Bifidobacterium animalis subsp. Animalis BN16 capable of stabilizing emotion and relieving anxiety, metagen and preparation method and application of bifidobacterium animalis subsp. Animalis BN16 and metagen

Abstract

The invention discloses bifidobacterium animalis subsp. Animalis BN16 for stabilizing emotion and relieving anxiety and a metagen, the metagen is prepared by fermenting a bifidobacterium animalis subsp. Animalis BN16 and probiotic fermentation composition, and the composition is prepared from the following materials in parts by weight: 35 parts of milled rice with embryo, 30 parts of corn kernels, 17 parts of lotus seeds, 10 parts of walnut kernels and 8 parts of white granulated sugar. And 400 parts of purified water. The bifidobacterium animalis subsp. Animalis BN16 is fermented and then processed to prepare the metagen, nutritional ingredients in the metagen are directly, effectively and fully biotransformed, tryptophan absorption can be promoted, the situations of insufficient nutrition transformation, insufficient absorption and the like caused by poor intestinal digestion ability of people with sub-health emotions are avoided, emotion stabilization is facilitated, and the health-care effect is achieved. Anxiety is relieved, and the anxiety period is shortened.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of general health, in particular to an animal bifidobacterium animalis subsp. animalis BN16, a postbiotic for stabilizing mood and relieving anxiety, and a preparation method and application thereof. BACKGROUND

[0002] Mood is the immediate psychological response of the human body to external stimuli, which can be expressed in various ways through external facial, limb, torso, etc. The changes in the mood response mode (such as experience, behavior, physiological indicators) of a person under a certain mood are not completely consistent. For example, the external performance of a person to anger is more obvious, causing a huge change in behavior performance, but the change in physiological signal may change relatively less dramatically; some people may have no obvious external performance, but the change in physiological indicators is particularly obvious. Anxiety is a common negative emotion, and persistent excessive anxiety can seriously damage the physical and mental health of the human body, accelerate aging, increase the feeling of losing control, damage self-confidence, and can induce diseases such as hypertension and coronary heart disease; acute anxiety attacks can lead to stroke, myocardial infarction, glaucoma, high-pressure headache, blindness, and accidental injuries. Therefore, having a stable, healthy, and non-excessive anxiety mood is crucial to health.

[0003] In recent years, the in-depth development of the scientific theory of the gut-brain axis has revealed the complex bidirectional communication mechanism between the gut microbiota and the central nervous system, which provides a solid scientific basis for using microorganisms to intervene in emotional problems. The gut microbiota affects brain function and emotional state through various pathways, including the neuroendocrine pathway (such as the hypothalamic-pituitary-adrenal axis), the immune regulation pathway (cytokine release), and the neural pathway (vagus nerve signaling). These findings have given birth to a new field - "psychobiotics", which are live microorganisms or their components that can produce mental health benefits through the gut-brain axis.

[0004] Postbiotics refers to inactivated microorganisms and / or their cellular components that are beneficial to the health of the host, including inactivated bacteria, cell wall components, and metabolic products, etc. Compared with live bacterial preparations, postbiotics has higher stability, longer shelf life, and better safety, etc., and shows broad application prospects in the field of mood regulation. SUMMARY

[0005] The present application aims to at least solve one of the above-mentioned technical problems in the prior art. To this end, the purpose of the present application is to provide an animal bifidobacterium animalis subsp. animalis BN16 and a postbiotic for stabilizing mood and relieving anxiety. The composition of the postbiotic of the present application selects four raw materials such as germinated brown rice, corn kernel, lotus seed, and walnut kernel as core components, which are not only rich in nutrients, but also contain B vitamins, GABA, minerals, V A , V E, glutamic acid, beta-sitosterol, nervonic acid and other brain-healthy ingredients, which is beneficial to supplement brain nutrition, and the best ratio is obtained through multiple experimental tests, and the obtained ratio is the best for the fermentation effect of the animal bifidobacterium animalis subspecies BN16. Embryo rice is rich in nutrients, containing rich carbohydrates, proteins, fats, B vitamins, GABA and relatively rich P, K, Mg, Ca, Zn, Mn, Fe, Na and Cu and other essential trace elements for the human body, as well as a small amount of Se element, V A , V E , can nourish brain neurons, relieve nervous tension, improve anxiety symptoms, and has the effects of helping to maintain the functions of the nervous system and the digestive system, brain health, nerve calming and the like. Corn kernels are sweet and flat in nature, and have the functions of regulating the middle and stimulating appetite, benefiting the lungs and calming the heart, clearing damp heat, benefiting the liver and gallbladder, and delaying aging. In addition, modern research shows that corn kernels contain a lot of glutamic acid, which has the effect of brain health. It can help and promote brain cells to breathe, and can remove waste in the body during physiological activities, helping to remove ammonia in brain tissue. Therefore, regular consumption of corn kernels can stimulate brain cells and enhance human brain power. Lotus seeds, also known as lotus seeds and lotus rice, are recorded in "Compendium of Materia Medica" as follows: "eating raw has the functions of clearing heat and cooling blood, and dispersing and gathering, eating cooked has the functions of lightening the body, tonifying qi, strengthening the body, clearing heat and removing stasis, nourishing the heart and calming the mind, tonifying the kidney, benefiting the bones, and cooling blood". Modern pharmacological research shows that lotus seeds contain 18 kinds of amino acids, of which 7 are essential amino acids for the human body, which can make up for the lack of lysine in cereals and improve the biological efficiency of proteins. In addition, beta-sitosterol contained therein has the effects of stabilizing emotions, calming, strengthening heart and anti-aging. Walnut kernels are warm in nature and sweet in taste. "Shennong's Herbal Classic" lists it as a top product for long-term consumption to benefit qi and prolong life. Modern pharmacological research shows that walnut kernels contain nervonic acid, which is a double-effect substance that can repair and unblock damaged brain nerve fibers and promote nerve cell regeneration. It is a core natural ingredient of brain nerve fibers and nerve cells. Regular consumption of walnut kernels is beneficial to preventing brain atrophy.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is: The first aspect of the present application provides an animal bifidobacterium animalis subspecies for stabilizing emotions and relieving anxiety, wherein the animal bifidobacterium is animal bifidobacterium animalis subspecies BN16, which is taxonomically named Bifidobacterium animalis subsp. animalis , and was preserved in the China General Microbiological Culture Collection Center on August 13, 2025, with the preservation number of CGMCC No. 35603.

[0007] The second aspect of the present application provides a probiotic for stabilizing emotions and relieving anxiety, which comprises the animal bifidobacterium animalis subspecies BN16 bacteria body and / or metabolites of BN16.

[0008] In a third aspect of the present application, a preparation method of a probiotic is provided, the preparation method comprising fermenting Bifidobacterium animalis subsp. animalis BN16 of the above aspect using a composition to obtain the probiotic; the composition comprising at least one of germinated brown rice, lotus seed, corn kernel, walnut kernel, white granulated sugar, and purified water.

[0009] In some embodiments of the present application, the microbial preparation is in a form of powder, pill, capsule, granule, tablet, oil drop, liquid preparation, or gel.

[0010] In some embodiments of the present application, the composition comprises the following components in mass parts: 20-40 parts of germinated brown rice, 10-30 parts of lotus seed, 20-40 parts of corn kernel, 10-30 parts of walnut kernel, 5-15 parts of white granulated sugar, and 200-400 parts of purified water.

[0011] In some embodiments of the present application, the composition comprises the following components in mass parts: 30-40 parts of germinated brown rice, 10-20 parts of lotus seed, 30-40 parts of corn kernel, 10-20 parts of walnut kernel, 5-10 parts of white granulated sugar, and 300-400 parts of purified water.

[0012] In some embodiments of the present application, the amount of Bifidobacterium animalis subsp. animalis BN16 added is 1% to 5% of the volume of the composition.

[0013] In some embodiments of the present application, the amount of Bifidobacterium animalis subsp. animalis BN16 added is 1% to 5% of the volume of the composition.

[0014] In some embodiments of the present application, the fermentation comprises constant pH fermentation and non-constant pH fermentation.

[0015] In some embodiments of the present application, the fermentation time is 30 to 40 hours, and the constant pH fermentation time is 15 to 25 hours.

[0016] In some embodiments of the present application, the pH is 6 to 7.

[0017] In some embodiments of the present application, the constant pH fermentation is constant pH fermentation for the first 15 to 25 hours.

[0018] In some embodiments of the present application, the preparation method further comprises homogenizing the fermentation broth after fermentation.

[0019] In some embodiments of the present application, the homogenization comprises homogenizing the fermentation broth at 2 to 8°C, heating to 50 to 80°C, and then homogenizing 1 to 3 times at a pressure of 100 to 150 MPa.

[0020] In some embodiments of the present application, the time for each homogenization is 5-20 minutes.

[0021] In some embodiments of the present application, the time for each homogenization is 5-10 minutes.

[0022] In some embodiments of the present application, the homogenization comprises homogenizing the fermentation broth for 5-10 minutes at a speed of 10000-15000 r / min and a temperature of 2-8℃, then heating to 50-80℃, and homogenizing 1-3 times under a pressure of 100-150 MPa; then heating to 80-100℃ and maintaining for 30 min.

[0023] In a fourth aspect of the present application, a product comprising at least one of the animal Bifidobacterium animalis subsp. animalis, the postbiotic and the postbiotic prepared by the preparation method described in the above aspects is provided.

[0024] In some embodiments of the present application, the product comprises a food or a drug.

[0025] In some embodiments of the present application, the dosage form of the food comprises a powder, a tablet, a granule, a capsule, a solution, an emulsion, a suspension.

[0026] In a fifth aspect of the present application, the animal Bifidobacterium animalis subsp. animalis, the postbiotic or the postbiotic prepared by the preparation method described in the above aspects is applied in any one of (1)-(5) as follows: (1) preparing a product for reducing prefrontal lobe damage; (2) preparing a product for preventing or relieving anxiety; (3) preparing a product for stabilizing mood; (4) preparing a product for improving tryptophan utilization rate; (5) preparing a product for reducing cortisol.

[0027] The present application has the following beneficial effects: The present application provides an animal Bifidobacterium animalis subsp. animalis for stabilizing mood and relieving anxiety, which is animal Bifidobacterium animalis subsp. animalis BN16, and a postbiotic prepared from the animal Bifidobacterium animalis subsp. animalis BN16.

[0028] The postbiotic preparation of the application is prepared by using two homogenization technologies in cooperation, that is, first homogenized by low temperature and high speed shearing, and then homogenized by high pressure for three times, which can not only disperse, miniaturize and homogenize the particles in the fermentation broth, and fully fuse the nutritional ingredients, but also fully break the animal bifidobacterium animalis subsp BN16 in the fermentation broth, release more nutrients such as protein, polysaccharide complex, vitamin, amino acid and short-chain fatty acid, improve the nutritional value of the animal bifidobacterium animalis subsp postbiotic, and promote the better absorption of the components in the postbiotic.

[0029] The animal bifidobacterium animalis subsp BN16 and the postbiotic prepared therefrom have the functions of reducing prefrontal lobe damage, reducing cortisol, promoting tryptophan absorption, etc., avoid the situation that the emotional sub-health population has poor intestinal digestion capacity, leading to insufficient nutrient transformation and insufficient absorption, etc., are beneficial to stabilizing emotions, relieving anxiety, shortening the cycle of anxiety, and the preparation process of the postbiotic is simple, the cost is low, and the postbiotic is easy to popularize and use. DETAILED DESCRIPTION

[0030] The content of the application will be further described in detail through specific examples. The raw materials, reagents or devices used in the examples and comparative examples are commercially available or can be obtained by existing technical methods unless otherwise specified. Unless otherwise specified, the test or test method is a conventional method in the art.

[0031] The unit "part" in the examples means "weight part", which is a weight unit.

[0032] Example 1 This example provides strain screening with the function of reducing prefrontal lobe damage, using the strains animal bifidobacterium animalis subsp BN16, animal bifidobacterium animalis subsp GDMCC No.1.4430 (preservation number: GDMCC No.1.4430), and animal bifidobacterium lactis subsp CICC 21715 (preservation number: CICC 21715) to conduct behavioral test and comet experiment on prefrontal lobe damage rat model. Alcohol-induced neuronal cell damage can cause DNA breakage in cells. In the comet experiment, the fragments will migrate away from the head to the anode, forming a comet-like tail. The longer the tail, the more serious the DNA damage and the more serious the cell damage. The strain animal bifidobacterium animalis subsp BN16 used in this example is taxonomically named Bifidobacterium animalis subsp. animalis , and was preserved in the China General Microbiological Culture Collection Center on August 13, 2025, with the address of No. 1, Beichen West Road, Haidian District, Beijing, and the preservation number of CGMCC No. 35603.

[0033] The specific steps are as follows.

[0034] (1) Preparation of medium B: weigh 15 g of proteose peptone, 2 g of yeast extract, 20 g of glucose, 0.5 g of soluble starch, 5 g of sodium chloride, 400 mL of tomato extract (preparation method of tomato extract: fresh tomatoes are washed, weighed, chopped, and heated in a water bath at 100°C with an equal weight of distilled water for 90 min, then filtered with gauze, and the pH is adjusted to 7.0), 80 mL of Tween 80, 0.3 g of liver powder, mix uniformly, add distilled water to 1 L, sterilize at 121°C for 15 min, cool to 37°C, add 1 mL of sterile cysteine salt solution (weigh 0.5 g of cysteine, add 1 mL of hydrochloric acid to dissolve all the cysteine), and adjust the pH to 6.8 to obtain medium B.

[0035] (2) Preparation of bacterial suspension: take the freeze-dried preservation tubes of the experimental strains of animal Bifidobacterium animalis subsp. animalis BN16, animal Bifidobacterium animalis subsp. animalis GDMCC No. 1.4430, and animal Bifidobacterium lactis CICC 21715 preserved at -80°C, thaw at room temperature, inoculate into medium B, activate for 2 generations, centrifuge at 4000 r / min and 4°C after 16 hours of culture of the 3rd generation, wash 3 times with sterile normal saline solution, resuspend with sterile normal saline, and adjust the viable bacterial count to 1.0 x 10 9 CFU / mL to obtain the bacterial suspension.

[0036] Animal experiment: 40 SPF male rats, 8 weeks old, weighing 190-200 g. All rats were fed in the SPF animal room, with room temperature of 25°C, humidity of 65%, 12-hour light-dark cycle, and free access to water and food. After 7 days of adaptive feeding in the normal incubator, the rats were randomly divided into 5 groups, 8 rats in each group, and were recorded as group A, group B, group C, group D, and group E.

[0037] The rats in groups B to E were placed in the conditional fear experiment box for 5 min, and then subjected to 0.8 mA inescapable foot shock every day for 10 s each time, with an interval of 10 s, 15 times a day, for 2 consecutive days. The rats in group A were placed in the conditional fear experiment box for free activity for 10 min without electric shock treatment for 2 consecutive days. Subsequently, all rats were transferred back to the normal incubator, and 10% (v / v) alcohol solution was used as the only liquid source for the rats in groups B to E during the dark cycle (20:00-8:00) for 14 consecutive days to establish the prefrontal injury rat model. The rats in group A were not given special treatment for 14 consecutive days.

[0038] During modeling, rats were synchronously subjected to gavage treatment, rats in group A and group B were gavaged with normal saline, rats in group C were gavaged with bacterial suspension of Bifidobacterium animalis subsp. animalis BN16, rats in group D were gavaged with bacterial suspension of Bifidobacterium animalis subsp. lactis CICC 21715, and rats in group E were gavaged with bacterial suspension of Bifidobacterium animalis subsp. lactis GDMCC No. 1.4430, the gavage dose was 2 mL, and the gavage was continuously performed for 14 days. On the 15th day, all rats were subjected to behavior test according to the rat behavior test method, and the average value of each group was obtained. The specific experimental steps are as follows.

[0039] The rats in each group were placed in the open field test room for 1 day of adaptation, and after the adaptation was completed, the rats were placed in a 50 cm x 50 cm x 50 cm open observation box for free activity for 5 minutes, and the activity distance and central area time of the rats during the test were recorded.

[0040] After the behavior test, the rats were anesthetized and subjected to whole-body perfusion, and the whole brain was taken out and washed in 4°C normal saline. The prefrontal cortex tissue was quickly separated, and the tailing of damaged neurons was determined according to the comet assay method. The comet tail length of each group was recorded, and the average value was obtained. The specific experimental steps are as follows.

[0041] (1) Single cell suspension preparation: The prefrontal cortex tissue was digested with 0.25% (w / v) trypsin for 15 min, and the digestion was stopped in DMEM medium containing 10% (v / v) FBS and blown to form a cell suspension. The cell suspension was filtered through a 200-mesh steel screen, and the cell density was adjusted to about 5 x 10 5 The cells were cultured in a 37°C, 5% CO2 incubator until the 5th day, and the fresh culture medium was replaced and subjected to ethanol treatment. After 24 hours of induction of the cells with ethanol (100 mM), the supernatant was discarded, and the cells were collected after 25% trypsin digestion. An appropriate amount of ice-cold PBS was added to prepare a cell suspension.

[0042] (2) Slab preparation: First layer: 100 μL of normal melting point agarose gel solution was added to the sand surface of the glass slide, and a cover glass was placed on it, and it was solidified at 4°C for 15 min. Second layer: The cover glass of the first layer of gel was removed, and the first layer of gel was gently scraped off. The cell suspension was mixed with 37°C low melting point agarose gel at a volume ratio of 1:1, and was added to the glass slide, and a cover glass was placed on it. It was solidified at 4°C for 10 min.

[0043] (3) Carefully remove the coverslips and immerse the slides in 4°C pre-cooled cell lysis solution (containing 2.5 mol / L NaCl, 100 mmol / L EDTA and 10 mmol / L Tris, before use add 1% (v / v) Triton X-100, 10% (v / v) dimethyl sulfoxide, pH 10) for 1 hour.

[0044] (4) Take out the slides and place them in a horizontal electrophoresis tank, add alkaline electrophoresis buffer (1 mM Na2EDTA, 300 mM NaOH) and stand for 30 min.

[0045] (5) Electrophorese at a voltage of 12 V / cm and a current of 300 mA for 20 min.

[0046] (6) After electrophoresis, take out the slides and neutralize them in 0.4 M Tris-HCI pH 7.5 buffer for 3 times, 5 min each time. Immerse the slides in anhydrous ethanol for 1 hour, then take out the slides and dry them. Add 50 μL EB aqueous solution to each slide and cover with a coverslip to stain.

[0047] (7) Under an inverted fluorescence microscope, the DNA is stained red, and neurons that have not undergone DNA fragmentation have only one round fluorescent head, while neurons that have undergone DNA fragmentation show fragments migrating away from the head towards the anode, forming a comet-like tail. Randomly observe 150 cells per sample and analyze using CASP software.

[0048] The results of the behavioral test and the comet experiment are shown in Table 1 below. Compared with Group A, the rats in Group B had significantly reduced movement distance and central area time in the open field, exhibited anxiety-like behavior, and had significantly increased comet tail length, indicating that the prefrontal neurons of the rats in Group B were damaged, proving that the prefrontal injury rat model was established in this example. Compared with Group B, the rats in Groups C to E had different degrees of increase in movement distance and central area time in the open field and different degrees of decrease in comet tail length, indicating that the degree of prefrontal injury in the rats was reduced and the anxiety symptoms were improved. Among them, the rats in Group C had the highest movement distance and central area time in the open field and the lowest comet tail length, indicating that the rats in Group C had the lowest degree of prefrontal injury and the best improvement in anxiety symptoms, i.e., the strain of animal bifidobacterium animalis subsp. BN16 used for gavage in Group C can reduce prefrontal injury and relieve anxiety, therefore, animal bifidobacterium animalis subsp. BN16 is selected as the best strain for reducing prefrontal injury.

[0049] Table 1 Effects of probiotics on prefrontal injury rats

[0050] Compared with Group B: P<0.0001.

[0051] Example 2 The present embodiment provides a preparation method of an animal bifidobacterium animalis ssp. lactis probiotic for stabilizing mood and relieving anxiety, and the specific steps are as follows.

[0052] (1) Select dry, no mildew, no insect infestation of embryo rice and lotus seeds, wash and drain, soak lotus seeds in water for 30 minutes, remove the core, and take the pulp. Select fresh, no mildew, no insect infestation of corn, wash and drain, and take the corn kernels. Select quality qualified walnut kernels and white granulated sugar.

[0053] (2) Take 35 parts of embryo rice in step (1), 17 parts of lotus seed pulp, 30 parts of corn kernels, 10 parts of walnut kernels, and 8 parts of white granulated sugar, mix evenly, then add 400 parts of purified water, break the wall at 31000 r / min for 3 minutes, adjust the pH to 6.8, then heat to 80℃, continue to break the wall for 3 minutes, then heat to 95℃, keep for 5 minutes, cool to 37℃, then get the material liquid A.

[0054] (3) Take 15g of proteose peptone, 2g of yeast extract, 20g of glucose, 0.5g of soluble starch, 5g of sodium chloride, 400mL of tomato extract (tomato extract preparation method: fresh tomatoes are washed, weighed and chopped, and then heated in a water bath at 100℃ with an equal amount of distilled water, stirred for 90min, then filtered with gauze, and the pH is adjusted to 7.0), 1mL of Tween 80, and 0.3g of liver powder, mix evenly, add distilled water to 1L, sterilize at 121℃ for 15min, cool to 37℃, add 1mL of sterile cysteine salt solution (take 0.5g of cysteine, add 1mL of hydrochloric acid to dissolve all the cysteine), adjust the pH to 6.8, and get the culture medium B.

[0055] (4) Take out the animal bifidobacterium animalis ssp. lactis BN16 freeze-dried preservation tube preserved at -80℃, thaw at room temperature, inoculate into culture medium B, activate 2 generations, centrifuge at 4000r / min, 4℃ after 16 hours of culture of the 3rd generation, wash 3 times with sterile normal saline solution, resuspend with material liquid A, and adjust the viable bacterial count to 2.5×10 9 CFU / mL, and get the bacterial suspension C.

[0056] (5) Take an appropriate amount of bacterial suspension, add it to the material liquid A at an addition amount of 2.5%, and anaerobic ferment at 37℃ for 36 hours, wherein the first 18 hours are constant pH fermentation (pH 6.8), and after fermentation, store in an environment at 4℃ for 12 hours, and get the fermentation liquid D.

[0057] (6) The fermentation liquid is homogenized for 10 min at a homogenization speed of 13000 r / min and a homogenization temperature of 4℃; then the temperature is raised to 60℃, and the liquid is homogenized 3 times at a pressure of 120 MPa; then the temperature is raised to 85℃, and the liquid is kept at this temperature for 30 min, to obtain the probiotic of animal bifidobacterium animalis.

[0058] Example 3 5-hydroxytryptamine (5-HT) is one of the main metabolites of tryptophan, and is an important neurotransmitter of the central and peripheral nervous system, which can regulate emotional stability and relieve anxiety. Tryptamine is produced in the human body only by the action of intestinal flora and tryptophan. Accumulation of tryptamine can reduce the utilization rate of tryptophan and severely reduce the generation of 5-HT, and can also affect the normal function of 5-HT. The present embodiment provides the screening of the composition of probiotic feed liquid A and the amount of bacterial suspension added, and detects the effect of probiotics prepared by different compositions of feed liquid A on the content of 5-HT and tryptophan in a rat depression model.

[0059] The specific experimental steps are as follows.

[0060] (1) The raw materials are screened and treated according to the method of Example 2, and the corresponding weight parts of germ rice, corn kernel, lotus seed, walnut kernel and white granulated sugar are selected according to the group setting of Table 2, mixed uniformly, then 400 parts of purified water are added, broken wall for 3 min at 31000 r / min, adjust pH to 6.8, then heat to 80℃, continue to break wall for 3 min, then raise the temperature to 95℃, keep for 5 min, cool to 37℃, then different groups of feed liquid A are obtained.

[0061] Table 2 Weight parts of each raw material in different groups of compositions

[0062] (2) Take 15 g of proteose peptone, 2 g of yeast extract, 20 g of glucose, 0.5 g of soluble starch, 5 g of sodium chloride, 400 mL of tomato extract (tomato extract preparation method: fresh tomatoes are washed, weighed and chopped, and an equal amount of distilled water is added to a water bath at 100℃, stirred for 90 min, then filtered with gauze, and the pH is adjusted to 7.0), 1 mL of Tween 80, and 0.3 g of liver powder, mix uniformly, add distilled water to 1 L, sterilize at 121℃ for 15 min, cool to 37℃, add sterile cysteine salt solution (take 0.5 g of cysteine, add 1 mL of hydrochloric acid to dissolve all the cysteine), adjust the pH to 6.8, and the culture medium B is obtained.

[0063] (3) Take out the animal Bifidobacterium animalis subsp. animalis BN16 freeze-dried preservation tube preserved at-80℃, thaw at room temperature, inoculate into culture medium B, activate 2 generations, centrifuge at 4000 r / min, 4℃ after 16 hours of culture of the 3rd generation, wash 3 times with sterile normal saline solution, resuspend with feed liquid A, adjust the viable bacterial count to 2.5x10 9 CFU / mL, and obtain bacterial suspension C.

[0064] (4) Take an appropriate amount of bacterial suspension C, add it into feed liquid A at an addition amount of 2.5%, anaerobic ferment at 37℃ for 36h, wherein the first 18 hours are constant pH fermentation, after fermentation, store at 4℃ for 12h, and obtain fermentation liquid D.

[0065] (5) Homogenize the fermentation liquid for 10min at a homogenization speed of 13000 r / min and a homogenization temperature of 4℃, then heat to 60℃, homogenize 3 times at a pressure of 120MPa, then heat to 85℃ and keep for 30min, and obtain animal Bifidobacterium animalis subsp. animalis postbiotic of different groups.

[0066] Meanwhile, set group 7: the preparation method of feed liquid A is the same as that of group 6, but no animal Bifidobacterium animalis subsp. animalis BN16 is added for fermentation, and an equal amount of purified water is used instead of bacterial suspension C for fermentation, and the final product obtained in step (5) is not called "animal Bifidobacterium animalis subsp. animalis postbiotic", but is called "sample".

[0067] Group 8: the preparation method of feed liquid A is the same as that of group 6, except that the addition amount of bacterial suspension C in step (5) is 5%.

[0068] Animal experiment: 80 SPF healthy male SD rats, weighing 200-210g, were fed in the SPF animal room, the room temperature was 25℃, the humidity was 65%, the light and dark cycle was 12 hours, and the water and food were free. After 7 days of adaptive feeding in the normal incubator, the rats were randomly divided into 10 groups, 8 rats in each group, and were recorded as F group, G group, H group, I group, J group, K group, L group, M group, N group and O group respectively.

[0069] The rats in G group to O group were fixed using a special restraint frame every day from 14:00 to 17:00, 2 soft adhesive strips on the restraint frame were used to bind the chest and abdomen of the rats respectively, and the activity of the rats was limited, which lasted for 21 days, during which sufficient water and food supply was ensured, and a depression rat model was established. The rats in F group were not treated and normally fed for 21 days.

[0070] During modeling, the rats were treated by gavage at the same time: The rats in F group and G group were gavaged with normal saline; The rats in H group were gavaged with animal Bifidobacterium animalis subsp. animalis postbiotic of group 1; Group I rats were administered with the Bifidobacterium animalis ssp. lactis postbiotic of Group 2 animals by gavage; Group J rats were administered with the Bifidobacterium animalis ssp. lactis postbiotic of Group 3 animals by gavage; Group K rats were administered with the Bifidobacterium animalis ssp. lactis postbiotic of Group 4 animals by gavage; Group L rats were administered with the Bifidobacterium animalis ssp. lactis postbiotic of Group 5 animals by gavage; Group M rats were administered with the Bifidobacterium animalis ssp. lactis postbiotic of Group 6 animals by gavage; Group N rats were administered with the sample of Group 7 animals by gavage; Group O rats were administered with the Bifidobacterium animalis ssp. lactis postbiotic of Group 8 animals by gavage; The gavage dose of each group was 2 mL, and the administration was performed continuously for 21 days.

[0071] On the 22nd day, the rats were subjected to the forced swimming test, i.e., the rats were placed in a glass cylinder with a water depth of 40 cm and a water temperature of 25°C, and observed for 6 min. The cumulative immobile time of each group of rats in the last 5 min was recorded, and the average value was calculated. The evaluation criteria for immobility were that the rats floated on the water surface, did not try to climb out of the cylinder, and only made some actions to keep their heads above water.

[0072] After the forced swimming test, all rats were normally fed for 1 day, and the diet and water were free. On the next morning at 9 o'clock, tryptophan was administered by gavage at a ratio of 50 mg / kg based on the weight of the rats, and the administration was performed continuously for 2 days. After 24 hours of fasting without water restriction, the rats were anesthetized, 5 mL of abdominal aortic blood was taken using a test tube without pyrogen and endotoxin, the blood sample was left to stand for 2 hours in an indoor environment, and then a low-temperature centrifuge was used for centrifugation at 3500 r / min for 10 minutes at 4°C. The supernatant was taken, and the 5-HT enzyme-linked immunosorbent assay kit (Shanghai Fusheng Industry Co., Ltd.) was used to determine the content of 5-hydroxytryptamine (5-HT) in the serum according to the instruction manual. The content of tryptamine in the serum was determined using the tryptamine content kit (Shanghai Xinyu Biological Technology Co., Ltd.) according to the instruction manual, and the average value of each group was calculated.

[0073] The experimental results are shown in Table 3 below. Compared with Group F, the immobile time of Group G rats in the forced swimming test was significantly increased, showing depressive symptoms, indicating that the depressive rat model was successful. The 5-HT content in the serum of Group G rats was significantly decreased, and the tryptamine content was significantly increased, indicating that the utilization ability of tryptophan in depressive rats was significantly reduced, and the emotional regulation ability was weaker.

[0074] Compared with group G, the immobility time of rats in groups H to M in the forced swimming test was reduced, the 5-HT content in serum was increased, and the tryptamine content was reduced, indicating that the utilization ability of rats to tryptophan was improved, and the depressive symptoms were improved. The animal bifidobacterium animalis subspecies postbiotic consumed by the rats had a certain promoting effect on improving depression. The difference lies in that the composition of the composition is different, and the improvement degree of the finally obtained postbiotic is also different. Among them, the immobility time of rats in group M in the forced swimming test is the lowest, the 5-HT content in serum is the highest, and the tryptamine content is the lowest, indicating that the utilization ability of rats to tryptophan is the strongest, and the depressive symptoms are improved best. The animal bifidobacterium animalis subspecies postbiotic consumed by the rats has the best effect on improving the utilization rate of tryptophan and is beneficial to stabilizing mood.

[0075] Compared with group M, the immobility time of rats in group N in the forced swimming test was significantly increased, the 5-HT content in serum was significantly reduced, and the tryptamine content was significantly increased, indicating that the product quality would be reduced without fermentation of animal bifidobacterium animalis subspecies BN16 to produce postbiotic. Compared with group M, there was no significant difference in the immobility time of rats in the forced swimming test, the 5-HT content in serum, and the tryptamine content, all of which could better improve the utilization rate of tryptophan and stabilize mood, indicating that the amount of bacterial suspension of group 6 has reached the saturation point of fermentation.

[0076] In summary, group 6 is selected as the best composition of postbiotic, and the obtained postbiotic can improve the utilization rate of tryptophan and is beneficial to stabilizing mood.

[0077] Table 3 Effect of animal bifidobacterium animalis subspecies postbiotic on depressive rats

[0078] Compared with group G: P<0.01, P<0.001, P<0.0001.

[0079] Example 4 This example provides optimization of the process conditions of animal bifidobacterium animalis subspecies postbiotic, and the specific experimental steps are as follows.

[0080] (1) Group 9: The preparation method of group 9 postbiotic is the same as that of group 6 postbiotic in example 3, and the difference lies in that the time of step (4) constant pH anaerobic fermentation in group 9 is adjusted to 0 h, and the total fermentation time is unchanged.

[0081] (2) Group 10: The preparation method of group 10 postbiotic is the same as that of group 6, and the difference lies in that the time of step (4) constant pH anaerobic fermentation in group 10 is adjusted to 36 h, and the total fermentation time is unchanged.

[0082] (3) Group 11: The preparation method of Group 11 is the same as that of Group 6, except that the homogenization in step (5) of Group 11 is not performed, i.e., step (5) is adjusted to "the fermentation broth is warmed to 85°C for 30 min, and then the probiotic of Bifidobacterium animalis subsp. animalis is obtained".

[0083] (4) Group 12: The preparation method of Group 12 is the same as that of Group 6, except that step (5) of Group 12 is modified to "the fermentation broth is homogenized for 10 min at a speed of 13000 r / min and a temperature of 4°C, and then warmed to 85°C for 30 min, and then the probiotic of Bifidobacterium animalis subsp. animalis is obtained".

[0084] (5) Group 13: The preparation method of Group 13 is the same as that of Group 6, except that step (5) of Group 13 is modified to "the fermentation broth is warmed to 60°C, and then homogenized for 3 times under a pressure of 120 MPa, and then warmed to 85°C for 30 min, and then the probiotic of Bifidobacterium animalis subsp. animalis is obtained".

[0085] (6) Group 14: The preparation method of Group 14 is the same as that of Group 6, except that step (5) is not performed.

[0086] After obtaining the probiotics of Bifidobacterium animalis subsp. animalis prepared by the above different processes, tests are performed, and the specific experimental steps are as follows: Six subjects, denoted as P group, are recruited, with an age of 21 years old and a gender of male. The inclusion criteria are that the subjects have no anxiety symptoms, the score of self-rating anxiety scale (SAS) is <40 points, and no antibiotic drugs have been consumed in the recent 1 month.

[0087] Forty-eight subjects, with an age of 21 years old and a gender of male, are recruited. The inclusion criteria are that the subjects have mild or moderate anxiety symptoms, the score of self-rating anxiety scale (SAS) is >50 points and <65 points, and no antibiotic drugs have been consumed in the recent 1 month. The subjects are evenly divided into 8 groups, each with 6 people, and are denoted as Q group, R group, S group, T group, U group, V group, W group, and X group, respectively.

[0088] The subjects in P group and Q group consume pure water; The subjects in R group consume the probiotic of Bifidobacterium animalis subsp. animalis of Group 6; The subjects in S group consume the probiotic of Bifidobacterium animalis subsp. animalis of Group 9; The subjects in T group consume the probiotic of Bifidobacterium animalis subsp. animalis of Group 10; The subjects in U group consume the probiotic of Bifidobacterium animalis subsp. animalis of Group 11; The subjects in group V eat the animal bifidobacterium animalis ssp. lactis postbiotic in group 12; The subjects in group W eat the animal bifidobacterium animalis ssp. lactis postbiotic in group 13; The subjects in group X eat the fermentation broth D in group 14.

[0089] The dose is 30 mL, and the subjects eat it half an hour after lunch every day for 30 days.

[0090] At 7 o'clock in the morning on the 1st day and the 31st day, the saliva of all the subjects is collected, and the collected saliva is centrifuged at 1000 r / min for 2 min, the upper cotton swab is discarded, and the lower saliva is taken to determine the content of cortisol (saliva) in the saliva by using a cortisol (saliva) detection kit (enzyme-linked immunoassay) (Germany DRG), and the average value of each group is obtained.

[0091] Cortisol is the final metabolite of the hypothalamic-pituitary-adrenal (HPA) axis and is a common stress factor. Anxiety can increase the level of cortisol. The experimental results are shown in Table 4. Compared with group P, the subjects in groups Q to X have a significantly increased content of saliva cortisol on the 1st day, indicating that their anxiety symptoms are more significant. Compared with group Q, the subjects in groups R to X have a decreased content of saliva cortisol on the 31st day, indicating that their anxiety symptoms are alleviated. The difference lies in that the postbiotics obtained by different processes have different alleviating effects. The subjects in group R have the lowest content of saliva cortisol on the 31st day, indicating that their anxiety symptoms are alleviated best. That is, the animal bifidobacterium animalis ssp. lactis postbiotic in group 6 has the best effect in alleviating anxiety, and after a specific fermentation process and homogenization process, the product quality can be greatly improved.

[0092] Table 4 Change in the content of saliva cortisol of the subjects

[0093] Compared with group Q: P < 0.01, P < 0.001, P < 0.0001.

[0094] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and all are included in the protection scope of the present application.

Claims

1. An animal subspecies of Bifidobacterium that stabilizes mood and relieves anxiety, characterized in that... The animal subspecies of Bifidobacterium animalis is Bifidobacterium animalis subspecies BN16, taxonomically named... Bifidobacterium animalis subsp.animalis It was deposited on August 13, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35603.

2. A post-electrophoresis agent for stabilizing emotions and alleviating anxiety, characterized in that, The metabiotic includes the animal subspecies BN16 of Bifidobacterium animalis as described in claim 1, and / or metabolites of BN16.

3. A method for preparing an epigenetic agent, characterized in that, The preparation method comprises fermenting the animal subspecies of Bifidobacterium animalis BN16 as described in claim 1 using a composition; the composition comprises at least one of germ rice, lotus seeds, corn kernels, walnut kernels, white sugar, and purified water.

4. The preparation method according to claim 3, wherein the composition comprises at least one of the following parts by weight: 20-40 parts germ rice, 10-30 parts lotus seeds, 20-40 parts corn kernels, 10-30 parts walnut kernels, 5-15 parts white sugar, and 200-400 parts purified water.

5. The preparation method according to claim 3, characterized in that, The fermentation includes constant pH fermentation and non-constant pH fermentation; the total fermentation time is 30-40 hours, of which constant pH fermentation time is 15-25 hours.

6. The preparation method according to claim 3, characterized in that, The preparation method further includes homogenizing the fermentation broth after fermentation; the homogenization includes homogenizing the fermentation broth at 2~8℃, heating it to 50~80℃, and then homogenizing it again 1~3 times under a pressure of 100~150MPa.

7. A product comprising at least one of the following: Bifidobacterium animalis subsp. as described in claim 1, the metagenin as described in claim 2, and the metagenin prepared by the method described in claim 3.

8. The product according to claim 7, characterized in that, The products include food or medicine.

9. The use of the animal subspecies of Bifidobacterium animalis according to claim 1, the metabiotic according to claim 2, or the metabiotic prepared by the preparation method according to claim 3 in any of the following (1) to (5): (1) Preparation of products for reducing prefrontal cortex damage; (2) To prepare products for the prevention or relief of anxiety; (3) Prepare products for stabilizing mood; (4) Prepare products to improve the utilization rate of tryptophan; (5) Prepare products for lowering cortisol.

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