Probiotic agent for resisting helicobacter pylori and preparation method thereof

Abstract

The invention provides a lactobacillus plantarum KU01, and the preservation number of the lactobacillus plantarum KU01 is CGMCC (China General Microbiological Culture Collection Center) No.31788. The lactobacillus plantarum KU01 has the advantages that the preservation number is CGMCC No.31788; the invention also provides a fermentation product of the plant lactobacillus and a probiotic preparation comprising the plant lactobacillus and the fermentation product thereof. According to the probiotic agent and the preparation method thereof, the anti-helicobacter pylori probiotic agent is obtained through peracid stress fermentation, peracid stress fermentation, bacterial liquid separation, embedding, compounding and sieving. According to the method, on the basis of the inherent anti-helicobacter pylori physiological activity of the probiotics, the anti-helicobacter pylori physiological activity of the probiotics and the fermentation product is enhanced through stress fermentation, the fermentation product and the probiotics are sequentially released through an embedding technology, and an exogenous iron source is matched; and the anti-helicobacter pylori physiological activity is further promoted. The probiotics provided by the invention can be applied to resisting helicobacter pylori.

Description

Technical Field

[0001] The present invention relates to a probiotic agent and a preparation method thereof, and in particular to a probiotic agent for Helicobacter pylori and a preparation method thereof. Background Art

[0002] Helicobacter pylori (Hp) is a spiral-shaped, motile, gram-negative bacterium with flagella. It is considered to be the main pathogen of peptic ulcer and chronic gastritis. It has also been confirmed to be a high-risk factor for gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma. It is also associated with gastrointestinal diseases such as gastroesophageal reflux disease and functional dyspepsia. As the first-line solution for eradicating Hp, the traditional triple therapy is two antibiotics plus a standard dose of proton pump inhibitors. Currently, quadruple therapy containing bismuth preparations is more commonly used in clinical practice, but both methods may cause gastrointestinal flora disorders and adverse drug reactions, the most serious of which is antibiotic-related diarrhea in the elderly. In addition, with the emergence of Hp resistance, its eradication rate has also decreased.

[0003] Probiotic preparations mainly refer to intestinal probiotics, which are a general term for the beneficial bacteria originally found in the intestines. By adding foreign bacteria beneficial to the human body from outside the body, the number of one or several probiotics in the intestines is directly increased, thereby regulating the balance of intestinal flora and exerting the functions of body regulation, collective defense, disease prevention, and disease treatment. The mechanism of action of probiotics on Hp includes regulating intestinal flora, forming mechanical and chemical biological barriers, forming anti-inflammatory factors, regulating immune mechanisms, and relieving visceral pain.

[0004] There are some public materials on anti-Helicobacter pylori microbial preparations and their preparation methods in the prior art, as follows. CN111820407 discloses an anti-Helicobacter pylori composition and its application, which is prepared by combining plant active ingredients, prebiotics and probiotics, and has the effect of inhibiting Helicobacter pylori through coordinated cooperation. This technical solution is to enhance the antibacterial effect on Helicobacter pylori by the conventional method of probiotics themselves, and physical combination of probiotics and active ingredients, and mutual cooperation, but the effect is limited.

[0005] CN113893275 discloses a pharmaceutical composition for treating Helicobacter pylori infection, wherein the extract of Melia azedarach bark is encapsulated with cyclodextrin as an active ingredient and used in combination with conventional triple therapy and quadruple therapy (i.e., proton pump inhibitor (PPI) plus two antibiotics or bismuth). Obviously, this method still relies on conventional triple therapy and quadruple therapy, and can only reduce other side effects such as drug resistance on the basis of improving the therapeutic effect.

[0006] CN111568919 discloses a chitosan-containing anti-Helicobacter pylori preparation, which is mainly composed of chitosan oligosaccharide, ε-polylysine, lysozyme and / or lactoferrin, and menthol, natural inulin, maltodextrin, starch, etc. as auxiliary materials, which are dissolved, mixed and dried. This technical solution is to enhance the antibacterial effect on Helicobacter pylori by the conventional method of probiotics themselves, and the physical combination and mutual synergy of probiotics and active ingredients, but the effect is limited.

[0007] CN108904546 discloses a probiotic combination preparation with inhibitory and killing effects on Helicobacter pylori and its preparation method, which is prepared by physically mixing prebiotics, probiotics, vitamins, sweeteners and fruit powder. This technical solution is to enhance the antibacterial effect on Helicobacter pylori in a conventional way of physical combination and mutual synergy of probiotics themselves and probiotics and active ingredients, but the effect is limited.

[0008] CN111632084 discloses a composite probiotic composition for inhibiting Helicobacter pylori and its application, which is made of Lactobacillus reuteri and Lactobacillus johnsonii as active bacteria, oligofructose, resistant dextrin, oligogalactose, licorice extract, strawberry extract and maltodextrin as auxiliary materials, and is physically mixed. The technical scheme is to use the probiotics themselves, and the conventional way of physical combination and mutual cooperation of probiotics and active ingredients to enhance the antibacterial effect on Helicobacter pylori, and the effect is limited. CN110075270 discloses a nisin enteric-coated tablet for inhibiting Helicobacter pylori and its preparation method, which is composed of nisin, colloidal pectin bismuth, furazolidone, spirulina, vitamin U, soy lecithin, diethyl phthalate, polyacrylic acid resin No. 2, Tween-80, castor oil, polyethylene glycol-6000, xanthan gum, talcum powder and starch. Wherein nisin is coated with acrylic resin No. 2 and the like. This technical solution does not include probiotics. On the one hand, as amino acid polypeptides, they are easily degraded and inactivated in the human body. At the same time, as food preservatives, they also have a strong inhibitory effect on beneficial lactobacilli and bacillus in the human body, which is not conducive to the competition between probiotics and Helicobacter pylori and the formation of population advantages. CN115119940 discloses the application of Lactobacillus acidophilus LA85 and Bifidobacterium lactis BLA80 in inhibiting Helicobacter pylori. The Lactobacillus acidophilus LA85 and Bifidobacterium lactis BLA80 in the technical solution are preserved exclusive bacteria with certain physiological effects such as anti-Helicobacter pylori. How to strengthen the physiological activity of the two probiotics and actually apply them is not clear in the technical solution.

[0009] CN106389863 discloses a Chinese medicine decoction for treating Helicobacter pylori infection and its preparation method, which is actually extracted from Chinese herbal medicines such as American ginseng, forsythia, oldenlandia diffusa, dandelion, and Poria. This technical solution does not contain probiotics, and the active ingredients are physically combined and synergistically combined to enhance the antibacterial effect on Helicobacter pylori, and the effect is limited.

[0010] CN114259055 discloses a composition for antagonizing Helicobacter pylori, which includes a Lactobacillus plantarum component and a roselle plant essential oil component. The Lactobacillus plantarum component includes a freeze-drying protective agent such as Lactobacillus plantarum, skimmed milk powder, trehalose, and a carrier such as corn starch; the roselle plant essential oil component is coated with β-cyclodextrin, maltodextrin, etc. The probiotics of this technical solution are not fortified, and the probiotics are directly exposed to gastric acid conditions when entering the stomach, which is very easy to inactivate. CN109609420 discloses a probiotic composition for resisting Helicobacter pylori and a preparation method thereof, which includes Lactobacillus plantarum, a bacterial powder carrier (microcrystalline cellulose, medicinal corn starch, inulin, magnesium stearate), and a Lactobacillus plantarum metabolite powder; the above raw materials are mixed and dried to obtain the result. This technical solution is to enhance the antibacterial effect on Helicobacter pylori in a conventional manner by physically combining the probiotics themselves and the probiotics with active ingredients and cooperating with each other, and the effect is limited.

[0011] CN111956671 discloses a composite probiotic for treating Helicobacter pylori, which is obtained by inoculating Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei and Rhamnosus into a medium prepared with apple juice, fresh goat milk and distilled water, and fermenting. This technical solution is to enhance the antibacterial effect on Helicobacter pylori by the conventional method of physical combination and mutual cooperation of probiotics themselves and probiotics and active ingredients, but the effect is limited.

[0012] It can be seen from the above-disclosed technical solutions that the current probiotic products against Helicobacter pylori mostly have active probiotics in the form of simple physical mixing, or supplemented with plant extracts, active ingredients and other substances. The antibacterial effect of probiotics on Helicobacter pylori mainly depends on the physically added probiotics or other active substances themselves and the synergistic effect, and there is no further development on how to further enhance the antibacterial efficacy of probiotics and their auxiliary ingredients.

[0013] Therefore, developing a probiotic agent against Helicobacter pylori and a preparation method thereof has become an urgent problem to be solved. Summary of the invention

[0014] The first object of the present invention is to provide a Lactobacillus plantarum having a strong anti-Helicobacter pylori effect.

[0015] The second object of the present invention is to provide a fermentation product of the plant lactobacillus.

[0016] The third object of the present invention is to provide a probiotic comprising the Lactobacillus plantarum and a fermentation product of the Lactobacillus plantarum.

[0017] The fourth object of the present invention is to provide a method for preparing the probiotic.

[0018] The fifth object of the present invention is to provide a use of the probiotic agent.

[0019] The present invention is achieved through the following technical solutions:

[0020] A Lactobacillus plantarum KU01, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee, with a deposit number of CGMCC No.31788, a strain number of KU01, and a deposit date of August 30, 2024. The deposit unit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101. The bacteria are alive when deposited.

[0021] A fermentation product is obtained by fermenting rosemary, turmeric and astragalus by lactobacillus plantarum through acid stress fermentation and alkaline stress fermentation.

[0022] A probiotic preparation, comprising the plant lactobacillus according to claim 1 and the fermentation product according to claim 2. The preparation method of the probiotic preparation comprises the following steps:

[0023] S1: fermentation under acid stress: mixing the plant lactobacillus, nutrient medium, rosemary, turmeric and astragalus, adding water, and fermenting under acidic conditions of pH 1.5 to 3 to obtain a first fermentation liquid;

[0024] S2: Alkaline stress fermentation: adjusting the pH value of the first fermentation liquid to alkaline, fermenting under alkaline conditions to obtain a second fermentation liquid;

[0025] S3 bacterial liquid separation: adjusting the pH value of the second fermentation liquid to 9.5-12, performing solid-liquid separation, microfiltration, collecting the permeate and the retentate respectively, and concentrating the permeate and the retentate respectively to obtain a permeate concentrate and a retentate concentrate, and drying the permeate concentrate to obtain a permeate concentrate dry powder;

[0026] S4 encapsulation: adding the retained concentrated solution to the first auxiliary material for microcapsule encapsulation to obtain wet probiotic microcapsules;

[0027] S5 compounding: uniformly mixing the wet probiotic microcapsules, the concentrated permeate dry powder and the second auxiliary material to obtain the probiotic preparation.

[0028] The inoculation amount of the probiotic seed solution is 1 to 5%;

[0029] The ratio of rosemary, turmeric and astragalus is 1-4:4-7:5-9;

[0030] The amount of water added is 20 to 40 times the total weight of the nutrient medium, rosemary, turmeric and astragalus.

[0031] The temperature of the peracid stress fermentation in S1 is 30-50°C, and the fermentation time is 24-48h;

[0032] The temperature of the alkaline stress fermentation in S2 is 30-50° C., and the fermentation time is 3-6 hours.

[0033] The retentate obtained by microfiltration is concentrated by membrane to a soluble solid content of 10-20%; the molecular weight cutoff of the concentration membrane is 100000-1000000Da;

[0034] The permeate obtained by the microfiltration is concentrated by a membrane to a soluble solid content of 15-30%; the molecular weight cut-off of the concentration membrane is 300-800Da.

[0035] The embedding in S4 comprises the steps of adding the mixed aqueous solution of the first auxiliary material to the retained concentrated solution, mixing evenly, dropping a calcium chloride solution into the solution, and placing the solution in a refrigerator at 4° C. for solidification for 3 to 6 hours.

[0036] The first auxiliary material includes pectin, β-cyclodextrin and an iron source;

[0037] The iron source is any one or both of ferrous carbonate and ferric pyrophosphate;

[0038] The second auxiliary material is at least three of resistant dextrin, psyllium husk powder, calcium carbonate, fucoidan and sodium alginate. The nutrient base is a mixture of glucose, soy protein, magnesium sulfate, dipotassium hydrogen phosphate and vitamin C.

[0039] The probiotic preparation is used to fight Helicobacter pylori.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The probiotics prepared by the method provided by the present invention have a high number of effective live bacteria, that is, the number of effective live bacteria is ≥3.5×10 9 CFU / g. The probiotics prepared by the method provided by the present invention, after fermentation under adverse conditions of Chinese medicinal materials, embedding, adding nutritional iron sources and compounding, have greatly improved the anti-Helicobacter pylori effect;

[0042] The probiotics prepared by the method provided by the present invention can pass through the stomach smoothly, can effectively resist gastric acid, and has a high survival rate, that is, the gastric survival rate is ≥85%.

[0043] The probiotics prepared by the method provided by the present invention can effectively resist the adverse effects of bile salts in the small intestine on probiotics, thereby enhancing their survival rate in the small intestine and improving the immune activity of probiotics, that is, the small intestine survival rate is ≥85%.

[0044] The probiotic prepared by the method provided by the present invention has a certain effect on improving iron deficiency anemia caused by or not caused by Helicobacter pylori.

[0045] The method provided by the invention has simple process, is applicable to various probiotics, and has strong industrialization capability. DETAILED DESCRIPTION

[0046] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0047] Screening of bacteria

[0048] (1) Strain isolation

[0049] Take 25mL of traditional food kimchi, dilute it in gradients, spread it on MRS medium, place it upside down in an anaerobic incubator, and culture it at 37℃ for 48 hours. After the colonies grow on the plate, observe the single colonies formed on each plate, and select the single colonies that meet the characteristics of lactic acid bacteria (round, milky white or slightly yellow, opaque or translucent, smooth protrusions, and neat edges) according to the shape, size, color, transparency, protrusions, and surface roughness of the colonies. Purify three times until the color, size, transparency, and edge shape of the colonies on the same plate are uniform.

[0050] (2) Gram staining of the purified bacteria, retaining Gram-positive bacteria, and performing strain screening:

[0051] A single colony of strain KU01 was picked up and placed in Lysis Buffer for Microorganism to Direct PCR, and lysed in a water bath at 80°C for 30 min to obtain a bacterial DNA template.

[0052] Universal primers for bacterial 16S rRNA gene amplification: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGACTTAACCCCAATCGC-3'.

[0053] The reaction system (25 μL) was: 1 μL each of universal primers 27F and 1492R for bacterial 16S rRNA gene amplification, 0.5 μL of FUA033 bacterial DNA template, 12.5 μL of 2×RapidTaq Master Mix, and finally dd H2O was used to make up the 25 μL reaction system.

[0054] PCR reaction program: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 15 s, 35 cycles; 72°C final extension for 5 min; 4°C storage. After PCR, the reaction mixture was electrophoresed in 1% agarose gel, and the electrophoresis buffer was 1×TAE (50 mmol / L Tris-acetic acid, 1 mmol / L EDTA, pH 8.0).

[0055] KU01 was sequenced and compared with the sequencing results, and was identified as belonging to Lactobacillus rhamnosus. The 16s DNA sequencing results of the bacteria are as follows:

[0056] GGAAGTGGCGGCGTGCTATACATGCAGTCGAACGAACTCTGGTATTGATTGGTGCTTG

[0057] CATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGC

[0058] CCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGAC

[0059] CGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCG

[0060] TATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAG

[0061] AGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGC

[0062] AGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAA

[0063] GAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGT

[0064] TCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGC

[0065] GGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGG

[0066] CGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAAC

[0067] TGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCG

[0068] TAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTG

[0069] AGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAA

[0070] CGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAA

[0071] GCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGG

[0072] CCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCA

[0073] GGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATAC

[0074] AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC

[0075] GAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCC

[0076] GGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCT

[0077] GGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAG

[0078] CTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGT

[0079] CGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTG

[0080] TACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCT

[0081] TTTAGGAACCAGCCGCCTAATGTGACAGGATG

[0082] The present invention provides a method for preparing an anti-Helicobacter pylori probiotic, comprising the following steps:

[0083] S1 Over-acid fermentation: a certain proportion of Lactobacillus plantarum, nutrient medium and Chinese medicinal materials are mixed and fermented under over-acid conditions;

[0084] S2 Over-alkaline stress fermentation: The pH value of the fermentation liquid after over-acid fermentation is adjusted to over-alkaline, and fermentation is carried out under over-alkaline conditions;

[0085] S3 bacterial liquid separation: adjust the pH value of the fermented liquid, separate the solid and liquid, and perform microfiltration to collect the permeate and the retentate, respectively, and perform membrane concentration to obtain the permeate concentrate and the retentate concentrate, and dry the permeate to obtain the concentrated permeate dry powder;

[0086] S4 encapsulation: adding the retained concentrated solution to the first auxiliary material for microcapsule encapsulation to obtain wet probiotic microcapsules;

[0087] S5 compounding: uniformly mixing the wet probiotic microcapsules, the concentrated permeate dry powder and the second auxiliary material;

[0088] S6 screening: After compounding, the mixture is screened to obtain a probiotic product against Helicobacter pylori.

[0089] The probiotics used in the present invention have inherent anti-Helicobacter pylori physiological activity, and then the probiotics are strengthened by forced fermentation to improve their anti-Helicobacter pylori physiological activity; since the fermentation products of rosemary, turmeric and astragalus have anti-Helicobacter pylori physiological activity, the fermentation products and probiotics can be released in sequence through the embedding technology, further promoting the anti-Helicobacter pylori physiological activity of the probiotics and their fermentation products.

[0090] In step S1, the peracid stress fermentation is to put the nutrient medium and the Chinese medicinal materials into a fermentation tank, then add 20 to 40 times of their total weight of sterile warm water, stir evenly and fully dissolve, adjust the pH value to 1.5 to 3 with hydrochloric acid, maintain a constant temperature of 30 to 50° C., add probiotics, and ferment under normal pressure, keep stirring during the fermentation, the stirring speed is 10 to 20 r / min, and the fermentation time is 24 to 48 hours;

[0091] In step S1, the nutrient base is a mixture of glucose, soy protein, magnesium sulfate, dipotassium hydrogen phosphate and vitamin C; in step S1, based on the quality of sterile warm water, the added amount of the nutrient base is 3-6% glucose, 2-7% soy protein, 0.01-0.04% magnesium sulfate, 0.15-0.3% dipotassium hydrogen phosphate and 0.01-0.05% vitamin C;

[0092] In step S1, the Chinese medicinal materials are a mixture of rosemary, turmeric and astragalus;

[0093] In step S1, the rosemary, turmeric and astragalus in the Chinese medicinal materials are pre-treated, and the pre-treatment is to mechanically chop and crush them respectively, pass them through a 80-200 mesh sieve, and take the sieved material;

[0094] Rosemary, turmeric and astragalus contain various effective active ingredients such as rosmarinic acid, ursolic acid, curcumin, baicalin, etc., which have a good effect in inhibiting Helicobacter pylori. When used in conjunction with probiotic fermentation, the probiotic product can be enhanced to have a better anti-Helicobacter pylori effect.

[0095] In step S1, based on the quality of sterile warm water, the amount of Chinese medicinal materials added is 1-4% rosemary, 4-7% turmeric and 5-9% astragalus;

[0096] In step S1, the inoculation amount of the probiotic seed solution is 1-5%;

[0097] In step S1, the hydrochloric acid is a sterile dilute hydrochloric acid aqueous solution with a mass fraction of 3 to 10%;

[0098] In step S2, the alkaline stress fermentation is to adjust the pH value to 9.5-12 with ammonia water after the fermentation in step (1), continue to maintain a constant temperature of 30-50° C., and ferment under normal pressure conditions, during which stirring is maintained at a stirring speed of 10-20 r / min, and the fermentation time is 3-6 h;

[0099] In step S2, the ammonia water is a sterile ammonia water solution with a mass fraction of 20-28%;

[0100] After fermentation under the stress of acidic conditions, it helps to improve the acid resistance of probiotics in the stomach and intestines, especially the ability to resist gastric acid, thereby improving the survival rate and proliferation ability of probiotics in the stomach and intestines, helping to form population advantages, resist Helicobacter pylori, and improve bile salt resistance;

[0101] After fermentation under alkaline stress, it helps to improve the ability of probiotics to resist and neutralize the "ammonia cloud" produced by Helicobacter pylori, so that probiotics can be closer to and more compatible with Helicobacter pylori, antagonize the proliferation of Helicobacter pylori, and then form a population advantage, which is beneficial for probiotics to maintain high activity, and further conducive to the probiotics' ability to antagonize Helicobacter pylori and resist bile salts;

[0102] More importantly, stress-induced fermentation can cause probiotics to produce unconventional secondary metabolites. It was unexpectedly discovered that the metabolites produced by stress-induced fermentation further enhanced the anti-Helicobacter pylori effect of probiotic products.

[0103] In step S3, the pH value is 6 to 7, and the acid or base used to adjust the pH is hydrochloric acid or ammonia water;

[0104] In step S3, the hydrochloric acid is a sterile dilute hydrochloric acid aqueous solution with a mass fraction of 3 to 10%;

[0105] In step S3, the ammonia water is a sterile ammonia water solution with a mass fraction of 20-28%;

[0106] In step S3, the solid-liquid separation is firstly passing through a 200-300 mesh screen, and then passing through a microfiltration membrane with a pore size of 0.1-20 μm, and collecting the retentate and the permeate respectively;

[0107] In step S3, the retentate is concentrated, and the membrane is concentrated to a soluble solid content of 10-20%; the molecular weight cutoff of the concentration membrane is 100000-1000000Da;

[0108] In step S3, the permeate is concentrated, and the membrane is concentrated to a soluble solid content of 15-30%; the molecular weight cutoff of the concentration membrane is 300-800Da.

[0109] In step S3, the drying is any one of vacuum freeze drying or vacuum heating drying; more preferably vacuum freeze drying; in step S4, the first auxiliary material is pectin, β-cyclodextrin, calcium chloride and iron source;

[0110] In step S4, the microcapsule embedding is to add the configured pectin, β-cyclodextrin and iron source mixed aqueous solution to the trapped concentrate, mix evenly, drop calcium chloride solution, and place in a 4 ° C refrigerator to solidify for 3 to 6 hours, filter with gauze, and wash with sterile saline to obtain wet probiotic microcapsules. By adding nutrient iron elements, the proliferation of probiotics can be further selectively promoted, and the proliferation of Helicobacter pylori can be curbed, thereby achieving the purpose of curbing the physiological activity of Helicobacter pylori, promoting the proliferation of probiotics and forming a population advantage in the stomach. At the same time, probiotics have the effect of alleviating iron deficiency anemia caused by / not caused by Helicobacter pylori. On the other hand, adding a nutrient iron source can subsidize the human body, thereby improving iron deficiency anemia caused by / not caused by Helicobacter pylori.

[0111] In step S3, the drying is any one of vacuum freeze drying or vacuum heating drying; vacuum freeze drying is more preferred; in step S4, the volume ratio of the mixed aqueous solution of pectin, β-cyclodextrin and iron source to the retained concentrated solution is 1:0.5-2; in step S4, the mass ratio of pectin, β-cyclodextrin and iron source in the first auxiliary material is 1:0.2-0.8:0.001-0.006, and the concentration of the mixed aqueous solution of pectin, β-cyclodextrin and iron source is 1.5-5%;

[0112] In step S4, the iron source is any one or more combinations of ferrous carbonate or ferric pyrophosphate; the iron source is the nutritional metal element of Helicobacter pylori and probiotics, and there is a competitive absorption relationship between them. Because probiotics are suitable for acidic environments, and Helicobacter pylori is more suitable for nearly neutral (pH 6-8) environments, especially the alkaline "ammonia cloud" produced around Helicobacter pylori is an alkaline environment. Therefore, ferrous carbonate and ferric pyrophosphate, which are not easily soluble in alkaline or neutral but soluble in acid, are selected as nutritional iron elements, which can inhibit the competitive absorption of Helicobacter pylori to nutritional iron sources, and promote the competitive absorption of probiotics to nutritional iron sources, thereby promoting the rapid formation of population advantages of probiotics.

[0113] In step S4, the concentration of the calcium chloride aqueous solution is 100-400 mmol / L;

[0114] In step (5), the second auxiliary material is any three or more of resistant dextrin, psyllium husk powder, calcium carbonate, fucoidan, and sodium alginate in any proportion;

[0115] In step (5), the mass ratio of the wet probiotic microcapsules, the concentrated permeate dry powder and the second auxiliary material is 1:0.5-3:1-4;

[0116] In step (5), the mixing is mechanical and physical mixing;

[0117] The concentrated permeate dry powder is added to the outer layer of the probiotic capsule. The secondary products of adverse fermentation in the concentrated permeate dry powder are rapidly dissolved in the stomach, and the urease of Helicobacter pylori is quickly and effectively destroyed, which is conducive to the subsequent probiotics to be closer to the "ammonia cloud" produced by Helicobacter pylori; then the probiotics in the microcapsule begin to release, and neutralize and destroy the "ammonia cloud" protective layer produced by Helicobacter pylori, thereby exposing the Helicobacter pylori under the "ammonia cloud" protective layer, so that the Helicobacter pylori is directly exposed to the environment of probiotics, gastric acid and secondary metabolites of probiotics, further inhibiting the proliferation of Helicobacter pylori and forming a probiotic population advantage.

[0118] In step (6), the sieving is through a 40-80 mesh sieve, and the sieved material is a probiotic product for resisting Helicobacter pylori. In summary, the present invention uses probiotics with certain resistance to Helicobacter pylori, and after mixing them in a certain proportion, they are fermented in acid and alkali adversity with Chinese medicinal materials with anti-Helicobacter pylori activity, so that the acid and alkali resistance of the probiotics is improved. Adversity fermentation will also force the probiotics to produce secondary metabolites with special functions. At the same time, adversity fermentation will also cause the Chinese medicinal materials to release their effective active ingredients.

[0119] The probiotics obtained by solid-liquid separation are embedded with embedding materials and nutritional iron sources, which, on the one hand, allow the probiotics to be slowly released in gastric acid, and on the other hand, the nutritional iron source inhibits the growth of Helicobacter pylori and promotes the proliferation of probiotics.

[0120] The concentrated permeate dry powder rich in secondary metabolites is mixed with probiotic capsules and other nutrients, which allows the secondary metabolites in the probiotic preparation to be released first. At this time, the secondary metabolites successively destroy the urease and ammonia cloud of Helicobacter pylori, and then expose the Helicobacter pylori protected by the "ammonia cloud" to gastric acid. Then the probiotics are released, so that the Helicobacter pylori is inhibited by the probiotics, and the probiotics form a population advantage.

[0121] The present invention is further described below in conjunction with specific embodiments.

[0122] Unless otherwise specified, the raw materials or chemical reagents used in the embodiments of the present invention were obtained through conventional commercial or homemade routes.

[0123] 1. Decapsulation method: Add 1 g of the prepared probiotic agent to 10 ml of 0.06 mol / L sodium citrate aqueous solution, shake in a shaker at 37°C and 250 r / min for 30 min, and take a sample to obtain the decapsulated probiotic agent.

[0124] 2. Detection method of effective viable bacteria count: After decapsulation according to method 1, GB 4789.35-2023 National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test

[0125] 3. Determination of in vitro simulated anti-Helicobacter pylori activity

[0126] (1) Setting up and preparing the control group and experimental group

[0127] Take 0.1g of the prepared microcapsule product, 0.1g of the microcapsule product prepared by the high-temperature inactivated sterilizer, and 0.1g of the microcapsule product prepared by the high-temperature inactivated product, respectively, put them into 50ml of simulated small intestinal fluid, shake them fully in a shaker at 37°C, and treat them at 50r / min for 3h before use.

[0128] (2) Determination of Anti-Helicobacter pylori Activity

[0129] Take 0.1mL of the prepared Helicobacter pylori fermentation liquid and add it to the Columbia blood agar medium at a temperature of 45℃~50℃, mix it evenly, and then quickly pour it into the plate with the Oxford cup placed in advance. After the culture medium is cooled and solidified, take out the Oxford cup, and inject 200μL of the control group and the experimental group to prepare the bacterial suspension in each well. Cover the plate lightly and place it in a 37℃ three-gas incubator (5% oxygen, 10% carbon dioxide, 85% nitrogen) for 48~72h. Observe and measure the diameter of the inhibition zone with the cross method of the vernier caliper. Amoxicillin solution with a concentration of 0.05mg / mL is used as the positive control, and the seed culture medium is used as the negative control.

[0130] 4. Determination of survival rate of simulated gastrointestinal system in vitro

[0131] (1) Preparation of simulated gastric fluid and determination of gastric survival rate

[0132] Mix 0.26 g potassium chloride, 0.02 g potassium dihydrogen phosphate, 1.05 g sodium bicarbonate, 1.40 g sodium chloride, 0.01 g magnesium chloride dihydrate, 0.04 g ammonium carbonate and 0.01 g calcium chloride dihydrate in 500 mL sterile water, adjust the pH to 2.00 with 1.00 mmol / L hydrochloric acid or 1.00 mmol / L sodium hydroxide, maintain the pepsin at 3.20 g / L, and mix thoroughly to obtain simulated gastric fluid.

[0133] Weigh 0.1 g of the prepared microcapsule product, put it into 50 ml of simulated gastric fluid, shake it fully in a shaker at 37°C, and after treating it at 150 r / min for 10 min, determine the number of viable bacteria and calculate the gastric survival rate.

[0134] Stomach survival rate = (final viable bacteria count / initial viable bacteria count) * 100%

[0135] (2) Preparation of simulated small intestinal fluid and determination of small intestinal survival rate

[0136] Mix 0.25 g potassium chloride, 0.01 g potassium dihydrogen phosphate, 3.27 g sodium bicarbonate, 1.12 g sodium chloride, 6.13 g magnesium chloride hexahydrate and 0.03 g calcium chloride dihydrate in 500 mL sterile water, adjust the pH to 7.40 with 1.00 mmol / L hydrochloric acid or 1.00 mmol / L sodium hydroxide, maintain the concentration of pancreatic enzyme at 10.00 g / L, and the concentration of bile salt at 3.00 g / L, and mix thoroughly to obtain simulated small intestinal fluid.

[0137] Take 0.1g of the prepared microcapsule product, put it into 50ml of simulated small intestinal fluid, shake it fully in a shaker at 37°C, and after treating it at 50r / min for 3h, determine the number of viable bacteria and calculate the survival rate.

[0138] Small intestine survival rate = (final viable bacteria count / initial viable bacteria count) * 100%

[0139] Example 1

[0140] (1) Fermentation under acid stress: Nutrient medium (5% glucose, 6% soy protein, 0.03% magnesium sulfate, 0.2% potassium dihydrogen phosphate and 0.03% vitamin C), Chinese medicinal materials (3% rosemary, 5% turmeric and 5% astragalus) and 25 times sterile water were put into the fermentation tank and dissolved, and the pH was adjusted to 2.0. After maintaining the temperature at 37°C, the inoculation amount of Lactobacillus plantarum seed liquid was 5%, and normal pressure fermentation was started for 30 hours.

[0141] (2) Alkaline stress fermentation: After the fermentation in (1) is completed, the pH is adjusted to 9.5 with ammonia water and the fermentation is continued for 5 hours to obtain the fermentation product.

[0142] (3) Bacterial liquid separation: The pH value of the fermented liquid was adjusted to 6.5, and it was first filtered with a 200-mesh filter bag, and then passed through a 10 μm microfiltration membrane to collect the permeate and retentate after the microfiltration membrane. The retentate was concentrated to 15.4% soluble solids using a membrane with a molecular weight cutoff of 800,000 Da, and the permeate was concentrated to 15.4% soluble solids using a membrane with a molecular weight cutoff of 500 Da. The permeate was then vacuum-freeze-dried to obtain a concentrated permeate dry powder;

[0143] (4) Encapsulation: The retained concentrated solution is added to the first auxiliary material for microcapsule encapsulation, wherein the volume ratio of the mixed aqueous solution of pectin, β-cyclodextrin and ferrous carbonate to the retained concentrated solution is 1:1; the mass ratio of pectin, β-cyclodextrin and ferrous carbonate in the first auxiliary material is 1:0.5:0.004, and the concentration of the mixed aqueous solution of pectin, β-cyclodextrin and iron source is 4%, thereby obtaining wet probiotic microcapsules;

[0144] (5) Compounding: the wet probiotic microcapsules are mixed with the concentrated permeate dry powder and the second auxiliary material in a mass ratio of 1:2:3, wherein the second auxiliary material is psyllium husk powder, fucoidan and calcium carbonate in a mass ratio of 1:2:1;

[0145] (6) Screening: After compounding, the mixture is freeze-dried in vacuum and screened through an 80-mesh sieve to obtain an anti-Helicobacter pylori probiotic product. The performance test results are shown in Table 1.

[0146] Example 2

[0147] (1) Fermentation under acid stress: Nutrient medium (6% glucose, 3% soy protein, 0.01% magnesium sulfate, 0.3% potassium dihydrogen phosphate and 0.02% vitamin C), Chinese medicinal materials (2% rosemary, 6% turmeric and 6% astragalus) and 35 times sterile water were put into the fermentation tank and dissolved, and the pH was adjusted to 2.5. After maintaining the temperature at 45°C, the inoculation amount of Lactobacillus plantarum seed liquid was 4%, and normal pressure fermentation was started for 30 hours.

[0148] (2) Alkaline stress fermentation: After the fermentation in (1) is completed, the pH is adjusted to 10 with ammonia water and the fermentation is continued for 4 hours to obtain the fermentation product.

[0149] (3) Separation of bacterial liquid: The pH value of the fermented liquid is adjusted to 6.3, and it is first filtered with a 200-mesh filter bag, and then filtered through a 10 μm microfiltration membrane. The permeate and the retentate after the microfiltration membrane are collected separately. The retentate is concentrated to 17.5% of soluble solids using a 600,000 Da membrane concentration, and the permeate is concentrated to 20.4% of soluble solids using a 500 Da membrane concentration. The permeate is then vacuum-freeze-dried to obtain a concentrated permeate dry powder;

[0150] (4) Encapsulation: The retained concentrated solution is added to the first auxiliary material for microcapsule encapsulation, wherein the volume ratio of the mixed aqueous solution of pectin, β-cyclodextrin and ferrous carbonate to the retained concentrated solution is 1:1.5; the mass ratio of pectin, β-cyclodextrin and ferrous carbonate in the first auxiliary material is 1:0.8:0.003, and the concentration of the mixed aqueous solution of pectin, β-cyclodextrin and iron source is 3%; obtaining wet probiotic microcapsules;

[0151] (5) Compounding: the wet probiotic microcapsules are mixed evenly with the concentrated permeate dry powder and the second auxiliary material in a mass ratio of 1:3:2, wherein the second auxiliary material is psyllium husk powder, fucoidan and sodium alginate in a mass ratio of 1:1:1;

[0152] (6) Screening: After compounding, the mixture is freeze-dried in vacuum and screened through an 80-mesh sieve to obtain an anti-Helicobacter pylori probiotic product. The performance test results are shown in Table 1.

[0153] Example 3

[0154] (1) Fermentation under acid stress: Nutrient medium (3% glucose, 6% soy protein, 0.04% magnesium sulfate, 0.2% potassium dihydrogen phosphate and 0.02% vitamin C), Chinese medicinal materials (2% rosemary, 7% turmeric and 6% astragalus) and 25 times sterile water were put into the fermentation tank and dissolved, and the pH was adjusted to 3. After maintaining the temperature at 40°C, the inoculation amount of Lactobacillus plantarum seed liquid was 4%, and normal pressure fermentation was started for 40 hours.

[0155] (2) Alkaline stress fermentation: After the fermentation in (1) is completed, the pH is adjusted to 9.5 with ammonia water and the fermentation is continued for 5 hours to obtain the fermentation product.

[0156] (3) Bacterial liquid separation: The pH value of the fermented liquid is adjusted to 6.6, and it is first filtered with a 200-mesh filter bag, and then filtered through a 20 μm microfiltration membrane. The permeate and the retentate after the microfiltration membrane are collected separately. The retentate is concentrated to 15.2% of soluble solids using a 500,000 Da membrane concentration, and the permeate is concentrated to 18.2% of soluble solids using a 500 Da membrane concentration. The permeate is then vacuum-freeze-dried to obtain a concentrated permeate dry powder;

[0157] (4) Encapsulation: The retained concentrated solution is added to the first auxiliary material for microcapsule encapsulation, wherein the volume ratio of the mixed aqueous solution of pectin, β-cyclodextrin and ferrous carbonate to the retained concentrated solution is 1:1.3; the mass ratio of pectin, β-cyclodextrin and ferrous carbonate in the first auxiliary material is 1:0.5:0.003, and the concentration of the mixed aqueous solution of pectin, β-cyclodextrin and iron source is 4%; wet probiotic microcapsules are obtained;

[0158] (5) Compounding: the wet probiotic microcapsules are mixed with the concentrated permeate dry powder and the second auxiliary material in a mass ratio of 1:3:2, wherein the second auxiliary material is psyllium husk powder, resistant dextrin and sodium alginate in a mass ratio of 3:1:1;

[0159] (6) Screening: After compounding, the mixture is freeze-dried in vacuum and screened through an 80-mesh sieve to obtain an anti-Helicobacter pylori probiotic product. The performance test results are shown in Table 1.

[0160] Comparative Example 1

[0161] The "superacid condition" in step 1 is replaced by "weak acid condition with a pH of 6.5", and the other processes and parameters are all carried out according to Example 1.

[0162] The performance test results are shown in Table 1.

[0163] Comparative Example 2

[0164] The "overalkaline condition" in step 2 is replaced by "weakly alkaline condition with a pH of 7.5", and the other processes and parameters are all carried out according to Example 1.

[0165] The performance test results are shown in Table 1.

[0166] Comparative Example 3

[0167] The "concentrated liquid powder" in step 5 is replaced by "white dextrin", and the other processes and parameters are all carried out according to Example 1.

[0168] The performance test results are shown in Table 1.

[0169] Comparative Example 4

[0170] The "ferrous carbonate" in step 4 is replaced by "white dextrin", and the other processes and parameters are all carried out according to Example 1.

[0171] The performance test results are shown in Table 1.

[0172] Comparative Example 5

[0173] The embedding process in step 4 was replaced by simple physical mixing, and the other processes and parameters were all carried out according to Example 1.

[0174] The performance test results are shown in Table 1.

[0175] Table 1 Performance test results of the embodiment

[0176]

[0177]

[0178] The applicant declares that the present invention illustrates the detailed preparation method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed preparation method, that is, it does not mean that the present invention must rely on the above-mentioned detailed preparation method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A Lactobacillus plantarum KU01, characterized in that: Its accession number is CGMCC No.31788.

2. A fermentation product, characterized in that: The product is obtained by subjecting the plant lactobacillus to acid stress fermentation and alkaline stress fermentation of rosemary, turmeric and astragalus.

3. A probiotic preparation, characterized in that: The invention comprises the plant lactobacillus as claimed in claim 1 and the fermentation product as claimed in claim 2.

4. The method for preparing the probiotic preparation according to claim 3, characterized in that: The steps include: S1: fermentation under acid stress: mixing the plant lactobacillus, nutrient medium, rosemary, turmeric and astragalus, adding water, and fermenting under acidic conditions of pH 1.5 to 3 to obtain a first fermentation liquid; S2: Alkaline stress fermentation: adjusting the pH value of the first fermentation liquid to alkaline, fermenting under alkaline conditions to obtain a second fermentation liquid; S3 bacterial liquid separation: adjusting the pH value of the second fermentation liquid to 9.5-12, solid-liquid separation, microfiltration, collecting the permeate and the retentate respectively, and performing membrane concentration on the permeate and the retentate to obtain the permeate concentrate and the retentate concentrate respectively, and drying the permeate concentrate to obtain the permeate concentrate dry powder; S4 encapsulation: adding the retained concentrated solution to the first auxiliary material for microcapsule encapsulation to obtain wet probiotic microcapsules; S5 compounding: uniformly mixing the wet probiotic microcapsules, the concentrated permeate dry powder and the second auxiliary material to obtain the probiotic preparation.

5. The method for preparing the probiotic preparation according to claim 1, characterized in that: The inoculation amount of the probiotic seed solution is 1 to 5%; The ratio of rosemary, turmeric and astragalus is 1-4:4-7:5-9; The amount of water added is 20 to 40 times the total weight of the nutrient medium, rosemary, turmeric and astragalus.

6. The method for preparing the probiotic preparation according to claim 1, characterized in that: The temperature of the peracid stress fermentation in S1 is 30-50°C, and the fermentation time is 24-48h; The temperature of the alkaline stress fermentation in S2 is 30-50° C., and the fermentation time is 3-6 hours.

7. The method for preparing the probiotic preparation according to claim 1, characterized in that: The retentate obtained by the microfiltration is concentrated to a soluble solid content of 10-20%; the molecular weight of the soluble solid is 100000-1000000Da; The permeate obtained by the microfiltration is concentrated to a soluble solid content of 15-30%; the molecular weight of the soluble solid is 300-800Da.

8. The method for preparing the probiotic preparation according to claim 1, characterized in that: The embedding in S4 comprises the steps of adding the mixed aqueous solution of the first auxiliary material to the retained concentrated solution, mixing evenly, dropping a calcium chloride solution into the solution, and placing the solution in a refrigerator at 4° C. for solidification for 3 to 6 hours.

9. The method for preparing the probiotic preparation according to claim 1, characterized in that: The first auxiliary material includes pectin, β-cyclodextrin and an iron source; The iron source is any one or both of ferrous carbonate and ferric pyrophosphate; The second auxiliary material is at least three of resistant dextrin, psyllium husk powder, calcium carbonate, fucoidan and sodium alginate; the nutrient base is a mixture of glucose, soy protein, magnesium sulfate, dipotassium hydrogen phosphate and vitamin C.

10. The use of the probiotic preparation according to claim 3, characterized in that: Used to fight Helicobacter pylori.

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