CN115322931B - Bacillus coagulans Wei Ciman capable of inhibiting activity of alpha-glucosidase and/or alpha-amylase and application thereof - Google Patents
Bacillus coagulans Wei Ciman capable of inhibiting activity of alpha-glucosidase and/or alpha-amylase and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The invention discloses a Wei Ciman bacillus coagulans capable of inhibiting alpha-glucosidase and/or alpha-amylase activities and application thereof. The Wei Ciman Bacillus coagulans SA9 strain was deposited at the Cantonese microorganism strain deposit center, 6/2022, accession number: GDMCC NO. 62517. The research of the invention shows that the SA9 strain has higher acid production capability and DPPH free radical removal capability, has higher inhibition effect on alpha-glucosidase which is a key enzyme causing blood sugar rise, and reaches more than 80% of acarbose efficiency of diabetes medicine; meanwhile, the strain also has excellent inhibition capability on alpha-amylase, the inhibition rate of fermentation liquor on the enzyme reaches 100%, the effect is equivalent to that of acarbose, but the strain has better safety and no toxic or side effect. The SA9 strain can be used as a high-efficiency strain for controlling blood sugar, can relieve diabetes and has positive effects on preventing and treating diabetes.
Description
Technical Field
The invention belongs to the technical field of microorganisms. More particularly, it relates to a strain of Wei Ciman bacillus coagulans capable of inhibiting alpha-glucosidase and/or alpha-amylase activities and uses thereof.
Background
Diabetes mellitus is a kind of endocrine system diseases caused by heredity or acquired environment, has a plurality of complications, long and complex illness state, greatly reduces the life quality of patients, causes huge economic burden to families and society, and has become a common chronic disease which third most impairs human health in the world, thus effectively preventing and treating diabetes mellitus. The existing research shows that the alpha-glucosidase in the digestive enzyme has higher activity, can promote the digestion and absorption of carbohydrate by intestinal tracts, can raise the glucose level in blood plasma, can delay the absorption of the carbohydrate by inhibiting the activity of the alpha-glucosidase, can increase insulin secretion, and can keep the glucose in blood plasma of diabetics at a lower level after meal. Therefore, the diabetes mellitus can be relieved by inhibiting the activity of alpha-glucosidase, the diabetes mellitus can be treated, and most of diabetes mellitus medicaments on the market belong to the enzyme inhibitor; or by inhibiting alpha-amylase to block the conversion of starch into sugar, thereby lowering the glucose level in the plasma for glycemic control. Such as acarbose, has the function of inhibiting both alpha-glucosidase and alpha-amylase.
In recent years, as the scientific community goes deep into research on probiotics, the probiotics play an increasing role in preventing and treating human diseases, and have great potential. Probiotics, a type of microorganism that has an improving effect on the health of a host after being ingested in sufficient amounts by the host. A plurality of researches show that probiotics, prebiotics or synbiotics and the like can obviously relieve clinical symptoms of diabetics. The document reports that the supernatants of lactobacillus rhamnosus GG and lactobacillus bifidus F-35 have good inhibition effect on the activity of alpha-glucosidase, and the inhibition rates respectively reach 29.41 percent and 21.82 percent. Chen et al found that rhamnose CCFM0528 had a good improvement in glucose tolerance in diabetic mice, with a decrease in blood glucose of about 43%. In addition, bacillus natto also proved to have excellent blood glucose lowering effect. Compared with chemotherapy, the probiotic has high safety and small toxic and side effects, and the biological therapy is more attractive.
The Bacillus coagulans Wei Ciman (Weizmannia coagulans) is a renamed Bacillus coagulans (Bacillus coagulans) and is one of the most popular probiotic bacteria strains studied at present, and a plurality of documents report that the bacteria strains have positive effects on preventing or treating various human diseases such as non-alcoholic fatty liver, constipation, irritable bowel syndrome and the like. The prior study reports that: the inhibition rate of the cell-free extract of bacillus coagulans JA845 on the activity of alpha-glucosidase reaches 39.51%; similarly, bacillus coagulans VHProbi C08 from Qingdao blue organism Co., ltd also has outstanding blood glucose reducing effect, and after 6 weeks of gastric lavage, the fasting blood glucose level of the bacillus coagulans pretreatment mice is reduced by 46.46% compared with the positive control group; however, the effect of the bacillus coagulans is to be improved, and no bacillus coagulans which can be used for simultaneously inhibiting the activity of alpha-glucosidase and the activity of alpha-amylase is known at present, so that in order to improve the inhibition efficiency, more bacillus coagulans with better effect and function are needed to be developed, and the bacillus coagulans can be used for preparing products for treating or assisting in treating diabetes and controlling blood sugar, and have important significance.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the problems, and provide a condensation Wei Ciman bacillus capable of inhibiting the activity of alpha-glucosidase and/or alpha-amylase and application thereof.
The invention aims to provide a strain of Wei Ciman bacillus coagulans SA 9.
Another object of the invention is to provide the use of said strain Wei Ciman Bacillus coagulans SA 9.
It is a further object of the present invention to provide an alpha-glucosidase and/or alpha-amylase inhibitor.
It is a further object of the present invention to provide a method of inhibiting alpha-glucosidase and/or alpha-amylase.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a condensation Wei Ciman bacillus (Weizmannia coagulans) SA9 strain capable of inhibiting the activity of alpha-glucosidase and/or alpha-amylase, which is stored in the Guangdong province strain collection at 6/2022, with the storage number: GDMCC NO. 62517. The SA9 strain is separated from mulberry fermentation residues, and has higher acid production capacity and DPPH free radical removal capacity; the SA9 strain has higher inhibition effect on key enzyme alpha-glucosidase causing blood sugar rise, can reach more than 80% of the acarbose efficiency of the diabetes drug, the inhibition rate of SA9 bacterial suspension (fermentation liquor) is 85%, the inhibition rate of cell metabolites of the thalli is 96%, and the cell-free extract is 87%; meanwhile, the SA9 strain also has excellent inhibition capability on alpha-amylase, the inhibition rate of fermentation liquor on the alpha-amylase can reach 100%, the effect is similar to that of acarbose, but the effect is better than that of acarbose, and the toxicity and side effects are avoided. Namely, the SA9 strain can inhibit the alpha-glucosidase and the alpha-amylase with high efficiency, can reduce the activity of the alpha-glucosidase and the alpha-amylase and block the transformation and the absorption of saccharides, and is beneficial to controlling the blood sugar and relieving diabetes.
Accordingly, the present invention provides the use of a strain of Bacillus coagulans SA9 and/or a fermentation broth thereof for inhibiting alpha-glucosidase and/or alpha-amylase, for preparing an alpha-glucosidase and/or alpha-amylase inhibitor, for preparing a product for alleviating or treating diabetes or for controlling blood glucose.
Preferably, the product is a food, a health product or a pharmaceutical product.
The invention provides an alpha-glucosidase and/or alpha-amylase inhibitor, which contains a Wei Ciman bacillus coagulans SA9 strain and/or fermentation liquor thereof.
Preferably, the fermentation broth is a fermentation supernatant, a cell-free extract and/or a bacterial cell metabolite.
Preferably, the OD of the fermentation broth is 0.5 to 1.0.
Preferably, SA9 strain in the fermentation broth is inoculated into a culture medium according to an inoculum size of 1-5%, and shake culture is carried out for 28-48 h at the temperature of 35-40 ℃ and under the condition of 120-200 r/min.
More preferably, the SA9 strain in the fermentation broth is inoculated into a culture medium according to an inoculum size of 1 percent, and shake culture is carried out for 48 hours at 37 ℃ and 180 r/min.
The invention provides a method for inhibiting alpha-glucosidase and/or alpha-amylase, which adopts a condensation Wei Ciman bacillus SA9 strain and/or fermentation liquor thereof to treat a sample.
The invention has the following beneficial effects:
the invention provides a condensation Wei Ciman bacillus (Weizmannia coagulans) SA9 strain capable of inhibiting alpha-glucosidase and/or alpha-amylase activity, wherein the SA9 strain has higher acid production capacity and DPPH free radical removal capacity, has higher inhibition effect on alpha-glucosidase which is a key enzyme causing blood sugar elevation, and can reach more than 80% of the acarbose efficiency of a diabetes drug, the inhibition rate of the SA9 bacterial suspension (fermentation liquor) on the alpha-glucosidase is 85%, the inhibition rate of bacterial cell metabolite is 96%, and the cell-free extract is 87%. Meanwhile, the SA9 strain also has excellent inhibition capability on alpha-amylase, the inhibition rate of fermentation liquor on the alpha-amylase can reach 100%, the effect is equivalent to that of acarbose, but the effect is better than that of acarbose, and the fermentation liquor has no toxic or side effect.
The SA9 strain provided by the invention can inhibit the activity reduction of alpha-glucosidase and alpha-amylase and block the transformation and absorption of saccharides, is beneficial to controlling blood sugar, relieving diabetes and has positive effects on the prevention and treatment of diabetes. In addition, the condensation Wei Ciman bacillus already enters the national edible probiotics directory, and the strain is high in safety, free of toxic and side effects and strong in stress resistance. The Wei Ciman bacillus coagulans SA9 can be applied to fermented foods, health-care products and medicines, and provides a new choice for daily blood sugar control of diabetics.
Drawings
FIG. 1 is a graph showing the results of acid production capacity measurement of different strains of primary screening;
FIG. 2 is a graph showing the results of the measurement of the ability of the different strains to initially screen and scavenge DPPH free radicals;
FIG. 3 is a phylogenetic tree diagram of the strain;
FIG. 4 is a graph showing the morphology of a bacterium of the condensation Wei Ciman;
FIG. 5 is a graph showing the results of inhibition of alpha-glucosidase activity by Bacillus coagulans Wei Ciman;
FIG. 6 is a graph showing experimental results of inhibition of alpha-amylase activity by Bacillus coagulans Wei Ciman;
FIG. 7 is a graph showing the results of a dose gradient inhibition experiment on the fermentation supernatant of Bacillus coagulans Wei Ciman; (stock solution, 4-fold dilution, 8-fold dilution in turn from left to right);
FIG. 8 is a graph showing the results of a graph of the experiment for inhibiting the concentration of a Wei Ciman bacterium coagulans broth in a dose gradient manner; (stock solution, 4-fold dilution, 8-fold dilution in this order from left to right).
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The following examples used the following media:
MRS broth: 10g of tryptone, 10g of beef extract, 4g of yeast extract powder, 20g of glucose, 2g of dipotassium hydrogen phosphate (anhydrous), 2g of tri-ammonium citrate (anhydrous), 5g of sodium acetate trihydrate, 0.2g of magnesium sulfate (containing heptahydrate), 0.05g of manganese sulfate (containing tetrahydrate), 1g of tween-80 and 6.8+/-0.2 of final pH.
Example 1 screening and identification of strains
(1) Isolation and purification of strains
The method comprises the steps of adopting commercial fresh mulberries, washing the mulberries cleanly, filling the mulberries into a fermentation tank, adding sugar water with different concentrations, and carrying out natural fermentation. After the fermentation was completed, an appropriate amount of fermentation residue was weighed and added to a conical flask containing 100mL of MRS liquid medium, which was placed in a shaking table at 37℃for 48 hours. Then, 1mL of the mixed bacterial solution was aspirated therefrom, and the solution was subjected to gradient dilution with sterile water, and 10 was applied to an MRS plate -2 、10 -3 、10 -4 、10 -5 、10 -6 Five dilutions were made. Different single colonies were then randomly picked from the plates and streaked onto MRS solid plates for purification. Culturing at 37℃for 48h. The obtained pure culture strain is washed by 20% glycerol, sucked into a glycerol pipe and stored in a refrigerator at the temperature of minus 20 ℃ for standby.
(2) Molecular biological identification of bacteria
The purified bacteria were subjected to DNA extraction by SDS method, and the conserved sequences of the bacteria were amplified using the universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-TACCTTGTTACGACTT-3'). And sending the amplified product to a sequencing company for sequencing, splicing the obtained sequences, and comparing the spliced sequences in an NCBI database to obtain 22 strains of Wei Ciman bacillus coagulans. After preliminary screening by the measurement of acid production capacity and DPPH free radical removal capacity, the results are shown in fig. 1 and 2, and 5 bacillus coagulans with good performances are comprehensively determined and respectively named as: SA19, SA9, SA12, SA4, SA6. The phylogenetic tree of 5 strains is shown in FIG. 3. As a result, it was found that the 16S rDNA sequence of the strain of the present invention has a high homology with Bacillus coagulans Wei Ciman (Weizmannia coagulans) in GenBank (note: the present invention was named and preserved by the latest Latin name by changing from Bacillus coagulans Bacillus coagulans to Bacillus coagulans Wei Ciman Weizmannia coagulans).
(3) Observation of the form of the fungus
The purified SA9 strain is inoculated into MRS liquid culture medium and cultured for 48 hours at 37 ℃. A small amount of bacterial liquid was taken and the bacterial body was observed by the gram staining method. As a result, as shown in FIG. 4, the cell morphology was in the form of a long rod, and the gram staining was positive and purple.
Based on the results of molecular biological identification and bacterial chromosome staining observation, the strain SA9 with higher acid production capacity and DPPH free radical removal capacity, which is obtained by separation of the invention, is identified as the Wei Ciman bacillus coagulans (Weizmannia coagulans) and is stored in the microorganism strain collection in Guangdong province at 6 months of 2022, with the storage number: GDMCC NO 62517, deposit address: guangzhou city first middle road No. 100 college No. 59 building 5.
EXAMPLE 2 experiments on inhibition of alpha-glucosidase Activity by Bacillus coagulans SA9
1. Solution preparation
1U/mL of alpha-glucosidase solution: precisely weighing 1mg of alpha-glucosidase, and adding 33mL of PBS solution to prepare 1U/mL of alpha-glucosidase solution;
5mmol/L p-nitrophenyl-alpha-D-glucopyranoside (PNPG): accurately weighing 0.015g of PNPG in a 50mL measuring flask, and adding a PBS solution to prepare 5mmol/L PNPG solution;
0.1mol/L Na 2 CO 3 solution: accurately weighing anhydrous Na 2 CO 3 0.53g, adding distilled water for dissolution and fixing the volume to 50 mL.
2. Preparation of different coagulans Wei Ciman bacilli
(1) Strain activation: the SA9 strain isolated and identified in example 1 was used, the strain was taken out from the refrigerator at-20℃and inoculated into a test tube for activation at an inoculum size of 1%, and the strain was used after two generations of activation.
(2) Preparation of fermentation broth (bacterial suspension): inoculating the activated strain SA9 of the bacillus coagulans Wei Ciman into 100mL of liquid culture medium according to an inoculum size of 1%, and shake culturing for 48h at 37 ℃ under the condition of 180r/min to obtain fermentation liquor.
(3) Preparing fermentation supernatant: centrifuging the fermentation broth at 4deg.C and 6000r/min for 0min, collecting supernatant, filtering with 0.22 μm water micro-filtration membrane to obtain fermentation supernatant sample, and refrigerating at 4deg.C.
(4) Preparation of somatic cell metabolites (CFS): centrifuging the above prepared fermentation broth at 4deg.C and 4000r/min for 15min, collecting thallus, washing with sterile 0.1mol/L PBS (pH=6.8) for 2 times, suspending the thallus in PBS, and adjusting the concentration of the thallus to OD 600 Shake culturing at 37deg.C and 180r/min for 24 hr at 1.0; centrifuging at 4deg.C and 6000r/min for 10min, collecting supernatant, filtering with 0.22 μm water system microfiltration membrane to obtain bacterial cell metabolite, and refrigerating at 4deg.C.
(5) Cell-free extract (CFE) was prepared: centrifuging the above prepared fermentation broth at 4deg.C and 4000r/min for 15min, collecting thallus, washing with sterile 0.1mol/L PBS (pH=6.8) for 2 times, suspending the thallus in PBS, and adjusting the concentration of the thallus to OD 600 1.0, lysozyme is added according to 1.5mg/mL, after the lysozyme is reacted for 3 hours at 37 ℃, ultrasonic disruption is carried out under ice bath (power 800W, ultrasonic time 15min, work 2s, intermittent 2 s); centrifuging the ultrasonic crushed solution at 4deg.C and 6000r/min for 15min, collecting supernatant, filtering with 0.22 μm water system microfiltration membrane to obtain cell-free extract, and refrigerating at 4deg.C for use.
3. Sample preparation
(1) Sample group: the bacterial suspension (fermentation broth), fermentation supernatant, bacterial cell metabolite and cell-free extract obtained by the above method were used, 50. Mu.L of the sample was mixed with 100. Mu.L of PNPG by pipetting, the mixture was taken out in a 37℃water bath for 10min, 100. Mu.L of an alpha-glucosidase solution (1U/mL) was then aspirated, the mixture was reacted at 37℃for 30min, the mixture was taken out, and 1mL of sodium carbonate (0.1M) was added to stop the reaction. The alpha-glucosidase activity was assayed at 400nm by pipetting 100. Mu.L to 96 well plates.
(2) Sample blank: the sample obtained above was taken out, 50. Mu.L of the sample was mixed with PNPG 100. Mu. L, PBS 100. Mu.L by pipetting, taken out in a water bath at 37℃for 10 minutes, then 100. Mu.L of an alpha-glucosidase solution (1U/mL) was pipetted, reacted at 37℃for 40 minutes, taken out, and 1mL of sodium carbonate (0.1M) was added to stop the reaction. The alpha-glucosidase activity was assayed at 400nm by pipetting 100. Mu.L to 96 well plates.
(3) Control group: 50. Mu.LPBS was pipetted into a 100. Mu.L mixture of PNPG, taken out in a 37℃water bath for 10min, then 100. Mu.L of an alpha-glucosidase solution (1U/mL) was pipetted, reacted at 37℃for 30min, taken out, and 1mL of sodium carbonate (0.1M) was added to stop the reaction. The alpha-glucosidase activity was assayed at 400nm by pipetting 100. Mu.L to 96 well plates.
(4) Blank group: mu.L of PBS was pipetted into and mixed with 100. Mu.L of PNPG, taken out in a water bath at 37℃for 40min, and 1mL of sodium carbonate (0.1M) was added to stop the reaction. The alpha-glucosidase activity was assayed at 400nm by pipetting 100. Mu.L to 96 well plates.
(5) Acarbose group: 50. Mu.L of the mixture was pipetted with 100. Mu.L of PNPG, mixed with the PNPG in a water bath at 37℃for 10 minutes, taken out, 100. Mu.L of an alpha-glucosidase solution (1U/mL) was pipetted, reacted at 37℃for 30 minutes, taken out, and 1mL of sodium carbonate (0.1M) was added to stop the reaction. The alpha-glucosidase activity was assayed at 400nm by pipetting 100. Mu.L to 96 well plates.
(6) The parameters and conditions for the different experimental groups are set forth in the following table, three parallel experiments were performed for each group:
(7) The inhibition rate of alpha-glucosidase is calculated as follows:
wherein: a is that c : is a control group;
A B : is a blank group;
A S : is a sample group;
A SB : is a blank group of samples.
4. Results
As shown in FIG. 5, the acarbose positive control group had an inhibition ratio of 100% of the alpha-glucosidase, an inhibition ratio of 85% of the alpha-glucosidase by the suspension of Bacillus coagulans SA9, an inhibition ratio of 29% of the fermentation supernatant, 96% of the cell metabolite, and 87% of the cell-free extract. It can be seen that the different components of Wei Ciman Bacillus coagulans SA9 all have an inhibitory effect on alpha-glucosidase.
EXAMPLE 3 alpha-amylase assay with Wei Ciman Bacillus coagulans SA9
1. Sample preparation
(1) Experimental samples: the preparation of the SA9 strain broth (bacterial suspension), fermentation supernatant, cell metabolites, cell-free extract, acarbose group and PBS group was the same as in example 2, and lactic acid solutions having ph3.5 and 4.0 were used.
(2) Starch medium: accurately weighing 6g of soluble starch and 3g of agar, placing into a 250mL conical flask, and sterilizing for later use.
(3) Alpha-amylase solution (1 mg/L): the required alpha-amylase (extracted from pig pancreas, purchased from market) was weighed accurately, 5mg dissolved in 5mL PBS buffer, and prepared ready for use.
2. Experimental method
Pouring a small amount of starch culture medium into a sterile flat plate, solidifying, placing oxford cup (with inner diameter of 6mm, outer diameter of 8mm, and height of 10 mm) on the surface of the culture medium by using forceps, pouring the culture medium into the flat plate again, and taking out the oxford cup after solidification. 3 oxford cups were placed per plate. 50. Mu.L of the above sample and 50. Mu.L of the alpha-amylase solution were mixed, respectively, and added to the wells. Wherein 50. Mu.L of PBS and 50. Mu.L of alpha-amylase solution are mixed to form a blank control group; 50. Mu.L of MRS and 50. Mu.L of alpha-amylase solution were mixed as a medium negative control group; 50. Mu.L of lactic acid and 50. Mu.L of alpha-amylase solution were mixed as an acidity negative control group. Culturing in an incubator at 37 ℃ for 24 hours. Taken out, observed after staining with dilute iodine solution, and the transparent ring size was measured using vernier calipers.
3. Results
The inhibition effect of different sample groups and alpha-amylase is shown in figure 6, the inhibition rate of the acarbose positive group to the alpha-amylase is 100%, no transparent ring is generated, and the bacterial suspension group also has no transparent ring; the diameter of the transparent circle of the fermentation supernatant group is 14.5; the diameter of the transparent ring of the lactic acid pH4.0 negative control group is 19.5mm, the diameter of the transparent ring of the MRS control group is 20.4mm, and the diameter of the transparent ring of the blank PBS control group is 20.6mm, which indicates that lactic acid and MRS components have no inhibition effect on alpha-amylase, namely, lactic acid and culture medium components of fermentation broth have no interference on experimental results. The inhibition effect of the condensation Wei Ciman bacillus SA9 fermentation liquor group on the alpha-amylase can be the same as that of acarbose, the enzyme activity is inhibited by 100%, and the inhibition rate of fermentation supernatant is about 50%. The cell metabolite and cell-free extract of the bacteria have no obvious effect.
In order to further illustrate the inhibition effect of the fermentation liquor group on alpha-amylase, a dosage gradient experiment is carried out on the fermentation liquor and fermentation supernatant of the condensation Wei Ciman bacillus, and a double dilution method is adopted, so that the results are shown in fig. 7 and 8, namely, raw liquor, 4-time dilution and 8-time dilution are sequentially carried out from left to right, and the diameters of transparent circles of the supernatant group are sequentially 15.0mm, 18.5mm and 20.7mm; the diameter of the transparent ring of the fermentation liquor group is 11.3mm, 12.1mm and 16.4mm in sequence; with the decrease of the concentration, the transparent circle is increased, the edge is clearer, the inhibition rate of fermentation supernatant fluid after 8 times dilution is almost 0, and the inhibition rate of fermentation liquid after 8 times dilution is still 50%, which shows that the inhibition of the condensation Wei Ciman bacillus SA9 and fermentation products thereof has dose dependency on the alpha-amylase.
In conclusion, the condensation Wei Ciman bacillus SA9 provided by the invention has good inhibition effect on alpha-glucosidase and alpha-amylase, can reduce the activity of the alpha-glucosidase and alpha-amylase and block the conversion and absorption of saccharides, is beneficial to controlling blood sugar and relieving diabetes, has positive effects on preventing and treating diabetes, and can be applied to products for reducing blood sugar level. At present, the bacillus coagulans Wei Ciman is listed in the edible fungus directory by a plurality of countries or regions, has the characteristics of high safety, high temperature resistance, spore production, processing resistance and the like, can promote the bacillus coagulans Wei Ciman to be presented in front of diabetics in various product forms, gives more treatment options to the diabetics, has positive significance in the aspect of controlling blood sugar of the diabetics and keeping a healthy and good psychological state, and in addition, the bacillus coagulans Wei Ciman has simple fermentation conditions, is easy to industrialize, has low cost and is a probiotic strain with huge market potential.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (9)
1. Wei Ciman Bacillus coagulans capable of inhibiting alpha-glucosidase and/or alpha-amylase activitiesWeizmannia coagulans) SA9 strain, which is stored in the collection of microorganism strains of the province of guangdong at 6/2022, with deposit number: GDMCC NO. 62517.
2. Use of the strain of bacillus coagulans SA9 and/or its fermentation broth according to claim 1 for inhibiting alpha-glucosidase and/or alpha-amylase for the purpose of non-diagnostic treatment.
3. Use of a strain of bacillus coagulans SA9 and/or a fermentation broth thereof according to claim 1 for the preparation of an alpha-glucosidase and/or alpha-amylase inhibitor.
4. Use of the strain of bacillus coagulans SA9 and/or its fermentation broth according to claim 1 for the preparation of a product for the alleviation or treatment of diabetes or for the control of blood glucose.
5. The use according to claim 4, wherein the product is a food or pharmaceutical product.
6. An alpha-glucosidase and/or alpha-amylase inhibitor comprising a strain of Wei Ciman bacillus coagulans SA9 and/or a fermentation broth thereof according to claim 1.
7. The inhibitor according to claim 6, wherein the OD of the fermentation broth 600 0.5 to 1.0.
8. The inhibitor according to claim 6, wherein the fermentation broth is prepared by the following steps: the SA9 strain is inoculated into a culture medium according to the inoculum size of 1 to 10 percent, and shake culture is carried out for 28 to 48 hours under the conditions of 35 to 40 ℃ and 120 to 200r/min, thus obtaining the strain.
9. A method for inhibiting α -glucosidase and/or α -amylase for non-disease diagnostic therapeutic purposes, characterized in that a sample is treated with the strain Wei Ciman bacillus coagulans SA9 and/or a fermentation broth thereof according to claim 1.
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| PCT/CN2023/113528 WO2024037588A1 (en) | 2022-08-19 | 2023-08-17 | Weizmannia coagulans capable of inhibiting activity of alpha-glucosidase and/or alpha-amylase and use thereof |
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| WO2001034168A1 (en) * | 1999-11-08 | 2001-05-17 | Ganeden Biotech, Inc. | Inhibition of pathogens by bacillus coagulans strains |
| CN107502575A (en) * | 2017-09-20 | 2017-12-22 | 中国农业科学院农产品加工研究所 | One plant of Lactobacillus plantarum with the high inhibitory activity of α glucuroides |
| CN108823118A (en) * | 2018-05-31 | 2018-11-16 | 四川师范大学 | One plant of Le gram bacterium that can inhibit alpha-glucosidase activity and its application |
| CN113425747A (en) * | 2021-07-26 | 2021-09-24 | 吉林省农业科学院 | Application of bacillus coagulans JA845 in preparation of enzyme activity inhibitor with hypoglycemic function |
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| KR102387028B1 (en) * | 2020-10-06 | 2022-04-14 | 이해익 | A novel Bacillus coagulans CC strain having high productivity for alpha glucosidase inhibitor |
| CN115322931B (en) * | 2022-08-19 | 2023-04-25 | 华南农业大学 | Bacillus coagulans Wei Ciman capable of inhibiting activity of alpha-glucosidase and/or alpha-amylase and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001034168A1 (en) * | 1999-11-08 | 2001-05-17 | Ganeden Biotech, Inc. | Inhibition of pathogens by bacillus coagulans strains |
| CN107502575A (en) * | 2017-09-20 | 2017-12-22 | 中国农业科学院农产品加工研究所 | One plant of Lactobacillus plantarum with the high inhibitory activity of α glucuroides |
| CN108823118A (en) * | 2018-05-31 | 2018-11-16 | 四川师范大学 | One plant of Le gram bacterium that can inhibit alpha-glucosidase activity and its application |
| CN113425747A (en) * | 2021-07-26 | 2021-09-24 | 吉林省农业科学院 | Application of bacillus coagulans JA845 in preparation of enzyme activity inhibitor with hypoglycemic function |
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