CN111887428A

CN111887428A - Highland barley probiotic preparation and preparation method thereof

Info

Publication number: CN111887428A
Application number: CN202010675298.1A
Authority: CN
Inventors: 马兴元; 仁青旺姆; 韩明勋; 郑文云; 倪旎; 刘畅; 刘地; 胡发彪
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2020-07-14
Filing date: 2020-07-14
Publication date: 2020-11-06

Abstract

The invention provides a highland barley probiotic preparation and a preparation method thereof, which utilizes bacillus licheniformis to ferment highland barley powder, and comprises the following steps: dissolving highland barley powder according to the mass percent of water of 1: 10-1: 20, adjusting pH to 7.0, inoculating bacillus licheniformis, fermenting at 35-38 ℃, centrifugally collecting fermentation liquor, mixing the fermentation product with sodium alginate solution, and then dripping calcium chloride solution to solidify into particles to prepare the microcapsule. The highland barley probiotics product has good stability and is beneficial to health.

Description

Highland barley probiotic preparation and preparation method thereof

Technical Field

The invention relates to the field of probiotic products, and in particular relates to a highland barley probiotic preparation and a preparation method thereof.

Background

The probiotic preparation can be widely applied to food and medicine, and has the function of coordinating intestines and stomach and the like. With the intensive research on gastrointestinal bacteria, more connection between probiotics and health is found. Thus, probiotic products tend to be more and more abundant and the market is larger. Besides the application in food and medicine, the probiotic preparation has also been applied in agriculture, animal husbandry, etc.

The highland barley is used as a main economic crop of people in Tibet, is planted in places with less environmental pollution, cold climate and higher altitude, has unique nutrition and characteristics, belongs to real green healthy food, has very good development trend in the years, and shows great future development prospect of more and more highland barley products on the market.

Disclosure of Invention

The first purpose of the invention is to provide a preparation method of the highland barley probiotic preparation with good stability.

The second purpose of the invention is to provide the highland barley probiotic microcapsule with good stability.

In order to realize the purpose, the invention provides a preparation method of a highland barley probiotic preparation, which utilizes bacillus licheniformis to ferment highland barley powder, and comprises the following steps of: dissolving highland barley powder according to the mass percent of water of 1: 10-1: 20, adjusting pH to 7.0, inoculating bacillus licheniformis, fermenting at 35-38 ℃, centrifugally collecting fermentation liquor, mixing the fermentation product with sodium alginate solution, and then dripping calcium chloride solution to solidify into particles to prepare the microcapsule.

As a preferred scheme, the mass percentage of the highland barley and the water is 1:15 when the highland barley powder is dissolved.

As a preferred scheme, the fermentation time is 72 h.

The invention provides a highland barley probiotic microcapsule prepared by the preparation method of the highland barley probiotic preparation.

In the fermentation process, fermentation conditions are optimized, single-factor experiments are respectively carried out on the material-water ratio, the initial pH, the inoculation amount of the bacillus licheniformis and the fermentation time, and the influence on the viable count in the fermentation liquid, the fermentation liquid form and the reducing sugar content is researched. Finally, determining the optimal fermentation conditions to prepare the probiotic microcapsule.

The inventors chose to use extrusion to prepare microcapsules. The microcapsules and microspheres of the present invention are the same concept. The prepared microcapsules are divided into two batches, one batch is directly fixed in calcium chloride, and the other batch is soaked in chitosan and then coated with a layer of chitosan. Subsequent experiments mainly evaluate various indexes of the two microcapsules, respectively experiment the apparent form, the survival rate and the acid and alkali resistance of the microcapsules, and finally, the optimal formula is selected to prepare the probiotic preparation. The method comprises the processes of preparing a colloidal solution, adding microorganisms, extruding a cell suspension through a needle or a nozzle, dripping a hardening solution into liquid drops, fixing and the like; the size of the microcapsules depends on the distance of the drop, the diameter of the pinhole and the concentration and composition of the hydrophilic colloid. The average particle size of the microcapsule is about 3mm, and after drying, shrinkage occurs, and the particle size becomes small, about 2.0 mm. The microcapsule is dried in an oven at 45 ℃ for two hours, the embedding rate of the sodium alginate microcapsule is 82.26 percent, and the embedding rate of the sodium alginate-chitosan microcapsule is 86.84 percent.

The invention has the advantages that the highland barley fermentation liquid is obtained by selecting the bacillus licheniformis to ferment the highland barley powder and optimizing the fermentation conditions, the fermentation product is mixed with the sodium alginate solution and then dripped into the calcium chloride to be solidified into particles to prepare the microcapsule, and the highland barley probiotic product has better stability and is beneficial to health.

Drawings

FIG. 1, experimental flow chart.

FIG. 2 shows the growth curve of Bacillus licheniformis.

FIG. 3, glucose standard curve.

FIG. 4 is a schematic diagram of the preparation process of sodium alginate microspheres.

FIG. 5, effect of feed-water ratio on reducing sugar content.

FIG. 6, effect of initial pH on reducing sugars.

Figure 7, microcapsule form before drying.

FIG. 8, the microcapsule size is measured by vernier caliper (left-before drying; right after drying).

FIG. 9 shows wet and dry microcapsules encapsulating Bacillus licheniformis.

Fig. 10, release of two microcapsules.

Detailed Description

Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.

The inventor screens a proper fermentation strain and provides a method for fermenting highland barley powder by taking bacillus licheniformis as the fermentation strain. The experimental procedure of the present invention is shown in FIG. 1.

The inventors chose to use extrusion to prepare microcapsules. The prepared microcapsules are divided into two batches, one batch is directly fixed in calcium chloride, and the other batch is soaked in chitosan and then coated with a layer of chitosan. Subsequent experiments mainly evaluate various indexes of the two microcapsules, respectively experiment the apparent form, the survival rate and the acid and alkali resistance of the microcapsules, and finally, the optimal formula is selected to prepare the probiotic preparation.

First, test materials

1. Main raw material

Highland barley flour (commercially available, from Tibet-Nikken).

2. Experimental strains

CGMCC1.0813 Bacillus licheniformis is provided by China general microbiological culture Collection center (CGMCC) and the culture temperature is 37 ℃.

3. Principal solution

0.5mol/L calcium chloride solution: 55.5g of anhydrous calcium chloride and ultrapure water are added to the solution to reach the constant volume of 1000mL, and the solution is sterilized by high-pressure steam at the temperature of 121 ℃ for 20 min. 0.4% chitosan solution: 4g of chitosan, 200mL0.5mol/L of CaCl2 and 10mL of glacial acetic acid are dissolved to 1000mL, and the pH value is adjusted to 5.0 by 5mol/L of NaOH; 2% sodium alginate solution: dissolving 10g of sodium alginate in 500mL of ultrapure water, heating at 60 ℃, stirring, fully dissolving, ultrasonically removing bubbles to ensure that the solution is uniform, and sterilizing for 20min by high-pressure steam at 121 ℃;

simulated Gastric Fluid (SGF): adding 2.0g NaCl and 3.2g purified pig source protease into 7mL concentrated hydrochloric acid, dissolving, adding distilled water into the solution to reach the constant volume of 1000mL, and the pH value is about 1.2;

simulated Intestinal Fluid (SIF): adding 6.8g KH2PO4 and 10g pancreatin into 800mL distilled water, mixing, adding distilled water to a constant volume of 1000mL, adjusting pH to 7.4 with 0.2N NaOH, and sterilizing with high pressure steam at 121 deg.C for 20 min.

Microsphere lysis solution: the pH was adjusted to 8.0 by adding 0.06M trisodium citrate solution to 0.2M sodium bicarbonate solution.

4. Main culture Medium

LB liquid medium-Yeast extract 0.5%; 1% of sodium chloride; tryptone 1%;

LB solid medium — 1.5% agar added on a liquid basis.

Weighing required reagents in a conical flask according to a proportion, adding water to dissolve completely, and covering a sealing film for autoclaving. The amount of liquid medium in each bottle is one fifth of the bottle capacity, for example 50ml in a 250ml conical bottle. While the solid medium is generally filled in 100ml in a 250ml bottle. The LB medium was sterilized at 121 ℃ for 25 to 30 minutes. After the solid medium was sterilized, the plate was poured on a clean bench. The petri dish is also sterilized and dried in advance. About 6 plates can be poured with 100ml of medium. During operation, the culture medium is preferably at about 50 ℃, and agar is not solidified and is not scalded. The flat plate can be wrapped with sealing film and stored in a refrigerator at 4 deg.C.

5. Primary reagent

Tryptone (OXOID, England), yeast extract (OXOID, England), sodium chloride (ARShanghai Linkun Chemicals Co., Ltd.), glucose (ARShanghai Linkun Chemicals Co., Ltd.), agar (AR Shanghai Tianlian Chemicals Co., Ltd.), inulin (Nanjing Zhonghao bioengineering Co., Ltd.), trehalose (Nanjing Zhonghao bioengineering Co., Ltd.), lactose (AR Shanghai Linkun Chemicals Co., Ltd.), beta-cyclodextrin (Guyao Bo Chemicals Co., Ltd.), soluble starch (AR Shanghai Linkun Chemicals Co., Ltd.), crystal violet dye (BR, Shanghai Alatin Chemicals Co., Ltd.), sodium alginate (Guyao Chemicals Co., Ltd.), chitosan (Shanghai Boo Biotech Co., Ltd.), ethanol (Tantai Kentum Technical Co., Ltd.), citric acid (AR Shanghai Linkun Chemicals Co., Ltd.), disodium hydrogen phosphate (AR shanghai Linfeng Chemicals, ltd.), hydrochloric acid (AR shanghai Linfeng Chemicals, ltd.), and sodium carboxymethylcellulose (national pharmaceutical group Chemicals, ltd.).

6. Main instrument equipment

Beaker, test tube, erlenmeyer flask, glass rod, petri dish, centrifuge tube, graduated cylinder, cuvette, alcohol lamp, weighted paper self-sealing bag, coating rod, inoculating loop, cover glass, slide glass, hemacytometer electronic balance (BSA223S-CW, Sartorius corporation), pH tester (PB-10sartoeius corporation), electron microscope (CX41OLYMPUS corporation), vertical pressure steam sterilizer (SYQ-DSX-280B shanghai shenshengan medical instrument factory), centrifuge (5810Reppendorf), forced air drying cabinet (shanghai-heng science instruments ltd), constant temperature shaking incubator (HZQ-X100A, shanghai-heng science ltd), refrigerator (BCD215DK, qingdahai hei gmbh), ultra clean bench (HJ-CJ-1D brand star instruments ltd), shaker (shanghai-shoji laboratory instruments ltd), ultraviolet-visible light spectrophotometer (UV-7504).

Second, analysis method

And (3) determining the content of reducing sugar:

(1) preparing a standard solution: and (4) standard sugar stock solution (20mg/mL) prepared by respectively weighing 1g of glucose dried at 96 + -2 deg.C for 2 hr, adding water to the solution to obtain a volume of 50mL, and sealing at 4 deg.C for one month. And (3) respectively sucking the sugar standard stock solution into a 10mL test tube, adding deionized water to fix the volume, and respectively corresponding to the concentration standard solutions of 0mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL and 1.0 mg/mL.

(2) Making a standard curve

0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of the glucose standard solution were pipetted into a 10mL stoppered tube, and the volume was adjusted to 1.0mL with distilled water. Adding 1.0mL of.5% phenol solution into the test solution, rapidly adding 5.0mL of concentrated sulfuric acid, standing the reaction solution for 10min, mixing the reaction solutions by using a vortex oscillator, placing the test tube in a water bath kettle at 30 ℃ for reacting for 20min, taking a proper amount of the reaction solution at 49nm, and measuring the absorbance. The glucose mass concentration is used as the abscissa and the absorbance is used as the ordinate, and a standard curve is prepared and shown in FIG. 3.

Third, Experimental methods

1. Preservation of strains

And (3) adopting an LB culture medium for culture, inoculating an LB solid slant culture medium after three passages, and placing the culture medium into a refrigerator at 4 ℃ for preservation by adopting a low-temperature regular planting method.

2. Bacillus licheniformis morphology, singleplex and growth curve

The bacillus licheniformis is shown to be rod-shaped and single-grown through the observation of an oil microscope of a microscope, and on an LB flat plate, the colony is round in shape, and the edge is rough.

The weight of each cell of Bacillus licheniformis was measured and the data are shown in Table 1.

TABLE 1 Bacillus licheniformis singleplex measurement

The culture time of the bacillus licheniformis is 18 h.

The concentration of Bacillus licheniformis was measured every 2 hours and the data are shown in Table 2.

TABLE 2 bacterial count of B.licheniformis at different times

The assay plots the growth curve of B.licheniformis as shown in FIG. 2.

As can be seen from FIG. 2, the concentration was significantly highest at 18 hours, so the incubation time for the spores of lichen was determined to be 18 hours. After 18h, the bacillus licheniformis is in a stable growth stage, the growth of the thallus is slow, microscopic examination can show that the thallus forms spores completely, and cysts are slightly enlarged; after 21h, the bacillus licheniformis starts to enter the decline period of growth, the bacterial number is obviously reduced, and partial spores start to fall off. Therefore, the inoculation age is 18-21 h, and the bacillus licheniformis enters the late logarithmic growth stage, so that the high cell activity can be maintained, and the cell number can be obtained as much as possible.

3. Preparation of probiotic seed fermentation broth

Scraping one ring of well-grown thallus from the strain preservation slant, inoculating into a 250ml triangular flask containing 50ml liquid seed culture medium, and standing and culturing at 37 deg.C for 15-24 hrs. Inoculating the activated seed culture solution into a 250ml triangular flask containing 100ml of production medium at an inoculation amount of 10%, and standing and culturing at 37 ℃ for 15-24hrs for later use.

4. Optimization of fermentation broth preparation

(1) Influence of fermentation conditions on the fermentation broth

Influence of feed-water ratio on reducing sugar content: accurately weighing 10 parts of equivalent highland barley powder, each 5g of highland barley powder is contained in 10 triangular bottles of 250mL and divided into 5 groups, and each group contains 2 parts of highland barley powder as a parallel sample according to the material-water ratio of 1:10, 1:12.5, 1:15, 1:17.5 and 1:20(g/ml) are added with water of 35 ℃, stirred to fully absorb water, sterilized at high temperature for 20min, cooled to about 40 ℃, adjusted to about 5.5 by 10 percent citric acid, then respectively added with 1.5 percent bacillus licheniformis liquid of 1.5 percent and fermented for 3 days at 37 ℃ for 180r/min to obtain highland barley enzymolysis liquid, and the content of reducing sugar is measured when the highland barley enzymolysis liquid is fermented for 24 hours to determine the optimum water ratio. The effect of feed-water ratio on fermentation broth is shown in table 3.

TABLE 3 influence of feed-water ratio on Bacillus licheniformis

The effect of feed water ratio on reducing sugar content is shown in FIG. 5.

As can be seen from FIG. 5, the reducing sugar content is 0.8% at a feed-water ratio of 1:15, and the preferred data is taken as a comprehensive consideration. Therefore, the optimum water ratio was determined to be 1: 15.

Effect of initial pH on reducing sugars: dissolving highland barley powder with the determined optimum water ratio, adding 35 ℃ water, stirring to fully absorb water, sterilizing at high temperature for 20min, cooling to about 40 ℃, adjusting the pH to 6.0, 6.5, 7.0, 7.5 and 8.0 respectively by 10 percent citric acid, respectively 5 groups of 10 samples, respectively adding 1.5 percent bacillus licheniformis liquid at 37 ℃, fermenting at 180r/min for 3 days to obtain highland barley enzymatic hydrolysate, measuring the reducing sugar content of the highland barley at 24h, and determining the optimum initial pH.

The effect of initial pH on reducing sugars is shown in FIG. 6.

As can be seen from FIG. 6, under other constant fermentation conditions, the acidity of the fermentation broth at the initial pH of 6.0 to 7.0 tends to increase with the increase of the initial pH, the acidity of the fermentation broth at the initial pH of 7.0 to 8.0 decreases with the increase of the initial pH, and the reducing sugar of the fermentation broth reaches the maximum value at the initial pH of 7.0. When the environment is acid or alkaline, the activity of enzymes in the probiotics is reduced, the growth capacity is weakened, the acid production capacity is weakened, the content of reducing sugar is small, and therefore the pH value 7.0 is determined to be the optimum fermentation starting pH value for the growth of the probiotics.

Effect of inoculum size on fermentation broth: dissolving highland barley powder with the determined optimum water ratio, adding 35 ℃ water, stirring to enable the highland barley powder to fully absorb water, sterilizing at high temperature for 20min, cooling to about 40 ℃, adjusting the pH to 7.0 by using 10% citric acid, then respectively adding 1%, 1.5% and 2% of three groups of bacillus licheniformis liquid with different inoculation amounts, fermenting at 37 ℃ for 3 days at 180r/min to obtain highland barley enzymolysis liquid, measuring the reducing sugar content in 24h, and determining the optimum inoculation amount of 1.0%. The results are shown in Table 4.

TABLE 4 Effect of inoculum size on fermentation broths

(2) Preparation of probiotic formulations

Preparing probiotic microcapsules by an extrusion method: 1.5 g of sodium alginate was weighed and dissolved sufficiently in 50ml of water. The fermentation broth, about 45ml, was collected by centrifugation, weighed, resuspended in 25ml of water to form a suspension, and poured into sodium alginate, thus prepared containing the bacteria. Preparing two 100ml 2% calcium chloride solutions in two beakers, sucking sodium alginate by a 5ml injector, slowly dripping into calcium chloride, and dripping 20ml into each cup of bacterial liquid to form the microcapsule. One cup of the solution is taken, the microcapsule is obtained by filtration, 100ml of 2 percent chitosan solution is prepared, and the microcapsule is immersed. After 30 minutes of fixation, two portions of the microcapsules were obtained by filtration. Drying in oven at 45 deg.C for two hours, weighing, and storing in self-sealing bag at normal temperature.

The preparation process of the sodium alginate microspheres is shown in figure 4.

(3) Evaluation of relevant Properties of probiotic products

Taking highland barley fermentation for 24h as an example, calculating to obtain the viable count of fermentation liquid to be 4.038 multiplied by 10⁸CFU/mL。

The method for determining the living bacteria survival rate of the probiotic product comprises the following steps:

1. and (3) measuring the number of bacteria in the fermentation liquor: in order to ensure accurate measurement of the number of viable bacteria, the flat plate count method was used, and 3 dilutions were set and made into gradients, each gradient being set to 3 replicates. Measuring quantity, typically 10⁴、10⁵And 10⁶Performing plate culture on the multiple diluted solution; the amount of actinomycetes is determined, and it is generally selectedBy 10³、10⁴And 10⁵Carrying out dilution; the amount of fungi is determined, typically 10²、10³And 10⁴And (5) diluting by times. If the number of colonies in the plate is too small, the counting error is inevitably too large; on the other hand, if the number of colonies in the plate is too large, it causes difficulty in counting. Therefore, the basic principle is to use a dilution plate with 30-300 colonies on the plate as a counting standard. Also, it is required that the standard deviation of the number of colonies on a plate in which the same dilution is repeated 3 times should be smaller than the allowable deviation. In the plate counting detection, if the number of 3 repeated colonies under the same dilution is within the range of 30-300, but the data difference is large, the sample to be detected needs to be abandoned and diluted by a series of 10 times, then bacterial solutions with three dilutions are selected, 0.2ml of the bacterial solutions is respectively taken and put into a sterile plate, then a proper amount of a culture medium which is melted and cooled to about 45 ℃ is poured, the culture medium is uniformly mixed with the bacterial solutions, cooled and solidified, then the culture medium is put into a 37 ℃ culture box or a greenhouse for culture, and the colonies are counted after growing out.

2. Survival rate of Bacillus licheniformis microcapsules: release buffer was first prepared, and 50ml of buffer was prepared with disodium hydrogen phosphate and citric acid, and the pH was adjusted to 7.2. 0.5 g of microcapsules are weighed into the buffer and released by shaking. 1ml of the solution was taken, diluted 105-fold in a gradient, and 100. mu.l of the solution was applied to LB plates and 3 plates. After overnight incubation, the average was calculated by counting colonies to obtain the actual CFU value. Knowing the wet weight of the bacteria in the microcapsules, the bacterial single weight, the dried total weight of the microcapsules and the dilution factor, the theoretical CFU value can be calculated, and the survival rate can be obtained. The microcapsule morphology before drying is shown in figure 7.

Characterization of the microcapsules: the regular spherical shape is not transparent because the bacteria are encapsulated, and the bad smell of the bacillus licheniformis can be effectively removed after the bacillus licheniformis is encapsulated into microcapsules. After the microcapsules were prepared, the particle size of 50 microspheres was measured with a vernier caliper. The particle size of the microcapsules measured by vernier caliper is shown in FIG. 8 (left-before drying; right after drying), and the wet and dry microcapsules encapsulating the Bacillus licheniformis are shown in FIG. 9.

After the microspheres are dried, shrinkage occurs, and the particle size becomes small, about 2.0 mm. The results are shown in Table 5.

TABLE 5 microcapsule particle size

The prepared microcapsules are divided into two batches, one batch is directly fixed in calcium chloride, and the other batch is soaked in chitosan and then coated with a layer of chitosan. The two microcapsules were analyzed for encapsulation efficiency and survival rate and the specific data are recorded in table 6 below.

TABLE 6 encapsulation efficiency and survival rate of two microcapsules

The embedding rate of the sodium alginate microcapsule is 82.26 percent, and the embedding rate of the sodium alginate-chitosan microcapsule is 86.84 percent.

Enteric release control effect of microcapsules: the microcapsules were placed in simulated gastric and intestinal juices and shaken. In gastric juice, the two microcapsule forms are not changed in size. And the sodium alginate microcapsule is gradually swelled and disintegrated in simulated intestinal fluid, and the disintegration of the sodium alginate microcapsule in the intestinal fluid is faster than that of the sodium alginate-chitosan microcapsule. To show more specifically the release of the microcapsules, the OD of the solution was measured every 5 minutes₆₀₀The specific measurement results are shown in Table 7.

TABLE 7 OD values of two kinds of microcapsules in simulated gastric and intestinal fluids at different times

The release of both microcapsules is plotted on the OD scale on the ordinate and the time on the abscissa as 10. The OD values of the two microcapsules in simulated gastric acid were substantially unchanged, indicating that no Bacillus licheniformis was released at all. In simulated intestinal fluid, the release of the sodium alginate microcapsule is obviously faster than that of the sodium alginate-chitosan microcapsule, and the release is much higher when the microcapsule finally tends to be stable. The measurement time was 3 hours.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a highland barley probiotic preparation is characterized in that bacillus licheniformis is utilized to ferment highland barley powder, and the highland barley is prepared by the following steps: dissolving highland barley powder according to the mass percent of water of 1: 10-1: 20, adjusting pH to 7.0, inoculating bacillus licheniformis, fermenting at 35-38 ℃, centrifugally collecting fermentation liquor, mixing the fermentation product with sodium alginate solution, and then dripping calcium chloride solution to solidify into particles to prepare the microcapsule.

2. The preparation method of the highland barley probiotic preparation according to claim 1, wherein the mass percentage of the highland barley and the water is 1:15 when dissolving the highland barley powder.

3. The preparation method of the highland barley probiotic preparation according to claim 1, characterized in that the fermentation time is 72 h.

4. Highland barley probiotic microcapsule prepared by the method for preparing highland barley probiotic preparation as shown in any one of claims 1-3.