Lactobacillus acidophilus CBBM004 and its application in intestinal health, kidney health, and blood sugar lowering products.

Abstract

This invention relates to *Lactobacillus acidophilus* CBBM004 and its application in intestinal health, kidney health, and blood sugar lowering products, belonging to the field of microbial application technology. The *Lactobacillus acidophilus* CBBM004 was deposited on March 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33903, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This *Lactobacillus acidophilus* was isolated from the human intestine. This strain has significant application effects in intestinal health, kidney health, and blood sugar lowering products. The application forms of this strain include powder, granules, capsules, tablets, pills, drops, or liquid dosage forms, providing new options for the development of related food, health products, or pharmaceutical products.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, and relates to Lactobacillus acidophilus CBBM004 and its application in intestinal health care, kidney health care and blood sugar lowering products. Background Technology

[0002] In modern society, with changes in people's lifestyles and adjustments in their dietary structure, 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.

[0003] In terms of gut health, a balanced gut microbiota is crucial for maintaining human health; an imbalanced gut microbiota can lead to various intestinal diseases. Currently, there are numerous probiotic products on the market for gut health, but they suffer from problems such as significant differences in the effectiveness of different strains and weak colonization capabilities, making it difficult to meet the gut health needs of diverse populations. Regarding kidney health, treatment options for kidney disease are relatively limited, and some medications have certain side effects. However, current research on probiotics specifically for kidney health is insufficient, lacking probiotic strains with clearly defined kidney-health benefits. In terms of lowering blood sugar, although some lactic acid bacteria have been proven to have blood sugar-lowering effects, the effects vary among different strains, and the mechanism of action is not yet fully understood. Existing blood sugar-lowering products are mainly drug-based, and long-term use may lead to adverse reactions; therefore, finding safe and effective natural blood sugar-lowering substances is of great significance.

[0004] The *Lactobacillus acidophilus* CBBM004 involved in this invention is a specially selected and cultivated strain with excellent intestinal colonization ability and the ability to regulate intestinal flora balance. Preliminary studies have shown that the strain involved in this invention performs excellently in regulating intestinal flora balance and reducing serum uric acid levels, and its metabolites have a significant inhibitory effect on related enzymes in lowering blood sugar. Based on this, products containing *Lactobacillus acidophilus* CBBM004 are being developed for application in intestinal health, kidney health, and blood sugar control, potentially providing new solutions for the prevention and treatment of related diseases. Summary of the Invention

[0005] The purpose of this invention is to provide Lactobacillus acidophilus CBBM004 and its application in intestinal health, kidney health, and blood sugar lowering products.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A type of Lactobacillus acidophilus CBBM004 was deposited on March 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33903, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This Lactobacillus acidophilus was isolated from the human intestine.

[0008] A *Lactobacillus acidophilus* CBBM004 inoculum, wherein the inoculum contains *Lactobacillus acidophilus* CBBM004 as described in claim 1, and the content of *Lactobacillus acidophilus* CBBM004 is not less than 1 × 10⁻⁶. 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 seed in MRS medium, inoculate the activated Lactobacillus acidophilus CBBM004 into MRS liquid medium at an inoculation amount of 2-5%, and culture at 37.0±1.0℃ under anaerobic or facultative anaerobic conditions for 12-24 h to obtain bacterial culture.

[0011] S3-2: After centrifuging the bacterial solution, collect the bacterial cells and add a protective agent at 0.5 to 1 times the amount of bacterial cells to obtain an emulsion; pre-freeze the emulsion at -40 to -60°C for 4 hours, and then freeze-dry it under vacuum at -30 to -50°C for 48 to 72 hours to obtain the Lactobacillus acidophilus CBBM004 bacterial powder.

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

[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 microbial agent includes powder, granules, capsules, tablets, pills, drops, or liquid dosage forms.

[0015] On the one hand, the present invention provides the application of Lactobacillus acidophilus CBBM004 in the preparation of intestinal health care products.

[0016] In another aspect, the present invention provides the application of Lactobacillus acidophilus CBBM004 in the preparation of kidney health care products.

[0017] In another aspect, the present invention provides the application of Lactobacillus acidophilus CBBM004 in the preparation of products that lower blood sugar.

[0018] Furthermore, the product is a pharmaceutical product comprising an effective dose of Lactobacillus acidophilus CBBM004 and pharmaceutically acceptable excipients.

[0019] Furthermore, the drug is one of tablets, granules, oral liquids, or capsules.

[0020] The beneficial effects of this invention are:

[0021] The *Lactobacillus acidophilus* CBBM004 of this invention can regulate the balance of intestinal flora. After entering the intestine, it increases the number of beneficial bacteria and inhibits the proliferation of harmful bacteria. Simultaneously, *Lactobacillus acidophilus* CBBM004 can also lower serum uric acid levels, thus protecting the kidneys. Furthermore, *Lactobacillus acidophilus* CBBM004 can lower blood glucose levels. During its growth and reproduction in the intestine, it consumes glucose and produces α-glucosidase inhibitors, a hypoglycemic substance, during fermentation, thereby achieving a blood glucose-lowering effect. In summary, *Lactobacillus acidophilus* CBBM004 has significant effects in regulating intestinal flora, protecting kidney function, and lowering blood glucose. Attached Figure Description

[0022] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0023] Figure 1 This is a colony morphology diagram of Lactobacillus acidophilus CBBM004. Detailed Implementation

[0024] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided.

[0025] The following content refers to Lactobacillus acidophilus CBBM004 ( Lactobacillus acidophilus The preservation information for strain CBBM004 is as follows: Classification and name: Lactobacillus acidophilus, Preservation date: March 20, 2025, Preservation address: No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101, Preservation number: CGMCCNo. 33903.

[0026] MRS medium formulation: 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 being pure water. An additional 1.5% agar powder is added to the solid MRS medium. The medium is then autoclaved at 121 °C for 15 min.

[0027] Example 1

[0028] Isolation, screening, purification and identification of strains

[0029] (1) Isolation, screening and purification of strains: Collect fecal samples from young people, take 0.5 g to 4.5 mL of sterile physiological saline, shake to disperse the sample, take 100 μL of sample for serial dilution, select appropriate gradient samples and spread them on MRS agar plates, place them under anaerobic conditions, and incubate at 37 ℃ for 36 h. Select colonies according to the following colony characteristics: white, round, moist, opaque, and with neat edges. Cultivate the initially screened colonies in MRS liquid medium to prepare colony culture solution, and at the same time take a part of the culture solution to prepare glycerol tube seed and store it in a -80 ℃ refrigerator.

[0030] (2) Strain identification: The colony culture was sent for identification. The identification unit was the Testing Center of General Biotechnology (Anhui) Co., Ltd.

[0031] The nucleic acid sequence of this strain was identified as follows:

[0032]

[0033] (3)Sequence alignment: By comparing with the local database, strains of Lactobacillus acidophilus that are closest at the species level were selected based on the 16S rRNA sequence. The Neighbor-Joining method was used to construct a phylogenetic tree through MEGA 6.0 software. According to the analysis results, CBBM004 was identified as Lactobacillus acidophilus with a similarity of 99.72%.

[0034] Example 2

[0035] Preparation of Lactobacillus acidophilus CBBM004 bacterial agent

[0036] (1)The glycerol tube seeds of Lactobacillus acidophilus CBBM004 were activated in MRS medium. The activated Lactobacillus acidophilus CBBM004 was inoculated into MRS liquid medium with an inoculation amount of 3%, and cultured anaerobically at 37 °C for 24 h to obtain a bacterial solution.

[0037] (2)After centrifuging the bacterial solution, the bacterial cells were collected, and a protective agent was added in an amount of 1 time the amount of the bacterial cells to obtain an emulsion. The emulsion was pre-frozen at -40 °C for 4 hours in a vacuum freeze dryer, and then continuously vacuum freeze-dried at -30 °C for 72 hours to obtain Lactobacillus acidophilus CBBM004 bacterial powder. Among them, the protective agent was in the following mass percentages: glucose 3%, trehalose 7%, maltodextrin 3%, sucrose 5%, and the rest was pure water.

[0038] In this example, by adjusting the ratio of Lactobacillus acidophilus CBBM004 bacterial powder to maltodextrin, a Lactobacillus acidophilus CBBM004 bacterial agent with a bacterial content of 1×10 9 cfu / g was obtained.

[0039] Example 3

[0040] Effect of Lactobacillus acidophilus on the intestinal flora of mice

[0041] Test animals: SPF-grade SD rats, half male and half female, with a body weight of 270 g ± 30 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number SCXK(Beijing)2012-0001; after the animals were purchased, the feeding environment temperature was (28 ± 2) °C, fed with normal feed and allowed to eat freely, and the animals were allowed to adapt to the environment for 14 days.

[0042] Test samples: The Lactobacillus acidophilus CBBM004 bacterial agent prepared in Example 2.

[0043] Test grouping: Random grouping was performed according to the random number table method, which were divided into a blank control group and an experimental group. There were a total of 20 SPF-grade SD rats, 10 in each group, half male and half female.

[0044] Administration method: The experimental group was given the Lactobacillus acidophilus CBBM004 bacterial agent prepared in Example 2 by gavage once a day for 7 consecutive days at a dose of 0.2 g / 10 g body weight. At the same time, the blank control group was given an equal volume of distilled water once a day for 7 days. During the administration period, each group was fed with normal feed.

[0045] Index measurement: On the 0th, 14th, and 28th days of the experiment, 16S rRNA gene sequencing analysis was performed through fecal sampling to evaluate the changes in beneficial bacteria (Lactobacillus and Bifidobacterium) and harmful bacteria (Escherichia coli). <^

[0046]

[0047] From the data in the above table, it can be seen that after 14 and 28 days of administration, the experimental group can significantly promote the proliferation of intestinal beneficial 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 bacterial agent of the present invention has good application prospects in the field of intestinal health care.

[0048] Example 4

[0049] Effect of Lactobacillus acidophilus on renal function of rats

[0050] Test animals: SPF-grade SD male rats, weighing 270 g ± 30 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the certificate number SCXK (Beijing) 2012-0001. After the animals were purchased, the feeding environment temperature was (28 ± 2) °C, and they were fed with normal feed and allowed to eat freely. They were acclimated to the environment for 7 days.

[0051] Test samples: The Lactobacillus acidophilus CBBM004 bacterial agent prepared in Example 2.

[0052] Administration method: Hypoxanthine and oxonic acid potassium were gavaged every day. The gavage doses of hypoxanthine and oxonic acid potassium were both 300 mg / kg, dissolved in 0.5 wt% sodium carboxymethyl cellulose solution to construct a hyperuricemia mouse model. The control group of mice was gavaged with the same volume of 0.5 wt% sodium carboxymethyl cellulose solution. After an interval of 2 hours, the therapeutic drug was gavaged. The grouping was as follows: The control group and the model group were gavaged with 200 μL of normal saline; the allopurinol group was gavaged with 5 mg / kg of allopurinol solution dissolved in normal saline, and the experimental group was gavaged with the Lactobacillus acidophilus CBBM004 bacterial agent prepared in Example 2 at a dose of 0.2 g / 10 g body weight for 14 consecutive days.

[0053] Index measurement: Blood was collected from the posterior vena cava of mice on day 0 and day 14 respectively. After the collected blood was left standing at room temperature for 2 hours, it was centrifuged at 3000 r / min for 15 minutes, and the serum was collected. The concentration of uric acid in the supernatant was detected using the Elabscience Uric Acid Colorimetric Assay Kit (E-BC-K016-M); the content of creatinine (Cr) in the mouse serum was detected using the Elabscience Creatinine (Cr) Colorimetric Assay Kit (E-BCK188-M); the content of urea (BUN) in the mouse serum was detected using the Elabscience Urea (BUN) Colorimetric Assay Kit (E-BC-K183-M).

[0054]

[0055] From the data in the above table, it can be seen that 14 days after administration, the experimental group could significantly reduce the blood uric acid level of mice. At the same time, the serum creatinine and urea concentrations in the experimental group of mice after administration were lower than those in the model group and allopurinol group, indicating that Lactobacillus acidophilus has the effect of protecting renal function, suggesting that the Lactobacillus acidophilus agent of the present invention has good application prospects in the field of kidney health care.

[0056] Example 5

[0057] Effect of Lactobacillus acidophilus on blood glucose of rats

[0058] Experimental animals: SPF-grade SD rats, half male and half female, with a body weight of 270 g ± 30 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the certificate number was SCXK (Beijing) 2012-0001; after the animals were purchased, the feeding environment temperature was (28 ± 2) °C, and they were fed with normal feed and allowed to eat and drink freely, and were adapted to the environment for 7 days.

[0059] Test samples: The Lactobacillus acidophilus CBBM004 agent prepared in Example 2.

[0060] Test grouping: Random grouping was carried out according to the random number table method, and they were divided into blank control group, model control group, positive control group, and experimental group. There were a total of 40 SPF-grade SD rats, 10 in each group, half male and half female.

[0061] Construction of T2DM (type 2 diabetes mellitus) rat model: Except for 10 mice in the blank control group, the other 150 rats were fed with a high-sugar diet for 4 weeks, then fasted but not water-restricted for 12 h, and streptozotocin (STZ) was intraperitoneally injected at one time at a dose of 45 mg / kg. When injecting, it was dissolved with a citric acid buffer solution with a pH of 4.5 at a concentration of 1%. 72 hours later, blood was collected from the tail vein at the same time for 3 consecutive days to detect the blood glucose levels of rats in each group. If the blood glucose levels detected 3 times were all greater than 16 mmol / L, it was considered that the T2DM model was successfully established.

[0062] Administration method: The experimental group was administered the *Lactobacillus acidophilus* CBBM004 bacterial agent prepared in Example 2 via gavage, once daily for 14 consecutive days, at a dose of 0.5 g / kg, with a volume of 0.1 mL / 10 g. Simultaneously, 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 administered 150 mg / (kg·d) metformin hydrochloride tablets (Beijing Jingfeng Pharmaceutical Group Co., Ltd., batch number: H11021518) via gavage. During the administration period, the blank group rats were fed a normal diet, while the other groups were fed a high-sugar diet.

[0063] Measurement of indicators: After 14 days of administration, rats in each group were fasted for 12 hours but allowed free access to water. Blood was then collected by tail cutting to measure blood glucose levels.

[0064]

[0065] As shown in the table above, after 14 days of administration, the experimental group showed a significant reduction in blood glucose concentration in rats, indicating that the Lactobacillus acidophilus agent of the present invention has good application prospects in the field of hypoglycemia.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention are still within the scope of the present invention.

Claims

1. A type of Lactobacillus acidophilus CBBM004, characterized in that, The Lactobacillus acidophilus CBBM004 was deposited on March 20, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33903, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The Lactobacillus acidophilus was isolated from the human intestine.

2. A Lactobacillus acidophilus CBBM004 inoculant, characterized in that, The bacterial agent contains *Lactobacillus acidophilus* CBBM004 as described in claim 1, wherein the content of *Lactobacillus acidophilus* CBBM004 is not less than 1 × 10⁻⁶. 9 cfu / g.

3. The Lactobacillus acidophilus CBBM004 inoculant according to claim 2, characterized in that, The preparation process of the microbial agent includes the following steps: S3-1: Activate Lactobacillus acidophilus CBBM004 glycerol tube seed in MRS medium, inoculate the activated Lactobacillus acidophilus CBBM004 into MRS liquid medium at an inoculation amount of 2-5%, and culture at 37.0±1.0℃ under anaerobic or facultative anaerobic conditions for 12-24 h to obtain bacterial culture. S3-2: After centrifuging the bacterial solution, collect the bacterial cells and add a protective agent at 0.5 to 1 times the amount of bacterial cells to obtain an emulsion; pre-freeze the emulsion at -40 to -60°C for 4 hours, and then freeze-dry it under vacuum at -30 to -50°C for 48 to 72 hours to obtain the Lactobacillus acidophilus CBBM004 bacterial powder.

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

5. The Lactobacillus acidophilus CBBM004 inoculant according to claim 3, characterized in that, The protective agent in S3-2 includes one or more of glucose, trehalose, maltodextrin, or sucrose.

6. The Lactobacillus acidophilus CBBM004 inoculant according to claim 2, characterized in that, The dosage forms of the microbial agents include powder, granules, capsules, tablets, pills, and liquids.

7. The use of Lactobacillus acidophilus CBBM004 as described in claim 1 in the preparation of a medicament that promotes the proliferation of beneficial intestinal bacteria and inhibits the growth of harmful intestinal bacteria. The beneficial bacteria are Lactobacillus and Bifidobacterium; the harmful bacteria are Escherichia coli.

8. The use of Lactobacillus acidophilus CBBM004 as described in claim 1 in the preparation of a drug for reducing blood uric acid and urea concentrations.

9. The use of Lactobacillus acidophilus CBBM004 as described in claim 1 in the preparation of a drug for lowering blood sugar.

10. The application according to any one of claims 7 to 9, characterized in that, The drug comprises an effective dose of Lactobacillus acidophilus CBBM004 and pharmaceutically acceptable excipients.

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