Bifidobacterium animalis subsp. Animalis AN-YQ56 with functions of inhibiting four highs and improving sleep quality, metabiotic microbial inoculum, preparation method and application of bifidobacterium animalis subsp. Animalis AN-YQ56 and metabiotic microbial inoculum

Abstract

The invention discloses a bifidobacterium animalis subsp. Animalis AN-YQ56 with the effects of inhibiting four highs and improving sleep quality, a metabiotic microbial inoculum, a preparation method and application of the bifidobacterium animalis subsp. Animalis AN-YQ56 and the metabiotic microbial inoculum. The strain is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M 20241567, the strain is classified and named as Bifidobacterium animalis subspecies AN-YQ56 Bifidobacterium animalis subsp.animalis AN-YQ56, and the preservation date is July 22, 2024, and the preservation number is CCTCC NO: M 20241567. No matter the strain AN-YQ56 is in a viable or inactivated state, body metabolism can be improved, diseases related to organs such as heart and cerebral vessels, livers and kidneys can be reduced, and occurrence of four highs can be effectively inhibited.

Description

Technical Field

[0001] The present application relates to the technical field of animal Bifidobacterium, and mainly to a strain of animal Bifidobacterium animal subspecies AN-YQ56 capable of inhibiting four highs and improving sleep quality, as well as a postbiotic bacterial agent, a preparation method and an application. Background Art

[0002] The "four highs" refer to high blood pressure, high blood sugar, high blood lipids and high uric acid. These chronic diseases are becoming more and more common in modern society, and they are interrelated. In recent years, cardiovascular and cerebrovascular diseases have become the number one killer of human health, seriously threatening human health. Most patients with the four highs often experience dizziness and insomnia, chest tightness and palpitations, shortness of breath and fatigue, eating more and getting hungry easily, obesity or weight loss, polyuria or increased nocturia, decreased vision due to fundus lesions, and numbness of the limbs due to cerebral infarction; some patients may develop angina pectoris, myocardial infarction, heart failure, cerebral ischemia, cerebral hemorrhage and renal failure, which seriously affect their daily lives. Summary of the invention

[0003] In view of this, the purpose of the present application is to provide a strain of animal Bifidobacterium animal subspecies AN-YQ56 that has the ability to inhibit four highs and improve sleep quality, so as to open up its practical application scenarios and fields.

[0004] In the first aspect, the present application screened and obtained an animal Bifidobacterium, which was deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241567, classified and named Bifidobacterium animalis subsp. animalis AN-YQ56, and the deposit date was July 22, 2024.

[0005] In the second aspect, the examples of the present application disclose the use of the animal Bifidobacterium animalis subspecies AN-YQ56 in the preparation of products for regulating hypertension, hyperglycemia, hyperlipidemia and hyperuricemia.

[0006] In the third aspect, the present application discloses a method for preparing the strain of Bifidobacterium animalis subspecies AN-YQ56 that has the ability to inhibit four highs and improve sleep quality as follows:

[0007] S1. Prepare seed suspension. Melt the glycerol tube of animal Bifidobacterium animalis subspecies AN-YQ56 stored at -20°C and inoculate 1 mL into 40-110 mL MRS liquid culture medium. Incubate at 31-37°C for 12-16 h. Wash with sterile physiological water by centrifugation for 3 times. Adjust the bacterial suspension to an appropriate concentration (10 6 -10 7 CFU / ml) for standby use;

[0008] S2, obtaining a fermentation broth, wherein the fermentation broth is obtained by transferring the seed suspension into the fermentation broth, and fermenting with aeration at 31-37° C. and stirring at 100-130 rpm for 36-72 hours;

[0009] S3, centrifugation collection, collect 20-30 ml of the fermented liquid after fermentation and place it in a sterile 50 ml centrifuge tube for centrifugation, discard the supernatant and collect the bacterial sludge;

[0010] S4, preparation of freeze-dried bacterial suspension, collecting centrifuged bacterial cells, adding a bacterial mud protective agent prepared by 18% milk powder, 16% trehalose, 2% glycerol, 0.5% xylo-oligosaccharide and ultrapure water, vortexing and mixing to obtain a freeze-dried bacterial suspension, and placing it at room temperature for 1 hour to allow the protective agent and the bacterial cells to fully fuse;

[0011] S5, vacuum freeze drying, pre-freeze in -80℃ refrigerator for 2h, and immediately put into freeze dryer after taking out to avoid damage to bacteria due to sudden temperature rise. The sample was placed in freeze dryer for 24h, and the content of animal Bifidobacterium animalis subspecies AN-YQ56 strain was 10 10 -10 12 CFU / g, the freeze dryer should be turned on in advance to reduce the freezing temperature to -55°C and the vacuum degree to 1000Pa during operation.

[0012] In the embodiment of the present application, the MRS liquid culture medium in step S1 is specifically: peptone 10g / L, beef extract 10g / L, yeast powder 5g / L, dipotassium hydrogen phosphate 2g / L, tricitrate 2g / L sodium acetate 5g / L, Tween-801mL / L, magnesium sulfate 0.2g / L, sulfuric acid 0.05g / L, glucose 20g / L, pH=6.5, sterilized at 121°C for 20min. In the embodiment of the present application, the ratio of air volume to tank volume in step S2 is 1:0.5~1 (v / vm).

[0013] In the embodiment of the present application, the centrifugation conditions in step S3 are 4000 r / min, 20 min, 4°C, or 6000 r / min, 10 min, 4°C.

[0014] In the embodiment of the present application, the volume of the protective agent added in step S4 is 1 / 5 of the culture medium before centrifugation.

[0015] In an embodiment of the present application, a food or medicine is also provided, which contains the animal Bifidobacterium animalis subspecies AN-YQ56.

[0016] In an embodiment of the present application, a postbiotic product is also provided, which contains the animal Bifidobacterium animalis subspecies AN-YQ56.

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

[0018] (1) The strain AN-YQ56 of the present invention can improve body metabolism and reduce the occurrence of cardiovascular, liver, kidney and other organ-related diseases.

[0019] (2) The strain AN-YQ56 of the present invention has the function of regulating the four highs regardless of whether it is in a live or inactivated state.

[0020] (3) The strain AN-YQ56 of the present invention can improve insomnia symptoms and enhance sleep quality.

[0021] (4) The strain of the present invention is also effective in an inactivated state, which can significantly broaden the application scenarios of the strain and represents a great technological advancement. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 This is an optical microscope photograph of Bifidobacterium animalis subspecies AN-YQ56.

[0024] Figure 2 Mouse fasting blood glucose graph.

[0025] Figure 3 UA detection in mouse blood. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the content of the present invention and are not used to limit the present invention.

[0027] Example 1 Screening Source Description of Bifidobacterium animalis subspecies AN-YQ56

[0028] The applicant has conducted a large number of screenings from infant feces and obtained several strains of animal Bifidobacterium with good health functions, among which the present invention is named animal Bifidobacterium animalis subspecies AN-YQ56.

[0029] Referring to the prior art, identification of strain AN-YQ56

[0030] Morphological identification

[0031] Bifidobacterium animalis subspecies AN-YQ56 is a Gram-positive bacillus with an optimum pH of 6.5-7.0 and an optimum growth temperature of 37°C-41°C. It has no spores, flagella, or capsules, is non-motile, and has a biochemical reaction characteristic of catalase negative. Bifidobacteria generally have a forked V, Y, or X shape, as well as a club shape, such as Figure 1 shown.

[0032] Physiological and biochemical identification

[0033] According to the Bergey's Bacteria Identification Manual and the Common Bacteria System Identification Manual, the strain AN-YQ56 was tested for its physiological and biochemical indicators. The specific results are shown in Table 1. Referring to the bacterial species identification manual, it can be seen that the strain AN-YQ56 meets the characteristics of Bifidobacterium bacteria and can be preliminarily determined to be animal Bifidobacterium.

[0034] Table 1 Identification results of physiological and biochemical characteristics of strain AN-YQ56

[0035]

[0036]

[0037] Molecular Biology Identification

[0038] The genome of strain AN-YQ56 was used as a template, and its 16SDNA sequence was amplified by PCR using universal bacterial primers, and the PCR product was verified by agarose electrophoresis. The PCR amplification product was sent to Shanghai Biotech for sequence determination, and the sequencing results of the obtained PCR products were compared with the 16SrDNA sequence in Genback. The sequence comparison showed that the homology between the AN-YQ56 sequence and Bifidobacterium animalis subsp.animalis CGMCC1.1852 reached 99%. Combined with the morphological characteristics and its physiological and biochemical characteristics, the strain AN-YQ56 was determined to be Bifidobacterium animalissubsp.animalis, and the strain AN-YQ56 was named Bifidobacterium animalis subsp.animalis AN-YQ56, and it was preserved in the China Center for Type Culture Collection with the preservation number of CCTCC NO: M 20241567.

[0039] Example 2 Preparation of bacterial powder

[0040] S1. Prepare seed suspension. Melt the glycerol tube of animal Bifidobacterium animalis subspecies AN-YQ56 stored at -20°C and take 1 mL to inoculate into 70 mL of MRS liquid medium containing 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast powder, 2 g / L dipotassium hydrogen phosphate, 2 g / L tricitrate, 5 g / L sodium acetate, 1 mL / L Tween-80, 0.2 g / L magnesium sulfate, 0.05 g / L sulfuric acid, 20 g / L glucose, pH = 6.5, sterilized at 121°C for 20 min, incubate at 37°C for 16 h, centrifuge and wash 3 times with sterile physiological water, adjust the bacterial suspension to a concentration of 10 with sterile water. 7 CFU / ml, for future use;

[0041] S2, obtain fermentation broth, the fermentation broth is obtained by transferring the seed suspension into the fermentation broth (skim milk powder (50g / L), soy protein isolate (8g / L) glucose (20g / L), beef extract (8g / L), NaCl (23g / L), MgSO4·7H2O (2g / L), Span S-80 (1g / L), adjusting pH=5.80, sterilizing at 121°C for 20min), aerating and fermenting at 37°C, 120rpm stirring conditions, fermenting for 48h, and the ratio of air volume to tank volume is 1:0.5 (v / vm);

[0042] S3, centrifugal collection, collect the fermented liquid after fermentation 30ml and place it in a sterile 50ml centrifuge tube for centrifugation at 4000r / min, 20min, 4°C, discard the supernatant and collect the bacterial sludge;

[0043] S4, preparation of freeze-dried bacterial suspension, collecting centrifuged bacterial cells, adding a bacterial mud protective agent prepared by 18% milk powder, 16% trehalose, 2% glycerol, 0.5% xylo-oligosaccharide and ultrapure water, vortexing and mixing, the volume of the protective agent added is 1 / 5 of the culture solution before centrifugation, and the freeze-dried bacterial suspension is obtained, and placed at room temperature for 1 hour to allow the protective agent and the bacterial cells to fully fuse;

[0044] S5. Vacuum freeze drying: pre-freeze in -80℃ refrigerator for 2h, and immediately put into freeze dryer after taking out to avoid damage to bacteria due to sudden temperature rise. Place the sample in freeze dryer and continue freeze drying for 24h to obtain animal Bifidobacterium animalis subspecies AN-YQ56 bacterial powder, in which the content of animal Bifidobacterium animalis subspecies AN-YQ56 strain is 10 12 CFU / g (the freeze dryer should be turned on in advance, the cold trap temperature should be lowered to -55℃ and the vacuum degree should be lowered to 1000Pa during operation).

[0045] Preparation of inactivated bacteria powder: except that the bacterial sludge obtained in the above step S3 is treated at 121° C. for 30 minutes to obtain inactivated bacteria, the remaining steps are the same as the above steps.

[0046] The bacterial powder can be further processed into food, medicine, and postbiotic products.

[0047] Comparative Example 1: To this end, the present application also provides a method using existing animal bifidobacteria as comparative example 1. The steps are the same as those in the above-mentioned Example 1, except that the strain used for fermentation is animal bifidobacterium (Bifidobacterium animalis subsp. animalis CGMCC 1.1852 (purchased from Shanghai Ruichu Biotechnology Co., Ltd., product number SHBCC D82494), and through the above steps, animal bifidobacterium CGMCC 1.1852 bacterial powder is also obtained.

[0048] Experiment 1 Application of Bifidobacterium animalis subspecies AN-YQ56 in regulating hypertension

[0049] Animal Experimentation:

[0050] The SPF-grade healthy Kunming mice used in this experiment were 74 male mice of uniform weight, 5 weeks old, with an initial weight of 22±2.7g, purchased from Beijing HFK Bioscience Co., Ltd. In order to adapt the mice to the surrounding environment, these 74 mice were adaptively raised for one week, and the feeding conditions were: humidity 4%, night / day cycle at a constant temperature of 24±2℃, free access to water and food.

[0051] Experimental grouping and model construction:

[0052] After 1 week of adaptive feeding, 10 mice were randomly selected as the normal group and raised normally. The rest of the mice were modeled. Starting from the 8th day, the modeled mice were gavaged with L-nitroarginine (L-NNA) at 9:00 every morning, with a gavage volume of 490 mg / kg / d. The normal group mice were gavaged with an equal volume of saline, and the modeling lasted for 28 consecutive days. Every 7 days during the gavage period, that is, at 15:00 on the 7th, 14th, 21st, and 28th day of the modeling, the blood pressure of the modeling group mice was measured using the BP-98A mouse non-invasive blood pressure measurement system, and the mouse tail systolic pressure was measured. Each mouse was measured three times and the average was calculated. When the average systolic blood pressure ≥180 mmHg, the modeling was successful.

[0053] Fifty mice with successful modeling were selected and randomly divided into a model group, a live bacteria example 2 group, a comparative example 1 group, an inactivated example 2 group and a positive control group, with 10 mice in each group. The normal group and the model group were free to eat basic feed and water, and the other four groups were treated. Among them, the live bacteria example 2 group: free to eat basic feed and water, and the feed was supplemented with 0.5% live bacteria of animal Bifidobacterium animalis subspecies AN-YQ56 (the content of animal Bifidobacterium animalis subspecies AN-YQ56 strain was 10 12CFU / g). Comparative Example 1 group: free access to basic feed and water, feed with 0.5% of live bacterial Bifidobacterium animalis CGMCC1.1852 added to the feed. Inactivated Example 2 group: free access to basic feed and water, feed with 0.5% of inactivated bacterial powder of Bifidobacterium animalis subspecies AN-YQ56 added to the feed. Positive control group: free access to basic diet and water, intragastric administration of captopril 25 mg / kg / d for 8 consecutive weeks, during which the body weight, food intake, heart rate, mental and activity status of the mice were regularly observed and recorded, and blood pressure changes were recorded. The pressure measurement method was the same as above. The statistical results are shown in Table 2.

[0054] Table 2

[0055]

[0056] Experiment 2 Application of Bifidobacterium animalis subspecies AN-YQ56 in regulating hyperglycemia

[0057] The SPF-grade healthy Kunming mice used in this experiment were 50 male mice of uniform weight, 5 weeks old, with an initial weight of 22±2.7g, purchased from Beijing HFK Bioscience Co., Ltd. In order to adapt the mice to the surrounding environment, these 50 mice were adaptively raised for one week, and the feeding conditions were: humidity 4%, a constant temperature of 24±2℃, a night / day cycle, free access to water and food.

[0058] Experimental grouping and model construction:

[0059] Fifty mice were randomly divided into five groups, with 10 mice in each group, and marked by ear clipping.

[0060] Except for 10 mice randomly selected from the normal group, which were free to eat basic feed (purchased from Cenpan Department Store, Donggang District, Rizhao City) and water, no intervention was performed. The living conditions of the mice were observed every day, and their body weight and feces were collected every week. The rest of the mice were modeled and fed with high-fat and high-sugar feed (purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.) every day from the 8th day, and streptozotocin (STZ) was injected intraperitoneally at 66 mg / kg every five days according to body weight for 28 days. After four weeks, the tails were cut and blood was collected to measure the fasting blood glucose of mice in the high-fat and high-sugar group, and FBG ≥ 11.1 mmol / L was used as the standard for the successful establishment of the T2DM pathological model.

[0061] After the modeling was completed, the other three groups except the model group were treated, among which the normal group and the model group were free to eat basic feed and water; the live bacteria Example 2 group: free to eat basic feed and water, and 0.5% of the live bacteria of animal Bifidobacterium animal subspecies AN-YQ56 was added to the feed. The comparative example 1 group: free to eat basic feed and water, and 0.5% of the live bacteria of animal Bifidobacterium CGMCC 1.1852 was added to the feed. The inactivated example 2 group: free to eat basic feed and water, and 0.5% of the inactivated animal Bifidobacterium animal subspecies AN-YQ56 bacterial powder was added to the feed. For 4 consecutive weeks, the body weight of each group of mice was recorded regularly, and the tail tip blood was collected to determine the fasting blood glucose level. After 4 weeks of administration, the fasting blood glucose level of each group is shown in Table 3 and Figure 2 shown.

[0062] Table 3

[0063]

[0064] Experiment 3 Application of Bifidobacterium animalis subspecies AN-YQ56 in regulating hyperlipidemia

[0065] The SPF-grade healthy Kunming mice used in this experiment were 50 male mice of uniform weight, 5 weeks old, with an initial weight of 22±2.7g, purchased from Beijing HFK Bioscience Co., Ltd. In order to adapt the mice to the surrounding environment, these 50 mice were adaptively raised for one week, and the feeding conditions were: humidity 4%, a constant temperature of 24±2℃, a night / day cycle, free access to water and food.

[0066] Experimental grouping and model construction:

[0067] Fifty mice were randomly divided into five groups, namely, a normal group, a model group, a live bacteria Example 2 group, a comparative example 1 group and an inactivated Example 2 group, with 10 mice in each group, and the mice were marked by ear clipping.

[0068] Except that the normal group freely ate basic feed and water, no intervention was performed, the living conditions of the mice were observed every day, and the body weight was weighed and feces were collected every week; model group: fed with high-fat and high-cholesterol feed (purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.); live bacteria Example 2 group: fed with high-fat and high-cholesterol feed supplemented with 0.5% live bacteria of animal Bifidobacterium animal subspecies AN-YQ56 powder; comparative example 1 group: fed with high-fat and high-cholesterol feed supplemented with 0.5% live bacteria of animal Bifidobacterium CGMCC 1.1852 powder; inactivated Example 2 group: fed with high-fat and high-cholesterol feed supplemented with 0.5% inactivated state of animal Bifidobacterium animal subspecies AN-YQ56 powder, for 8 consecutive weeks, on the 57th day, the mice were fasted but not watered for 12 hours, anesthetized, the mouse eyeballs were removed to take blood, and then dissected, the mouse blood was centrifuged at 4°C to obtain serum, and stored at -80°C for standby use. The serum TC (cholesterol), TG (triglyceride), and LDL-C (low-density lipoprotein cholesterol) levels were detected using kits, and the statistical results are shown in Table 4. Serum total cholesterol (TC) and triglyceride (TG) were purchased from Shanghai Shensuo Youfu Medical Diagnostic Products Co., Ltd., and the low-density lipoprotein cholesterol (LDL-C) kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0069] Table 4

[0070]

[0071] Experiment 4 Application of Bifidobacterium animalis subspecies AN-YQ56 in regulating hyperuricemia

[0072] The SPF-grade healthy Kunming mice used in this experiment were 60 male mice of uniform weight, 5 weeks old, with an initial weight of 22±2.7g, purchased from Beijing HFK Bioscience Co., Ltd. In order to adapt the mice to the surrounding environment, these 60 mice were adaptively raised for one week, and the feeding conditions were: humidity 4%, a constant temperature of 24±2℃, a night / day cycle, free access to water and food.

[0073] Experimental grouping and model construction:

[0074] 60 mice were randomly divided into 6 groups, with 10 mice in each group, and marked by ear clipping. Each group was administered with the following method:

[0075] Blank control group: 0.9% saline, intragastrically;

[0076] Model group: potassium oxonate 100 mg / kg·d intraperitoneal injection + hypoxanthine 500 mg / kg·d gavage;

[0077] Live bacteria Example 2 group: 10 mg / kg·d of bacterial powder prepared by live bacteria Example 2 group was intragastrically administered + 100 mg / kg·d of potassium oxonate was intraperitoneally injected + 500 mg / kg·d of hypoxanthine was intragastrically administered;

[0078] Comparative Example 1: 10 mg / kg·d of bacterial powder prepared in Comparative Example 1 was intragastrically administered + 100 mg / kg·d of potassium oxonate was intraperitoneally injected + 500 mg / kg·d of hypoxanthine was intragastrically administered;

[0079] Inactivation Example 2 Group: Inactivation Example 2 Group was administered with 10 mg / kg·d of bacterial powder prepared by intragastric administration + 100 mg / kg·d of potassium oxonate intraperitoneal injection + 500 mg / kg·d of hypoxanthine by intragastric administration; the administration was continued for 15 days.

[0080] Positive control group: allopurinol 10 mg / kg·d + potassium oxonate 100 mg / kg·d intraperitoneal injection + hypoxanthine 500 mg / kg·d gavage; continuous administration for 15 days.

[0081] Mouse sample collection: On the 16th day, after blood was collected from the eyeball, the collected blood was placed in an EP tube and allowed to rest for 30 minutes. The blood was centrifuged at 3500 rpm / min and 4°C for 10 minutes. The supernatant, a light yellow clear liquid, was taken as serum and frozen at -20°C for the detection of mouse blood biochemical indicators (UA). The statistical results are shown in Tables 5 and Figure 3 shown.

[0082] Table 5

[0083]

[0084]

[0085] According to the results in Tables 2-5, the use of animal Bifidobacterium CGMCC 1.1852 powder has almost no therapeutic effect on the hypertension, hyperglycemia, hyperlipidemia and hyperuricemia of mice. However, after treatment with live or inactivated powder of animal Bifidobacterium animal subspecies AN-YQ56, the hypertension, hyperglycemia, hyperlipidemia and hyperuricemia of mice returned to normal.

[0086] Experiment 5: Application of Bifidobacterium animalis subspecies AN-YQ56 in improving sleep quality

[0087] The SPF-grade healthy Kunming mice used in this experiment were 60 male mice of uniform weight, 5 weeks old, with an initial weight of 22±2.7g, purchased from Beijing HFK Bioscience Co., Ltd. In order to adapt the mice to the surrounding environment, these 60 mice were adaptively raised for one week, and the feeding conditions were: humidity 4%, a constant temperature of 24±2℃, a night / day cycle, free access to water and food.

[0088] Preparation of PCPA solution: Accurately weigh 1.0 g of PCPA, dissolve it in 30 mL of normal saline, then add 20 uL of polysorbate 80 to promote dissolution, and vortex to mix to obtain a PCPA suspension.

[0089] Experimental grouping and model construction:

[0090] 60 mice were randomly divided into 6 groups, namely, normal group, model group, live bacteria Example 2 group, comparative example 1 group, inactivated Example 2 group and positive control group, with 10 mice in each group, and marked by ear clipping.

[0091] Except for the normal group, the mice in the other groups were intraperitoneally injected with 350 mg / kg para-chlorophenylalanine (PCPA) suspension once a day for 3 days to establish an insomnia mouse model; after the modeling, the behavior of the mice was observed. When the mice showed increased activity, irritability, lack of circadian rhythm, uninterrupted activity during the day and night, the modeling was successful. After the modeling was successful, the mice in the normal group and the model group were free to eat basic feed and water, and the other three groups were treated. Live bacteria Example 2 Group: Free to eat basic feed and water, and add 0.5% of the live bacteria state animal Bifidobacterium animal subspecies AN-YQ56 feed. Comparative Example 1 Group: Free to eat basic feed and water, and add 0.5% of the live bacteria state animal Bifidobacterium CGMCC 1.1852 feed. Inactivation Example 2 group: free to eat basic feed and water, feed added with 0.5% inactivated animal Bifidobacterium animalis subspecies AN-YQ56 bacterial powder, positive control group: free to eat basic feed and water, and gavage diazepam 2.6 mg / kg once a day for 7 consecutive days. The general conditions of the mice, such as mental activity state, food intake, water intake, reaction to external stimuli and weight change, were observed every day.

[0092] After 7 days of administration, the mice were fasted for one day, anesthetized and killed, the skull was peeled off and the hippocampus was separated, the hippocampus was weighed, and 9 volumes of pre-cooled phosphate buffer were added to prepare tissue homogenate, and centrifuged at 12000r / min for 15min at 4°C, and the supernatant was stored at -80°C for later use. The ELISA method was used to determine the content of 5-HT in the hippocampus tissue, and the statistical results are shown in Table 6.

[0093] Table 6

[0094] Group 5-HT / (ng / mL) Normal group 176.21±20.28 Model Group 101.26±17.57 Live bacteria example group 175.53±18.65 Comparative group 107.34±16.81 Inactivation group 156.14±15.46 Positive control group 166.52±14.97

[0095] PCPA is a 5-HT synthesis inhibitor that can prevent the synthesis of 5-HT by inhibiting tryptophan hydroxylase. A decrease in 5-HT in the brain can lead to insomnia, depression and other symptoms. Therefore, this study used the method of intraperitoneal injection of PCPA to prepare an insomnia mouse model to explore the improvement effect of animal Bifidobacterium animal subspecies AN-YQ56 on insomnia mice. 5-HT has important physiological and neuroregulatory effects in hippocampal tissue. The results of this study showed that the content of 5-HT in the hippocampal tissue of mice in the live bacteria Example 2 group or the inactivated Example 2 group was significantly increased, indicating that the live bacteria or inactivated state of animal Bifidobacterium animal subspecies AN-YQ56 can promote the synthesis of 5-HT in mice and improve insomnia symptoms.

[0096] The above-mentioned embodiments only express the specific implementation of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A strain of Bifidobacterium animalis subsp. animalis AN-YQ56, which has the effects of inhibiting the four highs and improving sleep quality, is deposited in the China Center for Type Culture Collection, with the deposit number CCTCCNO: M20241567 and the deposit date of July 22, 2024.

2. Use of the animal Bifidobacterium animalis subspecies AN-YQ56 according to claim 1 in the preparation of products for regulating hypertension, hyperglycemia, hyperlipidemia and hyperuricemia.

3. Use of the animal Bifidobacterium animalis subspecies AN-YQ56 according to claim 1 in the preparation of a product for improving sleep quality.

4. The use according to claim 2 or 3, characterized in that: Among them, Bifidobacterium animalis subspecies animalis AN-YQ56 is in a live or inactivated state.

5. The method for preparing the bacterial powder of animal Bifidobacterium animalis subspecies AN-YQ56 capable of regulating four highs according to claim 1, characterized in that: Here are the steps: S1. Prepare seed suspension. Melt the glycerol tube of animal Bifidobacterium animalis subspecies AN-YQ56 stored at -20°C and inoculate 1 mL into 40-110 mL MRS liquid culture medium. Incubate at 31-37°C for 12-16 hours. Wash by centrifugation with sterile physiological water for 3 times. Adjust the bacterial suspension to a suitable concentration with sterile water for later use. S2, obtaining a fermentation broth, wherein the fermentation broth is obtained by transferring the seed suspension into a fermentation broth, and fermenting with aeration at 31-37° C. and 100-130 rpm for 36-72 hours; S3, centrifugation collection, collect the fermented liquid 20-30ml and place it in a sterile 50ml centrifuge tube for centrifugation, discard the supernatant, and collect the bacterial sludge; S4, preparation of freeze-dried bacterial suspension, collecting centrifuged bacterial cells, adding a bacterial mud protective agent prepared by 18% milk powder, 16% trehalose, 2% glycerol, 0.5% xylo-oligosaccharide and ultrapure water, vortexing and mixing to obtain a freeze-dried bacterial suspension, and placing it at room temperature for 1 hour to allow the protective agent and the bacterial cells to fully fuse; S5, vacuum freeze drying, pre-freeze the freeze-dried bacterial suspension in a -80 ℃ refrigerator for 2 hours, and immediately put it into a freeze dryer after taking it out to avoid damage to the bacteria due to a sudden rise in temperature. The sample is placed in a freeze dryer and freeze-dried for 24 hours to obtain animal Bifidobacterium animalis subspecies AN-YQ56 bacterial powder, in which the content of animal Bifidobacterium animalis subspecies AN-YQ56 strain is 10 10 -10 12 CFU / g, the freeze dryer should be turned on in advance to reduce the freezing temperature to -55°C and the vacuum degree to 1000Pa during operation.

6. The method according to claim 5, characterized in that: The MRS liquid culture medium in step S1 is specifically: peptone 10g / L, beef extract 10g / L, yeast powder 5g / L, dipotassium hydrogen phosphate 2g / L, tricitrate 2g / L, sodium acetate 5g / L, Tween-801mL / L, magnesium sulfate 0.2g / L, sulfuric acid 0.05g / L, glucose 20g / L, pH=6.5, sterilized at 121°C for 20min.

7. The method according to claim 5, characterized in that: In step S2, the ratio of the amount of air to the tank volume is 1:0.5-1 (v / vm).

8. The method according to claim 5, characterized in that: The centrifugation conditions in step S3 are 4000 r / min, 20 min, 4°C, or 6000 r / min, 10 min, 4°C.

9. The method according to claim 5, characterized in that: The volume of the protective agent added in step S4 is 1 / 5 of the culture medium before centrifugation. 10 . A food or a medicine, comprising the Bifidobacterium animalis subspecies animalis AN-YQ56 according to claim 1 .

11. A postbiotic preparation comprising the Bifidobacterium animalis subspecies AN-YQ56 according to claim 1.

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