CN116570552A - Carrier hydrogel for targeted slow release of probiotics and preparation method and application thereof - Google Patents
Carrier hydrogel for targeted slow release of probiotics and preparation method and application thereof Download PDFInfo
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- CN116570552A CN116570552A CN202310469611.XA CN202310469611A CN116570552A CN 116570552 A CN116570552 A CN 116570552A CN 202310469611 A CN202310469611 A CN 202310469611A CN 116570552 A CN116570552 A CN 116570552A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
-
- A—HUMAN NECESSITIES
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Abstract
The invention discloses a carrier hydrogel for targeting slow-release probiotics and a preparation method and application thereof, and belongs to the technical field of probiotic embedding materials. The carrier hydrogel of the targeted slow-release probiotics is prepared by taking cellulose and carboxymethyl cellulose extracted from the millettia speciosa champ as raw materials and epichlorohydrin as a cross-linking agent through a cross-linking reaction, has good swelling capacity and pH sensitivity, and can realize water loss shrinkage in the stomach and water absorption expansion in the intestinal tract, so that the targeted slow-release effect of the probiotics can be realized. Meanwhile, the hydrogel has good embedding effect and release rate on the LP-BY2, improves the abundance of the living LP-BY2 reaching the intestinal tract, enables the LP-BY2 to effectively exert the effect of reducing cholesterol, and lays a solid foundation for developing functional products for reducing serum cholesterol, such as health-care food or medicines and the like.
Description
Technical Field
The invention belongs to the technical field of probiotic embedding materials, and particularly relates to a carrier hydrogel for targeted slow release of probiotics, and a preparation method and application thereof.
Background
Radix seu herba Gei aleppici is root of Mesona fordii Millettia speciosa champ of genus Mesona of family Leguminosae, and is mainly distributed in Taiwan, guangdong, guangxi and Fujian etc. The root is used as a medicine, has homology of medicine and food, is rich in nutrient components such as cellulose, starch, protein and the like, and has the effects of improving immunity, protecting liver, resisting inflammation, resisting oxidation and the like.
At present, the eating modes of the beautiful millettia root are mostly slicing, soaking wine, soaking water or stewing soup, and the rest of the great millettia root can be selected to be thrown away for treatment, so that a certain degree of resource waste can be caused. In recent years, with the development of the millettia speciosa processing industry, the millettia speciosa residue is increased. Researches show that the residual millettia speciosa champ residue still contains the cellulose with extremely high content, so that the millettia speciosa champ residue is further fully and efficiently utilized, the problem of the waste of millettia speciosa champ resources can be solved, and the environmental problem caused by randomly discarding and stacking the millettia speciosa champ residue can be effectively solved. In addition, the economic benefit of the millettia speciosa champ industry can be improved, and the millettia speciosa champ has very important economic and social benefits.
pH-sensitive hydrogels are the most widely studied type of hydrogels at present, such as acrylic or methacrylic acid grafted polyvinyl alcohol hydrogels, which are incapable of releasing insulin in simulated gastric fluid, whereas large amounts of release of insulin can be observed in simulated intestinal fluid, indicating that the hydrogels have good pH sensitivity; the Chitosan (CS) and polyvinylpyrrolidone (PVP) are used as wall materials to prepare the cationic pH-sensitive hydrogel, and research shows that the amoxicillin-carrying CS/PVP hydrogel has better drug release property, and the amoxicillin-carrying CS/PVP hydrogel reacts for 3 hours under the low pH condition of pH 1.0 to release about 73 percent of amoxicillin. The polyelectrolyte chitosan and polyacrylic acid are used as wall materials to prepare the pH sensitive composite hydrogel, and the fact that the chitosan is used for partially replacing the acrylic acid is found to enable the hydrogel to have obvious pH sensitivity and reduce the synthesis cost. Most of the utilization of pH sensitive hydrogel is concentrated in the fields of drug controlled release, sustained release and the like, and huge blank still exists in the utilization of the field of lactic acid bacteria, so that the method has important significance.
At present, sodium alginate and protein substances are the most commonly used wall materials for microencapsulation of lactobacillus, and related documents and reports are also numerous, for example, sodium alginate is used as the wall materials to embed lactobacillus acidophilus Lactobacillus acidophilus ATCC 43121, and the result shows that the microencapsulated lactobacillus acidophilus Lactobacillus acidophilus ATCC 43121 has better gastric juice tolerance to pH 1.2 than naked bacteria; and the lactobacillus rhamnosus Lactobacillus rhamnosus ATCC 9595 is embedded by taking sodium alginate and lecithin as wall materials, so that the acid resistance, intestinal tract colonization capacity and the like of the lactobacillus are improved. Although sodium alginate and proteins are widely used as wall materials, there are a number of disadvantages: (1) under the extremely low pH value, sodium alginate molecules can be degraded, gel depolymerizes, gastric acid can not be effectively controlled to enter, the survival rate of lactic acid bacteria is affected, and the protection effect is reduced; and the sodium alginate microcapsule is almost completely disintegrated and released in the small intestine section, so that the embedded lactobacillus is difficult to reach the colon to play a role. (2) Protein is used as a wall material to prepare the microcapsule slowly, and embedding efficiency is low; proteins are easily enzymatically hydrolyzed by pepsin, so that lactic acid bacteria are inactivated by exposure to low pH gastric acid; and hydrophobic groups in protein molecules easily cause mutual aggregation among microcapsules, which is unfavorable for the preparation of the microcapsules.
At present, in China patent database, there are few researches on constructing lactobacillus-carrying hydrogel by using carboxymethyl cellulose and cellulose as wall materials, and there are few application pieces related to a targeting sustained-release probiotic carrier hydrogel, and only CN 115349639A, a preparation method and application thereof, CN 115737537A, a targeting sustained-release probiotic carrier hydrogel, a preparation method and application thereof, and the like are disclosed.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a carrier hydrogel for targeted slow release of probiotics and a preparation method thereof.
It is another object of the present invention to provide the use of the carrier hydrogel for targeted sustained release of probiotics.
In the present invention, carboxymethyl cellulose contains a large number of carboxyl groups (-COOH) in the molecular chain, so that the hydrogel is deionized under low pH condition to be in a shrinking state, and when the pH is raised, -COOH is ionized into-COO - Hydrogen bonding is disrupted, -COO - The electrostatic repulsive force between the groups is improved, so that the swelling ratio is increased. This difference in swelling over different pH environments allows the hydrogel to collapse under low pH gastric fluid conditions, and to swell in intestinal fluid to release lactic acid bacteria. And a large number of hydroxyl groups exist in cellulose molecules, and the cellulose molecules have highly ordered crystallization areas due to interaction force among hydrogen bond groups, so that the carboxymethyl cellulose hydrogel is endowed with a rigid structure. And the carboxymethyl cellulose and cellulose are not easily degraded and damaged by gastric acid and pepsin, and the hydrogel prepared by compounding the carboxymethyl cellulose and the cellulose has important significance for the survival rate and effective controlled release of lactobacillus in the gastrointestinal digestion processMeaning.
The aim of the invention is achieved by the following technical scheme:
the carrier hydrogel of the targeted slow-release probiotics is a crosslinked product with a porous network structure, which is obtained by taking bovine bristle dreg cellulose (MSCC) and bovine bristle dreg carboxymethyl cellulose (MSCCMC) as raw materials and crosslinking the raw materials.
The millettia speciosa champ cellulose is separated and extracted from millettia speciosa champ, and the millettia speciosa champ is derived from Guangdong wheat forest planting base.
The preparation method of the millettia speciosa champ cellulose comprises the following steps:
(1) Collecting the millettia speciosa champ, drying and crushing the millettia speciosa champ at 55-65 ℃ (preferably 60 ℃), and sieving the millettia speciosa champ by a sieve with 80-100 meshes (preferably a sieve with 100 meshes) to obtain millettia speciosa champ powder; evenly mixing the millettia speciosa powder with distilled water, wherein the mass ratio of the feed water to the water is 1:20, heating in a water bath at 90 ℃, stirring for 2-3 h (preferably 2 h), collecting the precipitate, repeating the operation for more than one time, centrifuging to obtain the precipitate, mixing the precipitate with distilled water, wherein the mass ratio of the water to the material is 1:20, adding alpha-amylase, wherein the addition amount of the alpha-amylase is 0.2% w/w of the weight of the millettia speciosa champ powder, stirring and reacting for 2-3 h in a water bath at 50-60 ℃ and preferably stirring and reacting for 2h in a water bath at 55 ℃, inactivating enzyme in a boiling water bath for 10-15 min (preferably 10 min), filtering to obtain filter residues, washing the filter residues with distilled water for multiple times until the filter residues are in a clear state, and then washing the filter residues with 95% ethanol for one time; drying the filter residue at 55-65 ℃ for 16-18 h (preferably at 60 ℃ for 16 h) to obtain the millettia speciosa champ crude cellulose;
(2) Uniformly mixing the coarse cellulose of the millettia speciosa champ and a sodium chlorite solution with the mass fraction of 7.5%, wherein the sodium chlorite solution needs to be adjusted to be 3.8-4.0 by an HCl solution in advance, and the mass ratio of the coarse cellulose of the millettia speciosa champ to the sodium chlorite solution is 1:20, heating in water bath at 70-80 ℃ for 2-3 h (preferably heating in water bath at 75 ℃ for 2 h), and continuously stirring during the heating; centrifuging, collecting and washing the precipitate for multiple times until the filtrate is neutral in pH, washing the filter residue once by using 95% ethanol, and drying at 55-65 ℃ for 16-18 h (preferably at 60 ℃ for 16 h) to obtain lignin-removed cellulose;
(3) Uniformly mixing the delignified cellulose with a KOH solution with the mass fraction of 10%, wherein the mass ratio of the delignified cellulose to the KOH solution is 1:20, continuously stirring at 25 ℃ for 12-14 hours (preferably 12 hours), filtering and collecting filter residues, taking distilled water as a washing liquid, washing the filter residues for a plurality of times until the filter residues are neutral in pH, washing the filter residues once by using 95% ethanol, finally drying at 60 ℃ for 16-18 hours (preferably 16 hours), crushing and sieving with a 80-100-mesh sieve (preferably a 100-mesh sieve) to obtain the millettia speciosa champ cellulose, and naming the millettia speciosa champ cellulose as MSCC.
The preparation method of the millettia speciosa champ carboxymethyl cellulose comprises the following steps:
(a) Weighing Oriental millettia root residue cellulose (MSCC), adding 90% isopropanol solution (v/v), mixing, and adding 30% H 2 O 2 Mixing the solution (v/v) and 50% NaOH solution (w/w) at room temperature under stirring for 2-3 h (preferably 2 h) to activate hydroxyl groups on cellulose; wherein, the ratio of the cow's-power dreg cellulose (MSCC) to the 90% isopropyl alcohol solution (v/v) is 1g:20mL; oriental millettia root residue cellulose (MSCC) with 30% concentration of H 2 O 2 The ratio of the solution (v/v) to the 50% NaOH solution (w/w) was 10g:1.2mL:16mL;
(b) And adding chloroacetic acid solution (w/w) with the concentration of 50% into the solution after 2-3 h (preferably 2 h), wherein the ratio of the niu dalton cellulose (MSCC) to the chloroacetic acid solution (w/w) with the concentration of 50% is 10g:14mL; and carrying out gradient heating to etherify, and the specific operation is as follows: placing the reaction solution at room temperature, continuously stirring and mixing for reaction for 25-35 min (preferably 30 min), then placing the reaction solution at 40-50 ℃ for continuously stirring and mixing for reaction for 25-35 min (preferably placing the reaction solution at 45 ℃ for continuously stirring and mixing for reaction for 30 min), then placing the reaction solution at 55-65 ℃ for stirring and mixing for reaction for 25-35 min (preferably placing the reaction solution at 60 ℃ for stirring and mixing for reaction for 30 min), and finally placing the reaction solution at 70-80 ℃ for stirring and mixing for reaction for 1-2 h (preferably placing the reaction solution at 75 ℃ for stirring and mixing for reaction for 1.5 h); after etherification reaction, neutralizing the etherification product with a small amount of acetic acid solution (v/v) with the concentration of 10%, filtering, repeatedly washing the product with absolute ethyl alcohol, and drying under the condition of 45 ℃ -55 ℃ (preferably 50 ℃); finally, grinding the product and sieving the product with a 80-100 mesh sieve (preferably a 100 mesh sieve) to obtain the millettia speciosa champ carboxymethyl cellulose, which is named as MSCCMC.
The probiotics are intestinal probiotics, and further are lactobacillus paracasei (Lactobacillus paracasei) BY2 with a preservation number of CGMCC No.22571, and are preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) of China academy of sciences of China No. 3 of the national academy of sciences of China, including the North Chen West Lu No. 1, the Korean area of Beijing, at 20 days of 2021.
The carrier hydrogel of the targeted slow-release probiotics is hydrogel with responsiveness to the pH of gastrointestinal fluid; wherein the composition is in a shrunken state in gastric juice pH and in a swelled state in intestinal juice pH.
The preparation method of the carrier hydrogel for targeting the slow-release probiotics comprises the following steps:
s1: 5wt% MSCCMC solution and 5wt% MSCC solution were mixed according to 4:0 to 0:4, preparing MSCCMC/MSCC solution according to the mass ratio;
s2: and (3) placing the MSCCMC/MSCC solution at room temperature, stirring to fully mix, slowly dripping a crosslinking agent Epichlorohydrin (ECH) during the stirring, and reacting under the water bath condition to obtain the MSCCMC/MSCC hydrogel.
In order to better achieve the object of the present invention, the method further comprises the steps of:
s3: and soaking the MSCCMC/MSCC hydrogel into the probiotic suspension, and performing vacuum freeze drying after complete swelling balance to obtain the carrier hydrogel of the targeted slow-release probiotics.
Preferably, in step S1, the preparation method of the 5wt% msccmc solution is as follows: mixing MSCCMC powder with NaOH/urea aqueous solution according to a mass ratio of 5:95, preparing; the method comprises the following steps: accurately weighing MSCCMC powder in a sample bottle, and mixing the MSCCMC powder with the sample bottle according to a mass ratio of 5:95 adding NaOH/urea aqueous solution, and standing at room temperature under high speed stirring overnight to dissolve completely, to prepare 5wt% MSCCMC solution;
preferably, in step S1, the preparation method of the 5wt% mscc solution is as follows: MSCC powder and NaOH/urea aqueous solution are mixed according to the mass ratio of 5:95, preparing; the method comprises the following steps: accurately weighing MSCC powder in a sample bottle, and mixing the MSCC powder with a mass ratio of 5:95 adding NaOH/urea aqueous solution, placing in a refrigerator at-80 ℃ to freeze for 1-2 h, then thawing in an ice-water bath and strongly stirring to completely dissolve the solution, and preparing MSCC solution with the concentration of 5 wt%;
wherein the NaOH/urea aqueous solution is 7wt% NaOH solution and 12wt% urea.
Preferably, in step S1, the mass ratio is 4:0 to 1:3, a step of; further 3:1 to 1:3, a step of; still further 3: 1-2: 2;
preferably, in step S1, the mass ratio is 4: 0. 3: 1. 2: 2. 1: 3. 0:4.
preferably, in the step S2, the temperature of the room temperature is 25 ℃ to 30 ℃; further 30 ℃;
The stirring time is 25-35 min; further for 30min;
preferably, in the step S2, the amount of the cross-linking agent is 1mL of the cross-linking agent added into each 10 mL-12 mL MSCCMC/MSCC solution;
preferably, in the step S2, the reaction is carried out for 1 to 3 hours under the water bath condition of 55 to 65 ℃; further reacting for 2 hours under the water bath condition of 60 ℃;
preferably, in step S3, the concentration of the probiotic bacteria in the probiotic bacteria suspension is 1×10 9 CFU/mL～1×10 11 CFU/mL; further 1X 10 10 CFU/mL。
Preferably, in the step S3, the time of vacuum freeze drying is 48 to 72 hours; further 48h.
The carrier hydrogel of the targeted slow-release probiotics is applied to preparation of medicines or health-care foods.
The application of the carrier hydrogel of the targeted slow-release probiotics in preparing products for reducing serum cholesterol.
Further, the product is a health food or a medicine.
Compared with the prior art, the invention has the following advantages and effects:
(1) The carrier hydrogel of the targeted slow-release probiotics is prepared by taking cellulose and carboxymethyl cellulose extracted from the millettia speciosa champ as raw materials and epichlorohydrin as a cross-linking agent through a cross-linking reaction, has good swelling capacity and pH sensitivity, and can realize water loss shrinkage in the stomach and water absorption expansion in the intestinal tract, so that the targeted slow-release effect of the probiotics can be realized. Meanwhile, the hydrogel has good embedding effect and release rate on the LP-BY2, improves the abundance of the living LP-BY2 reaching the intestinal tract, enables the LP-BY2 to effectively exert the effect of reducing cholesterol, and lays a solid foundation for developing functional products for reducing serum cholesterol, such as health-care food or medicines and the like.
(2) Of the 5 hydrogels in the invention, the GEL40 hydrogel has the highest embedding rate of 83.34 +/-1.77% and the strongest release capacity of 92.86+/-3.57%; when the concentration of bacteria released BY each hydrogel is controlled to be consistent, under the simulated gastrointestinal conditions containing 0.02% of bile salt, the survival rate of LP-BY2 in 5 hydrogels is maintained to be more than 78%, the survival rate is obviously higher than that of free LP-BY2 (70.27 +/-1.47%), the cholesterol reducing capacity is also maintained to be more than 38%, and the cholesterol reducing capacity is obviously higher than that of free LP-BY2 (34.565 +/-0.525%). Under simulated gastrointestinal conditions containing 0.3% bile salts, the survival rate of LP-BY2 in 5 hydrogels is also kept above 44%, which is significantly higher than that of free LP-BY2 (16.28+ -1.91%), and the cholesterol-lowering ability is also kept above 28%, which is significantly higher than that of free LP-BY2 (4.674 + -1.249%). Therefore, the hydrogel has higher embedding capacity, release capacity and protective capacity for LP-BY2, can ensure that the LP-BY2 still maintains higher survival rate and cholesterol reducing capacity after passing through the simulated gastrointestinal tract, and can be used for development and application in the aspect of health-care food or medicines.
Drawings
FIG. 1 is a flow chart of the preparation of LP-BY 2-loaded MSCCMC/MSCC hydrogels.
Fig. 2 is an infrared spectrum of MSCCMC.
Fig. 3 is a graph of determination of MSCCMC concentration.
Fig. 4 is a graph of determination of MSCC concentration.
FIG. 5 is an apparent morphology of MSCCMC/MSCC hydrogels before and after swelling.
FIG. 6 is a scanning electron microscope image of an MSCCMC/MSCC hydrogel, where (a) is GEL40, (b) is GEL31, (c) is GEL22, (d) is GEL13, and (e) is GEL04.
FIG. 7 is an infrared spectrum of MSCCMC/MSCC hydrogels; wherein MSCCMC, GEL40, GEL31, GEL22, GEL13 and GEL04 are sequentially arranged from top to bottom.
FIG. 8 is an X-ray diffraction pattern of MSCCMC/MSCC hydrogels; wherein, GEL40, GEL31, GEL22, GEL13 and GEL04 are sequentially arranged from bottom to top.
FIG. 9 is a thermal stability analysis of MSCCMC/MSCC hydrogels, wherein (a) is a TG plot and (b) is a DTG plot.
FIG. 10 is a graph showing the swelling capacity analysis of MSCCMC/MSCC hydrogels, wherein (a) is the swelling curve and (b) is the equilibrium swelling ratio.
FIG. 11 is a graph of the pH responsiveness of MSCCMC/MSCC hydrogels.
FIG. 12 is a graph showing the measurement of MSCCMC/MSCC hydrogel entrapment rate; and (3) injection: ' represents significant differences between GEL40, GEL31, GEL22 and GEL13 and GEL04 hydrogels, where p <0.05 and p <0.01.
FIG. 13 is a graph of the determination of MSCCMC/MSCC hydrogel release rate.
FIG. 14 is a graph of cholesterol lowering potential of LP-BY2 in hydrogels under different gastrointestinal environments; and (3) injection: 'a' and 'b', 'c' represent significant differences between each set of hydrogels and LP-BY2 under simulated gastrointestinal fluid conditions with 0.02% bile salts, wherein: 'b' represents p <0.05, and 'c' represents p <0.01; 'a' and 'C', 'D' represent significant differences between the respective sets of hydrogels and LP-BY2 under simulated gastrointestinal fluid conditions containing 0.3% bile salts, wherein 'C' represents p <0.01 and 'D' represents p <0.001; wherein, each group of treatment corresponds to 0.02% of bile salt (0.02% of bile salt) and 0.3% of bile salt (0.3% of bile salt) from left to right in sequence.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and apparatus used in the present invention are commercially available unless otherwise specified.
Preparation flow chart of the LP-BY 2-loaded MSCCMC/MSCC hydrogel is shown in FIG. 1.
Example 1: preparation of MSCCMC/MSCC hydrogel
1.1 materials and reagents
Great force of cow: and (5) purchasing the seedlings to a Guangdong wheat forest planting base.
Lactobacillus paracasei (Lactobacillus paracasei) BY2: pickled Chinese cabbage screened from Xinxing county of YunFu City, guangdong province has been preserved in China general microbiological culture Collection center (China General Microbiological Culture Collection Center, CMGCC) for 20 months in 2021, the preservation number is CGMCC No.22571, and the strain is disclosed in patent document 202110911715.2, namely a cholesterol-reducing lactobacillus paracasei and application thereof, and is abbreviated as LP-BY2.
Isopropanol, hydrogen peroxide, chloroacetic acid, acetic acid, absolute ethyl alcohol, sodium chloride, monopotassium phosphate, disodium hydrogen phosphate, concentrated hydrochloric acid, sodium hydroxide, cholesterol, pig bile salt, pepsin and trypsin.
1.2 preparation of reagents
Sterile physiological saline: 0.85% (w/v) NaCl solution was added and the mixture was sterilized at 121℃for 20 minutes.
NaOH/uric acid aqueous solution: 7wt% NaOH solution and 12wt% urea.
PBS solution: 0.8% (w/v) NaCl,0.02% (w/v) KH 2 PO 4 ，0.115％(w/v)Na 2 HPO 4 The pH was adjusted to 7.2.
Preparation of simulated gastrointestinal fluid:
simulating gastric juice: naCl 0.2g/100mL, pepsin (Pepsin) 0.35g/100mL, and 1mol/LHCl was used to adjust the pH to 3.0,0.45 μm Millipore PTFE filter membrane for filtration sterilization for later use.
Simulation of intestinal juice: naHCO (NaHCO) 3 1.1g/100mL, 0.2g/100mL NaCl, 0.1g/100mL Trypsin (Trypsin), 0.02g/100mL pig bile salt or 0.3g/100mL cholesterol, 10mg/100mL cholesterol, adjusting the pH value to 8.0,0.45 mu m, filtering and sterilizing by a Milpore PTFE filter membrane for later use.
1.3 preparation of MSCC
And (3) collecting the millettia speciosa champ, drying and crushing at 60 ℃, and sieving by a 100-mesh sieve to obtain millettia speciosa champ powder. Evenly mixing the millettia speciosa powder with distilled water, wherein the mass ratio of the feed water to the water is 1:20, heating in a water bath at 90 ℃, stirring for 2 hours, collecting the precipitate, repeating the operation for more than one time, centrifuging to obtain the precipitate, mixing the precipitate with distilled water, wherein the mass ratio of the material to the water is 1:20, adding alpha-amylase (the addition amount of the alpha-amylase is 0.2% (w/w) of the powder mass of the millettia speciosa champ, stirring and reacting for 2 hours at 55 ℃ in a water bath, inactivating enzyme in a boiling water bath for 10 minutes, filtering to obtain filter residues, washing the filter residues with distilled water for multiple times until the filter residues are in a clear state when the filter residues are washed for the last time, replacing the washing liquid with 95% ethanol, and washing the filter residues again. And drying the filter residues in a blast drying oven at 60 ℃ for 16 hours to obtain the millettia speciosa champ crude cellulose. Evenly mixing the coarse cellulose of the millettia speciosa champ with a sodium chlorite solution (the pH value of which is adjusted to 3.8-4.0 by using an HCl solution in advance) with the mass fraction of 7.5%, wherein the mass ratio of the coarse cellulose of the millettia speciosa champ to the sodium chlorite solution is 1:20, heating in water bath at 75 ℃ for 2h, and stirring continuously. And then centrifugally collecting and washing the precipitate for multiple times until the supernatant obtained by centrifugation is neutral in pH, then replacing the washing solution with 95% ethanol, washing the filter residue once again, and drying at 60 ℃ for 16 hours to obtain the lignocellulose-removing liquid. Uniformly mixing the delignified cellulose with a KOH solution with the mass fraction of 10%, wherein the mass ratio of the delignified cellulose to the KOH solution is 1:20, continuously stirring at 25 ℃ for 12 hours, filtering and collecting filter residues, taking distilled water as a washing liquid, washing the filter residues for a plurality of times until the filter liquor is neutral in pH, washing the filter residues again by using 95% ethanol, drying at 60 ℃ for 16 hours, crushing and sieving with a 100-mesh sieve to obtain the millettia speciosa champ cellulose, and naming the millettia speciosa champ cellulose as MSCC.
1.4 preparation of MSCCMC
10g MSCC is weighed into a beaker, 200mL of 90% isopropyl alcohol solution (v/v) is added and mixed evenly1.2mL of 30% H was added 2 O 2 The solution (v/v) and 16mL of 50% NaOH solution (w/w) were stirred and mixed at room temperature for 2 hours to activate the hydroxyl groups on the cellulose. Then 14mL of chloroacetic acid solution (w/w) with the concentration of 50% is added into the solution after 2h of reaction, and gradient heating is carried out to etherify the solution, and the specific operation is as follows: the reaction liquid is placed under the condition of room temperature to continue stirring and mixing for 30min, then is placed under the condition of 45 ℃ to continue stirring and mixing for 30min, is placed under the condition of 60 ℃ to continue stirring and mixing for 30min, and finally is placed under the condition of 75 ℃ to stir and mix for 1.5h. After etherification, the etherified product is neutralized and filtered by a small amount of acetic acid solution (v/v) with the concentration of 10%, and the product is repeatedly washed by absolute ethyl alcohol and then dried at 50 ℃. Finally, grinding the product, sieving the product with a 100-mesh sieve to obtain the millettia speciosa champ carboxymethyl cellulose, which is named as MSCCMC and is stored for standby under the condition of room temperature drying.
The infrared spectrogram of MSCCMC is shown in figure 2, and the infrared spectrogram of MSCCMC is similar to that of CMC standard. Two samples at 3464cm -1 The broad absorption peak at the site is caused by the stretching vibration of the hydroxyl group O-H, which indicates that the-OH group, the intramolecular and intermolecular hydrogen bonds are the main bonding bonds of the hydrogel; at 2926cm -1 The absorption peak appearing at the position is the stretching vibration peak of C-H in methyl, methylene and methine; at 1620cm -1 The absorption peak appearing at the position is an O-H bending vibration peak, which is an adsorbed water signal peak caused by the strong interaction between MSCCMC and water molecules; at 1163cm -1 And 1037cm -1 The absorption peaks appearing at these points correspond to the stretching vibration of the c=o group and the asymmetric stretching vibration of C1-O-C4 in the pyranose ring, respectively, which are typical characteristic absorption peaks of MSCCMC, indicating successful preparation of MSCCMC. And at 896cm -1 Is the characteristic absorption peak of the beta-glycosidic bond on the MSCCMC chain.
1.5MSCCMC and MSCC concentration optimization
Dissolution of niu da kura dreg carboxymethyl cellulose (MSCCMC): accurately weighing MSCCMC powder with different mass in a sample bottle, adding NaOH/urea water solution, and standing at room temperature for high-speed stirring overnight to completely dissolve the MSCCMC powder, thereby obtaining MSCCMC solutions with different mass fractions.
Dissolution of bovine total power dreg cellulose (MSCC): accurately weighing MSCC powder with different mass and a sample bottle, adding NaOH/urea aqueous solution, intermittently oscillating to form MSCC suspension, placing in a refrigerator with the temperature of-80 ℃ for freezing for 2 hours, thawing in an ice-water bath, and strongly stirring to obtain MSCC solutions with different mass fractions.
According to the method, MSCCMC and MSCC solutions with the concentration of 1-6wt% are prepared respectively, and the state of the sample before the reaction is recorded by photographing. Then MSCCMC and MSCC solutions with different concentrations are placed at 30 ℃ and stirred for 30min, during which time Epichlorohydrin (ECH) is slowly dripped, 1mL of cross-linking agent is added into each 10mL of solution, the reaction is carried out for 2h under the water bath condition of 60 ℃ to obtain hydrogel, and the state of the reacted sample is recorded by photographing.
Fig. 3 and 4 are morphology graphs of MSCCMC and MSCC concentration determinations. As shown in the figure, 1-2wt% MSCCMC and MSCC solution can not form gel state after adding epichlorohydrin for crosslinking, and still exist in the form of solution. And after the MSCCMC and MSCC solution with the concentration of 3-6wt% are crosslinked, gel is formed to stay at the bottom of the bottle. However, MSCCMC and MSCC hydrogels with a concentration of 3wt% all had severe collapse and no formation during removal; the MSCCMC and MSCC hydrogel with the concentration of 4 weight percent are seriously collapsed after being swelled by water absorption, and are difficult to form; for MSCCMC hydrogel with the concentration of 6wt%, the MSCCMC hydrogel with the concentration of 6wt% is too thick in the dissolution process and is not easy to stir and dissolve, and after epichlorohydrin is added into MSCC solution with the concentration of 6wt% to crosslink to form gel, the gel is easy to crack and has crisp texture. Thus, finally, a 5wt% MSCCMC and 5wt% MSCC solution were selected for subsequent experiments.
1.6 preparation of MSCCMC/MSCC hydrogel
As shown in FIG. 1, solutions of MSCCMC and MSCC having concentrations of 5wt% were prepared according to the method of 1.5, respectively, GEL40, GEL31, GEL22, GEL13 and GEL04 were prepared by mixing the two solutions in the ratio of Table 1, and 5 different hydrogels were placed in deionized water to soak for 3 days to remove unreacted reagents, monomers and oligomers, during which deionized water was continuously replaced.
TABLE 1 preparation parameters of hydrogels
| Hydrogel | 5wt% MSCCMC solution (g) | 5wt% MSCC solution (g) |
| GEL40 | 4 | 0 |
| GEL31 | 3 | 1 |
| GEL22 | 2 | 2 |
| GEL13 | 1 | 3 |
| GEL04 | 0 | 4 |
Example 2: structural analysis of MSCCMC/MSCC hydrogels
2.1 apparent morphology observations
After MSCCMC/MSCC solution reacts for 2 hours under the water bath condition of 60 ℃,5 different hydrogels are taken out of the mold respectively, the state of the hydrogels is recorded by photographing, and the obtained hydrogels are placed in sterile distilled water for swelling balance, and the state of the hydrogels after swelling is recorded by photographing.
The apparent morphology of the MSCCMC/MSCC hydrogels before and after swelling is shown in FIG. 5, and the 5 different MSCCMC/MSCC hydrogels swelled to different extents after absorbing water. With the increase of the MSCCMC content, the swelling capacity of the hydrogel is enhanced, wherein the MSCCMC content in GEL40 is the largest, the swelling degree is the largest, and the volume is the largest; and GEL04 does not contain MSCCMC, does not basically have swelling behavior, and basically does not change the volume.
2.2 scanning Electron microscope analysis (SEM)
Scanning electron microscopy (Scanning Electron Microscopy, SEM) was used to observe and analyze the cross-sectional morphology of 5 different hydrogel samples. And (3) placing the prepared hydrogel sample in distilled water for swelling, placing the hydrogel sample in liquid nitrogen for quick freezing after the swelling is balanced so as to keep the network structure of the hydrogel, and placing the hydrogel sample in a freeze dryer for freeze drying for 48-60 hours so that all the moisture in the hydrogel is completely volatilized. The 5 different freeze-dried hydrogels were carefully and rapidly sliced (taking care of preventing deformation of the hydrogel structure) with a sharp blade, each sample was stuck to a sample stage with a conductive adhesive, and after the section was subjected to a metal spraying treatment, the structure inside each hydrogel sample was observed.
As shown in the SEM image of the MSCCMC/MSCC hydrogel in FIG. 6, the hydrogel has a smooth three-dimensional network structure with continuous pores inside, dense pores are uniformly distributed, and the pore size distribution range is 50-1050 mu m. The continuous porous three-dimensional network structure provides more adsorption sites and interaction sites for lactic acid bacteria, and improves the adsorption performance of the hydrogel.
The 5 different hydrogels have different pore diameters of 1000-1050 μm, 700-750 μm, 400-450 μm, 200-250 μm and 50-100 μm respectively due to different MSCCMC and MSCC contents. The size of the hydrogel pore size increases along with the increase of the MSCCMC content and the decrease of the MSCC content, wherein the GEL40 contains the MSCCMC with the highest content, the degree of crosslinking among the hydrogels is relatively weaker, and the pore size is the largest; and GEL04 does not contain MSCCMC, contains MSCC with the highest content, has relatively less steric hindrance between molecules and epoxy chloropropane as a crosslinking agent, has relatively strong crosslinking degree and has the smallest pore diameter.
2.3 Fourier Infrared Spectroscopy (FTIR)
The freeze-dried hydrogel was mixed with potassium bromide and tabletted, and then subjected to FTIR scanning measurement, and the chemical structure of the hydrogel was analyzed by FTIR, revealing the formation of new functional groups or chemical bonds in the hydrogel and two independently existing network structures. The scanning wavelength range is 4000-400 cm -1 Resolution of 4cm -1 The scanning signals are accumulated for 32 times, and the scanning speed is 0.2cm/s.
FTIR analysis of MSCCMC/MSCC hydrogels As shown in FIG. 7, comparing the IR spectra of MSCCMC powder and various groups of hydrogels, it was found that characteristic absorption peak from 3514cm, which is characteristic of O-H, appears in MSCCMC -1 Migrate to 3451cm -1 This is due to the grafting of epichlorohydrin onto MSCCMC and MSCC. And when MSCCMC forms hydrogel, the hydrogel is 1620cm in length -1 And 1420cm -1 Characteristic absorption peaks of the stretching and bending vibrations belonging to the carboxyl group-COOH still appear, which indicates that the carboxyl group on MSCCMC does not participate in the crosslinking reaction, resulting in the carboxyl group still being present on the backbone of the hydrogel.
Compared with GEL40 hydrogel, GEL31, GEL22, GEL13 and GEL04 hydrogels were 1620cm in length -1 、1420cm -1 、1320cm -1 And 1051cm -1 The characteristic absorption peak intensities appearing there are reduced to different extents, because the carboxyl-COOH related characteristic absorption peak intensities appearing on the infrared absorption spectrum are reduced as the MSCCMC content in the hydrogel is reduced. In addition, the positions of the infrared absorption peaks of the MSCCMC powder and the hydrogels of each group were substantially identical, indicating that no new functional groups were generated during the preparation of the MSCCMC/MSCC hydrogels.
2.4X-ray diffraction analysis (XRD)
A certain amount of the lyophilized hydrogel was placed on an X-ray diffraction apparatus for XRD test under the following conditions: ni filtering, cu target, diffraction angle 2 theta=4 DEG-40 DEG, scanning speed 2 DEG/min, and measuring tube pressure and tube flow respectively at 40kV and 40mA.
XRD analysis of MSCCMC/MSCC hydrogel as shown in fig. 8, GEL40 exhibited a broad diffraction absorption peak with lower intensity at 2θ=21.5°, an XRD characteristic diffraction absorption peak conforming to cellulose II type, shoulder peaks with inconsistent intensity at 2θ=24.2° and 2θ=26.6° respectively, and a relatively weak diffraction absorption peak at 2θ=34.6°. The diffraction absorption peak intensities of GEL31, GEL22, GEL13 and GEL04 at 2θ=21.5°, 2θ=26.6° and 2θ=34.6° are attenuated to different extents compared with GEL40, because the crystallinity increases with the decrease of the MSCCMC content in the hydrogel, and the diffraction absorption peak intensity on the X-ray diffraction pattern increases gradually.
2.5 thermogravimetric analysis (TG)
To examine the thermal stability of the hydrogels, the hydrogels were subjected to thermogravimetric analysis, and the thermal stability of the hydrogels was measured by a thermogravimetric analyzer using a predetermined amount of lyophilized hydrogels. The measurement conditions are as follows: at N 2 The TG and DTG profile of the MSCCMC/MSCC hydrogel was shown in fig. 9, with protection, from room temperature to 500 ℃ at a ramp rate of 10 ℃/min.
As can be seen from the thermal decomposition TG curves of hydrogels in FIG. 9 (a), the weight loss of hydrogels at 50-500℃is divided into three stages, and the initial weight loss of GEL40, GEL31, GEL22, GEL13 and GEL04 occurs at 50-120℃at 50-115℃at 50-113℃at 50-107℃and at 50-106℃at 9.47%, 7.83%, 7.66%, 6.54% and 5.78% respectively, due to the evaporation of surface moisture from hydrogel samples. The second stage of weight loss occurs at 199-328 ℃, 206-334 ℃, 207-346 ℃, 207-376 ℃ and 299-442 ℃, respectively, and the weight loss rates are maximum, namely 51.82%, 53.53%, 61.96%, 66.46% and 79.98%, respectively, due to the thermal decomposition of oxygen-containing functional groups in the hydrogel polymer. The third stage of weight loss occurred after 330 ℃, 340 ℃, 350 ℃, 380 ℃ and 450 ℃ respectively, and the hydrogel samples were further decomposed. There was a significant difference in weight loss temperature and maximum weight loss rate between the 5 hydrogels, with an increase in MSCC content as the MSCCMC content was reduced, the weight loss temperature and maximum weight loss rate gradually increased, indicating that the increase in cellulose increased the thermal stability of the hydrogels.
Finally, the post-pyrolysis residue content of the 5 hydrogels (GEL 40, GEL31, GEL22, GEL13 and GEL 04) was 38.71%, 38.64%, 30.38%, 27.00% and 14.24%, respectively, because with the decrease in MSCCMC content, the steric hindrance of MSCC was smaller than that of MSCCMC, the original bonds and groups were more easily broken, and the crystallinity was decreased, so that GEL40 hydrogels with a large MSCCMC content had a higher residue content instead, while GEL04 hydrogels without MSCCMC had the lowest residue content.
As can be seen from the thermal drop Jie Feng DTG curve of the hydrogels in FIG. 9 (b), 5 hydrogels exhibited an endothermic peak at about 75deg.C, which corresponds to the loss of hydrogels caused by evaporation of surface water during the thermal decomposition TG assay. Whereas a sharp endothermic peak occurs between 270 deg.c and 370 deg.c, which is associated with the loss of weight in the second stage of the thermal decomposition TG diagram, due to the degradation of the hydrogel. The pyrolysis peaks between the 5 hydrogels (GEL 40, GEL31, GEL22, GEL13 and GEL 04) were 273.3 ℃, 287.4 ℃, 293.2 ℃, 300.8 ℃ and 365.7 ℃, respectively. As the MSCCMC content was reduced, the MSCC content increased and the pyrolysis peak of the hydrogel increased gradually, which is consistent with the previous thermal decomposition TG test results.
Example 3: functional analysis of MSCCMC/MSCC hydrogels
3.1 analysis of swelling Capacity of MSCCMC/MSCC hydrogels
The swelling properties of hydrogels are a key indicator for evaluating whether hydrogels can swell to release lactic acid bacteria at specific sites. The swelling performance of the MSCCMC/MSCC hydrogels was tested, 25mg of 5 different MSCCMC/MSCC hydrogels were weighed respectively, placed in a 50mL centrifuge tube, 40mL distilled water was added to make the hydrogels swell by absorbing water, the hydrogels were taken out at intervals, and after the surface water was gently absorbed, the weight of the hydrogel was weighed and recorded. When the hydrogel swells until the weight is unchanged, the swelling balance of the hydrogel is achieved, and the swelling rate of the hydrogel is recorded and calculated according to the following formula.
Swelling ratio SR (%) = (Wt-Wd) ×100/Wd
Wherein Wd represents the initial weight of the dried hydrogel; wt represents the weight after the swelling time t of the hydrogel.
As shown in FIG. 10, the swelling capacity of MSCCMC/MSCC hydrogels is about the same as the swelling trend of different hydrogels in the same solution of the medium, and the swelling trend is that the hydrogel rapidly absorbs water and expands within 30min, the swelling rate is improved to the highest level, and after 30min, the swelling rate of the hydrogels gradually slows down, reaches a peak about 120min and slowly approaches to swelling equilibrium.
As can be seen from the swelling curve and equilibrium swelling ratio of the hydrogel in fig. 10, as the content of MSCCMC increases, the content of carboxyl-COOH groups in the hydrogel increases, so that the hydrophilic property of the hydrogel is enhanced, the swelling ratio increases, so that the swelling capacity of the GEL40 hydrogel with the highest MSCCMC content is strongest, and the equilibrium swelling ratio is 31440 ±210%; the GEL04 hydrogel does not contain MSCCMC, lacks carboxyl-COOH groups, has extremely weak hydrophilic performance, basically does not generate swelling behavior, has worst swelling capacity and has balance swelling rate of 1950+/-30 percent, because the MSCCMC content is reduced, the increase of the MSCC content strengthens the cross-linking among three-dimensional networks of the hydrogel, and a network structure with smaller pore diameter, tighter structure and stronger physical rigidity is formed, so that the water adsorption capacity of the hydrogel is reduced, and the swelling capacity of the hydrogel is weakened.
3.2MSCCMC/MSCC hydrogel pH responsiveness analysis
The pH responsiveness of the hydrogel is a key indicator for evaluating the negative release of lactic acid bacteria by the hydrogel. The pH responsiveness of MSCCMC/MSCC hydrogels was tested and the swelling ratio of the dried hydrogels in solutions of different pH values was determined gravimetrically. Accurately weighing 25mg of the freeze-dried hydrogel, placing in a beaker, adding a proper amount of deionized water, swelling to be balanced at room temperature, taking out, immediately weighing and recording after the excessive water on the surface is absorbed by filter paper, and calculating the swelling rate of the hydrogel under different pH conditions according to the following formula.
Swelling ratio SR (%) = (Weq-Wd) ×100/Wd
Wherein Wd represents the initial weight of the dried hydrogel; weq represents the weight of the hydrogel after swelling equilibrium in solutions of different pH values.
The pH responsiveness of the MSCCMC/MSCC hydrogels is shown in fig. 11, and the pH responsiveness trends of different hydrogels in the same solution in the medium are approximately the same, and the swelling rate of the hydrogels reaches the highest at pH 8.0 as the pH increases, and then the pH responsiveness curves of the hydrogels increase and decrease more significantly as the content of MSCCMC increases.
This is because at low pH, hydrogen bonds between carboxyl-COOH groups in the hydrogel are strong, ionization is suppressed, and electrostatic repulsive force is reduced; at the same time add counter ion Cl - The combined action reduces the effective repulsive force between the hydrogel groups, resulting in a decrease in swelling rate at low pH; with increasing pH, -COOH ionizes to-COO - Hydrogen bonding is disrupted, -COO - The electrostatic repulsive force between the groups is improved, and thus the swelling ratio is increased; and when the pH is increased to 10.0, the swelling medium is subjected to Na as a counter ion + The charge shielding effect of (2) weakens the electrostatic repulsion between anions and the swelling ratio is reduced. The hydrogel forms a shrinkage state in gastric juice with the pH of 2.0, so that moisture is prevented from entering a gel network to take away lactobacillus, the lactobacillus is further prevented from being damaged by substances such as low-pH gastric acid, pepsin and the like, and the bacterial quantity and activity of the lactobacillus are greatly protected; meanwhile, the hydrogel absorbs water in intestinal juice with the pH value of 8.0 to expand, a large amount of water molecules enter a gel network to take away more lactic acid bacteria and are directionally released to specific parts of intestinal tracts, and the swelling difference of the hydrogel under different pH values enables the hydrogel to better realize the targeted release effect on the lactic acid bacteria.
The strength of the functions is related to the content of MSCCMC in the hydrogel, the GEL40 hydrogel contains the highest content of MSCCMC and is most influenced by carboxyl-COOH, and the water absorption capacity is the strongest, so that the GEL40 hydrogel keeps the highest swelling rate under different pH conditions; the GEL04 hydrogel does not contain MSCCMC, the swelling rate is not affected by different pH values basically, the swelling rate is kept low, and the pH response characteristic is not basically shown.
Example 4: preparation of LP-BY 2-loaded MSCCMC/MSCC hydrogel
4.1 Strain activation
Sterilizing the MRS liquid culture medium for 15min at 121 ℃ through an autoclave, cooling to room temperature, inoculating the LP-BY2 preserved in glycerol into the MRS liquid culture medium according to the inoculum size of 2%, culturing at the constant temperature of 37 ℃ for 24h, and continuing to activate for 2-3 times for standby.
4.2 preparation of cell suspensions:
inoculating LP-BY2 into MRS broth culture medium, shaking at 37deg.C 180r/min for overnight culture, centrifuging at 4deg.C 10000r/min for 10min, collecting thallus, washing with PBS solution, centrifuging for 2 times, suspending in PBS solution to obtain LP-BY2 suspension, subjecting LP-BY2 suspension to gradient dilution, and coating and counting BY plate counting method to control colony count at 1×10 10 CFU/mL, and stored in a refrigerator at 4 ℃.
4.3 preparation of LP-BY 2-loaded hydrogel
As shown in FIG. 1, the hydrogel was immersed at a concentration of 1X 10 10 After complete swelling balance in the LP-BY2 bacteria solution of CFU/mL, flushing the residual LP-BY2 on the surface BY deionized water, and carrying out vacuum freeze drying for 48 hours to obtain the freeze-dried MSCCMC/MSCC hydrogel carrying the LP-BY2, and placing the hydrogel in a refrigerator at the temperature of 4 ℃ for standby.
Example 5: determination of LP-BY2 entrapment Rate BY MSCCMC/MSCC hydrogel
Inoculating LP-BY2 into MRS broth culture medium with 2% inoculum size, culturing in an anaerobic incubator at 37 ℃ for 24-48 h, placing activated bacterial liquid at 4 ℃ for 1000r/min, centrifuging for 5min, removing supernatant, adding an equal amount of sterilized PBS to wash precipitate, centrifuging again, washing twice BY the same method, collecting precipitate, adding sterilized PBS, shaking uniformly, diluting to a proper gradient BY 10 times, performing coating counting, and calculating the total number W0 of initial colonies.
Weighing 25mg of freeze-dried hydrogel, placing the freeze-dried hydrogel in 15mL of bacterial liquid for swelling balance, flushing out the surface residual LP-BY2 BY deionized water after the hydrogel is completely swelled, collecting the residual bacterial liquid after swelling, measuring the total number of bacterial colonies of the residual solution BY adopting a plate counting method, and marking the total number as W1. The embedding rate of the hydrogel for LP-BY2 was calculated according to the following formula.
Embedding ratio (%) = (W0-W1) ×100/W0
The embedding effect of the MSCCMC/MSCC hydrogel on the LP-BY2 is shown in FIG. 12, and the encapsulation rate of the hydrogel on the LP-BY2 increases with the increase of the MSCCMC content, because the increase of the MSCCMC content, the increase of carboxyl-COOH groups and the increase of the swelling rate of the hydrogel lead to the higher embedding rate of the hydrogel on the increase of the number of the LP-BY2 entering the gel along with the water molecules. Similarly, yue et al, when studying the encapsulation efficiency of PPA hydrogels with respect to 5-fluorouracil (5-Fu), have also found that as the acrylic content increases, the carboxyl group content increases, resulting in an increase in the encapsulation efficiency of PPA hydrogels.
The MSCCMC content in the GEL40 hydrogel is highest in the 5 hydrogels, and the embedding rate is highest and is 83.34 +/-1.77%; in contrast, GEL04 hydrogel does not contain MSCCMC, contains the highest MSCC content, increases the crosslinking degree of the hydrogel, forms a network structure with smaller pore diameter, tighter structure and stronger physical rigidity, and has the lowest LP-BY2 quantity entering the hydrogel network structure through the diffusion of water molecules and the lowest embedding rate of 41.28+/-2.74 percent. Compared with the sodium alginate microcapsule, the embedding rate of the sodium alginate microcapsule to lactobacillus acidophilus Lactobacillus acidophilus GMCC1.2686 is only 55.5 plus or minus 0.4 percent at the highest. The embedding rate of the hydrogel GEL40 (83.34 +/-1.77%), GEL31 (77.46 +/-0.82%) and GEL22 (74.745 +/-0.085%) on the LP-BY2 is higher than that of the sodium alginate microcapsule on lactobacillus acidophilus Lactobacillus acidophilus CGMCC 1.2686.
Example 6: determination of LP-BY2 Release Rate BY MSCCMC/MSCC hydrogels
The final purpose of microencapsulation of lactic acid bacteria is to test the efficient delivery of human gastrointestinal tract, the lactic acid bacteria microcapsule can maintain the quantity and activity of lactic acid bacteria after being digested by simulated gastrointestinal fluid, and the lactic acid bacteria can be released to a large extent in the intestinal tract, which is an effective means for evaluating the functionality of the microcapsule. And the release capacity analysis of the hydrogel is an important index for evaluating whether the hydrogel can realize targeted release on lactobacillus.
Therefore, 5 different hydrogels are respectively subjected to in-vitro continuous simulated gastrointestinal fluid digestion analysis on release capacity, the simulated gastric fluid digestion time is set to be 2 hours in experiments, the simulated intestinal fluid digestion time is set to be 4 hours, the digestion process of food in the human digestive tract can be effectively simulated, firstly, 25mg of freeze-dried hydrogel is placed in a centrifuge tube containing 15mL of simulated gastric fluid, the freeze-dried hydrogel is placed in a condition of 180r/min at 37 ℃ for release, sampling is carried out every 30min, the sample is placed in a wavelength of 600nm for measuring the absorbance value, and the dissolution condition of LP-BY2 in the simulated gastric fluid is analyzed according to the change of absorbance.
After the hydrogel is placed in simulated gastric fluid for 2 hours, the microcapsule sample is placed in 10000r/min at 4 ℃ for centrifugation for 5 minutes, then the microcapsule sample is added into the simulated intestinal fluid with the same volume for 4 hours, sampling is carried out every 30 minutes, the sample is placed at a wavelength of 600nm for measuring the absorbance value of the sample, the dissolution condition of LP-BY2 in the simulated intestinal fluid is analyzed according to the absorbance change, and the Release Rate (RR) of the hydrogel to the LP-BY2 is calculated according to the following formula.
Release rate RR (%) =w t ×100/W 0
Wherein W is t The OD value of the LP-BY 2-loaded hydrogel releasing the LP-BY2 in different time periods is shown; w (W) 0 Indicating the OD value of the LP-BY 2-loaded hydrogel for accumulated release of the LP-BY2 in the total sustained release period.
The release rate of the MSCCMC/MSCC hydrogel to LP-BY2 is shown in FIG. 13, and 5 hydrogels maintain a low release rate (< 15%) in the simulated gastric digestion process due to the pH sensitivity characteristic of the MSCCMC/MSCC hydrogel, so that the hydrogel is shrunken under the low pH condition with the pH of 3.0, and the loss of lactic acid bacteria is prevented. In the digestion process of simulated intestinal juice, the 5 hydrogels are rapidly absorbed and expanded, and the release rate is greatly increased, because the hydrogen bond action between original carboxyl groups is destroyed under the intestinal juice condition of pH 8.0, the electrostatic repulsive force is improved, the hydrophilic capacity of the hydrogels is enhanced, and most lactic acid bacteria flow out along with water molecules.
The 5 hydrogels have different release capacities due to different MSCCMC contents. With the increase of MSCCMC content and carboxyl-COOH group content, the absorption and swelling capacity of the hydrogel is enhanced, the pH sensitivity is more obvious, and the release capacity is enhanced. Therefore, the GEL40 hydrogel with a high MSCCMC content has the strongest release capacity of 92.86+/-3.57%, while GEL04 contains the largest MSCCM content because of not containing the MSCCMC, has stronger three-dimensional network rigidity of the hydrogel, does not generate swelling behavior, basically has no pH sensitivity, and has the lowest release capacity of 31.58+/-5.27%.
Example 7: survival rate of LP-BY 2-loaded hydrogel in gastrointestinal environment and cholesterol reducing effect thereof
In order to realize the efficient delivery of lactobacillus in human gastrointestinal tract, the hydrogel has the capability of targeting release in intestinal tract, protecting lactobacillus from substances such as low pH gastric acid and pepsin in gastric juice, protecting bile salt, trypsin and the like in intestinal juice, and keeping high survival rate to reach specific parts of intestinal tract. The content of bile salt in different parts of a human body can be changed to a certain extent, usually 0.3-0.5%, and the content of bile salt in the small intestine can be fluctuated between 0.03-0.3%, and the LP-BY2 only can withstand the action of the bile salt in the small intestine and can exert the probiotics on specific parts after a certain number of viable bacteria are maintained.
Thus, 25mg of lyophilized LP-BY2 hydrogel (free LP-BY2 as control group) was added to 15mL of simulated gastric fluid, and after shaking for 10s, the mixture was placed in 180r/min at 37℃for shaking culture, sampled after 2h of treatment, diluted to a suitable gradient, counted BY the coating plate method, and assayed in parallel for 3 times.
Centrifuging the microcapsule sample at 10000r/min at 4deg.C for 10min, adding simulated intestinal juice with the same volume, shaking for 10s, shake culturing at 180r/min at 37deg.C, processing for 4 hr, sampling, diluting to appropriate gradient, coating, counting by plate method, and measuring for 3 times.
Meanwhile, the cholesterol reducing capacity of the LP-BY2 hydrogel is measured, 0.2mL of bacterial liquid cultured for 24h is respectively taken and added into a 10mL centrifuge tube, 4.8mL of absolute ethyl alcohol is slowly added, and the mixture is uniformly mixed BY shaking. Centrifuging for 10-15 min at 3000r/min at 4 ℃, carefully sucking 2mL of supernatant, slowly adding 2mL of ammonium ferric sulfate color developing agent, continuously blowing and mixing uniformly, respectively sucking 200 mu L of ammonium ferric sulfate color developing agent into a 96-hole ELISA plate, measuring the absorbance at 560nm wavelength, and respectively calculating the survival rate and cholesterol reducing capacity of LP-BY2 according to the following.
Survival (%) =lg CFUN1 ×100/lg CFUN0
Wherein N is 0 The number of viable bacteria colonies before treatment is represented; n (N) 1 The number of viable bacterial colonies treated with PBS solution or simulated gastric/intestinal fluid is indicated.
Cholesterol clearance (%) = (a) 0 -A 1 )×100/A 0
Wherein A is 0 Absorbance values representing control groups; a is that 1 The absorbance of the experimental group is shown.
The survival rate of the LP-BY 2-loaded hydrogel in the gastrointestinal environment and the cholesterol reducing effect thereof are shown in tables 2, 3 and 14, the free LP-BY2 has poor resistance to simulated gastrointestinal fluids under the condition of simulated gastrointestinal fluids containing 0.02% of bile salts, and the viable count is greatly reduced in the simulated digestion process. The viable count of free LP-BY2 is 7.115 +/-0.065 lg CFU.mL before digestion of simulated gastrointestinal fluid -1 After 2h of simulated gastric fluid digestion, the activity of the gastric fluid is obviously reduced to 6.23+/-0.03 lg CFU.mL -1 After 4 hours of digestion of simulated intestinal juice containing 0.02% of bile salt, the viable count is 5.00+/-0.15 lg CFU.mL -1 . The lactobacillus which still keeps activity is released into intestinal tracts to act with cholesterol, so as to achieve the effect of reducing cholesterol. The more live lactic acid bacteria are released, the more obvious the cholesterol lowering effect is. After continuous in vitro simulated gastrointestinal digestion, most of the lactic acid bacteria of free LP-BY2 are destroyed, and less part of the lactic acid bacteria are reserved, so that the capacity of reducing cholesterol in intestinal juice is relatively low, which is 34.565 +/-0.525%.
In contrast, 5 hydrogels significantly improved the survival rate (p < 0.05) and cholesterol lowering ability (p < 0.05) of LP-BY2 after digestion with gastrointestinal fluids containing 0.02% bile salts after microencapsulation of LP-BY 2. Under simulated gastrointestinal conditions with 0.02% bile salts, LP-BY2 in 5 hydrogels maintained over 78% survival, with sizes of GEL04 (90.02±0.20%) > GEL13 (87.99±0.04%) > GEL22 (84.54 ±0.32%) > GEL31 (79.73±1.28%) > GEL40 (78.32 ±0.15%). The method is characterized in that when the concentration of bacteria released by each hydrogel is consistent, the hydrogel can absorb water and expand more rapidly in intestinal juice along with the increase of the MSCCMC content, so that lactic acid bacteria are released more rapidly, the protective capability of the lactic acid bacteria is weakened, the survival rate of the lactic acid bacteria is greatly reduced, and the cholesterol reducing capability of the lactic acid bacteria is also greatly reduced. The GEL40 hydrogel has the highest MSCCMC content, and has stronger water absorption capacity and stronger release capacity, so that lactobacillus is more easily released to be damaged by gastrointestinal fluid, thus the survival rate of the lactobacillus is lower, and the cholesterol reducing capacity is also lower and is 38.165 +/-0.245%; the survival rate of LP-BY2 in the GEL31 hydrogel is slightly higher than that of GEL40, but the survival rate is not obviously different (p > 0.05), but the cholesterol reducing capacity is obviously higher than that of the GEL40 hydrogel (p < 0.05), and is 41.745 +/-0.205%; the survival rate and cholesterol reducing capacity of LP-BY2 in GEL22 and GEL13 hydrogels are obviously higher than those of GEL40 (p < 0.05), the GEL04 hydrogel contains highest MSCC content without MSCCMC, basically does not generate swelling behavior, the wrapped lactobacillus is not easy to be damaged BY gastrointestinal fluid, the survival rate of LP-BY2 in the hydrogel is still kept higher, the survival rate is obviously higher than that of GEL40 (p < 0.001), the cholesterol reducing capacity is also obviously higher than that of GEL40 (p < 0.001), and the cholesterol reducing capacity is 54.73+/-0.77%.
When the bile salt content in the simulated gastrointestinal fluid is increased to 0.3%, the resistance of free LP-BY2 to the simulated gastrointestinal fluid is greatly reduced. Before digestion with simulated gastrointestinal fluid, the viable count of free LP-BY2 was 7.425 + -0.05 lg CFU.mL -1 After 2h of simulated gastric fluid digestion, the activity of the gastric fluid is obviously reduced to 6.37+/-0.015 lg CFU.mL -1 After 4 hours of simulated intestinal juice digestion, the viable count is only 1.21+/-0.15 lg CFU.mL -1 Far lower than the minimum concentration (6-7 lg CFU.mL) for health in human body -1 ) The cholesterol reducing capacity is greatly reduced and is only 4.674 +/-1.249 percent. After being embedded BY MSCCMC/MSCC hydrogel, the survival rate (p) of LP-BY2 after being digested BY gastrointestinal fluid containing 0.3 percent of bile salt is obviously improved<0.01)And cholesterol lowering ability (p)<0.01). In 5 hydrogels, as GEL40 only contains MSCCMC, the swelling capacity is strong, LP-BY2 is easily influenced BY high-concentration bile salts and trypsin in intestinal juice, the survival rate is the lowest, 44.72 +/-1.49%, the cholesterol reducing capacity is relatively low, and 28.476 +/-1.792%; the survival rate of GEL31 was slightly higher than GEL40, but there was no significant difference between the two (p>0.05 52.21 + -1.38%, and its cholesterol-lowering ability 33.487 + -2.028%; the survival rate of LP-BY2 in GEL22, GEL13 and GEL04 is significantly higher than that of GEL40 (p <0.01 Also with various degrees of elevation in cholesterol lowering capacity, 40.446 ±1.105%, 43.231 ± 2.267% and 49.566 ±0.526%, respectively.
In conclusion, after digestion of simulated gastrointestinal fluids containing 0.02% bile salts and simulated gastrointestinal fluids containing 0.3% bile salts, 5 hydrogels can significantly improve the survival rate and cholesterol lowering ability of LP-BY 2. As the experiment controls the release amount of the free LP-BY2 to be consistent with that of each hydrogel, 5 hydrogels can absorb water and expand more rapidly in intestinal juice along with the increase of MSCCMC content, and then lactic acid bacteria are released more rapidly, the protective capability of the lactic acid bacteria is weakened, the survival rate of the lactic acid bacteria is greatly reduced, the cholesterol reducing capability of the lactic acid bacteria is also greatly reduced, and the sizes of the lactic acid bacteria are GEL04> GEL13> GEL22> GEL31> GEL40 respectively.
TABLE 2 Effect of MSCCMC/MSCC hydrogels on LP-BY2 survival in simulated gastrointestinal environments with 0.02% bile salts
Note that: ' represents a significant difference between each set of hydrogels and LP-BY2, where p <0.05 and p <0.01; ' # represents a significant difference between the other 4 hydrogels and GEL40, where # represents p <0.05, # represents p <0.01, # represents p <0.001;
TABLE 3 Effect of MSCCMC/MSCC hydrogels on LP-BY2 survival in simulated gastrointestinal environments with 0.3% bile salts
And (3) injection: ' represents a significant difference between each set of hydrogels and LP-BY2, where p <0.05 and p <0.01; ' # represents a significant difference between the other 4 hydrogels and GEL40, where # represents p <0.05, # represents p <0.01;
experiments prove that the carrier hydrogel for targeting slow-release probiotics has higher swelling capacity and pH sensitivity, so that the hydrogel is shrunken in the stomach to reduce the loss of the probiotics, and is water-absorbed and swelled in the intestinal tract to release the probiotics, so that the probiotics are slowly released and play a role in the intestinal tract. The hydrogel has higher embedding capacity, release capacity and probiotics protecting capacity on LP-BY2, can ensure that the LP-BY2 still maintains higher survival rate and cholesterol reducing capacity after stomach passes through simulated gastrointestinal tract, and can be used for developing health-care food or medicine.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The preparation method of the carrier hydrogel for targeting the slow-release probiotics is characterized by comprising the following steps of: the carrier hydrogel is a cross-linked product with a porous network structure, which is obtained by taking the millettia speciosa champ cellulose MSCC and the millettia speciosa champ carboxymethyl cellulose MSCCMC as raw materials and cross-linking the raw materials.
2. The method of manufacturing according to claim 1, characterized in that: the method specifically comprises the following steps:
s1: 5wt% MSCCMC solution and 5wt% MSCC solution were mixed according to 4:0 to 0:4, preparing MSCCMC/MSCC solution according to the mass ratio;
s2: and (3) placing the MSCCMC/MSCC solution at room temperature, stirring to fully mix, slowly dripping the crosslinking agent epichlorohydrin ECH during the stirring, and reacting under the water bath condition to obtain the MSCCMC/MSCC hydrogel.
3. The method of manufacturing according to claim 2, further comprising the step of:
s3: and soaking the MSCCMC/MSCC hydrogel into the probiotic suspension, and performing vacuum freeze drying after complete swelling balance to obtain the carrier hydrogel of the targeted slow-release probiotics.
4. A method of preparation according to claim 2 or 3, characterized in that:
in step S1, the mass ratio is 4:0 to 1:3, a step of; further 3:1 to 1:3, a step of; still further 3: 1-2: 2.
5. A method of preparation according to claim 2 or 3, characterized in that:
in step S1, the preparation method of the 5wt% MSCCMC solution is as follows: mixing MSCCMC powder with NaOH/urea aqueous solution according to a mass ratio of 5:95, preparing;
in step S1, the preparation method of the 5wt% MSCC solution is as follows: MSCC powder and NaOH/urea aqueous solution are mixed according to the mass ratio of 5:95, preparing;
wherein the NaOH/urea aqueous solution is 7wt% NaOH solution and 12wt% urea;
in the step S2, the room temperature is 25-30 ℃;
the stirring time is 25-35 min;
in the step S2, the using amount of the cross-linking agent is 1mL of the cross-linking agent added into each 10 mL-12 mL of MSCCMC/MSCC solution;
in the step S2, the reaction is carried out for 1 to 3 hours under the water bath condition of 55 to 65 ℃.
6. A method of preparation according to claim 3, characterized in that:
in step S3, the concentration of the probiotics in the probiotic suspension is 1×10 9 CFU/mL～1×10 11 CFU/mL；
In the step S3, the time of vacuum freeze drying is 48-72 h.
7. A process according to any one of claims 1 to 3, characterized in that:
the preparation method of the millettia speciosa champ cellulose comprises the following steps:
(1) Collecting the millettia speciosa champ, drying and crushing at 55-65 ℃, and sieving with a 80-100 mesh sieve to obtain millettia speciosa champ powder; evenly mixing the millettia speciosa powder with distilled water, wherein the mass ratio of the feed water to the water is 1:20, heating in a water bath at 90 ℃, stirring for 2-3 h, collecting the precipitate, repeating the operation for more than one time, centrifuging to obtain the precipitate, mixing the precipitate with distilled water, wherein the mass ratio of the material to the water is 1:20, adding alpha-amylase, wherein the addition amount of the alpha-amylase is 0.2% w/w of the weight of the millettia speciosa champ powder, stirring and reacting for 2-3 hours in a water bath at 50-60 ℃, inactivating enzyme in a boiling water bath for 10-15 minutes, filtering, taking filter residues, washing the filter residues with distilled water for multiple times until the filter residues are in a clear state, and then washing the filter residues with 95% ethanol for one time; drying the filter residue at 55-65 ℃ for 16-18 h to obtain the coarse cellulose of the millettia speciosa champ;
(2) Uniformly mixing the coarse cellulose of the millettia speciosa champ and a sodium chlorite solution with the mass fraction of 7.5%, wherein the sodium chlorite solution needs to be adjusted to be 3.8-4.0 by an HCl solution in advance, and the mass ratio of the coarse cellulose of the millettia speciosa champ to the sodium chlorite solution is 1:20, heating in water bath at 70-80 ℃ for 2-3 h, and continuously stirring during the period; centrifuging, collecting and washing the precipitate for multiple times until the filtrate is neutral in pH, washing the filter residue once by using 95% ethanol, and drying at 55-65 ℃ for 16-18 h to obtain lignin-removed cellulose;
(3) Uniformly mixing the delignified cellulose with a KOH solution with the mass fraction of 10%, wherein the mass ratio of the delignified cellulose to the KOH solution is 1:20, continuously stirring at 25 ℃ for 12-14 hours, filtering and collecting filter residues, taking distilled water as a washing liquid, washing the filter residues for a plurality of times until the filter residues are neutral in pH, washing the filter residues once by using 95% ethanol, finally drying at 60 ℃ for 16-18 hours, crushing and sieving with a 80-100-mesh sieve to obtain the millettia speciosa champ cellulose, and naming the millettia speciosa champ cellulose as MSCC;
the preparation method of the millettia speciosa champ carboxymethyl cellulose comprises the following steps:
(a) Weighing the millettia speciosa champ cellulose MSCC, adding an isopropanol solution with the concentration of 90% v/v, uniformly mixing, and then adding H with the concentration of 30% v/v 2 O 2 Placing the solution and NaOH solution with the concentration of 50% w/w under the condition of room temperature, stirring and mixing for 2-3 h to activate hydroxyl groups on cellulose; wherein, the ratio of the bovine lycra cellulose MSCC to the isopropanol solution with the concentration of 90% v/v is 1g:20mL; calf Dali dreg cellulose MSCC, H with concentration of 30% v/v 2 O 2 The ratio of the solution to the NaOH solution with the concentration of 50% w/w is 10g:1.2mL:16mL;
(b) Then adding chloroacetic acid solution with the concentration of 50% w/w into the solution after 2-3 h of reaction, wherein the ratio of the bovine total power dreg cellulose MSCC to the chloroacetic acid solution with the concentration of 50% w/w is 10g:14mL; and carrying out gradient heating to etherify, and the specific operation is as follows: the reaction solution is placed under the condition of room temperature to continue stirring and mixing for reaction for 25 min-35 min, then placed under the condition of 40 ℃ to 50 ℃ to continue stirring and mixing for reaction for 25 min-35 min, then placed under the condition of 55 ℃ to 65 ℃ to stir and mix for reaction for 25 min-35 min, and finally placed under the condition of 70 ℃ to 80 ℃ to stir and mix for reaction for 1 h-2 h; after etherification reaction, neutralizing an etherification product with a small amount of acetic acid solution with the concentration of 10% v/v, filtering, continuously adopting absolute ethyl alcohol to repeatedly wash the product, and then placing the product at 45-55 ℃ for drying treatment; finally, grinding the product and sieving the product with a 80-100-mesh sieve to obtain the millettia speciosa champ carboxymethyl cellulose which is named as MSCCMC.
8. A process according to any one of claims 1 to 3, characterized in that:
the probiotics are intestinal probiotics, and are further lactobacillus paracasei (Lactobacillus paracasei) BY2.
9. A carrier hydrogel of targeted sustained release probiotics, characterized in that the carrier hydrogel is prepared by the preparation method of any one of claims 1-8.
10. Use of a carrier hydrogel for targeting sustained release probiotics as claimed in claim 9, characterized in that:
the application of the carrier hydrogel of the targeted slow-release probiotics in preparing medicines or health foods; or alternatively, the first and second heat exchangers may be,
the application of the carrier hydrogel of the targeted slow-release probiotics in preparing products for reducing serum cholesterol.
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