CN119799571A - A strain of animal bifidobacterium with lipid-lowering and anti-aging effects and its application in enhancing the efficacy of transformed licorice - Google Patents
A strain of animal bifidobacterium with lipid-lowering and anti-aging effects and its application in enhancing the efficacy of transformed licorice Download PDFInfo
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Abstract
本发明公开了一株同时具有降脂与抗衰作用的动物双歧杆菌及其转化甘草增效的应用，属于微生物技术及医药技术领域。该菌株能够发酵甘草提取物，提升甘草次酸、甘草次酸3‑O‑葡萄糖醛酸苷、5‑O‑甲基染料木素等活性成分的含量从而增强其功效，具体表现为：(1)抑制高脂饮食下机体体重增加并降低血脂四项；(2)降低机体衰老相关分泌表型IL‑1β、IL‑6水平；(3)减少肝脏β‑半乳糖苷酶含量；(4)减少肝脏脂质堆积引起的损伤；(5)促进脂质代谢基因MGL、HSL、ATGL、PPAR‑α的表达，抑制脂质合成基因PPAR‑γ、Fasn的表达。因此动物双歧杆菌乳亚种CCFM1363与甘草提取物联合应用，具有广阔的降脂与抗衰老产品开发潜力。
The present invention discloses an animal bifidobacterium having both lipid-lowering and anti-aging effects and its application of transforming licorice to enhance synergy, belonging to the fields of microbial technology and pharmaceutical technology. The strain can ferment licorice extract, improve the content of active ingredients such as glycyrrhetinic acid, glycyrrhetinic acid 3-O-glucuronide, 5-O-methyl genistein, etc., so as to enhance its efficacy, specifically manifested as: (1) inhibiting body weight gain under high-fat diet and reducing four items of blood lipids; (2) reducing the level of IL-1β and IL-6, the secretory phenotype associated with body aging; (3) reducing liver β-galactosidase content; (4) reducing damage caused by liver lipid accumulation; (5) promoting the expression of lipid metabolism genes MGL, HSL, ATGL, PPAR-α, and inhibiting the expression of lipid synthesis genes PPAR-γ and Fasn. Therefore, animal bifidobacterium lactis subspecies CCFM1363 and licorice extract are used in combination, with broad potential for the development of lipid-lowering and anti-aging products.
Description
Technical Field
The invention relates to bifidobacterium animalis subspecies CCFM1363 and an application of anti-aging and lipid-lowering licorice prepared by the same in synergy of post-fermentation primordial and transformed licorice, belonging to the technical field of microorganisms and medicines.
Background
With the continued increase in aging population, anti-aging research has become a central issue in the biomedical and microbiological arts. Aging is a complex biological process commonly affected by multiple factors, and not only involves multiple layers of oxidative stress, chronic inflammation, metabolic function decline, immune function decline and the like, but also directly leads to gradual decline of body functions, and significantly increases the risk of suffering from chronic diseases, such as diabetes, cardiovascular diseases, neurodegenerative diseases and the like. Notably, unhealthy diets and lifestyles play an important role in them, they induce metabolic disorders in the body, promote poor accumulation of lipids, and thereby induce a series of diseases such as hyperlipidemia, cardiovascular diseases, fatty liver, and diabetes. These disease states in turn exacerbate the body's chronic inflammation, oxidative stress and DNA damage, forming a vicious circle, further accelerating the aging process.
In recent years, materials of homology to medicine and food and microbial metabolites thereof have received attention for their safety and effectiveness. Licorice is a common medicine and food source, has a wide application range, and related researches find that the licorice also has good effects on antioxidation, anti-inflammatory, anti-tumor, antivirus and the like, and in addition, the application potential of licorice extract is widely focused on anti-aging aspects. Triterpenoid saponins in licorice are considered as the main drug effect substances, and mainly comprise glycyrrhizic acid (GL), mono-glucuronic acid Glycyrrhetinic Acid (GAMG) and Glycyrrhetinic Acid (GA), wherein the content of glycyrrhizic acid is the highest.
Research shows that glycyrrhizic acid is inferior to active small molecules such as single glucuronic acid glycyrrhetinic acid and glycyrrhetinic acid in absorption and physiological activity. It is difficult to obtain a direct effect by directly using licorice extract, and the efficacy is poor when eating licorice extract. Therefore, how to promote the efficacy of the licorice extract is a key to the development of the licorice extract as a lipid-lowering and anti-aging related product.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the prior art, and provides the application of bifidobacterium animalis subsp (Bifidobacterium animalis subsp. Lactis) CCFM1363 in enhancing the efficacy of licorice extract in resisting aging and reducing blood fat.
The invention also aims to provide a fermented licorice extract and a composition for improving lipid-lowering and anti-aging effects.
It is still another object of the present invention to provide a method for promoting an increase in glycyrrhetinic acid content in a licorice extract.
The above object of the present invention is achieved by the following technical scheme:
The invention provides a bifidobacterium animalis subspecies (Bifidobacterium animalis subsp.lactis) CCFM1363, wherein the bifidobacterium animalis subspecies CCFM1363 has been deposited in the microorganism strain deposit center of Guangdong province in 12 months 06 of 2023, and the deposit number is GDMCC No:64111.
In one embodiment of the invention, the bifidobacterium animalis subspecies CCFM1363 of the invention have rounded, convex or lenticular colonies, are slightly white, opaque and have a soft surface that is smooth to a viscous liquid state after 48 hours of culture on MRS medium.
In one embodiment of the invention, the fermentation of the licorice extract by using the bifidobacterium animalis subspecies lactis CCFM1363 can significantly improve the content of glycyrrhetinic acid and promote the licorice extract to exert lipid-lowering and anti-aging effects. The method is characterized by comprising the following steps of (1) controlling the weight increase of an obese organism, (2) reducing the secretion phenotype level related to the aging of the organism, (3) reducing the content of beta-galactosidase in the organism, (4) inhibiting the accumulation and pathological changes of liver fat of the organism, (5) reducing the blood lipid level of the organism, and (6) effectively improving the expression of the lipid metabolism related genes MGL, HSL, ATGL, PPAR-alpha of the organism and reducing the expression of the lipid synthesis related genes PPAR-gamma and Fasn.
The invention also provides a metazoan prepared from the bifidobacterium animalis subspecies lactis CCFM1363, which is obtained by fermenting licorice extracts by the bifidobacterium animalis subspecies CCFM 1363.
In one embodiment of the present invention, the method for preparing the metagen includes the steps of:
(1) Activating bifidobacterium animalis subspecies of milk CCFM1363 to obtain bacterial liquid;
(2) Inoculating the bacterial liquid of the step (1) into a fermentation base material containing the licorice extract, and fermenting for 48 hours in an anaerobic environment, wherein the number of inoculated viable bacteria is 5 multiplied by 10 7 CFU/mL;
(3) After fermentation, the viable count is 1X 10 9 CFU/mL, and the fermentation broth is subjected to heat treatment and then high-pressure homogenization to obtain the microbial inoculum.
In one embodiment of the invention, step (1) is streaking bifidobacterium animalis subspecies of milk CCFM1363 on MRS solid medium, culturing the plates in anaerobic environment (preferably 37 ℃ inverted culturing for 48 h), and then picking single colony to be inoculated into MRS liquid medium (preferably 5 mL) for culturing (preferably 37 ℃ culturing for 48 h) to obtain bacterial liquid.
In one embodiment of the present invention, the concentration of licorice extract in the fermentation base containing licorice extract of step (2) is 5-15mg/mL (preferably 10 mg/mL).
In one embodiment of the present invention, the inoculum size of the bacterial liquid in the step (2) is 1-8% (v/v).
In one embodiment of the invention, the fermentation in step (2) is a 30-40 ℃ constant temperature fermentation for 24-72 hours (preferably 37 ℃ constant temperature fermentation for 48 hours).
In one embodiment of the present invention, the heat treatment conditions in step (3) are 60 to 70 ℃ for 25 to 35min (preferably 65 ℃ for 30 min).
In one embodiment of the present invention, the high pressure homogenization conditions in step (3) are 300 to 1500bar, and the strain is circulated 4 to 8 times (preferably 1200bar,6 times).
The invention also provides a synbiotic preparation prepared from the bifidobacterium animalis subspecies lactis CCFM 1363.
In one embodiment of the present invention, the method for preparing the synbiotic formulation includes the steps of:
(1) Activating bifidobacterium animalis subspecies of milk CCFM1363 to obtain bacterial liquid;
(2) Inoculating the bacterial liquid of the step (1) into an MRS culture medium, and inoculating the bacterial liquid with the viable count of 5 multiplied by 10 7 CFU/mL, and culturing in an anaerobic environment until the bacterial strain grows to the logarithmic phase to obtain the bifidobacterium animalis subspecies liquid;
(3) Collecting thallus from liquid culture medium by centrifugation, mixing with lyoprotectant (skim milk powder 12%), and lyophilizing to obtain lyophilized powder of strain;
(4) Mixing the lyophilized powder of strain and Glycyrrhrizae radix extract at a certain dose.
In one embodiment of the invention, step (1) is streaking bifidobacterium animalis subspecies of milk CCFM1363 on MRS solid medium, culturing the plates in anaerobic environment (preferably 37 ℃ inverted culturing for 48 h), and then picking single colony to be inoculated into MRS liquid medium (preferably 5 mL) for culturing (preferably 37 ℃ culturing for 48 h) to obtain bacterial liquid.
In one embodiment of the present invention, the concentration of licorice extract in the fermentation base containing licorice extract of step (2) is 5-15mg/mL (preferably 10 mg/mL).
In one embodiment of the present invention, the inoculum size of the bacterial liquid in the step (2) is 1-8% (v/v).
In one embodiment of the invention, the fermentation in step (2) is a 30-40 ℃ constant temperature fermentation for 24-72 hours (preferably 37 ℃ constant temperature fermentation for 48 hours).
In one embodiment of the present invention, the heat treatment conditions in step (3) are 60 to 70 ℃ for 25 to 35min (preferably 65 ℃ for 30 min).
In one embodiment of the present invention, the high pressure homogenization conditions in step (3) are 300 to 1500bar, and the strain is circulated 4 to 8 times (preferably 1200bar,6 times).
The invention provides a product containing the bifidobacterium animalis subspecies lactis CCFM1363 and/or its metazoan and/or its synbiotic preparation.
In one embodiment of the invention, the product is a food, pharmaceutical or health product.
In one embodiment of the invention, the product is a lyophilized powder of the metazoan or synbiotic formulation, or a reconstituted formulation containing the metazoan or synbiotic formulation lyophilized powder.
The invention provides a method for degrading glycyrrhizic acid, which comprises the step of adding bifidobacterium animalis subspecies of milk CCFM1363 into a culture medium containing the glycyrrhizic acid for fermentation.
The invention provides a method for preparing glycyrrhetinic acid, which comprises the step of adding bifidobacterium animalis subspecies of milk CCFM1363 into a culture medium containing glycyrrhizic acid for fermentation.
The invention provides a method for enhancing medicinal efficacy of liquorice, which is any one of the following steps:
(1) Adding the bifidobacterium animalis subspecies CCFM1363 into a culture medium containing liquorice products for fermentation;
(2) Mixing said bifidobacterium animalis subspecies lactis CCFM1363 with a licorice preparation.
In one embodiment of the invention, the licorice preparation comprises licorice or licorice extract.
The invention also provides application of the bifidobacterium animalis subspecies lactis CCFM1363 and/or its metazoan and/or its synbiotic preparation in preparation of lipid-lowering and/or anti-aging medicines.
In one embodiment of the invention, the anti-aging agent is used to alleviate aging-related disorders.
In one embodiment of the invention, the aging-related disorder comprises oxidative stress damage, chronic inflammation, liver lipid accumulation, hyperlipidemia or metabolic syndrome.
In one embodiment of the invention, the pharmaceutical product comprises at least one of the following functions:
(1) Reducing weight gain in a high fat diet;
(2) Reducing the level of IL-1 beta and IL-6, which are secretion phenotypes related to the aging of the organism;
(3) Reducing the content of beta-galactosidase in the organism;
(4) Inhibit liver fat accumulation and damage;
(5) Reducing blood lipid level of organism;
(6) Effectively improves the expression of the organism lipid metabolism related gene MGL, HSL, ATGL, PPAR-alpha and reduces the expression of the lipid synthesis related genes PPAR-gamma and Fasn.
The invention also provides an anti-aging medicine containing bifidobacterium animalis subspecies lactis CCFM1363 and licorice extract.
The invention also provides a medicine with the anti-aging effect, which contains a product obtained by fermenting the licorice extract by using the bifidobacterium animalis subspecies lactis CCFM 1363.
In one embodiment of the invention, the bifidobacterium animalis subspecies lactis CCFM1363 is added in an amount of at least 1X 10 9 CFU/mL in the product.
In one embodiment of the invention, the pharmaceutical product contains bifidobacterium animalis subspecies lactis CCFM1363, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
The beneficial effects are that:
The bifidobacterium animalis subspecies CCFM1363 has the capability of converting glycyrrhizic acid, the degradation rate of glycyrrhizic acid reaches 37.42% after fermentation in a fermentation base material rich in licorice extract, and active substances such as glycyrrhetinic acid, glycyrrhetinic acid 3-O-glucuronide, apiose isoliquiritigenin and the like are generated.
The bifidobacterium animalis subspecies CCFM1363 can promote the efficacy of the licorice extract to be exerted, and can relieve the degree of weight increase and the harm to liver functions under high-fat diet, improve the lipid metabolism capacity of the licorice extract and reduce the anti-aging capacity of organisms.
The bifidobacterium animalis subspecies CCFM1363 and the licorice extract are utilized to produce the product with improved anti-aging efficacy, the glycyrrhizic acid extract can be used as the raw material in the production process, the efficacy of the licorice extract is promoted, and the process has the advantages of safety, high efficiency, low cost, mild reaction and the like, and is suitable for large-scale industrial production. Therefore, the application of the bifidobacterium animalis subspecies CCFM1363 to the licorice extract-containing product has great application prospect and application foundation.
Preservation of biological materials
Bifidobacterium animalis subspecies (Bifidobacterium animalis subsp. Lacti) CCFM1363, taxonomic designation Bifidobacterium animalis subsp. Lacti, deposited at the collection of microorganisms and cell cultures, guangdong province, at day 12, 2023, accession number GDMCC No:64111, accession number Guangzhou, martyr, college 100, building 59.
Drawings
FIG. 1 is a liquid chromatogram of 0h and 48h of extract of fermented Glycyrrhrizae radix of Bifidobacterium animalis subspecies of Lactobacillus CCFM1363, and a liquid chromatogram of standard products of glycyrrhizic acid and glycyrrhetinic acid;
FIG. 2 weight gain of mice of different groups;
FIG. 3 levels of IL-1. Beta. And IL-6, a senescence-associated secretion phenotype of different groups of mice;
FIG. 4 beta-galactosidase content of different groups of mice;
FIG. 5H & E stained tissue sections of different groups of mice;
FIG. 6 liver oil red O stained tissue sections of different groups of mice;
FIG. 7 shows comparison of four blood lipid indicators TG, TC, LDL-c and HDL-c of different groups of mice;
FIG. 8 comparison of ALT and AST indicators for different groups of mice;
FIG. 9 expression of genes related to fat metabolism in mice of different groups.
", Indicates significant differences from the Model group (P < 0.05), and", indicates extremely significant differences from the Model group (P < 0.01). .
Detailed Description
The bifidobacterium animalis subspecies (Bifidobacterium animalis subsp.lacti) CCFM1363 used in the invention is deposited in the microorganism strain collection of Guangdong province at 12/06 of 2023, the deposition number is GDMCC No:64111, and the deposition address is No. 59 building 5 of the No. 100 university in Guangzhou martyr.
The C57BL/6J mice referred to in the examples below were purchased from Beijing Vietnam Liwa.
The following examples relate to the following media:
MRS liquid culture medium comprises 5.0g/L of yeast powder, 10.0g/L of beef extract, 10.0g/L of peptone, 20.0g/L of glucose, 2.0g/L of anhydrous sodium acetate, 2.0g/L of diamine hydrogen citrate, 2.6g/L of dipotassium hydrogen phosphate, 0.25g/L of manganese sulfate monohydrate, 0.5g/L, L g/L of magnesium sulfate heptahydrate-cysteine, 0.5g/L of tween-80 1mL/L and pH value of 6.2-6.4.
MRS solid culture medium comprises 5.0g/L of yeast powder, 10.0g/L of beef extract, 10.0g/L of peptone, 20.0g/L of glucose, 2.0g/L of anhydrous sodium acetate, 2.0g/L of diamine hydrogen citrate, 2.6g/L of dipotassium hydrogen phosphate, 0.25g/L of manganese sulfate monohydrate, 0.5g/L of magnesium sulfate heptahydrate, 0.5g/L of tween-80 1mL/L, L-cysteine, 20.0g/L of agar and pH of 6.2-6.4.
The fermentation base material (g/L) containing Glycyrrhrizae radix extract comprises Glycyrrhrizae radix extract 10g/L, peptone 10g/L, yeast extract 5g/L, beef extract 10g/L, glucose 10g/L, anhydrous sodium acetate 2g/L, citric acid diamine 2g/L、K2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.58g/L、MnSO4·7H2O 0.25g/L、 Tween-80 1g/L, distilled water 1000g/L, L-cysteine 0.5g/L.
The extraction method of Glycyrrhrizae radix extract comprises extracting with water at 90-95deg.C for 45min at 1:10 ratio, and extracting with water at 90-95deg.C for 30min at 1:5 ratio. The yield of the final licorice extract after drying was 12%, i.e., 1g licorice extract corresponds to 8.33g licorice material.
The following examples relate to the preparation of bifidobacterium animalis subspecies lactis-licorice extract synbiotic formulations in the following manner:
(1) Activating bifidobacterium animalis subspecies of milk CCFM1363 to obtain bacterial liquid;
(2) Inoculating the bacterial liquid of the step (1) into an MRS culture medium, and inoculating the bacterial liquid with the viable count of 5 multiplied by 10 7 CFU/mL, and culturing in an anaerobic environment until the bacterial strain grows to the logarithmic phase to obtain the bifidobacterium animalis subspecies liquid;
(3) Collecting thallus from liquid culture medium by centrifugation, mixing with lyoprotectant (skim milk powder 12%), and lyophilizing to obtain lyophilized powder of strain;
(4) Adjusting dosage according to mouse weight (in kg), and mixing to obtain 5×10 9 CFU animal bifidobacterium subspecies lactis CCFM1363+78mg/kg Glycyrrhrizae radix extract.
The following examples relate to the preparation of bifidobacterium animalis subspecies lactis-licorice fermentation metaplasia as follows:
(1) Marking off the bifidobacterium animalis subspecies CCFM1363 on an MRS solid culture medium, culturing for 48 hours at 37 ℃ in an inverted mode, and then picking single bacterial colonies, inoculating the single bacterial colonies into a 5mLMRS liquid culture medium, and culturing for 48 hours at 37 ℃ to obtain bacterial liquid;
(2) Inoculating the bacterial liquid of (1) into the fermentation base material containing the licorice extract, fermenting at 37 ℃ for 48 hours until the viable count reaches 1X 10 9 CFU/mL;
(3) After the fermentation is completed, the fermentation broth is subjected to heat treatment at 65 ℃ for 30min, and then subjected to 1200Bar and 6 times of high-pressure homogenization, thus obtaining the fermentation broth. The resulting metasolution was lyophilized and resuspended in physiological saline according to the required dose (78 mg/kg of licorice extract, calculated as the concentration of licorice extract before fermentation) prior to lavage.
Example 1 isolation screening, identification and preservation of bifidobacterium animalis subspecies lactobacilli CCFM1363
The method comprises the following specific steps:
1. Screening
The method comprises the steps of pretreating a sample from healthy human body excrement, storing the sample in a refrigerator at the temperature of-80 ℃ in 20% glycerol, taking out the sample for thawing, uniformly mixing and absorbing 0.5mL of the sample, adding the sample into 4.5mL of physiological saline, carrying out gradient dilution by the physiological saline, selecting proper gradient diluent to be coated on an MRS solid culture medium, culturing for 48 hours at the temperature of 37 ℃, picking a typical colony of animal bifidobacterium subspecies, carrying out streak purification on the MRS solid culture medium, picking a single colony, transferring the single colony to the MRS liquid culture medium for enrichment, and preserving 30% glycerol to obtain a strain, namely CCFM1363, wherein the typical colony of the animal bifidobacterium subspecies is convex, milky white and smooth.
2. Authentication
Extracting genome of the strain CCFM1363, amplifying and sequencing 16S rDNA of the strain CCFM1363 (the nucleotide sequence of the amplified 16S rDNA of the strain CCFM1363 is shown as SEQ ID NO.1 by Jin Weizhi biotechnology Co., ltd., suzhou), and comparing the sequence with nucleic acid sequence in NCBI, thus showing that the strain is bifidobacterium animalis subspecies, and is named as bifidobacterium animalis subspecies CCFM1363.
3. Preservation of bacterial species
Inoculating Bifidobacterium animalis subspecies CCFM1363 into 5mLMRS liquid culture medium, culturing at 37deg.C for 24 hr under anaerobic condition, collecting 1mL of bacterial liquid, centrifuging at 8000r/min for 3min in sterile centrifuge tube, discarding upper culture medium, and suspending bacterial mud in 30% glycerol solution for preservation at-80deg.C.
4. Preservation of strains
The bifidobacterium animalis subspecies CCFM1363 is preserved in the microorganism strain collection (GDMCC) of Guangdong province at 12/06 of 2023, the preservation number is GDMCC No:64111, and the preservation address is No. 59 building 5 of No. 100 university in Guangzhou martyr.
Example 2 transformation of Glycyrrhiza extract with Bifidobacterium animalis subspecies lactobacilli CCFM1363
1. The fermentation method comprises the following steps:
(1) The bifidobacterium animalis subspecies CCFM1363 of example 1 were streaked on MRS solid medium, the plates were incubated at 37℃for 48h in an inverted position, and single colonies were picked up and inoculated into 5mL of MRS liquid medium and incubated at 37℃for 48h to prepare seed solutions.
(2) Adding 4% (v/v) seed solution of Lactobacillus bifidus subspecies CCFM1363 (viable bacteria inoculation concentration is 5×10 7 CFU/mL) into fermentation base material containing Glycyrrhrizae radix extract, fermenting at 37deg.C for 48 hr at final concentration of Glycyrrhrizae radix extract in culture medium, centrifuging at 8000rpm for 5min in 2mL EP tube, removing thallus, and transferring supernatant into 5mL EP tube.
Diluting the fermentation broth with methanol for 3 times, shaking uniformly, and taking 1mL of sample to pass through a 0.22 mu m organic phase filtering membrane to obtain the liquid to be detected.
The control solution and the sample are sucked for solution sample injection, and detection is carried out according to the following liquid chromatography conditions, namely, using a Waters e2695 liquid chromatograph, a liquid column X Bridge RC18 (250 multiplied by 4.6mm,5 mu m), mobile phase: water (A), 0.1% acetonitrile (B) phosphate, detector: ultraviolet detector (UV) 237nm, column temperature box: 35 ℃, sample injection amount: 10 mu L, elution condition: flow rate 1.0mL/min, gradient elution program of 0min,38% B, 0-3 min, 38-50% B, 3-10 min, 50-52% B, 10-20 min, 52-85% B, 20-30 min, 85-90% B, 30-40 min, 90-38% B. The glycyrrhizic acid peak time is 6.8min, and the glycyrrhetinic acid peak time is 22.6min.
And calculating the content of glycyrrhizic acid and glycyrrhetinic acid in the fermentation broth by using an external standard method. The results are shown in Table 1 and FIG. 1.
TABLE 1 Bifidobacterium animalis subspecies lactis CCFM1363 content of the components after fermentation in Glycyrrhiza extract-containing Medium
| Content of each component (μg/mL) | Fermenting for 0h | Fermenting for 48h |
| Glycyrrhizic acid | 239.45±5.61 | 149.87±4.51 |
| Glycyrrhetinic acid | 0 | 7.15±1.51 |
Note that the glycyrrhetinic acid content was far below the detection lower limit of 100ppm at 0h fermentation.
The result shows that after 48 hours of fermentation is finished, the glycyrrhizic acid content in the fermentation medium is reduced by 37.41% compared with that before fermentation. Therefore, after the licorice extract is fermented by the bifidobacterium animalis subspecies lactis, the licorice extract is greatly converted into other active micromolecular substances such as glycyrrhetinic acid.
EXAMPLE 3 analysis of metabolites of Glycyrrhiza extract before and after fermentation and transformation by bifidobacterium animalis subspecies lactis CCFM1363
1. Metabolite extraction
Transferring 100. Mu.L of liquid sample into an EP tube, adding 400. Mu.L of extracting solution (methanol: acetonitrile=1:1 (v/v)) containing isotope labeled internal standard, vortex mixing for 30s, ultrasonic treatment for 10min (ice water bath), standing at-40 ℃ for 1h, centrifuging the sample at 4 ℃ and 12000rpm (centrifugal force 13800 (x g) and radius 8.6 cm) for 15min, collecting supernatant, centrifuging the supernatant at 4 ℃ and 12000rpm (centrifugal force 13800 (x g) and radius 8.6 cm) for 15min, collecting supernatant, loading the supernatant into a sample bottle for detection, and mixing equal amounts of the supernatant into QC sample for loading detection.
2. On-machine detection
The target compound was chromatographed on a Phenomenex Kinetex C (2.1 mm. Times.50 mm,2.6 μm) liquid chromatography column using a Vanquish (Thermo FISHER SCIENTIFIC) ultra performance liquid chromatograph for the non-polar metabolite project. The liquid chromatography phase A is an aqueous phase containing 0.01% acetic acid, and the phase B is isopropanol to acetonitrile (1:1, v/v). Column temperature 25 ℃, sample tray temperature 4 ℃ and sample injection volume 2 μl. Orbitrap Exploris 120A mass spectrometer is capable of primary and secondary mass spectrometry data acquisition under control of control software (Xcalibur, version 4.4, thermo). The detailed parameters are ：Sheath gas flow rate:50Arb,Aux gas flow rate:15Arb,Capillary temperature:320℃,Sweep Gas：1Arb,Vaporizer Temp：350℃.Full msresolution:60000,MS/MS resolution:15000,Collision energy:SNCE 20/30/40,SprayVoltage:3.8kV(positive) or-3.4 kV (negative).
Referring to the data, combining with an on-line Chinese medicine database, and collecting information such as compound name, molecular formula, molecular weight, retention time and the like. The database is imported into CompoundDiscoverer 3.3.3 software, the acquired data are analyzed by CompoundDiscoverer 3.3.3 software, and the possible structure of the compound is deduced by researching fragment ions, missing groups, mass spectrum cracking rules and the like of each molecular ion of the high collision energy channel. Screening differential metabolites according to P-value <0.05 and Log2 Fold Change >2, fermenting Glycyrrhrizae radix extract with Lactobacillus crispatus CCFM1362 for 48 hr, wherein the metabolites mainly comprise terpenes, shikimic acid, phenylpropionic acid, fatty acids, amino acids and short peptides.
TABLE 2 terpenes, shikimic acid and phenylpropionic acid detected in Glycyrrhiza extract fermentation broth before and after fermentation
| Molecular weight | Molecular formula | Name of the name | log2foldchange | Post fermentation change |
| 821.3975 | C42H62O16 | Glycyrrhizic acid | -0.956 | - |
| 629.3663 | C36H54O10 | Glycyrrhetinic acid 3-O-glucuronide | 8.563 | + |
| 471.3452 | C30H46O4 | Glycyrrhetinic acid | 5.878 | + |
| 551.1751 | C26H30O13 | Apiose isoliquiritigenin | 3.826 | + |
| 353.1008 | C20H16O6 | Semi-licorice isoflavone B | 4.436 | + |
| 287.0908 | C16H14O5 | Licochalcone B | 3.627 | + |
| 255.067 | C15H12O4 | Liquiritigenin | 3.026 | + |
| 285.075 | C16H12O5 | 5-O-methyl genistein | 5.572 | + |
| 195.0668 | C10H12O4 | Hydroxytyrosol acetate | 4.436 | + |
| 405.1717 | C25H26O5 | 6',8' -Diisopentenyl genistein | 2.161 | + |
| 301.0697 | C16H12O6 | Red clover extract | 3.627 | + |
| 455.354 | C30H48O3 | Ursolic acid | 3.314 | + |
Example 4 Effect of a synbiotic preparation prepared from Bifidobacterium animalis subspecies lactis CCFM1363 and Glycyrrhiza glabra fermented metazoan on weight control in high fat diet mice
48 Healthy male C57BL/6J mice of 6 weeks old are randomly divided into 6 groups, 8 groups are respectively a blank control group, a model group, a licorice extract group (78 mg/kg licorice extract), a bifidobacterium animalis subspecies lactis group (5X 10 9 CFU of bifidobacterium animalis subspecies CCFM 1363), a bifidobacterium animalis subspecies-licorice extract synbiotics preparation group (5X 10 9 CFU of bifidobacterium animalis subspecies CCFM1363, 78mg/kg licorice extract) and a bifidobacterium animalis subspecies-licorice fermented metaplas group (78 mg/kg licorice extract, calculated as the concentration of licorice extract before fermentation).
After the mice are adapted for a week for 13 weeks, a blank group is fed with low-fat low-sugar feed from the second week, the other groups are fed with high-fat feed, the molding cycle is 8 weeks, and from the 10 th week, each experiment group uses the mixture of strain freeze-dried powder and licorice extract or fermented freeze-dried powder (dissolved in physiological saline in corresponding dosage) to perform gastric lavage on the mice in an amount of 0.2 mL/day, and the blank group and the model group are subjected to gastric lavage with the same amount of physiological saline as a control until the experiment is ended. All groups were free drinking and ingestion.
Mice were observed for mental state at regular daily intervals following intervention, and were weighed weekly. The body weight of each group of mice after the dry period is shown in FIG. 2.
As can be seen from fig. 2, the weight gain rate of mice with the bifidobacterium lactis CCFM1363 and licorice extracts alone in the stomach is reduced by 14.632% and 14.152% compared with that of the model group, the effect is inferior to 22.455% and 40.072% of that of the synbiotic preparation and licorice fermented metazoan, after the drug intervention is finished, both the synbiotic preparation prepared by fermenting the licorice extracts with the bifidobacterium lactis CCFM1363 and the licorice fermented metazoan have the effect of relieving the weight gain of the mice, and the licorice fermented metazoan has the optimal weight control effect.
TABLE 3 grouping of experimental animals
Example 5 Effect of a synbiotic preparation prepared from Bifidobacterium animalis subspecies lactobacilli CCFM1363 and Glycyrrhiza glabra fermentation metazoan on the level of the secretion phenotype associated with aging in mice
The grouping, modeling and treatment method of mice were the same as in example 4.
The partially frozen liver tissue was weighed, PBS (pH 7.4, 4 ℃) was added at 1:9 (m/V), homogenized, centrifuged at 4℃and 12000 Xg for 30min, and the supernatants were collected and assayed for IL-1. Beta. And IL-6 levels according to the corresponding Elisa kit instructions.
As can be seen from FIG. 3, the IL-1β levels of mice treated with the Lactobacillus bifidus subspecies CCFM1363 and Glycyrrhrizae radix extract were reduced by 22.176% and 19.901% respectively, compared with the model group, and the effects were inferior to those of 31.220% and 57.129% of the equivalent dose synbiotic preparation and the post-fermentation radix Glycyrrhizae. In addition, at the IL-6 level, the IL-6 level of mice with bifidobacterium lactis subspecies CCFM1363 and licorice extracts alone in the stomach-infused animals is reduced by 10.644 percent and 5.129 percent respectively compared with the model group, and the effect is inferior to 40.382 percent and 25.249 percent of the equivalent dose of the synbiotic preparation and the biomass after licorice fermentation. After the medicine is dried, the synbiotic preparation prepared by fermenting the licorice extract by bifidobacterium animalis subspecies CCFM1363 and the prebiotics after licorice fermentation have the effect of reducing the secretion phenotype level related to the aging of mice, which shows that the medicine has stronger potential in the anti-aging field.
Example 6 Effect of a synbiotic preparation prepared from Bifidobacterium animalis subspecies lactobacilli CCFM1363 and Glycyrrhiza glabra fermentation metazoan on the beta-galactosidase (beta-GAL) content of mice
The grouping, modeling and treatment method of mice were the same as in example 4.
The partially frozen liver tissue was weighed, PBS (pH 7.4, 4 ℃) was added at 1:9 (m/V), homogenized, centrifuged at 4℃and 12000 Xg for 30min, and the supernatant was collected and assayed for age-related beta-galactosidase levels according to the corresponding Elisa kit instructions.
In senescent cells, the number and size of lysosomes increase, and beta-galactosidase acts as an enzyme directly related to the level of lysosomal mass, an important biomarker for characterizing the level of aging. As can be seen from FIG. 4, compared with the model group, the content of the beta-galactosidase in the mice of the bifidobacterium lactis subspecies CCFM1363 and the licorice extract of the individual gastric lavage animals is reduced by 7.782 percent and 17.056 percent, the effect is not as good as 24.644 percent and 29.431 percent of the effects of the co-formulation and the licorice fermented metagroup, after the drug intervention is finished, the co-formulation prepared by fermenting the licorice extract of the bifidobacterium lactis subspecies CCFM1363 and the licorice fermented metagroup have the effect of relieving the increase of the aging markers of the mice, and the licorice fermented metagroup shows the optimal anti-aging control effect.
Example 7 Effect of a synbiotic preparation prepared from Bifidobacterium animalis subspecies lactobacilli CCFM1363 and Glycyrrhiza glabra fermented metazoan on liver fat accumulation and lesions in high fat diet mice
The grouping, modeling and treatment method of mice were the same as in example 4.
After the end of the experiment, the mice were bled and sacrificed, and liver tissues were fixed in 4% paraformaldehyde solution for 24 hours, and hematoxylin-eosin (H & E) staining was performed to evaluate the degree of liver steatosis and inflammation. Frozen sections of fresh liver tissue embedded in OCT were cut and stained with oil red O to observe liver lipid accumulation. The stained tissue sections were observed with an optical microscope. The staining results are shown in FIGS. 5 and 6.
As can be seen from fig. 5 and 6, the liver tissue of the mice in the group of post-fermentation primordial administration of licorice prepared from the blank group and bifidobacterium animalis subspecies CCFM1363 is uniformly colored, the fat accumulation is less, and the liver cells are compact and uniform and take on a regular shape. The liver tissues of the other groups of mice have more fat accumulation, obvious pathological changes, a large number of fat vacuoles around cells, loose connection of liver cells, outflow of cell contents, swelling of cells and damage of cell integrity. Experimental results show that the mixture of the bifidobacterium animalis subspecies of the milk CCFM1363 and the licorice extract or the prepared licorice fermentation metagen effectively inhibits liver fat accumulation and lesions of high-fat diet mice, and particularly the metagen preparation obviously reduces the liver damage and fatty lesion degree.
Example 8 Effect of a synbiotic preparation prepared from Bifidobacterium animalis subspecies lactis CCFM1363 and a Glycyrrhiza uralensis fermented metazoan on blood lipid in high fat diet mice
The grouping, modeling and treatment method of mice were the same as in example 4.
After the experiment is finished, the mice are taken and killed, the whole blood of the mice is kept stand for 1h and centrifuged for 3000g and 15min to obtain serum, and a full-automatic biochemical analyzer is adopted to detect blood fat related indexes, wherein the related indexes comprise TG (triglyceride), TC (total cholesterol), HDL-C (high density lipoprotein cholesterol) and LDL-C (low density lipoprotein cholesterol), and the result is shown in figure 7.
As can be seen from fig. 7, the model group TC and TG content were significantly increased compared to the control group, indicating that lipid accumulation in mice caused an increase in blood lipid. The result of the pharmaceutical intervention shows that compared with a model group, the mice group of the bifidobacterium lactis CCFM1363 and the licorice extract of the single intragastric animal respectively have the TC respectively reduced by 6.301 percent and 9.032 percent, the TG respectively reduced by 20.152 percent and 22.222 percent, and the content of the synbiotic preparation and the licorice fermented synbiotic preparation group is reduced to different degrees, wherein the content of the fermented synbiotic of the bifidobacterium lactis CCFM1363 and the licorice extract of the animal respectively reduced by 21.075 percent and 29.885 percent in TC and TG indexes, and the content of the synbiotic preparation respectively reduced by 8.081 percent and 29.502 percent in TC and TG indexes;
Meanwhile, compared with a control group, the contents of LDL-c and HDL-c in a model group are obviously increased compared with a blank group, and the result of the pharmaceutical intervention shows that compared with the model group, the contents of LDL-c and HDL-c in a single intragastric animal bifidobacterium subspecies CCFM1363 mouse group are respectively reduced by 2.848 percent and 1.607 percent, and compared with the model group, the contents of LDL-c and HDL-c in a single intragastric licorice extract mouse group are respectively reduced by 15.913 percent and 0.952 percent, which are not as good as the effects of the metaplastic preparation and the radix glycyrrhizae after fermentation. The effect of reducing the levels of LDL-c and HDL-c of the synbiotic preparation reaches 21.441 percent and 1.964 percent, the effect of reducing the levels of LDL-c and HDL-c of the radix glycyrrhizae after fermentation reaches 22.111 percent and 18.869 percent respectively, which shows that the synbiotic preparation of bifidobacterium animalis subspecies CCFM1363 and radix glycyrrhizae extract and the synbiotic after fermentation of radix glycyrrhizae have good potential in reducing the blood lipid level, and further shows that the rise of the blood lipid caused by the accumulation of lipid can be reduced according to the four indexes of the blood lipid of mice.
Example 9 Effect of a synbiotic preparation prepared from Bifidobacterium animalis subspecies lactis CCFM1363 and Glycyrrhiza glabra post-fermentation prebiotics on liver function of high fat diet mice
The grouping, modeling and treatment method of mice were the same as in example 3.
After the experiment is finished, taking blood and dying the mice, standing the whole blood of the mice for 1h, centrifuging for 3000g and 15min to obtain serum, and detecting liver function related indexes by adopting a full-automatic biochemical analyzer, wherein the related indexes comprise ALT (glutamic pyruvic transaminase) and AST (glutamic oxaloacetic transaminase), and the result is shown in figure 8.
As can be seen from fig. 8, the ALT and AST contents of the model were significantly increased compared to the control group, indicating that lipid accumulation due to the high-fat model caused chronic inflammation in the liver of mice. ALT and AST contents in serum of mice of the synbiotic preparation and the licorice fermented metazoan intervention group are reduced to different degrees compared with a model group, which shows that inflammatory reaction caused by liver lipid accumulation can be reduced, wherein the AST contents 29.428 percent and 28.457 percent of the synbiotic preparation and the licorice fermented metazoan preparation are respectively reduced, and the ALT contents 42.588 percent and 37.825 percent are respectively reduced.
Example 10 Effect of a synbiotic preparation prepared from Bifidobacterium animalis subspecies lactis CCFM1363 and Glycyrrhiza glabra fermentation metazoan on expression of genes related to fat metabolism in high-fat diet mice
The grouping, modeling and treatment method of mice were the same as in example 4.
After the experiment is finished, the mice are taken and killed, total RNA in liver tissues of the mice is extracted by using an animal RNA extraction kit, and then the extracted RNA is reversely transcribed into cDNA by using a reverse transcription kit. Real-time fluorescent quantitative PCR was performed using cDNA as a template by using a fluorescent dye-ligation method, and mRNA expression levels of the target genes MGL, HSL, ATGL, PPAR-alpha, PPAR-gamma and Fasn were calculated by a 2 -ΔΔCt method using mouse GAPDH as an internal reference gene. Each group of genes of interest was expressed as its expression level relative to the blank (set to 1.0). Primers were synthesized by Shanghai Biotechnology Co., ltd, and the primer sequences are shown in Table 4.
TABLE 4 real-time fluorescent quantitative PCR detection primer sequences
As is clear from fig. 9, the relative expression levels of PPAR- γ and FASN in the model group were significantly improved, and the relative expression levels of PPAR- α and ATGL, HSL, MGL were reduced, compared with the blank group, indicating that the high-fat diet significantly promoted lipid synthesis in the liver of mice and inhibited lipid breakdown, and also explained the phenomenon of liver fat accumulation in the high-fat diet mice.
The synbiotic preparation prepared from the bifidobacterium animalis subspecies CCFM1363 and the licorice extract and the licorice fermentation postnatal preparation can improve MGL, HSL, ATGL, PPAR-alpha gene expression and reduce PPAR-gamma and Fasn gene expression to different degrees, thereby reducing organism lipid accumulation.
Compared with a model group, the PPAR-gamma and FASN levels are reduced by 16.732 percent and 39.631 percent after the intervention of the synbiotic preparation, the ATGL, mgl, hsl, PPAR-alpha gene levels are improved by 282.479 percent, 55.149 percent, 363.935 percent and 55.032 percent, the PPAR-gamma and FASN levels are reduced by 47.273 percent and 42.045 percent after the intervention of the synbiotic preparation after the fermentation of liquorice, and the effects of the synbiotic preparation and the liquorice after the fermentation are better than those of the bifidobacterium lactis CCFM1363 or the liquorice extract of the single gavaginary animal. It shows that the co-production preparation of animal bifidobacterium subspecies CCFM1363 and licorice extract or the fermentation post-production of licorice can raise MGL, HSL, ATGL, PPAR-alpha gene expression, reduce PPAR-gamma and Fasn gene expression and reduce body lipid accumulation.
The result comparison analysis shows that the bifidobacterium animalis subspecies CCFM 1363+licorice extract group can inhibit the weight increase under the high-fat diet, reduce the level of IL-1 beta and IL-6 of aging-related secretion phenotypes of mice, reduce the content of beta-galactosidase in livers, reduce the level of TC, TG, HDL-c and LDL-c in serum, reduce the damage of liver tissues caused by lipid accumulation, promote the expression of lipid metabolism-related genes MGL, HSL, ATGL, PPAR-alpha, reduce the expression of lipid synthesis-related genes PPAR-gamma and Fasn, improve the blood glucose steady-state level of the mice, and the effect is obviously better than that of single strains CCFM1363 and single licorice extract, and the bifidobacterium animalis subspecies CCFM1363 has the functions of realizing anti-aging and conversion of licorice synergism by multiple targets.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A strain of bifidobacterium animalis subspecies (Bifidobacterium animalis subsp. Lactis) CCFM1363, said bifidobacterium animalis subsp. Lactis CCFM1363 having been deposited at the microorganism strain deposit center in Guangdong province on 12 th year 06, accession number GDMCC No:64111.
2. A metazoan prepared from bifidobacterium animalis subspecies lactis CCFM1363 according to claim 1, wherein the metazoan is obtained by fermenting the bifidobacterium animalis subspecies lactis CCFM1363 in a culture medium containing licorice extract, inactivating the fermentation broth, and homogenizing.
3. A synbiotic formulation prepared from bifidobacterium animalis subspecies lactis CCFM1363 as claimed in claim 1 characterised in that the synbiotic formulation comprises bifidobacterium animalis subspecies CCFM1363 and a licorice preparation comprising licorice or licorice extract.
4. A product comprising the metaseduction of claim 2 or the synbiotic of claim 3, wherein the product comprises a food, pharmaceutical or health product.
5. The product of claim 4, wherein the product is a lyophilized powder of the metazoan or synbiotic formulation or a reconstituted formulation comprising the metazoan or synbiotic formulation lyophilized powder.
6. A method for degrading glycyrrhizic acid, characterized in that bifidobacterium animalis subspecies lactis CCFM1363 according to claim 1 is added to a medium containing glycyrrhizic acid for fermentation.
7. A method for preparing glycyrrhetinic acid, characterized in that bifidobacterium animalis subspecies lactis CCFM1363 according to claim 1 is added to a medium containing glycyrrhizic acid for fermentation.
8. A method for enhancing the medicinal efficacy of licorice, characterized in that the method is any one of the following:
(1) Adding the bifidobacterium animalis subspecies of milk CCFM1363 of claim 1 to a culture medium containing licorice preparations for fermentation;
(2) Mixing the bifidobacterium animalis subspecies lactis CCFM1363 of claim 1 with a licorice preparation;
the licorice preparation comprises licorice or licorice extract.
9. Use of a bifidobacterium animalis subspecies CCFM1363 and/or its derivatives and/or synbiotics as claimed in claim 1 for the manufacture of a lipid-lowering and/or anti-ageing medicament.
10. The use of claim 9, wherein the pharmaceutical product comprises at least one of the following functions:
(1) Reducing weight gain in a high fat diet;
(2) Reducing the level of IL-1 beta and IL-6, which are secretion phenotypes related to the aging of the organism;
(3) Reducing the content of beta-galactosidase in the organism;
(4) Inhibit liver fat accumulation and damage;
(5) Reducing blood lipid level of organism;
(6) Improving the expression of the organism lipid metabolism related gene MGL, HSL, ATGL, PPAR-alpha and reducing the expression of the lipid synthesis related gene PPAR-gamma, fasn.
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