Bifidobacterium animalis subsp. Lactis for relieving health problems caused by smoking and application of bifidobacterium animalis subsp. Lactis

Abstract

The invention belongs to the technical field of biology, and particularly relates to bifidobacterium animalis subsp. Lactis for relieving health problems caused by smoking and application of the bifidobacterium animalis subsp. Lactis. The bifidobacterium animalis subsp. Lactis CA-03 provided by the invention influences the number of macrophages and lymphocytes in pulmonary alveoli by regulating intestinal microorganisms, regulates the levels of key antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in the liver and regulates key inflammatory factors in serum, so as to improve the immune function of the lung. According to the present invention, the interleukin 6 (IL-6) and the interleukin 1beta (IL-1beta) are synthesized, such that the polyhedron of the interleukin 6 (IL-6) and the interleukin 1beta (IL-1beta) in the human body health maintenance is represented, and the broad application prospects in the health promotion and disease treatment aspects are provided.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a strain of animal Bifidobacterium lactis subspecies for alleviating health problems caused by smoking and an application thereof. Background Art

[0002] Smoking is one of the most serious problems in the world today. Smoking can cause a variety of diseases such as cancer, cardiovascular disease and chronic obstructive pneumonia. Studies have shown that at least 25% of continuous smokers develop into COPD patients, and 45% of deaths caused by smoking are attributed to COPD; bronchial asthma is a heterogeneous disease characterized by chronic inflammation of the airways involving multiple cells and cellular components. Further data analysis found that the risk of smokers developing asthma is 1.89 times that of non-smokers. Therefore, smoking is closely related to various diseases and seriously endangers the safety and health of the people. Therefore, it is necessary to develop new, effective means with fewer side effects to prevent or treat the occurrence of diseases.

[0003] The health of the gastrointestinal tract is affected by changes in the intestinal microbiota. Some studies have found that smoking and intestinal microbes are closely linked. There are a large number of toxic substances in the smoke inhaled by smoking, which may directly change the intestinal microbes by changing the human immune system, intestinal mucosa and oxygen ratio. Probiotics have been allowed to be used in the clinical treatment of many diseases. They are usually defined as a general term for a class of active microorganisms that can regulate and improve intestinal flora and have clear benefits to human health when the body takes in enough from the diet. Among them, Bifidobacterium is a genus of Gram-positive, non-motile, rod-shaped cells, one end of which is sometimes forked, and strictly anaerobic bacteria. It is widely present in the digestive tract, vagina and oral cavity of humans and animals. Bacteria of the genus Bifidobacterium are important members of the intestinal flora of humans and animals. Animal experiments and clinical trials have found that Bifidobacterium can regulate intestinal flora and inhibit the growth of intestinal spoilage bacteria by producing short-chain fatty acids such as acetic acid and lactic acid. However, how to use probiotics, especially bifidobacteria, to develop products to alleviate, prevent and treat health problems caused by smoking is a new and necessary topic. Summary of the invention

[0004] The present invention aims to solve the problems existing in the prior art and provides a strain of animal Bifidobacterium lactis subspecies CA-03 for alleviating health problems caused by smoking. The purpose is to use the strain to regulate intestinal microorganisms, the number of macrophages and lymphocytes in alveoli, the levels of superoxide dismutase SOD, catalase CAT and glutathione peroxidase GPx in liver tissue, and the levels of inflammatory factors TNF-α, IL-6 and IL-1β in serum, thereby alleviating, preventing and treating health problems caused by smoking.

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

[0006] A strain of Bifidobacterium animalis subsp. Lactis CA-03 that alleviates health problems caused by smoking. The Bifidobacterium animalis subsp. Lactis CA-03 is classified and named: Bifidobacterium animalis subsp. Lactis, and the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration, the preservation number is: CGMCC No.17743, the preservation time is: May 10, 2019, and the preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0007] The first technical solution provided by the present invention is the use of animal Bifidobacterium lactis subsp. Lactis CA-03 in the preparation of a product for preventing, alleviating and / or treating health problems caused by smoking. The product comprises animal Bifidobacterium lactis subsp. Lactis CA-03 and / or a fermentation metabolite of animal Bifidobacterium lactis subsp. Lactis CA-03, and the preservation number of animal Bifidobacterium lactis subsp. Lactis CA-03 is CGMCC No.17743.

[0008] In certain embodiments, the health issue is gastrointestinal health, lung health, liver health.

[0009] Furthermore, the intestinal health problems refer to intestinal flora imbalance and intestinal discomfort, the lung health problems refer to cough, sputum, low fever, chronic obstructive pulmonary disease, lung cancer, emphysema and chronic bronchitis, etc., and the liver health problems refer to abnormal liver metabolic function, liver inflammation and fibrosis and other liver diseases, etc.

[0010] In certain embodiments, the amount of Bifidobacterium animalis subspecies CA-03 added to the product is not less than 1.0×10 6 CFU / mL or 1.0×10 6 CFU / g.

[0011] In certain embodiments, the individual daily intake of Bifidobacterium animalis subspecies CA-03 is not less than 1.0×10 8 CFU / d.

[0012] In some embodiments, the application includes at least one of the following effects:

[0013] (1) Increase the content of beneficial bacteria in the individual intestine and reduce the content of harmful bacteria in the individual;

[0014] (2) Regulate the number of macrophages and lymphocytes in individual lung tissues;

[0015] (3) Regulate the levels of superoxide dismutase SOD, catalase CAT, and glutathione peroxidase GPx in individual liver tissue;

[0016] (4) Regulate the levels of inflammatory factors TNF-α, IL-6 and IL-1β in individual serum.

[0017] Furthermore, the beneficial bacteria include Lactobacillus reuteri, Parabacteroides distasonis and Bacteroides dorei; the harmful bacteria include Eggerthella lenta, Staphylococcus capitis and Escherichia coli.

[0018] In certain embodiments, the product is a food, a medicine or a health product.

[0019] In certain embodiments, the food comprises dairy products, soy products, or fruit and vegetable products.

[0020] In certain embodiments, the dairy product comprises fermented milk, a yogurt drink, sour cream or cheese.

[0021] In certain embodiments, the soy product includes soy milk, fermented bean curd, fermented black beans or bean paste.

[0022] In certain embodiments, the fruit and vegetable preparation comprises a cucumber, carrot, beet, celery or cabbage preparation.

[0023] In certain embodiments, the pharmaceutical product includes a pharmaceutically acceptable excipient.

[0024] In certain embodiments, the pharmaceutical excipients include at least one of fillers, binders, wetting agents, disintegrants, lubricants and flavoring agents commonly used in pharmacy.

[0025] In certain embodiments, the drug is in the form of tablets, powders, capsules or granules.

[0026] The second technical solution provided by the present invention is the use of animal Bifidobacterium lactis subspecies CA-03 in the preparation of a product for improving lung health problems, wherein the product comprises animal Bifidobacterium lactis subspecies CA-03 and / or a fermentation metabolite of animal Bifidobacterium lactis subspecies CA-03.

[0027] In certain embodiments, the lung health problem is caused by cigarette smoke.

[0028] The third technical solution provided by the present invention is the use of animal Bifidobacterium lactis subspecies CA-03 in the preparation of a product for improving liver health problems, wherein the product comprises animal Bifidobacterium lactis subspecies CA-03 and / or a fermentation metabolite of animal Bifidobacterium lactis subspecies CA-03.

[0029] In certain embodiments, the liver health problem is caused by cigarette smoke.

[0030] The fourth technical solution provided by the present invention is the use of animal Bifidobacterium lactis subspecies CA-03 in the preparation of a product that reduces the level of inflammatory factors in serum, wherein the product includes animal Bifidobacterium lactis subspecies CA-03 and / or a fermentation metabolite of animal Bifidobacterium lactis subspecies CA-03.

[0031] In certain embodiments, the elevated levels of inflammatory factors in serum are caused by cigarette smoke.

[0032] The fifth technical solution provided by the present invention is a product for regulating intestinal microorganisms, wherein the product comprises animal Bifidobacterium lactis subspecies CA-03 and / or a fermentation metabolite of animal Bifidobacterium lactis subspecies CA-03.

[0033] In certain embodiments, the amount of Bifidobacterium animalis subspecies CA-03 added to the product is not less than 1.0×10 6 CFU / mL or 1.0×10 6 CFU / g.

[0034] In certain embodiments, the individual daily intake of Bifidobacterium lactis subspecies CA-03 is not less than 1.0×10 8 CFU / d.

[0035] In certain embodiments, the product is a food, a medicine or a health product.

[0036] In certain embodiments, the food comprises dairy products, soy products, or fruit and vegetable products.

[0037] In certain embodiments, the dairy product comprises fermented milk, a yogurt drink, sour cream or cheese.

[0038] In certain embodiments, the soy product includes soy milk, fermented bean curd, fermented black beans or bean paste.

[0039] In certain embodiments, the fruit and vegetable preparation comprises a cucumber, carrot, beet, celery or cabbage preparation.

[0040] In certain embodiments, the pharmaceutical product includes a pharmaceutically acceptable excipient.

[0041] In certain embodiments, the pharmaceutical excipients include at least one of fillers, binders, wetting agents, disintegrants, lubricants and flavoring agents commonly used in pharmacy.

[0042] In certain embodiments, the drug is in the form of tablets, powders, capsules or granules.

[0043] The technical effects of the present invention are as follows:

[0044] The animal Bifidobacterium lactis subspecies CA-03 provided by the present invention regulates intestinal microorganisms, affects the number of macrophages and lymphocytes in the alveoli, regulates the levels of key antioxidant enzymes in the liver, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and regulates key inflammatory factors in serum, such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and interleukin 1β (IL-1β), showing its versatility in maintaining human health and having broad application prospects in promoting health and treating diseases.

[0045] Biological Deposit Description

[0046] A strain of Bifidobacterium animalis subsp. Lactis CA-03, classified and named: Bifidobacterium animalissubsp.Lactis; deposited by: China General Microbiological Culture Collection Center, abbreviated as CGMCC; deposit number: CGMCC No.17743; deposit date: May 10, 2019; deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the test result of the strain was survival, which has been disclosed in patent CN117511792A. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a heat map of the changes in intestinal microorganisms in the four groups of rats after 90 days of intervention, including blank groups A1-A20, control groups B1-B20, probiotic groups C1-C20, and comparison groups D1-D20. DETAILED DESCRIPTION

[0048] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0049] The raw materials used in the examples are:

[0050] 1. The optimized culture medium for lactic acid bacteria is Beijing Luqiao MRS broth medium.

[0051] 2. The composition of the protective agent solution is as follows: 15Kg skim milk, 12Kg lactose, 1Kg vitamin C, 1Kg sodium glutamate, and 1000L distilled water.

[0052] 3. Bifidobacterium animalis subspecies lactis Bb-12 was sourced from Chr. Hansen (Beijing) Trading Co., Ltd.

[0053] 4. Sprague-Dawley rats were obtained from Saiye Biotechnology.

[0054] Example 1

[0055] A bacterial powder of animal Bifidobacterium lactis subspecies CA-03 is prepared according to a conventional bacterial powder preparation process, and the preparation process comprises the following steps:

[0056] 1. Activation of strains: The animal Bifidobacterium lactis subsp. CA-03 strain stored at -80°C was inoculated into the corresponding liquid culture medium sterilized at 121°C for 15 minutes, and anaerobically cultured at 37°C for 18-24 hours. The activated strains were obtained by subculturing 1-2 times; animal Bifidobacterium lactis subsp. CA-03 was cultivated using a lactic acid bacteria optimized culture medium.

[0057] 2. Preparation of bacterial solution: Take each bacterial strain activated in step (1) and inoculate it into the optimized lactic acid bacteria culture medium, and culture it anaerobically at 37°C until the pH value reaches 4.5-4.8 to obtain seed solution.

[0058] 3. Inoculation and fermentation: The seed liquid of step (2) is inoculated into the lactic acid bacteria optimized culture medium prepared in step (2) at 1‰, and fermented for 18 hours under controlled fermentation conditions.

[0059] The fermentation conditions are controlled as follows: in the early stage of fermentation, the temperature is kept constant at 30°C, and the fermentation is carried out naturally until the pH reaches 5.0; then the fermentation temperature is adjusted to 37°C, and the pH is kept constant at 6.0 to maintain anaerobic fermentation. In this step, the pH is kept constant at 6.0 by adding a neutralizer, and the neutralizer is NaOH.

[0060] The anaerobic fermentation conditions are maintained by passing nitrogen through the chamber every two hours.

[0061] 4. Termination of fermentation: When the bacteria stop producing acid, the fermentation is terminated to obtain a high-density fermentation liquid of the strain, and the number of live bacteria in the fermentation liquid reaches 2×10 10 CFU / mL or more. The cessation of acid production is determined by the cessation of pH decrease and the cessation of neutralizing agent addition.

[0062] 5. Freeze drying: a. Bacteria concentration: The high-density fermentation broth is concentrated by centrifugation at 12000g;

[0063] b. Add protective agent: add 5 times the weight of protective agent solution to the bacterial concentrate;

[0064] c. Drying: The above-mentioned bacterial suspension after adding the protective agent is freeze-dried to obtain freeze-dried bacterial powder, and the bacterial powders of each strain are mixed according to the ratio (ratio of the number of colony forming units) to control the total number of viable bacteria in the mixed bacterial powder to reach 2.0×10 11 CFU / g and above.

[0065] The viable bacteria count in the animal Bifidobacterium lactis subsp. CA-03 powder prepared in this example reaches 200 billion to 1 trillion / g.

[0066] Comparative Example 1

[0067] A kind of animal Bifidobacterium lactis subspecies Bb-12 bacterial powder, the preparation process of which is the same as that of Example 1, and the number of viable bacteria in the prepared animal Bifidobacterium lactis subspecies Bb-12 bacterial powder reaches 200 billion to 1 trillion / g.

[0068] Example 2

[0069] 1. Experimental subjects

[0070] 80 healthy adult male Sprague-Dawley rats, aged 6-8 weeks, weighing approximately 200-250 g.

[0071] 2. Experimental Methods

[0072] 2.1 Grouping

[0073] The rats were randomly divided into the following four groups, with 20 rats in each group:

[0074] Blank group (normal air, no cigarette smoke exposure);

[0075] control group (cigarette smoke exposure);

[0076] probiotic group (cigarette smoke exposure + oral administration of Bifidobacterium animalis subsp. lactis CA-03);

[0077] Control group (cigarette smoke exposure + oral administration of Bifidobacterium animalis subsp. lactis Bb-12).

[0078] 2.2 Specific methods

[0079] SD rats were exposed to cigarette smoke in a whole-body exposure chamber (9 cigarettes / h, 2 hours each exposure, twice a day, 6 days a week) for 90 days. The probiotics were administered orally at a dose of 1×10 9 CFU / d.

[0080] 2.3 Index detection

[0081] At baseline and 90 days after the intervention, the lung health of the experimental rats in each group was evaluated, including the counts of macrophages and lymphocytes in the lung tissue, and the activity of major antioxidant enzymes in the liver tissue (such as superoxide dismutase SOD, catalase CAT, and glutathione peroxidase GPx); blood samples were collected to measure the levels of major inflammatory factors (TNF-α, IL-6, IL-1β) in the serum; and fecal samples were collected for fecal metagenomic analysis.

[0082] 2.4 Data Analysis

[0083] The data are expressed as mean ± standard deviation (SD).

[0084] ANOVA or nonparametric test was used to compare the differences among the groups, and p < 0.05 was considered statistically significant.

[0085] 3. Experimental results

[0086] 3.1 Intestinal flora results

[0087] Shotgun metagenomic analysis of rat feces revealed that 90 days of cigarette smoke exposure significantly altered the composition of the intestinal flora of the control rats compared with the control rats. The number of 11 intestinal microorganisms changed in the four groups ( Figure 1 : blank group A1-A20, control group B1-B20, probiotic group C1-C20, comparison group D1-D20).

[0088] Among them, the number of Eggerthella lenta, Staphylococcus capitis and Escherichia coli in the probiotic group was significantly lower than that in the control group and the comparison group, and there was no significant difference with the blank group. The beneficial intestinal bacteria including Lactobacillus reuteri, Parabacteroides distasonis and Bacteroides dorei were almost zero in the control group and the comparison group, while the content of these three bacteria in the probiotic group increased significantly, close to that of the blank group. The results showed that cigarette smoke changed the intestinal microbial composition of rats. Probiotic intervention significantly reduced the number of Edwardsiella tarda, Staphylococcus capitis and Escherichia coli, and significantly increased the number of beneficial bacteria Lactobacillus reuteri, Parabacteroides distasonis and Bacteroides dorei compared with the control group and the comparison group.

[0089] 3.2 Macrophage and lymphocyte counts in alveolar tissue

[0090] The cells were stained with Giemsa using a blood cell differential counter, and macrophages and lymphocytes were classified and counted under a microscope. The results are shown in Tables 1 and 2. After 90 days of intervention, the number of macrophages and lymphocytes in the probiotic group increased slightly compared with the blank group, but were significantly lower than the control group and the comparison group (p<0.05). This shows that the intervention of animal Bifidobacterium lactis subspecies CA-03 can alleviate the changes in lung tissue caused by cigarette smoke.

[0091] Table 1: Results of the number of macrophages in alveolar tissue of the four groups of rats before and after intervention

[0092]

[0093] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0094] Table 2: Results of lymphocyte counts in alveolar tissue of the four groups of rats before and after intervention

[0095]

[0096] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0097] 3.3 Results of major antioxidant enzymes in liver tissue

[0098] The contents of superoxide dismutase SOD, catalase CAT and glutathione peroxidase GPx in liver tissue were determined according to the Sigma-Aldrich kit method. The results showed that the contents of SOD, CAT and GPx in the control group and the comparison group decreased significantly after 90 days of intervention, while the contents of SOD, CAT and GPx in the probiotic group did not decrease significantly, and there was no significant difference with the blank group. This shows that the intervention of animal Bifidobacterium lactis subspecies CA-03 can alleviate the changes in liver tissue caused by cigarette smoke (see Table 3-5).

[0099] Table 3: Superoxide dismutase (SOD) indexes of the four groups of rats before and after intervention

[0100]

[0101] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0102] Table 4: Catalase CAT indexes in the four groups of rats before and after intervention

[0103]

[0104]

[0105] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0106] Table 5: Glutathione peroxidase GPx indexes in the four groups of rats before and after intervention

[0107]

[0108] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0109] 3.4 Levels of major inflammatory factors (TNF-α, IL-6, IL-1β) in serum samples

[0110] At baseline and after 90 days of intervention, the levels of inflammatory factors in rat serum were detected using ELISA kits (enzyme-linked immunosorbent assay kit, Xinxie Biotechnology). The results are shown in Tables 6-8. After 90 days of intervention, the inflammatory factors TNF-α, IL-6 and IL-1β in the control group and the comparison group increased significantly. The levels of inflammatory factors in the probiotic group rats remained almost unchanged, and there was no significant difference with the blank group. This shows that the intervention of animal Bifidobacterium lactis subspecies CA-03 can effectively alleviate the symptoms of changes in inflammatory factor levels caused by cigarette smoke.

[0111] Table 6: Tumor necrosis factor α (TNF-α) indexes in the four groups of rats before and after intervention

[0112]

[0113] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0114] Table 7: Interleukin 6 (IL-6) indexes of the four groups of rats before and after intervention

[0115]

[0116] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0117] Table 8: Interleukin 1β (IL-1β) indexes of the four groups of rats before and after intervention

[0118]

[0119]

[0120] Note: The same letters in the same column indicate no significant difference (P>0.05), and different letters indicate significant difference (P<0.05).

[0121] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of Bifidobacterium animalis subsp. Lactis CA-03 in the preparation of a medicament for preventing, alleviating and / or treating health problems caused by smoking, characterized in that: The medicine comprises animal Bifidobacterium lactis subspecies CA-03 and / or fermentation metabolites of animal Bifidobacterium lactis subspecies CA-03, and the preservation number of animal Bifidobacterium lactis subspecies CA-03 is CGMCC No.17743.

2. The use according to claim 1, characterized in that: The amount of animal Bifidobacterium lactis subspecies CA-03 added to the drug is not less than 1.0×10 6 CFU / mL or 1.0×10 6 CFU / g.

3. The use according to claim 1, characterized in that: The individual daily intake of the animal Bifidobacterium lactis subspecies CA-03 is not less than 1.0×10 8 CFU / d.

4. The use according to claim 1, characterized in that: The application includes at least one of the following functions: (1) Increase the content of beneficial bacteria in the individual intestine and reduce the content of harmful bacteria in the individual; (2) Regulate the number of macrophages and lymphocytes in individual lung tissues; (3) Regulate the levels of superoxide dismutase SOD, catalase CAT, and glutathione peroxidase GPx in individual liver tissue; (4) Regulate the levels of inflammatory factors TNF-α, IL-6 and IL-1β in individual serum.

5. The use according to claim 4, characterized in that: The beneficial bacteria include Lactobacillus reuteri, Parabacteroides distasonis and Bacteroides dorei; the harmful bacteria include Eggerthella lenta, Staphylococcus capitis and Escherichia coli.

6. Use of animal Bifidobacterium lactis subsp. CA-03 in the preparation of a medicament for improving lung health problems caused by cigarette smoke, characterized in that: The medicine comprises animal Bifidobacterium lactis subspecies CA-03 and / or fermentation metabolites of animal Bifidobacterium lactis subspecies CA-03, and the preservation number of animal Bifidobacterium lactis subspecies CA-03 is CGMCC No.17743.

7. Use of animal Bifidobacterium lactis subspecies CA-03 in the preparation of a medicament for improving liver health problems caused by cigarette smoke, characterized in that: The medicine comprises animal Bifidobacterium lactis subspecies CA-03 and / or fermentation metabolites of animal Bifidobacterium lactis subspecies CA-03, and the preservation number of animal Bifidobacterium lactis subspecies CA-03 is CGMCC No.17743.

8. Use of animal Bifidobacterium lactis subspecies CA-03 in the preparation of a drug for reducing the level of inflammatory factors in serum, characterized in that: The medicine comprises animal Bifidobacterium lactis subspecies CA-03 and / or fermentation metabolites of animal Bifidobacterium lactis subspecies CA-03, and the preservation number of animal Bifidobacterium lactis subspecies CA-03 is CGMCC No.17743.

9. The use according to any one of claims 6 to 8, characterized in that: The individual daily intake of the animal Bifidobacterium lactis subspecies CA-03 is not less than 1.0×10 8 CFU / d.

10. A product for regulating intestinal microorganisms, characterized in that: The product comprises Bifidobacterium animalis subspecies lactis CA-03 and / or fermentation metabolites of Bifidobacterium animalis subspecies lactis CA-03.

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