Bifidobacterium longum DM22-C-4-3 and application of bifidobacterium longum DM22-C-4-3 in preparation of product for relieving heat stress

Abstract

The invention provides bifidobacterium longum DM22-C-4-3 and application thereof in preparation of products for relieving heat stress, the bifidobacterium longum DM22-C-4-3 is obtained through bacterial culture, excrement, blood and tissue samples of mice are collected, related indexes are measured, statistical analysis is carried out, and results show that the bifidobacterium longum DM22-C-4-3 can be applied to preparation of products for relieving heat stress. By supplementing the bifidobacterium longum DM22-C-4-3, weight loss and testis mass reduction of mice caused by heat stress can be remarkably relieved, small intestine injury is improved, intestinal barriers are repaired, immunosuppressive inflammation is regulated, the oxidation resistance is enhanced, the integrity of liver tissues is protected, the content of short-chain fatty acids in intestinal tracts is increased, and the health of the mice is improved. The effect of relieving the heat stress is proved, so that a new choice is provided for preventing and relieving the heat stress harm.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to Bifidobacterium longum DM22-C-4-3 and application thereof in preparing a product for alleviating heat stress. Background Art

[0002] When the ambient temperature and humidity are too high, the human body cannot effectively dissipate heat. The accumulated heat in the body causes a rise in body temperature, resulting in heat stress. Under heat stress, the body experiences extreme fatigue, dizziness, thirst, and muscle cramps. Long-term exposure to this condition can affect the immune, endocrine, and cardiovascular systems. For example, heat stress can lead to psychological stress reactions such as helplessness, irritability, and difficulty concentrating, as well as physiological stress reactions such as increased heart rate, edema, cramps, syncope, and anorexia. In severe cases, it can lead to heat stroke, heat cramps, heart failure, kidney, intestinal, and cardiovascular diseases, and even death.

[0003] The intestine is not only the main organ for digestion and absorption of nutrients, but also constitutes an immune defense against external pathogens, bacteria, toxins and food antigens, which can prevent harmful components in the intestine from entering the human body through the blood circulation. The intestine is an important barrier to maintain the body's homeostasis, but current studies have shown that heat stress can lead to a decline in intestinal barrier function and increase intestinal permeability (ie, "leaky gut"). This may be because intestinal epithelial cells are affected by the stress response and the barrier function is damaged. Increased intestinal permeability can make it easier for toxins and pathogens to enter the blood, triggering a systemic inflammatory response. Exposure to heat stress can also lead to changes in the structure of the intestinal flora, especially a decrease in beneficial bacteria. Summary of the Invention

[0004] The present invention aims to provide Bifidobacterium longum DM22-C-4-3 and its application in preparing products for alleviating heat stress. Bifidobacterium longum DM22-C-4-3 is isolated from the bodies of centenarians, and its heat stress-alleviating effect is verified through animal experiments.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides Bifidobacterium longum DM22-C-4-3, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on January 2, 2025, with a deposit number of GDMCC No: 65713, and a deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou, and a taxonomic name: Bifidobacterium longum.

[0007] Furthermore, the Bifidobacterium longum DM22-C-4-3 was cultured on TPY agar medium at 37° C. for 24 hours. The colonies were milky white, 0.1-0.2 cm in diameter, with a smooth, moist, raised surface and neat edges. They were rod-shaped when observed under an optical microscope.

[0008] The present invention also provides use of the Bifidobacterium longum DM22-C-4-3 in preparing a product for alleviating heat stress.

[0009] Furthermore, the Bifidobacterium longum DM22-C-4-3 can significantly reduce weight loss caused by heat stress.

[0010] Furthermore, the Bifidobacterium longum DM22-C-4-3 can significantly alleviate the decrease in testicular weight caused by heat stress.

[0011] Furthermore, the Bifidobacterium longum DM22-C-4-3 can improve small intestinal damage caused by heat stress and repair the intestinal barrier.

[0012] Furthermore, the Bifidobacterium longum DM22-C-4-3 can inhibit the increase in the level of pro-inflammatory cytokines caused by heat stress.

[0013] Furthermore, the Bifidobacterium longum DM22-C-4-3 can significantly inhibit the increase in oxidative stress levels caused by heat stress, reduce the content of transaminase in the body, and alleviate liver damage.

[0014] Furthermore, the Bifidobacterium longum DM22-C-4-3 can increase the content of short-chain fatty acids in the intestine.

[0015] Furthermore, the short-chain fatty acids include acetic acid, propionic acid, butyric acid and isobutyric acid.

[0016] Furthermore, the product may be a probiotic preparation or a medicine.

[0017] The beneficial effects of the present invention are:

[0018] The present invention obtains Bifidobacterium longum DM22-C-4-3 through bacterial culture, collects mouse feces, blood and tissue samples, measures relevant indicators and performs statistical analysis. The results show that supplementation with Bifidobacterium longum DM22-C-4-3 can significantly reduce weight loss and testicular mass reduction in mice caused by heat stress, improve small intestinal damage, repair the intestinal barrier, regulate immunosuppressive inflammation, enhance antioxidant capacity, protect liver tissue integrity, and increase the content of intestinal short-chain fatty acids, confirming its effect on alleviating heat stress, thereby providing a new option for preventing and alleviating the hazards of heat stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Showing the changes in body weight and testicular weight of mice in the control group, model group and probiotic group.

[0020] Figure 2 Showing the changes in intestinal tissue of mice in the control group, model group and probiotic group.

[0021] Figure 3 Shown are the levels of proinflammatory cytokines in mice of the control group, model group, and probiotic group.

[0022] Figure 4 Shown are the oxidative stress levels of mice in the control group, model group, and probiotic group.

[0023] Figure 5 Showing the content of short-chain fatty acids in the intestines of mice in the control group, model group and probiotic group. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but will not limit the present invention in any form.

[0025] Example

[0026] 1. Materials and Methods

[0027] 1.1 Bacterial culture

[0028] The present invention provides a strain of Bifidobacterium longum DM22-C-4-3, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on January 2, 2025, with a deposit number of GDMCC No: 65713, and a deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100 Xianlie Middle Road, Guangzhou, and a taxonomic name: Bifidobacterium longum.

[0029] Bifidobacterium longum DM22-C-4-3 was cultured on TPY agar medium at 37°C for 24 hours. The colonies were milky white, 0.1-0.2 cm in diameter, with a smooth, moist, raised surface and neat edges. They were rod-shaped when observed under an optical microscope.

[0030] 1.2 Experimental animals and in vivo experimental design

[0031] C57BL / 6J male mice (6 weeks old) were obtained from Hunan Slake Jingda Biotechnology Co., Ltd. and housed under specific pathogen-free conditions with a 12-hour daylight cycle. Mice were allowed to acclimate for one week before the experiment and were divided equally into three groups, 6 in each group, based on body weight: ① control group (normal drinking water, daily gavage with sterile PBS); ② model group (normal drinking water, daily gavage with sterile PBS, and after 14 days, placed in a 38°C, 70% humidity environment for 12 hours daily, and gavage with sterile PBS daily for 14 days); and ③ probiotic group (normal drinking water, daily gavage with an equal amount of Bifidobacterium longum DM22-C-4-3, and after 14 days, placed in a 38°C, 70% humidity environment for 12 hours daily, and gavage with an equal amount of Bifidobacterium longum DM22-C-4-3 daily for 14 days). The gavage dose of Bifidobacterium longum DM22-C-4-3 was 5 Log10 CFU (dissolved in sterile PBS). During the entire experiment, mice in each group were fed the same feed and their body weight was measured every 4 days. The experiment lasted for a total of 30 days.

[0032] 1.3 Sample collection and measurement

[0033] The feces of the mice were collected once before and after the treatment, and stored at -80℃ in time. After the experiment, the blood, tissue samples and colon contents of the mice were collected under sterile conditions. The blood was centrifuged to obtain serum, and the serum and colon contents were stored at -80℃ until use. Serum samples were used to determine immune factor indicators (ELISA kit, Shanghai Xinyu Biotechnology Co., Ltd., Nanjing Jiancheng Biotechnology Co., Ltd.). Colon contents were used to determine short-chain fatty acids. The liver after dissection was divided into two parts, one was placed in a sterile centrifuge tube for quick freezing in liquid nitrogen and then frozen at -80℃, and the other was rinsed with 0.85% saline and fixed in paraformaldehyde solution for tissue sectioning (Wuhan Sevier Biotechnology Co., Ltd.); the small intestine after dissection was placed in paraformaldehyde for fixation to make intestinal sections (Wuhan Sevier Biotechnology Co., Ltd.). After dissection, the testicles were taken and photographed, and the size of the testicles in each group was compared and weighed. Short-chain fatty acids were determined using gas chromatography-mass spectrometry (GC-MS) from Agilent Technologies (column: Agilent DB-WAX: 0.25 mm × 0.25 μm × 50 cm; inlet temperature: 250°C; gas interface temperature: 250°C; carrier gas flow rate: 1.5 mL / min; split ratio = 3:1; split ratio: 1 μL).

[0034] 1.4 Statistical analysis

[0035] All statistical analyses were performed using one-way ANOVA with Graphpad Pism 9.5 software. Significant differences were considered based on a p-value threshold of 0.05 (*p < 0.05, **p < 0.01). Data are presented as mean ± standard error (SE). Graphs were plotted using Graphpad Pism 9.5.

[0036] 2. Results

[0037] 2.1 Supplementation of Bifidobacterium longum DM22-C-4-3 significantly alleviated heat stress-induced weight loss and testicular mass reduction in mice

[0038] A high temperature and high humidity environment was used to induce heat stress in mice, and the effect of supplementation with Bifidobacterium longum DM22-C-4-3 was evaluated by recording the weight and testicular mass of the mice. During the experiment, the weight of the mice in the model group gradually decreased and was significantly lower than that of the control group. On the contrary, supplementation with Bifidobacterium longum DM22-C-4-3 was able to significantly prevent the weight loss induced by the high temperature and high humidity environment, and was significantly higher than that of the model group. Not only that, on the 30th day, it was found that the high temperature and high humidity treatment caused a significant decrease in the testicular weight of mice, while supplementation with Bifidobacterium longum DM22-C-4-3 significantly alleviated the decrease in testicular weight of mice, as shown in Figure 3. Figure 1 shown.

[0039] 2.2 Supplementation of Bifidobacterium longum DM22-C-4-3 improves small intestinal damage and repairs the intestinal barrier in heat-stressed mice induced by high temperature and high humidity

[0040] The results of hematoxylin-eosin stained paraffin sections showed that the intestinal tissue morphology of the control group mice was normal, with clear structure, intact mucosa, and no obvious inflammatory cell infiltration. However, the intestinal tissue of the model group mice was ulcerated and there was no obvious intestinal gland structure. Supplementation of Bifidobacterium longum DM22-C-4-3 significantly improved the pathological condition of the intestinal tissue of the mice. Although small areas of ulcers were visible, the intestinal gland structure was still maintained and the mucosal epithelium was intact. Figure 2 shown.

[0041] 2.3 Supplementation of Bifidobacterium longum DM22-C-4-3 can improve inflammation in heat-stressed mice induced by high temperature and high humidity

[0042] The levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in mouse serum showed that the levels of pro-inflammatory cytokines in the model group mice increased significantly after high temperature and high humidity treatment, while the levels of pro-inflammatory cytokines were significantly inhibited after supplementation with Bifidobacterium longum DM22-C-4-3. Figure 3 These results indicate that supplementation with Bifidobacterium longum DM22-C-4-3 can modulate host immunity and inhibit the level of inflammation induced by high temperature and humidity treatment.

[0043] 2.4 Supplementation of Bifidobacterium longum DM22-C-4-3 can improve oxidative stress and liver damage in heat-stressed mice induced by high temperature and high humidity

[0044] The levels of alanine aminotransferase (ALT), glutamate aminotransferase (AST), malondialdehyde (MDA), and superoxide dismutase (SOD) in the serum of mice showed that the oxidative stress level of the liver of the model group mice increased significantly and the liver was obviously damaged after high temperature and high humidity treatment. However, supplementation with Bifidobacterium longum DM22-C-4-3 significantly inhibited the oxidative stress level in the mice, reduced the content of transaminases in the body, and alleviated liver damage. Figure 4 These results indicate that supplementation with Bifidobacterium longum DM22-C-4-3 can enhance host antioxidant capacity and protect liver tissue integrity.

[0045] 2.5 Supplementation of Bifidobacterium longum DM22-C-4-3 can improve the content of short-chain fatty acids in the intestine of mice with heat stress induced by high temperature and high humidity

[0046] We tested the content of acetic acid, propionic acid, butyric acid, and isobutyric acid in the cecal contents of mice. The results showed that the content of propionic acid, butyric acid, and isobutyric acid in the intestine of heat-stressed mice induced by high temperature and high humidity decreased significantly, but after intervention with Bifidobacterium longum DM22-C-4-3, the content of acetic acid, propionic acid, butyric acid, and isobutyric acid in heat-stressed mice was significantly increased. Figure 5 These results indicate that supplementation with Bifidobacterium longum DM22-C-4-3 can increase the content of short-chain fatty acids in the intestine.

[0047] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Bifidobacterium longum DM22-C-4-3, characterized in that The Bifidobacterium longum DM22-C-4-3 was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on January 2, 2025, with a deposit number of GDMCC No: 65713, and a deposit address of: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a taxonomic name: Bifidobacterium longum.

2. The Bifidobacterium longum DM22-C-4-3 according to claim 1, characterized in that The Bifidobacterium longum DM22-C-4-3 was cultured on TPY agar medium at 37° C. for 24 hours. The colonies were milky white, 0.1-0.2 cm in diameter, with a smooth, moist, raised surface and neat edges. They were rod-shaped when observed under an optical microscope.

3. Use of the Bifidobacterium longum DM22-C-4-3 according to claim 1 or 2 in the preparation of a product for alleviating heat stress.

4. The use according to claim 3, characterized in that The Bifidobacterium longum DM22-C-4-3 can significantly reduce the weight loss caused by heat stress.

5. The use according to claim 3, characterized in that The Bifidobacterium longum DM22-C-4-3 can significantly alleviate the decrease in testicle weight caused by heat stress.

6. The use according to claim 3, characterized in that The Bifidobacterium longum DM22-C-4-3 can improve small intestinal damage caused by heat stress and repair the intestinal barrier.

7. The use according to claim 3, characterized in that The Bifidobacterium longum DM22-C-4-3 can inhibit the increase in the level of pro-inflammatory cytokines caused by heat stress.

8. The use according to claim 3, characterized in that The Bifidobacterium longum DM22-C-4-3 can significantly inhibit the increase in the level of oxidative stress caused by heat stress, reduce the content of transaminase in the body, and alleviate liver damage.

9. The use according to claim 3, characterized in that The Bifidobacterium longum DM22-C-4-3 can increase the content of short-chain fatty acids in the intestine.

10. The use according to claim 9, characterized in that The short-chain fatty acids include acetic acid, propionic acid, butyric acid and isobutyric acid.

Images