CN117384973A - Method for fermenting glucan post-raw meal by using cheese bacillus and application of glucan post-raw meal - Google Patents
Method for fermenting glucan post-raw meal by using cheese bacillus and application of glucan post-raw meal Download PDFInfo
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- CN117384973A CN117384973A CN202311685463.1A CN202311685463A CN117384973A CN 117384973 A CN117384973 A CN 117384973A CN 202311685463 A CN202311685463 A CN 202311685463A CN 117384973 A CN117384973 A CN 117384973A
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- glucan
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- glycinin
- fermenting
- lactobacillus paracasei
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to a method for fermenting glucan metapowder by using Lactobacillus paracasei, and an application thereof. The invention uses the paracasei IOB413 to ferment glycinin-glucan post-raw flour product nutrition component detection and sensitization and digestibility detection, and the results show that the paracasei IOB413 ferment glycinin-glucan post-raw flour has the advantages of low sensitization and good digestibility.
Description
Technical Field
The application relates to the technical field of biological fermentation, in particular to a preparation method and application of glycinin-glucan metapowder fermented by lactobacillus paracasei IOB 413.
Background
Soy proteins have good nutritional and functional properties, and are increasingly used as functional ingredients in food processing, and soy protein products with outstanding functional properties are more required, and the combination of the properties of polysaccharides and the properties of proteins gives proteins new functional properties. The dextran has good water solubility and stability, and has effects of scavenging free radicals, resisting radiation, dissolving cholesterol, and preventing hyperlipidemia. Through extensive literature research, it has been found that the protein-sugar grafting reaction does not require any chemical reagents and can be carried out spontaneously by heating alone. The research shows that the protein-sugar graft has higher stability, obviously improved solubility and emulsification property, can obviously improve the functional properties of soybean protein digestion, sensitization and the like, and has wide application prospect. The protein-sugar grafting opens up a new way for researching and developing a novel solid fermentation base material, improving the functional characteristics of application products and preparing food functional ingredients with ideal functional properties.
The common grafting reaction mainly comprises two methods of dry grafting and wet-heat grafting, but from the aspect of practical operation, the dry grafting and the wet-heat grafting have obvious advantages and disadvantages, and the dry-heat method has long reaction time and strict requirements on reaction conditions. The wet-heat method reaction is fast, but the reaction between the protein and the polysaccharide can not be realized mainly in the reaction between simple protein and monosaccharide or disaccharide, which is not beneficial to the industrialization of the commodity. Therefore, it is necessary to properly adjust the grafting reaction conditions of the protein and polysaccharide to obtain a glycinin-glucan product of excellent quality.
In recent years, the incidence of intestinal diseases is continuously improved due to the problems of rapid pace of life, increased working pressure, irregular diet, lack of exercise and the like of people. Among them, office groups, especially white-collar people, become high-incidence groups plagued by intestinal problems. Intestinal tract diseases are one of chronic diseases which afflict people, have long latency and sudden onset, and are closely related to daily life and eating habits. And the treatment and conditioning time of intestinal diseases is long, the treatment effect is not obvious in a short period, and side effects and drug resistance are easy to generate after long-term administration of the medicine.
More and more researchers try to prevent intestinal diseases and maintain intestinal health in a dietary intervention mode. The metazoan refers to inactivated microorganisms and/or thallus components with definite genetic background beneficial to host health, and researches show that the metazoan can restore intestinal microecological balance, can achieve the aim of effectively assisting in treating diseases, and has the obvious advantages of safe use, no residue, no drug resistance, promotion of nutrient absorption, inhibition of growth of intestinal pathogenic bacteria, improvement of intestinal microecology and the like. The prior literature reports that the application of the strain in the aspect of intestinal health mainly realizes the technical means that the strain is used for improving intestinal microecology balance, relieving intestinal inflammation and treating diseases and assisting in preventing intestinal diseases by methods of preparing a composition and the like, but the technical means that the strain is directly used for improving intestinal microecology balance, relieving intestinal inflammation and treating diseases do not consider the technical problems of absorption and metabolism of a microbial inoculum and optimization of intestinal action and further treatment effect, and the technical means are based on the advantages of a metaproduct in the aspects of intestinal absorption efficiency and intestinal flora regulation.
Therefore, it is urgent to find and develop a post-fermentation metazoan microecological product of lactobacillus paracasei which has good functionality and can also improve intestinal microecological balance, alleviate or assist in treating intestinal diseases.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a preparation method of post-raw metapowder of Lactobacillus paracasei IOB413 and glycinin-glucan products, which are used for improving intestinal microecological balance and relieving or assisting in treating intestinal diseases.
The cheese-making bacillus paracasei IOB413 (Lacticasseibacillus paracasei) used in the invention is separated from natural fermented acid dough in the house of residents in Tianjin, the strain has been subjected to strain preservation and physical and chemical index detection, bacteria identification detection is carried out in China food fermentation industry institute Limited company at 19 days 2021, the colony is white, round, moist and opaque, and the edge is regular, and the colony is preserved in China general microbiological culture collection center (CGMCC) at 29 days 2018, 6 and 29, and the preservation number is CGMCC No.16022.
The technical scheme of the invention is as follows:
the invention provides a preparation method of post-fermentation soybean globulin-glucan metapowder by using Lactobacillus paracasei IOB413, a biological fermentation technical product obtained according to the method and application of the product in preparation of related medicines for preventing and assisting in treating intestinal diseases.
A method for fermenting glucan post-raw meal by using cheese bacillus, which is characterized by comprising the following steps of: fermenting with Lactobacillus paracasei IOB413, adding glycinin-dextran into the fermentation substrate, fermenting, and drying to obtain metapowder.
The invention provides a preparation method of post-production meta-powder of glycinin-glucan products by fermenting Lactobacillus paracasei IOB413, which comprises the following specific steps:
step (1) the strain of the Lactobacillus paracasei IOB413 (preservation number CGMCC No. 16022) is activated and cultured;
the fermentation substrate in the step (2) is a glycinin-glucan product obtained by grafting reaction of soybean powder and polysaccharide;
and (3) fermenting the activated Lactobacillus paracasei IOB413 and the self-made glycinin-glucan product, and drying at 55 ℃ for 24 hours to obtain the Lactobacillus paracasei IOB413 fermented glycinin-glucan metaflour.
The culture temperature in the step (1) is 36+/-2 ℃ and the activation time is 20+/-2 hours;
the grafting modification parameters of the fermentation substrate soybean meal in the step (2) are as follows: and (3) uniformly mixing glycinin and glucan in a mass ratio of 1:1.5 in purified water by adopting ultrasonic waves, and regulating the pH value to 7.0 to obtain a mixture dispersion liquid. And then stirring the mixture dispersion liquid in a water bath at 80 ℃ for reaction for 2 hours at 200r/min, rapidly cooling to room temperature after the reaction is finished, and freeze-drying to obtain the glycinin-glucan grafted product.
The fermentation parameter in the step (3) is that the feed-liquid ratio is 1:1.75, inoculum size 5X 10 7 CFU/mL, culture time is 24+ -2 h, and activity of the obtained solid state fermentation product is 6.78X10 10 CFU/g；
Component measurement of the post-soy globulin-glucan raw meal fermented by the lactobacillus paracasei IOB413 in the step (3): polysaccharides, organic acids, and sensitizers;
verifying the effect of the post-fermentation soybean globulin-glucan metameal of the lactobacillus paracasei IOB413 in the step (3) on relieving intestinal inflammation, preventing intestinal diseases and assisting in treatment;
the invention provides a post-production powder product of glycinin-glucan by fermenting the Lactobacillus paracasei IOB413, which is obtained by a preparation method of the post-production powder of the Lactobacillus paracasei IOB 413.
The invention provides application of the post-fermentation glycinin-glucan metapowder of the Lactobacillus paracasei IOB413 in medicines related to relieving intestinal inflammation, preventing and assisting in treating intestinal diseases.
The beneficial effects of the invention are as follows:
1. the invention provides a preparation method of post-production powder of glycinin-glucan by fermenting Lactobacillus paracasei IOB413, which is used for completing the preparation of the post-production powder of glycinin-glucan by fermenting Lactobacillus paracasei IOB413 and obtaining a corresponding post-production product by grafting modification of a fermentation substrate and high-efficiency fermentation parameters.
2. The invention uses the paracasei IOB413 to ferment glycinin-glucan post-raw flour product nutrition component detection and sensitization and digestibility detection, and the results show that the paracasei IOB413 ferment glycinin-glucan post-raw flour has the advantages of low sensitization and good digestibility.
3. According to the invention, the research on the inhibition of intestinal inflammation by the post-consumer soy globulin-glucan powder product fermented by the Lactobacillus paracasei IOB413 shows that the post-consumer soy globulin-glucan powder fermented by the Lactobacillus paracasei IOB413 has the effects of relieving intestinal inflammation, and preventing and assisting in treating intestinal diseases.
Drawings
FIG. 1 is a graph showing the rate of sensitization decrease. Wherein, sample 1: glycinin-dextran, sample 2: IOB413 fermented soybean metameal, sample 3: IOB413 fermented glycinin-glucan metameal.
FIG. 2 is a bar graph of the molecular weight distribution of the extrameal digests of glycinin-dextran and IOB413 post fermentation, with left hand representation in each bar graph: glycinin-dextran, representing: IOB413 fermented soybean metameal, right representation: IOB413 fermented glycinin-glucan metameal.
Fig. 3 is a diagram of different group weights.
FIG. 4 is a graph showing changes in levels of intestinal inflammatory factor (TNF-. Alpha.) expression in mice of different groups.
FIG. 5 is a graph showing changes in levels of intestinal inflammatory factor (IL-6) expression in mice of different groups.
FIG. 6 is a graph showing changes in levels of intestinal inflammatory factor (IL-1β) in different groups of mice.
FIG. 7 is a graph showing the relative expression levels of ZO-1 in different groups of mice.
FIG. 8 is a graph showing the relative expression levels of Occidins in different groups of mice.
FIG. 9 is a graph showing the relative expression levels of intestinal epithelial cell mucin 2 (MUC 2) in mice of different groups.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The cheese-making bacillus used in the invention IOB413 (Lacticasseibacillus paracasei) is isolated from natural fermented sour dough in the house of the residents in Tianjin, and the strain has been subjected to strain preservation and physical and chemical index detection, and bacteria identification detection is carried out in the company Limited of Chinese food fermentation industry at 3 month 19 in 2021, and the colony is white, round, moist, opaque and clean in edge.
Preservation information of the Lactobacillus paracasei IOB413 used in the present invention: preservation date: 2018, 6, 29, deposit unit: china general microbiological culture Collection center, preservation address: the microbiological institute of China, national academy of sciences, no. 3, beijing, chaoyang, north Chen West way 1, classification and naming: lactobacillus paracasei Lactobacillus paracasei with a preservation number of CGMCC No.16022.
Animal experiments prove that an intestinal inflammation model is established by adopting SPF-grade androstane groups, and after the fermentation of glycinin-glucan by the Lactobacillus paracasei IOB413, the stomach is irrigated with raw meta-powder to serve as a verification experiment;
the detection indexes of the experimental mice are as follows: the changes of the expression level of the mouse morphology, the mouse weight and the mouse intestinal inflammatory factors TNF-alpha and IL-1 beta are detected, and the changes of the expression level of the zonulin ZO-1 and occludin are detected.
Example 1
Preparation method of fermentation substrate soybean powder graft modification
The glycinin-glucan grafted product is prepared by adopting a dry heat grafting reaction method and modifying to a certain extent. And (3) uniformly mixing glycinin and glucan in a mass ratio of 1:1.5 in purified water by adopting ultrasonic waves, and regulating the pH value to 7.0 to obtain a mixture dispersion liquid. And then stirring the mixture dispersion liquid in a water bath at 80 ℃ for reaction for 2 hours at 200r/min, rapidly cooling to room temperature after the reaction is finished, and freeze-drying to obtain the glycinin-glucan grafted product.
Example 2
Preparation method of post-production element of fermented soybean by cheese bacillus paracasei IOB413
(1) Strain activation: inoculating the strain in the freezing tube into a slant culture medium, and placing the slant culture medium at 36+/-2 ℃ for culturing, wherein the activation time is 20+/-2 hours. The activated bacterial colony is white, round, moist in surface, opaque, neat in edge, rod-shaped in microscopic thallus, uniform and thick in individual.
The slant culture medium is MRS culture medium, and the formula is as follows: 10.0 g of peptone, 5.0 g of beef extract, 4.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of Tween 80 and dipotassium hydrogen phosphate (7H) 2 O) 2.0, sodium acetate (3H) 2 O) 5.0 g, triammonium citrate 2.0 g, magnesium sulfate (7H) 2 O) 0.2, manganese sulfate (4H) 2 O) 0.05, 15.0 g of agar, ph=6.2±0.2, and the water addition amount is 1L.
(2) Determining the age of seed liquid: inoculating a ring of fresh slant strain into 25mL bottle containing 2% soybean powder at 36+ -2deg.C, and hermetically standing for 20+ -2 hr.
(3) Determination of solid state process parameters: the material-liquid ratio is 1:1.75, inoculating Lactobacillus paracasei IOB413 on solid medium at an inoculum size of 5×10 7 CFU/mL, 36+ -2deg.C, culturing for 24+ -2 h to obtain solid state fermentation product with activity of 6.78X10 10 CFU/g。
(4) The preparation process of the metapowder comprises the following steps: inoculating the seed solution with optimal fermentation condition to soybean powder, standing at 36+ -2deg.C under sealed condition for 24+ -2 hr, fermenting, and drying at 55deg.C for 24 hr to obtain the final product.
Example 3
Preparation method of post-prebiotics by fermenting glycinin-glucan through lactobacillus paracasei IOB413
(1) Strain activation: inoculating the strain in the freezing tube into a slant culture medium, and placing the slant culture medium at 36+/-2 ℃ for culturing, wherein the activation time is 20+/-2 hours. The activated bacterial colony is white, round, moist in surface, opaque, neat in edge, rod-shaped in microscopic thallus, uniform and thick in individual.
The slant culture medium is MRS culture medium, and the formula is as follows: 10.0 g of peptone, 5.0 g of beef extract, 4.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of tween 80, 2.0 g of dipotassium hydrogen phosphate (7H 2O), 5.0 g of sodium acetate (3H 2O), 2.0 g of tri-ammonium citrate, 0.2 g of magnesium sulfate (7H 2O), 0.05 of manganese sulfate (4H 2O), 15.0 g of agar, pH=6.2+/-0.2 and 1L of water addition amount.
(2) Determining the age of seed liquid: a ring of fresh slant strain is inoculated into 25mL of 2% glycinin-glucan bottle, 36+ -2 ℃, and the culture time is 20+ -2 h.
(3) Determination of solid state process parameters: the material-liquid ratio is 1:1.75, the Lactobacillus paracasei IOB413 is cultured on a solid culture medium with an inoculum size of 5X 107CFU/mL and a temperature of 36+ -2deg.C for 24+ -2 h, and the activity of the obtained solid fermentation product is 6.78X106 CFU/g.
(4) The preparation process of the metapowder comprises the following steps: inoculating the seed solution with optimal fermentation condition to glycinin-dextran, sealing and standing at 36+ -2deg.C for 24+ -2 h, inactivating at 55deg.C after fermentation, and drying to obtain the final product.
Example 4
Determination of nutrient content of raw meal after fermentation of Lactobacillus paracasei IOB413
The reference standards for nutrient component detection are as follows: the detection method of the soybean peptide is GB/T22492-2008 soybean peptide powder; the detection method of the polysaccharide is a phenol-sulfuric acid method for measuring crude polysaccharide in the SN/T24260-2015 outlet plant source food; the organic acid is determined in GB 5009.157-2016 food, and the detection results are shown in the table.
TABLE 5 results of nutrient detection
As is clear from Table 5, during the fermentation of soybeans and glycinin-glucan, lactobacillus paracasei IOB413 is continuously accumulated to increase the content of bioactive substances such as soybean peptide, polysaccharide, organic acid and the like in the post-raw meal obtained by fermentation, so that the growth of beneficial bacteria in intestinal tracts can be promoted, the disturbance of intestinal flora can be eliminated, and various gastrointestinal protection functions such as gastrointestinal tract functions can be enhanced, thereby effectively relieving symptoms such as intestinal inflammation and the like.
Example 5
Method for detecting sensitization and digestibility of raw meal after fermentation of glycinin-glucan and Lactobacillus paracasei IOB413
(1) Sensitization assay
Diluting the glycinin-glucan, the post-fermentation soybean meal of the Lactobacillus paracasei IOB413 and the post-fermentation soybean meal of the Lactobacillus paracasei IOB413 by a certain multiple by using a diluent, adding a diluted sample into a 96-hole ELISA plate of an ELISA kit, adding an antibody working solution, gently shaking uniformly, and incubating for 30 min at 37 ℃; discarding the liquid, adding a washing liquid, keeping for 10 s, washing for 4 times, and beating with water-absorbing paper; after beating to dry, the mixed color developing solution is added, incubated for 15 min at 37 ℃, and finally, the stop solution is added, and after the mixture is evenly mixed with light shaking, the OD value is immediately read by an enzyme labeling instrument 450 nm. The allergen concentration in the sample was calculated according to the kit standard curve.
(2) Digestion assay
A certain amount of pepsin was weighed and a 2.5% by mass pepsin solution was prepared from a 0.2% na Cl solution having ph=2. An amount of trypsin was weighed out and used with 0.68% kh at ph=7 2 PO 4 And 0.062% Na OH mixed solution to prepare 0.5% pancreatin solution. Adjusting p H values of the soybean globulin-dextran, the post-soybean meal fermented by the Lactobacillus paracasei IOB413 and the post-soybean meal fermented by the Lactobacillus paracasei IOB413 to 2, water-bathing at 37 ℃ for 10 min, adding pepsin solution, and water-bathing at 37 ℃ for 60 min. After the gastric enzyme digestion is finished, the pH value of the sample is adjusted to 7, pancreatin is added in a water bath at 37 ℃ for 10 min, the digestion is carried out for 120 min at 37 ℃, the digestion is carried out in a boiling water bath until the temperature is 90 ℃ for 10 min, and the corresponding digestion product is obtained after freeze drying.
(3) Sensitization and digestibility assay results analysis
As can be seen from fig. 1 and 2, compared with glycinin-glucan, the post-production soybean meal fermented by the lactobacillus paracasei IOB413 and the post-production soybean meal fermented by the lactobacillus paracasei IOB413 can reduce sensitization, wherein the sensitization rate of the post-production soybean meal fermented by the lactobacillus paracasei IOB413 is reduced by about 10%, which is helpful for improving intestinal tract immunoregulation ability and keeping intestinal flora balance. Meanwhile, the molecular mass distribution in the protease digestion product of the post-fermentation glycinin-glucan raw meal of the lactobacillus paracasei IOB413 is obviously changed, so that the digestibility of the product is improved, the absorption is facilitated, the growth of beneficial bacteria in the intestinal tract is promoted, the balance of intestinal flora is regulated, and the intestinal inflammation is relieved.
Example 6
Research on inhibition of intestinal inflammation by post-raw meta-powder of Lactobacillus paracasei IOB413
(1) Experimental materials
Experimental animals: SPF-class healthy male mice, weighing 20+ -2 g.
The experimental product comprises the following components: glycinin-glucan, post-production powder of post-production soybean by fermenting with Lactobacillus paracasei IOB413
(2) Raising environment
12 h, light/12 h is circulated at night, the temperature is 22+/-2 ℃, the humidity is 40% -60%, and the food and beverage can be drunk freely.
(3) Mouse modeling and grouping
Mouse modeling: mice were fed adaptively for 7d, and the mice were randomly divided into a blank group, a model group, an experimental control group, an experimental 1 group, an experimental 2 group, and an experimental 3 group. The DSS solution of 30 g/L was consumed freely for a continuous period of 7d. Distilled water is drunk from day 8, distilled water is continuously drunk for 7d, the period of 14 d is 1, the total period of the whole model construction stage is 3, and the total period is 42 d. All mice fed normal maintenance feed, mice in the negative control group are not interfered during the experiment period, the corresponding experimental samples are subjected to gastric lavage intervention in the last period, and the blank group and the model group are subjected to gastric lavage by using physiological saline.
Verification experiment in mice: after modeling is successful, the model-making mice are randomly divided into a model group, an experiment 1 group, an experiment 2 group, an experiment 3 group, 10 model groups, 0.3 ml/group, 1 time/day, 7d of continuous gastric lavage, an experiment 1 group, a gastric lavage unfermented glycinin-dextran sample, 0.1 mL/group, 1 time/day, and 7d of continuous gastric lavage; experiment group 2 is the post-fermented soybean metapowder of the cheese bacillus of intragastric administration IOB413, the intragastric dosage is 0.1 mL per unit, 1 time per day, and the continuous intragastric administration is carried out for 7 days; experiment 3 is that the post-production powder of the glycinin-glucan product fermented by the cheese bacillus paracasei IOB413 is subjected to gastric lavage at a dosage of 0.1 mL per unit, 1 time per d and 7 days after continuous gastric lavage.
(4) Experimental mouse index detection
Morphology observation of mice: during the experimental period, the fur state, the weight, the feces and the like of the mice are observed before the stomach is irrigated every day, and records are made.
Detection of mouse inflammatory factors and anti-inflammatory factors: the content of the inflammatory factors TNF-alpha, IL-1 beta and anti-inflammatory factors IL-10 cytokines in the intestinal tract of the mice is detected by using the kit.
Detection of protein expression: detecting the expression level of the tight junction proteins ZO-1, occlutin and MUC-2 in the colorectal by RT-PCR technology
(5) Experimental results
Mouse morphology: by observing the activity and mental condition of mice, mice in a blank group, a model group and an experimental group are normal in activity and good in mental state.
(1) Body weight of mice
As shown in fig. 3, the death rate of mice during the experiment was zero, the weight of mice induced by 30 g/L DSS was significantly reduced compared to the mice in the blank group, the prognosis of the dried soybean post-meal fermented by glycinin-dextran, lactobacillus paracasei IOB413 and glycinin-dextran post-meal fermented by lactobacillus paracasei IOB413 was increased, and the increase of mice in the group of mice with weight bacteria was more remarkable compared to the group of models.
(2) Mouse inflammatory factor
As shown in fig. 4, 5 and 6, comparing the changes of the expression levels of the intestinal inflammatory factors TNF-alpha, IL-1 beta and IL-6 of different groups of mice, compared with the model group, the soybean post-meal fermented by glycinin-dextran and lactobacillus paracasei IOB413 and the dried post-meal fermented by lactobacillus paracasei IOB413 are found to have a certain effect of relieving the intestinal inflammation of the mice, and the effect of the post-meal fermented by lactobacillus paracasei IOB413 and the post-meal fermented by lactobacillus paracasei IOB413 is more remarkable.
(3) Protein expression level
As shown in fig. 7 and 8, the relative expression levels of zonula occludens (ZO-1) and Occludin (Occludin) were significantly reduced in the mice in the model group compared to the blank group, probably due to the apparent DSS-induced inflammation of colitis, destruction of intestinal epithelial cells, resulting in increased permeability. After the soybean post-baking powder is dried and pre-dried by the soybean globulin-glucan and the lactobacillus paracasei IOB413, the relative expression quantity of ZO-1 and Occludin is increased, which indicates that the relative expression quantity of ZO-1 and Occludin in the mouse intestinal epithelial cells can be improved by the soybean globulin-glucan post-baking powder fermented by the lactobacillus paracasei IOB413 and the soybean globulin-glucan post-baking powder fermented by the lactobacillus paracasei IOB413, so that the intestinal inflammation of the mouse is effectively relieved, and the effect of the soybean globulin-glucan post-baking powder fermented by the lactobacillus paracasei IOB413 is obvious.
As shown in fig. 9, the relative expression of intestinal epithelial cell mucin 2 (MUC 2) in mice from the model group was significantly elevated compared to the blank group, which may be a significant inflammation of DSS-induced colitis, and destruction of intestinal epithelial cells. After the soybean post-production powder is fermented by the gastric lavage soybean globulin-glucan and the paracasei bacillus IOB413, and the relative expression level of MUC2 is obviously reduced after the soybean globulin-glucan post-production powder is fermented by the paracasei bacillus IOB413, the relative expression level of MUC2 in the mouse intestinal epithelial cells can be reduced by the paracasei bacillus IOB413 and the paracasei bacillus IOB413, so that the effect of the paracasei bacillus IOB413 on fermenting the soybean globulin-glucan post-production powder is optimal.
In conclusion, the preparation method of glycinin-glucan is optimized, and the problems of long reaction time, strict control of reaction conditions and the like of the traditional dry grafting method are solved. The sensitization and digestibility of the post-processed soybean glycinin-glucan powder by the Lactobacillus paracasei IOB413 and the research of relieving intestinal inflammation are studied, and the result shows that compared with the post-processed soybean powder by the Lactobacillus paracasei IOB413, the sensitization of the post-processed soybean glycinin-glucan powder by the Lactobacillus paracasei IOB413 is reduced, the digestibility is improved, the intestinal inflammation can be relieved, and the effect of improving the intestinal flora disorder is remarkable. The post-processed soybean globulin-glucan powder product obtained by the method disclosed by the invention can provide a certain pharmacological basis for auxiliary treatment of intestinal inflammation and a new thought for developing application of the post-processed soybean globulin-glucan powder product fermented by the cheese bacillus IOB413 in intestinal disease treatment medicines.
Claims (7)
1. A method for fermenting glucan post-raw meal by using cheese bacillus, which is characterized by comprising the following steps of: fermenting with Lactobacillus paracasei IOB413, adding glycinin-dextran into the fermentation substrate, fermenting, and drying to obtain metapowder.
2. The method of fermenting glucan post-consumer powder with lactobacillus paracasei as claimed in claim 1, wherein: the fermentation medium is MRS medium, and the adding amount of glycinin-dextran is 2-5%.
3. The method of fermenting glucan post-consumer powder with lactobacillus paracasei as claimed in claim 1, wherein: the method comprises the following steps of
Step (1) activation and cultivation of the strain of Lactobacillus paracasei IOB 413;
step (2) preparing glycinin-dextran;
and (3) fermenting the activated lactobacillus paracasei IOB413 and the glycinin-glucan product, and drying the fermentation product at 55 ℃ for 24 hours to obtain the post-production soybean globulin-glucan powder by fermenting the lactobacillus paracasei IOB 413.
4. A method of fermenting glucan post-consumer powder with lactobacillus paracasei as defined in claim 3, wherein: the culture temperature in the step (1) is 36+/-2 ℃ and the activation time is 20+/-2 hours.
5. A method of fermenting glucan post-consumer powder with lactobacillus paracasei as defined in claim 3, wherein: the method for preparing glycinin-dextran in the step (2) comprises the following steps:
mixing glycinin and glucan in a mass ratio of 1:1.5 in purified water by adopting ultrasonic for 1h to uniformly mix the glycinin and the glucan, and regulating the pH value to 7.0 to obtain a mixture dispersion liquid;
and then stirring the mixture dispersion liquid in a water bath at 80 ℃ for reaction for 2 hours at 200r/min, rapidly cooling to room temperature after the reaction is finished, and freeze-drying to obtain the glycinin-glucan grafted product.
6. A method of fermenting glucan post-consumer powder with lactobacillus paracasei as defined in claim 3, wherein: the fermentation parameter in the step (3) is that the feed-liquid ratio is 1:1.75, inoculum size 5X 10 7 CFU/mL, culture time is 24+ -2 h, and activity of the obtained solid state fermentation product is 6.78X10 10 CFU/g。
7. Use of the metapowder prepared according to any one of claims 1 to 6 for preparing a medicament for alleviating inflammation of the intestinal tract, and for preventing and adjunctively treating intestinal diseases.
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| WO2006039768A1 (en) * | 2004-09-17 | 2006-04-20 | Universidade Estadual De Campinas-Unicamp | Fermented functional food on the basis of soy containing probiotics and prebiotics and process of production thereof |
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| CN117050920A (en) * | 2023-10-10 | 2023-11-14 | 天津创源生物技术有限公司 | Preparation method and application of active ingredient of post-raw powder of cheese bacillus paracasei IOB413 |
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| WO2006039768A1 (en) * | 2004-09-17 | 2006-04-20 | Universidade Estadual De Campinas-Unicamp | Fermented functional food on the basis of soy containing probiotics and prebiotics and process of production thereof |
| CN115969957A (en) * | 2022-12-21 | 2023-04-18 | 浙江佰穗莱生命健康科技有限公司 | Compound probiotic preparation for inflammatory bowel diseases and preparation method thereof |
| CN117050920A (en) * | 2023-10-10 | 2023-11-14 | 天津创源生物技术有限公司 | Preparation method and application of active ingredient of post-raw powder of cheese bacillus paracasei IOB413 |
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