CN111004732B - Bacillus coagulans capable of promoting motilin secretion and application thereof - Google Patents
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Abstract
The invention discloses bacillus coagulans capable of promoting motilin secretion and application thereof, and belongs to the technical field of microorganisms. The bacillus coagulans provided by the invention has a preservation number of GDMCC No.60538, and experiments show that the bacillus coagulans can obviously improve the serum motilin level and the small intestine propulsion rate of a gastrointestinal motility inhibition model mouse, obviously shorten the time for discharging the first-particle black feces, and has more obvious effect in the form of spore powder. The bacillus coagulans provided by the invention has the effects of up-regulating motilin and promoting gastrointestinal motility, so that the bacillus coagulans has good prevention and treatment effects on gastrointestinal motility disorder related diseases caused by functional constipation, infantile anorexia, functional dyspepsia, reflux esophagitis and other motilin disorders.
Description
Technical Field
The invention relates to bacillus coagulans capable of promoting motilin secretion and application thereof, belonging to the technical field of microorganisms.
Background
Motilin (MTL) is an important excitatory gastrointestinal hormone, whose chemical nature is a linear polypeptide consisting of 22 amino acids, which is present in the gastrointestinal mucosa and nervous tissues and has important regulatory effects on gastrointestinal motility. Motilin can strongly stimulate gastric contraction and obvious segmental motion of small intestine, and the contraction sequence is transmitted from the near end to the far end of the gastrointestinal tract, thereby completing the cleaning effect on the contents in the gastrointestinal cavity. Abnormal motilin secretion inevitably affects gastrointestinal motility, causes dysfunction of the digestive tract and produces various adverse clinical symptoms. For example, decreased motilin secretion can cause the contraction of gastrointestinal smooth muscle to be weakened, and intestinal peristalsis to be inhibited, so that constipation can occur; inadequate secretion of motilin causes delayed gastric emptying, weakened small intestine movement, and symptoms such as nausea and satiety after meals, and is one of the important mechanisms causing diseases such as infantile anorexia, functional dyspepsia, reflux esophagitis and the like.
For gastrointestinal dysfunction caused by insufficient secretion of motilin, gastrointestinal motilin-activating medicaments such as an oral motilin receptor agonist, human growth hormone releasing peptide, a dopamine receptor blocker, a 5-hydroxytryptamine receptor agonist and the like are mainly used for treatment at present, but the medicament treatment has certain toxic and side effects on a body. For example, the common motilin receptor agonist erythromycin can be clinically used for treating diseases such as gastroesophageal reflux caused by hyposecretion of motilin, but belongs to the field of antibiotics, and the use of the conventional motilin receptor agonist erythromycin is avoided as much as possible; cisapride belongs to the most widely used 5-hydroxytryptamine receptor agonist gastrointestinal motility promoting drugs at present, and has the effects of prolonging QT interval and causing severe arrhythmia, so that the clinical use of cisapride is limited in a plurality of countries in Europe and North America.
In view of the problems associated with conventional therapies, it is important to find a new method for effectively modulating motilin. The probiotic strategy is to regulate the intestinal micro-ecological balance by orally taking a sufficient number of viable bacteria preparations and to play a role in gastrointestinal tract function health care by the improved intestinal flora. The probiotics belongs to food-grade microorganisms, has the advantages of strong functionality and high safety, and can prevent and treat diseases on the premise of no toxic or side effect compared with medicines. Oral probiotic bacteria are therefore a potentially effective strategy for modulating the level of motilin secretion. However, the efficacy of probiotics is strain-dependent, so that screening a probiotic strain with a specific efficacy requires extensive and long-term experimental studies.
At present, some patents relate to health-care food with the function of regulating motilin and a preparation method thereof, for example, patent CN105943682A discloses a traditional Chinese medicine preparation consisting of codonopsis pilosula, bighead atractylodes rhizome, honey-fried astragalus, honey-fried licorice and the like, which can effectively promote the secretion of motilin and improve gastrointestinal motility and has no toxic or side effect; patent CN107266599A discloses an extraction method of flammulina velutipes polysaccharide and application thereof in relieving constipation, wherein the extract can obviously increase the motilin level in serum. However, there are a few patent applications relating to probiotics having the function of regulating the secretion level of motilin, for example, patent CN105132329A discloses Lactobacillus fermentum Lee having the efficacy of down-regulating the level of motilin. No probiotic patent application related to the function of up-regulating the secretion level of motilin is available at present.
Disclosure of Invention
The first purpose of the invention is to provide an application of bacillus coagulans in preparing a biological preparation for promoting the secretion of motilin, wherein the bacillus coagulans is preserved in Guangdong province microorganism strain preservation center in 2019, 18.01, and the preservation address is Guangzhou city Michelia Tokyo 100 large institute No. 59 building, 5 building, Guangdong province microorganism research institute, and the preservation number is GDMCC No. 60538.
In one embodiment of the invention, the biological agent comprises a bacillus coagulans biological agent in the form of vegetative cells or a bacillus coagulans biological agent in the form of spores.
In one embodiment of the invention, the bacillus coagulans biological agent with vegetative cell form is prepared by activating and culturing bacillus coagulans in an MRS culture medium, centrifuging to obtain bacillus coagulans, washing, collecting bacteria, and resuspending with a protective agent to obtain the bacillus coagulans biological agent; or drying the resuspended thallus again to obtain the bacillus coagulans powder biological preparation.
In one embodiment of the present invention, the method for preparing a bacillus coagulans biological agent with a spore cell form comprises the following steps:
(1) activating and culturing bacillus coagulans in an MRS culture medium;
(2) inoculating the activated strain into a spore production culture medium for culturing, heating in a water bath, centrifuging to obtain a precipitate, washing the precipitate, collecting spores, or resuspending the collected spores with a protective agent, and drying to obtain bacillus coagulans spore powder.
In one embodiment of the present invention, step (1) is to inoculate Bacillus coagulans in an amount of 1% -10% (10)6-108CFU/mL) into MRS culture medium, and culturing at 35-40 deg.C for 16-22h to obtain activated Bacillus coagulans strain.
In one embodiment of the present invention, the culture conditions in step (2) are such that the activated above-mentioned bacterial species are present in an amount of 1-4% (10)6-108CFU/mL) is inoculated into a spore production culture medium and cultured for 45-50h at the temperature of 35-42 ℃ and at the speed of 180-250 r/min.
In one embodiment of the invention, the water bath heating condition of step (2) is 80-85 ℃ for 10-15min, and the centrifugation condition is 5500-6000g for 10-15 min.
In one embodiment of the invention, the drying comprises freeze drying or spray drying.
In one embodiment of the invention, the biological agent takes skimmed milk powder, trehalose or sucrose as a protective agent.
In one embodiment of the invention, the biological agent is used for regulating the deficiency of motilin levels caused by functional constipation, child anorexia, functional dyspepsia or reflux esophagitis.
The invention has the beneficial effects that:
(1) the bacillus coagulans NJ23 provided by the invention belongs to food grade, is safe to human bodies and has no toxic or side effect.
(2) The bacillus coagulans NJ23 provided by the invention can obviously improve the level of motilin in serum, and has the effect of relieving diseases such as functional constipation, infantile anorexia, functional dyspepsia, reflux esophagitis and the like.
(3) The bacillus coagulans NJ23 spore preparation provided by the invention has high tolerance to heat, is suitable for being taken with hot water or added into food which needs to be processed at higher temperature, and has very wide application prospect.
Biological material preservation
Bacillus coagulans (Bacillus coagulans) NJ23, which is classified and named as Bacillus coagulans, is stored in Guangdong province microorganism strain storage center in 2019, 18.01.7.9, has the storage address of Guangzhou city Midduo No. 100 college No. 59 building, Guangdong province microorganism research institute, and has the storage number of GDMCC No. 60538.
Drawings
FIG. 1 shows the morphology of a Bacillus coagulans NJ23 colony.
FIG. 2 shows the forms of vegetative cells and spore cells of Bacillus coagulans NJ23, observed with 10 Xeyepiece and 100 Xoleoscope.
Figure 3 is the rate of intestinal transit for each group of mice, with the difference in the letters on the bar graph indicating significant differences between groups (p < 0.05).
FIG. 4 is the time of the first black stool in each group of mice, and the difference in the letters on the bar chart indicates significant difference between groups (p < 0.05).
FIG. 5 is a graph of motilin levels in serum of mice in each group, with bar differences indicating significant differences between groups (p < 0.05).
FIG. 6 shows the rate of intestinal transit in mice of groups perfused with different preparations of Bacillus coagulans NJ23, where the difference in the letters on the bar graph indicates significant differences between groups (p < 0.05).
FIG. 7 is a graph showing the time of first-grain black stool formation in various groups of mice with different preparations of Bacillus coagulans NJ23 by gavage, and the difference in the letters on the bar graph indicates significant difference between the groups (p < 0.05).
FIG. 8 is a graph showing the motilin levels in serum of mice in groups receiving different preparations of B.coagulans NJ23, where the difference in the letters on the bar graph indicates significant differences between groups (p < 0.05).
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically indicated, are commercially available.
(I) culture Medium
The formula of the MRS culture medium is as follows: 10g of peptone, 10g of beef extract, 20g of glucose, 5g of yeast extract, 2g of anhydrous sodium acetate, 0.25g of manganese sulfate monohydrate, 1mL of Tween 80, 2.6g of dipotassium hydrogen phosphate trihydrate, 0.5g of magnesium sulfate heptahydrate and 2g of diammonium citrate are added into 1L of distilled water, and the pH is adjusted to 6.8.
The formula of the spore production culture medium is as follows: 0.7g of yeast extract, 1g of peptone, 1g of glucose, 0.2g of ammonium sulfate, 0.2g of magnesium sulfate heptahydrate, and 1g of dipotassium hydrogen phosphate were added to 1L of distilled water.
EXAMPLE 1 preparation of a liquid Bacillus coagulans vegetative cell biological preparation
(1) Strain activation
Inoculating the bacillus coagulans into an MRS culture medium in an inoculation amount of 2mL/100mL, culturing at 37 ℃ for 18h to obtain activated bacillus coagulans, and continuously activating for 2 generations.
As shown in fig. 1 and 2, the bacteriological characteristics of bacillus coagulans NJ23 are specifically: colony characteristics: the strain grows well on an MRS culture medium to form beige, opaque and round colonies; the characteristics of the thallus are as follows: the bacteria are rod-shaped, the two ends are blunt, the bacteria are arranged singly, in pairs or in a chain shape, the gram staining reaction is positive, and the spores are grown.
(2) Preparation of biological agent
Inoculating the activated culture into MRS culture medium at an inoculum size of 2mL/100mL, culturing at 37 deg.C for 18h, centrifuging at 4 deg.C for 20min at 3000g, collecting precipitate, washing with sterile physiological saline for 3 times, collecting precipitate, and resuspending with protectant to obtain thallus concentration of 5 × 108cfu/mL, liquid Bacillus coagulans vegetative cell biologicals, stored at-20 ℃. The protective agent is 30% sucrose solution.
EXAMPLE 2 preparation of a Bacillus coagulans vegetative cell powder biological agent
(1) Strain activation
The same as in example 1.
(2) Preparation of biological agent
Inoculating the activated culture into an MRS culture medium according to the inoculation amount of 2mL/100mL, culturing for 18h at 37 ℃, centrifuging for 20min at 3000g at 4 ℃, taking a precipitate, washing for 3 times by using sterile normal saline, collecting the precipitate, using 100g/L skim milk powder as a protective agent for heavy suspension, pre-freezing for 3h at-20 ℃, and then freeze-drying to obtain the bacillus coagulans vegetative cell bacterial powder biological preparation.
EXAMPLE 3 preparation of a liquid Bacillus coagulans spore biological preparation
(1) Strain activation
The same as in example 1.
(2) Preparation of biological agent
Inoculating the activated strain into spore production medium at an inoculation amount of 2mL/100mL, shake-culturing at 37 deg.C for 48h at 250r/min, heating in water bath at 85 deg.C for 13min, centrifuging at 6000g for 10min to obtain precipitate, washing with sterile normal saline for 3 times, collecting spore, and resuspending in sterile normal saline to spore concentration of 5 × 108cfu/mL to obtain liquid bacillus coagulans spore biological preparation, and storing at-4 ℃.
EXAMPLE 4 preparation of a Bacillus coagulans spore powder biological preparation
(1) Strain activation
The same as in example 1.
(2) Preparation of biological agent
Inoculating the activated strain into a spore production culture medium with the inoculation amount of 2mL/100mL, carrying out shake-flask culture at 37 ℃ for 48h at 250r/min, heating in a water bath at 85 ℃ for 13min, centrifuging for 10min at 6000g, taking a precipitate, washing for 3 times by using sterile normal saline, collecting spores, carrying out heavy suspension by using 100g/L skimmed milk powder as a protective agent, pre-freezing for 3h at-20 ℃, and then carrying out freeze drying to obtain the bacillus coagulans spore powder biological preparation.
The formulation of the spore-forming medium was the same as in example 3.
EXAMPLE 5 application of liquid Bacillus coagulans spore biologics
1 animal Experimental design
40 male SPF-grade BALB/c mice at 7 weeks of age were randomly divided into 8 groups, blank group, made group, Bacillus coagulans WX12 group, Bacillus coagulans LY11 group and Bacillus coagulans NJ23 group. After 7 days of acclimation, the blank group was perfused with sterile normal saline every day, and the other 4 groups of mice were perfusedLoperamide hydrochloride (10mg/kg b.w) to form a mouse gastrointestinal motility inhibition model, and the perfusing sterile normal saline of a blank group and a model group after 1h, and the perfusing bacillus coagulans biological agent (5 multiplied by 10) of a bacillus coagulans treatment group8cfu/mL), the gavage volume was 0.2mL for 14 consecutive days. Wherein a bacillus coagulans biological agent was prepared by the method of example 3.
2 detection index and method
2.1 measurement of gastrointestinal motility in mice
On day 13, the mice were fasted overnight, the mice in the blank group were gavaged with sterile normal saline, the other groups were gavaged with loperamide hydrochloride (10mg/kg b.w), and 1h later, all the mice were gavaged with ink containing the corresponding test formulation, and the gavage volume was 0.2 mL. The animals were then immediately transferred to a clean, empty cage and food and water were ingested ad libitum. The time from the gavage of the ink to the excretion of the first black stool was recorded.
On day 14, the mice were fasted overnight, the gavage method was the same as that on day 13, and 30min later, the mice were anesthetized by intraperitoneal injection of ketamine (100mg/kgb.w), after blood withdrawal, cervical vertebrae were removed, sacrificed, abdomens were opened, the entire small intestine from the pylorus to the cecum was carefully removed, and the distance traveled by the ink and the total length of the small intestine were measured. The small intestine propulsion rate (%) ═ ink travel distance/small intestine full length × 100%.
2.2 measurement of mouse serum motilin levels
Standing the collected blood of the mouse for 2h, centrifuging at 3000 Xg for 15min to obtain serum, performing experiments according to the instructions of a corresponding mouse Motilin (MTL) ELISA kit, and calculating the concentration of the motilin in the serum of the mouse according to a standard curve.
3 results
3.1 gastrointestinal motility of groups of mice
The results of the detection of the motility of the small intestine and the whole intestinal tract are shown in the attached figure 3 (table 1) and the attached figure 4 (table 2), and the results show that the bacillus coagulans biological agent can effectively increase the small intestine propulsion rate of the mice and shorten the time for the mice to excrete the first black feces. Among them, bacillus coagulans NJ23 was more effective (p <0.05), and its ability to promote small and whole intestinal motility was significantly better than bacillus coagulans WX12 and bacillus coagulans LY11(p < 0.05).
TABLE 1 Small intestine Productivity of the groups
TABLE 2 time of first-row black stool for each group
3.2 serum motilin levels in mice of each group
The experimental results are shown in fig. 5 (table 3), and the results show that the bacillus coagulans biological agent can significantly increase the mouse serum motilin level (p < 0.05). Wherein the effect of the bacillus coagulans NJ23 is more remarkable (p <0.05), and the recovery capability of the bacillus coagulans NJ23 on the serum motilin level of a mouse is remarkably better than that of the bacillus coagulans WX12 and bacillus coagulans LY11(p < 0.05).
TABLE 3 serum motilin levels in groups of mice
The experimental results show that the bacillus coagulans NJ23 biological agent can obviously adjust the content of motilin in animals and improve the gastrointestinal motility of the animals. Proves that the traditional Chinese medicine composition has good prevention and treatment effects on diseases related to the motilin imbalance, such as functional constipation, infantile anorexia, functional dyspepsia, reflux esophagitis and the like.
Example 6 comparison of the effectiveness of the use of different forms of Bacillus coagulans NJ23 biologics
1 animal Experimental design
48 male SPF-grade BALB/c mice at the age of 7 weeks are randomly divided into a blank group, a modeling group, a liquid spore group, a spore powder group, a liquid vegetative cell group and a vegetative cell powder group, and each group comprises 8 mice. After 7 days of adaptation, the blank group was perfused with sterile normal saline every day, and the other 5 groups of mice were perfused with loperamide hydrochloride (10mg/kg b.w) to cause mice gastrointestinal motility inhibition modelType, after 1h, blank group and model group are filled with stomach sterile normal saline, bacillus coagulans NJ23 treatment group is filled with stomach corresponding bacillus coagulans NJ23 biological agent (5 multiplied by 10)8cfu/mL), the gavage volume was 0.2mL for 14 consecutive days. Wherein a liquid bacillus coagulans biological formulation was prepared by the method of example 3, a bacillus coagulans biological formulation was prepared by the method of example 4, a liquid vegetative cell group bacillus coagulans biological formulation was prepared by the method of example 1, and a bacillus coagulans biological formulation was prepared by the method of example 2.
2 detection index and method
The same as in example 5.
3 results
3.1 gastrointestinal motility of groups of mice
The results of the detection of the motility of the small intestine and the whole intestine are shown in the attached figure 6 (table 4) and the attached figure 7 (table 5), and the results show that the bacillus coagulans NJ23 biological agents in different preparation forms can effectively increase the small intestine propulsion rate of the mice and shorten the time for the mice to excrete the first black feces. The effect of bacillus coagulans NJ23 spore powder is more obvious (p < 0.05).
TABLE 4 intestinal motility in groups of mice
TABLE 5 time to first-row black stool for each group of mice
3.2 serum motilin levels in mice of each group
The experimental results are shown in fig. 8 (table 6), and the results show that different preparations of bacillus coagulans NJ23 biological agent can effectively increase the mouse serum motilin level (p < 0.05). The effect of bacillus coagulans NJ23 spore powder is more obvious (p < 0.05).
TABLE 6 motilin levels in groups of mice
The experimental results show that the bacillus coagulans NJ23 biological preparations in different preparation forms have different abilities of regulating the motilin of animals, wherein the bacillus coagulans is more than liquid spores and more than vegetative cell powder and more than liquid vegetative cells, because the biological activities of the bacillus coagulans in different preparation forms are different, and the high activity is the premise and the basis for the probiotics to play the probiotic effect. Therefore, the method of preparing the biological preparation of bacillus coagulans NJ23 with motilin-regulating function of the present invention is preferably the method of example 4.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (12)
1. The application of Bacillus coagulans (Bacillus coagulans) in preparing a biological agent for promoting the secretion of motilin is characterized in that the Bacillus coagulans is preserved in Guangdong province microorganism strain preservation center in 2019, 18.01.7.4, the preservation address is No. 59 building No. 5 building No. Guangdong province microorganism research institute of Michelia Tokoro, Guangzhou city, and the preservation number is GDMCC No. 60538.
2. The use of claim 1, wherein the biological agent comprises a bacillus coagulans biological agent in the form of vegetative cells or a bacillus coagulans biological agent in the form of spores.
3. The use of claim 2, wherein the bacillus coagulans biological agent with vegetative cell morphology is prepared by activating bacillus coagulans in MRS culture medium, culturing, centrifuging to obtain bacillus coagulans, washing, collecting bacteria, and resuspending with protectant to obtain bacillus coagulans biological agent; or drying the resuspended thallus to obtain the bacillus coagulans powder biological preparation.
4. The use according to claim 2, wherein the bacillus coagulans biological agent having a cellular morphology of spores is prepared by a method comprising the steps of:
(1) activating and culturing bacillus coagulans in an MRS culture medium;
(2) inoculating the activated strain into a spore production culture medium for culturing, heating in a water bath, centrifuging to obtain a precipitate, washing the precipitate, collecting spores, or resuspending the collected spores with a protective agent, and drying to obtain bacillus coagulans spore powder.
5. The use according to claim 4, wherein the Bacillus coagulans strain obtained in step (1) is activated by inoculating the Bacillus coagulans strain into MRS medium at an inoculation amount of 1% -10% and culturing at 35-40 ℃ for 16-22 h.
6. The use as claimed in claim 4, wherein the culture conditions in step (2) are such that the activated strain is inoculated into the spore production medium at an inoculum size of 1-4% and cultured at 35-42 ℃ for 45-50h at 180-250 r/min.
7. The use as claimed in claim 4, wherein the water bath heating condition in step (2) is 80-85 ℃ for 10-15min, and the centrifugation condition is 5500-6000g centrifugation for 10-15 min.
8. Use according to claim 3, wherein the drying is freeze-drying or spray-drying.
9. Use according to claim 4, wherein the drying is freeze-drying or spray-drying.
10. Use according to any one of claims 1 to 9, wherein the biological agent is skimmed milk powder, trehalose or sucrose as a protectant.
11. The use according to any one of claims 1 to 9, wherein the biological agent is for the modulation of insufficient motilin levels due to functional constipation, child anorexia, functional dyspepsia or reflux esophagitis.
12. The use according to any one of claims 1 to 9, wherein the biological agent is skimmed milk powder, trehalose or sucrose as a protective agent, and the biological agent is used for regulating insufficient motilin levels caused by functional constipation, infantile anorexia, functional dyspepsia or reflux esophagitis.
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| CN114908023B (en) * | 2022-06-17 | 2023-08-25 | 江南大学 | Bacillus coagulans for improving relative abundance of actinomycota in intestinal tract and inhibiting expression quantity of proinflammatory factors |
| CN116731937B (en) * | 2023-08-15 | 2023-11-10 | 天津创源生物技术有限公司 | Preparation method and application of Wittman coagulans IOB502 zymocyte powder |
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