature of 45-46°C.

[0022] (22) Two-step enzymatic hydrolysis: In the first step, 50-56 U / g of cellulase was used for hydrolysis at 40°C and pH 5.0 for 1 h; in the second step, xylanase and pectinase were used, with the mass ratio of xylanase to pectinase and cellulase being 2:1:5, and hydrolysis was performed at 50°C and pH 5.5 for 2 h. The enzymatic hydrolysis product was obtained after enzyme inactivation treatment at 60°C.

[0023] As a further technical solution, after the two-step enzymatic hydrolysis in step (22), the enzymatic hydrolysis product is filtered and concentrated. The filtration is carried out using a polyethersulfone filter membrane with a pore size of 0.45 μm, and the product is concentrated under reduced pressure at a vacuum degree of -0.08 MPa and a temperature of 50°C to 1 / 5 of the original volume to obtain an Astragalus extract concentrate.

[0024] As a further technical solution, step (3) adopts three-stage dissolved oxygen control, specifically: during the proliferation period, the dissolved oxygen is 5.0-5.8%, the temperature is 37°C, and the duration is 0-12h; during the spore production period, the dissolved oxygen is ≤1.0%, the temperature is 37°C, and the duration is 12-24h; during the metabolic period, the dissolved oxygen is 2.0-2.2%, the temperature is 42°C, and the duration is 24-36h.

[0025] As a further technical solution, the method for preparing the culture medium containing the astragalus extract in step (2) in step (3) is as follows: take 200-230 mL of the treated astragalus extract concentrate in step (2), add 5-8 g of glucose, 10-12 g of peptone, and 3-4 g of sodium chloride, then dilute to 1 L with deionized water, and adjust the pH to 7.0±0.2.

[0026] As a further technical solution, in step (4), the mass ratio of sodium alginate to chitosan in the sodium alginate-chitosan matrix is 4:1-2, and the pH-sensitive enteric coating dissolves in an environment of pH ≥ 6.8.

[0027] As a further technical solution, in step (4), a sodium alginate-chitosan matrix is used to embed the bacterial cells, and a specific method of coating the outer layer of the embedded bacterial cells with a pH-sensitive enteric coating includes:

[0028] (1) Centrifuge the bacterial solution after symbiotic fermentation, discard the supernatant, and collect the bacterial precipitate;

[0029] The centrifugation parameters were: 8000 rpm, 10 min;

[0030] (2) Prepare embedding solution: Weigh 4 g of sodium alginate and dissolve it in 100 mL of deionized water. Heat to 60°C and stir until completely dissolved. After cooling to room temperature, add 1-2 g of chitosan. Adjust the pH to 5.5 with 1% acetic acid solution and stir evenly.

[0031] (3) Add the bacterial precipitate to the above embedding solution to make the bacterial concentration 1×1010 CFU / mL, after mixing evenly, drop into 0.2 mol / L calcium chloride solution and cross-link and cure for 30-35 minutes to form embedded microspheres;

[0032] (4) The embedded microspheres were washed three times with deionized water, and then placed in a pH-sensitive enteric coating solution. The coating was performed by spray coating at a temperature of 40°C and a pressure of 0.3 MPa. After coating, the microspheres were dried at 50°C for 2 h.

[0033] As a further technical solution, the concentration of the pH-sensitive enteric coating solution is 10%, and the solvent is ethanol.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention significantly improves the activity and stress resistance of the strain through multi-stage synergistic activation, including temperature gradient control, nutritional enhancement, and stress adaptation. Among them, 42°C heat shock treatment can induce the strain to produce heat shock proteins, enhancing its resistance to gastric acid; sodium butyrate stress adaptation promotes the strain's adaptability to the high-concentration butyric acid environment in the intestine, ultimately increasing the number of viable bacteria. Butyric acid produced by metabolism is the main energy source for intestinal epithelial cells, which can promote the proliferation and differentiation of intestinal mucosal cells and repair the intestinal barrier; acetic acid and propionic acid can lower the intestinal pH value and inhibit the growth of harmful bacteria such as Escherichia coli and Salmonella.

[0036] The composite enzymatic hydrolysis system provided by the present invention performs step-by-step enzymatic hydrolysis of astragalus, increasing the extraction rate of astragalus polysaccharides (such as astragaloside IV). As a prebiotic, astragalus polysaccharides can specifically promote the proliferation of Clostridium butyricum. Meanwhile, astragalus flavonoids and other components have antioxidant activity, can scavenge intestinal free radicals, and alleviate inflammatory responses. Supercritical carbon dioxide degreasing pretreatment removes fat-soluble impurities in astragalus, preventing their inhibitory effect on bacterial growth, while also improving the purity of polysaccharides.

[0037] The three-stage oxygen-controlled fermentation employed in this invention further optimizes the process: the proliferation phase promotes rapid bacterial growth; the sporulation phase induces spore formation, enhancing stress resistance; and the metabolic phase optimizes the short-chain fatty acid synthesis pathway, increasing butyrate production. Combined with the nutrient matrix in the Astragalus extract, this achieves a dynamic balance between bacterial growth and metabolite production.

[0038] The multi-layer encapsulation technology used in this method will have a significant protective effect; the sodium alginate-chitosan matrix forms microspheres through ionic cross-linking, with a high encapsulation efficiency, which can block the damage of gastric acid and digestive enzymes to bacteria; the outer pH-sensitive enteric coating (dissolved at pH ≥ 6.8) ensures that the microspheres are released in specific parts of the intestine, thereby improving the survival rate in the gastric acid environment and increasing the intestinal colonization rate.

[0039] The prebiotics prepared by this invention possess multi-dimensional intestinal regulating mechanisms. The antibacterial and mucosal repair functions of Clostridium butyricum synergize with the prebiotic effects and antioxidant activity of astragalus polysaccharides to form a multi-layered protective system, more effectively improving intestinal flora imbalance than single probiotics or prebiotics. Multi-layer encapsulation technology addresses the issues of probiotic inactivation by gastric acid and difficulty in intestinal colonization. Combined with spore induction through three-stage fermentation, the product maintains a high level of viable bacterial count after six months of storage at room temperature, significantly extending its shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a process flow chart for preparing intestinal regulating prebiotics using Clostridium butyricum and Astragalus. DETAILED DESCRIPTION

[0041] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0042] The original strain of Clostridium butyricum of the present invention is classified and named Clostridium butyricum F06, with a preservation number of CCTCCNO: M2019962, reference document: application publication number 202111200171.5, Clostridium butyricum and its application.

[0043] The present invention provides a process for preparing an intestinal regulating prebiotic from Clostridium butyricum and Astragalus membranaceus, comprising the following steps:

[0044] (1) Multi-stage synergistic activation of Clostridium butyricum: The original strain is treated through three stages: temperature gradient control, nutritional enhancement, and stress adaptation;

[0045] (2) Complex enzymatic hydrolysis of Astragalus extract: using a cellulase-xylanase-pectinase system for step-by-step enzymatic hydrolysis;

[0046] (3) Symbiotic fermentation: inoculating the activated strain from step (1) into a culture medium containing the astragalus extract from step (2) to perform a three-stage oxygen-controlled fermentation;

[0047] (4) Multi-layer encapsulation: The bacteria are encapsulated in a sodium alginate-chitosan matrix, and the outer layer of the encapsulated bacteria is coated with a pH-sensitive enteric coating.

[0048] Multi-stage synergistic activation of Clostridium butyricum

[0049] In the present invention, the activation treatment includes:

[0050] (11) Temperature gradient control: The original strain was prepared into a bacterial solution with a concentration of OD600 The bacterial suspension was prepared by refrigerating at 4°C ± 0.5°C for 12-14 hours and then inoculating into RCM medium at an inoculum size of 5-5.2% (v / v) and incubating at 37°C under anaerobic conditions for 18 hours. Finally, the cultured bacterial suspension was transferred to fresh RCM medium at an inoculum size of 5-5.2% (v / v) and heat-shocked at 42°C ± 0.5°C, shaking at 150 rpm, and aeration at 1.5-1.8 L / min for 2 hours; the aeration was nitrogen.

[0051] (12) Nutritional fortification: RCM medium and fortified Clostridium medium were used alternately for culturing, with the inoculum volume of each culture being 5-5.2% (v / v); the RCM medium was cultured at 37°C under anaerobic conditions for 12-14 hours; the fortified Clostridium medium was cultured at 37°C under anaerobic conditions for 12-14 hours;

[0052] In the present invention, the components and dosage of RCM culture medium are as follows per liter: peptone 10.0g, beef powder 10.0g, yeast powder 3.0g, glucose 5.0g, soluble starch 1.0g, sodium acetate 3.0g, sodium chloride 5.0g, L-cysteine hydrochloride 0.5g, agar 0.5g, pH 6.8±0.2 (25°C).

[0053] The composition and dosage of the enhanced Clostridium culture medium are basically the same as those of the RCM culture medium. Per liter, the following contents are: peptone 10.0 g, beef powder 10.0 g, yeast powder 3.0 g, glucose 5.0 g, soluble starch 1.0 g, sodium acetate 3.0 g, sodium chloride 5.0 g, L-cysteine hydrochloride 0.5 g, agar 0.5 g, corn flour 5 g, potassium permanganate 0.05 g, pH 6.8±0.2 (25°C).

[0054] In actual preparation, weigh the corresponding amount of each ingredient into 1L of distilled water or deionized water, heat and boil until completely dissolved, then autoclave at 121℃ for 15 minutes and cool;

[0055] (13) Stress adaptation: During the culture phase in the enhanced Clostridium medium, sodium butyrate was added every 2 hours from the start of the culture. The first addition of sodium butyrate was sufficient to make the concentration of sodium butyrate in the culture medium reach 10 g / L. The concentration of sodium butyrate was increased by 5 g / L each time thereafter until the concentration reached 40-45 g / L. The culture was continued for two alternating cycles.

[0056] (14) During the entire activation process, the volume of the culture system at each stage remained consistent, all at 1 L.

[0057] Compound enzymatic hydrolysis astragalus extract

[0058] In the present invention, the specific preparation method of the composite enzymatic hydrolysis astragalus extract includes:

[0059] (21) Pretreatment: After the Astragalus root is sliced, it is defatted using supercritical carbon dioxide at a pressure of 30-35 MPa and a temperature of 45-46°C.

[0060] (22) Two-step enzymatic hydrolysis: In the first step, 50-56 U / g of cellulase was used for hydrolysis at 40°C and pH 5.0 for 1 h; in the second step, xylanase and pectinase were used, with the mass ratio of xylanase to pectinase and cellulase being 2:1:5, and hydrolysis was performed at 50°C and pH 5.5 for 2 h. The enzymatic hydrolysis product was obtained after enzyme inactivation treatment at 60°C.

[0061] In the present invention, after the two-step enzymatic hydrolysis in step (22), the enzymatic hydrolysis product is filtered and concentrated. The filtration is performed using a polyethersulfone filter membrane with a pore size of 0.45 μm, and the product is concentrated under reduced pressure at a vacuum degree of -0.08 MPa and a temperature of 50°C to 1 / 5 of the original volume to obtain an Astragalus extract concentrate.

[0062] Symbiotic fermentation

[0063] In the present invention, step (3) adopts three-stage dissolved oxygen control, specifically: proliferation period, dissolved oxygen is 5.0-5.8%, temperature is 37°C, and duration is 0-12h; sporulation period, dissolved oxygen ≤1.0%, temperature is 37°C, and duration is 12-24h; metabolic period, dissolved oxygen is 2.0-2.2%, temperature is 42°C, and duration is 24-36h.

[0064] In the present invention, the method for preparing the culture medium containing the astragalus extract in step (2) in step (3) is as follows: taking 200-230 mL of the astragalus extract concentrate after treatment in step (2), adding 5-8 g of glucose, 10-12 g of peptone, and 3-4 g of sodium chloride, and then diluting the volume to 1 L with deionized water, and adjusting the pH to 7.0±0.2.

[0065] Multilayer embedding

[0066] In the present invention, in step (4), the mass ratio of sodium alginate to chitosan in the sodium alginate-chitosan matrix is 4:1-2, and the pH-sensitive enteric coating dissolves in an environment of pH ≥ 6.8.

[0067] In the present invention, in step (4), a sodium alginate-chitosan matrix is used to embed the bacterial cells, and a specific method of coating the outer layer of the embedded bacterial cells with a pH-sensitive enteric coating includes:

[0068] (1) Centrifuge the bacterial solution after symbiotic fermentation, discard the supernatant, and collect the bacterial precipitate; the centrifugation parameters are: 8000 rpm, 10 min;

[0069] (2) Prepare embedding solution: Weigh 4 g of sodium alginate and dissolve it in 100 mL of deionized water. Heat to 60°C and stir until completely dissolved. After cooling to room temperature, add 1-2 g of chitosan. Adjust the pH to 5.5 with 1% acetic acid solution and stir evenly.

[0070] (3) Add the bacterial precipitate to the embedding solution to a bacterial concentration of 1 × 1010 CFU / mL. After mixing evenly, add dropwise to a 0.2 mol / L calcium chloride solution and cross-link and solidify for 30-35 minutes to form embedded microspheres.

[0071] (4) The embedded microspheres were washed three times with deionized water, and then placed in a pH-sensitive enteric coating solution. The coating was performed by spray coating at a temperature of 40°C and a pressure of 0.3 MPa. After coating, the microspheres were dried at 50°C for 2 h.

[0072] In the present invention, the concentration of the pH-sensitive enteric coating solution is 10%, and the solvent is ethanol.

[0073] The preparation process provided by the present invention improves the activity and stability of Clostridium butyricum through multi-stage synergistic activation, the composite enzymatic hydrolysis system improves the extraction rate of the effective ingredients of Astragalus, the three-stage oxygen-controlled fermentation optimizes the growth of the bacteria and the generation of metabolites, and the multi-layer embedding technology improves the survival rate of the bacteria in the gastrointestinal tract, thereby realizing the efficient preparation of intestinal regulating prebiotics.

[0074] In order to further illustrate the present invention, the following examples are given below to provide a detailed description.

[0075] Example 1

[0076] (1) Multi-stage synergistic activation of Clostridium butyricum:

[0077] (11) Temperature gradient control: The original strain was prepared into a bacterial solution with a concentration of OD 600 The bacterial suspension was prepared by refrigerating at 4°C for 12 hours and then inoculating into RCM medium at a volume of 5% (v / v) and incubating at 37°C under anaerobic conditions for 18 hours. Finally, the cultured bacterial suspension was transferred to fresh RCM medium at a volume of 5% (v / v) and heat-shocked at 42°C, shaking at 150 rpm, and aeration at 1.5 L / min for 2 hours.

[0078] (12) Nutritional fortification: RCM medium and fortified Clostridium medium were used alternately for cultivation, switching every 12 hours, with the inoculum volume of each culture being 5% (v / v); RCM medium was cultured at 37°C under anaerobic conditions for 12 hours; fortified Clostridium medium was cultured at 37°C under anaerobic conditions for 12 hours;

[0079] (13) Stress adaptation: During the culture phase in the enhanced Clostridium medium, sodium butyrate was added every 2 hours from the start of the culture. The concentration of sodium butyrate in the culture medium reached 10 g / L after the first addition, and the concentration was increased by 5 g / L each time thereafter until the concentration reached 40 g / L. The culture was continued for two alternating cycles.

[0080] (2) Compound enzymatic hydrolysis of Astragalus extract:

[0081] (21) Pretreatment: After the Astragalus root was sliced, it was defatted using supercritical carbon dioxide at a pressure of 30 MPa and a temperature of 45°C.

[0082] (22) Two-step enzymatic hydrolysis: In the first step, 50 U / g of cellulase was used for hydrolysis at 40°C and pH 5.0 for 1 h; in the second step, xylanase and pectinase were used, with the mass ratio of xylanase, pectinase, and cellulase being 2:1:5, for hydrolysis at 50°C and pH 5.5 for 2 h, and the enzymes were inactivated at 60°C;

[0083] (23) Filtration and concentration: The enzymatic hydrolysis product was filtered through a polyethersulfone filter membrane with a pore size of 0.45 μm and concentrated under reduced pressure to 1 / 5 of the original volume at a vacuum degree of -0.08 MPa and a temperature of 50°C to obtain an astragalus extract concentrate;

[0084] (3) Symbiotic fermentation:

[0085] (31) Culture medium preparation: Take 200 mL of Astragalus extract concentrate, add 5 g of glucose, 10 g of peptone, and 3 g of sodium chloride, dilute to 1 L with deionized water, and adjust the pH to 7.0;

[0086] (32) Three-stage oxygen-controlled fermentation: proliferation phase, dissolved oxygen 5.0%, temperature 37°C, duration 0-12 h; sporulation phase, dissolved oxygen 0.8%, temperature 37°C, duration 12-24 h; metabolic phase, dissolved oxygen 2.0%, temperature 42°C, duration 24-36 h;

[0087] (4) Multi-layer embedding:

[0088] (41) Centrifugal collection of bacteria: The fermentation broth was centrifuged at 8000 rpm for 10 min and the precipitate was collected;

[0089] (42) Prepare embedding solution: Weigh 4 g of sodium alginate and dissolve it in 100 mL of deionized water. Heat to 60 °C to dissolve. After cooling, add 1 g of chitosan and adjust the pH to 5.5 with 1% acetic acid.

[0090] (43) Preparation of embedded microspheres: Add bacterial precipitate to embedding solution to make the bacterial concentration 1×10 10 CFU / mL, add 0.2 mol / L calcium chloride solution and cross-link for 30 min;

[0091] (44) Enteric coating: The embedded microspheres were washed three times with deionized water, placed in a 10% pH-sensitive enteric coating ethanol solution, spray-coated at 40 °C and 0.3 MPa, and dried at 50 °C for 2 h.

[0092] Example 2

[0093] The difference from Example 1 is:

[0094] (12) During the nutrient enrichment phase, RCM medium and enriched Clostridium medium were used alternately every 13 hours;

[0095] (22) In the first enzymatic hydrolysis step, 53 U / g of cellulase was used;

[0096] (31) Take 215 mL of concentrated Astragalus extract and add 6.5 g of glucose;

[0097] (42) The amount of chitosan added was 1.5 g.

[0098] Example 3

[0099] The difference from Example 1 is:

[0100] (11) heat shock ventilation was 1.8 L / min;

[0101] (13) The final concentration of sodium butyrate reached 45 g / L;

[0102] (21) Degreasing pressure 35 MPa, temperature 46°C;

[0103] (32) Dissolved oxygen 2.2% during the metabolic period;

[0104] (44) Coating pressure 0.35 MPa.

[0105] Example 4

[0106] The difference from Example 1 is:

[0107] (11) Refrigerated resuscitation time: 14 hours;

[0108] (22) Second step enzymatic hydrolysis pH 5.6;

[0109] (32) Dissolved oxygen 0.5% during sporulation period;

[0110] (42) The mass ratio of chitosan to sodium alginate is 1:4.

[0111] Example 5

[0112] The difference from Example 1 is:

[0113] (12) Incubation time of fortified Clostridium culture medium is 14 h;

[0114] (22) The mass ratio of xylanase, pectinase, and cellulase is 2:1:5.5;

[0115] (31) The pH of the culture medium was adjusted to 7.1;

[0116] (43) Cross-linking curing time: 35 min.

[0117] Comparative Example 1

[0118] The preparation process of Example 1 was adopted, except that the temperature gradient control in step (1) was omitted, and the original strain was directly inoculated into RCM medium, cultured at 37°C under anaerobic conditions for 18 h, and then subjected to nutritional enhancement and stress adaptation.

[0119] Comparative Example 2

[0120] The preparation process of Example 1 was adopted, except that: in step (2), a single cellulase was used for enzymatic hydrolysis, the enzyme dosage was 50 U / g, and the hydrolysis was carried out at 40° C. and pH 5.0 for 3 h, and the second enzymatic hydrolysis was not performed.

[0121] Comparative Example 3

[0122] The preparation process of Example 1 was adopted, except that the multi-layer embedding process in step (4) was omitted, and the fermented bacterial liquid was directly centrifuged to collect the bacterial bodies without embedding and enteric coating.

[0123] test

[0124] Test 1: Determination of viable bacteria count and short-chain fatty acid content in fermentation broth

[0125] Experimental methods

[0126] Determination of viable bacteria count: Refer to GB4789.35-2016 "National Food Safety Standard Food Microbiology Examination Bifidobacterium Examination", use the dilution spread plate method, use reinforced clostridial medium (RCM) for culture, and count after anaerobic culture at 37℃ for 48 hours.

[0127] Short-chain fatty acid (SCFA) content determination: Gas chromatography was used (GB5009.168-2016 "National Food Safety Standard - Determination of Fatty Acids in Food"). 10 mL of fermentation broth was centrifuged at 12,000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter membrane and then analyzed by gas chromatography. The chromatographic column was DB-FFAP (30 m × 0.25 mm × 0.25 μm). The column temperature was programmed: initial temperature 100°C, held for 2 minutes, then increased to 180°C at a rate of 10°C / min and held for 5 minutes; the injection port temperature was 250°C, the detector temperature was 280°C, the carrier gas was nitrogen, the flow rate was 1.0 mL / min, and the injection volume was 1 μL. The results are as follows:

[0128] Table 1

[0129] sample Viable bacteria count (CFU / mL) Short-chain fatty acid content (mmol / L) Example 1 <![CDATA[(1.25±0.08)×10 9 ]]> 78.5±4.2 Example 2 <![CDATA[(1.32±0.09)×10 9 ]]> 82.3±3.8 Example 3 <![CDATA[(1.41±0.11)×10 9 ]]> 85.6±5.1 Example 4 <![CDATA[(1.28±0.07)×10 9 ]]> 79.8±4.5 Example 5 <![CDATA[(1.35±0.10)×10 9 ]]> 81.2±3.9 Comparative Example 1 <![CDATA[(3.25±0.21)×10 8 ]]> 35.6±2.3 Comparative Example 2 <![CDATA[(5.12±0.34)×10 8 ]]> 48.7±3.1 Comparative Example 3 <![CDATA[(8.76±0.52)×10 8 ]]> 62.4±4.0

[0130] As can be seen from Table 1, the viable cell count and short-chain fatty acid content of Examples 1-5 are significantly higher than those of Comparative Examples 1-3. Comparative Example 1 omits the temperature gradient control, resulting in the strain not being fully activated, low metabolic activity, and a significant decrease in the viable cell count and SCFA production. Comparative Example 2 uses a single enzymatic hydrolysis, and the extraction of effective ingredients such as astragalus polysaccharides is insufficient, and the culture medium is insufficiently nutritious, affecting the growth and metabolism of the bacteria. Comparative Example 3 does not undergo embedding treatment, and the bacteria may be affected by environmental factors during the fermentation process, resulting in the death of some bacteria, and a decrease in the viable cell count and metabolite content. The multi-stage activation, composite enzymatic hydrolysis and multi-layer embedding technology of the present invention work synergistically to effectively improve the activity of the bacteria and the production of metabolites.

[0131] Experiment 2: Astragalus polysaccharide extraction rate and antioxidant activity determination

[0132] Experimental methods

[0133] Astragalus polysaccharide extraction yield was determined using the phenol-sulfuric acid method (GB / T31326-2014, "Plant-derived Beverages"). 1 mL of the enzymatic hydrolysis concentrate was diluted to 10 mL with water. 1 mL was placed in a test tube and 1 mL of 5% phenol solution was added. Mix thoroughly, then quickly add 5 mL of concentrated sulfuric acid. Shake well, cool to room temperature, and measure absorbance at 490 nm. The polysaccharide content was calculated using a glucose standard curve. The extraction yield (%) was calculated as (polysaccharide mass / astragalus raw material mass) × 100%.

[0134] Antioxidant activity determination: DPPH free radical scavenging rate determination method (GB / T23776-2018 "Tea Sensory Evaluation Method") was used. Take 2 mL of sample solution, add 2 mL of 0.1 mmol / L DPPH ethanol solution, shake well, and react in the dark for 30 minutes. Measure the absorbance at 517 nm. Simultaneously measure the sample blank and reagent blank. The DPPH scavenging rate (%) is [1-(A sample - A sample blank) / A reagent blank] × 100%. The results are as follows:

[0135] Table 2

[0136]

[0137]

[0138] As can be seen from Table 2, the astragalus polysaccharide extraction rate and DPPH clearance rate of the embodiment are significantly higher than those of the comparative example. Due to the lack of temperature gradient control, the activation of the thalline is insufficient in comparative example 1, which may affect the utilization of the astragalus components in subsequent fermentation, but the main reason is that comparative example 2 adopts a single cellulose enzymatic hydrolysis, which cannot effectively decompose the components such as xylan and pectin in the astragalus, resulting in a low polysaccharide extraction rate and a small content of antioxidant active substances. Although the enzymatic hydrolysis process of comparative example 3 is complete, it is not embedded, and the utilization efficiency of the thalline to the astragalus components may be reduced during the fermentation process, resulting in a polysaccharide extraction rate and antioxidant activity slightly lower than those of the embodiment. The composite enzymatic hydrolysis system of the present invention can more comprehensively decompose the astragalus cell wall, release effective ingredients such as polysaccharides, and combine multi-stage fermentation to improve the extraction rate and antioxidant performance of active substances.

[0139] Experiment 3: Simulation test of bacterial resistance to gastric acid and intestinal colonization ability

[0140] Experimental methods

[0141] Gastric acid resistance test: simulated gastric fluid preparation: 0.2 mol / L hydrochloric acid solution, add pepsin (10 g / L), adjust the pH to 1.2. Take 1 mL of the embedded bacterial suspension (containing 1×10 8 CFU / mL), 9 mL of simulated gastric fluid was added, and the cells were cultured at 37°C with shaking for 2 h. Samples were taken every 0.5 h, and the number of viable bacteria was determined by the dilution plate method. The survival rate (%) was calculated as (number of viable bacteria after treatment / initial number of viable bacteria) × 100%.

[0142] Simulating Intestinal Colonization Ability: Prepare simulated intestinal fluid: 0.2 mol / L disodium hydrogen phosphate solution, adjust pH to 7.4, and add trypsin (10 g / L). Add 1 mL of the bacterial solution after the gastric acid resistance test to 9 mL of simulated intestinal fluid. Incubate at 37°C with shaking for 4 hours. Samples were taken every hour and the viable bacterial count was determined. The intestinal colonization rate (%) was calculated as (number of viable bacteria after intestinal fluid culture / number of viable bacteria after gastric acid treatment) × 100%. The results are as follows:

[0143] Table 3

[0144] sample Gastric acid 2h survival rate (%) Intestinal colonization rate (%) Example 1 85.6±4.3 92.3±3.8 Example 2 88.2±3.5 94.5±2.9 Example 3 91.5±5.1 96.7±4.1 Example 4 86.3±3.9 93.1±3.2 Example 5 89.4±4.2 95.2±3.5 Comparative Example 1 32.5±2.1 56.7±4.3 Comparative Example 2 45.6±3.2 68.9±5.1 Comparative Example 3 12.3±1.5 28.4±2.7

[0145] As can be seen in Table 3, the survival rate and intestinal colonization rate of the bacteria in the example under gastric acid conditions were significantly higher than those in the control example. The multi-layer encapsulation technology of the present invention utilizes a sodium alginate-chitosan matrix to form microspheres, which are coated with a pH-sensitive enteric coating. This allows the microspheres to maintain structural integrity in gastric acid conditions, reducing damage to the bacteria. Upon reaching the intestines, the enteric coating dissolves, releasing the microspheres and improving colonization rates, thereby enhancing the intestinal regulating prebiotic effect.

[0146] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. The preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification.

Claims

1. A process for preparing intestinal regulating prebiotics from Clostridium butyricum and Astragalus membranaceus, characterized in that: The following steps are involved: (1) Multi-stage synergistic activation of Clostridium butyricum: The original strain is treated through three stages: temperature gradient control, nutritional enhancement, and stress adaptation; (2) Complex enzymatic hydrolysis of Astragalus extract: using a cellulase-xylanase-pectinase system for step-by-step enzymatic hydrolysis; (3) Symbiotic fermentation: inoculating the activated strain from step (1) into a culture medium containing the astragalus extract from step (2) to perform a three-stage oxygen-controlled fermentation; (4) Multi-layer encapsulation: The bacteria are encapsulated in a sodium alginate-chitosan matrix, and the outer layer of the encapsulated bacteria is coated with a pH-sensitive enteric coating.

2. The process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus according to claim 1, characterized in that: The activation treatment in step (1) comprises: (11) Temperature gradient control: The original strain was prepared into a bacterial solution with a concentration of OD 600 The bacterial suspension was prepared by refrigerating at 4°C ± 0.5°C for 12-14 hours and then inoculating into RCM medium at an inoculum size of 5-5.2% (v / v) and incubating at 37°C under anaerobic conditions for 18 hours. Finally, the cultured bacterial suspension was transferred to fresh RCM medium at an inoculum size of 5-5.2% (v / v) and heat-shocked at 42°C ± 0.5°C, shaking at 150 rpm, and aeration at 1.5-1.8 L / min for 2 hours; the aeration was nitrogen. (12) Nutritional fortification: RCM medium and fortified Clostridium medium were used alternately for culturing, with the inoculum volume of each culture being 5-5.2% (v / v); the RCM medium was cultured at 37°C under anaerobic conditions for 12-14 hours; the fortified Clostridium medium was cultured at 37°C under anaerobic conditions for 12-14 hours; (13) Stress adaptation: During the culture phase in the enhanced Clostridium medium, sodium butyrate was added every 2 hours from the start of the culture. The first addition of sodium butyrate was sufficient to make the concentration of sodium butyrate in the culture medium reach 10 g / L. The concentration of sodium butyrate was increased by 5 g / L each time thereafter until the concentration reached 40-45 g / L. The culture was continued for two alternating cycles. (14) During the entire activation process, the volume of the culture system at each stage remained consistent, all at 1 L.

3. The process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus according to claim 2, characterized in that: In step (12), the R3M culture medium and the enhanced Clostridium culture medium are alternately used every 12 hours.

4. The process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus according to claim 1, characterized in that: The specific preparation method of the compound enzymatic hydrolysis astragalus extract in step (2) comprises: (21) Pretreatment: After the Astragalus root is sliced, it is defatted using supercritical carbon dioxide at a pressure of 30-35 MPa and a temperature of 45-46°C. (22) Two-step enzymatic hydrolysis: In the first step, 50-56 U / g of cellulase was used for hydrolysis at 40°C and pH 5.0 for 1 h; in the second step, xylanase and pectinase were used, with the mass ratio of xylanase to pectinase and cellulase being 2:1:5, and hydrolysis was performed at 50°C and pH 5.5 for 2 h. The enzymatic hydrolysis product was obtained after enzyme inactivation treatment at 60°C.

5. The process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus according to claim 4, characterized in that: After the two-step enzymatic hydrolysis in step (22), the enzymatic hydrolysis product is filtered and concentrated. The filtration is performed using a polyethersulfone filter membrane with a pore size of 0.45 μm, and the product is concentrated under reduced pressure at a vacuum degree of -0.08 MPa and a temperature of 50°C to 1 / 5 of the original volume to obtain an Astragalus extract concentrate.

6. The process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus according to claim 1, characterized in that: Step (3) adopts three-stage dissolved oxygen control, specifically: proliferation period, dissolved oxygen is 5.0-5.8%, temperature is 37°C, and duration is 0-12h; sporulation period, dissolved oxygen ≤1.0%, temperature is 37°C, and duration is 12-24h; metabolic period, dissolved oxygen is 2.0-2.2%, temperature is 42°C, and duration is 24-36h.

7. The process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus according to claim 1, characterized in that: The method for preparing the culture medium containing the astragalus extract in step (2) in step (3) is as follows: take 200-230 mL of the astragalus extract concentrate after treatment in step (2), add 5-8 g of glucose, 10-12 g of peptone, and 3-4 g of sodium chloride, then dilute to 1 L with deionized water, and adjust the pH to 7.0±0.

2.

8. The process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus according to claim 1, characterized in that: In step (4), the mass ratio of sodium alginate to chitosan in the sodium alginate-chitosan matrix is 4:1-2, and the pH-sensitive enteric coating dissolves in an environment of pH ≥ 6.

8.

9. The process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus according to claim 1, characterized in that: In step (4), the method of using a sodium alginate-chitosan matrix to embed the bacterial cells and coating the outer layer of the embedded bacterial cells with a pH-sensitive enteric coating comprises: (1) Centrifuge the bacterial solution after symbiotic fermentation, discard the supernatant, and collect the bacterial precipitate; The centrifugation parameters were: 8000 rpm, 10 min; (2) Prepare embedding solution: Weigh 4 g of sodium alginate and dissolve it in 100 mL of deionized water. Heat to 60°C and stir until completely dissolved. After cooling to room temperature, add 1-2 g of chitosan. Adjust the pH to 5.5 with 1% acetic acid solution and stir evenly. (3) Add the bacterial precipitate to the above embedding solution to make the bacterial concentration 1×10 10 CFU / mL, after mixing evenly, drop into 0.2 mol / L calcium chloride solution and cross-link and cure for 30-35 minutes to form embedded microspheres; (4) The embedded microspheres were washed three times with deionized water, and then placed in a pH-sensitive enteric coating solution. The coating was performed by spray coating at a temperature of 40°C and a pressure of 0.3 MPa. After coating, the microspheres were dried at 50°C for 2 h.

10. The process for preparing intestinal regulating prebiotics by using Clostridium butyricum and Astragalus according to claim 9, characterized in that: The concentration of the pH-sensitive enteric coating solution is 10%, and the solvent is ethanol.

Images