CN116162570B - Probiotic composition and preparation method and application thereof - Google Patents

Probiotic composition and preparation method and application thereof Download PDF

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CN116162570B
CN116162570B CN202310047780.4A CN202310047780A CN116162570B CN 116162570 B CN116162570 B CN 116162570B CN 202310047780 A CN202310047780 A CN 202310047780A CN 116162570 B CN116162570 B CN 116162570B
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lactobacillus
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lactobacillus paracasei
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CN116162570A (en
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满朝新
程莎莎
张宇
丁弋芯
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Northeast Agricultural University
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Abstract

The invention discloses a probiotic composition, a preparation method and application thereof, wherein the probiotic composition comprises the following viable bacteria with the ratio of 1:2 embedding Lactobacillus paracasei @Lactobacillus paracasei) JY062 and Bao Maige Lactobacillus gasseriLactobacillus gasseri) JM1 and the probiotic composition has a viable count of at least 10 10 CFU/g. The invention shows better action effect than single strain through probiotic combination. Meanwhile, in order to promote the more effective colonization and proliferation of probiotics in the gastrointestinal tract, the probiotic composition is further embedded and protected, so that the probiotic composition plays a role in stabilizing the gastrointestinal tract. The invention provides a probiotic composition capable of relieving functional dyspepsia, and provides a new scheme for the application of follow-up probiotics in functional foods and medicines for relieving functional dyspepsia.

Description

Probiotic composition and preparation method and application thereof
Technical Field
The invention relates to a probiotic composition, a preparation method and application thereof, in particular to a probiotic composition capable of relieving functional dyspepsia, a preparation method and application thereof, and belongs to the technical field of probiotics.
Background
Functional dyspepsia is one of the most common functional Gastrointestinal (GI) diseases affecting 10% to 30% of the population worldwide. Functional dyspepsia can be subdivided into Postprandial Distress Syndrome (PDS) and Epigastric Pain Syndrome (EPS). According to the roman IV diagnostic criteria published in 2016, there are four major symptoms of functional dyspepsia: postprandial satiety, early satiety, epigastric pain, and epigastric burning. PDS presents as postprandial satiety and early satiety, whereas EPS presents as epigastric pain and epigastric burning, these two subtypes correspond to 67.5% and 48.2% of functional dyspepsia patients, respectively. Wherein the functional dyspepsia is known to be ubiquitous in asian populations. Wherein gastric dyskinesia, psychotic factors, mucosal inflammation, intestinal flora, gastrointestinal hormones, etc. are all considered to play a role. Based on this, prescribed treatments including prokinetic drugs, tricyclic antidepressants, eradication of helicobacter pylori, histamine-2 type receptor antagonists, proton pump inhibitors or selective serotonin reuptake inhibitors, and the like are widely used. However, drug therapy has clinical limitations such as low response and frequent recurrence after withdrawal, with adverse side effects.
The search for healthy therapies and natural substances that reduce the effects of functional dyspepsia has attracted considerable attention in recent years. At present, limitation and side effects of drug treatment can be avoided by alleviating functional dyspepsia through probiotics, and the method is widely paid attention to. More researches show that lactobacillus plantarum CQPC05, bifidobacterium lactis TY-S01, lactobacillus rhamnosus and other strains play an important role in relieving functional dyspepsia by affecting neurotransmitter production pathways, improving gastrointestinal tract regulating peptides, balancing intestinal microbiota and regulating intestinal metabolites. The probiotic compounds including lactobacillus acidophilus LA11-Onlly, lactobacillus rhamnosus LR22, lactobacillus reuteri LE16, lactobacillus plantarum LP-Onlly and bifidobacterium animalis BI516 can promote the colonization of specific strains, improve gastrointestinal tract movement and facilitate the improvement of functional dyspepsia.
The function of probiotics has been well explained, such as regulating intestinal microbiota, alleviating digestive tract dyskinesia and ameliorating inflammation. Further studies on alleviating functional dyspepsia with probiotic compositions of lactobacillus paracasei and lactobacillus grignard are few. The invention further researches the mechanism of relieving the functional dyspepsia by discussing the regulation effects of single probiotics and the compound thereof on the gastric emptying rate, the small intestine propulsion rate, gastrointestinal tissue pathology, gastrointestinal regulatory peptides and Short Chain Fatty Acids (SCFAs) of the functional dyspepsia mice, and provides a functional probiotic combined preparation for relieving the functional dyspepsia.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a probiotic composition which has the function of relieving functional dyspepsia and is superior to a positive drug (mosapride).
Meanwhile, the invention provides a preparation method of the probiotic composition, and the probiotic composition obtained by the preparation method shows higher total bacterial count.
Meanwhile, the invention provides application of the probiotic composition in preparation of a medicament for relieving functional dyspepsia.
In order to solve the technical problems, the invention adopts the following technical scheme:
a probiotic composition comprising a viable count ratio of 1:2 embedding Lactobacillus paracasei @Lactobacillus paracasei) JY062 and Bao Maige Lactobacillus gasseriLactobacillus gasseri) JM1 and the probiotic composition has a viable count of at least 10 10 CFU/g。
The embedded lactobacillus paracasei JY062 is the lactobacillus paracasei JY062 embedded with the protective agent; bao Maige Lactobacillus gasseri JM1 is Lactobacillus gasseri JM1 having the protective agent embedded therein.
The protective agent solution comprises 0.02-0.1 wt% of L-ascorbic acid, 1-5 wt% of glycerol, 5-10 wt% of polydextrose, 2-10 wt% of gelatin and the balance of distilled water; the ratio w/w of the protective agent solution to the thallus is (0.8-2): 1.
a method of making a probiotic composition comprising the steps of:
step one, preparing a protective agent colloid solution according to the proportion, and obtaining the protective agent solution after sterilization treatment;
step two, lactobacillus paracasei JY062 and lactobacillus gasseri JM1 are respectively mixed with the protective agent solution prepared according to the proportion to obtain bacterial colloid;
step three, taking a ratio of 1 according to the number of living bacteria: 2, uniformly mixing the lactobacillus paracasei JY062 colloid and the lactobacillus gasseri JM1 colloid to obtain a mixed bacterium colloid of the lactobacillus paracasei JY062 and the lactobacillus gasseri JM1 embedded with the protective agent;
adding sodium alginate with the concentration of 1-3% w/v into deionized water, wherein the pH value is 6.5-7.0, stirring for 2-3 hours at the temperature of 23-25 ℃ at 1000-1200 rpm until the sodium alginate is completely dissolved, standing the solution for at least 30min, and removing bubbles to obtain a sodium alginate solution;
fifthly, mixing the mixed bacteria colloid according to the volume ratio of 1: (10-20) adding the emulsion into a sodium alginate solution, stirring at 400-800 rpm/min for 15-30 min, and emulsifying for 15-30 min to obtain an emulsion;
step six, a peristaltic pump is used, the flow speed is 4-7 mL/min, the titration height is 5-10 cm, and the emulsion is dripped into 1-3% w/v CaCl arranged on a magnetic stirrer 2 Fixing in the solution for 0.5-2 h, filtering with gauze, and washing with sterile physiological saline for at least 3 times to obtain a composition of the lactobacillus paracasei JY062 and the lactobacillus grignard JM1 embedding the protective agent;
and step seven, obtaining the probiotic composition after freeze drying.
Step one, sterilizing treatment is as follows: sterilizing at 105deg.C for 15min.
The stirring speed of the magnetic stirrer is as follows: 500-1000 rpm/min.
Use of a probiotic composition in the manufacture of a medicament for alleviating functional dyspepsia.
The invention has the following beneficial effects:
since the gastrointestinal tract is a complex environment, the invention shows better effect than single strain through probiotic combination. Meanwhile, in order to promote the more effective colonization and proliferation of probiotics in the gastrointestinal tract, the probiotic composition is further embedded and protected, so that the probiotic composition plays a role in stabilizing the gastrointestinal tract. The invention provides a probiotic composition capable of relieving functional dyspepsia, and provides a new scheme for the application of follow-up probiotics in functional foods and medicines for relieving functional dyspepsia.
Lactobacillus paracasei and Lactobacillus gasseri and combinations thereof are used in this study, but currently there are few functional studies on the combination of both to alleviate functional dyspepsia, and functional strains to alleviate gastrointestinal motility can be enriched. And the strain proportion of the lactobacillus paracasei and lactobacillus grignard composition is determined, and the functional effect of the lactobacillus paracasei and lactobacillus grignard composition is better than that of single bacteria. Thus, a probiotic combination that alleviates functional dyspepsia may be provided for a probiotic formulation.
The composition of the Lactobacillus paracasei JY062 and Lactobacillus gasseri JM1 has the function of relieving functional dyspepsia. The effect of embedding the composite probiotics is most remarkable and is superior to that of a positive medicament (mosapride).
Drawings
FIG. 1 shows the effect of single Lactobacillus paracasei JY062 and Lactobacillus gasseri JM1 and compositions thereof on functional dyspepsia mice according to the present invention, (A) change in gastric emptying rate of mice, (B) change in intestinal motility of mice, and (C) change in water content of feces of mice;
FIG. 2 is a graph of HE staining results (200X) of sections of antrum, small intestine and colon according to the invention;
FIG. 3 is a graph showing the levels of the mouse gastrointestinal peptide and neurotransmitter of the present invention, (A) the level of the gastrointestinal peptide MTL, (B) the level of the gastrointestinal peptide GAS, (C) the level of the gastrointestinal peptide PYY, (D) the level of the gastrointestinal peptide VIP, (E) the level of neurotransmitter 5-HT, and (F) the level of neurotransmitter NO;
FIG. 4 shows the expression of c-kit protein level in the colon and the expression of IOD/AREA in the colon, (A) expression of c-kit protein level in the colon (200X), (B) IOD/AREA in the colon;
FIG. 5 shows the relative expression of mouse-related genes of the present invention, (A) mRNA of Aqp4, (B) mRNA of Aqp8, (C) mRNA of Thp1, (D) 5-HT 4 mRNA of R is expressed relatively. (E) mRNA relative expression of SERT, (F) mRNA relative expression of c-kit, (G) mRNA relative expression of SCF, (H) mRNA relative expression of NOS, and (I) mRNA relative expression of VIPR 1;
FIG. 6 shows the content of SCFAs, (A) acetic acid, (B) acrylic acid, (C) butyric acid, (D) isobutyric acid, (E) valeric acid, and (F) isovaleric acid in the cecum of the mice of the present invention.
Detailed Description
The invention will be further described with reference to the accompanying drawings.
Lactobacillus paracasei JY062 (original Lactobacillus paracasei TD 062) was isolated from Tibetan traditional fermented dairy products and described in Zhang Yu, zhao Guyuan, zheng Jiapeng, manchurian, jiangjun. An adhesion and tolerance evaluation of high-yield exopolysaccharide hypoglycemic Lactobacillus paracasei JY062 (TD 062) [ J ]. China dairy industry, 2022,50 (04): 4-8+13.
Lactobacillus gasseri JM1, isolated from feces of healthy infants, is described in Sun Linlin A study of the immunomodulatory effects of Lactobacillus gasseri and its regulatory pathways [ D ]. Northeast university of agriculture.
Example 1
1.1 Bacterial strains and culture conditions
(1) Bacterial strain source: lactobacillus paracasei JY062 (Lactobacillus paracasei TD 062) and Lactobacillus gasseri JM1 were isolated from the feces of conventional fermented milk products and healthy infants, respectively.
(2) Strain activation: lactobacillus paracasei JY062 isolated from traditional dairy products and lactobacillus gasseri JM1 isolated from feces of healthy infants were treated with 3:1 (fungus liquid: glycerin) and freezing at-20deg.C to obtain frozen solution, wherein the fungus liquid has a concentration of 10 8 CFU/mL. The frozen stock solution of each strain is inoculated into MRS broth culture medium respectively with an inoculum size of 5% (v/v), and is subjected to anaerobic culture at 37 ℃ for 20 hours to obtain first-generation bacterial solution.
MRS broth: 1% (w/v) peptone, 0.8% (w/v) beef powder, 0.4% (w/v) yeast powder, 2% (w/v) glucose, 0.2% (w/v) dipotassium hydrogen phosphate, 0.2% (w/v) diammonium hydrogen citrate, 0.5% (w/v) sodium acetate, 0.02% (w/v) magnesium sulfate, 0.004% (w/v) manganese sulfate, 0.01% (w/v) tween 80, and the balance distilled water.
(3) Strain passage: the first-generation bacterial liquid is inoculated into 12% (w/v) MRS broth culture medium with an inoculum size of 5% (v/v) respectively, and is subjected to anaerobic culture for 20 hours at 37 ℃ to obtain the second-generation bacterial liquid.
(4) And (3) performing expansion culture: the second-generation bacterial solutions were inoculated into 12% (w/v) skim milk powder medium at an inoculum size of 5% (v/v), and subjected to anaerobic culture at 37℃for 16 h.
Skim milk powder medium: 12% (w/v) skim milk powder, the remainder being distilled water.
(5) And (3) collecting thalli: and (5) centrifuging at 5000rpm for 5min respectively, and obtaining the thalli.
(6) Pre-freezing and freeze-drying, and collecting freeze-dried powder. Wherein, the single strains are respectively freeze-dried to obtain non-embedded freeze-dried powder of each single strain; the ratio of viable bacteria number of the composite probiotics is 1 by unencapsulated lactobacillus paracasei JY062 and lactobacillus gasseri JM 1:2, mixing and freeze-drying in proportion; for the composite probiotics embedded with the protective agent, the ratio of the viable bacteria number is 1:2, uniformly mixing the lactobacillus paracasei JY062 colloid and the lactobacillus gasseri JM1 colloid to obtain a mixed bacterium colloid of the lactobacillus paracasei JY062 and the lactobacillus gasseri JM1 embedded with the protective agent, and obtaining the composite probiotics embedded with the protective agent after freeze drying.
(1) Composition and proportion of protective agent
The ratio (w/w) of the protectant solution to the cells was 1.5:1, the protective agent solution is 0.05% (w/w) L-ascorbic acid, 3% (w/w) glycerol, 8% (w/w) polydextrose, 5% (w/w) gelatin, 83.95% (w/w) distilled water.
(2) Embedding and protecting technology
The thallus is mixed with a protective agent colloid solution (105 ℃ C., 15min sterilization treatment) prepared according to a certain proportion to obtain a mixed bacterium colloid. Sodium alginate was added to deionized water at a concentration of 2% (w/v), pH 6.5, and stirred at 1200rpm at 23℃for 2h until sodium alginate was completely dissolved. The solution was allowed to stand for 30min to remove air bubbles. Mixing the mixed bacteria colloid according to the volume ratio of 1:10 is added into sodium alginate solution, stirred for 15min at 500rpm/min and emulsified for 15min. 2% (w/v) CaCl was added dropwise to a magnetic stirrer with a stirring speed of 500rpm/min using a peristaltic pump at a flow rate of 5mL/min and a titration height of 5cm 2 In the solution, after fixing for 1h, the solution was washed 3 times with sterile physiological saline after filtration with gauze.
(3) Embedding and protection principle
The existence of the protective agent can effectively resist inactivation or death caused by the process in the freeze-drying process, so that the death rate of the bacteria in the freeze-drying process is reduced, and the embedding rate of the probiotics is increased. Compared with a single protective agent, the compound protective agent has more obvious protective effect in freeze drying, which mainly means that the compound protective agent can act at different sites at the same time, thereby reducing the influence of external environment on probiotics to the greatest extent. The specific protectant is L-ascorbic acid, glycerol, polydextrose and gelatin.
L-ascorbic acid: antioxidants and semi-permeable protectants. It can pass through cell wall, not cell membrane, and can induce separation of cell cytoplasm wall of thallus before freezing, and form a buffer layer between cell membrane and cell wall to prevent ice crystal growth and provide mechanical protection for membrane. Meanwhile, the probiotic has the function of eliminating oxygen free radicals and increases the stress resistance of probiotics.
Glycerol: permeability protectants. It can inhibit cell dehydration through cell membrane and water molecule combination, weaken ice crystal generation capacity, and increase membrane plasticity; functional groups such as hydroxyl groups contained in glycerol can be combined with water in microbial cells, and the functional groups are combined with molecules such as phospholipid in the cells to form hydrogen bonds, so that a relatively stable structure is formed around the cells; mechanical damage to cells due to moisture curing can also be reduced by passivating the proteins.
Polydextrose: polysaccharide impermeable protective agents. The microbial agent cannot pass through the cell wall, can be adsorbed on the surface of microorganisms to form a viscous layer, increases the solution viscosity to inhibit the growth speed of ice, and reduces the freeze-drying damage of thalli. The hydrogen bond of polysaccharide can be combined with glycoprotein on the cell membrane of microorganism, so that the functional protein, polysaccharide, etc. of the cell are relatively stable; a vitrified protective shell is formed around the cell membrane to protect the spatial structure of the probiotics.
Gelatin: polymeric impermeable protectants. The gelatin can increase the compactness of the protective agent, and along with the increase of the gelatin content in the protective agent, the gelatin concentration is gradually increased, which further promotes the gelation reaction between the gelatin and the sodium alginate, thereby improving the compactness and the tolerance of the sodium alginate microcapsule.
Sodium alginate: the preparation method has the advantages of safety, low cost, biocompatibility, bioavailability and the like, has a buffer function, can protect probiotics from the gastrointestinal environment such as gastric acid and the like, and becomes one of the most commonly used embedded immobilized cell carriers of the probiotics.
(7) Tolerance to artificial gastrointestinal fluids
After the thalli is collected, the initial total bacterial count of the thalli is measured, meanwhile, the same amount of thalli is treated by different protection embedding combinations, the total bacterial count and the survival rate of the thalli are measured after freeze drying, and the tolerance of each freeze-dried sample to the artificial simulated gastrointestinal fluid is detected. Treating in artificial simulated gastric juice for 2-3 hours, then treating in artificial simulated intestinal juice for 4-7 hours, and calculating the total bacterial count and survival rate.
(1) Gastric juice is simulated manually: the pepsin concentration is 0.35%, the NaCl content is 0.3g/100mL, the simulated artificial gastric fluid is adjusted to pH3.0 by 0.1000 mol/L HCl, and the filter membrane of 0.22 mu m is used for filtration and sterilization.
(2) Manually simulating intestinal juice: trypsin concentration of 0.1%, naHCO 3 The simulated artificial intestinal juice with the concentration of 1.1 percent and the NaCl content of 0.3g/100mL is adjusted to pH8.0, and a 0.22 mu m filter membrane is filtered and sterilized for standby.
(3) The protective agent combination is specifically as follows:
combination 0: directly freeze-drying without protection embedding treatment.
Combination 1:3% glycerol, 8% polydextrose, 5% gelatin.
Combination 2:0.05% L-ascorbic acid, 8% polydextrose, 5% gelatin.
Combination 3:0.05% L-ascorbic acid, 3% glycerol, 5% gelatin.
Combination 4:0.05% L-ascorbic acid, 3% glycerol, 8% polydextrose.
Combination 5:1% L-ascorbic acid, 3% glycerol, 8% polydextrose, 5% gelatin.
Combination 6:0.05% L-ascorbic acid, 10% glycerol, 8% polydextrose, 5% gelatin.
Combination 7:0.05% L-ascorbic acid, 3% glycerol, 20% polydextrose, 5% gelatin.
Combination 8:0.05% L-ascorbic acid, 3% glycerol, 8% polydextrose, 20% gelatin.
Combination 9:0.05% L-ascorbic acid, 3% glycerol, 8% polydextrose, 5% gelatin.
1.2 Mouse model and Experimental design
Male KM mice (n= 56,6-7 weeks) were from velariwa laboratory animal technologies limited. Mice were kept at room temperature of 22.+ -. 2 ℃ and humidity of 55.+ -. 5% in an environment with a light/dark cycle of 12 hours. All smallThe rats were free to obtain water and food for the first week to adapt to the environment. After 7 days, mice were divided into 7 groups (n=8), including normal control group (NC), model group (M), lactobacillus paracasei JY062 group (JY), lactobacillus grignard JM1 group (JM), complex probiotic group (CP), complex probiotic group with embedded protectant (ECP) and mosapride (Mos). Each of the other groups except NC group was continuously perfused with loperamide of 5 mg/(kg·d) body weight for 7 days. Then, JY group, JM group, CP group and ECP group were administered at a dose of 10 mg/(kg.d) body weight of 10, respectively 9 CFU/mL of lactobacillus paracasei JY062, lactobacillus casei JM1, complex probiotics (lactobacillus paracasei: lactobacillus griseus=1:2) and complex probiotics with embedded protectant (lactobacillus paracasei: lactobacillus griseus=1:2), were gastric lavaged for two weeks, whereas Mos groups were continuously gastric lavaged with mosapride for two weeks at a dose of 3 mg/(kg·d) body weight, while NC and M groups were given Phosphate Buffered Saline (PBS) at the same dose (i.e., 10 mg/(kg·d)). After the experiment is finished, the cervical vertebra dislocation method of the mice is adopted.
The lactobacillus paracasei JY062, lactobacillus gasseri JM1, the compound probiotics and the compound probiotics embedded with protective agent are all lyophilized bacteria, and the lyophilized bacteria are resuspended into 10 by PBS 9 CFU/mL, the amount of the gastric lavage bacterial suspension was determined based on the body weight of the mice at a dose of 10 mg/(kg.d) body weight.
Each group of lavage is 10 9 CFU/mL total bacterial suspension. A bacterial suspension of 10 for the single bacterial group 9 CFU/mL; the bacterial suspension of the composite bacteria is 10 9 The ratio of the number of living bacteria of CFU/mL is 1:2, and the ratio of the number of living bacteria of CFU/mL is 1/3 and the ratio of living bacteria of CFU/mL are 2/3 for each strain.
1.3 physiological index
Mice were fasted for 24 hours, then injected with 0.5mL of ink intragastrically, and treated by cervical dislocation after 30 minutes.
(1) Weigh the ink weight%a 1 ) Total weight of stomach harmonizinga 2 ) Cleaning the stomach content and weighing the dry stomach net weighta 3 ）。
(2) From pylorus to pylorus measured with rulerDistance between front ends of inkL 1 ) And distance from pylorus to ileocecumL 2 ）。
(3) The moisture content of the feces was determined by lyophilization. Weigh the flat plate weight [ (]b 1 ) Total weight of plate and feces before lyophilizationb 2 ) Total weight of plate and feces after lyophilizationb 3 ). The calculation formula is as follows.
1.4 histological analysis
Gastric sinus, small intestine and colon samples were collected under sterile conditions. After immersing some tissues in 4% paraformaldehyde for 48 hours, histological features were analyzed by Hematoxylin and Eosin (HE) staining of 5mm thick sections.
1.5 determination of gastrointestinal tract modulating peptides
Motilin (MTL), gastrin (GAS), peptide YY (PYY), vasoactive Intestinal Peptide (VIP), 5-hydroxytryptamine (5-HT) were assayed for their concentration in serum by ELISA kit. Nitric Oxide (NO) is determined by a NO content biochemical kit. All procedures were performed as indicated by the kit manufacturer.
1.6 immunohistochemical staining analysis
The colon sections were deparaffinized in xylene, antigen recovered with citrate antigen recovery, cooled to room temperature, blocked with goat serum for 1h, incubated overnight with anti-c-kit antibody at 4 ℃, then incubated with biotinylated secondary antibody for 1h at room temperature, labeled with horseradish enzyme, and incubated for 30min at 37 ℃. The diaminobenzidine color development solution was added for color development, and observed under an optical microscope.
1.7 Reverse Transcription (RT) and quantitative real-time PCR (qPCR)
To investigate the expression of the relevant genes (Aqp 4, aqp8, thp1, 5-HT4R, SERT, c-kit, SCF, VIPR1, NOS), total RNA from colon tissue was isolated with Simple P Total RNA Extraction Kit and cDNA was synthesized using PrimeScript ™ RT kit. All procedures were performed according to the manufacturer's instructions. mRNA levels were determined using Quantum studio 3 real-time PCR system and TB Green Premix Ex Taq ™ II. The relative expression level of mRNA was calculated by the 2-DeltaCt method. The primers used in this study are listed in Table 1.
TABLE 1 specific RT-qPCR primers for target genes
1.8 Determination of SCFAs
The cecal content of the mice was homogenized and sonicated into a suspension and centrifuged at 5000rpm for 20min. Adding 50% H to the supernatant (0.8. 0.8 mL) 2 SO 4 (0.1. 0.1 mL) and an extract (0.8. 0.8 mL, containing internal standard 2-methylpentanoic acid, internal standard content 25. 25 mg/L, methyl tert-butyl ether), shaking uniformly for 10 min, sonicating in ice bath water for 10 min, centrifuging at 10000 rpm for 15min, and transferring the supernatant to a glass bottle for GC-MS analysis.
2.1 Freeze-drying and artificial simulation of influence of gastrointestinal fluid on total bacterial count of bacterial body
The total bacterial count after treatment with combination 9 is shown to be higher, see table 2 below, and after the combination 9 is subjected to a freeze-drying process, the total bacterial count is 10.22+/-0.043 CFU (here, the total bacterial count is CFU, not the bacterial count per unit mass, CFU/g, the sample in the measurement of the total bacterial count of the fixed bacterial sludge (namely, the initial total bacterial count in table 2) is wet weight), and then after the artificial simulated gastrointestinal fluid treatment is carried out, the total bacterial count is 9.07+/-0.046 CFU, and the survival rates are 89.69 +/-0.00% and 79.59 +/-0.006%, respectively. The combination 1-combination 4 are respectively the combination of three protective agents, and compared with the combination 1-combination 4, the combination 9 has higher total bacteria number and survival rate after being treated by the combination 9 than the combination 1-combination 4, which indicates that the combination of 4 protective agents has certain promoting effect; the combination 5-combination 8 is a combination of 4 kinds of protective agents, wherein the addition amount of one protective agent in each combination is obviously higher than the optimal addition range, and compared with the combination 5-combination 8, the combination 9 has higher total bacterial count and survival rate after being treated by the combination 9 than the combination 5-combination 8, which indicates that the protective effect of the protective agent on bacterial activity is reduced when the addition amount of the protective agent is higher.
TABLE 2 viable count cases of different combinations of treatment with embedded freeze-dried and artificial simulated gastrointestinal fluids
Note that: the survival rate is the ratio of the total bacterial count to the initial total bacterial count.
2.2 Effect on mouse physiological index
As shown in a and B of fig. 1, the gastric emptying rate and intestinal propulsion rate of group M were significantly lower than NC (P < 0.05), but the trend was reversed after probiotic intervention, the effect of compound probiotic intervention was superior to single bacterial intervention. Of these, CP, ECP, mos had no significant difference (P > 0.05) from NC, but the gastric emptying rate was significantly higher in ECP than in CP and Mos, while small intestine propulsion rate was not significantly different (P > 0.05) between CP, ECP, mos, but the ECP was higher than in other groups. Meanwhile, the moisture content of the excrement is increased after intervention, the low level (P < 0.05) of M groups is obviously improved (figure 1C), and the effect of the composite probiotics embedded with the protective agent is optimal after intervention. In conclusion, the lactobacillus paracasei JY062 and the lactobacillus gasseri JM1 can relieve functional dyspepsia to a certain extent, so that the composite probiotics embedded with the protective agent are more effective, and the trend of the composite probiotics is better than that of mosapride in physiological indexes.
2.3 histological analysis of mice
Damage to the antrum, small intestine and colon has a direct or indirect effect on gastrointestinal function. Histological analysis of HE staining was performed on each of the above tissues (fig. 2). The results show that the local mucosal epithelial cells of the M groups of antrum are erosive and shed (red arrows), and the lower layer is infiltrated by a large amount of inflammatory cells (blue arrows). In addition, the villus structure of the small intestine is poor, the length is obviously shortened, and inflammatory cells are present (blue arrows). Furthermore, inflammatory cells infiltrate the colonic mucosa, and submucosal edema is evident (black arrow). Through the intervention of the Lactobacillus paracasei JY062 and the Lactobacillus gasseri JM1, the damage of the antrum, the intestinal tract and the colon is relieved to different degrees, the improvement effect of the CP group is better than that of the JY group and the JM group, and the ECP group and the Mos group are close to the NC group. And the presence of panda cells (yellow arrows) and goblet cells (green arrows) in the small intestine and colon may be related to the body's stress response to the external environment. The result shows that the intervention of single lactobacillus paracasei JY062, lactobacillus gasseri JM1 and compound probiotics can reduce the damage degree, and further facilitate the intestinal tract to play a role so as to relieve functional dyspepsia.
2.4 effects on mouse gastrointestinal active peptides and neurotransmitters
The levels of the gastrointestinal active peptide (MTL, GAS, PYY, VIP) and neurotransmitters (5-HT, NO) were determined. The results show (figures 3A and B) that the content of MTL and GAS is improved after probiotic intervention, significantly higher than in group M (P < 0.05), and the intervention effect of the complex probiotics is higher than that of the single bacteria, optimal for ECP group, better than that of Mos group. PYY and VIP are inhibitory modulators of gastrointestinal hormones, which inhibit gastric acid secretion and gastrointestinal motility. The PYY levels were significantly elevated in the M group compared to the NC group (P < 0.05), and this trend was reversed after probiotic intervention, especially in the CP and ECP groups (fig. 3C). Only the ECP and Mos groups significantly differed from the M group on VIP (P < 0.05) (fig. 3D), indicating that the complex probiotics with embedded protectants and mosapride dry prognosis can reduce VIP levels. Excitatory factor 5-HT and inhibitory factor NO are critical in regulating the gastrointestinal tract. The results show that the levels of 5-HT increased significantly after probiotic intervention (P < 0.05) compared to group M, whereas NO showed the opposite trend, with a more pronounced degree in the ECP group (FIGS. 3E and F). Therefore, lactobacillus paracasei JY062 and lactobacillus gasseri JM1 can relieve functional dyspepsia by regulating the level of gastrointestinal regulatory peptide, so that the effect of the composite probiotics embedded with the protective agent is most obvious.
2.5 Effect on mouse c-kit protein
c-kit is a transmembrane receptor, producing the primary signal that activates the interstitial cells (ICCs) of Cajal. Tissue sections (200×) see fig. 4A, the brownish yellow portion indicates positive expression of antibody-labeled c-kit protein. The brown-yellow portion of the M group was severely shallower compared to the NC group, while the brown-yellow portions of the JY group and JM group were deepened, especially the MIX group (i.e., the CP group and the ECP group). The average optical density (IOD/AREA) results showed (fig. 4B) that the c-kit protein levels were significantly higher in the complex probiotic group compared to the M group compared to the single bacterial group, and the ECP group was significantly higher than the CP group (P < 0.05). The c-kit protein levels of the ECP group showed no significant difference (P > 0.05) from those of the Mos group, but tended to be higher than those of the Mos group. Therefore, the single strain and the compound strain of the lactobacillus paracasei JY062 and the lactobacillus griseus JM1 can increase the expression of c-kit protein, and the compound probiotics effect of the embedded protective agent is good, so that the increase of the dry prognosis ICC amount can be further promoted, and the promotion of intestinal power is facilitated to relieve the functional dyspepsia.
2.6 Effect on mouse-associated Gene expression
Aquaporins are proteins on the cell membrane that selectively transport water molecules. The expression levels of Aqp4 and Aqp8 were significantly increased in the M group compared to the NC group (P<0.05 And the prognosis was reversed in the strain and positive drug intervention (fig. 5A and B), where the expression of Aqp4 and Aqp8 was significantly lower in the ECP group than in the Mos group, improving intestinal lumen water uptake. Meanwhile, the water content in the feces shows an ascending trend. After probiotic intervention, mRNA expression of Tpp 1 and 5-HT4R was up-regulated, mRNA expression of SERT was down-regulated, thp1 and 5-HT4R had more pronounced effects in ECP group (FIG. 5C-E), promoting 5-HT and 5-HT 4 And R is combined. Wherein 5-HT is synthesized by Enterochromaffin Cells (EC) mainly under the action of tryptophan hydroxylase 1 (Tpp 1) and released into the lamina propria, acting by activating the corresponding 5-HT receptor, 5-HT 4 R is mainly responsible for regulating intestinal secretion. The 5-HT is inactivated by the action of 5-hydroxytryptamine transporter (SERT) and the reaction is terminated. Downregulation of mRNA expression of SERT may promote a sufficient response to 5-HT. mRNA expression of c-kit and SCF increased following probiotic intervention (FIGS. 5F and G), while protein expression of the above c-kit increased. Dimers formed by binding of Stem Cell Factor (SCF) and ICC's tyrosine kinase receptor c-kit promote proliferation and differentiation of ICC. And ICC is an important pacemaker cell in the intestine, responsible for slow wave potential generation and smooth muscle contraction. Reduced expression of NOS and VIPR1 (fig. 5H and I) promoted a decrease in NO and VIP, but VIP changes were not significant in the JY, JM and CP groups, and other mechanisms of influence or associated with probiotic damage in the gastrointestinal tract may be present. To sum upSingle and composite strains of lactobacillus paracasei JY062 and lactobacillus gasseri JM1 can be used to facilitate the relief of functional dyspepsia by improving the expression of the regulator mRNA.
2.7 Effect on mouse SCFAs
Current studies indicate that SCFAs levels can affect intestinal motility in mice, and that some probiotics have been shown to regulate intestinal SCFAs production. The content of SCFAs (acetic acid A, propionic acid B, butyric acid C, isobutyric acid D, valeric acid E and isovaleric acid F) in the M groups is obviously reduced (P < 0.05), the SCFAs are improved to different degrees after intervention, probiotics are easier to promote the production of the SCFAs than mosapride, and the effect of the compound probiotics is better than the intervention effect of single bacteria, so that the ECP groups are most obvious. SCFAs promote intestinal peristalsis by promoting the absorption of water and electrolyte, participate in glucose production, and provide energy for a wide range of metabolism and intestinal peristalsis. Thus, the complex strain of Lactobacillus paracasei JY062 and Lactobacillus gasseri JM1 can alleviate functional dyspepsia by increasing SCFAs.
Example 2
The effect of the protective agent-embedded complex probiotics (lactobacillus paracasei: lactobacillus grignard = 1:1) on treating functional dyspepsia was inferior to that of example 1.
Example 3
The effect of the protective agent-embedded complex probiotics (lactobacillus paracasei: lactobacillus grignard = 1:3) on the treatment of functional dyspepsia was inferior to that of example 1.
Example 4
This embodiment differs from embodiment 1 only in that:
(1) composition and proportion of protective agent
The ratio (w/w) of the protectant solution to the cells was 0.8:1, the protective agent solution is 0.02% (w/w) L-ascorbic acid, 1% (w/w) glycerol, 5% (w/w) polydextrose, 2% (w/w) gelatin, 91.98% (w/w) distilled water.
(2) Embedding and protecting technology
The thallus is mixed with a protective agent colloid solution (105 ℃ C., 15min sterilization treatment) prepared according to a certain proportion to obtain a mixed bacterium colloid. Adding seaweed into deionized waterSodium alginate, 1% (w/v) at pH 7.0, was stirred at 1000rpm at 25℃for 3h until sodium alginate was completely dissolved. The solution was allowed to stand for 60 min to remove air bubbles. Mixing the mixed bacteria colloid according to the volume ratio of 1:20 is added into sodium alginate solution, stirred at 400rpm/min for 30min and emulsified for 30min. Using peristaltic pump, flow rate was 4mL/min, titer height was 10cm, and 1% (w/v) CaCl was added dropwise to a magnetic stirrer at a stirring speed of 1000rpm/min 2 After fixing in solution for 0.5h, the solution was washed 4 times with sterile physiological saline after filtration with gauze.
Example 5
This embodiment differs from embodiment 1 only in that:
(1) composition and proportion of protective agent
The ratio (w/w) of the protective agent solution to the thalli is 2:1, the protective agent solution is 0.1% (w/w) L-ascorbic acid, 5% (w/w) glycerol, 10% (w/w) polydextrose, 10% (w/w) gelatin, 74.9% (w/w) distilled water.
(2) Embedding and protecting technology
The thallus is mixed with a protective agent colloid solution (105 ℃ C., 15min sterilization treatment) prepared according to a certain proportion to obtain a mixed bacterium colloid. Sodium alginate was added to deionized water at a concentration of 3% (w/v), pH 7.0, and stirred at 1100rpm at 24℃for 2.5h until the sodium alginate was completely dissolved. The solution was allowed to stand for 40 min to remove air bubbles. Mixing the mixed bacteria colloid according to the volume ratio of 1:15 is added into the sodium alginate solution, stirred for 20min at 700rpm/min and emulsified for 20min. 3% (w/v) CaCl was dropped onto a magnetic stirrer with a stirring speed of 800rpm/min using a peristaltic pump at a flow rate of 7mL/min and a titration height of 8cm 2 In the solution, after fixing for 2 hours, the solution is washed 5 times with sterile physiological saline after being filtered by gauze.
The foregoing is only a preferred embodiment of the invention, it being noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (5)

1. A probiotic composition comprising a viable cell count ratioThe value is 1:2 embedding Lactobacillus paracasei @Lactobacillus paracasei) JY062 and Bao Maige Lactobacillus gasseriLactobacillus gasseri) JM1 and the probiotic composition has a viable count of at least 10 10 CFU/g；
The embedded lactobacillus paracasei JY062 is the lactobacillus paracasei JY062 embedded with the protective agent; bao Maige Lactobacillus gasseri JM1 is Lactobacillus gasseri JM1 embedded with protective agent;
the protective agent solution comprises 0.02-0.1 wt% of L-ascorbic acid, 1-5 wt% of glycerol, 5-10 wt% of polydextrose, 2-10 wt% of gelatin and the balance of distilled water; the ratio w/w of the protective agent solution to the thallus is 0.8-2: 1, a step of;
the composition has the function of relieving functional dyspepsia and is superior to positive medicine mosapride.
2. A method of preparing a probiotic composition according to claim 1, comprising the steps of:
preparing a protective agent colloid solution according to a proportion, wherein the protective agent colloid solution comprises 0.02-0.1wt% of L-ascorbic acid, 1-5wt% of glycerol, 5-10wt% of polydextrose, 2-10wt% of gelatin and the balance of distilled water; the ratio w/w of the protective agent solution to the thallus is 0.8-2: 1, a step of; after sterilization treatment, a protective agent solution is obtained;
step two, lactobacillus paracasei JY062 and lactobacillus gasseri JM1 are respectively mixed with the protective agent solution prepared according to the proportion to obtain bacterial colloid;
step three, taking a ratio of 1 according to the number of living bacteria: 2, uniformly mixing the lactobacillus paracasei JY062 colloid and the lactobacillus gasseri JM1 colloid to obtain a mixed bacterium colloid of the lactobacillus paracasei JY062 and the lactobacillus gasseri JM1 embedded with the protective agent;
adding sodium alginate with the concentration of 1-3% w/v into deionized water, wherein the pH value is 6.5-7.0, stirring for 2-3 hours at the temperature of 23-25 ℃ at 1000-1200 rpm until the sodium alginate is completely dissolved, standing the solution for at least 30min, and removing bubbles to obtain a sodium alginate solution;
fifthly, mixing the mixed bacteria colloid according to the volume ratio of 1: adding 10-20 parts of sodium alginate solution, stirring at 400-800 rpm/min for 15-30 min, and emulsifying for 15-30 min to obtain emulsion;
step six, a peristaltic pump is used, the flow speed is 4-7 mL/min, the titration height is 5-10 cm, and the emulsion is dripped into 1-3% w/v CaCl arranged on a magnetic stirrer 2 Fixing in the solution for 0.5-2 h, filtering with gauze, and washing with sterile physiological saline for at least 3 times to obtain a composition of the lactobacillus paracasei JY062 and the lactobacillus grignard JM1 embedding the protective agent;
and step seven, obtaining the probiotic composition after freeze drying.
3. The method of claim 2, wherein the sterilizing process comprises: sterilizing at 105deg.C for 15min.
4. The method according to claim 2, wherein the magnetic stirrer has a stirring speed of: 500-1000 rpm/min.
5. Use of a probiotic composition according to claim 1 for the preparation of a medicament for alleviating functional dyspepsia.
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