Lactic acid bacteria microcapsule and preparation method thereof

Abstract

This invention discloses a lactic acid bacteria microcapsule and its preparation method, relating to the field of lactic acid bacteria microcapsule technology. The preparation method includes the following steps: adding pullulan polysaccharide to a distillers' grains gluten solution for a conjugation reaction, followed by freeze-drying to obtain a conjugated product; adding cell wall powder, the conjugated product, okra polysaccharide, and blackcurrant anthocyanin water extract for ultrasonic dispersion to obtain an inner coating wall material solution; adding lactic acid bacteria sludge to the inner coating wall material solution, stirring evenly, homogenizing, and ultrasonicating to obtain an inner coating emulsion; adding hydroxypropyl methylcellulose phthalate and sodium alginate to deionized water, and adding calcium chloride solution to obtain an outer wall material solution; mixing the inner coating emulsion and the outer wall material solution, homogenizing under high pressure, drying, vacuum sealing, protecting from light, and storing in an environment of 4-5℃ to obtain lactic acid bacteria microcapsules.

Description

Technical Field

[0001] This invention relates to the field of lactic acid bacteria microcapsule technology, specifically to a lactic acid bacteria microcapsule and its preparation method. Background Technology

[0002] Lactic acid bacteria, as common probiotics, are widely used in the food and pharmaceutical industries. However, they are sensitive to gastric acid, oxidative environments, and storage conditions, and are easily inactivated by external stimuli, limiting their practical application effectiveness. Traditional lactic acid bacteria microcapsules often use single-layer wall materials, which are often unstable and prone to aggregation and delamination during storage. They also have weak antioxidant capacity, failing to protect lactic acid bacteria from oxidative damage, and poor resistance to gastric acid, leading to significant loss of lactic acid bacteria in the gastric environment and hindering their ability to reach the intestines to exert their effects. Furthermore, some manufacturing processes may damage the wall material structure or lactic acid bacteria activity due to high temperatures or uneven dispersion, further reducing the product's usability.

[0003] Therefore, inventing a lactic acid bacteria microcapsule is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a lactic acid bacteria microcapsule and its preparation method to solve the problems raised in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for preparing lactic acid bacteria microcapsules includes the following steps: S1: Alkaline protease is added to a 15% w / v Saccharomyces cerevisiae suspension, heated to 50-55℃ and stirred for 20-24h, ultrasonically treated for 30-45min, centrifuged, the precipitate is collected, freeze-dried, and pulverized to obtain cell wall powder.

[0007] Furthermore, the alkaline protease is of the type Alcalase;

[0008] Furthermore, in the preparation of the cell wall powder, the amount of alkaline protease added is 0.03-0.06g of alkaline protease per 100-200mL of 15%w / v Saccharomyces cerevisiae suspension.

[0009] S2: Grind the blackcurrant fruit into powder and add it to deionized water. Heat to 45-46℃ and mix evenly. Place in an environment of 40-41℃ and sonicate for 30-45 minutes. Centrifuge in an environment of 4-5℃ and collect the supernatant to obtain blackcurrant anthocyanin water extract.

[0010] Furthermore, in the preparation process of the blackcurrant anthocyanin water extract, 100-150g of blackcurrant fruit is added to every 300mL of deionized water.

[0011] S3: Add distillers' grains gluten to deionized water, adjust the pH of the system to 9.9-10.1 using 4 mol / L sodium hydroxide solution, stir for 2-3 h, and refrigerate at 4-5℃ for 8-12 h to obtain a distillers' grains gluten solution; add the distillers' grains gluten solution to pullulan, stir at room temperature for 4-5 h, adjust the pH of the system to 9.9-10.1 using 4 mol / L sodium hydroxide solution, heat to 89-91℃ and react for 175-180 min, cool, adjust the pH of the system to neutral using 6 mol / L hydrochloric acid solution, and freeze-dry to obtain the conjugate;

[0012] Furthermore, during the preparation of the conjugate, the concentration of the distillers' grains gluten solution is 9-11 mg / mL, and the mass ratio of pullulan to distillers' grains gluten is 2:1.

[0013] S4: Add cell wall powder, conjugate, and okra polysaccharide to deionized water, heat to 37-38℃ and stir evenly, add blackcurrant anthocyanin water extract, and ultrasonically disperse to obtain inner layer coated wall material liquid.

[0014] Furthermore, in the preparation process of the inner coating wall material liquid, the proportions of each component by mass are as follows: 15-20 parts cell wall powder, 10-20 parts conjugate, 5-10 parts okra polysaccharide, 5-10 parts blackcurrant anthocyanin water extract, and 100-150 parts deionized water.

[0015] S5: Add the lactic acid bacteria mud to the inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain the inner layer coating emulsion.

[0016] Furthermore, the number of live bacteria in the lactic acid bacteria sludge is 1×10⁻⁶. 8 -5×10 9 CFU / g;

[0017] Furthermore, in the preparation process of the inner layer coating emulsion, the mass ratio of lactic acid bacteria mud to inner layer coating wall material liquid is 1:(10-20);

[0018] Furthermore, the inner layer coating emulsion has a particle size of 100-200 nm;

[0019] S6: Add hydroxypropyl methylcellulose phthalate and sodium alginate to deionized water, adjust the pH of the system to 7.9-8.1 with 4 mol / L sodium hydroxide solution, add 0.05 mol / L calcium chloride solution, and stir at 30-31℃ for 30-35 min to obtain the outer wall material solution;

[0020] Furthermore, in the preparation process of the outer wall material solution, the mass ratio of hydroxypropyl methylcellulose phthalate to sodium alginate is (1-2):1;

[0021] S7: Mix the inner layer coating emulsion with the outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store in an environment of 4-5℃ to obtain lactic acid bacteria microcapsules.

[0022] Furthermore, in the preparation process of the lactic acid bacteria microcapsules, the mass ratio of the inner coating emulsion to the outer wall material solution is (5-10):1;

[0023] Furthermore, the drying process employs a fluid bed dryer with the following parameters: inlet air temperature of 30-50℃, outlet air temperature of 30-50℃, flow rate of 25-60mL / min, and time of 15-20min.

[0024] Furthermore, the bulk density of the lactic acid bacteria microcapsules is 0.14-0.16 g / cm³. 3 .

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. This invention constructs a stable ester ether network by forming a conjugate between glutenin from brewer's yeast and pullulan via Maillard reaction; β-glucan from the cell wall of brewer's yeast forms a protective barrier, and okra polysaccharide fills the gaps in the cell wall material; combined with ultrasonic dispersion and high-pressure homogenization to ensure uniform particle size, and freeze-drying to preserve structural integrity, this invention improves the storage stability, mechanical resistance to breakage, and emulsification uniformity of microcapsules, and reduces storage stratification and agglomeration problems.

[0027] 2. This invention directly scavenge free radicals by adding blackcurrant anthocyanins, and synergistically reduces self-oxidative degradation with the conjugate; the conjugate itself also has a certain antioxidant capacity, which together protects lactic acid bacteria from oxidative damage; enhances the overall antioxidant activity of the microcapsule, delays the oxidation of lactic acid bacteria cell membrane, and prolongs the survival period of live bacteria.

[0028] 3. This invention utilizes a multi-layered inner and outer coating of lactic acid bacteria. The outer layer of hydroxypropyl methylcellulose phthalate is insoluble in the gastric acid environment, forming a physical barrier; the inner layer of yeast cell wall, okra polysaccharide, and conjugate slows down the penetration of gastric acid; and the hydroxypropyl methylcellulose phthalate dissolves in the intestinal environment. This ensures the survival of lactic acid bacteria in gastric acid, avoids premature release, and achieves targeted release into the intestine. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1: A method for preparing lactic acid bacteria microcapsules, comprising the following steps: S1: 0.04g alkaline protease is added to 100mL of 15%w / v Saccharomyces cerevisiae suspension, heated to 50℃ and stirred for 20h, sonicated for 30min, centrifuged, the precipitate is collected, freeze-dried, and pulverized to obtain cell wall powder;

[0031] S2: Grind 100g of blackcurrant fruit into powder and add it to 300mL of deionized water. Heat to 45℃ and mix evenly. Place in a 40℃ environment and sonicate for 30min. Centrifuge in a 4℃ environment and collect the supernatant to obtain blackcurrant anthocyanin water extract.

[0032] S3: Add distillers' grains gluten to deionized water, adjust the pH of the system to 10 using 4 mol / L sodium hydroxide solution, stir for 2 h, and refrigerate at 4℃ for 12 h to obtain a 10 mg / mL distillers' grains gluten solution; add the distillers' grains gluten solution to pullulan, stir at room temperature for 4 h, adjust the pH of the system to 10 using 4 mol / L sodium hydroxide solution, heat to 90℃ and react for 180 min, cool, adjust the pH of the system to neutral using 6 mol / L hydrochloric acid solution, freeze dry to obtain the conjugate;

[0033] The mass ratio of pullulan to distillers' grains gluten is 2:1.

[0034] S4: Add 15g cell wall powder, 10g conjugate, and 5g okra polysaccharide to 100g deionized water, heat to 37℃ and stir evenly, add 5g blackcurrant anthocyanin water extract, and ultrasonically disperse to obtain inner layer coating wall material liquid.

[0035] S5: Add 1g of lactic acid bacteria mud to 10g of inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain inner layer coating emulsion.

[0036] S6: Add 1g of hydroxypropyl methylcellulose phthalate and 1g of sodium alginate to 10g of deionized water, adjust the pH of the system to 8 using 4mol / L sodium hydroxide solution, add 5mL of 0.05mol / L calcium chloride solution, and stir at 30℃ for 30min to obtain the outer wall material solution.

[0037] S7: Mix 5g of inner layer coating emulsion with 1g of outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store at 4℃ to obtain lactic acid bacteria microcapsules.

[0038] The drying process uses a fluid bed dryer with the following parameters: inlet air temperature of 30℃, outlet air temperature of 50℃, flow rate of 25mL / min, and time of 15min.

[0039] Example 2: A method for preparing lactic acid bacteria microcapsules, comprising the following steps: S4: 20g cell wall powder, 20g conjugate, and 10g okra polysaccharide are added to 150g of deionized water, heated to 37°C and stirred evenly, 10g blackcurrant anthocyanin water extract is added, and ultrasonically dispersed to obtain an inner layer coating wall material liquid.

[0040] S5: Add 1g of lactic acid bacteria mud to 10g of inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain inner layer coating emulsion.

[0041] S6: Add 1g of hydroxypropyl methylcellulose phthalate and 1g of sodium alginate to 10g of deionized water, adjust the pH of the system to 8 using 4mol / L sodium hydroxide solution, add 5mL of 0.05mol / L calcium chloride solution, and stir at 30℃ for 30min to obtain the outer wall material solution.

[0042] S7: Mix 5g of inner layer coating emulsion with 1g of outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store at 4℃ to obtain lactic acid bacteria microcapsules.

[0043] The remaining steps are the same as in Example 1.

[0044] Example 3: A method for preparing lactic acid bacteria microcapsules, comprising the following steps: S4: 15g cell wall powder, 10g conjugate, and 5g okra polysaccharide are added to 100g deionized water, heated to 37°C and stirred evenly, 5g blackcurrant anthocyanin water extract is added, and ultrasonically dispersed to obtain an inner layer coating wall material liquid.

[0045] S5: Add 1g of lactic acid bacteria mud to 20g of inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain inner layer coating emulsion.

[0046] S6: Add 1g of hydroxypropyl methylcellulose phthalate and 1g of sodium alginate to 10g of deionized water, adjust the pH of the system to 8 using 4mol / L sodium hydroxide solution, add 5mL of 0.05mol / L calcium chloride solution, and stir at 30℃ for 30min to obtain the outer wall material solution.

[0047] S7: Mix 5g of inner layer coating emulsion with 1g of outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store at 4℃ to obtain lactic acid bacteria microcapsules.

[0048] The remaining steps are the same as in Example 1.

[0049] Example 4: A method for preparing lactic acid bacteria microcapsules, comprising the following steps: S4: 15g cell wall powder, 10g conjugate, and 5g okra polysaccharide are added to 100g deionized water, heated to 37°C and stirred evenly, 5g blackcurrant anthocyanin water extract is added, and ultrasonically dispersed to obtain an inner layer coating wall material liquid.

[0050] S5: Add 1g of lactic acid bacteria mud to 10g of inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain inner layer coating emulsion.

[0051] S6: Add 2g of hydroxypropyl methylcellulose phthalate and 1g of sodium alginate to 10g of deionized water, adjust the pH of the system to 8 using 4mol / L sodium hydroxide solution, add 5mL of 0.05mol / L calcium chloride solution, and stir at 30℃ for 30min to obtain the outer wall material solution.

[0052] S7: Mix 5g of inner layer coating emulsion with 1g of outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store at 4℃ to obtain lactic acid bacteria microcapsules.

[0053] The remaining steps are the same as in Example 1.

[0054] Example 5: A method for preparing lactic acid bacteria microcapsules, comprising the following steps: S4: 15g cell wall powder, 10g conjugate, and 5g okra polysaccharide are added to 100g deionized water, heated to 37°C and stirred evenly, 5g blackcurrant anthocyanin water extract is added, and ultrasonically dispersed to obtain an inner layer coating wall material liquid.

[0055] S5: Add 1g of lactic acid bacteria mud to 10g of inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain inner layer coating emulsion.

[0056] S6: Add 1g of hydroxypropyl methylcellulose phthalate and 1g of sodium alginate to 10g of deionized water, adjust the pH of the system to 8 using 4mol / L sodium hydroxide solution, add 5mL of 0.05mol / L calcium chloride solution, and stir at 30℃ for 30min to obtain the outer wall material solution.

[0057] S7: Mix 10g of inner layer coating emulsion with 1g of outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store at 4℃ to obtain lactic acid bacteria microcapsules.

[0058] The remaining steps are the same as in Example 1.

[0059] Comparative Example 1: A method for preparing lactic acid bacteria microcapsules, comprising the following steps: S1: 0.04 g alkaline protease is added to 100 mL of 15% w / v Saccharomyces cerevisiae suspension, heated to 50 °C and stirred for 20 h, sonicated for 30 min, centrifuged, the precipitate is collected, freeze-dried, and pulverized to obtain cell wall powder;

[0060] S2: Grind 100g of blackcurrant fruit into powder and add it to 300mL of deionized water. Heat to 45℃ and mix evenly. Place in a 40℃ environment and sonicate for 30min. Centrifuge in a 4℃ environment and collect the supernatant to obtain blackcurrant anthocyanin water extract.

[0061] S3: Add distillers' grains gluten to deionized water, adjust the pH of the system to 10 using 4 mol / L sodium hydroxide solution, stir for 2 h, and refrigerate at 4℃ for 12 h to obtain a 10 mg / mL distillers' grains gluten solution; add the distillers' grains gluten solution to pullulan, stir at room temperature for 4 h, adjust the pH of the system to 10 using 4 mol / L sodium hydroxide solution, heat to 90℃ and react for 180 min, cool, adjust the pH of the system to neutral using 6 mol / L hydrochloric acid solution, freeze dry to obtain the conjugate;

[0062] The mass ratio of pullulan to distillers' grains gluten was 0.5:1.

[0063] S4: Add 15g cell wall powder, 10g conjugate, and 5g okra polysaccharide to 100g deionized water, heat to 37℃ and stir evenly, add 5g blackcurrant anthocyanin water extract, and ultrasonically disperse to obtain inner layer coating wall material liquid.

[0064] S5: Add 1g of lactic acid bacteria mud to 10g of inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain inner layer coating emulsion.

[0065] S6: Add 1g of hydroxypropyl methylcellulose phthalate and 1g of sodium alginate to 10g of deionized water, adjust the pH of the system to 8 using 4mol / L sodium hydroxide solution, add 5mL of 0.05mol / L calcium chloride solution, and stir at 30℃ for 30min to obtain the outer wall material solution.

[0066] S7: Mix 5g of inner layer coating emulsion with 1g of outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store at 4℃ to obtain lactic acid bacteria microcapsules.

[0067] The drying process uses a fluid bed dryer with the following parameters: inlet air temperature of 30℃, outlet air temperature of 50℃, flow rate of 25mL / min, and time of 15min.

[0068] Comparative Example 2: A method for preparing lactic acid bacteria microcapsules, comprising the following steps: S1: 0.04 g alkaline protease is added to 100 mL of 15% w / v Saccharomyces cerevisiae suspension, heated to 50 °C and stirred for 20 h, sonicated for 30 min, centrifuged, the precipitate is collected, freeze-dried, and pulverized to obtain cell wall powder.

[0069] S2: Grind 100g of blackcurrant fruit into powder and add it to 300mL of deionized water. Heat to 45℃ and mix evenly. Place in a 40℃ environment and sonicate for 30min. Centrifuge in a 4℃ environment and collect the supernatant to obtain blackcurrant anthocyanin water extract.

[0070] S3: Add distillers' grains gluten to deionized water, adjust the pH of the system to 10 using 4 mol / L sodium hydroxide solution, stir for 2 h, and refrigerate at 4℃ for 12 h to obtain a 10 mg / mL distillers' grains gluten solution; add the distillers' grains gluten solution to pullulan, stir at room temperature for 4 h, adjust the pH of the system to 10 using 4 mol / L sodium hydroxide solution, heat to 90℃ and react for 180 min, cool, adjust the pH of the system to neutral using 6 mol / L hydrochloric acid solution, freeze dry to obtain the conjugate;

[0071] The mass ratio of pullulan to distillers' grains gluten is 1:1.

[0072] S4: Add 15g cell wall powder, 10g conjugate, and 5g okra polysaccharide to 100g deionized water, heat to 37℃ and stir evenly, add 5g blackcurrant anthocyanin water extract, and ultrasonically disperse to obtain inner layer coating wall material liquid.

[0073] S5: Add 1g of lactic acid bacteria mud to 10g of inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain inner layer coating emulsion.

[0074] S6: Add 1g of hydroxypropyl methylcellulose phthalate and 1g of sodium alginate to 10g of deionized water, adjust the pH of the system to 8 using 4mol / L sodium hydroxide solution, add 5mL of 0.05mol / L calcium chloride solution, and stir at 30℃ for 30min to obtain the outer wall material solution.

[0075] S7: Mix 5g of inner layer coating emulsion with 1g of outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store at 4℃ to obtain lactic acid bacteria microcapsules.

[0076] The drying process uses a fluid bed dryer with the following parameters: inlet air temperature of 30℃, outlet air temperature of 50℃, flow rate of 25mL / min, and time of 15min.

[0077] Comparative Example 3: A method for preparing lactic acid bacteria microcapsules, comprising the following steps: S1: 0.04 g alkaline protease is added to 100 mL of 15% w / v Saccharomyces cerevisiae suspension, heated to 50 °C and stirred for 20 h, sonicated for 30 min, centrifuged, the precipitate is collected, freeze-dried, and pulverized to obtain cell wall powder;

[0078] S2: Grind 100g of blackcurrant fruit into powder and add it to 300mL of deionized water. Heat to 45℃ and mix evenly. Place in a 40℃ environment and sonicate for 30min. Centrifuge in a 4℃ environment and collect the supernatant to obtain blackcurrant anthocyanin water extract.

[0079] S3: Add distillers' grains gluten to deionized water, adjust the pH of the system to 10 using 4 mol / L sodium hydroxide solution, stir for 2 h, and refrigerate at 4℃ for 12 h to obtain a 10 mg / mL distillers' grains gluten solution; add the distillers' grains gluten solution to pullulan, stir at room temperature for 4 h, adjust the pH of the system to 10 using 4 mol / L sodium hydroxide solution, heat to 90℃ and react for 180 min, cool, adjust the pH of the system to neutral using 6 mol / L hydrochloric acid solution, freeze dry to obtain the conjugate;

[0080] The mass ratio of pullulan to distillers' grains gluten is 4:1.

[0081] S4: Add 15g cell wall powder, 10g conjugate, and 5g okra polysaccharide to 100g deionized water, heat to 37℃ and stir evenly, add 5g blackcurrant anthocyanin water extract, and ultrasonically disperse to obtain inner layer coating wall material liquid.

[0082] S5: Add 1g of lactic acid bacteria mud to 10g of inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain inner layer coating emulsion.

[0083] S6: Add 1g of hydroxypropyl methylcellulose phthalate and 1g of sodium alginate to 10g of deionized water, adjust the pH of the system to 8 using 4mol / L sodium hydroxide solution, add 5mL of 0.05mol / L calcium chloride solution, and stir at 30℃ for 30min to obtain the outer wall material solution.

[0084] S7: Mix 5g of inner layer coating emulsion with 1g of outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store at 4℃ to obtain lactic acid bacteria microcapsules.

[0085] The drying process uses a fluid bed dryer with the following parameters: inlet air temperature of 30℃, outlet air temperature of 50℃, flow rate of 25mL / min, and time of 15min.

[0086] Comparative Example 4: A method for preparing lactic acid bacteria microcapsules, comprising the following steps: S1: Grind 100g of blackcurrant fruit into powder and add it to 300mL of deionized water, heat to 45℃ and mix evenly, place in an environment of 40℃ and sonicate for 30min, place in an environment of 4℃ and centrifuge, collect the supernatant to obtain blackcurrant anthocyanin water extract.

[0087] S2: Add distillers' grains gluten to deionized water, adjust the pH of the system to 10 using 4 mol / L sodium hydroxide solution, stir for 2 h, and refrigerate at 4℃ for 12 h to obtain a 10 mg / mL distillers' grains gluten solution; add the distillers' grains gluten solution to pullulan, stir at room temperature for 4 h, adjust the pH of the system to 10 using 4 mol / L sodium hydroxide solution, heat to 90℃ and react for 180 min, cool, adjust the pH of the system to neutral using 6 mol / L hydrochloric acid solution, freeze dry to obtain the conjugate;

[0088] The mass ratio of pullulan to distillers' grains gluten was 0.5:1.

[0089] S3: Add 10g of conjugate and 5g of okra polysaccharide to 100g of deionized water, heat to 37℃ and stir evenly, add 5g of blackcurrant anthocyanin water extract, and ultrasonically disperse to obtain the inner layer coating wall material liquid.

[0090] S4: Add 1g of lactic acid bacteria mud to 10g of inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain inner layer coating emulsion.

[0091] S5: Add 1g of hydroxypropyl methylcellulose phthalate and 1g of sodium alginate to 10g of deionized water, adjust the pH of the system to 8 using 4mol / L sodium hydroxide solution, add 5mL of 0.05mol / L calcium chloride solution, and stir at 30℃ for 30min to obtain the outer wall material solution.

[0092] S6: Mix 5g of inner layer coating emulsion with 1g of outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store at 4℃ to obtain lactic acid bacteria microcapsules.

[0093] The drying process uses a fluid bed dryer with the following parameters: inlet air temperature of 30℃, outlet air temperature of 50℃, flow rate of 25mL / min, and time of 15min.

[0094] Comparative Example 5: A method for preparing lactic acid bacteria microcapsules, comprising the following steps: S1: 0.04 g alkaline protease is added to 100 mL of 15% w / v Saccharomyces cerevisiae suspension, heated to 50 °C and stirred for 20 h, sonicated for 30 min, centrifuged, the precipitate is collected, freeze-dried, and pulverized to obtain cell wall powder;

[0095] S2: Grind 100g of blackcurrant fruit into powder and add it to 300mL of deionized water. Heat to 45℃ and mix evenly. Place in a 40℃ environment and sonicate for 30min. Centrifuge in a 4℃ environment and collect the supernatant to obtain blackcurrant anthocyanin water extract.

[0096] S3: Add 15g cell wall powder and 5g okra polysaccharide to 100g deionized water, heat to 37℃ and stir evenly, add 5g blackcurrant anthocyanin water extract, and ultrasonically disperse to obtain inner layer coating wall material liquid.

[0097] S4: Add 1g of lactic acid bacteria mud to 10g of inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain inner layer coating emulsion.

[0098] S5: Add 1g of hydroxypropyl methylcellulose phthalate and 1g of sodium alginate to 10g of deionized water, adjust the pH of the system to 8 using 4mol / L sodium hydroxide solution, add 5mL of 0.05mol / L calcium chloride solution, and stir at 30℃ for 30min to obtain the outer wall material solution.

[0099] S6: Mix 5g of inner layer coating emulsion with 1g of outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store at 4℃ to obtain lactic acid bacteria microcapsules.

[0100] The drying process uses a fluid bed dryer with the following parameters: inlet air temperature of 30℃, outlet air temperature of 50℃, flow rate of 25mL / min, and time of 15min.

[0101] Performance testing: Antioxidant test: ABTS / DPPH scavenging rate: After the sample was extracted with DMSO, it was reacted with ABTS working solution at 37℃ for 6 min and DPPH ethanol solution at room temperature in the dark for 30 min. The absorbance at 734nm / 517nm was measured and the scavenging rate was calculated according to the formula.

[0102] Gastrointestinal fluid resistance test: Gastric fluid resistance: The sample was incubated with gastric fluid containing pepsin at simulated pH 2.0 for 2 hours at 37°C. After neutralization, the sample was serially diluted, plated on MRS agar, and the survival rate was calculated.

[0103] Intestinal fluid release: Samples treated with gastric fluid were incubated with simulated intestinal fluid containing pancreatic enzymes / bile salts at pH 7.2 for 4 hours at 37°C. Samples were taken periodically and counted to calculate the cumulative release rate.

[0104] The experimental results are shown in Table 1 below.

[0105] Table 1. Test data for lactic acid bacteria microcapsules

[0106]

[0107] Conclusion: The lactic acid bacteria microcapsules prepared in this application have excellent antioxidant properties and gastric acid resistance.

[0108] Comparative Example 1: Insufficient polysaccharides resulted in low grafting degree and low antioxidant activity;

[0109] Comparative Example 2 showed lower grafting degree and emulsification stability than Example 1, resulting in reduced antioxidant activity and gastric acid resistance.

[0110] In Comparative Example 3, excessive polysaccharides resulted in insufficient interfacial coverage, reduced solubility, and high viscosity aggregation, leading to decreased antioxidant activity and gastric acid resistance.

[0111] Comparative Example 4 lacked the inner wall protection of cell wall powder, resulting in reduced resistance to gastric acid.

[0112] Comparative Example 5 lacks the conjugate and the ester ether network formed by the Mandela reaction, resulting in a significant reduction in performance.

[0113] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing lactic acid bacteria microcapsules, characterized in that: Includes the following steps: S1: Add alkaline protease to a 15-20% w / v Saccharomyces cerevisiae suspension, heat to 50-55℃ and stir for 20-24h, sonicate for 30-45min, centrifuge, collect the precipitate, freeze dry, and pulverize to obtain cell wall powder. S2: Grind the blackcurrant fruit into powder and add it to deionized water. Heat to 45-46℃ and mix evenly. Place in an environment of 40-41℃ and sonicate for 30-45 minutes. Centrifuge in an environment of 4-5℃ and collect the supernatant to obtain blackcurrant anthocyanin water extract. S3: Add distillers' grains gluten to deionized water, adjust the pH of the system to 9.9-10.1, stir for 2-3 hours, and refrigerate at 4-5℃ for 8-12 hours to obtain a distillers' grains gluten solution; add the distillers' grains gluten solution to pullulan, stir at room temperature for 4-5 hours, adjust the pH of the system to 9.9-10.1, heat to 89-91℃ and react for 175-180 minutes, cool, adjust the pH of the system to neutral, and freeze-dry to obtain the conjugate; S4: Add cell wall powder, conjugate, and okra polysaccharide to deionized water, heat to 37-38℃ and stir evenly, add blackcurrant anthocyanin water extract, and ultrasonically disperse to obtain inner layer coated wall material liquid. S5: Add the lactic acid bacteria mud to the inner layer coating wall material liquid, stir evenly, homogenize, and sonicate to obtain the inner layer coating emulsion. S6: Add hydroxypropyl methylcellulose phthalate and sodium alginate to deionized water, adjust the pH of the system to 7.9-8.1, add 0.05-0.06 mol / L calcium chloride solution, and stir in an environment of 30-31℃ for 30-35 min to obtain the outer wall material solution; S7: Mix the inner layer coating emulsion with the outer layer wall material solution, homogenize under high pressure, dry, vacuum seal, protect from light, and store in an environment of 4-5℃ to obtain lactic acid bacteria microcapsules; During the preparation of the conjugate, the concentration of the distillers' grains gluten solution was 9-11 mg / mL, and the mass ratio of pullulan to distillers' grains gluten was 2:

1. In the preparation of the inner coating wall material liquid, the proportions of each component by mass are as follows: 15-20 parts cell wall powder, 10-20 parts conjugate, 5-10 parts okra polysaccharide, 5-10 parts blackcurrant anthocyanin water extract, and 100-150 parts deionized water.

2. The method for preparing lactic acid bacteria microcapsules according to claim 1, characterized in that: In the preparation of cell wall powder, the amount of alkaline protease added is 0.03-0.06g of alkaline protease per 100-200mL of 15%w / v Saccharomyces cerevisiae suspension.

3. The method for preparing lactic acid bacteria microcapsules according to claim 1, characterized in that: In the preparation of blackcurrant anthocyanin water extract, 100-150g of blackcurrant fruit is added to every 300mL of deionized water.

4. The method for preparing lactic acid bacteria microcapsules according to claim 1, characterized in that: In the preparation of the inner coating emulsion, the mass ratio of lactic acid bacteria mud to inner coating wall material liquid is 1:(10-20).

5. The method for preparing lactic acid bacteria microcapsules according to claim 1, characterized in that: In the preparation of the outer wall material solution, the mass ratio of hydroxypropyl methylcellulose phthalate to sodium alginate is (1-2):

1.

6. The method for preparing lactic acid bacteria microcapsules according to claim 1, characterized in that: In the preparation of lactic acid bacteria microcapsules, the mass ratio of inner coating emulsion to outer wall material solution is (5-10):

1.

7. Lactic acid bacteria microcapsules prepared by the method for preparing lactic acid bacteria microcapsules according to any one of claims 1-6.

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