Lactobacillus plantarum microcapsule with high folate production and preparation method thereof

Abstract

The application belongs to the field of food biotechnology, and particularly relates to a high-folate Lactobacillus plantarum ZFM4 microcapsule and a preparation method thereof. The method comprises the following steps: centrifuging and resuspending a fermentation liquor of high-folate Lactobacillus plantarum ZFM4 to obtain concentrated bacterial liquid; uniformly mixing the concentrated bacterial liquid with a composite wall material and then adding the mixture into a calcium chloride solution to obtain wet Lactobacillus plantarum single-layer microcapsules; the composite wall material is composed of sodium alginate, cassava starch and water; and wet double-layer Lactobacillus plantarum microcapsules are obtained by adding the wet Lactobacillus plantarum single-layer microcapsules into a chitosan solution. The high-folate Lactobacillus plantarum microcapsule prepared by the method has good acid resistance, high survival rate of lactic acid bacteria and high embedding rate, and has a high application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food biotechnology, and particularly relates to a high-folate-producing Lactobacillus plantarum ZFM4 microcapsule and a preparation method thereof. BACKGROUND

[0002] Natural folate exists mainly in the form of tetrahydrofolate, 5-methyltetrahydrofolate and 10-formyltetrahydrofolate, and is polyglutamylated. Bacterial folate synthesis genes are ubiquitous in 512 gastrointestinal reference genomes, of which 13% contain all the genes required for complete de novo synthesis of folate, and another 39% have the ability to synthesize folate in the presence of para-aminobenzoic acid (pABA). It is expected that lactic acid bacteria generally cannot produce folate without pABA. Lactobacillus plantarum is an exception among lactobacilli, as it is capable of high-yield folate production in the presence of para-aminobenzoic acid (pABA). The increased expression of key folate synthesis genes is evident during the exponential phase, and the increased folate polyglutamylation occurs in the late stationary phase. Lactic acid bacteria can maintain intestinal microecological balance by producing active substances to inhibit the growth of pathogenic bacteria, improve the intestinal barrier, and regulate the immune response of the body.

[0003] Most of the current lactic acid bacteria have poor tolerance to the gastrointestinal environment of the human body, and only a small part can reach the human intestinal tract and remain active. Microencapsulation is a common method currently used to improve the tolerance of lactic acid bacteria to adverse environments, and can enable a sufficient number of live bacteria to reach the intestine to exert their probiotic effects. Common microencapsulation methods, such as spray drying and emulsification, have poor protective ability for lactic acid bacteria, while sharp-hole granulation has mild preparation conditions and high embedding rate, and is widely used in the microencapsulation preparation of lactic acid bacteria. It can also be combined with freeze-drying to further improve the embedding stability and the activity of lactic acid bacteria.

[0004] Traditional microcapsule technology has certain shortcomings in the application of the food field, as follows:

[0005] Microcapsules prepared by emulsification often have residual vegetable oil on the surface of the capsules, which affects their sensory properties, and the emulsifiers and surfactants essential in the preparation process may be toxic to lactic acid bacteria cells, affecting the strain and the human body.

[0006] Spray drying has low operation difficulty and high production efficiency, and is suitable for industrial production, but most lactic acid bacteria do not have tolerance to high temperature, and high inlet air temperature and outlet air temperature of spray drying will significantly reduce the survival rate of lactic acid bacteria;

[0007] Complex coacervation is simple and fast to prepare, but the scope of application is too narrow, and microcapsule adhesion easily occurs during the preparation process, resulting in different microcapsule morphologies and poor protection performance;

[0008] The orifice granulation method is widely used in the embedding of lactic acid bacteria microcapsules due to low cost and strong protection performance of the prepared microcapsules, but has the disadvantage of difficult large-scale production.

[0009] The application of CN114774309A provides a lactobacillus plantarum microcapsule, characterized in that it comprises a lactobacillus plantarum core material and a composite wall material, the composite wall material comprises soybean protein isolate and sodium alginate. The mass percentage concentration of soybean protein isolate is 2%-5%, and the mass percentage concentration of sodium alginate is 1%-4%. The volume ratio of soybean protein isolate and sodium alginate is 4-1:1.

[0010] The application of CN113826904A provides a high-activity probiotic microcapsule and a preparation method thereof. The probiotic microcapsule comprises a core material solution and a wall material solution. The core material solution is composed of probiotic lactobacillus plantarum Grx16 and a freeze-dried protective agent aqueous solution. The wall material solution is prepared from a natural high molecular material solution. The freeze-dried protective agent aqueous solution is composed of sucrose, inulin, and skimmed milk. The natural high molecular material solution comprises sodium alginate and whey protein isolate. SUMMARY

[0011] The technical problem to be solved by the present application is to provide a high-folate-producing lactobacillus plantarum microcapsule, which is embedded with lactobacillus plantarum by a composite wall material to improve the tolerance of the microcapsule to external adverse environments.

[0012] To solve the above technical problems, the present application provides a preparation method of high-folate-producing lactobacillus plantarum ZFM4 microcapsule, comprising the following steps:

[0013] 1) Centrifuge and resuspend the obtained fermentation broth of high-folate-producing lactobacillus plantarum ZFM4 to obtain concentrated bacterial solution. The bacterial content of the concentrated bacterial solution is (2.5±0.5)×10 9 CFU / ml;

[0014] The preservation information of the lactobacillus plantarum ZFM4 is as follows:

[0015] Preservation name (taxonomic name): lactobacillus plantarum ZFM4, preservation unit: China Center for Type Culture Collection, preservation address: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M2016629, preservation date: November 10, 2016;

[0016] 2) mixing the concentrated bacterial solution with the complex wall material to form a mixed solution (mixing time is about 10±5 minutes), under stirring, the mixed solution is added into the calcium chloride solution (added by syringe and the flow rate is controlled, the adding time is controlled in 15±5 minutes), continue stirring until no solid is produced (the total time from the beginning of adding the mixed solution to stopping stirring is about 60±10 minutes), to obtain the wet Lactobacillus plantarum single-layer microcapsules;

[0017] The complex wall material is composed of sodium alginate, cassava starch and water; in 100ml of the complex wall material, there are 1.50±0.1g of sodium alginate and 0.80±0.1g of cassava starch;

[0018] The volume ratio of the complex wall material to the concentrated bacterial solution is (3.5±0.1):1;

[0019] The concentration of the calcium chloride solution is (0.2±0.02)g / 100ml, and the volume ratio of the calcium chloride solution to the mixed solution is (1±0.1):1.

[0020] As an improvement of the preparation method of the high-folate-producing Lactobacillus plantarum ZFM4 microcapsules of the present application, the step 3) further comprises the following steps:

[0021] First, the chitosan powder with high deacetylation degree (deacetylation degree ≥75%, preferably deacetylation degree ≥90%) is dissolved in an ice acetic acid solution with a volume concentration of 0.5-1.5% (preferably 1%), to obtain a chitosan solution with a chitosan concentration of (0.9±0.1)g / 100ml;

[0022] Under stirring, the wet Lactobacillus plantarum single-layer microcapsules obtained in step 2) are slowly added (slowly adding time is about 15±5 minutes) into the chitosan solution, stirring until no solid is produced (the total time from the beginning of adding the wet Lactobacillus plantarum single-layer microcapsules to stopping stirring is about 60±10 minutes), and the obtained product is collected, which is the wet double-layer Lactobacillus plantarum microcapsules; the volume ratio of the chitosan solution to the wet Lactobacillus plantarum single-layer microcapsules is (1±0.1):1.

[0023] As a further improvement of the preparation method of the high-folate-producing Lactobacillus plantarum ZFM4 microcapsules of the present application, the wet microcapsules are freeze-dried under the protection of a freeze-drying protective agent;

[0024] The wet microcapsules are the wet Lactobacillus plantarum single-layer microcapsules obtained in step 2) or the wet double-layer Lactobacillus plantarum microcapsules obtained in step 3);

[0025] The freeze-drying is: after adding freeze-drying protective agent in the wet state microcapsule, quick freezing (quick freezing at -80±10℃), freeze-drying (freeze-drying at -80±10℃ to constant weight), obtaining the Lactobacillus plantarum dry single-layer microcapsule / Lactobacillus plantarum dry double-layer microcapsule;

[0026] The freeze-drying protective agent is composed of trehalose, skimmed milk powder, sorbitol and mannitol, wherein the trehalose is (6±0.5) % of the weight of the wet state microcapsule, the skimmed milk powder is (3±0.3) % of the weight of the wet state microcapsule, the sorbitol concentration is (2±0.2) % of the weight of the wet state microcapsule, and the mannitol is (3±0.3) % of the weight of the wet state microcapsule.

[0027] As a further improvement of the preparation method of the high-folate Lactobacillus plantarum ZFM4 microcapsule of the present application, the step 1) is:

[0028] The Lactobacillus plantarum ZFM4 (frozen preserved Lactobacillus plantarum ZFM4) is inoculated and activated for fermentation culture under the condition of 37±1℃ and 160±20r / min for 36±1h, and a fermentation broth is obtained.

[0029] The fermentation broth is centrifuged at 8000±1000rpm and 4±0.5℃ for 8±2min, the supernatant is removed, then the bacterial slurry is washed with normal saline (2-4 times), and finally the bacterial body is resuspended with sterile normal saline to obtain a concentrated bacterial solution.

[0030] As a further improvement of the preparation method of the high-folate Lactobacillus plantarum ZFM4 microcapsule of the present application, the step 1) is: inoculating the Lactobacillus plantarum ZFM4 (frozen preserved Lactobacillus plantarum ZFM4) into MRS liquid medium for activation and fermentation culture at an inoculation amount of 1.5% (volume %).

[0031] As a further improvement of the preparation method of the high-folate Lactobacillus plantarum ZFM4 microcapsule of the present application:

[0032] The stirring speed of step 2) is 130±30r / min,

[0033] The stirring speed of step 3) is 600±50r / min.

[0034] The present application also simultaneously provides the high-folate Lactobacillus plantarum ZFM4 microcapsule prepared by any of the above methods, which is any of the following: wet state Lactobacillus plantarum single-layer microcapsule, wet state double-layer Lactobacillus plantarum microcapsule, Lactobacillus plantarum dry single-layer microcapsule, and Lactobacillus plantarum dry double-layer microcapsule.

[0035] The Lactobacillus plantarum ZFM4 used in the present application has a high folate yield of up to 10.31ng / mL.

[0036] The application uses sodium alginate and cassava starch as a compound wall material, and finds that the survival rate of the microcapsules obtained by embedding is significantly improved in simulated saliva, gastric juice and pancreatic juice, and the microcapsules can be well released in simulated intestinal juice, and the survival rate of the embedded high-folate Lactobacillus plantarum ZFM4 can still reach 90.15% and 88.21% after 2h of simulated gastrointestinal juice digestion. The microcapsules prepared by the method can effectively improve the various tolerances of Lactobacillus plantarum ZFM4, and can provide theoretical support for the microencapsulation of lactic acid bacteria.

[0037] The high-folate Lactobacillus plantarum microcapsules prepared by the method have good acid resistance, high survival rate of lactic acid bacteria and high embedding rate, and have a high application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0038] The specific embodiments of the application will be further described in detail below with reference to the accompanying drawings.

[0039] Figure 1 Effect of sodium alginate concentration on the embedding rate and viable count of high-folate Lactobacillus plantarum ZFM4 microcapsules;

[0040] Figure 2 Effect of cassava starch concentration on the embedding rate and viable count of high-folate Lactobacillus plantarum ZFM4 microcapsules;

[0041] Figure 3 Effect of calcium chloride concentration on the embedding rate and viable count of high-folate Lactobacillus plantarum ZFM4 microcapsules;

[0042] Figure 4 Effect of chitosan concentration on the embedding rate and viable count of high-folate Lactobacillus plantarum ZFM4 microcapsules;

[0043] Figure 5 Survival rate of high-folate Lactobacillus plantarum ZFM4 single-layer and double-layer microcapsules and bacterial suspension at different storage temperatures;

[0044] Figure 6 Microcapsule survival rate of high-folate Lactobacillus plantarum ZFM4 single-layer and double-layer microcapsules and bacterial suspension in simulated saliva;

[0045] Figure 7 Viable count survival rate of high-folate Lactobacillus plantarum ZFM4 microcapsules in simulated gastric juice;

[0046] Figure 8 Viable count survival rate of high-folate Lactobacillus plantarum ZFM4 microcapsules in simulated intestinal juice;

[0047] Figure 9 Release of high-folate Lactobacillus plantarum ZFM4 microcapsules in simulated intestinal juice;

[0048] Figure 10 Figure 1 is the storage stability result of the high folate-producing Lactobacillus plantarum ZFM4 microcapsule of the present application; Figure A is at -20℃, Figure B is at 4℃, and Figure C is at 25℃;

[0049] Figure 11 Figure 2 is the morphological diagram of the four microcapsules;

[0050] A, wet single-layer microcapsule (wet microcapsule of Lactobacillus plantarum single-layer microcapsule); B, dry single-layer microcapsule (dry single-layer microcapsule of Lactobacillus plantarum); C, wet double-layer microcapsule (wet microcapsule of Lactobacillus plantarum double-layer microcapsule); D, dry double-layer microcapsule (dry double-layer microcapsule of Lactobacillus plantarum);

[0051] Figure 12 Figure 3 is the scanning electron microscope diagram of the high folate-producing Lactobacillus plantarum ZFM4 microcapsule of the present application;

[0052] Figure 12 Figure 4 is the microstructure diagram of the high folate-producing Lactobacillus plantarum ZFM4 microcapsule of the present application; middle: A, overall appearance of dry single-layer microcapsule (×40); B, overall appearance of dry double-layer microcapsule (×60); C, surface appearance of dry single-layer microcapsule (×10,000); D, surface appearance of dry double-layer microcapsule (×10,000); E, cross-sectional appearance of dry single-layer microcapsule (×10,000); F, cross-sectional appearance of dry double-layer microcapsule (×10,000). DETAILED DESCRIPTION

[0053] The present application is further described below in conjunction with specific examples, but the scope of protection of the present application is not limited to this:

[0054] The present application provides an embedding technology of high folate-producing bacterial strains. The present application is further described by taking Lactobacillus plantarum as an example. The microcapsule technology can effectively improve the stability of lactic acid bacteria and improve the survival rate of lactic acid bacteria in various adverse environments.

[0055] Example 1, preparation of culture medium and solution

[0056] MRS liquid culture medium: weigh 10 g of proteose peptone, 10 g of calf extract, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 2 g of citric acid diammonium, 2 g of potassium phosphate dibasic, 0.05 g of manganese sulfate, 0.5 g of magnesium sulfate, 1 mL of Tween-80, dissolve in ultrapure water, and make up to 1 L, 121℃ high pressure sterilization for 15 min.

[0057] Lactic acid bacteria screening medium: add 25 g of calcium carbonate (i.e., calcium carbonate content 2.5%) to 1 L of MRS liquid culture medium as lactic acid bacteria screening medium.

[0058] LB liquid medium: 10 g of tryptone, 10 g of sodium chloride, 5 g of yeast extract were weighed and dissolved in ultrapure water, and then the volume was made up to 1 L. The solution was sterilized at 121℃ for 15 min and was ready for use.

[0059] Simulated gastric juice: 0.52 g of potassium chloride, 0.04 g of potassium dihydrogen phosphate, 2.10 g of sodium bicarbonate, 2.80 g of sodium chloride, 0.02 g of magnesium chloride dihydrate, 0.08 g of ammonium carbonate and 0.02 g of calcium chloride dihydrate were weighed, and 3.20 g of pepsin was added. The solution was dissolved in ultrapure water and the volume was made up to 1 L. The pH of the original simulated gastric juice was adjusted to 2.0, 2.5 and 3.0, respectively, and was ready for use.

[0060] Simulated intestinal juice: 0.51 g of potassium chloride, 0.02 g of potassium dihydrogen phosphate, 6.54 g of sodium bicarbonate, 2.24 g of sodium chloride, 12.26 g of magnesium chloride hexahydrate and 0.06 g of calcium chloride dihydrate were weighed, and 10.00 g of pancreatin was added. The pH was adjusted to 8.0, and the volume was made up to 1 L with ultrapure water.

[0061] Example 2, strain activation and preparation of bacterial suspension

[0062] Lactobacillus plantarum ZFM4 strain (CCTCC NO: M 2016629) was inoculated into MRS liquid medium at an inoculation amount of 1.5% (volume %) and was cultured at 37℃ and 160 r / min for 36 h. The culture solution was centrifuged at 8,000 rpm and 4℃ for 8 min, and the supernatant was removed. The bacterial slurry was washed with physiological saline for 3 times, and finally the bacterial body was resuspended with sterile physiological saline to obtain a concentrated bacterial solution containing 2.5×10 9 CFU / ml of bacteria, which was stored in a refrigerator at 4℃ for standby use.

[0063] Example 3, biocompatibility of microcapsule wall material and strain

[0064] In order to determine whether sodium alginate and cassava starch have toxic effects on Lactobacillus plantarum or inhibit the growth of Lactobacillus plantarum, different contents of sodium alginate and cassava starch were added to the autoclaved MRS liquid medium. The concentration of sodium alginate was 1.0% (1 g / 100 ml), 2.0% and 3.0%, and the concentration of cassava starch was 1.0% (1 g / 100 ml), 2.0% and 3.0%. Then, Lactobacillus plantarum was inoculated at a content of 1% (volume %). The MRS medium containing only 1% Lactobacillus plantarum was used as a blank control (i.e., the concentration of sodium alginate was 0 and the concentration of cassava starch was 0). The culture was incubated at 37℃ for 24 h. After the incubation, 0.1 mL of each culture medium was taken and diluted at different gradients (diluted by 10 times). The diluted solution was spread on MRS flat plate culture, and after 24 h of culture, the number of colonies was counted. Each group was tested in triplicate.

[0065] I. The effect of sodium alginate concentration on the biocompatibility of Lactobacillus plantarum ZFM4:

[0066] As shown in Table 1, the viable count was slightly higher after adding sodium alginate than without adding sodium alginate. When the concentration of sodium alginate was 3.0%, the growth activity of Lactobacillus plantarum was the strongest, and the viable count was about (3.05±0.26)xlO 9 CFU / mL, and the growth activity of Lactobacillus plantarum ZFM4 also increased when the concentration of sodium alginate was 1.0% and 2.0%, indicating that the biocompatibility between the two was good, and sodium alginate could be used as the wall material for Lactobacillus plantarum microcapsules.

[0067] Table 1, the effect of sodium alginate concentration on the activity of Lactobacillus plantarum

[0068]

[0069] II. The effect of cassava starch concentration on the biocompatibility of Lactobacillus plantarum:

[0070] As shown in Table 2, the growth activity of Lactobacillus plantarum increased after adding cassava starch, but the viable count was the highest when the concentration of cassava starch was 2.0%. Thereafter, the promotion effect of the growth activity of Lactobacillus plantarum decreased with the increase of the concentration of cassava starch, but the biocompatibility between the two was still good, and cassava starch could be used for microencapsulation of Lactobacillus plantarum.

[0071] Table 2, the effect of cassava starch concentration on the activity of Lactobacillus plantarum

[0072]

[0073] Example 4, Lactobacillus plantarum single-layer microcapsule preparation condition optimization

[0074] Wet single-layer microcapsules were prepared by sharp-hole granulation, and a micro-injection pump was used for single-layer microencapsulation of Lactobacillus:

[0075] Sodium alginate solution (sodium alginate + water) was mixed with cassava starch to form a composite wall material. A magnetic stirrer was set to 130 r / min, and the composite wall material was stirred uniformly for 10 min. The prepared bacterial suspension (i.e., concentrated bacterial solution) was added to the composite wall material at a volume ratio of 1:3.5, and stirred for 10 min to form a mixed solution.

[0076] The mixed solution was taken up using a sterile syringe, the syringe was mounted on a microsyringe pump, the appropriate flow rate was adjusted, and the mixed solution was steadily dropped into an equal volume of calcium chloride solution under stirring conditions (the dropwise addition time was controlled for about 15 minutes). After the dropwise addition was completed, stirring was continued for 45 min (so that the ion crosslinking was completed and the microcapsules were fixed and shaped), and then the prepared wet capsules were washed with sterile normal saline, and the washing process was repeated 3 times. The prepared wet capsules were stored in a 4°C chromatography cabinet.

[0077] Description: Under stirring conditions, the mixed solution was added dropwise to the calcium chloride solution for continuous stirring, at which time wet solid microcapsules were slowly produced. After the mixed solution was completely added, stirring was continued until no wet solid microcapsules were produced, and then the stirring was stopped. The stirring time was about 45 min.

[0078] Optimization I: Effect of sodium alginate concentration on the embedding yield and viable cell count of Lactobacillus plantarum microcapsules

[0079] The sodium alginate concentration in the composite wall material was set to be 0.5% (0.5 g / 100 mL), 1%, 1.5%, 2%, and 2.5%, respectively, the cassava starch concentration was 0.8% (0.8 g / 100 mL), the calcium chloride concentration in the calcium chloride solution was set to be 0.2% (0.2 g / 100 mL), and the volume ratio of the concentrated bacterial solution to the composite wall material was set to be 1:3.5, and the embedding yield and viable cell count were determined.

[0080] Figure 1 As shown in the table, when the sodium alginate concentration was 0.5%, the viable cell count and embedding yield of the microcapsules were the lowest, which were (1.12±0.06) ×10 8 CFU / mL and 40.13±0.21%, respectively. When the sodium alginate concentration was increased to 1.5%, the viable cell count and embedding yield of the microcapsules were the highest, which were (1.87±0.04) ×10 8 CFU / mL and 67.84±0.34%, respectively. When the sodium alginate concentration was increased to 2.0%, the viable cell count and embedding yield of the prepared microcapsules decreased, which was presumably due to the fact that a too high concentration of sodium alginate would cause the viscosity of the wall material to be too large, which would easily block the nozzle of the syringe and would also increase the difficulty of mixing with the bacterial suspension, resulting in a decrease in the protective performance of the microcapsules and a decrease in the viable cell count and embedding yield of the microcapsules.

[0081] Optimization II: Effect of cassava starch concentration on the embedding yield and viable cell count of Lactobacillus plantarum microcapsules

[0082] The concentration of cassava starch in the composite wall material was set to 0.2% (0.2 g / 100 mL), 0.4%, 0.6%, 0.8%, and 1.0%, the concentration of sodium alginate was set to 1.5% (1.5 g / 100 mL), the concentration of calcium chloride in the calcium chloride solution was set to 0.2%, and the volume ratio of concentrated bacterial solution to composite wall material was set to 1:3.5, and the embedding rate and viable bacterial count were determined.

[0083] Figure 2 As shown in the table, when the concentration of cassava starch was 0.8%, the viable bacterial count and embedding yield of the Lactobacillus plantarum microcapsules were the highest, which were (4.19±0.04)×10 9 CFU / mL and 79.13±0.35%, respectively. During the experiment, it was found that when the concentration of cassava starch was 1.0%, the viable bacterial count and embedding yield were significantly decreased, and the composite wall material formed was not suitable for the preparation of microcapsules.

[0084] Optimization III: Effect of calcium chloride concentration on the embedding yield and viable bacterial count of Lactobacillus plantarum microcapsules:

[0085] The concentration of calcium chloride in the calcium chloride solution was set to 0.1% (0.1 g / 100 mL), 0.15%, 0.2%, 0.25%, and 0.3%, the concentration of sodium alginate in the composite wall material was set to 1.5%, the concentration of cassava starch was set to 0.8%, and the volume ratio of concentrated bacterial solution to composite wall material was set to 1:3.5, and the embedding rate and viable bacterial count were determined.

[0086] Figure 3 As shown in the table, when the concentration of calcium chloride was below 0.20%, its effect on the Lactobacillus plantarum microcapsules increased with the increase of its own concentration; when the concentration of calcium chloride increased to 0.20%, the viable bacterial count and embedding yield of Lactobacillus plantarum were (3.47±0.06)×10 9 CFU / mL and 72.92±0.21%, respectively, and then tended to be stable, that is, even if the concentration of calcium chloride increased, the viable bacterial count and embedding rate remained basically unchanged, therefore, the concentration of 0.20% calcium chloride was selected as the condition for the subsequent preparation of microcapsules.

[0087] Optimization IV: Effect of volume ratio of bacterial solution to composite wall material on the embedding yield and viable bacterial count of Lactobacillus plantarum microcapsules:

[0088] The volume ratio of bacterial solution to composite wall material was set to 1:2, 1:2.5, 1:3, 1:3.5, and 1:4, the concentration of sodium alginate in the composite wall material was set to 1.5%, the concentration of cassava starch was set to 0.8%, the concentration of calcium chloride in the calcium chloride solution was set to 0.2%, and the embedding rate and viable bacterial count were determined.

[0089] When the volume ratio of composite wall material-bacteria liquid reaches 3.5:1, the proportion of bacteria liquid in the mixed liquid decreases, so the number of living bacteria decreases, but the proportion of wall material in the mixed liquid increases, forming a high-strength microcapsule wall, ensuring the protection effect and improving the embedding rate of microcapsules.

[0090] Optimization of the best process parameters by response surface:

[0091] On the basis of the results of single-factor experiments, the preparation process of Lactobacillus plantarum single-layer microcapsules was further optimized. Box-Behnken principle was used, with the concentration of sodium alginate (A), the concentration of cassava starch (B), and the volume ratio of wall material-bacteria liquid (C) as independent variables. The embedding rate of Lactobacillus plantarum microcapsules Y1 (%) and the number of living bacteria embedded per unit mass of Lactobacillus plantarum Y2 (Log(CFU / mL)) were used as response values. The specific experimental factors and levels are shown in Table 3.

[0092] Table 3, design table of microcapsule process factors and levels

[0093]

[0094] The optimal process for preparing Lactobacillus plantarum microcapsules was obtained as follows: the concentration of sodium alginate was 1.48%, the concentration of cassava starch was 0.82%, and the volume ratio of wall material-bacteria liquid was 3.5:1. Under these conditions, the predicted embedding rate of Lactobacillus plantarum microcapsules was 89.5%, and the predicted number of living bacteria was 7.84×10 9 CFU / mL. Considering the actual operation, the above-mentioned optimal process conditions were modified as follows: the concentration of sodium alginate was 1.50%, the concentration of cassava starch was 0.80%, and the volume ratio of wall material-bacteria liquid was 3.5:1. Under the modified conditions, three repeated experiments were conducted for verification, and the embedding rate of Lactobacillus plantarum microcapsules was 88.12±0.24%, and the number of living bacteria was 7.61×10 9 ±0.46 CFU / mL, which was close to the theoretical predicted value, indicating that the above model was feasible for the preparation of Lactobacillus plantarum ZFM4 microcapsules.

[0095] Example 5, preparation of Lactobacillus plantarum ZFM4 single-layer microcapsules and double-layer microcapsules

[0096] 1) The concentrated bacteria liquid obtained in Example 2 with a bacteria content of 2.5×10 9 CFU / ml was used;

[0097] 2) The sodium alginate, cassava starch, and water were uniformly mixed to form a composite wall material. In 100 ml of the composite wall material, there were 1.50 g of sodium alginate, 0.80 g of cassava starch, and the rest was water.

[0098] The concentrated bacterial solution was added to the complex wall material (3.5:1 volume ratio of complex wall material to concentrated bacterial solution) in a magnetic stirrer (130 r / min) to uniformly mix (mixing time about 10 minutes) to form a mixed solution.

[0099] A calcium chloride solution with a concentration of 0.2 g / 100 mL was prepared, and the volume ratio of the calcium chloride solution to the mixed solution was set to 1:1.

[0100] The stirring speed was set to 130 r / min, a sterile syringe with a low pore size (about 1.2 mm) was used to draw the mixed solution, the syringe was mounted on a microsyringe pump, and the appropriate flow rate was adjusted. The mixed solution was steadily dropped into the calcium chloride solution under stirring conditions (dropping time about 15 minutes), and stirring was continued for 45 min after the dropping was completed (i.e., stirring until no solid was produced), at which time the ion crosslinking was completed and the microcapsules were fixed and formed. Then, the microcapsules were washed with sterile normal saline, and the process was repeated three times to obtain the prepared Lactobacillus plantarum single-layer microcapsule wet capsules.

[0101] 3) 0.9 g of chitosan powder with a high degree of deacetylation (deacetylation degree ≥ 90%) was weighed and added to 100 ml of 1% ice acetic acid (1 ml of ice acetic acid was mixed with 99 ml of water to obtain a 1% ice acetic acid solution) to dissolve, obtaining a chitosan solution with a concentration of 0.9 g / 100 ml;

[0102] The volume ratio of the chitosan solution to the wet Lactobacillus plantarum single-layer microcapsules was set to 1:1.

[0103] The stirring speed was set to 600 r / min, and the wet Lactobacillus plantarum single-layer microcapsules obtained in step 2) were slowly added (slowly added for about 15 minutes) to the chitosan solution under stirring conditions. Stirring was continued until no solid product was produced (the total time from slow addition to stopping stirring was about 1 h), and the resulting product was collected. Finally, the product was rinsed with sterile normal saline three times to obtain the Lactobacillus plantarum double-layer microcapsule wet capsules, which were stored in a 4°C refrigerator.

[0104] Optimization V: Effect of chitosan concentration in chitosan solution on the embedding yield and viable count of Lactobacillus plantarum microcapsules:

[0105] The chitosan concentration in the chitosan solution was set to 0.3%, 0.6%, 0.9%, 1.2%, and 1.5%, respectively, and the embedding yield and viable count were determined.

[0106] Figure 4 As shown, the addition of chitosan had a positive effect on the embedding yield and viable count of the microcapsules. When the addition amount was 0.3-0.9%, the embedding yield and viable count increased, and when the addition amount reached 0.9%, the embedding yield reached a maximum value of 93.10±0.31%, and the viable count also reached a maximum value of (8.84±0.31)×109 CFU / mL, compared with single-layer embedding, the embedding rate increased by 5.01±0.07%, and the embedding rate no longer increased and the number of viable bacteria decreased with the continuous increase of the concentration of chitosan. The reason is that chitosan has antibacterial effect, and high concentration may cause damage to Lactobacillus plantarum. Therefore, 0.9% chitosan is selected for double-layer embedding.

[0107] Optimization VI, Lactobacillus plantarum microcapsule freeze-drying test and freeze-drying protectant condition optimization test

[0108] The prepared Lactobacillus plantarum double-layer microcapsule wet capsules were freeze-dried to form dry microcapsules, and the survival rate of the microcapsules after drying was determined:

[0109] 5 g of fresh wet capsules (Lactobacillus plantarum double-layer microcapsule wet capsules) were taken, added with freeze-drying protectants, frozen in a-80°C refrigerator, and then dried by a freeze dryer. After drying, the obtained dry microcapsules were placed in a centrifuge tube, sealed with a sealing film, and stored at a suitable temperature. Different proportions of freeze-drying protectants were added before pre-freezing, and the survival rate of Lactobacillus plantarum ZFM4 in the freeze-dried microcapsules was determined to evaluate the effect of the freeze-drying protectants. The preconditions before single-factor test were: trehalose concentration % (2, 4, 6, 8, 10), skim milk % (1, 2, 3, 4, 5), sorbitol % (1, 2, 3, 4, 5), and mannitol % (1, 2, 3, 4, 5). The survival rate of the microcapsules was determined, and the best freeze-drying protectant process combination was determined by orthogonal test. See Tables 4 and 5.

[0110] Table 4, orthogonal test factors

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[0112]

[0113] Table 5, orthogonal experiment results

[0114]

[0115] Example 6, freeze-drying of wet Lactobacillus plantarum microcapsules (wet Lactobacillus plantarum single-layer microcapsules / wet Lactobacillus plantarum double-layer microcapsules) under the protection of freeze-drying protectants;

[0116] The freeze-drying protectants are composed of trehalose, skim milk powder, sorbitol, and mannitol; 6 g of trehalose, 3 g of skim milk powder, 2 g of sorbitol, and 3 g of mannitol are added to every 100 ml of wet Lactobacillus plantarum microcapsules (wet Lactobacillus plantarum single-layer microcapsules / wet Lactobacillus plantarum double-layer microcapsules).

[0117] The wet-state Lactobacillus plantarum microcapsules (Lactobacillus plantarum single-layer microcapsules / Lactobacillus plantarum double-layer microcapsules) are placed in a -80°C refrigerator for quick freezing after adding a freeze-drying protective agent, and then are dried by a freeze dryer (-80°C freeze-drying until constant weight). After drying is completed, the dry-state Lactobacillus plantarum single-layer microcapsules / dry-state Lactobacillus plantarum double-layer microcapsules are obtained.

[0118] Example 7, heat resistance test of Lactobacillus plantarum microcapsules

[0119] The dry-state Lactobacillus plantarum single-layer microcapsules / dry-state Lactobacillus plantarum double-layer microcapsules obtained in Example 6 are used as an experimental group, and the concentrated bacterial solution obtained in Example 2 is used as a control group, and are respectively placed at 55°C, 66°C and 75°C for 30 min for a heat resistance test. After 30 min, plate counting is performed, then viable count is determined to determine the survival rate of viable bacteria, so as to evaluate the heat protection effect of the microcapsules on Lactobacillus plantarum. The obtained results are as follows Figure 5 .

[0120] Example 8, tolerance test of Lactobacillus plantarum microcapsules in a simulated in-vitro digestion system

[0121] In this test, the tolerance of Lactobacillus plantarum microcapsules in the human body is studied by using an in-vitro simulated digestion model. The dry-state Lactobacillus plantarum single-layer microcapsules / dry-state Lactobacillus plantarum double-layer microcapsules obtained in Example 6 are used as an experimental group, and the concentrated bacterial solution obtained in Example 2 is used as a control group. The in-vitro simulated digestion model in this experiment is improved according to the method of Minekus et al.

[0122] Simulated saliva tolerance test: A magnetic stirrer is placed in a 37°C incubator simulating the body temperature of a human body, and the rotation speed is set to 120 r / min. 20 mL of simulated saliva is taken, 1 ml of the microcapsules (prepared from 1 ml of bacterial suspension) or 1 ml of the bacterial suspension of the control group is added, and the treatment is performed for 8 min. The survival rate is calculated every 1 min during the treatment, and the test is repeated three times. The obtained results are as follows Figure 6 . Microencapsulation can improve the tolerance of Lactobacillus plantarum ZFM4 in saliva.

[0123] Simulated gastric juice tolerance test: A magnetic stirrer is placed in a 37°C incubator simulating the body temperature of a human body, and the rotation speed is set to 120 r / min. 20 mL of simulated gastric juice is taken, 1 ml of the microcapsules (prepared from 1 ml of bacterial suspension) or 1 ml of the bacterial suspension of the control group is added, and the treatment is performed for 120 min. The survival rate is calculated every 10 min during the treatment, and the test is repeated three times. The obtained results are as follows Figure 7 .

[0124] Simulation of pancreatic juice resistance test: a magnetic stirrer was placed in a 37 °C incubator simulating the human body temperature, the speed was set at 120 r / min, 20 mL of simulated pancreatic juice was taken respectively, 1 mL of test group microcapsules (prepared from 1 mL of bacterial suspension) or control group bacterial suspension was added, and the treatment was carried out for 120 min. The survival rate was calculated every 10 min during the treatment, and the test was repeated three times. The results are as follows Figure 8 .

[0125] Example 9, release test of Lactobacillus plantarum microcapsules in simulated intestinal juice

[0126] The Lactobacillus plantarum dry single-layer microcapsules / Lactobacillus plantarum dry double-layer microcapsules obtained in Example 6 were subjected to simulated human digestion process, the simulated intestinal juice was placed in a 37 °C incubator simulating the human body temperature, and then the Lactobacillus plantarum dry single-layer microcapsules / Lactobacillus plantarum dry double-layer microcapsules were added. The viable bacterial count was determined every 15 min, and the sustained-release effect of the microcapsules was judged by the size of the viable bacterial count. The double-layer microcapsule structure has a thicker wall, a longer dissolution time, and a longer complete release time than the single-layer microcapsule. The double-layer microcapsule has better protection performance, so the viable bacterial count after complete release is higher. The results are as follows Figure 9 .

[0127] Example 10, storage stability test of Lactobacillus plantarum microcapsules

[0128] The Lactobacillus plantarum dry single-layer microcapsules / Lactobacillus plantarum dry double-layer microcapsules obtained in Example 6 were stored at -20 °C, 4 °C, and 25 °C, respectively. The samples were taken at 0, 1, 3, 7, 14, and 21 d for plate counting, and the viable bacterial count was determined to determine the corresponding viable bacterial survival rate. The results are as follows Figure 10 .

[0129] Example 11, surface structure of Lactobacillus plantarum microcapsules

[0130] The morphological diagrams of wet capsules (i.e., Lactobacillus plantarum single-layer microcapsule wet capsules and Lactobacillus plantarum double-layer microcapsule wet capsules obtained in Example 5) and dried microcapsules (i.e., Lactobacillus plantarum dry single-layer microcapsules / Lactobacillus plantarum dry double-layer microcapsules obtained in Example 6) are as follows Figure 11 .

[0131] The overall appearance, surface appearance, and cross-sectional appearance of the Lactobacillus plantarum dry single-layer microcapsules / Lactobacillus plantarum dry double-layer microcapsules are as follows Figure 12 .

[0132] Finally, it should be noted that the above enumeration is only several specific embodiments of the present application. Obviously, the present application is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or inferred by those of ordinary skill in the art from the disclosure of the present application should be considered within the scope of the present application.

Claims

1. A process for the preparation of high folate yielding Lactobacillus plantarum ZFM4 microcapsules, characterized by The method comprises the following steps: 1), centrifuging and resuspending the fermentation liquor of Lactobacillus plantarum ZFM4 with high folic acid yield to obtain concentrated bacterial liquor; the bacterial content of the concentrated bacterial liquor is (2.5±0.5)×10 9 CFU / ml; The preservation number of the Lactobacillus plantarum ZFM4 is CCTCC NO: M 2016629; 2) The concentrated bacterial solution and the composite wall material are uniformly mixed to form a mixed solution, and the mixed solution is added to the calcium chloride solution under stirring, and the stirring is continued until no solid is produced, to obtain the wet single-layer Lactobacillus plantarum microcapsule; The composite wall material is composed of sodium alginate, cassava starch and water; in 100 ml of the composite wall material, 1.50±0.1 g of sodium alginate and 0.80±0.1 g of cassava starch are contained; The volume ratio of the composite wall material to the concentrated bacterial solution is (3.5±0.1):

1. The concentration of the calcium chloride solution is (0.2±0.02) g / 100 mL, and the volume ratio of the calcium chloride solution to the mixed solution is (1±0.1):

1. 3) First, the high-deacetylation degree chitosan powder is dissolved in an ice acetic acid solution with a volume concentration of 0.5-1.5%, to obtain a chitosan solution with a chitosan concentration of (0.9±0.1) g / 100 ml. Under stirring, the wet single-layer Lactobacillus plantarum microcapsule obtained in step 2) is slowly added to the chitosan solution, and the stirring is continued until no solid is produced, and the obtained product is collected, which is the wet double-layer Lactobacillus plantarum microcapsule; the volume ratio of the chitosan solution to the wet single-layer Lactobacillus plantarum microcapsule is (1±0.1):

1.

2. The process for the preparation of high folate producing Lactobacillus plantarum ZFM4 microcapsules as claimed in claim 1, wherein: The wet double-layer Lactobacillus plantarum microcapsule obtained in step 3) is freeze-dried under the protection of a freeze-drying protective agent. The freeze-drying is as follows: after the wet microcapsule is added with the freeze-drying protective agent, it is rapidly frozen, and then freeze-dried, to obtain the dry double-layer Lactobacillus plantarum microcapsule. The freeze-drying protective agent is composed of trehalose, skimmed milk powder, sorbitol and mannitol; the trehalose is (6±0.5)% of the weight of the wet microcapsule, the skimmed milk powder is (3±0.3)% of the weight of the wet microcapsule, the sorbitol concentration is (2±0.2)% of the weight of the wet microcapsule, and the mannitol is (3±0.3)% of the weight of the wet microcapsule.

3. The process for the preparation of high folate producing Lactobacillus plantarum ZFM4 microcapsules as claimed in claim 2, wherein: The step 1) is as follows: The Lactobacillus plantarum ZFM4 is inoculated and activated for fermentation culture, and the activation fermentation culture conditions are 37±1℃ and 160±20 r / min for 36±1 h; the fermentation liquid is obtained. The fermentation liquid is centrifuged at 8000±1000 rpm and 4±0.5℃ for 8±2 min, the supernatant is removed, then the bacterial slurry is washed with normal saline, and finally the bacterial body is resuspended with sterile normal saline to obtain the concentrated bacterial solution.

4. The process for the preparation of high folate producing Lactobacillus plantarum ZFM4 microcapsules as claimed in claim 3, wherein: The step 1) is as follows: the Lactobacillus plantarum ZFM4 is inoculated into the MRS liquid culture medium according to an inoculation amount of 1.5% for activation fermentation culture.

5. The preparation method of the high-folate Lactobacillus plantarum ZFM4 microcapsule according to any one of claims 1-4, characterized in that: The stirring speed in step 2) is 130±30 r / min, The stirring speed in step 3) is 600±50 r / min.

6. The high-folate Lactobacillus plantarum ZFM4 microcapsule prepared by any one of the methods in claims 1-5.

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