Hypolipidemic complex microbial inoculant and application thereof

Abstract

The invention discloses a blood fat reducing complex microbial inoculant and application thereof, and belongs to the technical field of medicines. The composite bacterial agent is prepared by compounding pediococcus acidilactici freeze-dried powder and lactococcus gasseri freeze-dried powder, the active bacteria in the pediococcus acidilactici freeze-dried powder are pediococcus acidilactici CICC 20720, and the active bacteria in the lactococcus gasseri freeze-dried powder are lactococcus gasseri CICC 21038. The invention also discloses a preparation method of the composite bacterial agent, and the composite bacterial agent is prepared by compounding the pediococcus acidilactici freeze-dried powder and the lactococcus gasseri freeze-dried powder, the active bacteria in the pediococcus acidilactici freeze-dried powder are pediococcus acidilactici CICC 20720, and the active bacteria in the lactococcus gasseri freeze-dried powder are pediococcus gasseri CICC 21038. The mass ratio of the pediococcus acidilactici freeze-dried powder to the lactococcus gasseri freeze-dried powder in the complex microbial inoculant is (1-3): (1-3). Tests show that the complex microbial inoculant prepared by the invention can significantly reduce the blood fat level in serum and also has the effect of improving hypothyroidism.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a lipid-lowering composite bacterial agent and application thereof. Background Art

[0002] Hyperlipidemia usually refers to elevated triglycerides and / or total cholesterol, elevated low-density lipoprotein cholesterol, and decreased high-density lipoprotein cholesterol in plasma. As people's living standards and eating habits change, hyperlipidemia occurs in people of different ages and genders, especially in people aged 50 to 69, and has a clear genetic tendency. The occurrence of hyperlipidemia is affected by many factors, including gene mutations; environmental factors, such as poor eating habits, lack of physical activity, obesity, etc.; and other diseases such as diabetes, nephrotic syndrome, liver disease, etc.

[0003] Currently, the methods used to lower blood lipids are mainly through Western medicine, Chinese medicine or a combination of the two, but long-term use of drugs leads to gastrointestinal burden and may also lead to drug resistance. Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of the flora in a certain part of the host. By using probiotics to change the health of the human intestine, and then promote the absorption and utilization of nutrients, it has a certain effect on improving hyperlipidemia caused by environmental factors. In addition, there are many reports in the prior art on the use of probiotics to lower blood lipids, such as: CN107714738A A probiotic complex with the functions of improving immunity, lowering blood lipids, and promoting metabolism and its preparation method and application, by compounding Lactobacillus acidophilus, Saccharomyces cerevisiae, and Licheniformis, which are synergistic with the culture medium components to complement each other, so that it has significant effects in improving immunity, lowering blood lipids, and promoting metabolism; CN107684567A A composite probiotic preparation for improving obesity and lowering blood lipids, which discloses Bifidobacterium breve: Lactobacillus acidophilus: Lactobacillus casei: Streptococcus thermophilus Thermophilus) is compounded according to the ratio of live bacteria count of 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5. The obtained composite probiotic preparation can be given to obese mothers during pregnancy and lactation to improve the flora disorder and blood lipid levels of the offspring. Its effect on the flora and blood lipid improvement of the offspring can last until adulthood. It can be seen that the existing technology has achieved the effect of lowering blood lipids to a certain extent by compounding different strains, but on the one hand, it is for the selection of specific strains, and on the other hand, it is effective for the increase of blood lipids caused by specific factors. This also shows that the blood lipid-lowering effects of different strain combinations are different and uneven. Therefore, enriching blood lipid-lowering probiotic agents is of great significance for the treatment of hyperlipidemia caused by different factors. Summary of the invention

[0004] The purpose of the present invention is to provide a lipid-lowering composite bacterial agent and its application to solve the problems existing in the above-mentioned prior art. By compounding Pediococcus acidilactici and Lactococcus garvieae, the blood lipid level in serum can be significantly reduced, providing a new strain combination for the screening of lipid-lowering bacterial agents.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a composite bacterial agent. The composite bacterial agent is prepared by compounding lyophilized powder of Pediococcus acidilactici and lyophilized powder of Lactococcus garvieae, wherein the active bacteria in the lyophilized powder of Pediococcus acidilactici are Pediococcus acidilactici CICC 20720, and the active bacteria in the lyophilized powder of Lactococcus garvieae are Lactococcus garvieae CICC 21038.

[0007] Optionally, the mass ratio of freeze-dried powder of Pediococcus acidilactici to freeze-dried powder of Lactococcus gasseri in the composite bacterial agent is (1-3): (1-3).

[0008] Optionally, the Pediococcus acidilactici freeze-dried powder is obtained by fermenting Pediococcus acidilactici CICC 20720 to obtain Pediococcus acidilactici fermentation broth; centrifuging the Pediococcus acidilactici fermentation broth, collecting the supernatant, and freeze-drying the supernatant;

[0009] The Lactococcus gasseri freeze-dried powder is obtained by fermenting Lactococcus gasseri CICC 21038 to obtain Lactococcus gasseri fermentation liquid; centrifuging the Lactococcus gasseri fermentation liquid, collecting the supernatant, and freeze-drying the supernatant.

[0010] The present invention also provides application of the composite bacterial agent in preparing a drug for lowering blood lipids.

[0011] The present invention also provides application of the composite bacterial agent in preparing a medicine for treating hypothyroidism.

[0012] The present invention also provides a blood lipid lowering medicine, comprising the composite bacterial agent.

[0013] The present invention also provides a medicine for improving hypothyroidism, comprising the composite bacterial agent.

[0014] The present invention discloses the following technical effects:

[0015] The present invention finds that the compounding of lactic acid Pediococcus and Lactococcus gasseri has a significant lipid-lowering effect. It was found in the process of single strain verification that the lipid-lowering effect was not obvious, but after compounding, the total cholesterol, triglyceride, and low-density lipoprotein content in serum were significantly reduced, and the high-density lipoprotein content was increased, so that the above indicators were restored to the content in normal serum without significant difference. The inventor inferred from years of research on probiotic strains that the two strains may have similar environmental and nutritional requirements and can coexist in the same environment after compounding. In addition, lactic acid Pediococcus can produce acid, regulate gastrointestinal flora, and maintain intestinal microecological balance; it has an antagonistic effect on pathogenic microorganisms in animals, can competitively inhibit pathogenic microorganisms, enhance the immune function of animal bodies, produce beneficial metabolites such as organic acids, bacteriocins, and fat-soluble substances such as coenzyme Q10, etc., and can also activate the activity of acid proteases, participate in the metabolism of the body, and prevent the production of harmful substances. As a kind of lactic acid bacteria, Lactococcus gasseri can promote intestinal health, improve immunity, etc., and importantly, the beneficial metabolites produced by Pediococcus acidilactici and the acidic environment can also be used to rapidly propagate and colonize in vivo, and Pediococcus acidilactici can be combined to form beneficial flora, jointly inhibit harmful bacteria, and regulate the indicators that affect the increase of blood lipids such as cholesterol and triglycerides. In short, the blood lipid content in serum can be significantly reduced by compounding of two strains of bacteria in a specific ratio, and it is also unexpectedly found that it has a significant effect on improving hypothyroidism. The composite bacterial agent prepared by the present invention is compounded by Pediococcus acidilactici and Lactococcus gasseri in a specific ratio, and the effect of synergistically reducing blood lipids and improving hypothyroidism is played, which provides a new bacterial source combination for enriching clinical lipid-lowering composite bacteria, and also provides a new direction and method for the treatment of hypothyroidism. DETAILED DESCRIPTION

[0016] 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.

[0017] It should be understood that the terms described in the present invention are only for describing a particular embodiment 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. The intermediate value in any stated value or stated range, and each smaller range between 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.

[0018] 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.

[0019] 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 those skilled in the art. The present invention description and examples are exemplary only.

[0020] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0021] The strains used in the following examples are: Pediococcus acidilactici, with a collection number of CICC 20720, and Lactococcus garvieae, with a collection number of CICC 21038. Both strains were purchased from the China Industrial Microbiological Culture Collection Center, and the public can purchase the strains from the above platform.

[0022] Example 1 A composite bacterial agent

[0023] (1) Strain fermentation

[0024] The preserved lactic acid Pediococcus was activated with MRS solid medium (15.0 g agar was added to 1 L MRS liquid medium) at 40°C for 16 h to obtain activated bacteria; then the activated bacteria were inoculated into MRS liquid medium (Thermo Fisher) for expansion culture (3 rings of activated bacteria were inoculated per 100 mL MRS liquid medium), and the culture conditions were 40°C and 150 rpm shaking culture for 5 days to obtain lactic acid Pediococcus fermentation liquid with a bacterial count of >10 8 cfu / mL; then, the fermentation broth of Pediococcus acidilactici was centrifuged, the supernatant was collected, and freeze-dried to obtain freeze-dried powder of Pediococcus acidilactici.

[0025] The preserved Lactococcus gasseri was activated with solid fermentation medium at 37°C for 20h to obtain activated bacteria; then the activated bacteria was inoculated into liquid fermentation medium for expansion culture (3 rings of activated bacteria were inoculated per 100mL liquid fermentation medium), and the culture conditions were 37°C and 120rpm shaking culture for 4d to obtain Lactococcus gasseri fermentation liquid with a bacterial count of >108 cfu / mL; then, the fermentation liquid of Lactococcus gasseri was centrifuged, the supernatant was collected, and freeze-dried to obtain freeze-dried powder of Lactococcus gasseri. Among them, the solid fermentation medium is: yeast extract powder 7.5g, glucose 10.0g, tomato juice (squeezed whole juice) 100.0mL, peptone 7.5g, KH2PO42.0g, Tween 80 0.5mL, agar 15.0g, distilled water 900mL, pH7.0. The above components were sterilized at 121℃ for 15min. No agar was added to the liquid fermentation medium.

[0026] (2) Preparation of composite bacterial agents

[0027] The above-obtained freeze-dried powder of Pediococcus acidilactici and freeze-dried powder of Lactococcus garvieae were mixed uniformly under sterile conditions according to the ratio described in Table 1, and packaged to obtain a composite bacterial agent. The total bacterial count in the composite bacterial agent was determined by the plate count method.

[0028] Table 1 Results of total bacterial count determination at different ratios

[0029]

[0030] From the experimental data in Table 1, it can be seen that the total bacterial count of the composite bacterial agents obtained by compounding freeze-dried powder of Pediococcus acidilactici and freeze-dried powder of Lactococcus gasseri with different ratios is different. This shows that the compounding ratio of the two strains affects the survival of live bacteria. The above results show that when the mass ratio of freeze-dried powder of Pediococcus acidilactici to freeze-dried powder of Lactococcus gasseri is 2.5:1, the total bacterial count is the highest; when the mass ratio of freeze-dried powder of Pediococcus acidilactici to freeze-dried powder of Lactococcus gasseri is 1:1, the total bacterial count is the lowest.

[0031] Example 2 Effect of the composite bacterial agent on blood lipid levels in mice

[0032] Healthy BALB / c mice (200±5g) were selected and randomly divided into 11 groups, with 10 mice in each group, namely: blank control group, high-fat model group, positive control group, low-dose group of freeze-dried powder of Pediococcus acidilactici, high-dose group of freeze-dried powder of Pediococcus acidilactici, low-dose group of freeze-dried powder of Lactococcus garrisonii, high-dose group of freeze-dried powder of Lactococcus garrisonii, composite bacterial agent 1 (low-dose group), composite bacterial agent 1 (high-dose group), composite bacterial agent 2 (low-dose group), and composite bacterial agent 2 (high-dose group). The blank control group was fed with ordinary feed, and the high-fat model group and other groups were fed with high-fat feed (purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., 60% fat energy high-fat feed). Each group was gavaged at regular intervals every day, and the blank control group and the high-fat model group were gavaged with clean water once a day; the positive control group took 10 mg of fluvastatin once a day; the low-dose group (100 mg / kg) and the high-dose group (250 mg / kg) were gavaged with the corresponding bacterial agent once a day according to the required dosage; the drug was administered continuously for 4 weeks. During the experiment, the mice were allowed to eat and drink freely, the state of the mice was observed, and the amount of food intake was recorded. After the experiment, the mice were fasted for 12 hours but not water, and then weighed, blood was collected from the eyeballs, and the serum was separated by centrifugation (3000r / min, 15min) after 37℃ water bath for 30min. The total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) in the serum were measured. The results are shown in Table 2.

[0033] Table 2 Effects of each treatment group on TC, TG, LDL-C and HDL-C

[0034]

[0035] In the above Table 2, compound bacterial agent 1 is a compound bacterial agent prepared by using a mass ratio of 1:1 between freeze-dried powder of lactic acid and freeze-dried powder of Lactococcus gasseri, and compound bacterial agent 2 is a compound bacterial agent prepared by using a mass ratio of 2.5:1 between freeze-dried powder of lactic acid and freeze-dried powder of Lactococcus gasseri. The purpose is to verify that the compound bacterial agent with the lowest bacterial content can also play a role, and the two compound combinations with the lowest and highest bacterial content are used for the experiment.

[0036] It can be seen from the experimental data in Table 2 that after administration of the composite bacterial agent prepared by the present invention, whether it is the low-dose group and the high-dose group with relatively low total bacterial content, or the low-dose group and the high-dose group with relatively high total bacterial content, the TC, TG and LDL-C contents are significantly reduced compared with the high-fat model group, and the HDL-C content is increased; and there is no significant difference compared with the normal control group and the positive control group. The single freeze-dried powder of lactic acid and the freeze-dried powder of Lactococcus gasseri can improve the condition of high blood lipid content to a certain extent, but the effect is not obvious, and it is obviously not as good as the composite bacterial agent in lowering blood lipids.

[0037] Example 3 Experimental study on the effect of composite bacterial agent on the treatment of hypothyroidism

[0038] Experimental animals: Healthy BALB / c male mice (200±5g) were selected and randomly divided into 5 groups, with 10 mice in each group.

[0039] Animal model: The mice were fed 0.03% methylthiouracilum (MTU) in drinking water for 5 weeks to establish a hypothyroidism mouse model.

[0040] The groups are as follows:

[0041] (1) Normal control group: normal mice fed with purified water;

[0042] (2) Negative control group: hypothyroidism mouse model was fed with pure water daily;

[0043] (3) Positive control group: hypothyroidism mouse model was fed thyroid tablets daily according to the instructions;

[0044] (4) Low-dose group: The mice were fed with a composite bacterial agent at a dosage of 100 mg / kg per day, once in the morning and once in the evening. The composite bacterial agent was a mixture of freeze-dried powder of Pediococcus acidilactici and freeze-dried powder of Lactococcus gasseri in a mass ratio of 1:1.

[0045] (5) High-dose group: The mice were fed with a composite bacterial agent at a dosage of 250 mg / kg per day, once in the morning and once in the evening. The composite bacterial agent was a mixture of freeze-dried powder of Pediococcus acidilactici and freeze-dried powder of Lactococcus gasseri in a mass ratio of 1:1.

[0046] During the experiment, the subjects were free to eat and drink. After 2 weeks of administration, thyroid tissue was taken for hormone content determination. Enzyme-linked immunosorbent assay kit was used to detect hormone indicators: thyroid T4 and triiodothyronine T3. The results are shown in Table 3.

[0047] Table 3

[0048] Grouping T4 content (μg / L) T3 (ng / mL) Normal control group 45.87±0.15 1.35±0.06 Negative control group 23.35±0.20 0.64±0.10 Positive control group 47.85±0.17 1.15±0.05 Low dose group 38.14±0.15 0.71±0.03 High dose group 45.11±0.16 1.20±0.06

[0049] It can be seen from the experimental data in Table 3 that feeding the composite bacterial agent prepared by the present invention can significantly restore the T4 and T3 levels of the hypothyroidism mouse model, and the effect of the high-dose group is more obvious. Compared with the normal control group and the positive control group, there is no significant difference between the low-dose group and the high-dose group, and the effects of the low-dose group and the high-dose group are significantly higher than those of the negative control group, indicating that the composite bacterial agent prepared by the present invention can significantly improve the symptoms of hypothyroidism.

[0050] It can be seen from the above embodiments and experimental results that the composite bacterial agent prepared by compounding Pediococcus acidilactici and Lactococcus gestate in the present invention has an obvious effect of lowering blood lipids. During the experiment, it was found that the mental fatigue, slow reaction, and preference for gathering in mice caused by hyperlipidemia were significantly improved. Therefore, the inventors thought that the hypothyroidism condition that may be caused by hyperlipidemia had the same symptoms. Therefore, a hypothyroidism model was constructed and experimental verification was carried out. The present invention unexpectedly found that the prepared composite bacterial agent also had a significant improvement effect on hypothyroidism.

[0051] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composite bacterial agent, characterized in that: The composite bacterial agent is prepared by compounding freeze-dried powder of Pediococcus acidilactici and freeze-dried powder of Lactococcus garvieae, wherein the active bacteria in the freeze-dried powder of Pediococcus acidilactici are Pediococcus acidilactici CICC 20720, and the active bacteria in the freeze-dried powder of Lactococcus garvieae are Lactococcus garvieae CICC 21038.

2. The composite bacterial agent according to claim 1, characterized in that The mass ratio of freeze-dried powder of Pediococcus acidilactici to freeze-dried powder of Lactococcus garvieae in the composite bacterial agent is (1-3): (1-3).

3. The composite bacterial agent according to claim 1, characterized in that The lactic acid Pediococcus lyophilized powder is obtained by fermenting lactic acid Pediococcus CICC 20720 to obtain lactic acid Pediococcus fermentation liquid; centrifuging the lactic acid Pediococcus fermentation liquid, collecting the supernatant, and freeze-drying the supernatant; The Lactococcus gasseri freeze-dried powder is obtained by fermenting Lactococcus gasseri CICC 21038 to obtain Lactococcus gasseri fermentation liquid; centrifuging the Lactococcus gasseri fermentation liquid, collecting the supernatant, and freeze-drying the supernatant.

4. Use of the composite bacterial agent according to any one of claims 1 to 3 in the preparation of a drug for lowering blood lipids.

5. Use of the composite bacterial agent according to any one of claims 1 to 3 in the preparation of a medicament for treating hypothyroidism.

6. A drug for lowering blood lipids, characterized in that: The invention comprises the composite bacterial agent according to any one of claims 1 to 3.

7. A drug for improving hypothyroidism, characterized in that: The invention comprises the composite bacterial agent according to any one of claims 1 to 3.

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