Bihalite-containing probiotic preparation for improving gastrointestinal environment and preparation method of bihalite-containing probiotic preparation

Abstract

The invention provides a halite-containing probiotic preparation for improving gastrointestinal environment and a preparation method, the halite-containing probiotic preparation comprises the following components in parts by weight: 60-70 parts of a probiotic composition, the probiotic composition comprises lactobacillus casei Zhang, animal bifidobacterium subsp. Lactis V9, lactobacillus plantarum P-01, animal bifidobacterium subsp. Lactis B-01 and casei paracasei LC-11, the weight ratio is 3: 2: 2: 1: 1. 1.2 to 3.5 parts of nanoscale refined halite; 10 to 15 parts of a microcapsule embedding material; 0.6 to 1.2 parts of an antioxidant system; 15 to 30 parts of auxiliary materials; the probiotic preparation disclosed by the invention has the characteristics of obvious strain combination advantage, nano-scale halite protection and synergism, accurate release of a multi-layer microcapsule structure, synergistic effect of an antioxidant system and the like, the survival rate of probiotics, gastrointestinal tract tolerance and intestinal colonization capacity are remarkably improved, and a new technical scheme is provided for improving gastrointestinal health.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial preparations, and particularly relates to a halite-containing probiotic preparation for improving the gastrointestinal environment and a preparation method thereof. Background Art

[0002] Probiotics are active microorganisms beneficial to the host. By improving the balance of the host intestinal flora, promoting digestion and absorption, and enhancing immune function, they are widely used in the fields of functional foods and medical health. However, traditional probiotic preparations face many challenges during storage and oral administration: First, the activity of probiotics is easily affected by external factors such as temperature, humidity, and oxygen, resulting in a reduced survival rate; second, probiotics have poor stability in the gastric acid environment, and a large number of probiotics lose their activity before reaching the intestine; third, the colonization ability of existing probiotic preparations is limited, and it is difficult to form a long-term flora in the intestine.

[0003] In recent years, natural minerals as probiotic enhancers have attracted wide attention. As a natural mineral resource, halite is rich in various trace elements and minerals and has high medicinal and nutritional value. Research shows that the mineral elements in halite can promote the growth of microorganisms and enhance the stability of cell membranes. However, there are few studies on combining halite with probiotics at present, and in the existing technology, the particle size of halite is relatively large, the bioavailability is low, and the combination method with probiotics is simple, unable to achieve comprehensive protection and functional enhancement of probiotics.

[0004] In addition, existing probiotic preparations mostly use single or simply compounded strains, and the synergistic effect is not good; the embedding technology is simple and cannot achieve precise positioning and release; the antioxidant protection system is not perfect and it is difficult to cope with the challenges of the complex gastrointestinal environment. Therefore, the development of a new type of probiotic preparation with high survival rate, strong tolerance, and good colonization ability is of great significance for improving human gastrointestinal health. Summary of the Invention

[0005] The present invention provides a halite-containing probiotic preparation for improving the gastrointestinal environment and a preparation method thereof, aiming to solve the technical problems of low survival rate, poor gastric acid tolerance, and weak intestinal colonization ability of existing probiotic preparations. By means of optimized multi-strain compounding, nano-scale halite mineral enhancement, multi-layer microencapsulation protection, and construction of a synergistic antioxidant system, the survival rate, stability, and functional activity of probiotics are significantly improved, realizing the efficient colonization and long-term action of probiotics in the intestine, thereby effectively improving the human gastrointestinal environment.

[0006] The solution of the present invention is as follows: In the first aspect, the present invention provides a halite-containing probiotic preparation for improving the gastrointestinal environment, and the preparation contains the following components in parts by weight: Probiotic composition: 60 - 70 parts, the probiotic composition comprising: Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01, and Lactobacillus paracasei LC-11, composed according to a weight ratio of 3:2:2:1:1; Halite: 1.2 - 3.5 parts, the halite being purified nano-scale refined halite, with the particle diameter controlled within the range of 100 - 200 nm; Microcapsule embedding material: 10 - 15 parts, the microcapsule embedding material being composed of sodium alginate, gum arabic, chitosan, and gelatin according to a weight ratio of 5:2:2:1; Antioxidant system: 0.6 - 1.2 parts, the antioxidant system including: a synergistic complex of vitamin E, vitamin C, citric acid, and anthocyanins, according to a weight ratio of 1:2:1:0.5; Excipients: 15 - 30 parts, the excipients including: inulin, fructooligosaccharide, resistant dextrin, erythritol, silicon dioxide, magnesium stearate, composed according to a weight ratio of 20:15:5:2:1:1.

[0007] Furthermore, the form of the preparation is granule, oral powder, enteric-coated capsule, chewable tablet, or enteric-coated tablet.

[0008] In a second aspect, the present invention provides a method for preparing a halite-containing probiotic preparation for improving the gastrointestinal environment, for preparing the halite-containing probiotic preparation of the first aspect. The method includes the following steps: Step S1, strain activation: Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01, and Lactobacillus paracasei LC-11 are respectively activated and cultured in a dedicated MRS medium at 37 ± 1°C for 18 - 24 hours; the dedicated MRS medium is a medium added with 0.5 - 1% of a halite mineral extract.

[0009] Step S2, scale-up culture: The activated strains are respectively inoculated into a sterilized MRS liquid medium and cultured at a constant temperature of 37 ± 1°C for 16 - 20 hours to obtain the scale-up culture solutions of each strain; 0.5 - 1% of an acid-resistant precursor substance is added to the MRS liquid medium.

[0010] Step S3, Fermentation and Culture: Inoculate the enlarged culture solutions of each strain obtained in Step S2 into the sterilized fermentation medium at a volume ratio of 1:2. The fermentation medium includes the following raw materials in parts by weight: 10 - 15 parts of lactose, 10 - 12 parts of whey protein, 5 - 8 parts of yeast extract, 2 - 3 parts of ammonium citrate, 1 - 2 parts of dipotassium hydrogen phosphate, 0.5 - 1 part of magnesium sulfate, 0.1 - 0.3 part of vitamin B complex mixture, 12 - 15 parts of skim milk powder, and the balance is water. Anaerobically ferment at 37 ± 1°C for 18 - 24 hours to make the viable count in the bacterial liquid reach 1×10¹¹ CFU / mL or more.

[0011] Step S4, Bacterial Cell Collection: Collect the bacterial cells from the fermented bacterial liquid by high-speed centrifugation. The centrifugation conditions are 6000 - 8000 r / min and the centrifugation time is 15 - 20 min; then perform secondary concentration using membrane filtration concentration technology to improve the purity and density of the bacterial cells.

[0012] Step S5, Preparation of Freeze-Drying Protectant: Sterilize the aqueous solution of the freeze-drying protectant with a mass concentration of 10 - 15% at 115°C for 15 minutes and then cool it to 4°C for standby. The freeze-drying protectant includes the following raw materials in parts by weight: 8 - 10 parts of trehalose, 5 - 7 parts of maltodextrin, 3 - 5 parts of gelatin, 1 - 2 parts of sodium ascorbate, and 1 - 2 parts of L-glutamate; Step S6, Bacterial Cell Resuspension: Uniformly mix the bacterial cells collected in Step S4 with the aqueous solution of the freeze-drying protectant with a mass concentration of 10 - 15% prepared in Step S5 to obtain a bacterial cell suspension.

[0013] Step S7, Treatment with Big Green Salt: Add the sterilized big green salt to the bacterial cell suspension obtained in Step S6. The addition amount of big green salt is 1.2 - 3.5 parts, and perform pulsed ultrasonic treatment at 4°C for 2 - 4 hours to obtain a mixed solution.

[0014] Step S8, Freeze-Drying: Perform programmed pre-freezing on the mixed solution obtained in Step S7. The pre-freezing temperature is -40°C and the pre-freezing time is 2 - 4 hours; then perform primary drying under the conditions of -30°C to -10°C, a vacuum degree of 10 - 30 Pa, and a drying time of 16 - 20 hours; finally perform post-drying under the conditions of 10 - 25°C, a vacuum degree of 5 - 10 Pa, and a drying time of 4 - 6 hours to obtain the freeze-dried powder of probiotics containing big green salt.

[0015] Step S9, Microencapsulation: After mixing the prepared probiotic freeze-dried powder containing halite of each strain obtained in Step S8 according to a weight ratio of 3:2:2:1:1, take 60 - 70 parts and mix it evenly with 10 - 15 parts of microencapsulation embedding material and 0.6 - 1.2 parts of antioxidant system, and carry out microencapsulation by three-dimensional pulsed vibration spray drying technology. The spray drying conditions are: inlet air temperature 120 - 150 °C, outlet air temperature 50 - 65 °C, atomization pressure 0.2 - 0.3 MPa, and nitrogen is introduced for protection during the drying process to obtain microencapsulated probiotic powder containing halite with a multi-layer structure.

[0016] Step S10, Auxiliary Material Mixing: Mix the microencapsulated probiotic powder containing halite obtained in Step S9 with 15 - 30 parts of auxiliary materials to prepare a probiotic preparation containing halite.

[0017] Further, in Step S1, the preparation method of the halite mineral extract is as follows: Select natural halite ore, wash it and crush it into fine powder with a particle size of more than 200 meshes; mix the fine powder with a phosphate buffer solution with a pH value of 6.8 - 7.2 according to a weight ratio of 1:8, carry out stirring extraction at 50 - 55 °C, the stirring speed is 200 - 300 r / min, and the extraction time is 4 - 6 hours; after the extraction is completed, centrifuge at a speed of 8000 r / min for 15 minutes, and collect the supernatant; sequentially filter and sterilize the supernatant through 0.45 μm and 0.22 μm microporous filter membranes; measure the concentration of the filtrate, determine the total mineral content by the conductivity method, and adjust the mineral concentration to 10 - 12 g / L; sub-pack the prepared halite mineral extract, sterilize it at 121 °C for 15 minutes and then cool it to room temperature for storage and standby.

[0018] Further, the acid-resistant precursor substance described in Step S2 is composed of the following components by weight: 2.5 parts of L-alanine, 1.5 parts of sodium L-glutamate, 0.8 part of cysteine hydrochloride, 0.4 part of magnesium chloride hexahydrate, 0.15 part of manganese sulfate monohydrate, 0.1 part of zinc sulfate heptahydrate, and 0.05 part of vitamin B6 hydrochloride.

[0019] Further, in Step S4, the membrane filtration and concentration technology includes the following specific steps: A polysulfone hollow fiber ultrafiltration membrane with a molecular weight cut-off of 15 kDa is used, and the effective membrane area is 0.5 - 0.8 m²; the operating pressure is controlled at 0.07 - 0.09 MPa, and the transmembrane pressure difference is maintained at 0.04 - 0.06 MPa; the filtration temperature is controlled at 4 - 6 °C, and a double-layer jacket refrigeration system is adopted; the flow rate of the bacterial liquid is controlled at 65 - 75 mL / min, and the tangential flow mode is adopted during filtration to reduce membrane fouling; a filtration circulation system is set up, and backwashing is carried out every 20 minutes, with the backwashing pressure being 0.12 - 0.15 MPa and the time being 15 - 20 seconds; it is concentrated to 15 - 20% of the original volume to make the bacterial density reach more than 5×10¹² CFU / mL; after concentration, the bacteria are washed twice with an isotonic phosphate buffer solution of pH 7.0 to remove the residues of the fermentation medium.

[0020] Further, the fermentation culture in step S3 is carried out under the condition that the pH value in the tank is maintained at 5.8 - 6.2, and a sterile glucose solution and an amino acid mixture are supplemented once every 6 hours during the fermentation process, and the supplementation amount is 1 - 2% of the volume of the initial medium.

[0021] Further, the big green salt is obtained by the following method: 1) Wash and crush the natural green salt ore into fine powder with a mesh size of more than 100 meshes; 2) Mix the fine powder with deionized water according to a weight ratio of 1:5, and carry out ultrasonic-assisted extraction for 2 - 3 hours at 60 - 70 °C; 3) Centrifuge the extract at a high speed, with the centrifugation conditions being 10000 r / min for 15 minutes, and collect the supernatant; 4) Rotate and evaporate the supernatant under reduced pressure to 1 / 3 of the initial volume, and then let it stand at 4 °C for 12 hours to precipitate impurities; 5) Filter and remove the precipitate with a 0.22 μm microporous membrane, and then purify the filtrate by nano reverse osmosis membrane filtration technology in gradients; 6) Prepare the purified solution by low-temperature vacuum freeze spray drying technology to obtain nano-scale refined big green salt powder.

[0022] Further, in step S7, the big green salt solution is prepared as follows: Take nano-scale refined big green salt powder, and accurately weigh 10 - 12 g; add the weighed big green salt powder to 100 mL of sterile ultrapure water, stir and dissolve it at 40 - 45 °C, with the stirring speed being 300 - 350 r / min and the time being 20 - 30 minutes; The dissolved large green salt solution is processed by a high-pressure homogenizer at a pressure of 80 - 100 MPa and circulated 3 - 5 times; the particle size is measured using dynamic light scattering (DLS) to ensure that the particle diameter is distributed in the range of 100 - 200 nm and the polydispersity index (PDI) is less than 0.3; after filtration and sterilization through a 0.22 μm polyethersulfone membrane, the large green salt solution is stored at 4°C for standby use with a validity period of 7 days.

[0023] Furthermore, the three-dimensional pulsed vibration spray drying technology specifically includes: Using a spray drying device equipped with a piezoelectric ceramic vibrator, the vibrator generates vibrations simultaneously in three directions: X, Y, and Z; The vibration frequency in the X direction is set to 30 ± 2 Hz, and the amplitude is 1.2 ± 0.2 mm; the vibration frequency in the Y direction is set to 35 ± 2 Hz, and the amplitude is 1.0 ± 0.2 mm; the vibration frequency in the Z direction is set to 40 ± 2 Hz, and the amplitude is 0.8 ± 0.2 mm; The spray system uses a two-fluid nozzle with an inner diameter of 0.7 mm and an outer diameter of 1.5 mm, and the nitrogen purity is ≥99.999%; The single-pulse feeding time is 1.0 ± 0.1 seconds, and the pulse interval time is 1.5 ± 0.1 seconds; The main tower temperature is 85 ± 5°C, the drying air flow rate is 85 ± 5 m³ / h, nitrogen is introduced for protection during the drying process, and the oxygen content is controlled below 3%; The particle size distribution of the collected microencapsulated probiotic powder is 15 - 25 μm, and the moisture content is controlled below 3.5%.

[0024] Compared with the prior art, the beneficial effects of the present invention are: The survival rate of the large green salt-containing probiotic preparation prepared by the present invention reaches over 82% after 2 hours in the gastric acid environment (pH 2.0), which is more than twice that of ordinary probiotic preparations (the survival rate is about 40%); after storage at -20°C for 12 months, the viable count decreases by no more than 0.5 logarithmic order; the intestinal colonization experiment shows that the improvement effect of the intestinal flora is significant after taking for 7 days and can last for more than 28 days, providing a new preparation that is efficient, stable, and long-lasting for improving gastrointestinal health. Detailed implementation manners

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention are described clearly and completely below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0026] It should be noted that Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01, and Lactobacillus paracasei LC-11 of the present invention are all existing bacterial strains, and the used strains are all obtained by purchase.

[0027] Lactobacillus casei Zhang is an acid- and bile-acid-tolerant probiotic isolated from koumiss. The strain of this bacterium was deposited on April 21, 2006, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 13, Beiyitiao, Zhongguancun, Haidian District, Beijing, China, with the deposit number CGMCC No. 1697; Bifidobacterium animalis subsp. lactis V9 is a probiotic strain isolated from the intestines of healthy Mongolian children in 2005. The Bifidobacterium animalis subsp. lactis V9 is deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 5470; Lactobacillus plantarum P-01, with the microbial deposit number CGMCC No. 17748, is deposited in Beijing Ketuo Hengtong Biotechnology Co., Ltd. The Lactobacillus plantarum P-01 is isolated from naturally fermented yak milk in Inner Mongolia grassland; Bifidobacterium animalis subsp. lactis B-01 is deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 17742; Lactobacillus paracasei LC-11 was deposited on March 18, 2019, at the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 17341.

[0028] Example 1 A probiotic preparation containing halite for improving the gastrointestinal environment, the preparation contains the following components by weight: Probiotic composition: 60 parts, the probiotic composition comprising: Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01, and Lactobacillus paracasei LC-11, composed in a weight ratio of 3:2:2:1:1.

[0029] Halite: 1.2 parts, the halite being purified nanoscale refined halite, with the particle diameter controlled within the range of 100 - 200 nm.

[0030] Microcapsule embedding material: 10 parts, the microcapsule embedding material composed of sodium alginate, gum arabic, chitosan, and gelatin in a weight ratio of 5:2:2:1.

[0031] Antioxidant system: 0.6 parts, the antioxidant system including a synergistic complex of vitamin E, vitamin C, citric acid, and anthocyanins, in a weight ratio of 1:2:1:0.5.

[0032] Excipients: 30 parts, the excipients including inulin, fructooligosaccharide, resistant dextrin, erythritol, silicon dioxide, and magnesium stearate, composed in a weight ratio of 20:15:5:2:1:1.

[0033] Example 2 A probiotic preparation containing halite for improving the gastrointestinal environment, the preparation comprising the following components by weight: Probiotic composition: 70 parts, the probiotic composition comprising: Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01, and Lactobacillus paracasei LC-11, composed in a weight ratio of 3:2:2:1:1.

[0034] Halite: 3.5 parts, the halite being purified nanoscale refined halite, with the particle diameter controlled within the range of 100 - 200 nm.

[0035] Microcapsule embedding material: 15 parts, the microcapsule embedding material composed of sodium alginate, gum arabic, chitosan, and gelatin in a weight ratio of 5:2:2:1.

[0036] Antioxidant system: 1.2 parts, the antioxidant system including a synergistic complex of vitamin E, vitamin C, citric acid, and anthocyanins, in a weight ratio of 1:2:1:0.5.

[0037] Excipients: 15 parts, the excipients including inulin, fructooligosaccharide, resistant dextrin, erythritol, silicon dioxide, and magnesium stearate, composed in a weight ratio of 20:15:5:2:1:1.

[0038] Example 3 A probiotic preparation containing halite for improving gastrointestinal environment, the preparation comprises the following components by weight: Probiotic composition: 65 parts, the probiotic composition comprises: Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01 and Lactobacillus paracasei LC-11, which are composed according to the weight ratio of 3:2:2:1:1.

[0039] Halite: 2.8 parts, the halite is purified nano-level refined halite, and the particle diameter is controlled within the range of 100-200nm.

[0040] Microcapsule embedding material: 12 parts, the microcapsule embedding material is composed of sodium alginate, gum arabic, chitosan and gelatin according to the weight ratio of 5:2:2:1.

[0041] Antioxidant system: 1 part, the antioxidant system includes: a synergistic complex of vitamin E, vitamin C, citric acid and anthocyanin, according to the weight ratio of 1:2:1:0.5.

[0042] Excipients: 25 parts, the excipients include: inulin, fructooligosaccharide, resistant dextrin, erythritol, silicon dioxide, magnesium stearate, which are composed according to the weight ratio of 20:15:5:2:1:1.

[0043] Example 4 A probiotic preparation containing halite for improving gastrointestinal environment, the preparation comprises the following components by weight: Probiotic composition: 65 parts, the probiotic composition comprises: Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01 and Lactobacillus paracasei LC-11, which are composed according to the weight ratio of 3:2:2:1:1.

[0044] Halite: 3 parts, the halite is purified nano-level refined halite, and the particle diameter is controlled within the range of 100-200nm.

[0045] Microcapsule embedding material: 15 parts, the microcapsule embedding material is composed of sodium alginate, gum arabic, chitosan and gelatin according to the weight ratio of 5:2:2:1.

[0046] Antioxidant system: 0.8 part, the antioxidant system includes: a synergistic complex of vitamin E, vitamin C, citric acid and anthocyanin, according to the weight ratio of 1:2:1:0.5.

[0047] Excipients: 20 parts, the excipients include: inulin, fructooligosaccharide, resistant dextrin, erythritol, silicon dioxide, magnesium stearate, which are composed according to the weight ratio of 20:15:5:2:1:1.

[0048] After testing, after incubating the halite-containing probiotic preparations of Examples 1-4 in artificial gastric juice at pH 2.0 for 1 hour, the survival rates were all higher than 90%.

[0049] Example 5 The specific operation of the preparation method of a halite-containing probiotic preparation for improving the gastrointestinal environment in Example 1 is as follows: Step S1, strain activation: Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01, and Lactobacillus paracasei LC-11 were respectively activated and cultured in MRS medium supplemented with 0.8% halite mineral extract at 37 ± 1 °C for 20 hours. The halite mineral extract was prepared according to the method of Claim 4, and the final mineral concentration was 11.5 g / L.

[0050] Step S2, scale-up culture: The activated strains were respectively inoculated into sterilized MRS liquid medium supplemented with 0.8% acid-resistant precursor substances and cultured at a constant temperature of 37 ± 1 °C for 18 hours to obtain the scale-up culture solutions of each strain.

[0051] Step S3, fermentation culture: The scale-up culture solutions of each strain obtained in Step S2 were inoculated into the sterilized fermentation medium at a volume ratio of 1:2. The fermentation medium was formulated according to the following parts by weight: 12 parts of lactose, 11 parts of whey protein, 6 parts of yeast extract, 2.5 parts of ammonium citrate, 1.5 parts of dipotassium hydrogen phosphate, 0.8 part of magnesium sulfate, 0.2 part of vitamin B complex mixture, 14 parts of skim milk powder, and water up to 1000 parts. Anaerobic fermentation was carried out at 37 ± 1 °C for 20 hours, and the pH value in the tank was maintained at 6.0 ± 0.2. A sterile glucose solution and an amino acid mixture were added every 6 hours, and the added amount was 1.5% of the initial medium volume. After fermentation, the viable cell counts of each strain reached: Lactobacillus casei Zhang 5.3×10¹¹ CFU / mL, Bifidobacterium animalis subsp. lactis V9 4.7×10¹¹ CFU / mL, Lactobacillus plantarum P-01 4.9×10¹¹ CFU / mL, Bifidobacterium animalis subsp. lactis B-01 4.1×10¹¹ CFU / mL, Lactobacillus paracasei LC-11 3.8×10¹¹ CFU / mL.

[0052] Step S4, cell collection: The fermented bacterial liquid was centrifuged at 6000 - 8000 r / min for 15 - 20 min to collect the cells; then a membrane filtration concentration technology was used for secondary concentration to improve the cell purity and density.

[0053] Step S5, preparation of lyophilization protectant: A lyophilization protectant aqueous solution with a mass concentration of 10 - 15% is sterilized at 115°C for 15 minutes and then cooled to 4°C for standby. Among them, the lyophilization protectant includes the following raw materials in parts by weight: 8 parts of trehalose, 7 parts of maltodextrin, 3 parts of gelatin, 2 parts of sodium ascorbate, and 2 parts of L-sodium glutamate; Step S6, cell resuspension: The cells collected in Step S4 are uniformly mixed with the lyophilization protectant aqueous solution with a mass concentration of 10 - 15% prepared in Step S5 to obtain a cell suspension.

[0054] Step S7, halite treatment: Sterilized halite is added to the cell suspension obtained in Step S6. The addition amount of halite is 1.2 parts, and pulsed ultrasonic treatment is carried out at 4°C for 2 - 4 hours to obtain a mixed solution.

[0055] Step S8, lyophilization: The mixed solution obtained in Step S7 is subjected to programmed pre-freezing. The pre-freezing temperature is -40°C and the pre-freezing time is 2 hours; then main drying is carried out under the conditions of -30°C to -10°C, a vacuum degree of 10 Pa, and a drying time of 20 hours; finally, post-drying is carried out under the conditions of 25°C, a vacuum degree of 5 Pa, and a drying time of 4 hours to obtain a probiotic freeze-dried powder containing halite.

[0056] Step S9, microencapsulation: The probiotic freeze-dried powders containing halite of each strain prepared in Step S8 are mixed according to a weight ratio of 3:2:2:1:1. Then, 60 parts are taken and mixed evenly with 10 parts of microencapsulation embedding material and 0.6 part of antioxidant system, and microencapsulation is carried out by three-dimensional pulsed vibration spray drying technology. The spray drying conditions are: inlet air temperature 120°C, outlet air temperature 65°C, atomization pressure 0.2 MPa, and nitrogen is introduced for protection during the drying process to obtain a microencapsulated probiotic powder containing halite with a multi-layer structure.

[0057] Step S10, auxiliary material mixing: The microencapsulated probiotic powder containing halite obtained in Step S9 is mixed with 30 parts of auxiliary materials to prepare a probiotic preparation containing halite.

[0058] Example 6 An experiment on the gastric acid tolerance of a probiotic preparation containing halite A comparative experiment on the gastric acid tolerance of the probiotic preparation containing halite prepared in Example 5 and the control group probiotic preparation is carried out. The control group probiotic preparation uses the same strain composition as the present invention (Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01, and Paracasei paracasei LC-11, composed according to a weight ratio of 3:2:2:1:1), but does not add halite and does not carry out microencapsulation treatment, and other production process parameters are the same as those in Example 5.

[0059] Take 1 g of each of the two preparations and dissolve them separately in 10 mL of artificial gastric juice (pH 2.0). Incubate at 37°C and sample at 0, 30, 60, 90, and 120 minutes respectively. Determine the viable count by the plate counting method. The results are shown in Table 1: Table 1 Comparison of the survival rates of different probiotic preparations in artificial gastric juice (%)

[0060] The results show that after incubation in artificial gastric juice at pH 2.0 for 2 hours, the survival rate of the probiotic preparation containing halite prepared by the present invention is still as high as 82.6%, far higher than 39.5% of the control probiotic preparation, an increase of 109%. This proves that the addition of halite and microencapsulation treatment in the present invention significantly improves the gastric acid tolerance of probiotics.

[0061] Example 7 A stability experiment of a probiotic preparation containing halite Store the probiotic preparation containing halite prepared in Example 5 and the control probiotic preparation under different temperature conditions. Sample at 0, 3, 6, 9, and 12 months respectively and determine the viable count. The results are shown in Table 2 and Table 3: Table 2 Changes in viable count of different probiotic preparations stored at 4°C (lgCFU / g)

[0062] Table 3 Changes in viable count of different probiotic preparations stored at -20°C (lgCFU / g)

[0063] The results show that after storage at -20°C for 12 months, the viable count of the probiotic preparation containing halite prepared by the present invention only decreases by 0.12 logarithmic orders, while that of the control probiotic preparation decreases by 1.13 logarithmic orders; after storage at 4°C for 12 months, the viable count of the preparation of the present invention decreases by 0.27 logarithmic orders, and that of the control probiotic preparation decreases by 1.69 logarithmic orders. This proves that the preparation of the present invention has excellent storage stability.

[0064] Example 8 An experiment on the intestinal colonization ability of a probiotic preparation containing halite Sixty healthy volunteers (30 males and 30 females, aged 25 - 45 years old) were selected and randomly divided into two groups, with 30 people in each group. Group A took the big green salt probiotic preparation prepared in Example 5 of the present invention every day (5 g each time, twice a day); Group B took the control group probiotic preparation every day (5 g each time, twice a day), and stopped taking after 7 consecutive days of administration. Fecal samples were collected before administration (day 0), during administration (days 3 and 7), and after stopping administration (days 14 and 28), and the changes in intestinal flora were analyzed by 16S rDNA sequencing and colony counting method.

[0065] Table 4 Detection amounts of target strains in feces after taking different probiotic preparations (lgCFU / g feces)

[0066] Table 5 Effects of taking different probiotic preparations on intestinal flora balance (relative abundance %)

[0067] Table 6 Changes in intestinal environment indicators before and after taking different probiotic preparations

[0068] The results showed that after taking the big green salt probiotic preparation prepared in the present invention for 7 days, the colonization levels of each target strain in the intestine were significantly higher than those of the control group probiotic preparation; 21 days after stopping administration (day 28), the residual amounts of the target strains in Group A in the intestine were still significantly higher than those in Group B, and the proportion of beneficial flora remained at a relatively high level. The preparation of the present invention simultaneously improved the intestinal pH value, the content of short-chain fatty acids and reduced the intestinal inflammation index, and these improvement effects were still obvious 21 days after stopping administration, proving that the preparation of the present invention has significant intestinal colonization ability and lasting effect of improving the intestinal environment.

[0069] Example 9 Clinical efficacy evaluation of a big green salt probiotic preparation Ninety volunteers with gastrointestinal discomfort symptoms (such as abdominal distension, irregular defecation, mild abdominal pain, etc.) were selected and randomly divided into three groups, with 30 people in each group. Group A took the big green salt probiotic preparation prepared in Example 5 of the present invention every day (5 g each time, twice a day); Group B took the control group probiotic preparation every day (5 g each time, twice a day); Group C took a placebo (5 g each time, twice a day), and took it continuously for 14 days. The improvement of gastrointestinal symptoms was evaluated by questionnaire survey and clinical examination.

[0070] Table 7 Improvement effects of different preparations on gastrointestinal discomfort symptoms (symptom score, full score 10 points, the lower the score, the lighter the symptom)

[0071] The results showed that after 14 days of taking the probiotic preparation containing halite according to the present invention, the gastrointestinal discomfort symptoms of the volunteers were significantly improved. The comprehensive symptom index decreased from 7.4 points to 2.3 points, and the improvement rate reached 69%. After 14 days of stopping taking (on the 28th day), the symptom improvement effect was still maintained, and the comprehensive symptom index was 2.7 points. In contrast, after 14 days of taking the probiotic preparation of the control group in Group B, the comprehensive symptom index decreased from 7.2 points to 3.7 points, and the improvement rate was 49%. After 14 days of stopping taking, the symptoms rebounded significantly, and the comprehensive symptom index rose to 4.7 points. The symptom improvement in Group C, the placebo group, was not obvious.

[0072] These results further prove that the probiotic preparation containing halite of the present invention has a significant effect on improving the gastrointestinal environment, and the effect is lasting, providing an efficient and stable solution for gastrointestinal health problems.

[0073] The present invention is illustrated by specific embodiments. Those skilled in the art should understand that various transformations and equivalent substitutions can be made to the present invention without departing from the scope of the present invention. In addition, various modifications can be made to the present invention for specific situations or materials without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed, but should include all embodiments falling within the scope of the claims of the present invention.

Claims

1. A probiotic preparation containing halite for improving the gastrointestinal environment, characterized in that, The preparation contains the following components by weight: Probiotic composition: 60 - 70 parts, and the probiotic composition contains: Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01, and Lactobacillus paracasei LC-11, which are composed according to the weight ratio of 3:2:2:1:1; Sal glauberitum: 1.2 - 3.5 parts, and the Sal glauberitum is purified nano-level refined Sal glauberitum, and the particle diameter is controlled within the range of 100 - 200 nm; Microcapsule embedding material: 10 - 15 parts, and the microcapsule embedding material is composed of sodium alginate, gum arabic, chitosan, and gelatin according to the weight ratio of 5:2:2:1; Antioxidant system: 0.6 - 1.2 parts, and the antioxidant system includes: a synergistic complex of vitamin E, vitamin C, citric acid, and anthocyanin, according to the weight ratio of 1:2:1:0.5; Excipients: 15 - 30 parts, and the excipients include: inulin, fructooligosaccharide, resistant dextrin, erythritol, silicon dioxide, magnesium stearate, which are composed according to the weight ratio of 20:15:5:2:1:

1.

2. The probiotic preparation containing halite according to claim 1, wherein The form of the preparation is granule, oral powder, enteric capsule, chewable tablet, or enteric tablet.

3. A preparation method of a probiotic preparation containing halite for improving the gastrointestinal environment, which is used to prepare the probiotic preparation containing halite according to any one of claims 1-2, characterized in that, The method includes the following steps: Step S1, strain activation: Lactobacillus casei Zhang, Bifidobacterium animalis subsp. lactis V9, Lactobacillus plantarum P-01, Bifidobacterium animalis subsp. lactis B-01, and Lactobacillus paracasei LC-11 are respectively activated and cultured in a dedicated MRS medium at 37 ± 1 °C for 18 - 24 hours; the dedicated MRS medium is a medium added with 0.5 - 1% of Sal glauberitum mineral extract; Step S2, subculture: The activated strains are respectively inoculated into a sterilized MRS liquid medium and cultured at a constant temperature of 37 ± 1 °C for 16 - 20 hours to obtain the subcultured broth of each strain; 0.5 - 1% of acid-resistant precursor substances are added to the MRS liquid medium; Step S3, fermentation culture: The subcultured broth of each strain obtained in Step S2 is inoculated into a sterilized fermentation medium according to the volume ratio of 1:

2. Among them, the fermentation medium includes the following raw materials in parts by weight: 10 - 15 parts of lactose, 10 - 12 parts of whey protein, 5 - 8 parts of yeast extract, 2 - 3 parts of ammonium citrate, 1 - 2 parts of dipotassium hydrogen phosphate, 0.5 - 1 part of magnesium sulfate, 0.1 - 0.3 part of vitamin B complex mixture, 12 - 15 parts of skim milk powder, and the balance of water. Anaerobic fermentation is carried out at 37 ± 1 °C for 18 - 24 hours to make the viable count in the bacterial liquid reach more than 1 × 10¹¹ CFU / mL; Step S4, cell collection: The fermented bacterial liquid is centrifuged at 6000 - 8000 r / min for 15 - 20 min to collect the cells; then a membrane filtration concentration technology is used for secondary concentration to improve the purity and density of the cells; Step S5, preparation of freeze-drying protectant: Aqueous solution of freeze-drying protectant with a mass concentration of 10 - 15% is sterilized at 115°C for 15 minutes and then cooled to 4°C for standby. Among them, the freeze-drying protectant includes the following raw materials in parts by weight: 8 - 10 parts of trehalose, 5 - 7 parts of maltodextrin, 3 - 5 parts of gelatin, 1 - 2 parts of sodium ascorbate, and 1 - 2 parts of L-glutamate; Step S6, cell resuspension: The cells collected in Step S4 are uniformly mixed with the aqueous solution of freeze-drying protectant with a mass concentration of 10 - 15% prepared in Step S5 to obtain a cell suspension; Step S7, halite treatment: Sterilized halite is added to the cell suspension obtained in Step S6. The addition amount of halite is 1.2 - 3.5 parts, and pulsed ultrasonic treatment is carried out at 4°C for 2 - 4 hours to obtain a mixed solution; Step S8, freeze-drying: The mixed solution obtained in Step S7 is subjected to programmed pre-freezing. The pre-freezing temperature is -40°C and the pre-freezing time is 2 - 4 hours; then main drying is carried out under the conditions of -30°C to -10°C, a vacuum degree of 10 - 30 Pa, and a drying time of 16 - 20 hours; finally, post-drying is carried out under the conditions of 10 - 25°C, a vacuum degree of 5 - 10 Pa, and a drying time of 4 - 6 hours to obtain freeze-dried probiotic powder containing halite; Step S9, microencapsulation: The freeze-dried probiotic powder containing halite of each strain prepared in Step S8 is mixed according to a weight ratio of 3:2:2:1:

1. Then, 60 - 70 parts are taken and mixed evenly with 10 - 15 parts of microcapsule embedding material and 0.6 - 1.2 parts of antioxidant system. Microencapsulation is carried out by three-dimensional pulsed vibration spray drying technology. The spray drying conditions are: inlet air temperature 120 - 150°C, outlet air temperature 50 - 65°C, atomization pressure 0.2 - 0.3 MPa, and nitrogen is introduced for protection during the drying process to obtain microencapsulated probiotic powder containing halite with a multi-layer structure; Step S10, auxiliary material mixing: The microencapsulated probiotic powder containing halite obtained in Step S9 is mixed with 15 - 30 parts of auxiliary materials to prepare a probiotic preparation containing halite.

4. The preparation method according to claim 3, wherein In the said Step S1, the preparation method of the halite mineral extract is as follows: Select natural halite ore, wash it and crush it into fine powder with a mesh size of more than 200 meshes; Mix the fine powder with a phosphate buffer solution with a pH value of 6.8 - 7.2 according to a weight ratio of 1:8, and carry out stirring extraction at 50 - 55°C. The stirring speed is 200 - 300 r / min, and the extraction time is 4 - 6 hours; After extraction, centrifuge at a speed of 8000 r / min for 15 minutes, and collect the supernatant; The supernatant is filtered and sterilized successively through 0.45 μm and 0.22 μm microporous filter membranes; The concentration of the filtrate is measured, and the total mineral content is determined by the conductivity method, and the mineral concentration is adjusted to 10 - 12 g / L; The prepared halite mineral extract is sub-packed, sterilized at 121°C for 15 minutes and then cooled to room temperature for storage and standby.

5. The preparation method according to claim 3, characterized in that, The acid-resistant precursor substance described in step S2 is composed of the following components by weight: 2.5 parts of L-alanine, 1.5 parts of sodium L-glutamate, 0.8 part of cysteine hydrochloride, 0.4 part of magnesium chloride hexahydrate, 0.15 part of manganese sulfate monohydrate, 0.1 part of zinc sulfate heptahydrate, and 0.05 part of vitamin B6 hydrochloride.

6. The preparation method according to claim 3, characterized in that, In the said step S4, the membrane filtration and concentration technology includes the following specific steps: Use a polysulfone hollow fiber ultrafiltration membrane with a molecular weight cut-off of 15 kDa and an effective membrane area of 0.5 - 0.8 m²; control the operating pressure at 0.07 - 0.09 MPa and maintain the transmembrane pressure difference at 0.04 - 0.06 MPa; control the filtration temperature at 4 - 6 °C and use a double-layer jacket refrigeration system; control the flow rate of the bacterial liquid at 65 - 75 mL / min and adopt a tangential flow mode during filtration to reduce membrane fouling; set up a filtration circulation system, perform backwashing every 20 minutes, with a backwashing pressure of 0.12 - 0.15 MPa and a time of 15 - 20 seconds; concentrate to 15 - 20% of the original volume to make the bacterial density reach above 5×10¹² CFU / mL; after concentration, wash the bacteria twice with an isotonic phosphate buffer solution of pH 7.0 to remove the residues of the fermentation medium.

7. The preparation method according to claim 3, wherein The fermentation culture in step S3 is carried out under the condition that the pH value in the tank is maintained at 5.8 - 6.

2. During the fermentation process, a sterile glucose solution and an amino acid mixture are added every 6 hours, and the addition amount is 1 - 2% of the initial medium volume.

8. The preparation method according to claim 3, characterized in that, The big green salt is obtained by the following method: 1) Wash and crush the natural green salt ore into fine powder with a mesh size of over 100 meshes; 2) Mix the fine powder with deionized water at a weight ratio of 1:5 and perform ultrasonic-assisted extraction at 60 - 70 °C for 2 - 3 hours; 3) Centrifuge the extract at a high speed, with the centrifugation conditions of 10000 r / min for 15 minutes, and collect the supernatant; 4) Rotate and evaporate the supernatant under reduced pressure to 1 / 3 of the initial volume, and then let it stand at 4 °C for 12 hours to precipitate impurities; 5) Filter and remove the precipitate with a 0.22 μm microporous membrane, and then purify the filtrate by nano reverse osmosis membrane filtration technology; 6) Prepare the purified solution by low-temperature vacuum freeze spray drying technology to obtain nano-scale refined big green salt powder.

9. The preparation method according to claim 8, wherein In step S7, the big green salt solution is prepared as follows: Take nano-scale refined big green salt powder and accurately weigh 10 - 12 g; add the weighed big green salt powder to 100 mL of sterile ultrapure water, stir and dissolve it at 40 - 45 °C, with a stirring speed of 300 - 350 r / min and a time of 20 - 30 minutes; Process the dissolved big green salt solution through a high-pressure homogenizer at a pressure of 80 - 100 MPa and cycle it 3 - 5 times; use dynamic light scattering (DLS) to measure the particle size to ensure that the particle diameter distribution is in the range of 100 - 200 nm and the polydispersity index (PDI) is less than 0.3; after filtering and sterilizing with a 0.22 μm polyethersulfone membrane, store the big green salt solution at 4 °C for standby, and the validity period is 7 days.

10. The preparation method according to claim 3, characterized in that, The three-dimensional pulsed vibration spray drying technology specifically includes: Using a spray drying device equipped with a piezoelectric ceramic vibrator, the vibrator generates vibrations simultaneously in the three directions of X, Y, and Z; The vibration frequency in the X direction is set to 30 ± 2 Hz, and the amplitude is 1.2 ± 0.2 mm; the vibration frequency in the Y direction is set to 35 ± 2 Hz, and the amplitude is 1.0 ± 0.2 mm; the vibration frequency in the Z direction is set to 40 ± 2 Hz, and the amplitude is 0.8 ± 0.2 mm; The spray system uses a two-fluid nozzle with an inner diameter of 0.7 mm and an outer diameter of 1.5 mm, and the nitrogen purity is ≥99.999%; The single-pulse feeding time is 1.0 ± 0.1 seconds, and the pulse interval time is 1.5 ± 0.1 seconds; The main tower temperature is 85 ± 5 °C, the drying air flow rate is 85 ± 5 m³ / h, nitrogen is introduced for protection during the drying process, and the oxygen content is controlled below 3%; The particle size distribution of the collected microencapsulated probiotic powder is 15 - 25 μm, and the moisture content is controlled below 3.5%.

Images