A composite probiotic for lowering cholesterol and preparation method thereof

Abstract

The present invention relates to a composite probiotic agent for reducing cholesterol and a preparation method thereof. The composite probiotic agent capable of reducing cholesterol comprises at least two of Lactobacillus johnsonii CICC 6084, Bifidobacterium animalis CICC 6250 and Lactobacillus reuteri CICC 6132. The three probiotics are subjected to high-density anaerobic fermentation, freeze drying and other steps to obtain probiotic freeze-dried bacterial powder. Any two of the three probiotic freeze-dried bacterial powders have significant synergistic effects in reducing serum cholesterol in vitro or in vivo compared with a single bacterial agent. The composite bacterial agent composed of the three probiotic freeze-dried bacterial powders also has significant synergistic effects compared with any two of them. The obtained composite probiotic agent is applied to the preparation of cholesterol-reducing agents, which has great application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a composite probiotic agent for reducing cholesterol and a preparation method thereof. Background Art

[0002] Cholesterol is an unsaturated sterol and the main sterol in the human body. Its physiological function is mainly used to synthesize cell plasma membranes, steroid hormones and bile acids. In addition to food, 90% of endogenous cholesterol in the human body is synthesized by acetyl coenzyme A in the liver, and is also restricted by the amount of cholesterol in the fifteen types of cholesterol. Under normal circumstances, almost all of the body's cholesterol is synthesized by the liver and the distal small intestine, so people pay less attention to the study of hypocholesterolemia. Plasma cholesterol is mainly present in low-density lipoprotein (LDL), followed by high-density lipoprotein cholesterol (HDL) and very low-density lipoprotein (VLDL), and the least content is in chylomicrons (CM). Although cholesterol is an important nutrient in the human body, its high content will have adverse effects on health, such as hypercholesterolemia, atherosclerosis and cardiovascular disease.

[0003] Statins are the most commonly used cholesterol-lowering drugs in clinical practice, but long-term use can cause side effects such as myalgia, muscle weakness, and increased risk of diabetes. Therefore, the development of alternatives with similar efficacy to cholesterol-lowering drugs such as statins and without side effects has become a hot topic in current research and development. Probiotics, as a class of active microorganisms that can have a beneficial effect on host health after the body ingests a certain amount from the diet, include lactic acid bacteria, streptococci, and bifidobacteria. In addition to the common probiotic effects of regulating the balance of intestinal flora, activating immunity, and nutrition, some probiotics such as Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium, and Lactobacillus rhamnosus, as well as fermented products containing probiotics, have the effect of lowering cholesterol and regulating blood lipids. However, there are currently limited reports on cholesterol-lowering probiotics and their composite bacterial agents, and the development of cholesterol-lowering probiotics and their composite bacterial agents is of great significance. Summary of the invention

[0004] In view of the technical problems in the prior art such as lack of alternatives to cholesterol drugs with less side effects and lack of composite probiotic agents that can remove cholesterol both in vivo and in vitro, a composite probiotic agent that can lower cholesterol and a preparation method thereof are provided.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The invention provides a composite probiotic agent capable of lowering cholesterol. The composite probiotic agent comprises at least two of Lactobacillus johnsonii CICC 6084, Bifidobacterium animalis CICC 6250 and Limosilactobacillus reuteri CICC 6132.

[0007] Preferably, the composite probiotic agent comprises Lactobacillus johnsonii CICC 6084 and Bifidobacterium animalis CICC6250, Lactobacillus johnsonii CICC 6084 and Lactobacillus reuteri CICC 6132, Bifidobacterium animalis CICC 6250 and Lactobacillus reuteri CICC 6132.

[0008] Preferably, the composite probiotic agent comprises Lactobacillus johnsonii CICC 6084, Bifidobacterium animalis CICC6250 and Lactobacillus reuteri CICC 6132.

[0009] Preferably, the composite probiotic agent is freeze-dried bacterial powder.

[0010] Preferably, the number of viable bacteria in the freeze-dried bacterial powder is 1.0×10 11 CFU / g and above.

[0011] Preferably, the viable count of Lactobacillus johnsonii CICC 6084 in the freeze-dried bacterial powder is 3.2×10 11 CFU / g, and the viable count of Lactobacillus reuteri CICC 6132 was 3.1×10 11 CFU / g, the number of viable bacteria of Bifidobacterium animalis CICC 6250 was 2.9×10 11 CFU / g.

[0012] Preferably, the composite probiotic agent further comprises a freeze-drying protectant.

[0013] Preferably, the lyoprotectant comprises any one of trehalose, maltodextrin, sucrose and skimmed milk powder.

[0014] Preferably, the freeze-drying protective agent components are: 60 g / L trehalose, 50 g / L maltodextrin, 40 g / L sucrose, 50 g / L skim milk powder and solvent purified water.

[0015] The present invention also provides a method for preparing the probiotic composite bacterial agent, the method comprising the following steps:

[0016] (1) After activating and expanding the probiotic strains, the strains are inoculated into a liquid culture medium for high-density anaerobic fermentation;

[0017] (2) After the fermentation liquid is centrifuged, the bacterial sludge is collected;

[0018] (3) mixing the bacterial sludge collected by centrifugation with a freeze-drying protective agent, and freeze-drying the mixture to obtain freeze-dried bacterial powder, i.e., a probiotic bacterial agent;

[0019] (4) The different probiotic agents are fully mixed according to the proportion to obtain a composite probiotic agent.

[0020] Preferably, in step (1), Lactobacillus johnsonii CICC 6084 and Lactobacillus reuteri CICC 6132 are inoculated in MRS liquid culture medium, and Bifidobacterium animalis strain CICC 6250 is inoculated in RCM liquid culture medium.

[0021] Preferably, in step (1), the inoculation amount is 1% by volume.

[0022] Preferably, in step (2), the centrifugation conditions are 4°C, 8000 r / min, and 10 min.

[0023] Preferably, in step (3), the mixing ratio of the bacterial sludge to the lyophilization protective agent is 1:1 by weight.

[0024] The present invention also provides the use of the probiotic composite bacterial agent in the preparation of a cholesterol-lowering product

[0025] Preferably, the product is selected from medicines.

[0026] Compared with the prior art, the present invention has achieved the following beneficial technical effects:

[0027] (1) Through in vitro screening among known commercial probiotic strains, three probiotic strains with significant cholesterol-lowering function were obtained.

[0028] (2) The three probiotic strains obtained were subjected to high-density fermentation, freeze-drying and other steps to prepare probiotic freeze-dried powder, and the number of viable bacteria in the freeze-dried powder can reach 10 11 CFU / g and above.

[0029] (3) Through in vivo verification in a high cholesterol rat model, it was found that a combination of two probiotics had a significant synergistic effect in lowering serum cholesterol compared with a single probiotic, and a combination of three probiotics had a significant synergistic effect compared with a combination of two probiotics. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0031] Unless otherwise specified, the experimental methods involved in the examples are all conventional methods.

[0032] Strains:

[0033] Lactobacillus johnsonii, strain collection numbers CICC 10861, CICC 6084, CICC 6252, CICC 10862;

[0034] Bifidobacterium animalis, strain deposit numbers CICC 21711, CICC6174, CICC 21713, CICC 6250;

[0035] Limosilactobacillus reuteri, strain deposit numbers CICC 6126, CICC 6132, CICC 6127, CICC 6124, CICC 6128, CICC 6125;

[0036] The above strains were purchased from China Industrial Microbiology Culture Collection Center.

[0037] Culture medium:

[0038] MRS medium: casein peptone (trypsin digestion) 10.0 g, beef extract powder 10.0 g, yeast extract powder 5.0 g, glucose 20.0 g, Tween 80 1.0 g, sodium acetate 5.0 g, ammonium citrate tribasic 2.0 g, K2HPO4 2.0 g, MgSO4·7H2O 0.2 g, MnSO4·H2O

[0039] 0.05g, agar 15.0g, distilled water 1000.0mL, pH 6.2-6.5, 121℃, sterilize for 15min.

[0040] RCM medium: beef extract powder 10.0 g, peptone 5.0 g, yeast extract powder 3.0 g, glucose 5.0 g, soluble starch 1.0 g, NaCl 5.0 g, NaAc 3.0 g, L-cysteine ​​hydrochloride 0.5 g, agar 15.0 g, distilled water 1000.0 mL, pH 6.8, 121°C, sterilization for 15 min.

[0041] Feed (for rats):

[0042] Basic feed formula (w / w): corn 26%, soybean meal 22%, flour 20%, wheat bran 16%, rice flour 10%, fish meal 2%, bone meal 2.5%, salt 1%, lysine 0.2%, methionine 0.2%, vitamin AD 0.1%.

[0043] High cholesterol feed formula (w / w): 50% basic feed, 15% lard, 20% sucrose, 5% egg yolk powder, 5% casein, 2% salt, and 3% cholesterol.

[0044] Example 1 Determination of cholesterol removal in vitro by Lactobacillus johnsonii

[0045] High-pressure sterilized MRS liquid medium was added with bile salt (3 mg / mL), sodium thioglycolate (0.2%, w / v) and cholesterol (120 μg / mL) that were sterilized by filtration. Activated Lactobacillus johnsonii strains CICC 10861, CICC 6084, CICC 6252, and CICC10862 were inoculated into the above medium at a 1% inoculum amount, cultured anaerobically at 37°C for 24 hours, and centrifuged (4000 r / min, 4°C, 10 min) to obtain the supernatant. Each group of experiments was repeated 3 times, and the mean was calculated. The cholesterol content in the supernatant was determined by the o-phthalaldehyde method, and the cholesterol removal rate was calculated according to formula (I).

[0046] (I) (D1 and D2 are the cholesterol contents in the supernatant before and after fermentation, respectively).

[0047] The results are shown in Table 1. Among them, Lactobacillus johnsonii CICC 6084 has a higher cholesterol removal rate, up to 43.8% ± 0.023 (**, p < 0.01), and the cholesterol removal rates of other strains of Lactobacillus johnsonii in vitro are lower or almost negligible. Therefore, in the end, Lactobacillus johnsonii CICC 6084 was selected as the constituent strain of the subsequent composite bacterial agent.

[0048] Table 1 In vitro cholesterol removal rate of Lactobacillus johnsonii

[0049] Group Cholesterol removal rate CICC 10861 1.2％±0.018 CICC 6084 43.8％±0.023** CICC 6252 8.5％±0.022 CICC 10862 1.7％±0.034

[0050] Example 2 Determination of cholesterol removal by animal bifidobacterium in vitro

[0051] High-pressure sterilized RCM liquid culture medium was added with filtered sterilized bile salt (3 mg / mL), sodium thioglycolate (0.2%, w / v) and cholesterol (120 μg / mL). Activated animal Bifidobacterium strains CICC 21711, CICC 6174, CICC 21713, CICC6250 were inoculated into the above culture medium at a 1% inoculum amount, cultured anaerobically at 37°C for 24 hours, and centrifuged (4000r / min, 4°C, 10min) to obtain the supernatant. Each group of experiments was repeated 3 times, and the mean was calculated. The cholesterol content in the supernatant was determined by the o-phthalaldehyde method, and the cholesterol removal rate was calculated according to formula (I).

[0052] The results are shown in Table 2. Animal Bifidobacterium CICC 6250 has a higher cholesterol removal rate, up to 37.2% ± 0.014 (**, p < 0.01), and the cholesterol removal rates of other strains of animal Bifidobacterium in vitro are lower or almost negligible. Therefore, finally, animal Bifidobacterium CICC 6250 was selected as the constituent strain of the subsequent composite bacterial agent.

[0053] Table 2 In vitro cholesterol removal rate of animal Bifidobacterium

[0054] Group Cholesterol removal rate CICC 21711 1.3％±0.019 CICC 6174 9.3％±0.015 CICC 21713 1.5％±0.026 CICC 6250 37.2％±0.014**

[0055] Example 3 Determination of cholesterol removal in vitro by Lactobacillus reuteri

[0056] High-pressure sterilized MRS liquid culture medium was added with filtered sterilized bile salt (3 mg / mL), sodium thioglycolate (0.2%, w / v) and cholesterol (120 μg / mL). Activated Lactobacillus reuteri strains CICC 6126, CICC 6132, CICC 6127, CICC6124, CICC 6128, CICC 6125 were inoculated into the above culture medium at a 1% inoculum amount, cultured anaerobically at 37°C for 24 hours, and centrifuged (4000r / min, 4°C, 10min) to obtain the supernatant. Each group of experiments was repeated 3 times, and the mean was calculated. The cholesterol content in the supernatant was determined by the o-phthalaldehyde method, and the cholesterol removal rate was calculated according to formula (I).

[0057] The results are shown in Table 3. Among them, Lactobacillus reuteri CICC 6132 has a higher cholesterol removal rate, up to 40.3% ± 0.012 (**, p < 0.01), and the cholesterol removal rates of other strains of animal Bifidobacterium in vitro are low or almost negligible. Therefore, in the end, Lactobacillus reuteri CICC 6132 was selected as the constituent strain of the subsequent composite bacterial agent.

[0058] Table 3 In vitro cholesterol removal rate of Lactobacillus reuteri

[0059] Group Cholesterol removal rate CICC 6126 2.1％±0.005** CICC 6132 40.3％±0.012** CICC 6127 1.6％±0.019 CICC 6124 5.2％±0.023 CICC 6128 1.9％±0.017 CICC 6125 1.4％±0.031

[0060] Example 4 Preparation of bacterial agent

[0061] After Lactobacillus johnsonii CICC 6084 and Lactobacillus reuteri CICC 6132 were activated and expanded, they were inoculated into 2L MRS liquid culture medium at a volume ratio of 1%, and high-density anaerobic fermentation was carried out in a 5L fully automatic fermenter. The temperature was controlled at 37°C, the pH of the fermentation liquid was 6.5, the stirring speed was 180r / min, and the fermentation was carried out for 26 hours to obtain the fermentation liquid. The fermentation liquid was placed in a centrifuge and centrifuged at 4°C and 8000r / min for 10 minutes to collect the bacterial mud. The bacterial mud collected by centrifugation was mixed evenly with the freeze-drying protective agent at a mass ratio of 1:1, placed in a freeze dryer, and freeze-dried to obtain the freeze-dried bacterial powder A1 of Lactobacillus johnsonii CICC6084 and the freeze-dried bacterial powder C1 of Lactobacillus reuteri CICC 6132. The number of viable bacteria in the freeze-dried powders A1 and C1 was 3.2×10 11 CFU / g, 3.1×10 11 CFU / g.

[0062] After the animal Bifidobacterium strain CICC 6250 was activated and expanded, it was inoculated into 2LRCM liquid culture medium at a volume ratio of 1%, and high-density anaerobic fermentation was carried out in a 5L fully automatic fermenter. The temperature was controlled at 37°C, the pH of the fermentation liquid was 6.8, the stirring speed was 180r / min, and the fermentation was carried out for 26 hours to obtain the fermentation liquid. The fermentation liquid was placed in a centrifuge and centrifuged at 4°C and 8000r / min for 10 minutes to collect the bacterial mud. The bacterial mud collected by centrifugation was mixed evenly with the freeze-drying protective agent at a mass ratio of 1:1, placed in a freeze dryer, and freeze-dried to obtain the freeze-dried bacterial powder B1 of animal Bifidobacterium strain CICC 6250. The number of viable bacteria in the freeze-dried bacterial powder B1 was 2.9×10 11 CFU / g.

[0063] The freeze-drying protective agent components include: 60 g / L trehalose, 50 g / L maltodextrin, 40 g / L sucrose, 50 g / L skim milk powder, and the solvent is purified water.

[0064] Preparation of composite bacterial agents A1B1, A1C1 and B1C1: The freeze-dried bacterial powders A1, B1 and C1 prepared above were fully mixed at a ratio of 1:1 (w / w) to obtain composite bacterial agents A1B1, A1C1, A1C2, B1C1 and B1C2.

[0065] Preparation of composite bacterial agent A1B1C1: The freeze-dried bacterial powders A1, B1 and C1 prepared above were fully mixed at a ratio of 1:1:1 (w / w / w) to obtain composite bacterial agent A1B1C1.

[0066] Example 5 Determination of the effect of bacterial agent on cholesterol removal in vivo

[0067] A total of 90 rats were randomly divided into 9 groups, 10 rats in each group, and fed with different diets, including normal control group (ND): given basal diet; high cholesterol diet group (HFD): given high cholesterol diet; high cholesterol diet (HFD) + probiotics group: given high cholesterol diet + probiotics (1 mL / 100 g body weight by intragastric administration, the probiotics prepared in Example 4 were prepared with physiological saline to 1.5 × 10 9 CFU / ml), and fed continuously for 6 weeks. Before the end of the experiment, after fasting for 12 hours but not water, blood was collected from the orbits, and serum was separated from the obtained blood to determine the content of LDL cholesterol, HDL cholesterol and total cholesterol in the serum and the ratio of HDL / LDL. The results are shown in Table 4. Compared with the normal control group (ND) and the high cholesterol diet group (HFD), the probiotics intervention group can significantly remove total cholesterol and LDL-cholesterol in the body, among which the composite bacteria A1B1, A1C1 and B1C1 are better than single bacteria in removing total cholesterol and LDL-cholesterol in the body; and the composite bacteria A1B1C1 is the best in removing total cholesterol and LDL-cholesterol in the body compared with single bacteria and composite bacteria A1B1, A1C1 and B1C1, and there is a synergistic effect between the composite bacteria.

[0068] Table 4 Determination of cholesterol removal by bacterial agents in vivo

[0069]

[0070] Note: Compared with the normal group, ** P<0.05; compared with the model group, # P<0.05; compared with single bacterial agent, a P<0.05; compared with the two kinds of bacterial compound agents, b P<0.05.

[0071] The present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned steps, that is, it does not mean that the present invention must rely on the above-mentioned steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A composite probiotic agent for lowering cholesterol, characterized in that: The composite probiotic bacterial agent comprises at least two of Lactobacillus johnsonii CICC 6084, Bifidobacterium animalis CICC 6250, and Limosilactobacillus reuteri CICC 6132.

2. The composite probiotic agent according to claim 1, characterized in that: The composite probiotic bacterial agent comprises Lactobacillus johnsonii CICC 6084, Bifidobacterium animalis CICC 6250 and Lactobacillus reuteri CICC 6132.

3. The composite probiotic agent according to any one of claims 1 to 2, wherein the composite probiotic agent is freeze-dried bacterial powder.

4. The composite probiotic agent according to claim 3, wherein the number of viable bacteria in the freeze-dried bacterial powder is 1.0×10 11 CFU / g or more; and / or, the number of viable Lactobacillus johnsonii CICC 6084 in the freeze-dried bacterial powder is 3.2×10 11 CFU / g, and the viable count of Lactobacillus reuteri CICC 6132 was 3.1×10 11 CFU / g, the number of viable bacteria of Bifidobacterium animalis CICC 6250 was 2.9×10 11 CFU / g.

5. The composite probiotic preparation according to any one of claims 1 to 2, further comprising a freeze-drying protective agent.

6. The composite probiotic agent according to claim 5, wherein the freeze-drying protective agent comprises any one of trehalose, maltodextrin, sucrose and skim milk powder.

7. The composite probiotic agent according to claim 6, wherein the freeze-drying protective agent component is: 60 g / L trehalose, 50 g / L maltodextrin, 40 g / L sucrose, 50 g / L skim milk powder and solvent purified water.

8. A method for preparing the probiotic composite agent according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: (1) After activating and expanding the probiotic strains, the strains are inoculated into a liquid culture medium for high-density anaerobic fermentation; (2) After the fermentation liquid is centrifuged, the bacterial sludge is collected; (3) Evenly mixing the bacterial sludge collected by centrifugation with a freeze-drying protective agent, and freeze-drying to obtain freeze-dried bacterial powder, i.e., a probiotic bacterial agent; (4) The different probiotic agents are fully mixed according to the proportion to obtain a composite probiotic agent.

9. The preparation method according to claim 8, characterized in that: In the step (1), Lactobacillus johnsonii CICC6084 and Lactobacillus reuteri CICC 6132 are inoculated in MRS liquid culture medium, and Bifidobacterium animalis strain CICC6250 is inoculated in RCM liquid culture medium, and the inoculation amount is 1% by volume; In step (2), the centrifugation conditions are 4° C., 8000 r / min for 10 min; in step (3), the mixing ratio of the bacterial sludge and the lyophilization protective agent is 1:1 by weight.

10. Use of the probiotic composite bacterial preparation according to any one of claims 1 to 7 or the probiotic composite bacterial preparation obtained by the preparation method according to any one of claims 8 to 9 in the preparation of a cholesterol-lowering drug.

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