Probiotic protection composition and probiotic tablet

Abstract

The invention relates to a probiotic tablet which comprises probiotic powder and a probiotic protection composition, and the probiotic protection composition comprises araboxylan, a filling agent and a lubricating agent. The araboxylan is used as a probiotic protective agent to construct the probiotic protective composition, high pressure in tablets can be reduced, damage of factors such as low pH and high bile salt in human gastrointestinal tracts to the probiotics can be reduced, and the survival rate of the probiotics in the tabletting process is increased. According to the present invention, the arabinoxylan and other auxiliary materials are matched for use, such that the friability of the tablet is controlled to be less than 0.5%, the water activity is controlled to be 0.1%, the viable bacteria stability can be significantly improved, and the quality requirement of the viable bacteria number during the shelf life of the product can be completely ensured.

Description

[0001] This application claims priority to Chinese patent application 202411216900.X filed on September 2, 2024, and the entire contents of this patent application are incorporated herein by reference in their entirety. Technical Field

[0002] The invention relates to the technical field of microbial preparations, and in particular to a probiotic protection composition and a probiotic tablet with high bacterial activity. Background Art

[0003] In 2001, the World Health Organization and the Food and Agriculture Organization of the United Nations jointly defined "probiotics". When ingested in appropriate quantities, live bacteria that can play a beneficial role in the health of the host are called probiotics. In recent years, scientific research has shown that probiotics colonize the host's intestines and play a beneficial role in the host by improving the balance of intestinal microecology, such as regulating host immunity and improving host metabolism. Related research results have brought broad application prospects for probiotics in the field of biomedicine. In recent years, many probiotic drugs around the world have been in clinical research. In probiotic products, "live strains" and "sufficient numbers" are the two core elements of probiotics. However, as microorganisms, probiotics are very fragile and easily affected by environmental factors. At present, many probiotic products die in large numbers during production, processing and environmental storage, and the number of live bacteria is low. At the same time, surviving probiotics need to pass through multiple biological barriers to reach the human intestine. The tolerance and adhesion ability of probiotics to the gastrointestinal tract are necessary conditions for them to exert their physiological effects. Among the many defense mechanisms of the human body, the strong acid environment provided by gastric juice and the high bile salt environment provided by intestinal juice have the greatest impact on the activity of microorganisms. After bacteria enter the digestive tract from the mouth, the gastric acid, bile salts, digestive enzymes, electrolytes, etc. contained in the stomach and small intestine environment will affect the activity of microorganisms, affect the composition of the biofilm, change the fluidity of the biofilm, affect the microorganism's absorption of nutrients and the excretion of metabolic products, and cause damage to or even inactivate the microorganisms.

[0004] Tablets are the most common dosage form in the field of medicine and food because of their advantages such as convenient oral administration, accurate dosage, and convenient storage. In recent years, tablets have also been one of the key dosage forms for domestic and foreign manufacturers to develop probiotic products. However, during the preparation process of probiotic tablet products, factors such as pressure and temperature during tableting can cause the cell wall of probiotics to rupture and die, resulting in a low number of live probiotics in tablet products. In addition, environmental factors such as oxygen and moisture during the shelf life, as well as the low pH and high bile salt environment of the gastrointestinal tract in the subject's body, can have a serious impact on the bacterial activity of probiotics. Therefore, whether the strain can withstand the high pressure in the preparation, various environmental factors during the shelf life, and the complex environment of low pH and high bile salt in the body, and successfully reach the intestine to survive, reproduce and have metabolic activity, is one of the key indicators for testing product quality.

[0005] The viability of probiotics is the key to their biological activity. A sufficient number of probiotics alive and reaching the intestine is one of the basic conditions for probiotics to exert their efficacy. Therefore, the development of a probiotic tablet and its preparation method can ensure the viability of probiotic tablet products, and the friability and water activity of the tablet products meet actual needs, which is of practical significance. Summary of the invention

[0006] In order to overcome the above-mentioned deficiencies of the prior art, the primary purpose of the present invention is to provide a probiotic protection composition, which is used in the preparation of probiotic tablets. The probiotic tablets have a high survival rate of probiotics and meet the requirements of tablet friability and water activity of probiotic products.

[0007] In a first aspect, the present application provides a probiotic protection composition, which includes a probiotic protection agent, a filler, and a lubricant.

[0008] In certain embodiments, the probiotic protectant comprises arabinoxylan.

[0009] In certain embodiments, the arabinoxylan is feruloylarabinoxylan acylated with ferulic acid.

[0010] In certain embodiments, the content of ferulic acid in the feruloylarabinoxylan is ≥ 0.02%; preferably, the content of ferulic acid in the arabinoxylan is ≥ 5.04%.

[0011] In certain embodiments, the probiotic protective agent further comprises resistant dextrin, and / or sodium alginate.

[0012] In certain embodiments, the bulking agent comprises a sugar alcohol.

[0013] In certain embodiments, the sugar alcohol is mannitol, mannitol, sorbitol, and / or xylitol.

[0014] In certain embodiments, the lubricant includes at least one of magnesium stearate, silicon dioxide, and microcrystalline cellulose.

[0015] In certain embodiments, the addition ratio of the probiotic protective agent, filler, and lubricant is (80-120):(344-404):(36-66); preferably, the addition ratio is 100:385:40.

[0016] In a second aspect, the present application provides the use of arabinoxylan in the preparation of a probiotic protection composition, wherein the probiotic protection composition is used to protect probiotics from, and / or reduce the damage to the activity of probiotics caused by adverse factors; the adverse factors include high pressure factors in the tableting of probiotic tablets, strong acid factors in the human stomach, and / or high bile salt factors in the human intestine.

[0017] Those skilled in the art can understand that unfavorable factors, also known as adverse factors, are external factors that affect the activity of probiotics. High pressure factor refers to the pressure applied by the tablet press to the material being pressed during the tableting of probiotic tablets. In order to press the tablets into shape and ensure that the tablets have a certain hardness, a higher pressure needs to be applied to the material; strong acid factor refers to the strongly acidic environment of gastric juice in the human stomach, and its pH value is usually in the range of 0.9-3.5; high bile salt factor refers to the high bile salt environment of intestinal juice in the human small intestine, and its concentration is usually in the range of 0.003%-3%.

[0018] In certain embodiments, the high pressure is 10-30 kN.

[0019] In certain embodiments, the strong acid has a pH of 2.0.

[0020] In certain embodiments, the probiotic protection composition further comprises a filler and a lubricant.

[0021] In certain embodiments, the arabinoxylan is feruloylarabinoxylan acylated with ferulic acid.

[0022] In certain embodiments, the content of ferulic acid in the feruloylarabinoxylan is ≥ 0.02%; preferably, the content of ferulic acid in the arabinoxylan is ≥ 5.04%.

[0023] In certain embodiments, the bulking agent comprises a sugar alcohol.

[0024] In certain embodiments, the sugar alcohol is mannitol, mannitol, sorbitol, and / or xylitol.

[0025] In certain embodiments, the lubricant includes at least one of magnesium stearate, silicon dioxide, and microcrystalline cellulose.

[0026] In certain embodiments, the addition ratio of the probiotic protective agent, filler, and lubricant is (80-120):(344-404):(36-66); preferably, the addition ratio is 100:385:40.

[0027] In a third aspect, the present application provides a probiotic tablet comprising the probiotic protection composition and probiotic powder described in the aforementioned aspects.

[0028] In certain embodiments, the probiotic is selected from Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus mucosus, Lactobacillus plantarum, Lactobacillus unisomeris, Lactobacillus spp., Streptococcus, Lactococcus, Propionibacterium, Propionibacterium, Leuconostoc, Pediococcus, Weizmannella, Zoococcus, Staphylococcus, Akkermansia, Faecalibacterium prausnitzii, or any combination thereof.

[0029] In certain embodiments, the probiotic of the genus Bifidobacterium is selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, or any combination thereof.

[0030] In certain embodiments, the probiotic of the genus Lactobacillus is selected from: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacilus kefiranofaciens subsp. Kefiranofaciens, or any combination thereof.

[0031] In certain embodiments, the probiotic bacteria of the genus Lactobacillus are selected from the group consisting of Lactobacillus casei, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus rhamnosus, or any combination thereof.

[0032] In certain embodiments, the probiotic bacteria of the genus Lactobacillus are selected from: Limosilactobacillus fermentum, Limosilactobacillus reuteri, or a combination thereof.

[0033] In certain embodiments, the probiotic of the genus Lactobacillus is Lactobacillus plantarum.

[0034] In certain embodiments, the probiotic of the genus Lactobacillus is Ligilactobacillus salivarius.

[0035] In certain embodiments, the probiotic Streptococcus is Streptococcus salivarius subsp. thermophilus.

[0036] In certain embodiments, the probiotic bacteria of the genus Propionibacterium is Propionibacterium freudenreichii subsp. shermanii.

[0037] In certain embodiments, the probiotic bacteria of the genus Propionibacterium are Propionibacterium acidipropionici.

[0038] In certain embodiments, the probiotic bacteria of the genus Lactococcus are selected from: Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. Lactis biovar diacetylactis, Lactococcus cermoris, or any combination thereof.

[0039] In certain embodiments, the probiotic bacteria of the genus Akkermansia are selected from the group consisting of Akkermansia muciniphila and Akkermansia glycaniphila, or a combination thereof.

[0040] In certain embodiments, the probiotic of the genus Faecalibacterium prausnitzii is Faecalibacterium prausnitzii.

[0041] In certain embodiments, the addition ratio of the probiotic powder, the probiotic protective agent, the filler, and the lubricant is (50-100): (80-120): (344-404): (36-66); preferably, the addition ratio is 75:100:385:40.

[0042] In a fourth aspect, the present application provides a method for maintaining the activity of probiotics in a tablet, comprising adding the probiotic protection composition described in the above aspects into a probiotic tablet.

[0043] In a fifth aspect, the present application provides a method for preparing a probiotic tablet, the preparation method comprising the following steps:

[0044] a. The components of the probiotic protection composition in the aforementioned aspect are dried separately at 70-90 ℃ to control the moisture content of each component to ≤4%;

[0045] b. After the components of the probiotic protection composition are cooled to room temperature, the components are evenly mixed with the probiotic powder and tableted using a tablet press.

[0046] In certain embodiments, the drying temperature is 85°C.

[0047] In certain embodiments, the moisture content of each component in the probiotic protection composition after drying is ≤2%.

[0048] In certain embodiments, the tabletting pressure is 10-30 kN.

[0049] In certain embodiments, the tableting pressure is 20 KN.

[0050] In certain embodiments, the tablet friability and water activity are checked regularly (eg, every 10,000 tablets), and when the tablet friability is ≤0.5% and the water activity is ≤0.1, it indicates that the tableting is normal.

[0051] Definition of terms

[0052] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0053] As used herein, the term "probiotics" refers to live bacteria that, when taken in appropriate amounts, can have a beneficial effect on the health of the host. The "probiotics" include but are not limited to the strains mentioned in the "List of Bacteria That Can Be Used in Food" and the "List of Bacteria That Can Be Used in Infant Food" issued by the National Health Commission.

[0054] As used herein, the term "Arabinoxylan (AX)" refers to a natural polysaccharide widely found in the bran of cereals such as corn, rice, and wheat. The basic structure of arabinoxylan is a linear skeleton with (1,4)-β-D-pyranose residues and α-L-arabinofuranosyl attached to the side chains. As a dietary fiber, arabinoxylan has been reported by many studies to play a role in host health. The "arabinoxylan" has been approved as a new food raw material by the National Health Commission.

[0055] As used herein, the term "feruloylarabinoxylan" refers to a complex polysaccharide formed by esterification of ferulic acid (FA, a naturally occurring phenolic acid) with arabinoxylan molecules, with ferulic acid being ester-bonded to the arabinosyl side chains. The presence of feruloyl groups enhances the physicochemical properties of arabinoxylan. Arabinoxylan in cereal bran is usually present in the form of feruloylarabinoxylan, but the feruloyl bonds are broken during the alkaline extraction process from cereal skin, resulting in the feruloyl groups in the extracted arabinoxylan being usually less than 1%.

[0056] Advantageous Effects of the Invention

[0057] The present invention uses arabinoxylan in combination with other auxiliary materials to construct a probiotic protection composition. Compared with the prior art, the probiotic protection composition of the present application has at least the following beneficial effects: (1) The present invention adds the probiotic protection composition to the probiotic tablet, and the arabinoxylan in the composition can effectively help the probiotics resist the high pressure in the tableting process, thereby improving the survival rate of the probiotics during the tableting process; (2) The present invention adds the probiotic protection composition to the probiotic tablet, which can control the friability of the tablet to below 0.5%, the water activity to 0.1%, and can significantly improve the stability of the live bacteria, fully ensuring the quality requirements of the number of live bacteria during the shelf life of the product; (3) The present invention adds the probiotic protection composition to the probiotic tablet, and the arabinoxylan in the composition can effectively help the probiotics resist the low pH and high bile salt environment in the human gastrointestinal tract, thereby significantly improving the survival rate of the live bacteria passing through the lower digestive tract of the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The figure shows the survival rate of probiotics in probiotic tablets containing different probiotic protection compositions after tablet compression, where different letters between groups indicate significant differences, P<0.05;

[0059] Figure 2 The results show that probiotic tablets containing different probiotic protection compositions can absorb water and swell in a buffer solution, as well as simulate the digestion and disintegration effects of gastric and intestinal fluids;

[0060] Figure 3 The figure shows the survival rate of probiotics in probiotic tablets containing different probiotic protection compositions after digestion in simulated gastric and intestinal fluids, where different letters between groups indicate significant differences, P<0.05;

[0061] Figure 4 The probiotic survival rate of probiotic tablets containing different probiotic protective compositions after 1, 3, 6, 9 and 12 months of storage simulating the product shelf life is shown. Specific implementation methods

[0063] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0064] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references.

[0065] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0066] Example 1. Preparation of arabinoxylan

[0067] 1. Preparation of Feruloylarabinoxylan (L-AX) with Low Ferulic Acid Content

[0068] Disperse defatted corn bran in distilled water (volume (mL) = corn bran mass (g) ÷ 0.15), add 1.0-3.0 mol / L NaOH to adjust the pH to 6.5-7.5, and keep mechanical stirring in a 100°C water bath for 1 hour to obtain a bran suspension. Disperse and dissolve amylase (enzyme amount is 1.5-3g enzyme / 100g corn bran) in water and centrifuge to obtain an amylase solution. Add the amylase solution to the bran suspension, and then place it in a 90-94°C water bath and mechanically stir for 0.5-1.5h to remove the starch in the bran suspension. After cooling the bran suspension from which the starch has been removed to room temperature, add 1.5-4.0 mol / L HCl to adjust the pH of the bran suspension to 5.5-6.5. Disperse and dissolve protease (enzyme amount is 1-2g enzyme / 100g corn bran) in water and centrifuge to obtain a protease solution. The protease solution is added to the bran suspension with a pH of 5.5 to 6.5, and then placed in a water bath at 45 to 55°C for mechanical stirring for 4 to 7 hours to remove the protein in the bran suspension. Finally, the bran suspension is heated and boiled for 10 to 20 minutes to inactivate the enzyme, centrifuged at 3000-5000 rpm for 10 minutes, the precipitate is dispersed in an oven at 50 to 55°C for drying, crushed, and sieved through a 60-mesh sieve to obtain water-insoluble dietary fiber.

[0069] Take water-insoluble dietary fiber, add 1-1.5mol / L NaOH solution (volume (mL) = 10×water-insoluble dietary fiber mass (g)), stir at 55-60°C for 1-2h. Then, slowly add 30% H2O2 solution (volume (mL) = 0.1-0.2×water-insoluble dietary fiber mass (g)), stir at 60°C for 4-6h, centrifuge at 3000-5000rpm for 10min to obtain supernatant. Wash the precipitate with 1-1.5mol / L NaOH solution twice and centrifuge to obtain supernatant, collect all supernatants and adjust the pH to 6.0-8.0 with 5-6mol / L HCl solution, add 3-5 times volume of ethanol, stir appropriately, let stand at 4°C for 24h, centrifuge at 4700rpm for 10min, dry the precipitate in a 53°C oven to obtain feruloyl arabinoxylan (L-AX) with low feruloyl content, crush and set aside.

[0070] 2. Preparation of feruloylarabinoxylan (M-AX) with medium ferulic acid content

[0071] Dissolve equal amounts of ferulic acid (FA) and N,N′-carbonyldiimidazole (CDI) in a certain amount of dimethyl sulfoxide (DMSO), and stir at 55-65°C for 14-18 hours to activate FA. Dissolve 3-4 times the mass of L-AX in DMSO, and then add the L-AX solution to the activated FA solution to obtain a reaction mixture.

[0072] The obtained reaction mixture solution was kept at 85-93°C for 5-6 hours, cooled to room temperature, and 3-5 times the volume of isopropanol was added and stirred evenly for sufficient alcohol precipitation, and centrifuged at 4800rpm for 10 minutes to obtain a precipitate. The precipitate was washed with isopropanol 2-4 times to remove unreacted FA and CDI, and centrifuged at 4700rpm for 10 minutes. After the precipitate still containing DMSO was redissolved in DMSO, the solution was dialyzed in a 2500-3500Da dialysis bag with flowing distilled water for 3 days to remove all DMSO, and freeze-dried to obtain feruloylarabinoxylan (M-AX) with medium ferulic acid content.

[0073] 3. Preparation of feruloylarabinoxylan (H-AX) with high ferulic acid content

[0074] The preparation method of feruloylarabinoxylan with high ferulic acid content is basically the same as the preparation method of feruloylarabinoxylan with medium ferulic acid content, and the only difference is the added mass ratio of FA, CDI and L-AX. In the preparation of the feruloylarabinoxylan with medium ferulic acid content, the mass ratio of FA:CDI:AX in the reaction mixed solution is 1:1:(3-4), while in the preparation of the feruloylarabinoxylan with high ferulic acid content, the mass ratio of FA:CDI:AX in the reaction mixed solution is 1:1:(1-2). In this way, feruloylarabinoxylan with high ferulic acid content (H-AX) is prepared.

[0075] 4. Detection of ferulic acid content in feruloylarabinoxylan

[0076] Ferulic acid content was determined by high performance liquid chromatography.

[0077] Extraction of total ferulic acid (bound and free forms): weigh 100 mg of sample in a 50 mL centrifuge tube, add 10 mL of 2 mol / L NaOH for hydrolysis, protect from light at room temperature, seal and incubate for two hours after nitrogen filling to release ferulic acid groups. Adjust the pH to 2.0 by adding 6 mol / L HCl solution, then extract FA with three times the volume of ethyl acetate, extract three times in total. Then, combine the ethyl acetate extracts, evaporate to dryness with a vacuum rotary evaporator, add a certain amount of methanol, and obtain a total ferulic acid extract.

[0078] Extraction of free ferulic acid: Weigh 100 mg of sample into a 50 mL centrifuge tube, add 10 mL of distilled water to dissolve, then add 6 mol / L HCl solution to adjust the pH to 2.0, and then extract FA with three times the volume of ethyl acetate, extracting three times in total. Then, combine the ethyl acetate extracts, evaporate to dryness using a vacuum rotary evaporator, and add a certain amount of methanol to obtain a free ferulic acid extract.

[0079] High performance liquid chromatography detection: The extract was filtered through a 0.22 μm organic filter membrane into a sample bottle, and ferulic acid was quantified using a high performance liquid chromatograph (Agilent 1260, USA) equipped with a diode array detector. The chromatographic analysis conditions were: C18 column; mobile phase was 1.0% acetic acid (A) and acetonitrile (B), isocratic elution: 0-13 min, 80% A + 20% B; detection wavelength was 320 nm; column temperature was 20 ° C; flow rate was 0.8 mL / min; injection volume was 5 μL.

[0080] The contents of total ferulic acid and free ferulic acid were calculated by the peak areas corresponding to the gradient concentrations of ferulic acid standards, and the feruloyl content of AX was obtained by subtracting the free FA content from the total FA content.

[0081] The results were: the feruloyl contents of L-AX, M-AX and H-AX were 0.20 mg / g, 50.40 mg / g and 80.75 mg / g, respectively, accounting for 0.02%, 5.04% and 8.08%, respectively.

[0082] Example 2. Screening of probiotic tablet formulations

[0083] 1. Formula composition of probiotic tablets

[0084] Design probiotic tablets according to the addition amount of each component in Table 1 below

[0085] Table 1 Probiotic formula composition and addition amount (weight percentage)

[0086] 1 2 3 4 5 6 7 8 9 10 11 Fungus powder 50 50 50 50 100 75 50 50 75 75 50 L-AX 550 0 0 80 120 100 0 0 30 200 0 M-AX 0 550 0 0 0 0 120 0 0 0 0 H-AX 0 0 550 0 0 0 0 120 0 0 0 Mannitol 0 0 0 404 0 0 364 364 455 285 484 Sorbitol 0 0 0 0 344 0 0 0 0 0 0 Xylitol 0 0 0 0 0 385 0 0 0 0 0 Silicon dioxide 0 0 0 6 6 40 6 6 40 40 0 Microcrystalline Cellulose 0 0 0 54 30 0 60 60 0 0 60 Magnesium Stearate 0 0 0 6 0 0 0 0 0 0 6

[0087] Note: L-AX, M-AX and H-AX were prepared by the applicant according to Example 1, mannitol, xylitol, silicon dioxide, microcrystalline cellulose and magnesium stearate were purchased by the applicant from the market, and the probiotic powder was a mixed powder of 4 probiotic powders, and its components and addition ratios were: Lactobacillus paracasei Lpc-37: Lactobacillus rhamnosus HN001: Lactobacillus gasseri Lg36: Bifidobacterium breve M-16V = 1:1:1:4.

[0088] 2. Preparation method of probiotic tablets

[0089] Each material (except probiotic powder) in each tablet formulation in Table 1 was dried separately at 85°C to control the water content of each component to be less than 2%;

[0090] After the materials are cooled to room temperature, the materials in the formula are mixed evenly and compressed using a tablet press with a compression pressure of 20 kN to obtain probiotic tablets 1-11.

[0091] 3. Friability and water activity test of probiotic tablets

[0092] The tablet friability test method refers to the "Chinese Pharmacopoeia"; the water activity test method refers to GB 5009.238-2016.

[0093] 4. Probiotic activity test after probiotic tablet compression

[0094] Referring to GB4789.35-2023, the viable bacterial counts N1 (CFU) and N2 (CFU) of the bacterial powder before tableting and the tablets after tableting were tested respectively, and the survival rate of probiotics was calculated according to the following formula:

[0095] The survival rate of probiotics after tableting (%) = (N2 / N1) × 100%.

[0096] 5. Shelf life stability test of probiotic tablets

[0097] The probiotic tablets were sealed in aluminum foil bags and stored at 4°C for 12 months. The viable count N3 of the tablets after 12 months of storage was determined according to GB4789.35-2023, and the probiotic survival rate was calculated according to the following formula:

[0098] The survival rate of probiotics after 12 months of simulated shelf life (%) = (N3 / N1) × 100%.

[0099] 6. Test results

[0100] The friability, water activity, survival rate of probiotic tablets after tableting, and survival rate of probiotic tablets after a simulated shelf life of 12 months are shown in Table 2:

[0101] Table 2 Test results

[0102]

[0103] Referring to Table 2, by comparing the survival rate of probiotic tablets 1-8 and 9-11, it can be seen that arabinoxylan can effectively protect probiotics from the huge pressure of tableting and improve the survival rate of probiotics during tableting. The amount of arabinoxylan added will directly affect the survival rate of probiotics during tableting. This may be due to the special skeleton structure of feruloylarabinoxylan, which can resist the huge pressure during tableting and protect the probiotics. By comparing the survival rate of probiotic tablets between tablets 4-8, it can be seen that with the increase of the ferulic acid content in feruloylarabinoxylan, its ability to protect the probiotics in tableting becomes stronger and stronger.

[0104] From the simulated shelf life probiotic survival results in Table 2, it can be seen that after the simulated 12-month shelf life storage of tablets 1-3, the probiotics were less than 1%, indicating that the probiotics survival rate of the probiotic tablets was low after the shelf life storage when only arabinoxylan was used as a tablet excipient. After the simulated 12-month shelf life storage of tablet 10, the probiotics also dropped from 88.72% to 2.58%, indicating that the addition of arabinoxylan will also affect the probiotics tablets during the shelf life. The applicant further tested and analyzed and found that because arabinoxylan is a hygroscopic component, when the tablet contains only one excipient, arabinoxylan, the probiotic tablet is easy to absorb moisture, resulting in the inactivation of probiotics.

[0105] In addition, by comparing the friability test results of tablets 1-11, it can be seen that when the probiotic tablets contain only one auxiliary material, arabinoxylan, or the amount of arabinoxylan added is too high, the friability of the tablet product is very high and cannot meet the requirements of the tablet product. It is necessary to use arabinoxylan in combination with fillers and lubricants.

[0106] Example 3 Comparison of the effects of probiotic tablets with different protective agents

[0107] The prior application CN10973635A disclosed that the auxiliary material composition containing resistant dextrin can protect the probiotics in the tableting process. In addition, literature research found that sodium alginate is also often used as a probiotic protective agent. Therefore, the present invention designs probiotic tablets according to the addition amount of each component in Table 3 below, and studies the protective effect of different protective agents on probiotics.

[0108] 1. Formula composition and preparation of probiotics

[0109] Table 3 Tablet formulation

[0110]

[0111] Probiotic tablets were prepared according to the ingredients and addition amounts in Table 3. The sources and preparation methods of the ingredients in the tablets were the same as those in Example 2.

[0112] The probiotic powder is a composite powder of 15 probiotic powders, and its composition and addition ratio are as follows: Lactobacillus rhamnosus Lr322 8.7%; Bifidobacterium animalis lactis subspecies BL-04 7.3%; Lactobacillus plantarum Lp115 7.3%; Lactobacillus rhamnosus GG 9.6%; Streptococcus salivarius thermophilic subspecies St21 5.9%; Lactobacillus casei Lc-11 7.8%; Lactobacillus paracasei Lpc-37 2.9%; Lactobacillus rhamnosus HN001 2.0%; Bifidobacterium animalis lactis subspecies HN019 2.3%; Lactobacillus gasseri Lg36 2.3%; Lactobacillus delbrueckii subspecies bulgaricus Lb-87 3.7%; Lactobacillus reuteri mucosa 1E1 3.7%; Bifidobacterium longum infantis subspecies M-63 3.4%; Bifidobacterium breve M-16V 7.3% and Bifidobacterium longum subsp. longum BB536 5.7%.

[0113] 2. Test of bacterial activity after probiotic tablet compression

[0114] 2.1 Experimental methods

[0115] The test method for the bacterial activity of probiotics after tableting is the same as that in Example 2.

[0116] 2.2 Experimental Results

[0117] The results showed that sodium alginate, resistant dextrin and arabinoxylan all had a certain degree of pressure protection during the probiotic tableting process. The pressure protection of resistant dextrin was significantly higher than that of sodium alginate, but lower than that of feruloyl arabinoxylan. In addition, the pressure protection of arabinoxylan with different ferulic acid contents was significantly different (P<0.05). With the increase of the degree of substitution of ferulic acid, the pressure protection effect of arabinoxylan on probiotics was better (see Figure 1 ).

[0118] 3. Disintegration test of probiotic tablets in simulated gastrointestinal fluid digestion

[0119] 3.1 Experimental methods

[0120] The probiotic tablets were placed in an appropriate amount of 0.01 mol / L phosphate buffer with a pH of 7.2, and waited for quiescence. The size of the tablets was measured every 15 minutes, and the morphology was photographed with a stereo microscope to determine the maximum swelling time, and the water absorption and swelling characteristics of the probiotic tablets were measured. At the same time, the disintegration characteristics of the probiotic tablets in the human gastrointestinal digestion process were simulated by using gastric juice simulation liquid and intestinal fluid simulation liquid with pH values ​​of 2.0 and 7.2, respectively. The composition of the simulated gastric juice and simulated intestinal fluid is:

[0121] Simulated gastric fluid (500mL): 8.625mL KCl (37.3g / L), 1.125mL KH2PO4 (68g / L), 15.625mL NaHCO3 (84g / L), 14.75mL NaCl (117g / L), 0.5mL MgCl2(H2O)6 (30.5g / L), 0.625mL (NH4)2CO3 (48g / L), 1.625mL HCl (6mol / L), 0.25mL CaCl2(H2O)2 (0.3g / L), 400mg pepsin, 1000mg lipase.

[0122] Simulated intestinal fluid (500mL): 8.5mL KCl (37.3g / L), 1mL KH2PO4 (68g / L), 53.125mL NaHCO3 (84g / L), 12mL NaCl (117g / L), 1.375mL MgCl2(H2O)6 (30.5g / L), 0.875mL HCl (6mol / L), 2mLCaCl2(H2O)2 (0.3g / L), 6250mg pancreatic enzyme, 15mg bile salts.

[0123] 3.2 Experimental Results

[0124] As for the water absorption and swelling properties of tablets, resistant dextrin, sodium alginate and L-AX all reached the maximum swelling at 30 min, M-AX reached the maximum swelling at 60 min, and H-AX still maintained its intact shape at 300 min.

[0125] When the pH conditions of human gastrointestinal digestion are simulated, the resistant dextrin begins to disintegrate in the presence of gastric fluid simulation liquid, and the resistant dextrin disintegrates completely after the addition of intestinal fluid simulation liquid. At the same time, sodium alginate and L-AX also begin to disintegrate during the gastric fluid simulation liquid. Sodium alginate disintegrates rapidly after the addition of intestinal fluid simulation liquid, while L-AX still disintegrates relatively slowly. M-AX did not disintegrate significantly in the presence of gastric fluid simulation liquid, but began to disintegrate in the presence of intestinal fluid simulation liquid, and the degree of disintegration was lower than that of L-AX. H-AX did not disintegrate significantly during the entire simulated digestion stage. This shows that with the increase of the degree of substitution of ferulic acid, the effect of arabinoxylan in resisting disintegration of probiotic tablets during the digestion process is better, and it is more conducive to protecting probiotics (see Figure 2 ).

[0126] 4. Probiotic activity detection after simulated digestion of tablets

[0127] 4.1 Experimental methods

[0128] Place the probiotic tablets in a centrifuge tube, add 0.6mL of oral simulated liquid to simulate for 2 minutes, then add 1.2mL of gastric fluid simulated liquid to simulate for 180 minutes, and finally add 2.4mL of small intestine simulated liquid to simulate for 240 minutes. The simulation stages are all at 37°C and 120rpm. After the simulated digestion is completed, take an appropriate amount of the suspension after the tablets are dispersed, refer to GB4789.35-2023 to detect the number of viable bacteria (N4), and calculate the probiotic survival rate according to the following formula:

[0129] Probiotic survival rate after model digestion (%) = (N4 / N1) × 100%

[0130] 4.2 Experimental Results

[0131] Regarding the survival rate of probiotic tablets in the simulated human digestion process, the resistant dextrin had the lowest survival rate, sodium alginate and L-AX had no significant difference, and H-AX was significantly higher than M-AX (P<0.05). This indicates that as the degree of substitution of ferulic acid increases, the protective effect of arabinoxylan on probiotic tablets during the digestion stage becomes better (see Figure 3 ).

[0132] 5. Detection of probiotic activity after simulated shelf life

[0133] 5.1 Detection Method

[0134] The processing method for simulating the shelf life is the same as that in Example 2, specifically detecting the survival rate of the probiotics in the probiotic tablets stored for 1, 3, 6, 9, and 12 months in the simulated shelf life.

[0135] 5.2 Test results

[0136] Table 4 Probiotic activity during simulated shelf life storage

[0137] 0 month January March June September December Tablet A 69.97％±2.75 58.30％±3.85 27.79％±0.67 18.94％±1.68 4.41％±1.21 0.54％±0.07 Tablet B 54.43％±5.06 48.80％±4.89 32.23％±0.94 27.60％±1.22 21.49％±0.97 13.71％±0.37 Tablet C 77.46％±2.15 61.44％±1.03 32.71％±1.21 24.26％±2.54 19.42％±1.76 13.09％±0.55 Tablet D 88.54％±7.29 78.61％±1.90 72.89％±2.65 59.76％±3.41 52.36％±2.30 40.37％±1.50 Tablet E 97.71％±2.49 86.05％±3.90 81.55％±3.87 68.60％±2.43 57.50％±2.01 44.16％±1.75

[0138] The test results are shown in Table 4 and Figure 4 For the survival rate of probiotic tablets stored at 4°C, it was found that the survival rate of probiotics gradually decreased with the increase of storage time. For the survival rate of probiotics after 12 months of storage, the resistant dextrin had the lowest survival rate, sodium alginate and L-AX had no significant difference, and H-AX was significantly higher than M-AX. This shows that as the degree of substitution of ferulic acid increases, the protective effect of arabinoxylan on probiotics during storage is better.

[0139] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A probiotic protection composition, comprising a probiotic protective agent, a filler, and a lubricant; the probiotic protective agent comprises arabinoxylan.

2. The probiotic protection composition according to claim 1, characterized in that: The arabinoxylan is feruloylarabinoxylan acylated with ferulic acid; preferably, the ferulic acid content in the feruloylarabinoxylan is ≥ 0.02%; further preferably, the ferulic acid content in the feruloylarabinoxylan is ≥ 5.04%.

3. The probiotic protection composition according to claim 1, characterized in that: The probiotic protective agent also includes resistant dextrin and / or sodium alginate.

4. The probiotic protection composition according to claim 1, having one or more of the following characteristics: (1) The filler comprises a sugar alcohol; preferably, the sugar alcohol is mannitol, sorbitol, xylitol and / or mannitol; (2) The lubricant includes at least one of magnesium stearate, silicon dioxide, and microcrystalline cellulose; (3) The added weight fraction ratio of the probiotic protective agent, filler and lubricant is (80-120):(344-404):(36-66); preferably, the added ratio is 100:385:

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5. The use of arabinoxylan in preparing a probiotic protection composition, characterized in that: The probiotic protection composition is used to protect probiotics from, and / or reduce the damage to the activity of probiotics caused by adverse factors; the adverse factors include high pressure factors in the compression of probiotic tablets, moisture factors in the environment, strong acid factors in the human gastrointestinal tract, and / or high bile salt factors in the human intestine.

6. The use according to claim 5, characterized in that: The arabinoxylan is feruloylarabinoxylan acylated with ferulic acid; preferably, the ferulic acid content in the feruloylarabinoxylan is ≥ 0.02%; further preferably, the ferulic acid content in the feruloylarabinoxylan is ≥ 5.04%.

7. A probiotic tablet comprising the probiotic protection composition according to any one of claims 1 to 6 and probiotic powder.

8. The probiotic tablet according to claim 7, characterized in that The probiotics are selected from the group consisting of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus mucosus, Lactobacillus plantarum, Lactobacillus unifying, Lactobacillus radiatus, Streptococcus, Lactococcus, Propionibacterium, Propionibacterium, Leuconostoc, Pediococcus, Weizmannella, Zoococcus, Staphylococcus, Akkermansia, Faecalibacterium prausnitzii, or any combination thereof; Preferably, in certain embodiments, the probiotic of the genus Bifidobacterium is selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, or any combination thereof; Preferably, in certain embodiments, the probiotic bacteria of the genus Lactobacillus are selected from: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacilus kefiranofaciens subsp. Kefiranofaciens, or any combination thereof; Preferably, the probiotic bacteria of the genus Lactobacillus are selected from: Lactobacillus casei, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus rhamnosus, or any combination thereof; Preferably, the probiotic bacteria of the genus Lactobacillus are selected from: Limosilactobacillusfermentum, Limosilactobacillus reuteri, or a combination thereof; Preferably, in certain embodiments, the probiotic bacteria of the genus Lactobacillus is Lactobacillus plantarum; Preferably, the probiotic of the genus Lactobacillus is Lactobacillus salivarius; Preferably, the probiotic bacteria of the genus Streptococcus is Streptococcus salivarius subsp. thermophilus; Preferably, the probiotic bacteria of the genus Propionibacterium is Propionibacterium freudenreichii subsp. shermanii; Preferably, the probiotic bacteria of the genus Propionibacterium are propionibacterium acidipropionici; Preferably, the probiotic bacteria of the genus Lactococcus are selected from: Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. Lactisbiovar diacetylactis, Lactococcus cermoris, or any combination thereof. Preferably, the probiotic bacteria of the genus Akkermansia are selected from the group consisting of Akkermansia muciniphila and Akkermansia glycaniphila, or a combination thereof. Preferably, the probiotic bacteria of the genus Faecalibacterium prausnitzii is Faecalibacterium prausnitzii.

9. The probiotic tablet according to claim 7, characterized in that: The added weight fraction ratio of the probiotic powder, the probiotic protective agent, the filler and the lubricant is (50-100): (80-120): (344-404): (36-66); Preferably, the addition ratio is 75:100:385:

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10. A method for maintaining the activity of probiotics in a probiotic tablet, characterized in that: The probiotic protection composition according to any one of claims 1 to 9 is added to the probiotic tablet.

11. A method for preparing a probiotic tablet, characterized in that: The method comprises the following steps: (a) drying each component of the probiotic protection composition according to any one of claims 1 to 9 separately at 70-90° C. to control the water content of each component to ≤ 4%; (b) after all components in the probiotic protection composition are cooled to room temperature, the probiotic powder and the probiotic protection composition are uniformly mixed, and tabletted using a tabletting device to prepare a probiotic tablet; Preferably, the drying temperature is 85°C; Preferably, the moisture content of each component in the probiotic protection composition after drying is ≤2%; Preferably, the tabletting pressure is 10-30 kN; Preferably, the tablet has a friability of ≤0.5% and a water activity of ≤0.1.

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