Lactobacillus acidophilus CBBM004 and application of lactobacillus acidophilus CBBM004 in intestinal health care, kidney health care and hypoglycemic products

Abstract

The invention relates to lactobacillus acidophilus CBBM004 and application thereof in intestinal health care, kidney health care and hypoglycemic products, and belongs to the technical field of microorganism application. The lactobacillus acidophilus CBBM004 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 20, 2025, the preservation number is CGMCC No.33903, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the lactobacillus acidophilus is separated from human intestinal tracts. The strain has a remarkable application effect in intestinal health care, kidney health care and hypoglycemic products. The application forms of the strain comprise powder, granules, capsules, tablets, pills, drops or liquid dosage forms, and a new choice is provided for development of related food, health care products or medicine products.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial application, and relates to a Lactobacillus acidophilus CBBM004 and its application in intestinal health care, kidney health care and blood sugar lowering products. Background Art

[0002] In modern society, with the changes in people's lifestyles and adjustments in their dietary structures, the incidence of chronic diseases such as intestinal diseases, kidney diseases and diabetes is on the rise, seriously affecting people's quality of life and health level.

[0003] In terms of intestinal health, the balance of intestinal flora is crucial to maintaining human health. An unbalanced intestinal flora may lead to a variety of intestinal diseases. There are many intestinal health probiotic products on the market, but there are problems such as large differences in the effects of strains and weak colonization ability, which makes it difficult to meet the intestinal health needs of different populations. In terms of kidney health, the treatment options for kidney disease are relatively limited, and some drugs have certain side effects. However, the current research on probiotics for kidney health is not in-depth enough, and there is a lack of probiotic strains with clear kidney health effects. In terms of lowering blood sugar, although some lactic acid bacteria have been proven to have a blood sugar lowering effect, the effects of different strains vary, and the mechanism of action has not yet been fully clarified. Existing blood sugar lowering products are mainly based on drugs, and long-term use may cause adverse reactions. Therefore, it is of great significance to find safe and effective natural blood sugar lowering substances.

[0004] The Lactobacillus acidophilus CBBM004 strain described in this invention is a specially selected and cultivated strain with excellent intestinal colonization and ability to regulate intestinal flora balance. Preliminary studies have shown that this strain excels in regulating intestinal flora balance and lowering serum uric acid levels. Its metabolites also significantly inhibit related enzymes in lowering blood sugar. Based on this, the development of products containing Lactobacillus acidophilus CBBM004 for applications in intestinal health, kidney health, and blood sugar reduction is expected to provide new solutions for the prevention and treatment of related diseases. Summary of the Invention

[0005] The present invention aims to provide a Lactobacillus acidophilus CBBM004 and its application in intestinal health care, kidney health care and blood sugar lowering products.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A Lactobacillus acidophilus CBBM004, wherein the Lactobacillus acidophilus CBBM004 was deposited in the General Microbiology Center of the China Culture Collection Administration on March 20, 2025, with a deposit number of CGMCC No. 33903 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Lactobacillus acidophilus was isolated from the human intestine.

[0008] A Lactobacillus acidophilus CBBM004 bacterial agent, the bacterial agent containing the Lactobacillus acidophilus CBBM004 according to claim 1, the content of the Lactobacillus acidophilus CBBM004 being not less than 1×10 9 cfu / g.

[0009] Furthermore, the preparation process of the bacterial agent includes the following steps:

[0010] S3-1: Activate Lactobacillus acidophilus CBBM004 glycerol tube seeds in MRS medium, inoculate the activated Lactobacillus acidophilus CBBM004 into MRS liquid medium at an inoculum size of 2-5%, and culture at 37.0±1.0°C under anaerobic or facultative anaerobic conditions for 12-24 hours to obtain a bacterial solution;

[0011] S3-2: After centrifuging the bacterial liquid, the bacteria are collected, and a protective agent is added according to 0.5 to 1 times the amount of the bacteria to obtain an emulsion; the emulsion is pre-frozen at a temperature of -40 to -60°C for 4 hours, and then continuously vacuum freeze-dried at a temperature of -30 to -50°C for 48 to 72 hours to obtain the Lactobacillus acidophilus CBBM004 bacterial powder.

[0012] Furthermore, the formula of the MRS liquid culture medium in the S3-1 is as follows: in percentage by mass, 2-4% glucose monohydrate, 2-4% lactose, 1.5-3% peptone, 1-2% yeast peptone, 1.5-3% yeast extract, 0.2-0.4% dipotassium hydrogen phosphate, 0.2-0.4% diammonium hydrogen citrate, 0.5-1% anhydrous sodium acetate, 0.1-0.2% Tween-80, 0.06-0.12% magnesium sulfate heptahydrate, 0.02-0.04% manganese sulfate monohydrate, and the balance pure water.

[0013] Furthermore, the protective agent in S3-2 includes one or more of glucose, trehalose, maltodextrin or sucrose.

[0014] Furthermore, the dosage form of the bacterial agent includes powder, granules, capsules, tablets, pills, drops or liquid dosage forms.

[0015] In one aspect, the present invention provides a use of Lactobacillus acidophilus CBBM004 in preparing intestinal health products.

[0016] In another aspect, the present invention provides a use of Lactobacillus acidophilus CBBM004 in preparing a kidney health product.

[0017] In another aspect, the present invention provides a use of Lactobacillus acidophilus CBBM004 in preparing a product for lowering blood sugar.

[0018] Furthermore, the product is food, which includes but is not limited to cereal products and dairy products.

[0019] The cereal products include but are not limited to bread, noodles, biscuits, and steamed buns; the dairy products include but are not limited to milk, cheese, ice cream, and butter.

[0020] Furthermore, the product is a health product or a fermented product. The health product includes but is not limited to nutritional supplements or functional foods, and the fermented product includes but is not limited to fermented dairy products, fermented soy products or fermented fruit and vegetable products.

[0021] Furthermore, the nutritional supplements include but are not limited to calcium tablets, fish oil capsules, and probiotic powder, and the functional foods include but are not limited to dietary fiber powder, low-sugar meal replacement bars, antioxidant juices, and enzyme drinks.

[0022] Furthermore, the fermented dairy products include but are not limited to yogurt, sour cream, and cheese; the fermented soy products include but are not limited to soy milk, fermented black beans, and soy paste; the fruits and vegetables in the fermented fruit and vegetable products include but are not limited to cucumbers, carrots, beets, celery, and cabbage.

[0023] Furthermore, the product is a medicine, comprising an effective dose of Lactobacillus acidophilus CBBM004 and pharmaceutically acceptable excipients.

[0024] Furthermore, the medicine is in the form of tablets, granules, oral liquid, and capsules.

[0025] Beneficial effects of the present invention:

[0026] Lactobacillus acidophilus CBBM004 in the present invention can regulate the balance of intestinal flora. After entering the intestinal tract, it can increase the number of beneficial bacteria and suppress the breeding of harmful bacteria. At the same time, Lactobacillus acidophilus CBBM004 can also reduce serum uric acid levels and has a protective effect on the kidneys. In addition, Lactobacillus acidophilus CBBM004 can also reduce blood sugar content. It consumes glucose during the growth and reproduction process in the intestine and can also produce the hypoglycemic substance of α-glucosidase inhibitor during the fermentation process, thereby achieving the effect of lowering blood sugar. In summary, the Lactobacillus acidophilus CBBM004 has a significant effect in regulating intestinal flora, protecting renal function and lowering blood sugar. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] To facilitate understanding by those skilled in the art, the present invention is further described below with reference to the accompanying drawings.

[0028] Figure 1 This is the colony morphology of Lactobacillus acidophilus CBBM004. DETAILED DESCRIPTION

[0029] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0030] The Lactobacillus acidophilus CBBM004 strain involved in the following content has the following preservation information: classification name: Lactobacillus acidophilus, preservation date: March 20, 2025, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Preservation Number: CGMCC No. 33903.

[0031] MRS medium formula: 3% glucose monohydrate, 3% lactose, 2% peptone, 1% yeast peptone, 2% yeast extract, 0.3% dipotassium hydrogen phosphate, 0.2% diammonium hydrogen citrate, 0.8% anhydrous sodium acetate, 0.1% Tween-80, 0.1% magnesium sulfate heptahydrate, 0.03% manganese sulfate monohydrate, and the balance pure water. 1.5% agar powder is additionally added to the solid MRS medium and sterilized under high pressure at 121°C for 15 minutes.

[0032] Example 1

[0033] Isolation, screening, purification and identification of strains

[0034] (1) Isolation, screening and purification of strains: Fecal samples from young people were collected, and 0.5 g was taken and added to 4.5 mL of sterile saline. The sample was fully shaken and dispersed. 100 μL of the sample was taken for gradient dilution. Appropriate gradient samples were selected and spread on MRS agar plates. The plates were placed under anaerobic conditions and cultured at 37°C for 36 h. Colonies were selected according to the following colony characteristics: white, round, moist, opaque, and with neat edges. The preliminarily screened colonies were cultured in MRS liquid culture medium to prepare colony culture fluid. At the same time, part of the culture fluid was taken to prepare glycerol tube seeds and stored in a -80°C refrigerator.

[0035] (2) Strain identification: The colony culture medium is sent for identification. The identification unit is the Testing Center of General Biotechnology (Anhui) Co., Ltd.

[0036] The nucleic acid sequence of the strain was identified as:

[0037]

[0038] (3) Sequence Alignment: By comparing with a local database, strains closest to Lactobacillus acidophilus at the species level were selected based on 16S rRNA sequences. A phylogenetic tree was constructed using the Neighbor-Joining method using MEGA 6.0 software. Based on the analysis results, CBBM004 was confirmed to be Lactobacillus acidophilus with a similarity of 99.72%.

[0039] Example 2

[0040] Preparation of Lactobacillus acidophilus CBBM004 bacterial agent

[0041] (1) Activating Lactobacillus acidophilus CBBM004 glycerol tube seeds in MRS medium, inoculating the activated Lactobacillus acidophilus CBBM004 into MRS liquid medium at an inoculum size of 3%, and culturing under anaerobic conditions at 37°C for 24 hours to obtain a bacterial solution;

[0042] (2) After centrifuging the bacterial solution, the bacterial cells were collected, and a protective agent was added at a ratio of 1 times the amount of the bacterial cells to obtain an emulsion; the emulsion was pre-frozen in a vacuum freeze dryer at -40°C for 4 hours, and then vacuum freeze-dried at -30°C for 72 hours to obtain Lactobacillus acidophilus CBBM004 bacterial powder. The protective agent comprises, by mass percentage, 3% glucose, 7% trehalose, 3% maltodextrin, 5% sucrose, and the remainder is pure water.

[0043] In this embodiment, by adjusting the ratio of Lactobacillus acidophilus CBBM004 powder and maltodextrin, the bacterial content was 1×10 9 cfu / g of Lactobacillus acidophilus CBBM004 agent.

[0044] Example 3

[0045] Effects of Lactobacillus acidophilus on intestinal flora in mice

[0046] Experimental animals: SPF-grade SD rats, half male and half female, weighing 270 g ± 30 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with certificate number SCXK (Beijing) 2012-0001. After purchase, the animals were kept at a temperature of (28 ± 2) °C and fed a normal diet ad libitum, and acclimatized to the environment for 14 days.

[0047] Test sample: Lactobacillus acidophilus CBBM004 bacterial agent prepared in Example 2.

[0048] Experimental Grouping: The rats were randomly divided into blank control group and experimental group according to the random number table method. A total of 20 SPF SD rats were used, with 10 rats in each group, half of which were male and half were female.

[0049] Administration: The experimental group was administered the Lactobacillus acidophilus CBBM004 inoculum prepared in Example 2 by oral gavage once daily for 7 consecutive days at a dose of 0.2 g / 10 g body weight. Simultaneously, the blank control group was administered an equal volume of distilled water once daily for 7 consecutive days. During the administration period, all groups were fed with standard feed.

[0050] Measurements: On days 0, 14, and 28 of the experiment, fecal samples were collected for 16S rRNA gene sequencing analysis to assess changes in beneficial bacteria (Lactobacillus and Bifidobacterium) and harmful bacteria (Escherichia coli).

[0051]

[0052]

[0053] As can be seen from the data in the above table, after 14 and 28 days of administration, the experimental group can significantly promote the proliferation of beneficial intestinal bacteria and effectively inhibit the growth of harmful bacteria, proving its good efficacy in regulating the balance of intestinal flora, indicating that the Lactobacillus acidophilus agent of the present invention has good application prospects in the field of intestinal health.

[0054] Example 4

[0055] Effects of Lactobacillus acidophilus on renal function in rats

[0056] Experimental animals: SPF-grade SD male rats, weighing 270 g ± 30 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with a certificate number of SCXK (Beijing) 2012-0001. After purchase, the animals were kept at a temperature of (28 ± 2) °C and fed with normal feed ad libitum for 7 days to adapt to the environment.

[0057] Test sample: Lactobacillus acidophilus CBBM004 bacterial agent prepared in Example 2.

[0058] Administration: A hyperuricemia mouse model was established by daily gavage of hypoxanthine and potassium oxonate at a dose of 300 mg / kg each dissolved in 0.5 wt% sodium carboxymethylcellulose solution. A control group of mice received the same volume of 0.5 wt% sodium carboxymethylcellulose solution. Two hours later, mice were gavaged with the therapeutic drug and divided into the following groups: the control and model groups received 200 μL of normal saline; the allopurinol group received 5 mg / kg of allopurinol solution dissolved in normal saline. The experimental group received the Lactobacillus acidophilus CBBM004 inoculum prepared in Example 2 at a dose of 0.2 g / 10 g body weight. Treatment was continued for 14 days.

[0059] Measurements: Blood was collected from the retro-ocular vein of mice on days 0 and 14. The collected blood was allowed to rest at room temperature for 2 hours and then centrifuged at 3000 rpm for 15 minutes to collect serum. Uric acid concentration in the supernatant was measured using an Elabscience uric acid colorimetric test kit (E-BC-K016-M); serum creatinine content was measured using an Elabscience creatinine (Cr) colorimetric test kit (E-BCK188-M); and serum urea content was measured using an Elabscience urea (BUN) colorimetric test kit (E-BC-K183-M).

[0060]

[0061] As can be seen from the data in the above table, after 14 days of administration, the experimental group was able to significantly reduce the blood uric acid level of mice. At the same time, after administration, the serum creatinine and urea concentrations of the experimental group mice were lower than those in the model group and the allopurinol group, indicating that Lactobacillus acidophilus has the effect of protecting renal function, indicating that the Lactobacillus acidophilus agent of the present invention has good application prospects in the field of kidney health.

[0062] Example 5

[0063] Effects of Lactobacillus acidophilus on blood glucose in rats

[0064] Experimental animals: SPF-grade SD rats, half male and half female, weighing 270 g ± 30 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., with certificate number SCXK (Beijing) 2012-0001. After purchase, the animals were kept at a temperature of (28 ± 2) °C and fed with normal feed ad libitum for 7 days to adapt to the environment.

[0065] Test sample: Lactobacillus acidophilus CBBM004 bacterial agent prepared in Example 2.

[0066] Experimental Grouping: The rats were randomly divided into blank control group, model control group, positive control group and experimental group according to the random number table method. A total of 40 SPF SD rats were used, with 10 rats in each group, half male and half female.

[0067] T2DM (Type 2 Diabetes Mellitus) rat model construction: 150 rats, except for 10 control mice, were fed a high-glucose diet for 4 weeks and then fasted for 12 hours without water. A single intraperitoneal injection of streptozotocin (STZ) at 45 mg / kg was administered, dissolved in a 1% pH 4.5 citrate buffer. Seventy-two hours later, tail vein blood was collected for measurement at the same time for three consecutive days. A successful T2DM model was established if blood glucose levels remained above 16 mmol / L on all three occasions.

[0068] Administration: The experimental group was administered with the Lactobacillus acidophilus CBBM004 agent prepared in Example 2 by oral gavage, once daily for 14 consecutive days, at a dose of 0.5 g / kg (dosage volume of 0.1 mL / 10 g). Meanwhile, the blank control group and the model control group were given an equal volume of distilled water once daily for 14 consecutive days. The positive control group was given 150 mg / (kg·d) metformin hydrochloride tablets (Beijing Jingfeng Pharmaceutical Group Co., Ltd., batch number: National Medicine Standard H11021518) by oral gavage. During the administration period, the blank group rats were fed with a normal diet, while the other groups were fed with a high-sugar diet.

[0069] Index measurement: After 14 days of drug administration, rats in each group were fasted but not watered for 12 hours, and then tails were cut to draw blood for measuring blood glucose levels.

[0070]

[0071] As can be seen from the data in the above table, after 14 days of administration, the experimental group can significantly reduce the blood glucose concentration of rats, indicating that the Lactobacillus acidophilus agent of the present invention has good application prospects in the field of lowering blood glucose.

[0072] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A Lactobacillus acidophilus CBBM004, characterized in that The Lactobacillus acidophilus (Lactobacillus acidophilus) CBBM004 was deposited in the General Microbiology Center of the China Culture Collection Administration on March 20, 2025, with the deposit number CGMCC No. 33903 and the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Lactobacillus acidophilus was isolated from the human intestine.

2. A Lactobacillus acidophilus CBBM004 bacterial agent, characterized in that The bacterial agent contains the Lactobacillus acidophilus CBBM004 according to claim 1, and the content of the Lactobacillus acidophilus CBBM004 is not less than 1×10 9 cfu / g.

3. The Lactobacillus acidophilus CBBM004 according to claim 2, characterized in that The preparation process comprises the following steps: S3-1: Activate Lactobacillus acidophilus CBBM004 glycerol tube seeds in MRS medium, inoculate the activated Lactobacillus acidophilus CBBM004 into MRS liquid medium at an inoculum size of 2-5%, and culture at 37.0±1.0°C under anaerobic or facultative anaerobic conditions for 12-24 hours to obtain a bacterial solution; S3-2: After centrifuging the bacterial liquid, the bacteria are collected, and a protective agent is added according to 0.5 to 1 times the amount of the bacteria to obtain an emulsion; the emulsion is pre-frozen at a temperature of -40 to -60°C for 4 hours, and then continuously vacuum freeze-dried at a temperature of -30 to -50°C for 48 to 72 hours to obtain the Lactobacillus acidophilus CBBM004 bacterial powder.

4. The Lactobacillus acidophilus CBBM004 bacterial agent according to claim 2, characterized in that The formula of the MRS liquid culture medium in the S3-1 is as follows: in percentage by mass, 2-4% glucose monohydrate, 2-4% lactose, 1.5-3% peptone, 1-2% yeast peptone, 1.5-3% yeast extract, 0.2-0.4% dipotassium hydrogen phosphate, 0.2-0.4% diammonium hydrogen citrate, 0.5-1% anhydrous sodium acetate, 0.1-0.2% Tween-80, 0.06-0.12% magnesium sulfate heptahydrate, 0.02-0.04% manganese sulfate monohydrate, and the balance pure water.

5. The Lactobacillus acidophilus CBBM004 bacterial agent according to claim 2, characterized in that The protective agent in S3-2 includes one or more of glucose, trehalose, maltodextrin or sucrose.

6. The Lactobacillus acidophilus CBBM004 bacterial agent according to claim 2, characterized in that The dosage form of the bacterial agent includes powder, granule, capsule, tablet, pill, drop or liquid dosage form.

7. A use of the Lactobacillus acidophilus CBBM004 according to claim 1 in the preparation of products for intestinal health care, kidney health care, and blood sugar reduction.

8. The product according to claim 7, characterized in that The product is a food, which includes but is not limited to cereal products and dairy products.

9. The product according to claim 7, characterized in that The products are health products or fermented products. The health products include but are not limited to nutritional supplements or functional foods. The fermented products include but are not limited to fermented dairy products, fermented soy products or fermented fruit and vegetable products.

10. The product according to claim 7, characterized in that The product is a medicine, comprising an effective dose of Lactobacillus acidophilus CBBM004 and pharmaceutically acceptable excipients.

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