CN112043723A - Application of bacillus amyloliquefaciens exopolysaccharide - Google Patents
Application of bacillus amyloliquefaciens exopolysaccharide Download PDFInfo
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- CN112043723A CN112043723A CN202010848299.1A CN202010848299A CN112043723A CN 112043723 A CN112043723 A CN 112043723A CN 202010848299 A CN202010848299 A CN 202010848299A CN 112043723 A CN112043723 A CN 112043723A
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- bacillus amyloliquefaciens
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
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Abstract
The invention discloses application of bacillus amyloliquefaciens exopolysaccharide, in particular application in preparing medicines or health-care products for preventing and treating ulcerative colitis and application in preparing fermented milk or acidic milk beverages. The exopolysaccharide inhibits the colonization of pathogenic bacteria and promotes the growth of probiotics by regulating the structure of the intestinal flora of mice, thereby improving the ulcerative colitis. Meanwhile, the extracellular polysaccharide effectively improves the gel property and related physicochemical indexes of the fermented milk, enhances the flavor and the tissue state of the fermented milk, and improves the quality of the fermented milk. The method has simple process and convenient operation, and can enrich the application of the microbial polysaccharide.
Description
Technical Field
The invention relates to bacillus amyloliquefaciens exopolysaccharide capable of relieving ulcerative colitis and an application method thereof, belonging to the field of food processing.
Background
Inflammatory Bowel Disease (IBD), consisting of Crohn's Disease (CD) and Ulcerative Colitis (UC), is an idiopathic and chronic inflammatory disease of the gastrointestinal tract. IBD has a high incidence and affects people around the world. Although the cause of UC is still unclear, there is evidence that environmental, dietary habits, intestinal epithelial damage, and gut microbiota dysregulation are potential causes of UC pathogenesis. In recent years, UC has been increasing in incidence and is becoming younger. Although the medical treatment level is improved in recent years, the development of safe and effective natural medicines without side effects for treating UC still has important significance.
The probiotic extracellular polysaccharide refers to secondary metabolites secreted outside cell walls during the growth and metabolism process of some probiotics, and can be divided into two types according to the strength of the combination of the secondary metabolites and the cell walls, wherein one type loses the combination of the secondary metabolites and permeates into a culture medium to form mucus, and is called as mucus polysaccharide; the other adheres to the cell wall to form a capsule, called capsular polysaccharide. In the natural environment, exopolysaccharides generally play a role in protecting microorganisms, and can prevent cells from desiccation and dehydration, infection by bacteriophage, damage by antibiotics or toxic substances, damage by protozoa, and also stabilize osmotic pressure, support cell structures, participate in cell information transmission and cell formation, and the like. Exopolysaccharides (EPS) have physicochemical properties such as water solubility, moisture retention, thickening and emulsifying properties, and are therefore widely used in the food industry, for example, they can improve the viscosity, texture and mouthfeel of fermented milk, and are potential food stabilizers. The extracellular polysaccharide is a long-chain and high-molecular substance secreted by microorganisms into the external environment, and the EPS also has special physiological activities, such as cholesterol reduction, tumor resistance, organism immunity regulation and the like, and is an important component of functional health care products and medical products. In addition, some studies have shown that exopolysaccharides as prebiotics promote the growth and activity of beneficial microorganisms in the gut, and improve host health by regulating the gut flora. Exopolysaccharides produced by different microorganisms have different probiotic properties due to the difference in producing bacteria and tissue structure.
At present, the patents on the exopolysaccharide of the microorganism mainly relate to the extraction of the exopolysaccharide, the yield optimization of the exopolysaccharide, the oxidation resistance of the exopolysaccharide and the like, such as: chinese invention with application number CN201911155787.8 discloses a chlorella extracellular polysaccharide with antioxidant activity, which separates polysaccharide secreted in the extracellular space from the chlorella fermentation culture medium environment, and the obtained chlorella extracellular polysaccharide has scavenging effect on DPPH free radical, superoxide anion, hydroxyl free radical and ABTS free radical; chinese invention with patent number CN108330086B discloses an extracellular polysaccharide producing space lactobacillus plantarum SS18-33 and application thereof in improving biological antioxidant activity; the Chinese invention with the application number of CN201911141437.6 discloses a method for extracting rhizobium japonicum extracellular polysaccharide. There is no mention of the alleviating effect of exopolysaccharides on colitis.
Disclosure of Invention
Aiming at the problem of application of microbial polysaccharide, the invention aims to provide application of exopolysaccharide of bacillus amyloliquefaciens. The results of animal experiments show that: compared with the mice in the DSS model group, the DSS +80EPS group mice and the DSS +160EPS group mice have obviously relieved diseases, the contents of endotoxin, D-lactic acid, diamine oxidase and TNF-alpha in serum are reduced, and the content of IL-10 is increased. EPS-K4 stimulates the secretion of mucin by goblet cells, thereby protecting the intestinal epithelial barrier integrity. In addition, EPS-K4 can inhibit the colonization of pathogenic bacteria and promote the growth of probiotics by regulating the structure of mouse intestinal flora, thereby improving the symptoms of ulcerative colitis.
In order to achieve the purpose, the invention adopts the following technical scheme:
an application of Bacillus amyloliquefaciens exopolysaccharide in preparing the medicines or health-care products for preventing and treating ulcerative colitis is disclosed.
Preferably, the polysaccharide is prepared into an aqueous solution with the concentration of more than 160 mg/kg.
Application of Bacillus amyloliquefaciens exopolysaccharide in preparing fermented milk or acidic milk beverage is provided.
The polysaccharide is prepared by fermenting Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DMBA-K4, the strain is preserved in Guangdong province microorganism strain preservation center with the preservation number of GDMCC NO: 60644.
Preferably, the addition amount of the polysaccharide in the fermented milk or the acidic milk beverage is 0.1-0.8%.
Preferably, the addition amount of the polysaccharide is 0.3-0.4%.
A preparation method of bacillus amyloliquefaciens exopolysaccharide capable of relieving ulcerative colitis comprises the following steps:
(1) activating strains: activating the Bacillus amyloliquefaciens DMBA-K4 of claim 1 to obtain a seed fermentation liquid with a bacterial content of 108～109CFU/mL；
(2) And (3) amplification culture: and (3) mixing the seed fermentation liquor according to the volume ratio of 3 +/-1: 100, inoculating into an expanded culture medium, and culturing on a shaking table to obtain a bacillus amyloliquefaciens DMBA-K4 fermentation broth; the pH of the expanded culture medium is 7.5 +/-0.5, the carbon source is sucrose, and the nitrogen source is tryptone and yeast extract in a mass ratio of 1 (1-2);
(3) and (3) removing thalli: centrifuging the fermentation liquor of the bacillus amyloliquefaciens DMBA-K4, removing precipitated thalli, and keeping supernatant;
(4) alcohol precipitation: adding ethanol into the supernatant, standing, centrifuging, removing supernatant, dissolving the precipitate with appropriate amount of distilled water, and dialyzing in dialysis bag;
(5) protein removal: adding sevage reagent into the liquid obtained in the step (4), placing the mixture on a shaking table at room temperature, shaking to enable the protein to be adsorbed in an organic phase, then centrifuging, retaining an aqueous phase, and repeating the operation until the protein is completely removed.
Preferably, in the step (2), the formulation of the expanding medium is as follows according to the mass-to-volume ratio (w/v): 0.4-0.6% of sodium chloride, 0.7-0.8% of yeast extract, 0.7-0.8% of tryptone, 0.3-0.5% of sucrose and the balance of water.
Preferably, the expanded culture medium is sterilized for 15-20 min under 0.08-0.10 MPa, and is cooled to 36-38 ℃.
Preferably, in the step (2), the rotating speed of the shaking table is 150-180 r/min, the temperature is 28-32 ℃, and the time is 12 +/-4 hours.
Preferably, in the steps (3) and (4), the centrifugation time is 15-20 min, the centrifugation temperature is 4 ℃, and the rotation speed is 10000 r/min; and (5) centrifuging for 1min under the centrifugation condition of 4000 Xg.
Preferably, in the step (4), the ethanol concentration is 95%, and the volume ratio of ethanol: the supernatant was kept at 3:1 at 4 ℃ for 24 h.
Preferably, in the step (4), the dialysis time is 24-48 h.
Preferably, in step (5), the volume ratio of sevage reagent: dialysate 1:4, where sevage reagent is chloroform: n-butanol 4: 1.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, the yield of the probiotic extracellular polysaccharide is improved by optimizing the formula of the culture medium and the conditions of culture and fermentation.
(2) The exopolysaccharide inhibits the colonization of pathogenic bacteria and promotes the growth of probiotics by regulating and controlling the structure of intestinal flora of mice, thereby improving ulcerative colitis. The exopolysaccharide can remarkably relieve severe pathological symptoms of ulcerative colitis mice such as weight loss, colon length reduction, anus hematochezia and the like. It can inhibit the growth of harmful bacteria in intestinal tract, such as: proteobacteria (Proteobacteria), Akkermansia (Akkermansia), enterococci (Enterococcus), Escherichia coli (Escherichia-Shigella), and Streptococcus (Streptococcus) and promote probiotics such as: colonization by Lactobacillus plantarum (Lactobacillus). In addition, it can stimulate the secretion of mucin in the intestinal tract, and is favorable to maintaining the integrity of intestinal tract barrier.
(3) The method adopts sevage reagent, removes protein in the polysaccharide extracting solution through adsorption, has mild reaction conditions, and has small damage degree to the polysaccharide structure.
(4) The exopolysaccharide effectively improves the gel property and related physicochemical indexes of the fermented milk, enhances the flavor and the tissue state of the fermented milk, and improves the quality of the fermented milk.
The results show that: the bacillus amyloliquefaciens exopolysaccharide EPS-K4 is an immunostimulant for treating ulcerative colitis, and has a good improvement effect on the quality characteristics of fermented milk.
The bacillus amyloliquefaciens is DMBA-K4, is preserved in Guangdong province microbial strain preservation center in 2019, 4 and 16 months, is called GDMCC for short, has the preservation number of GDMCC NO:60644, and has the preservation address of No. 59 building of Dazhou college No. 100 of Jiulianglu in Guangzhou city, and the institute of microbiology in Guangdong province. The strain is disclosed in Chinese patent CN 110607254A, and belongs to the prior art.
Drawings
FIG. 1 is a graph showing the change in body weight of mice in example 1.
FIG. 2 is a comparative photograph of the colon of the mouse of example 1.
FIG. 3 is a graph comparing the colon lengths of mice of example 1.
FIG. 4 is a graph showing the effect of disease Activity index DAI scoring in mice of example 1.
FIG. 5 is a pathological section image (magnification X200) of H & E and AB-PAS staining of mice of example 1.
FIG. 6 is a graph showing the secretion of cytokines and toxic substances in the serum of mice in example 1 (A represents TNF-. alpha., B represents IFN-. gamma., C represents IL-10, D represents endotoxin ET, E represents diamine oxidase DAO, and F represents D-lactic acid D-LA).
FIG. 7 is a graph of Principal axis Analysis (PCoA) of the 16S rDNA sequencing of the colon contents of the mice in example 1.
FIG. 8 is a graph showing the significantly different genera of the mouse intestinal flora in example 1 (A for Lactobacillus, B for Akkermansia, C for Proteobacteria, D for Enterococcus, E for Escherichia-Shigella, and F for Streptococcus).
FIG. 9 is a graph showing the water separation rate of the refreshing acidic milk beverage containing EPS-K4 of example 3 stored at 4 ℃ for 15 days.
Detailed Description
The present invention will be described in further detail with reference to examples, but the scope of the invention to be claimed is not limited thereto.
Example 1: the bacillus amyloliquefaciens exopolysaccharide EPS-K4 can effectively relieve the ulcerative colitis symptoms of mice
(a) Taking 32 male BalB/C mice with the age of 6-8 weeks, weighing 18-22 g, adaptively feeding for 7 days, and dividing the mice into four groups according to 8 mice per group:
blank group (control): freely drinking distilled water, and irrigating with distilled water for 7 days, wherein the irrigation amount is 200 μ L/d, and the administration is once a day;
and (4) DSS group: freely drinking Dextran Sodium Sulfate (DSS) water solution, and perfusing stomach with distilled water, wherein the perfusion volume is 200 μ L/d, and the administration is carried out once a day for 7 days;
DSS +80EPS group: freely drinking DSS aqueous solution, and irrigating the stomach with Bacillus amyloliquefaciens extracellular polysaccharide EPS-K4 aqueous solution, wherein the irrigation amount is 80mg/kg/d, and the total irrigation amount is 7 days once a day;
DSS +160EPS group: freely drinking DSS aqueous solution, and irrigating stomach with Bacillus amyloliquefaciens extracellular polysaccharide EPS-K4 aqueous solution, wherein the irrigation amount is 160mg/kg/d, once a day, and 7 days in total;
(b) the weight change of the mice was recorded daily, the weight loss rate of the mice was calculated, the stool characteristics of the mice were recorded, and the stool of the mice was collected for testing the fecal occult blood status and Disease Activity Index (DAI) of the mice. The DAI scoring criteria are shown in table 1.
TABLE 1 DAI scoring criteria
(c) The mice were dissected and the effect of bacillus amyloliquefaciens exopolysaccharide EPS-K4 on DSS-induced ulcerative colitis conditions was evaluated by measuring the length of the colon in the mice, serum cytokines and toxin substances, pathological sections, and 16S rDNA sequencing of the intestinal contents of the mice.
As shown in FIG. 1, the weight of the mice in the blank group is relatively stable due to normal diet and drinking water, and even the weight of the mice slightly increases after 7 days of culture; in the former 5 days, the weight of mice in the DSS group, the DSS +80EPS group and the DSS +160EPS group is obviously reduced compared with that in the blank group. On days 6 and 7, the weight loss of mice in two treatment groups is obviously improved under the action of EPS-K4. Therefore, 4% DSS solution can induce a colonic inflammatory response in mice, resulting in weight loss. EPS-K4 can significantly improve the disease, and the dosage of EPS-K4 affects the action and effect of EPS-K.
The length and shape of the colon of each group of mice are shown in fig. 2 and fig. 3. The DSS group showed a reduction in colon length compared to the blank group, which is one of the major conditions of ulcerative colitis, indicating successful modeling of mice with 4% DSS solution for ulcerative colitis. After the stomach-filling treatment of EPS-K4, the colon length of the mice in the high-dose group can be kept at the same level as that in the blank group. The colon length of the mice in the low-dose group has no significant difference with that in the DSS group, and the result shows that the EPS-K4 with the concentration of 160mg/kg has a significant effect on maintaining the colon length of the mice.
Disease score index profiles for each group of mice are shown in figure 4. Compared with the normal blank group, the mice in the DSS model group have the symptoms of obvious weight reduction, slow movement, cachexia and the like on the 5 th day of DSS modeling, the feces are in a mucus state, and anus hematochezia appears, so the DAI level is higher. The DAI level of the high-dose treatment group is significantly different from that of the model group (P <0.01), and the symptoms of the mice in the group can be considered to be obviously relieved.
The colon status of mice was assessed using histopathology (fig. 5A). The results of H & E stained sections showed that, after DSS treatment, the colon of DSS group mice had severe ulceration with a massive crypt deletion. Compared with the DSS group, the EPS-K4 treatment can obviously relieve the damage condition, and the intestinal epithelial cells of the mice in the DSS +160EPS group are effectively repaired. The results of AB/PAS staining (FIG. 5B) show that high dose (160mg/kg) treatment inhibited the loss of mucus layer goblet cells and stimulated mucin secretion, thereby protecting the intestinal epithelial barrier. Therefore, it can be considered that: EPS-K4 can relieve the symptoms of ulcerative colitis by stimulating intestinal epithelial mucin secretion.
To assess the role of EPS-K4 in immunomodulation, cytokine levels in the sera of groups of mice were determined by ELISA and the results are shown in FIG. 6(A, B, C). The TNF-alpha and IFN-gamma of the DSS model group were significantly higher than those of the blank group (P <0.05), while the anti-inflammatory factor IL-10 was significantly lower than that of the blank group (P < 0.05). EPS-K4 treatment significantly improved this. Compared with the DSS model group, the level of TNF-alpha in the DSS +160EPS group is remarkably reduced, and the level of IL-10 is remarkably increased (P <0.05), but the effect on IFN-gamma is not remarkable. FIG. 6(D, E, F) is a graph showing the level of toxin substance in serum from each group of mice, which reflects the integrity of the intestinal barrier of the mice. In the DSS model group, three indexes (ET, DAO and D-LA) are obviously higher than those of other groups, which indicates that the intestinal tracts of mice in the DSS group are seriously damaged and toxin substances permeate into blood. The intestinal tract integrity of mice in the DSS +160EPS group is good, and the level of three toxin substances in serum is obviously lower than that in the DSS model group.
The intestinal microbial composition structure is closely related to ulcerative colon disease. Total DNA was extracted from the contents of the mouse colon using the HiPure fecal DNA kit and analyzed by the Gene Denovo technique. The 16S rDNA V3-V4 region of the ribosomal RNA gene was amplified by PCR. The primer is 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT. Valid tags with a similarity of > 97% are clustered into Operation Taxonomic Units (OTUs).
PCA (based on Bray-Curtis distance) representing beta diversity describes different clusters of microbial composition for each group (fig. 7). The DSS +160EPS group had parallels to the microbial composition of the blank group. FIG. 8 shows the differential genus of intestinal flora of mice in each group. The intestinal flora of the mice in the DSS group had significantly higher relative levels in Proteobacteria, Akkermansia, Enterococcus, Escherichia-Shigella, and Streptococcus. While EPS-K4 significantly reduced the relative abundance of these microorganisms. Enterococcus and Escherichia-Shigella are typical pathogenic bacteria that increase the risk of gastroenteritis and digestive tract cancer. And the Streptococcus is relatively abundant in the intestinal flora of patients with intestinal diseases. Lactobacillus is a typical probiotic bacterium, and a secondary metabolite, namely short-chain fatty acid, secreted in the intestinal tract has a good improving effect on ulcerative colitis. Compared with the DSS group, the EPS-K4 can obviously improve the relative abundance of Lactobacillus. The results show that EPS-K4 can regulate the composition structure of microorganisms in the intestinal tract, inhibit the growth of pathogenic bacteria and promote the colonization of probiotics, thereby relieving the symptoms of ulcerative colitis.
Example 2: application of bacillus amyloliquefaciens exopolysaccharide EPS-K4 in fermented yogurt
The method comprises the following steps of weighing 12.5% of whole milk powder, 8.5% of white sugar and 40.3% of bacillus amyloliquefaciens exopolysaccharide EPS-K by weight, fixing the volume to 100% by using production beverage water, uniformly mixing, preheating to 60 ℃, homogenizing, cooling to 41 ℃, adding 30g of zymocyte powder into each ton of milk, fermenting for 5.0 hours at 41 ℃, and enabling the acidity of the yoghourt to reach 65 DEG T after the fermentation is finished. The fermented yoghurt prepared according to the invention has significantly improved hardness, viscosity and cohesiveness (table 2), and has better yoghurt mouthfeel.
TABLE 2 texture Properties of EPS-K4 fermented yogurts
Example 3: application of bacillus amyloliquefaciens exopolysaccharide EPS-K4 in refreshing acidic milk beverage
Heating raw milk in water bath kettle at 60 deg.C, adding 6% white sugar, stirring, sterilizing at 90 deg.C for about 15min, cooling to 42 deg.C, inoculating starter, fermenting in constant temperature incubator until pH of raw milk is 4.6, and refrigerating in refrigerator for aging.
0-0.5% of bacillus amyloliquefaciens exopolysaccharide EPS-K4, 10% of white granulated sugar and 3% of citric acid are fully mixed, dissolved in production beverage water at 70 ℃ (the constant volume is 100%), stirred for 10min for full dissolution, sterilized for 3min at the temperature of more than 90 ℃, and cooled to room temperature for later use.
Mixing the raw milk and the stabilizer according to a ratio of 1:2, shearing by adopting a laboratory high-shear emulsifying machine at 15000r/min for 7min, homogenizing under high pressure (the technical conditions of primary pressure of 30MPa and secondary pressure of 15 MPa), and canning to prepare the refreshing acidic milk beverage containing the bacillus amyloliquefaciens extracellular polysaccharide EPS-K4. The refreshing acidic milk beverage prepared according to the invention has pure fragrance, remarkably low water-separating rate after being stored for 15 days at 4 ℃, and good protein stability (figure 9).
Example 4: yield of extracellular polysaccharide EPS-K4 of bacillus amyloliquefaciens
A preparation method of bacillus amyloliquefaciens exopolysaccharide capable of relieving ulcerative colitis comprises the following steps:
(1) activating strains: the slant conservation of the bacillus amyloliquefaciens DMBA-K4 is placed in an LB culture medium for two times of activation respectively to obtain seed fermentation liquor, and the bacteria content of the seed fermentation liquor is 108～109CFU/mL；
(2) And (3) amplification culture: and (3) mixing the seed fermentation liquor according to the volume ratio of 3: 100 portions of the mixture are inoculated into an enlarged culture medium, and the mixture is cultured on a shaking table for 12 hours, the temperature of the shaking table is 30 ℃, and the rotating speed is 180 r/min. Obtaining the fermentation liquor of the bacillus amyloliquefaciens DMBA-K4;
(3) and (3) removing thalli: centrifuging the fermentation liquor of the bacillus amyloliquefaciens DMBA-K4 for 15min, setting the temperature of a centrifuge to be 4 ℃ and the rotating speed to be 10000r/min, removing precipitated thalli, and keeping supernatant;
(4) alcohol precipitation: adding 95% ethanol 3 times volume ratio into the supernatant, standing at 4 deg.C for 24 hr, centrifuging for 15min, decanting the supernatant, dissolving the precipitate with appropriate amount of distilled water, and dialyzing in dialysis bag for 24 hr;
(5) protein removal: 1/4 volume ratio sevage reagent (chloroform: n-butanol 4:1) was added to the liquid obtained in step (4), and the mixture was shaken in a shaker at room temperature for 30min to adsorb the protein in the organic phase, and then centrifuged at 4000 Xg for 1min to retain the aqueous phase. Repeating for 4-5 times until protein is completely removed, and freeze-drying and storing.
The formula of the amplification medium is as follows according to the mass-to-volume ratio (w/v): 0.5% of sodium chloride, 0.75% of yeast extract, 0.75% of tryptone and 0.4% of sucrose.
The expanding culture medium comprises: the raw materials are subjected to constant volume to 100 parts by using distilled water, the pH value is adjusted to 7.5, the raw materials are stirred and dissolved uniformly, sterilized for 15-20 min at 0.08-0.10 MPa, and cooled to 36-38 ℃ for later use.
The centrifugal temperature is 4 ℃, and the centrifugal rotating speed is 10000 r/min.
The actual extracellular polysaccharide yield is 175.46mg/mL by performing verification tests on the culture fermentation conditions.
Example 5: yield of extracellular polysaccharide EPS-K4 of bacillus amyloliquefaciens
A bacillus amyloliquefaciens exopolysaccharide capable of relieving ulcerative colitis and an application method thereof comprise the following steps:
(1) activating strains: the slant conservation of the bacillus amyloliquefaciens DMBA-K4 is respectively placed in an LB culture medium for two times of activation to obtain seed fermentation liquor, and the bacteria content of the seed fermentation liquor is 108～109CFU/mL；
(2) And (3) amplification culture: and (3) mixing the seed fermentation liquor according to the volume ratio of 3: 100 portions of the mixture are inoculated into an enlarged culture medium, and the mixture is cultured on a shaking table for 8 hours, the temperature of the shaking table is 30 ℃, and the rotating speed is 180 r/min. Obtaining the fermentation liquor of the bacillus amyloliquefaciens DMBA-K4;
(3) and (3) removing thalli: centrifuging the fermentation liquor of the bacillus amyloliquefaciens DMBA-K4 for 15min, setting the temperature of a centrifuge to be 4 ℃ and the rotating speed to be 10000r/min, removing precipitated thalli, and keeping supernatant;
(4) alcohol precipitation: adding 95% ethanol 3 times volume ratio into the supernatant, standing at 4 deg.C for 24 hr, centrifuging for 15min, decanting the supernatant, dissolving the precipitate with appropriate amount of distilled water, and dialyzing in dialysis bag for 24 hr;
(5) protein removal: 1/4 volume ratio sevage reagent (chloroform: n-butanol 4:1) was added to the liquid obtained in step (4), and the mixture was shaken in a shaker at room temperature for 30min to adsorb the protein in the organic phase, and then centrifuged at 4000 Xg for 1min to retain the aqueous phase. Repeating for 4-5 times until protein is completely removed, and freeze-drying and storing.
The formula of the amplification medium is as follows according to the mass-to-volume ratio (w/v): 0.5% of sodium chloride, 0.75% of yeast extract, 0.75% of tryptone and 0.4% of sucrose.
The expanding culture medium comprises: the raw materials are subjected to constant volume to 100 parts by using distilled water, the pH value is adjusted to 8.0, the raw materials are stirred and dissolved uniformly, sterilized for 15-20 min at 0.08-0.10 MPa, and cooled to 36-38 ℃ for later use.
The centrifugal temperature is 4 ℃, and the centrifugal rotating speed is 10000 r/min.
The actual extracellular polysaccharide yield is 124.22mg/mL by performing verification tests on the culture fermentation conditions.
Example 6: yield of extracellular polysaccharide EPS-K4 of bacillus amyloliquefaciens
A bacillus amyloliquefaciens exopolysaccharide capable of relieving ulcerative colitis and an application method thereof comprise the following steps:
(1) activating strains: the slant conservation of the bacillus amyloliquefaciens DMBA-K4 is respectively placed in an LB culture medium for two times of activation to obtain seed fermentation liquor, and the bacteria content of the seed fermentation liquor is 108～109CFU/mL；
(2) And (3) amplification culture: and (2) mixing the seed fermentation liquor according to the volume ratio of 2: 100 portions of the mixture are inoculated into an enlarged culture medium, and the mixture is cultured on a shaking table for 16 hours, the temperature of the shaking table is 30 ℃, and the rotating speed is 180 r/min. Obtaining the fermentation liquor of the bacillus amyloliquefaciens DMBA-K4;
(3) and (3) removing thalli: centrifuging the fermentation liquor of the bacillus amyloliquefaciens DMBA-K4 for 15min, setting the temperature of a centrifuge to be 4 ℃ and the rotating speed to be 10000r/min, removing precipitated thalli, and keeping supernatant;
(4) alcohol precipitation: adding 95% ethanol 3 times volume ratio into the supernatant, standing at 4 deg.C for 24 hr, centrifuging for 15min, decanting the supernatant, dissolving the precipitate with appropriate amount of distilled water, and dialyzing in dialysis bag for 24 hr;
(5) protein removal: 1/4 volume ratio sevage reagent (chloroform: n-butanol 4:1) was added to the liquid obtained in step (4), and the mixture was shaken in a shaker at room temperature for 30min to adsorb the protein in the organic phase, and then centrifuged at 4000 Xg for 1min to retain the aqueous phase. Repeating for 4-5 times until protein is completely removed, and freeze-drying and storing.
The formula of the amplification medium is as follows according to the mass-to-volume ratio (w/v): 0.5% of sodium chloride, 0.75% of yeast extract, 0.75% of tryptone and 0.4% of sucrose.
The expanding culture medium comprises: the raw materials are subjected to constant volume to 100 parts by using distilled water, the pH value is adjusted to 7.5, the raw materials are stirred and dissolved uniformly, sterilized for 15-20 min at 0.08-0.10 MPa, and cooled to 36-38 ℃ for later use.
The centrifugal temperature is 4 ℃, and the centrifugal rotating speed is 10000 r/min.
The actual extracellular polysaccharide yield is 123.65mg/mL by performing verification tests on the culture fermentation conditions.
Example 7: yield of extracellular polysaccharide EPS-K4 of bacillus amyloliquefaciens
A bacillus amyloliquefaciens exopolysaccharide capable of relieving ulcerative colitis and an application method thereof comprise the following steps:
(1) activating strains: the slant conservation of the bacillus amyloliquefaciens DMBA-K4 is respectively placed in an LB culture medium for two times of activation to obtain seed fermentation liquor, and the bacteria content of the seed fermentation liquor is 108～109CFU/mL；
(2) And (3) amplification culture: and (3) mixing the seed fermentation liquor according to the volume ratio of 4: 100 portions of the mixture are inoculated into an enlarged culture medium, and the mixture is cultured on a shaking table for 12 hours, the temperature of the shaking table is 30 ℃, and the rotating speed is 180 r/min. Obtaining the fermentation liquor of the bacillus amyloliquefaciens DMBA-K4;
(3) and (3) removing thalli: centrifuging the fermentation liquor of the bacillus amyloliquefaciens DMBA-K4 for 15min, setting the temperature of a centrifuge to be 4 ℃ and the rotating speed to be 10000r/min, removing precipitated thalli, and keeping supernatant;
(4) alcohol precipitation: adding 95% ethanol 3 times volume ratio into the supernatant, standing at 4 deg.C for 24 hr, centrifuging for 15min, decanting the supernatant, dissolving the precipitate with appropriate amount of distilled water, and dialyzing in dialysis bag for 24 hr;
(5) protein removal: 1/4 volume ratio sevage reagent (chloroform: n-butanol 4:1) was added to the liquid obtained in step (4), and the mixture was shaken in a shaker at room temperature for 30min to adsorb the protein in the organic phase, and then centrifuged at 4000 Xg for 1min to retain the aqueous phase. Repeating for 4-5 times until protein is completely removed, and freeze-drying and storing.
The formula of the amplification medium is as follows according to the mass-to-volume ratio (w/v): 0.5% of sodium chloride, 0.75% of yeast extract, 0.75% of tryptone and 0.4% of sucrose.
The expanding culture medium comprises: the raw materials are subjected to constant volume to 100 parts by using distilled water, the pH value is adjusted to 7.0, the raw materials are stirred and dissolved uniformly, sterilized for 15-20 min at 0.08-0.10 MPa, and cooled to 36-38 ℃ for later use.
The centrifugal temperature is 4 ℃, and the centrifugal rotating speed is 10000 r/min.
The actual extracellular polysaccharide yield is 146.96mg/mL by performing verification tests on the culture fermentation conditions.
Claims (7)
1. An application of Bacillus amyloliquefaciens exopolysaccharide in preparing the medicines or health-care products for preventing and treating ulcerative colitis is disclosed.
2. The use according to claim 1, wherein the polysaccharide is prepared by fermentation using Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DMBA-K4, which is deposited at the Guangdong collection of microorganisms with the accession number GDMCC NO: 60644.
3. Use according to claim 1 or 2, wherein the polysaccharide is formulated in an aqueous solution at a concentration of 160mg/kg or more.
4. Application of Bacillus amyloliquefaciens exopolysaccharide in preparing fermented milk or acidic milk beverage is provided.
5. The use according to claim 4, wherein the polysaccharide is prepared by fermentation using Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DMBA-K4, which is deposited at the Guangdong collection of microorganisms with the accession number GDMCC NO: 60644.
6. The use according to claim 4 or 5, wherein the polysaccharide is added in an amount of 0.1 to 0.8%.
7. The use according to claim 6, wherein the polysaccharide is added in an amount of 0.3 to 0.4%.
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| CN112813009A (en) * | 2021-03-02 | 2021-05-18 | 浙江华缔药业集团有限责任公司 | Bacillus amyloliquefaciens and application thereof |
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