Lactobacillus crispatus capable of efficiently inhibiting alpha-glucosidase and application of lactobacillus crispatus

Abstract

The invention discloses lactobacillus crispatus capable of efficiently inhibiting alpha-glucosidase and application of the lactobacillus crispatus. The lactobacillus crispatus JQH7 strain provided by the invention is separated and identified from human excrement, has relatively strong self-aggregation capability which can reach about 96% in 24 hours, can be better adhered to biological or non-biological surfaces, and is beneficial to effective colonization in an intestinal environment. Meanwhile, the JQH7 strain has certain antioxidant capacity, and the scavenging rate of a bacterial suspension of the JQH7 strain on DPPH free radicals is 49.18%; the JQH7 strain has the capability of efficiently inhibiting alpha-glucosidase, the effect of the JQH7 strain is equivalent to that of a clinical medicine acarbose, and compared with the acarbose, the JQH7 strain is safe in source, free of side effects, low in price, easy to obtain and easy to prepare. The invention provides more sources and choices for preparing preparations for efficiently inhibiting alpha-glucosidase and hypoglycemic products, provides a new thought for adjuvant therapy of diabetes, and is beneficial to improving and controlling the blood sugar of diabetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and more specifically, relates to a strain of Lactobacillus crispatus capable of effectively inhibiting alpha-glucosidase and application thereof. Background Art

[0002] Diabetes is a systemic disease caused by relative or absolute insufficiency of insulin secretion in the body, characterized by metabolic disorders, and mainly manifested as persistent hyperglycemia. Current studies have shown that by inhibiting the activity of α-glucosidase, a key digestive enzyme, the hydrolysis of carbohydrates and the release of glucose can be slowed, thereby delaying the increase in blood sugar after meals in diabetic patients. Currently, commonly used drugs that inhibit α-glucosidase in clinical practice include acarbose and metformin, but long-term use can cause side effects of gastrointestinal discomfort such as abdominal distension and diarrhea. Therefore, it is particularly important to explore safe and effective treatment methods.

[0003] Lactobacillus crispatus is a facultative anaerobic bacterium that can be isolated from human feces and widely colonized in the intestines and vagina. As a probiotic, Lactobacillus crispatus plays an important role in inhibiting harmful bacteria, maintaining intestinal health, enhancing the immune system and anti-inflammation. Studies have shown that Lactobacillus crispatus can inhibit the growth of bacteria by producing antibacterial substances such as hydrogen peroxide. In addition, Lactobacillus crispatus can grow slowly in an acidic environment, which helps maintain the balance of intestinal microecology. It can also promote the proliferation and repair of intestinal mucosal cells, maintain intestinal barrier function, and prevent increased intestinal permeability. At the same time, Lactobacillus crispatus can enhance the body's resistance by regulating the intestinal immune system, activate immune cells and produce immunomodulatory factors to resist the invasion of pathogens and reduce inflammatory responses.

[0004] At present, the research and application of Lactobacillus crispatus are mainly concentrated in the field of female reproductive health, such as inhibiting the growth of vaginal pathogens, alleviating vaginitis and preventing intrauterine adhesions. However, there are still relatively few studies on other probiotic functions of Lactobacillus crispatus, especially in terms of hypoglycemic effect. The prior art discloses a strain of Lactobacillus crispatus MY7, whose fermentation supernatant has an inhibition rate of 18.16% on α-glucosidase, and its inhibitory effect is poor. At present, Lactobacillus crispatus with efficient hypoglycemic effect has not been screened, and there are almost no literature reports on the research of its metabolites in inhibiting α-glucosidase. Therefore, it is of great significance to screen out a strain of Lactobacillus crispatus that can efficiently inhibit α-glucosidase. This not only helps to expand the functional characteristics of Lactobacillus crispatus, but also provides new ideas and strategies for the prevention and treatment of diabetes for probiotics and their metabolites. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the poor inhibitory effect of existing Lactobacillus crispatus on α-glucosidase and the lack of new bacterial species resources that effectively inhibit α-glucosidase, and to provide a strain of Lactobacillus crispatus that effectively inhibits α-glucosidase and its application.

[0006] The first object of the present invention is to provide a Lactobacillus crispatus JQH7 strain.

[0007] The second object of the present invention is to provide the application of Lactobacillus crispatus JQH7 strain.

[0008] The third object of the present invention is to provide an α-glucosidase inhibitor or a DPPH free radical scavenging preparation.

[0009] The fourth object of the present invention is to provide a blood sugar lowering or anti-oxidation product.

[0010] The fifth object of the present invention is to provide a method for inhibiting α-glucosidase and / or scavenging DPPH free radicals for non-disease diagnosis and treatment purposes.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention provides a Lactobacillus crispatus JQH7 strain, which has been deposited in Guangdong Provincial Culture Collection Center on November 4, 2024, with a deposit number of GDMCC No: 65414.

[0013] The present invention isolates and identifies a strain of Lactobacillus crispatus JQH7 that efficiently inhibits alpha-glucosidase from human feces. The colony morphology of the strain is round, milky white, and smooth and moist on the surface, and the strain is a Gram-positive bacterium. The nucleotide sequence of 16S rDNA of the JQH7 strain is shown in SEQ ID NO.1, and the total length is about 1455bp. The JQH7 strain has a strong self-aggregation ability, which can reach about 96% in 24 hours, can better adhere to biological or non-biological surfaces, is conducive to effective colonization in the intestinal environment, and enables the Lactobacillus crispatus JQH7 strain to better play its role.

[0014] Studies have shown that the JQH7 strain has a high inhibitory effect on α-glucosidase, a key enzyme that causes elevated blood sugar levels. Its effect is comparable to that of the diabetes drug acarbose. The inhibition rate of the strain fermentation supernatant on α-glucosidase is 96.8%, the inhibition rate of the bacterial suspension is 38.5%, and the inhibition rate of bacterial cell metabolites is 44.7%. That is, the JQH7 strain can produce substances that inhibit α-glucosidase. These substances may come from the metabolic activities of the strain, gene expression products, and its enzyme system composition. They can effectively inhibit α-glucosidase, delay the decomposition of carbohydrates and the release of glucose, thereby helping to control blood sugar levels and can be used to alleviate or treat diabetes. At the same time, the JQH7 strain also has the ability to scavenge DPPH free radicals. The scavenging rate of its bacterial suspension on DPPH free radicals is 49.18%. It has a certain antioxidant capacity and can be used to prepare antioxidant products.

[0015] Therefore, the present invention provides the use of Lactobacillus crispatus JQH7 strain and / or its bacterial liquid and metabolites in inhibiting α-glucosidase or scavenging DPPH free radicals for the purpose of non-disease diagnosis and treatment.

[0016] The invention provides application of Lactobacillus crispatus JQH7 strain and / or its bacterial liquid and metabolites in preparing alpha-glucosidase inhibitors or in preparing DPPH free radical scavenging preparations.

[0017] The present invention provides application of Lactobacillus crispatus JQH7 strain and / or its bacterial liquid and metabolites in preparing products for alleviating or treating diabetes or products for controlling blood sugar.

[0018] The present invention provides application of Lactobacillus crispatus JQH7 strain and / or its bacterial liquid and metabolites in preparing antioxidant products.

[0019] Furthermore, the product is food or medicine.

[0020] More preferably, the product is a health product.

[0021] The invention provides an alpha-glucosidase inhibitor or a DPPH free radical scavenging preparation, which contains a Lactobacillus crispatus JQH7 strain and / or a bacterial liquid and a metabolite thereof.

[0022] The invention also provides a blood sugar lowering or anti-oxidation product containing the above preparation.

[0023] In addition, the present invention provides a method for inhibiting α-glucosidase and / or removing DPPH free radicals for non-disease diagnosis and treatment purposes, using Lactobacillus crispatus JQH7 strain and / or its bacterial solution and metabolites to process samples.

[0024] Preferably, the fermentation supernatant of Lactobacillus crispatus JQH7 strain is used for treatment.

[0025] More preferably, the JQH7 strain fermentation broth is obtained by inoculating the JQH7 strain into an optimized culture medium, culturing at 37° C. for 48 hours, and collecting the supernatant by centrifugation.

[0026] More preferably, the OD of the fermentation broth is 1 to 1.5.

[0027] The present invention has the following beneficial effects:

[0028] The present invention identifies a new Lactobacillus crispatus JQH7 strain, which is derived from human feces, grows faster, has a strong self-aggregation ability, can reach about 96% in 24 hours, can better adhere to biological or non-biological surfaces, is conducive to effective colonization in the intestinal environment, and enables the Lactobacillus crispatus JQH7 strain to play a better role. At the same time, the JQH7 strain has a certain antioxidant ability, and the bacterial suspension has a DPPH free radical scavenging rate of 49.18%; and it also has the ability to efficiently inhibit alpha-glucosidase, and its effect is equivalent to the effect of the clinical drug acarbose. Compared with acarbose, the source of the Lactobacillus crispatus JQH7 strain is safe, has no side effects, is cheap and easy to obtain, and is easy to prepare, which provides more sources and options for preparing preparations and hypoglycemic products that efficiently inhibit alpha-glucosidase, and can be stably colonized in the intestinal environment through its excellent self-aggregation ability, and exerts its hypoglycemic effect of inhibiting alpha-glucosidase, thereby providing a safe and reliable natural health care option for diabetic patients.

[0029] In addition, as an edible fungus, Lactobacillus crispatus can be widely used in food, improve the nutritional value of food and promote human health. It provides a scientific basis for the further development of functional probiotics or postbiotic resources based on Lactobacillus crispatus JQH7, and provides new ideas for the auxiliary treatment of diabetes. It also opens up new paths for nutritional intervention and health management, which helps to improve blood sugar control and overall health status of diabetic patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a Gram-type microscopic image of Lactobacillus crispatus JQH7.

[0031] Figure 2 This is the phylogenetic tree of Lactobacillus crispatus JQH7.

[0032] Figure 3 This is the growth curve of Lactobacillus crispatus JQH7.

[0033] Figure 4 This is the self-aggregation result diagram of Lactobacillus crispatus JQH7.

[0034] Figure 5 This is a graph showing the antioxidant results of Lactobacillus crispatus JQH7.

[0035] Figure 6 This is the result of inhibiting α-glucosidase by Lactobacillus crispatus JQH7. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] The culture medium formula used in the following examples is as follows:

[0039] MRS solid culture medium: peptone 10g / L, yeast extract 5g / L, beef extract 10g / L, anhydrous glucose 20g / L, dipotassium hydrogen phosphate 2g / L, anhydrous sodium acetate 5g / L, triammonium citrate 2g / L, magnesium sulfate 0.1g / L, manganese sulfate 0.05g / L, Tween 80 1g / L, agar, 15g / L, prepared with distilled water.

[0040] Optimized culture medium: peptone 10g / L, yeast extract 5g / L, beef extract 10g / L, anhydrous glucose 20g / L, dipotassium hydrogen phosphate 2g / L, anhydrous sodium acetate 5g / L, triammonium citrate 2g / L, magnesium sulfate 0.1g / L, manganese sulfate 0.05g / L, cysteine ​​0.5g / L, Tween 80 1g / L, prepared with distilled water.

[0041] Example 1 Isolation and screening of strains

[0042] 1. Isolation of strains

[0043] 1 g of fecal sample (from a centenarian) was diluted with 99 mL of sterile saline and placed in a conical flask with an appropriate amount of glass beads. The sample was shaken and cultured at 200 rpm for 30 min at room temperature to mix the sample evenly and disperse the microorganisms fully. The sample concentration in this conical flask was 10 -2 .

[0044] In the clean bench, sterile saline was used to dilute the concentration gradient to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 and 10 -9 Dilution gradient, 10-7 , 10 -8 and 10 -9 The sample dilution was spread on the sterilized MRS solid culture medium and cultured at 37°C. A single colony was then picked for partitioning, streaking, purification and culture, and repeated twice. Finally, a single colony was obtained and stored for later use.

[0045] 2. Microscopic examination and molecular biological identification

[0046] The purified strain was activated in MRS medium for 24 h and then analyzed under a microscope using a Gram staining kit.

[0047] The colony morphology of the strain is round, milky white, and has a smooth and moist surface; the microscopic examination results are as follows Figure 1 As shown in the figure, the results showed that the strain was Gram-positive, the bacteria were purple, slender rods, slightly bent into chains, and belonged to Gram-positive bacteria (G + ).

[0048] The DNA of the purified bacteria was extracted by SDS method, and the conserved sequences of bacteria were amplified using universal primers 27f and 1492r (27f: 5'-AGTTTGATCMTGGCTCAG-3' and 1492r: 5'-GGTTACCTTGTTACGACTT-3'). The amplified products were sent to Sangon Sequencing Company for sequencing, and the obtained sequences were spliced ​​and compared in the NCBI database. The evolutionary tree was constructed using the neighbor-joining method of Mega software.

[0049] The nucleotide sequence of 16S rDNA of JQH7 strain is shown in SEQ ID NO.1, with a total length of about 1455 bp. The results of repeated comparisons and constructed phylogenetic trees show that the 16S rRNA gene sequence of the isolated strain has the highest homology with Lactobacillus crispatus, with a similarity of 100%. The phylogenetic tree is shown in Figure 2 As shown; Based on the above microscopic identification results, the taxonomic status of the isolated new strain was finally determined to be Lactobacillus crispatus, and the strain was named Lactobacillus crispatus JQH7 strain, and was deposited in Guangdong Provincial Microbiological Culture Collection on November 4, 2024, with a deposit number of GDMCC No: 65414, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0050] Example 2 Growth performance test of strains

[0051] 1. Strain growth curve test

[0052] Take the activated Lactobacillus crispatus JQH7 twice and inoculate it into 50mL optimized culture medium at a rate of 2%, culture it at 37°C, take appropriate amount of fermentation liquid at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28h to measure the absorbance value (OD 600nm ), the test was performed three times in parallel and the average value was taken to draw the growth curve.

[0053] The results are as follows Figure 3 As shown, when the culture time is 2-8h, it is the logarithmic growth phase of strain JQH7, and when it is 12-28h, it is the stable phase.

[0054] 2. Test of strain self-aggregation ability

[0055] Take the twice activated Lactobacillus crispatus JQH7 and inoculate it into 50mL optimized culture medium at 2%, culture it at 37℃ for 24h, then centrifuge it at 8000rpm for 15min at 4℃, and collect the supernatant. Wash the remaining bacterial sludge twice with sterile PBS solution, and then resuspend it with PBS solution to adjust the viable bacterial count to about 1.0×10 8 CFU / mL to obtain bacterial suspension.

[0056] 1 mL of bacterial suspension was taken and the absorbance was measured at 600 nm, recorded as A. The bacterial suspension was left to stand at 37°C for 2, 4, 6, 8, 10, 12, and 24 h, and the upper clear liquid was taken and the absorbance was measured at 600 nm, recorded as B. The self-aggregation ability was calculated by the following formula, and the average value was obtained by three parallel measurements.

[0057]

[0058] Where: A is the absorbance of the bacterial suspension at the beginning; B is the absorbance of the supernatant at different times.

[0059] The results of the test are as follows Figure 4 As shown, the self-aggregation ability of strain JQH7 reached about 70% after 8 hours of standing, and about 96% after 24 hours of standing. Self-aggregation ability is usually related to the ability of microorganisms to adhere to biological or non-biological surfaces. The results show that strain JQH7 can form biofilms and effectively colonize in the intestinal environment, thereby better playing a probiotic role.

[0060] Example 3 Antioxidant capacity test of strains

[0061] Weigh 7.896 mg DPPH, dilute to 100 mL with anhydrous ethanol, that is, prepare 0.2 mmol / L, and store in a refrigerator at 4 ° C in the dark. Take the strain JQH7 bacterial suspension prepared in Example 2 as the sample group, PBS solution as the negative control group, and 1 mg / mL vitamin C solution as the positive control. Take 4 mL of the solution to be tested, add 4 mL of 0.2 mmol / L DPPH in anhydrous ethanol solution, mix well, react at room temperature in the dark for 30 minutes, and measure the absorbance at 517 nm, which is recorded as B. Use 4 mL of anhydrous ethanol instead of DPPH as the control group, measure the absorbance at 51 nm, and record it as C. Use 4 mL of anhydrous ethanol instead of the sample as blank zero adjustment, and measure the absorbance at 517 nm, which is recorded as A. Calculate the DPPH free radical scavenging rate according to the following formula.

[0062]

[0063] Wherein: A is the absorbance value of the blank group; B is the absorbance value of the sample group; C is the absorbance value of the control group.

[0064] The results of the test are as follows Figure 5 As shown, vitamin C was used as the positive control group, and its scavenging rate for DPPH free radicals was 99.18%; PBS buffer was used as the blank control group, and its scavenging rate for free radicals was lower. When determining the antioxidant capacity of the bacterial suspension, the influence of the buffer composition could be excluded; and the scavenging rate for DPPH free radicals by the strain JQH7 group was 49.18%, indicating that the strain had a certain antioxidant effect and the ability to scavenge DPPH free radicals.

[0065] Experimental Example 4 Test of the ability of strains to inhibit α-glucosidase

[0066] 1. Sample preparation

[0067] The JQH7 strain was inoculated into the optimized culture medium at an inoculation rate of 2%, and the culture was carried out at 37° C. for 24 h to obtain the strain fermentation liquid, which was subjected to refrigerated centrifugation at 8000 rpm for 15 min at 4° C. to collect the fermentation supernatant.

[0068] The remaining bacterial sludge was washed twice with sterile PBS solution and then resuspended with PBS solution to adjust the number of viable bacteria to about 1.0×10 8 CFU / mL, that is, bacterial suspension.

[0069] The bacterial suspension was cultured at 37°C for 24 hours to obtain the bacterial metabolic products.

[0070] 2. Experimental steps

[0071] Sample group: 25 μL of 20 mmol / L p-nitrophenyl-α-D-pyranoglucoside (PNPG) and 25 μL of the test solution were added to a 96-well plate, incubated at 37°C for 10 min, and then 50 μL of 0.2 U / mL α-glucosidase was added.

[0072] Sample control group: 25 μL PNPG and 25 μL test solution were added to a 96-well plate, incubated at 37° C. for 10 min, and then 50 μL pH=6.8 PBS buffer solution was added.

[0073] Blank group: 25 μL PNPG and 25 μL PBS solution were added to a 96-well plate, incubated at 37°C for 10 min, and then 50 μL α-glucosidase was added.

[0074] Blank control group: 25 μL PNPG and 25 μL PBS solution were added to a 96-well plate, incubated at 37°C for 10 min, and then 50 μL PBS solution was added.

[0075] Finally, each group was reacted at 37°C for 20 min, and then 100 μL of 0.1 mol / L Na2CO3 solution was added to terminate the reaction. The absorbance of each reaction solution was detected at a wavelength of 405 nm using an ELISA reader, and the inhibition rate was calculated using the following formula.

[0076]

[0077] Wherein: A is the absorbance of the blank group after reaction; B is the absorbance of the blank control group after reaction; C is the absorbance of the sample group after reaction; D is the absorbance of the sample control group after reaction.

[0078] According to the above operation, the inhibition rate of α-glucosidase of the fermentation supernatant, bacterial suspension, bacterial metabolites, acarbose and optimized culture medium of Lactobacillus crispatus JQH7 strain was measured respectively, wherein acarbose (1 mg / mL) was used as the positive group and the optimized culture medium was used as the negative group.

[0079] 3. Results

[0080] Test results such as Figure 6As shown, the inhibition rate of acarbose on α-glucosidase in the positive control group was 99.9%, and the inhibition rate of acarbose on α-glucosidase in the negative group was 8.8%, indicating that the optimized culture medium did not contain components that inhibited α-glucosidase. The inhibition rate of the fermentation supernatant of Lactobacillus crispatus JQH7 strain on α-glucosidase was 96.8%, the inhibition rate of the bacterial suspension on α-glucosidase was 38.5%, and the inhibition rate of the bacterial cell metabolites on α-glucosidase was 44.7%. It can be seen that the different metabolites prepared by the strain JQH7 have a certain inhibitory effect on α-glucosidase, among which the effect of the fermentation supernatant of the JQH7 strain is equivalent to that of the clinical drug acarbose, and has the effect of efficiently inhibiting α-glucosidase.

[0081] In summary, the present invention identifies a new strain of Lactobacillus crispatus JQH7, which is derived from human feces, is safe in source, cheap and easy to obtain, grows faster, has a strong self-aggregation ability, can reach about 96% in 24h, can better adhere to biological or non-biological surfaces, is conducive to effective colonization in the intestinal environment, so that the Lactobacillus crispatus JQH7 strain can play a better role; and has a certain antioxidant capacity and excellent α-glucosidase inhibition ability. Since Lactobacillus crispatus has been officially included in the list of edible fungi, it has a wide range of application potential in the food field. Therefore, the different metabolites (fermentation supernatant, bacterial suspension, bacterial metabolites) of the strain JQH7 provided by the present invention have different degrees of inhibitory effects on α-glucosidase, have a positive promoting effect on modulating blood sugar and improving the symptoms of hyperglycemia caused by diabetes, and can be widely used in products for auxiliary treatment of diabetes and control of blood sugar. In addition, due to its rich probiotic properties, Lactobacillus crispatus can promote intestinal health, enhance immunity, antioxidant function, anti-inflammatory function, etc. It also has important application value in food processing, health product development and functional food addition.

[0082] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus crispatus JQH7, characterized in that: The strain was deposited in Guangdong Provincial Culture Collection Center on November 4, 2024, with the deposit number GDMCCNo: 65414.

2. Use of the Lactobacillus crispatus JQH7 strain and / or its bacterial liquid and metabolites according to claim 1 in inhibiting α-glucosidase or scavenging DPPH free radicals for purposes other than disease diagnosis and treatment.

3. Use of the Lactobacillus crispatus JQH7 strain and / or its bacterial liquid and metabolites according to claim 1 in the preparation of α-glucosidase inhibitors or in the preparation of DPPH free radical scavenging preparations.

4. Use of the Lactobacillus crispatus JQH7 strain and / or its bacterial liquid and metabolites according to claim 1 in preparing products for alleviating or treating diabetes, or products for controlling blood sugar.

5. Use of the Lactobacillus crispatus JQH7 strain and / or its bacterial liquid and metabolites according to claim 1 in the preparation of antioxidant products.

6. The use according to claim 4 or 5, characterized in that: The product is a food or a medicine.

7. An α-glucosidase inhibitor or a DPPH free radical scavenging preparation, characterized in that: Contains the Lactobacillus crispatus JQH7 strain and / or its bacterial liquid and metabolites as described in claim 1.

8. A blood sugar lowering or anti-oxidation product, characterized in that: Containing the preparation according to claim 7.

9. A method for inhibiting α-glucosidase and / or scavenging DPPH free radicals for non-disease diagnosis and treatment purposes, characterized in that: The sample is treated with the Lactobacillus crispatus JQH7 strain and / or its bacterial liquid and metabolites described in claim 1.

10. The method according to claim 9, characterized in that: The fermentation supernatant of Lactobacillus crispatus JQH7 strain was used for treatment.

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