Microbial preparation for preventing and treating anxiety after long-term heat stress as well as preparation method and application of microbial preparation

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to a microbial preparation RN010 for preventing and treating anxiety after long-term heat stress as well as a preparation method and application of the microbial preparation RN010. The viable count of the fecal bacillus przewalskii is 10 < 9 >-10 < 11 > CFU / g; the metabolite is a dry metabolite substance obtained by fermenting, culturing and inactivating the fecal bacillus przewalskii; the composite protective agent is prepared from inulin, medium chain triglyceride, trehalose, vitamin powder and emulsifying and galactomannan. The components are compounded in a specific weight part range to obtain the microbial preparation RN010, the various components are matched with one another, the action effects of the components are fully exerted, and a behavioral detection result shows that the anxiety behavior of a heat stress mouse is remarkably reduced, and various pathophysiological and physiological indexes are improved; the traditional Chinese medicine composition has a certain relieving effect on mouse anxiety caused by heat stress.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and specifically relates to a microbial preparation for preventing and treating anxiety after long-term heat stress, and a preparation method and application thereof. Background Art

[0002] Since the 21st century, the increase in the frequency, intensity and duration of extreme heat weather has become an important risk factor affecting public health and mass health. About half of the world's population, including more than 1 billion workers, are exposed to high temperatures for a long time. The mortality rate of people with underlying mental illness during hot weather is three times that of healthy people, while the incidence of mental illnesses such as anxiety, sleep disorders, depression, and schizophrenia in healthy people will increase significantly. Among them, anxiety disorder is the most common mental disorder associated with long-term heat stress. Anxiety manifests as strong worry and fear about events that have not occurred, lacking a clear objective object. Patients often feel persistent tension and fear, accompanied by autonomic nervous symptoms, muscle tension and motor restlessness. Excessive thinking can also lead to sleep disorders and inattention, which occur earlier than other mental illnesses, and will further lead to depression, compulsion, and even more serious suicidal consequences, seriously affecting normal social work and life.

[0003] The Global Burden of Disease Study shows that mental disorders, mainly anxiety disorders, are the sixth leading cause of disability and the largest disease burden among people aged 15 to 34. However, as the clinical first choice for anti-anxiety treatment, selective serotonin reuptake inhibitors and serotonin norepinephrine reuptake inhibitors have long treatment cycles, poor efficacy, low patient compliance, and are often accompanied by side effects such as nausea, headache, diarrhea, and constipation. Therefore, in the context of global warming, anxiety disorders related to long-term heat stress are becoming increasingly serious, and there is no satisfactory treatment. This situation highlights the urgent need for effective treatments to meet the growing public health challenges.

[0004] The gastrointestinal tract is one of the main interfaces between the host and the external environment, with an area of ​​about 250-400 square meters. The intestinal microbiota is composed of bacteria, archaea and eukaryotes that colonize the gastrointestinal tract, forming a symbiotic relationship with the host and co-evolving. These intestinal microorganisms play an important role in maintaining the body's energy balance, immune regulation, and internal environment homeostasis. In addition, the signals generated by the intestinal flora can be transmitted to the central nervous system through the vagus nerve, endocrine, immune and metabolic pathways, thereby affecting emotional state and cognitive function.

[0005] Studies have shown that the α diversity of intestinal flora in patients with anxiety and depression is significantly lower than that in healthy individuals. The relative abundance of normal intestinal flora such as Firmicutes, Soft-walled Bacteria, Eubacterium coprostanoligenes, Ruminococcaceae UCG-014 and Prevotella 9 is negatively correlated with the severity of anxiety. Bercik et al. further confirmed through animal experiments that mice that received fecal microbiota transplants from anxious mice showed anxiety-like behavioral phenotypes. These research results all suggest that the occurrence of anxiety may be closely related to intestinal flora imbalance. Supplementation with probiotics can also improve anxiety symptoms. The results of a double-blind clinical trial further proved that probiotic treatment can significantly reduce patients' anxiety, depression and stress levels. Therefore, it is of great practical significance to develop a microbial preparation that can effectively relieve anxiety symptoms caused by heat stress. Summary of the invention

[0006] The purpose of the present invention is to provide a microbial preparation for preventing and treating anxiety after long-term heat stress, and a preparation method and application thereof. The preparation can effectively relieve the anxiety symptoms caused by heat stress, and at the same time can improve pathological and physiological indicators such as weight changes and organ damage caused by heat stress, which has positive significance for the relief and treatment of anxiety symptoms after long-term heat stress.

[0007] The objective of the present invention is achieved through the following technical solutions: The present invention provides a microbial preparation for preventing and treating anxiety after long-term heat stress. The microbial preparation RN010 comprises the following components: Faecalibacterium prausnitzii, postbiotics and a composite protective agent.

[0008] Furthermore, the viable count of Faecalibacterium prausnitzii is 10 9 -10 11 CFU / g.

[0009] Furthermore, the postbiotic refers to the dry matter of metabolites obtained by inactivating the Faecalibacterium prausnitzii after fermentation and culture.

[0010] Furthermore, the composite protective agent is composed of the following components by weight: 20-30 parts of inulin, 10-20 parts of medium-chain triglycerides, 3-8 parts of trehalose, 1-5 parts of cellulose powder, 0.5-3 parts of emulsifier and 0.1-0.5 parts of galactomannan.

[0011] The present invention also provides a method for preparing the microbial preparation, comprising the following steps: (1) inoculating the Faecalibacterium prausnitzii into a YCFA liquid culture medium according to a certain proportion and shaking and culturing until the desired concentration and activity peak are reached, then centrifuging and collecting the cells, and resuspending the cells obtained by centrifugation in sterile water; (2) inoculating the Faecalibacterium prausnitzii into a YCFA liquid culture medium in proportion, culturing at a constant temperature and shaking until fermentation is complete, collecting the fermentation liquid, inactivating the fermentation liquid, and then spray drying to obtain the dry matter of bacterial metabolites, namely, postbiotics; (3) sterilizing inulin, medium chain triglyceride, trehalose, cellulose powder, emulsifier and galactomannan under ultraviolet light, mixing and weighing them according to the proportions, and obtaining a composite protective agent; (4) The bacterial cells resuspended in sterile water obtained in step (1), the postbiotics obtained in step (2), and the composite protective agent obtained in step (3) are mixed, vacuum freeze-dried, and pulverized to obtain a microbial preparation.

[0012] Furthermore, the YCFA liquid culture medium comprises: 10.0 g / L tryptone; 2.5 g / L yeast extract; 4.0 g / L sodium bicarbonate; 2.0 g / L glucose; 2.0 g / L maltose; 2.0 g / L cellobiose; 1.0 g / L cysteine ​​hydrochloride; 0.001 g / L resazurin; 0.45 g / L dipotassium hydrogen phosphate; 0.45 g / L potassium dihydrogen phosphate; 0.9 g / L sodium chloride; 0.044 g / L magnesium sulfate; 0.09 g / L calcium chloride, and is sterilized at 121°C for 15 min after adding water.

[0013] Furthermore, in step (1), the ratio is 1:30; the shaking culture conditions are a temperature of 37°C, a sealed shaking culture speed of 200 r / min, and a culture time of 14 hours+; the ideal concentration is a live bacterial count of 10 9 -10 11 CFU / g.

[0014] Furthermore, in step (2), the ratio is 1:30; the shaking culture conditions are a temperature of 37°C, a sealed shaking culture speed of 200 r / min, and a culture of more than 24 hours; the inactivation is a high-temperature inactivation at 85°C for 15 minutes, and the inlet temperature of the spray drying is 180°C and the outlet temperature is 60°C.

[0015] Furthermore, in step (3), the weight ratio of inulin, medium-chain triglycerides, trehalose, cellulose powder, emulsifier and galactomannan is 25:15:5:3:1:0.1.

[0016] The present invention also provides a use of the microbial preparation in the preparation of a drug for preventing and treating anxiety after long-term heat stress, wherein the drug has at least the following functions: (1) Significantly improved the anxiety-like behavior of heat-stressed mice; (2) Significantly improved the body weight changes and organ damage indicators of heat stressed mice.

[0017] Beneficial effects: 1. The present invention develops a new probiotic compounding method and a new heat stress-induced anxiety treatment strategy, that is, the strain and its postbiotics as well as inulin, medium-chain triglycerides, trehalose, cellulose powder, emulsifier, and galactomannan are compounded in proportion and used. It is found that the compounded products can cooperate with each other and promote each other, and have a significant alleviating effect on anxiety caused by heat stress.

[0018] 2. The bacterial body of Faecalibacterium prausnitzii (DSM 17677, Ningbo Mingzhou Biotechnology Co., Ltd.) and its fermented metabolites are produced without any addition of any substances during the entire fermentation and post-treatment process. In addition to the bacterial components such as teichoic acid and peptidoglycan, and metabolites such as polypeptides, polyphenols, short-chain fatty acids, and extracellular polysaccharides of Li Yongle probiotics, the nutritional components such as dietary fiber, small molecule active peptides, and polysaccharides of the fermentation substrate are also utilized.

[0019] 3. High temperature environment can cause anxiety-like behavior, weight changes and organ damage in mice. Microbial preparations can alleviate the anxiety-like behavior caused by heat stress in mice and reverse the weight changes and organ damage caused by heat stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments 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 work.

[0021] Figure 1 The figures are verification diagrams of anxiety-like behaviors induced by long-term heat stress and the protective effect of the microbial preparation of the present invention on anxiety caused by long-term heat stress, wherein: (A) schematic diagram of elevated plus maze experiment; (B) trajectory diagram of elevated plus maze; (C) bar graph of inter-group difference analysis of open arm residence time; (D) schematic diagram of open field experiment; (E) trajectory diagram of open field experiment; (F) bar graph of inter-group difference analysis of residence time in the center area of ​​open field; KW test, n=8, significant difference is indicated by *, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; 14dHS: long-term heat stress group, Ctrl: negative control group, ABX_14dHS: antibiotic cocktail bowel cleansing treatment group; FPM_14dHS: microbial preparation treatment group of the present invention.

[0022] Figure 2The figure is a graph showing the changes in the body weight of mice during 2 weeks of long-term heat stress, wherein 14dHS: long-term heat stress group, ABX_14dHS: antibiotic cocktail bowel cleansing treatment group; FPM_14dHS: microbial preparation treatment group of the present invention.

[0023] Figure 3 HE staining images of lung, liver and kidney tissue sections of mice in the blank control group, bowel cleansing treatment group and microbial preparation intervention group after long-term heat stress, including (A) lung tissue section and HE staining and scoring, (B) kidney tissue section and HE staining and scoring, (C) liver tissue section and HE staining and scoring, 14dHS: long-term heat stress group, Ctrl: negative control group, ABX_14dHS: antibiotic cocktail bowel cleansing treatment group; FPM_14dHS: lung tissue damage covers three aspects: alveolar and pulmonary interstitial edema, inflammatory cell infiltration, and alveolar structural disorder (no such lesions are 0 points, mild lesions are 1 point, moderate lesions are 2 points, and severe lesions are 3 points); kidney tissue damage assessment includes tubular atrophy, epithelial cell degeneration, epithelial cell necrosis, interstitial edema, renal interstitial fibrosis and inflammatory cell infiltration (normal is 0 points, mild damage is 1 point, moderate damage is 2 points, and severe damage is 3 points); pathological damage of liver tissue is evaluated using the SUZUKI scoring system. Scoring criteria include sinusoidal congestion and cytoplasmic vacuolization: if such lesions are not seen, 0 points are given, if they occur occasionally, 1 point, if they are mild, 2 points, if they are moderate, 3 points, and if they are severe, 4 points; the scoring basis for the proportion of hepatic parenchymal cell necrosis is as follows: if such lesions are absent, 0 points are given, if they are rarely seen, 1 point, if the proportion of lesion cells is less than 30%, 2 points, if the proportion is 30%-60%, 3 points, and if it is more than 60%, 4 points. The Student T test for independent samples was used between the two groups. * indicates P <0.05, ** P <0.01, *** P <0.001. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The test methods used in the following implementations, unless otherwise specified, are all commonly used experimental methods in this field; Unless otherwise specified, the experimental materials used in the following examples are all commonly used experimental materials in this field.

[0030] The microbial preparation for improving heat stress anxiety in the present invention comprises the following components by weight: Faecalibacterium prausnitzii viable bacteria count 10 9 -10 11 CFU / g, inulin 20-30 parts, medium chain triglycerides 10-20 parts, trehalose 3-8 parts, cellulose powder 1-5 parts, emulsifier 0.5-3 parts, postbiotics 1 part, galactomannan 0.1-0.5 parts.

[0031] As a preferred embodiment, the microbial preparation comprises the following raw materials: 25 parts of inulin, 15 parts of medium-chain triglycerides, 5 parts of trehalose, 3 parts of cellulose powder, 1 part of emulsifier, 1 part of postbiotics, and 0.1 part of galactomannan.

[0032] The preparation method of the microbial preparation for improving heat stress anxiety in the present invention comprises the following steps: (1) Inoculate Faecalibacterium prausnitzii into YCFA liquid culture medium and shake culture. Observe the turbidity of the culture medium. When the bacterial solution reaches the ideal concentration, centrifuge at 5000 r / min for 5 minutes to collect the bacterial cells, and resuspend the obtained cells in sterile water. (2) Inoculate Faecalibacterium prausnitzii into YCFA liquid culture medium and culture at a constant temperature with shaking for 24 h+ until fermentation is complete, then collect the fermentation broth, inactivate it at 85°C for 15 min, and then spray dry it with an inlet air temperature of 180°C and an outlet air temperature of 60°C to obtain the dry matter of bacterial metabolites, namely postbiotics.

[0033] (3) The concentrations of the components in the YCFA liquid culture medium are as follows: casein peptone 10.0 g / L; yeast extract 2.5 g / L; resazurin 0.001 g / L; dipotassium hydrogen phosphate 0.45 g / L; potassium dihydrogen phosphate 0.45 g / L; sodium chloride 0.9 g / L; sodium bicarbonate 4.0 g / L; magnesium sulfate 0.044 g / L; and calcium chloride 0.09 g / L. After adding water, sterilize at 121°C for 15 min.

[0034] (4) Sterilize inulin, medium chain triglycerides, trehalose, cellulose powder, emulsifier and galactomannan under ultraviolet light, mix them according to the above weight proportions and weigh them for later use to obtain a composite protective agent.

[0035] (5) The bacterial cells, postbiotics and composite protective agent resuspended in sterile water are mixed, vacuum freeze-dried and crushed to obtain a microbial preparation.

[0036] Example 1 Preparation method of microecological preparation This embodiment provides a microbial preparation for improving heat stress anxiety, comprising the following components, measured by weight: Faecalibacterium prausnitzii viable bacteria count 10 11 CFU / g, inulin 25 parts, medium chain triglycerides 15 parts, trehalose 5 parts, cellulose powder 3 parts, emulsifier 1 part, postbiotics 1 part, galactomannan 0.1 part.

[0037] The preparation method of the microbial preparation is as follows: (1) Activation culture: Carefully selected strains: Faecalibacterium prausnitzii was inoculated into YCFA liquid culture medium at a ratio of 1:30 and cultured at 37°C with a sealed shaking culture at a speed of 200 r / min. After culturing for 14 h+ until the ideal concentration and activity peak were reached, the cells were collected by centrifugation and resuspended in sterile water.

[0038] (2) Preparation of postbiotics: Inoculate Faecalis prausnitzii into liquid culture medium, culture at 37°C with shaking for 24 h+ until fermentation is complete, collect the fermentation broth, inactivate it at 85°C for 15 min, and then spray dry it with an inlet air temperature of 180°C and an outlet air temperature of 60°C.

[0039] (3) YCFA liquid culture medium composition: tryptone 10.0 g / L; yeast extract 2.5 g / L; sodium bicarbonate 4.0 g / L; glucose 2.0 g / L; maltose 2.0 g / L; cellobiose 2.0 g / L; cysteine ​​hydrochloride 1.0 g / L; resazurin 0.001 g / L; potassium dihydrogen phosphate 0.45 g / L; potassium dihydrogen phosphate 0.45 g / L; sodium chloride 0.9 g / L; magnesium sulfate 0.044 g / L; calcium chloride 0.09 g / L. After adding water, sterilize at 121°C for 15 min.

[0040] (4) Preparation of composite protective agent: Inulin, medium chain triglycerides, trehalose, cellulose powder, emulsifier, and galactomannan are sterilized under ultraviolet light for more than 30 minutes, and weighed according to the weight ratio of 25 parts of inulin, 15 parts of medium chain triglycerides, 5 parts of trehalose, 3 parts of cellulose powder, 1 part of emulsifier, and 0.1 part of galactomannan for later use.

[0041] (5) The bacterial cells, postbiotics and composite protective agent resuspended in sterile water are mixed, vacuum freeze-dried and crushed into a probiotic powder preparation.

[0042] (6) Dissolve the bacterial powder preparation again, make gradient dilutions and plate counts, and record the live bacteria content in various bacterial powders. The live bacteria count in the microbial preparation is 10 9 -10 11 CFU / g.

[0043] Example 2 Effect of microbial preparations on improving heat stress anxiety in mice Thirty six-week-old C57BL / 6 mice were selected as experimental animals. The heat stress model was induced by simulating 42°C heat stroke model in mice using Tianjin Test RGX-400P artificial climate chamber. The animals were housed in SPF animal room for conventional feeding.

[0044] After 1 week of adaptive feeding, the mice were randomly divided into 3 groups: blank control group (control group), climate chamber simulated heatstroke environment group (model group), and heatstroke model group with added microbial preparations (treatment group). The model group and treatment group were intervened in the artificial climate chamber to simulate the heatstroke environment for 1 hour every day for 2 weeks. The treatment group was gavaged with 50 mg / kg of probiotic powder prepared with sterile distilled water every day, and the control group and model group mice were gavaged with the same amount of sterile distilled water every day.

[0045] Among them, microbial preparations have an impact on anxiety-related behavioral indicators such as Figure 1 Anxiety-related behavioral testing of mice: An open field and elevated maze tests were used to evaluate the behavioral symptoms of anxiety in mice, and Smart3.0 software was used for data analysis.

[0046] Mouse tissue collection: At the end of each day's heat stress experiment, the three groups of mice were weighed after fasting for 12 hours overnight, and the effect of microbial preparations on the weight changes of mice after long-term heat stress was statistically analyzed. After gas anesthesia, the mice were killed by cervical dislocation, and the lung, liver, and kidney tissues were quickly removed for HE staining. The organ pathology score was used to evaluate the improvement of the microbial preparation on the damage of various organs after long-term heat stress in mice.

[0047] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. 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, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A microbial preparation for preventing and treating anxiety after long-term heat stress, characterized in that: The microbial preparation RN010 includes the following components: Faecalibacterium prausnitzii, postbiotics and a composite protective agent.

2. The microbial preparation for preventing and treating anxiety after long-term heat stress according to claim 1, characterized in that: The viable count of Faecalibacterium prausnitzii is 10 9 -10 11 CFU / g.

3. The microbial preparation for preventing and treating anxiety after long-term heat stress according to claim 1, characterized in that: The postbiotics refer to the dry matter of metabolites obtained by inactivating the Faecalibacterium prausnitzii after fermentation and culture.

4. The microbial preparation for preventing and treating anxiety after long-term heat stress according to claim 1, characterized in that: The composite protective agent is composed of the following components by weight: 20-30 parts of inulin, 10-20 parts of medium-chain triglycerides, 3-8 parts of trehalose, 1-5 parts of cellulose powder, 0.5-3 parts of emulsifier and 0.1-0.5 parts of galactomannan.

5. A method for preparing a microbial preparation according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) inoculating the Faecalibacterium prausnitzii into a YCFA liquid culture medium according to a certain proportion and shaking and culturing until the desired concentration and activity peak are reached, then centrifuging and collecting the cells, and resuspending the cells obtained by centrifugation in sterile water; (2) inoculating the Faecalibacterium prausnitzii into a YCFA liquid culture medium in proportion, culturing at a constant temperature and shaking until fermentation is complete, collecting the fermentation liquid, inactivating the fermentation liquid, and then spray drying to obtain the dry matter of bacterial metabolites, namely, postbiotics; (3) sterilizing inulin, medium chain triglyceride, trehalose, cellulose powder, emulsifier and galactomannan under ultraviolet light, mixing and weighing them according to the proportions, and obtaining a composite protective agent; (4) The bacterial cells resuspended in sterile water obtained in step (1), the postbiotics obtained in step (2), and the composite protective agent obtained in step (3) are mixed, vacuum freeze-dried, and pulverized to obtain a microbial preparation.

6. The preparation method according to claim 5, characterized in that: The YCFA liquid culture medium comprises: 10.0 g / L tryptone; 2.5 g / L yeast extract; 4.0 g / L sodium bicarbonate; 2.0 g / L glucose; 2.0 g / L maltose; 2.0 g / L cellobiose; 1.0 g / L cysteine ​​hydrochloride; 0.001 g / L resazurin; 0.45 g / L dipotassium hydrogen phosphate; 0.45 g / L potassium dihydrogen phosphate; 0.9 g / L sodium chloride; 0.044 g / L magnesium sulfate; and 0.09 g / L calcium chloride. After adding water, sterilize at 121°C for 15 min.

7. The preparation method according to claim 5, characterized in that: In step (1), the ratio is 1:30; the shaking culture conditions are a temperature of 37°C, a sealed shaking culture speed of 200 r / min, and a culture time of 14 hours+; the ideal concentration is a live bacterial count of 10 9 -10 11 CFU / g.

8. The preparation method according to claim 5, characterized in that: In step (2), the ratio is 1:30; the shaking culture conditions are a temperature of 37°C, a sealed shaking culture speed of 200 r / min, and a culture of more than 24 hours; the inactivation is a high-temperature inactivation at 85°C for 15 minutes, the inlet temperature of the spray drying is 180°C, and the outlet temperature is 60°C.

9. The preparation method according to claim 5, characterized in that: In step (3), the weight ratio of inulin, medium-chain triglycerides, trehalose, cellulose powder, emulsifier and galactomannan is 25:15:5:3:1:0.

1.

10. Use of the microbial preparation according to any one of claims 1 to 4 in the preparation of a drug for preventing and treating anxiety after long-term heat stress, characterized in that: The drug has at least the following functions: (1) Significantly improved the anxiety-like behavior of heat-stressed mice; (2) Significantly improved the body weight and organ damage of heat stressed mice.

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