CN116970501B

CN116970501B - Triplex solid fermentation preparation and preparation method and application thereof

Info

Publication number: CN116970501B
Application number: CN202310944965.5A
Authority: CN
Inventors: 宋亚囝; 包莉媛; 张禾宁; 甄学微; 周飒; 罗学刚
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2023-07-31
Filing date: 2023-07-31
Publication date: 2024-05-14
Anticipated expiration: 2043-07-31
Also published as: CN116970501A

Abstract

The invention discloses a triple fungus solid fermentation preparation, a preparation method and application thereof, and relates to the technical field of biology. The preparation method comprises the step of carrying out solid state fermentation on the raw materials for preparing the medicated leaven by utilizing a microorganism combination to prepare the triple fungus solid state fermentation preparation. The microorganism combination comprises Kluyveromyces marxianus (Kluyveromyces marxianus) JSA16, lactobacillus plantarum (Lactiplantibacillus plantarum) BY3 and Bacillus subtilis (Bacillus subtilis) BK3. The invention firstly uses Kluyveromyces marxianus, lactobacillus plantarum and bacillus subtilis to carry out mixed solid state fermentation, and develops a triple fungus solid state fermentation preparation which can relieve food retention and promote digestion and has the effect of relieving colonitis.

Description

Triplex solid fermentation preparation and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a triple fungus solid fermentation preparation and a preparation method and application thereof.

Background

Probiotics are living microorganisms that are capable of providing health benefits to a host when administered in amounts and contain an active amount during the useful life and are safe for their intended use. Many studies have shown that probiotics can enhance human health in a variety of ways, which can improve intestinal microbial composition, regulate human immune function, and prevent or reduce the occurrence of a variety of diseases.

Solid state fermentation is one of fermentation processes, mainly refers to one or more microbial fermentation processes in an environment without any free water, or with water of a certain humidity but not dissolved in a solid substrate, and the whole process is mainly biological reaction; in a broader definition, solid state fermentation can be regarded as a process comprising culturing microorganisms in the presence of a liquid phase of a maximum substrate concentration or inert carrier. Compared with other fermentation processes, the solid fermentation medium has wide and easily obtained raw materials, and most of the raw materials are residues in the agricultural food industry; the wastewater yield is low, the use of chemical substances such as defoamer and the like can be avoided, and the environmental protection advantage is realized; the cost of the culture medium is low, the water consumption is low, the conversion rate of the product is high, and the method has good economic benefit.

The solid state fermentation can simultaneously convert macromolecules and anti-nutritional factors which are difficult to utilize in agricultural residues such as bran, plant meal cake and the like into nutritional feed which can be absorbed and utilized. The nutrition characteristic, digestibility, palatability and safety of the feed can be improved after fermentation treatment.

Massa Medicata Fermentata was originally recorded in Ben Cao gang mu, and is also discussed in Yao xing Lun. The solid fermentation preparation is a starter prepared by mixing wheat bran, flour, bitter apricot kernel, red bean, fresh sweet wormwood, fresh polygonum hydropiper and fresh xanthium in a certain proportion and then fermenting by microorganisms, and is mainly used for treating weakness of spleen and stomach, chest and abdominal distention, food stagnation, infantile indigestion and the like.

The traditional fermentation strains of the medicated leaven are mostly mould such as mucor, aspergillus, and the like, the traditional fermentation process is long in time, the quantity and the types of microorganisms are not easy to control, the difference among product batches is large, and other microorganisms in natural fermentation can generate harmful metabolites such as aflatoxin, and the like, which are easy to cause black and peculiar smell of the yeast, toxin, and the like; traditional medicated leaven is mostly used for aiding digestion and has single purpose. Therefore, it is necessary to develop a process of combined fermentation by adopting pure-bred and safe multiple strains, so as to enhance the application scene of the fermented materials of the medicated leaven, and achieve the effects of good sensory quality and high application value of the solid state fermentation preparation of the medicated leaven.

Disclosure of Invention

The invention aims to provide a triple fungus solid fermentation preparation, a preparation method and application thereof, and aims to solve the problems in the prior art.

In order to achieve the above object, the present invention provides the following solutions:

The invention provides a microorganism combination for improving fermentation quality of medicated leaven, which comprises Kluyveromyces marxianus (Kluyveromyces marxianus) JSA16, lactobacillus plantarum (Lactiplantibacillus plantarum) BY3 and bacillus subtilis (Bacillus subtilis) BK3;

the Kluyveromyces marxianus JSA16 is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 2023, wherein the preservation address is number 3 of West Song No.1 of the Korean area North Star of Beijing, and the preservation number is CGMCC No.26422;

The lactobacillus plantarum BY3 is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the 12 th month of 2022, and has a preservation address of the No. 1 or 3 of the West-Lou No. 1 of the Korean area North Star of Beijing, and a preservation number of the CGMCC No.26262;

The bacillus subtilis BK3 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the 12 th month of 2022, and has a preservation address of number 3 of West Song No. 1 of the Korean region North Star of Beijing, and a preservation number of CGMCC No. 26160.

The invention also provides application of the microorganism combination in preparation of a triple fungus solid fermentation preparation.

The invention also provides a preparation method of the triple fungus solid fermentation preparation, which comprises the step of preparing the triple fungus solid fermentation preparation by using the microorganism combination to perform solid fermentation on the raw materials for preparing the medicated leaven.

Further, the medicated leaven comprises 50 parts of wheat bran, 25 parts of flour, 1 part of bitter apricot seed, 1 part of red bean, 5 parts of flaccid knotweed herb, 5 parts of sweet wormwood herb and 5 parts of cocklebur fruit according to parts by weight.

Further, the preparation method comprises the following steps:

(1) Pulverizing testa Tritici, flour and semen Armeniacae amarum, sterilizing and drying, adding water decoction of herba Polygoni Capitati, herba Artemisiae Annuae and herba Xanthii, and mixing to obtain solid fermentation preparation material;

(2) Inoculating the kluyveromyces marxianus JSA16, the lactobacillus plantarum BY3 and the bacillus subtilis BK3 into the solid fermentation preparation raw materials to prepare a yeast block, and performing solid fermentation to obtain the triple bacteria solid fermentation preparation.

Further, in the step (2), the inoculation mass fractions of the kluyveromyces marxianus JSA16, the lactobacillus plantarum BY3 and the bacillus subtilis BK3 with respect to the solid state fermentation preparation raw material are 3%, 3% and 12%, respectively.

Further, in the step (2), the temperature of the solid state fermentation is 30 ℃ and the time is 4d.

The invention also provides a triple bacteria solid fermentation preparation prepared by the preparation method.

The invention also provides application of the triple fungus solid fermentation preparation in preparing feed, health care product or medicine for relieving food retention and/or promoting digestion.

The invention also provides application of the triple fungus solid fermentation preparation in preparing feed, health care products or medicines for preventing colonitis.

The invention discloses the following technical effects:

1. The invention firstly uses Kluyveromyces marxianus, lactobacillus plantarum and bacillus subtilis to carry out mixed solid fermentation, and develops a triple-fungus solid fermentation preparation which can relieve food stagnation caused by high-protein diet, play a certain role in promoting digestion, and also can increase secretion of gastrointestinal hormone and maintain balance of intestinal tract environment; the solid fermentation preparation of the triple bacteria also has the function of preventing colonitis; in addition, the triple fungus solid fermentation preparation has soft smell and sour flavor; golden yellow, good quality and wide market application scene.

2. The three strains used in the invention are all good functional strains, the Kluyveromyces marxianus JSA16 is derived from traditional fermented yak milk, the growth is rapid, the protease activity is realized, the fermented wheat bran product has fruit fragrance, and the polypeptide content is improved; the lactobacillus plantarum BY3 is derived from traditional pickle of northeast farmhouse, has the advantages of high growth speed, low temperature resistance and the like, and can inhibit the growth of pathogenic bacteria BY a large amount of organic acid generated in the fermentation process; the bacillus subtilis BK3 is derived from traditional pickle of northeast farmhouse, has rapid growth, strong stress resistance and a large amount of enzymes in metabolites, and has higher protease activity and cellulase activity required by wheat bran decomposition.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diameter of a lactobacillus zone of inhibition;

FIG. 2 shows the protease activity of Bacillus;

FIG. 3 is a graph of yeast growth curve;

FIG. 4 is a graph showing diameter of a circle of enzymatic hydrolysis of yeast;

FIG. 5 is a colony morphology and strain morphology map;

FIG. 6 is a biological evolutionary tree of JSA 16;

FIG. 7 is a biological phylogenetic tree of BY 3;

FIG. 8 is a biological evolutionary tree of BK 3;

FIG. 9 shows the content of the fermented soluble polypeptide in different combinations;

FIG. 10 is a graph showing the content of different combinations of fermentation reducing sugars;

FIG. 11 shows the amounts of fermented soluble polypeptide content with different inoculum sizes;

FIG. 12 is a graph of the fermentation reducing sugar content at different inoculum sizes;

FIG. 13 shows the L-glutamine content of the fermented seed amount;

FIG. 14 is a flow chart of the preparation method of example 4;

FIG. 15 is a diagram of the final solid fermentation preparation of the triple bacteria of example 4;

FIG. 16 is a schematic diagram of the experimental group of food retention model animals in example 5;

FIG. 17 is a graph showing the effect of a solid fermentation preparation of triple bacteria on stool parameters of mice in animal experiments;

FIG. 18 is a graph showing the effect of a triple solid fermentation preparation on the rate of intestinal thrust in mice;

FIG. 19 is a graph showing the results of the effect of a triple fungus solid state fermentation formulation on mouse gastrin;

Fig. 20 is a schematic diagram of experimental groups of animals in the colitis model of example 6;

FIG. 21 is a graph showing the effect of a solid fermentation preparation of triple bacteria on mouse hematochezia;

FIG. 22 is a graph showing the effect of a triple fungus solid state fermentation formulation on colon length in mice;

FIG. 23 is a graph showing the effect of the solid fermentation preparation of triple bacteria on IL-6, TNF-alpha and IL-10 in mice;

FIG. 24 is a slice diagram of the effect of a triple fungus solid state fermentation formulation on the colon morphology of mice.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

EXAMPLE 1 preliminary screening of strains

The invention adopts oxford cup method, uses wheat bran as fermentation substrate, uses escherichia coli (3.97X10 ⁸ CFU/mL) and staphylococcus aureus (3.53X10 ⁸ CFU/mL) as indicator bacteria, and determines the content of escherichia coli according to GB4789.3-2016 method; GB4789.10-2016 shows the measurement of Staphylococcus aureus content, as shown in FIG. 1. Wherein the bacteriostasis circle of the lactobacillus plantarum BY3, the lactobacillus plantarum S6 and the lactobacillus casei LH23 is superior to other strains.

Bacillus with protease activity was selected by Fu Lin Fen method, and the result is shown in FIG. 2. Wherein the content of the soluble polypeptide of the bacillus pumilus Y2 and the bacillus subtilis BK3 after fermentation is superior to that of other strains.

Yeast with good growth curve is screened by adopting the measurement of the growth curve and the protease transparent circle experiment, and the results are shown in FIG. 3 and FIG. 4. Wherein the growth curve of Kluyveromyces marxianus JSA16 and Saccharomyces cerevisiae J2 is superior to other strains.

The wheat bran is used as a fermentation substrate, single-strain fermentation is carried out, sensory evaluation is carried out on the fermented wheat bran, the sensory evaluation criteria are shown in table 1 and table 2, wherein the feed after BY3 fermentation has apple vinegar fragrance, the feed after JSA16 fermentation has wine fragrance, the feed after LH23 fermentation has bean fragrance and slight cheese fragrance, and the fermentation feeds of BY3 and J2 are free of caking.

TABLE 1

TABLE 2

And synthesizing the results of single-strain fermentation, wherein the finally determined fermentation strains are bacillus subtilis BK3, bacillus pumilus Y2, lactobacillus plantarum BY3, lactobacillus plantarum S6, lactobacillus casei LH23, kluyveromyces marxianus JSA16 and kluyveromyces marxianus J2.

The identification process of the JSA16, BY3 and BK3 is as follows:

The JSA16 colony is round, large and thick, and has neat edges and milky white (see figure 5); gram staining to G ⁺, extracting strain genome from the thallus ellipse by using a fungus genome DNA rapid extraction kit, and carrying out PCR amplification by using ITS1 and ITS4 as primers by using the genome as a template. The amplified PCR products were sequenced and spliced to DNA sequences, and sequence homology was analyzed using the BLAST search system at NCBI (fig. 6). JSA16 was finally identified as Kluyveromyces marxianus (Kluyveromyces marxianus).

BY3 colonies are smaller, round, white, opaque, protruding, moist and flat in edge (see FIG. 5); gram staining to G ⁺, extracting bacterial genome with bacterial genome DNA fast extraction kit, and PCR amplification with 27F and 1492R as primer. The amplified PCR products were sequenced and spliced to DNA sequences, and sequence homology was analyzed using the BLAST search system at NCBI (fig. 7). BY3 was finally identified as Lactobacillus plantarum (Lactiplantibacillus plantarum).

BK3 colonies were large, or white, rough, irregular in edge, and the colony surfaces had protrusions and wrinkles (see FIG. 5); gram staining to obtain G ⁺, extracting bacterial genome with bacterial genome DNA fast extraction kit, and PCR amplification with 27F and 1492R as primer. The amplified PCR products were sequenced and spliced to DNA sequences, and sequence homology was analyzed using the BLAST search system at NCBI (fig. 9). BK3 was finally identified as Bacillus subtilis (Bacillus subtilis).

Strain preservation:

Kluyveromyces marxianus JSA16 is preserved in China general microbiological culture Collection center (CGMCC) for 2023, wherein the preservation address is number 3 of West Song No. 1 of the Korean area North Star of Beijing city, and the preservation number is CGMCC No.26422; the lactobacillus plantarum BY3 is preserved in China general microbiological culture collection center (CGMCC) at the 12 th month of 2022 with the preservation address of the China general microbiological culture Collection center (CGMCC) of the North Star Xiyu No. 1, no. 3 of the Korean area of Beijing city and the preservation number of the CGMCC No.26262; the bacillus subtilis BK3 is preserved in China general microbiological culture Collection center (CGMCC) at the 12 th month of 2022 with the preservation address of North Star Xili No. 1,3 in the Korean region North Star in Beijing city and the preservation number of CGMCC No.26260.

Example 2 determination of Mixed fermentation combinations

Wheat bran is used as a fermentation substrate, and the proportion of mixed fermentation inoculation is as follows: lactic acid bacteria: bacillus=3% (w/w): 3% (w/w): 3% (w/w), fermenting at 30deg.C for 6d, and fermenting three bacteria in parallel. The content of soluble polypeptide is shown in figure 7, compared with an unfermented feed group, the content of soluble polypeptide of the combination jsa16+by3+bk3, jsa16+by3+y2 and j2+by3+bk3 is improved, wherein the content of soluble polypeptide of the combination jsa16+by3+bk3 after fermentation is the highest, the difference is obvious (P < 0.05), the content of soluble polypeptide of the feed after fermentation of the combination jsa16+s6+y2 is obviously improved, and the difference is extremely obvious (P < 0.01); the reducing sugar content is shown in fig. 10, the reducing sugar content of the combinations jsa16+by3+bk3, j2+by3+bk3 and j2+by3+y2 is increased compared to the unfermented feed group, and the difference is very significant (P < 0.01). The reducing sugar and the soluble polypeptide are used as detection indexes to determine the mixed bacteria fermentation combination into four groups of JSA16+BY3+BK3, JSA16+BY3+Y2, J2+BY3+BK3 and J2+BY3+Y2.

Example 3 determination of optimal Mixed fermentation combinations and inoculum size

The seven groups of combinations are fermented in different inoculation proportions by taking the content of soluble polypeptide (figure 11) and the content of reducing sugar (figure 12) of the fermented feed and the content of L-glutamine (figure 13) as detection indexes and wheat bran as fermentation substrates. Mixed bacteria fermentation inoculation proportion yeast (w/w): lactic acid bacteria (w/w): bacillus (w/w) was 3%:3%:3% and 3%:3%:12%, fermenting at 30deg.C for 6d, and fermenting three bacteria in parallel.

The final mixed bacteria fermentation combination is determined to be 3% (w/w) of Kluyveromyces marxianus JSA16, 3% (w/w) of lactobacillus plantarum BY3 and 12% (w/w) of bacillus subtilis BK3, and the final mixed bacteria fermentation combination is specifically characterized in that the total acid content of the fermented wheat bran is 1.16g/100g, the acidity pH value is 4.17, the total acid yield is obviously improved, the mixed bacteria can be inhibited, the content of soluble polypeptide is improved to 3.15 times, the content of reducing sugar is improved to 2.84 times, and the content of L-glutamine is improved to 1.64 times.

EXAMPLE 4 preparation of solid fermentation preparation

As shown in fig. 14, the preparation method of the triple fungus solid state fermentation preparation comprises the following steps:

(1) Preparing triple bacteria: activating Kluyveromyces marxianus JSA16, lactobacillus plantarum BY3 and bacillus subtilis BK3 for three times, culturing for 7 hours until the bacterial body amount is not less than 10 ⁷ CFU/mL, and centrifuging at 4 ℃ and 4000r/min for 15 minutes to collect bacterial bodies.

(2) Preparing raw materials of a solid fermentation preparation: 50 parts of wheat bran, 25 parts of flour, 1 part of bitter apricot kernel and 1 part of red bean, crushing the mixture by a high-speed crusher, sterilizing the crushed mixture for 15min, and drying the crushed mixture at 45 ℃;5 parts of flaccid knotweed herb, 5 parts of sweet wormwood herb, 5 parts of siberian cocklebur fruit, adding 10 times of purified water, decocting and leaching for 1h, and concentrating by a rotary evaporator for 5 times; mixing the above materials to obtain solid fermentation preparation.

(3) Inoculating: under a sterile environment, inoculating triple bacteria into the solid fermentation preparation raw material, wherein Kluyveromyces marxianus JSA16 is 3% (w/w), lactobacillus plantarum BY3 is 3% (w/w), bacillus subtilis BK3 is 12% (w/w), and inoculating the solid fermentation preparation raw material BY 20mL of sterile water in a heavy suspension manner.

(4) Fermentation: the total water content of the solid fermentation preparation is controlled to be 45%, the solid fermentation preparation is prepared into a curved block under the aseptic environment, twelve layers of gauze are covered and placed into a constant temperature incubator at 30 ℃, the humidity is controlled to be 80%, the solid fermentation preparation is fermented for 4 days until the solid fermentation preparation emits sour flavor, the content changes of each component before and after fermentation are shown in a table 3, and a finished product is shown in fig. 15.

(5) And (3) drying: and (3) drying the solid fermentation preparation after fermentation for 12 hours at the temperature of 75 ℃ in a forced air drying oven until the surface of the solid fermentation preparation is micro-burnt, so as to obtain the triple bacteria solid fermentation preparation.

(6) Decocting: adding 10 times of purified water into the solid fermentation preparation of the triple bacteria, boiling, filtering with four layers of gauze, and forming a solid fermentation preparation decoction with solid content of 0.1g/mL for later use.

TABLE 3 Table 3

Example 5 animal experiment group of digestion promoting effect of Triplex solid fermentation preparation on indigestible mice

As shown in fig. 16, animal experiment tests for digestion promotion of the triple fungus solid fermentation preparation prepared in example 5:

The experimental animals were 18 male Kunming mice, fed at room temperature, fed with free diet, and after 7 days of adaptive feeding, were randomly divided into 3 groups, namely, a normal group (NC), a high protein induced food accumulation model group (HPD) and a solid state fermentation preparation group (LSQ), 6 each. The NC group is fed with normal feed, and the HPD group and the LSQ group are both fed with high-protein feed; the NC group and the HPD group drink normal water, and the LSQ group drinks the solid fermentation preparation decoction prepared in example 5 for four weeks. The following tests were then performed:

1. determination of the Effect of solid fermentation preparations on the Small intestine Propulsion of mice

The effect of the triple fungus solid fermentation preparation on the small intestine propulsion of the mice is determined by adopting a gastric lavage semi-solid nutrition paste mode. Four weeks of animal experiments, after the experiments, the mice are fasted for 12 hours without water control, 0.4mL of semi-solid nutrition paste is filled into the stomach, after 20 minutes, the mice are killed by cervical dislocation, the parts from the stomach to the ileocecum of the mice are taken, the parts from the stomach to the ileocecum of the mice are straightened under the condition of no additional external force, the distance from the pylorus of the stomach to the front edge of the semi-solid nutrition paste and the total length of the small intestine from the pylorus to the ileocecum of the mice are measured, and the propulsion rate of the small intestine of the mice is calculated.

As shown in fig. 17-18, experimental data indicate that continuous four-week high protein diet, mice in the HPD group had a slower intestinal transit rate and mice in the LSQ group had an improved intestinal transit rate. Specifically, LSQ group mice showed a significant increase in intestinal transit rate and significant variability (P < 0.05) compared to HPD group mice. This shows that the triple fungus solid fermentation preparation has the effect of promoting the small intestine propulsion on high protein diet mice, thereby having the beneficial effect of promoting digestion.

2. Determination of the Effect of triple solid fermentation preparation on mouse gastrin

ELISA method is used to measure the effect of the solid fermentation preparation of the triple bacteria on the gastrin of the mice. Four weeks after the animal experiment, the mice were sacrificed after the end of the experiment, the whole stomach of the mice was taken, and the gastric residues were removed. 8 grinding beads and 900 mu LPBS were added to a 1.5mL EP tube, and 0.1g of stomach tissue was placed therein; fully homogenizing by using a homogenizer added with liquid nitrogen, and centrifuging for 10min by using a high-speed refrigerated centrifuge 8000 r/min; the supernatant was aspirated and gastrin was determined using a gastrin ELISA kit.

Gastrin is an excitatory gastrointestinal hormone, which is mainly secreted by G cells of the antrum and duodenum, and promotes pepsin secretion and gastrointestinal motility, thereby promoting gastrointestinal motility. As shown in fig. 19, experimental data indicate that the LSQ group mice have significantly elevated gastrin levels and significantly different (P < 0.01) levels compared to the HPD group mice on a continuous four week high protein diet. The triple fungus solid fermentation preparation has the effects of regulating the secretion of gastrointestinal hormone to promote gastrointestinal movement and improve gastric motility disorder on high-protein diet mice, thereby having the beneficial effect of promoting digestion.

In summary, the in vivo experiment in this example shows that the small intestine propulsion rate and gastrin content of the solid state fermentation preparation group are improved compared with the model group by the four-week high protein diet; and has significant differences; in addition, the feces of the mice in the model group are dark, and compared with the solid fermentation preparation group, the feces have lighter color, obviously improved water content and healthfulness. The solid fermentation preparation prepared by the method has a certain effect of relieving food stagnation caused by high-protein diet and has the effect of promoting digestion. For food stagnation caused by high protein diet digestive system diseases such as dyspepsia have important practical significance.

Example 6 animal experiment group of the protective action of Triplex solid fermentation preparation on mice with colitis

As shown in fig. 20, animal experiment groups and operations of the triple fungus solid state fermentation preparation for protecting colon effect:

The experimental animals were 18 male C57BL/6 mice, fed at room temperature, fed with free diet, and after 7 days of adaptive feeding, were randomly divided into 3 groups, namely, 6 groups of normal group (NC), dextran Sodium Sulfate (DSS) -induced colitis model group (DSS) and solid state fermentation preparation group (LSQ). Normal group and model group were perfused with 0.3mL physiological saline per day, solid state fermentation preparation group was perfused with 0.3mL concentrated solution (0.4 g/mL) of the triple fungus solid state fermentation preparation prepared in example 5 per day, feed and drinking water conditions were the same, and at week 3 of the experiment, mice of model group and solid state fermentation preparation group were replaced with 2.5% dss drinking water, and feeding was continued for 7 days. The following tests were then performed:

1. Observing the influence of the solid fermentation preparation of the triple bacteria on the hematochezia condition of mice with enteritis

Mice were observed daily for hematochezia after molding by feeding dextran sodium sulfate.

Symptoms of mouse colitis are mainly manifested as ulcerative bleeding of the colon, and blood adheres to feces or to outflow. As shown in fig. 21, five days after molding, mice in DSS group had hematochezia, and gradually aggravated, and LSQ group hematochezia was improved, which indicates that the triple bacteria solid fermentation preparation can form protection in intestinal tract, and alleviate colitis symptoms.

2. Determination of the Effect of the triple solid fermentation preparation on colon Length of mice with colon inflammation

After four weeks of animal experiments, the cervical dislocation method is used for completely shearing the cecum to the anus after the mice are killed, straightening the mice without additional external force, and measuring the length from the lower end of the cecum to the upper end of the anus, namely the colon length.

Colonic shortening is one of the main manifestations of the acute colitis model, and an increase in colonic length can prove an improvement in colonic inflammation in mice. As shown in fig. 22, experimental data indicate that LSQ group mice have significantly longer colon lengths than DSS group mice. This shows that the triple fungus solid state fermentation preparation has certain effect of relieving and protecting the mice with the enteritis.

3. Determination of the Effect of Triplex solid fermentation preparation on inflammatory factor IL-6 in mice with enteritis

ELISA method is used for measuring the influence of the triple fungus solid fermentation preparation on the mouse inflammatory factor IL-6. Four weeks of animal experiment, collecting blood from eyeballs of mice after the experiment is finished, solidifying the blood at room temperature for 30min, and centrifuging at 3000r/min for 15min; the supernatant was aspirated and the content was determined using an IL-6ELISA kit.

IL-6 is a cytokine capable of modulating and activating immune cells, thereby mediating an inflammatory response in which IL-6 is expressed in large amounts and the content of IL-6 in serum is increased. As shown in fig. 23, experimental data indicate that IL-6 levels in serum were significantly elevated in DSS mice compared to NC mice; the IL-6 content in serum was significantly reduced in mice of LSQ group compared to mice of DSS group. This shows that the triple fungus solid fermentation preparation can play a certain role in protecting inflammation of mice with colon inflammation.

4. Determination of the Effect of Triplex solid fermentation preparation on inflammatory factor TNF-alpha in mice with enteritis

ELISA method is used for measuring the influence of the triple fungus solid fermentation preparation on the mouse inflammatory factor TNF-alpha. Four weeks of animal experiment, collecting blood from eyeballs of mice after the experiment is finished, solidifying the blood at room temperature for 30min, and centrifuging at 3000r/min for 15min; the supernatant was aspirated and the content was determined using TNF- α ELISA kit.

During the onset of colitis, the increased TNF- α content is induced by the massive production of mononucleated and macrophages. As shown in fig. 23, experimental data indicate that TNF- α levels in the serum of DSS mice are significantly elevated compared to NC group; compared with the DSS mice, the serum of the LSQ mice has obviously reduced TNF-alpha content. This shows that the triple fungus solid fermentation preparation can play a certain role in recovering inflammation of mice with colon inflammation.

5. Determination of the Effect of Triplex solid fermentation preparation on inflammatory factor IL-10 in mice with enteritis

ELISA method is used for measuring the influence of the triple fungus solid fermentation preparation on the mouse inflammatory factor IL-10. Four weeks of animal experiment, collecting blood from eyeballs of mice after the experiment is finished, solidifying the blood at room temperature for 30min, and centrifuging at 3000r/min for 15min; the supernatant was aspirated and the content was determined using an IL-10ELISA kit.

IL-10 is one of the anti-inflammatory factors, has the functions of inhibiting the development of inflammation and immunosuppression, and is beneficial to restoring the colonic mucosa environment. Blood IL-10 levels increased when the symptoms of colitis were restored. As shown in fig. 23, the experimental data demonstrate that the serum IL-10 level was significantly increased in the DSS group mice compared to the NC group, and the serum IL-10 level was increased in the LSQ group mice compared to the DSS group mice. This shows that the triple fungus solid fermentation preparation can play a certain role in recovering inflammation of mice with colon inflammation.

6. Effect of solid state fermentation preparation on colon morphology of mice with colon inflammation

The colonic morphology changes of the colitis mice were observed using paraffin sections and H & E staining. Cutting the lower end colon tissue of the mouse to be not more than 1cm, fixing the lower end colon tissue by using 10% neutral formaldehyde solution, eluting by ethanol and transparent by dimethylbenzene, and then immersing the lower end colon tissue into paraffin for embedding; slicing and dewaxing again by using xylene; hematoxylin eosin is dyed, dehydrated, transparent and sealed, and observed by using a forward fluorescent microscope.

As shown in fig. 24, the colon goblet cells of the DSS group mice were deleted, and had more pronounced inflammatory cell infiltration, crypt disappeared, intestinal mucosa structure was destroyed, the colon goblet cells of the LSQ group mice were aligned, inflammatory symptoms were relieved, and gradually approached the normal colon morphology. The solid fermentation preparation of the triple bacteria can restore the intestinal tract of a colonitis mouse and has a certain protection effect on the colon.

In summary, the in vivo experiments in this example show that by inducing colitis by drinking DSS for one week, the colon length of the solid state fermentation formulation group is elongated and the level of IL-6 is reduced compared to the model group; and has significant differences; in addition, the colon section of the mice in the model group shows obvious inflammatory infiltration, goblet cells are lost, colon epithelial cells are destroyed, and the colon morphology of the mice is obviously recovered compared with the solid fermentation preparation group. This shows that the prepared triple fungus solid fermentation preparation plays a certain role in protecting mice with acute colitis induced by DSS. The method has important practical significance for preventing and treating the colonitis of animals.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A microbial combination for improving fermentation quality of medicated leaven, which is characterized BY comprising kluyveromyces marxianus (Kluyveromyces marxianus) JSA16, lactobacillus plantarum (Lactiplantibacillus plantarum) BY3 and bacillus subtilis (Bacillus subtilis) BK 3;

The Kluyveromyces marxianus JSA16 is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 2023 and a preservation address of North Star Xiyu No. 1, 3 in the Korean region of Beijing city, and a preservation number of CGMCC No.26422;

The lactobacillus plantarum BY3 is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the 12 th month of 2022, and has a preservation address of the No. 1 and No. 3 of the West road of North Star in the Korean area of Beijing, and a preservation number of CGMCC No.26262;

The bacillus subtilis BK3 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the 12 th month of 2022, and has a preservation address of number 3 of West Song No. 1 of the Korean region North Star of Beijing city and a preservation number of CGMCC No.26260.

2. Use of the combination of microorganisms of claim 1 for the preparation of a triple bacterial solid state fermentation formulation.

3. The preparation method of the triple fungus solid state fermentation preparation is characterized by comprising the step of carrying out solid state fermentation on a raw material for preparing medicated leaven by utilizing the microorganism combination of claim 1 to prepare the triple fungus solid state fermentation preparation.

4. The preparation method according to claim 3, wherein the raw materials for preparing the medicated leaven comprise 50 parts by weight of wheat bran, 25 parts by weight of flour, 1 part by weight of bitter apricot seed, 1 part by weight of red bean, 5 parts by weight of polygonum hydropiper, 5 parts by weight of sweet wormwood and 5 parts by weight of xanthium sibiricum.

5. A method of preparation according to claim 3, characterized in that the method of preparation comprises the steps of:

6. The production method according to claim 5, wherein in the step (2), the inoculation mass fractions of the kluyveromyces marxianus JSA16, the lactobacillus plantarum BY3, and the bacillus subtilis BK3 with respect to the solid state fermentation preparation raw material are 3%, and 12%, respectively.

7. The method according to claim 5, wherein in the step (2), the solid state fermentation is performed at a temperature of 30℃for a period of 4d.

8. A triple fungus solid state fermentation preparation prepared according to the preparation method of any one of claims 3-7.

9. Use of the triple fungus solid state fermentation preparation of claim 8 in the preparation of feed, health food or medicament for relieving food retention and/or promoting digestion.

10. Use of the triple fungus solid state fermentation preparation of claim 8 in the preparation of a medicament for relieving colitis.