A polygonatum probiotic fermentation broth, its preparation method and application

Abstract

The present invention discloses a polygonatum probiotic fermentation broth, its preparation method and application, belonging to the field of microbial technology. The polygonatum probiotic fermentation broth disclosed by the present invention is obtained by fermenting polygonatum with Lactobacillus plantarum NX-1. The lipid-lowering effect of the polygonatum probiotic fermentation broth disclosed by the present invention is superior to that of unfermented raw polygonatum, nine-processed polygonatum, ginseng-seven-ginkgo-polygonatum tea, and Tongren Qingzhi tea.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and more specifically, to a polygonatum probiotic fermentation broth, a preparation method thereof, and an application thereof. Background Art

[0002] With the improvement of modern living standards, people have ingested excessive amounts of high-fat foods, leading to an increasing number of diseases related to lipid metabolism disorders such as obesity, hyperlipidemia, and non-alcoholic fatty liver. For example, according to data provided by the World Health Organization, approximately 1.9 billion people aged 18 or older are overweight; among them, approximately 650 million people are diagnosed with obesity. And obese individuals are more likely to suffer from diseases such as hyperlipidemia and non-alcoholic fatty liver. However, there are currently no specific drugs with good efficacy and quick results for problems such as obesity and hyperlipidemia. Instead, more reliance is placed on a healthy lifestyle and eating habits.

[0003] Polygonatum is a perennial herb of the genus Polygonatum in the family Liliaceae and is an edible and medicinal ingredient. Its main medicinal part is its rhizome. Polygonatum contains active ingredients such as polysaccharides, saponins, flavonoids, polyphenols, alkaloids, amino acids, and trace elements. Some studies have found that polygonatum can alleviate the abnormal increase in the levels of TC, TG, and LDL-C in the serum and liver of mice fed a high-fat diet, the abnormal decrease in HDL-C, and the significant enlargement of the liver, showing a good lipid-lowering effect. However, raw polygonatum has strong irritation and contains some harmful components and is not suitable for direct consumption.

[0004] Lactobacillus plantarum is a type of lactic acid bacterium. Its optimal growth temperature is 30 - 35°C, anaerobic or facultatively anaerobic. The bacterial strain is straight or curved rod-shaped, single, sometimes paired or in chains, and the optimal pH is about 6.5. It belongs to homofermentative lactic acid bacteria. Lactobacillus plantarum is a relatively common lactic acid bacterium in traditional fermented foods (such as pickles and fermented bean curd) and has beneficial effects such as reducing serum cholesterol content and preventing cardiovascular diseases.

[0005] Therefore, providing a polygonatum probiotic fermentation broth, a preparation method thereof, and an application thereof is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a polygonatum probiotic fermentation broth, a preparation method thereof, and an application thereof. Lactobacillus plantarum NX-1 is used to ferment polygonatum to improve the lipid-lowering activity of polygonatum.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A polygonatum probiotic fermentation broth is obtained by fermenting polygonatum with Lactobacillus plantarum NX-1, and the preservation number of Lactobacillus plantarum NX-1 is CGMCC No. 20109.

[0009] Furthermore, the preparation method of the polygonatum probiotic fermentation broth is as follows:

[0010] (1) Seed liquid preparation:

[0011] Culture Lactobacillus plantarum NX-1 until the OD 600 is 1.8 - 2.2 to obtain the Lactobacillus plantarum NX-1 seed liquid, and store it in a 4°C refrigerator for later use;

[0012] (2) Preparation of polygonatum aqueous solution:

[0013] Dry raw polygonatum until constant weight, pulverize it to obtain polygonatum powder, and prepare a 5% - 15% (mass ratio) polygonatum aqueous solution with pure water. After sterilization, store it for later use;

[0014] (3) Fermentation:

[0015] Inoculate the Lactobacillus plantarum NX-1 seed liquid obtained in step (1) into the sterilized polygonatum aqueous solution in step (2). The inoculation amount is 2% (volume ratio), and statically ferment at 32°C - 42°C for 2 - 4 days to obtain the fermentation broth; centrifuge the fermentation broth, take the supernatant to obtain the polygonatum probiotic fermentation broth.

[0016] Furthermore, the specific steps of the seed liquid preparation in step (1) are as follows: Use the streak plate method to inoculate Lactobacillus plantarum NX-1 onto an MRS plate, and place it in a 37°C constant temperature incubator for 24 h; then pick a single colony from the plate and inoculate it into an MRS liquid medium, and statically culture at 37°C until the OD 600 is 1.8 - 2.2 to obtain the Lactobacillus plantarum NX-1 seed liquid, and store it in a 4°C refrigerator for later use.

[0017] Furthermore, the specific steps of the preparation of the polygonatum aqueous solution in step (2) are as follows: Take raw polygonatum, place it in a 60°C forced air drying oven and dry it until constant weight. Pulverize the dried raw polygonatum into uniformly granulated powder with a pulverizer, and prepare a 5% - 15% (mass ratio) polygonatum aqueous solution with pure water. After sterilization at 118°C for 15 min, store it for later use.

[0018] Furthermore, the centrifugation conditions in step (3) are: 4°C, 4000 rpm / min for 20 min.

[0019] Furthermore, the application of the polygonatum probiotic fermentation broth or the polygonatum probiotic fermentation broth prepared by the preparation method in the preparation of lipid-lowering products.

[0020] Furthermore, the application of the polygonatum probiotic fermentation broth or the polygonatum probiotic fermentation broth prepared by the preparation method in the preparation of products for increasing cholesterol binding rate and pancreatic lipase inhibition rate.

[0021] The present invention applies microbial fermentation technology to ferment polygonatum sibiricum, and various exogenous active enzymes or beneficial secondary metabolites can be produced through microbial fermentation metabolism, thereby improving the lipid-lowering activity of polygonatum sibiricum. First, the cell wall-breaking effect of the composite enzyme during the fermentation process is conducive to the dissolution of active ingredients (such as polysaccharides and saponins) in polygonatum sibiricum; second, the enzymes produced by the secondary metabolism during the growth of microorganisms can modify the structure of the substrate, thereby generating new compounds and improving the activity of the product; at the same time, through fermentation treatment, the irritation or other harmful components of raw polygonatum sibiricum can also be removed, reducing its toxic and side effects.

[0022] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a polygonatum sibiricum probiotic fermentation broth, its preparation method and application. The lipid-lowering effect of the polygonatum sibiricum probiotic fermentation broth is superior to that of unfermented raw polygonatum sibiricum, nine-processed polygonatum sibiricum, ginseng-seven-ginkgo-polygonatum sibiricum tea, and Tongren Qingzhi tea. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0024] Figure 1 The drawings are of the fat in zebrafish of different groups of the present invention. Detailed Embodiments

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0026] Lactobacillus plantarum NX-1, preservation number CGMCC No. 20109 (see patent number 202010699291.3);

[0027] Lactobacillus casei LS01, preservation number CGMCC No. 22006 (see patent number 202110619616.7);

[0028] Lactobacillus casei LS02, preservation number CGMCC No. 22007 (see patent number 202211460414.3);

[0029] Lactobacillus acidophilus LA-03, deposit number CGMCC No. 22239 (see patent number 202110619616.7).

[0030] Example 1

[0031] A method for preparing a fermented solution of polygonatum and probiotics, the specific steps are as follows:

[0032] (1) Preparation of the bacterial strain solution:

[0033] Using the streak plate method, inoculate Lactobacillus plantarum NX-1 onto an MRS plate, place it in a constant temperature incubator at 37 °C for 24 h, then pick a single colony from the plate and inoculate it into 200 mL of MRS liquid medium, and incubate it statically at 37 °C until the OD 600 reaches 2.0 to obtain the Lactobacillus plantarum NX-1 seed solution, and place it in a refrigerator at 4 °C for later use.

[0034] (2) Preparation of the polygonatum aqueous solution:

[0035] Take an appropriate amount of fresh polygonatum, place it in a forced air drying oven at 60 °C and dry it to constant weight, then use a pulverizer to pulverize the dried fresh polygonatum into uniformly sized powder, prepare a 10% (mass ratio) polygonatum aqueous solution with pure water, dispense it into 500 mL Erlenmeyer flasks (200 mL per flask), sterilize it at 118 °C for 15 min, and then keep it for later use.

[0036] (3) Fermentation:

[0037] Inoculate the Lactobacillus plantarum NX-1 seed solution into the sterilized 10% polygonatum aqueous solution, with an inoculation amount of 2% (volume ratio), ferment statically at 37 °C for 3 days, then centrifuge the fermented solution at 4000 rpm / min at 4 °C for 20 min, and take the supernatant as the fermented solution of polygonatum and probiotics.

[0038] Example 2

[0039] A method for preparing a fermented solution of polygonatum and probiotics, the specific steps are as follows:

[0040] (1) Preparation of the bacterial strain solution:

[0041] Using the streak plate method, inoculate Lactobacillus plantarum NX-1 onto an MRS plate, place it in a constant temperature incubator at 37 °C for 24 h, then pick a single colony from the plate and inoculate it into 200 mL of MRS liquid medium, and incubate it statically at 37 °C until the OD 600 reaches 1.8 to obtain the Lactobacillus plantarum NX-1 seed solution, and place it in a refrigerator at 4 °C for later use.

[0042] (2) Preparation of polygonatum sibiricum aqueous solution:

[0043] Take an appropriate amount of fresh polygonatum sibiricum, place it in a forced-air drying oven at 60 °C and dry it to a constant weight. Then use a pulverizer to pulverize the dried fresh polygonatum sibiricum into uniformly sized powder. Prepare a 5% (mass ratio) polygonatum sibiricum aqueous solution with pure water, dispense it into 500 mL Erlenmeyer flasks (200 mL per flask), and sterilize it at 118 °C for 15 min for later use.

[0044] (3) Fermentation:

[0045] Inoculate the Lactobacillus plantarum NX-1 seed solution into the sterilized 5% polygonatum sibiricum aqueous solution with an inoculation amount of 2% (volume ratio), and ferment it statically at 32 °C for 2 days. Then centrifuge the fermentation broth at 4000 rpm / min and 4 °C for 20 min, and take the supernatant as the polygonatum sibiricum probiotic fermentation broth.

[0046] Example 3

[0047] A method for preparing a polygonatum sibiricum probiotic fermentation broth, the specific steps are as follows:

[0048] (1) Preparation of strain solution:

[0049] Use the streak plate method to inoculate Lactobacillus plantarum NX-1 onto an MRS plate, place it in a constant temperature incubator at 37 °C for 24 h, and then pick a single colony from the plate and inoculate it into 200 mL of MRS liquid medium, and culture it statically at 37 °C until the OD 600 reaches 2.2 to obtain the Lactobacillus plantarum NX-1 seed solution, and place it in a 4 °C refrigerator for later use.

[0050] (2) Preparation of polygonatum sibiricum aqueous solution:

[0051] Take an appropriate amount of fresh polygonatum sibiricum, place it in a forced-air drying oven at 60 °C and dry it to a constant weight. Then use a pulverizer to pulverize the dried fresh polygonatum sibiricum into uniformly sized powder. Prepare a 15% (mass ratio) polygonatum sibiricum aqueous solution with pure water, dispense it into 500 mL Erlenmeyer flasks (200 mL per flask), and sterilize it at 118 °C for 15 min for later use.

[0052] (3) Fermentation:

[0053] Inoculate the Lactobacillus plantarum NX-1 seed solution into the sterilized 15% polygonatum sibiricum aqueous solution with an inoculation amount of 2% (volume ratio), and ferment it statically at 42 °C for 4 days. Then centrifuge the fermentation broth at 4000 rpm / min and 4 °C for 20 min, and take the supernatant as the polygonatum sibiricum probiotic fermentation broth.

[0054] Comparative Example 1

[0055] A method for preparing a polygonatum sibiricum probiotic fermentation broth, the specific steps are as follows:

[0056] (1) Preparation of bacterial strain solution:

[0057] Using the streak plate method, Lactobacillus plantarum NX-1, Lactobacillus casei LS01, Lactobacillus casei LS02, and Lactobacillus acidophilus LA-03 were respectively inoculated onto MRS plates and placed in an incubator at 37°C for 24 h. Then, single colonies were picked from the plates and inoculated into 200 mL of MRS liquid medium, and statically cultured at 37°C until the OD 600 reached 2.0 to obtain the seed solutions of the corresponding strains, which were placed in a refrigerator at 4°C for standby.

[0058] (2) Preparation of polygonatum aqueous solution:

[0059] An appropriate amount of fresh polygonatum was placed in a forced-air drying oven at 60°C and dried to a constant weight. Then, the fresh polygonatum was pulverized into uniformly granulated powder using a pulverizer, and the polygonatum powder was formulated into a 2% (mass ratio) polygonatum aqueous solution with pure water, which was dispensed into 500 mL Erlenmeyer flasks (200 mL per flask), sterilized at 118°C for 15 min, and then used for standby.

[0060] (3) Fermentation:

[0061] The seed solutions of Lactobacillus plantarum NX-1, Lactobacillus casei LS01, Lactobacillus casei LS02, and Lactobacillus acidophilus LA-03 were respectively inoculated into the sterilized 2% polygonatum aqueous solution with an inoculation amount of 2% (volume ratio), and statically fermented at 37°C for 2 days. Then, the fermentation broth was centrifuged at 4000 rpm / min and 4°C for 20 min, and the supernatant was taken as the polygonatum probiotic fermentation broth.

[0062] Comparative Example 2

[0063] A method for preparing a polygonatum probiotic fermentation broth, the specific steps are as follows:

[0064] (1) Preparation of bacterial strain solution:

[0065] Using the streak plate method, Lactobacillus plantarum NX-1 was inoculated onto an MRS plate and placed in an incubator at 37°C for 24 h. Then, a single colony was picked from the plate and inoculated into 200 mL of MRS liquid medium, and statically cultured at 37°C until the OD 600 reached 2.0 to obtain the seed solution of Lactobacillus plantarum NX-1, which was placed in a refrigerator at 4°C for standby.

[0066] (2) Preparation of polygonatum aqueous solution:

[0067] An appropriate amount of fresh polygonatum was placed in a forced-air drying oven at 60°C and dried to a constant weight. Then, the fresh polygonatum was pulverized into uniformly granulated powder using a pulverizer, and the polygonatum powder was formulated into a 2% (mass ratio) polygonatum aqueous solution with pure water, which was dispensed into 500 mL Erlenmeyer flasks (200 mL per flask), sterilized at 118°C for 15 min, and then used for standby.

[0068] (3) Fermentation:

[0069] Inoculate the Lactobacillus plantarum NX-1 seed solution into the sterilized 2% polygonatum sibiricum aqueous solution, with an inoculation amount of 2% (volume ratio), and ferment statically at 37°C for 1 - 6 days respectively. Then, centrifuge the fermentation broth samples at 4000 rpm / min and 4°C for 20 min, and take the supernatant as the polygonatum sibiricum probiotic fermentation broth.

[0070] Comparative Example 3

[0071] A preparation method of polygonatum sibiricum probiotic fermentation broth, the specific steps are as follows:

[0072] (1) Preparation of strain solution:

[0073] Use the streak plate method to inoculate Lactobacillus plantarum NX-1 onto the MRS plate, place it in a constant temperature incubator at 37°C for 24 h, and then pick single colonies from the plate and inoculate them into 200 mL of MRS liquid medium, and culture statically at 37°C until the OD 600 is 2.0 to obtain the Lactobacillus plantarum NX-1 seed solution, and place it in a 4°C refrigerator for later use.

[0074] (2) Preparation of polygonatum sibiricum aqueous solution:

[0075] Take an appropriate amount of raw polygonatum sibiricum, place it in a forced-air drying oven at 60°C and dry it to constant weight, then use a pulverizer to pulverize the raw polygonatum sibiricum into uniformly granular powder, and prepare a 2% (mass ratio) polygonatum sibiricum aqueous solution with pure water, and sub-pack it into 500 mL Erlenmeyer flasks (200 mL / flask), sterilize at 118°C for 15 min and then reserve for later use.

[0076] (3) Fermentation:

[0077] Inoculate the Lactobacillus plantarum NX-1 seed solution into the sterilized 2%, 5%, and 15% polygonatum sibiricum aqueous solutions respectively, with an inoculation amount of 2% (volume ratio), ferment statically at 37°C for 3 days, then centrifuge the fermentation broth at 4000 rpm / min and 4°C for 20 min, and take the supernatant as the polygonatum sibiricum probiotic fermentation broth.

[0078] Comparative Example 4

[0079] Preparation of control sample:

[0080] ① Preparation of raw polygonatum sibiricum solution: Take an appropriate amount of raw polygonatum sibiricum, place it in a forced-air drying oven at 60°C and dry it to constant weight, then use a pulverizer to pulverize the raw polygonatum sibiricum into uniformly granular powder, and prepare 2%, 5%, 10%, 15% (mass ratio) polygonatum sibiricum aqueous solutions with pure water, and sub-pack it into 500 mL Erlenmeyer flasks (200 mL / flask), sterilize at 118°C for 15 min, and cool for later use.

[0081] ② Preparation of nine-steamed polygonatum sibiricum red juice solution: Take 20 g of nine-steamed polygonatum sibiricum, add an appropriate amount of water, boil and soak for 30 min, filter through a 60-mesh sieve, and make the filtrate up to 200 mL.

[0082] ③ Ginseng, notoginseng, ginkgo and polygonatum sibiricum tea: Take 20 g of ginseng, notoginseng, ginkgo and polygonatum sibiricum tea, add an appropriate amount of water, boil and soak for 30 min, filter through a 60-mesh sieve, and make the filtrate up to 200 mL.

[0083] ④ Tongren lipid-clearing tea: Take 20 g of Tongren lipid-clearing tea, add an appropriate amount of water, boil and soak for 30 min, filter through a 60-mesh sieve, and make the filtrate up to 200 mL.

[0084] Experimental Example 1

[0085] Toxicity experiment (zebrafish lethality experiment):

[0086] Randomly select 5-dpf zebrafish larvae with normal development and place them in a cell culture plate, with 30 fish in each group and 3 replicates. The normal group is treated with fish-raising water, and the test groups are treated with raw polygonatum sibiricum solutions (prepared in Comparative Example 4) and polygonatum sibiricum probiotic fermentation broth (prepared in Example 1) at different concentrations (1%, 0.8%, 0.5%) for 48 h, and then observe the survival rate of zebrafish. The results are shown in Table 1.

[0087] Table 1

[0088]

[0089] Experimental Example 2

[0090] Perform in vitro detection of lipid-lowering activity on the products prepared in Examples 1-3 and Comparative Examples 1-4.

[0091] ① Cholesterol binding rate detection (o-phthalaldehyde colorimetric method): Take 0.4 mL of polygonatum sibiricum probiotic fermentation broth (or control sample), add 2 mL of 0.2 mg / mL cholesterol solution, react at 37 °C for 2 h, then add 0.2 mL of 1 mg / mL o-phthalaldehyde solution and 4 mL of mixed acid (prepared by mixing glacial acetic acid and concentrated sulfuric acid at 1:1), water bath at 37 °C for 10 min, centrifuge at 4000 rpm for 20 min, and measure the absorbance value of the supernatant at 550 nm.

[0092] Cholesterol binding rate % = (1 - A1 / A0) * 100

[0093] A1 is the absorbance value of the sample group, and A0 is the absorbance value of the blank group (using pure water instead of the polygonatum sibiricum probiotic fermentation broth in the sample group).

[0094] ② Detection of pancreatic lipase inhibition rate: Take 0.25 mL of reaction substrate (1% Triton X-100, 5 mM sodium acetate, 0.1% p-nitrophenyl laurate aqueous solution), add 0.1 mL of the fermented solution of polygonatum and probiotics (or control sample) and 0.2 mL of 0.1 M Tris buffer, then add 0.15 mL of 5 mg / mL lipase solution (the background group adds an equal amount of Tris buffer instead), react at 37 °C for 2 h, centrifuge at 10000 rpm for 1 min, and then take the supernatant to detect the absorbance value at 420 nm.

[0095] % of pancreatic lipase inhibition rate = (1 - (A1 - A2) / A0) * 100

[0096] A1 is the absorbance value of the sample group, A2 is the absorbance value of the background group, and A0 is the absorbance value of the blank group (using pure water to replace the fermented solution of polygonatum and probiotics in the sample group).

[0097] The results of cholesterol binding rate and pancreatic lipase inhibition rate of the fermented solutions of polygonatum and probiotics prepared in Examples 1-3 are shown in Table 2.

[0098] Table 2

[0099]

[0100] The results of cholesterol binding rate and pancreatic lipase inhibition rate of the fermented solution of polygonatum and probiotics prepared in Comparative Example 1 are shown in Table 3.

[0101] Table 3

[0102]

[0103] The results of cholesterol binding rate and pancreatic lipase inhibition rate of the fermented solution of polygonatum and probiotics prepared in Comparative Example 2 are shown in Table 4.

[0104] Table 4

[0105]

[0106] The results of cholesterol binding rate and pancreatic lipase inhibition rate of the fermented solution of polygonatum and probiotics prepared in Comparative Example 3 are shown in Table 5.

[0107] Table 5

[0108]

[0109] The results of cholesterol binding rate and pancreatic lipase inhibition rate of the product prepared in Comparative Example 4 are shown in Table 6.

[0110] Table 6

[0111]

[0112] Test Example 3

[0113] The zebrafish high-fat model was evaluated for the polygonatum probiotic fermentation broth prepared in Example 1, the raw polygonatum solution, the nine-steamed polygonatum solution, the ginseng, seven-leaf ginseng, gingko and polygonatum tea, and the Tongren Qingzhi tea prepared in Comparative Example 4.

[0114] ① Model construction: Randomly select 5dpf (days-post fertilization) zebrafish larvae with normal development and place them in a cell culture plate, with 30 fish in each group and 3 replicates; the normal group is fed with ordinary feed (purchased from Shanghai Slake Co., Ltd.); the model group, the positive control group (atorvastatin calcium, purchased from Shanghai Yuanye), and the test article groups (polygonatum probiotic fermentation broth, nine-steamed polygonatum solution, ginseng, seven-leaf ginseng, gingko and polygonatum tea, Tongren Qingzhi tea, raw polygonatum solution) are all fed with high-fat feed (purchased from Shanghai Yuanye) for 48 h.

[0115] ② Intervention: After the construction of the hyperlipidemia model is completed, E3 Water (fish-raising water, purchased from Shanghai Slake Co., Ltd.) is added to the normal group, the model group, the positive control group, and the test article groups; 20 μl of fresh fish-raising water is added to the normal group and the model group, 20 μl of atorvastatin calcium solution (0.24 μg / ml) is added to the positive control group, and the test article groups (polygonatum probiotic fermentation broth, raw polygonatum, nine-steamed polygonatum, ginseng, seven-leaf ginseng, gingko and polygonatum tea, Tongren Qingzhi tea) are respectively fed with equal amounts of polygonatum probiotic fermentation broth, raw polygonatum, nine-steamed polygonatum, ginseng, seven-leaf ginseng, gingko and polygonatum tea, and Tongren Qingzhi tea for 48 h.

[0116] ③ Oil red O staining: After the intervention, the zebrafish are washed twice with E3 Water and fixed with 4% paraformaldehyde solution at 4°C, rinsed twice with PBS, dehydrated with a gradient of 1,2-propanediol, then the zebrafish are transferred into a 24-well plate, oil red O staining solution is added, and then decolorized with a gradient of 1,2-propanediol. Observe and photograph the fat in the zebrafish under a stereomicroscope. Use Image J software to analyze the oil red O staining of fat in the zebrafish and count the corresponding gray value (S). The calculation formula for the relative fat level of zebrafish is as follows:

[0117] Relative fat level of zebrafish (%) = (S of the model group, positive control group, test article groups) / S of the normal group × 100%

[0118] ④ Data statistics: The experimental data are statistically analyzed using GraphPad Prism 6.0 software and are all expressed as mean ± SEM. Analyzed by t-test, compared with the normal group: #P < 0.05, ##P < 0.01, P < 0.001; compared with the model group: &P < 0.05, &&P < 0.01, &&&P < 0.001; analyzed by one-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001.

[0119] The test results are shown in Table 7 andFigure 1 。

[0120] Table 7

[0121]

[0122] The depth of Oil Red O staining reflects the level of fat content in zebrafish. Table 7 and Figure 1 show that, compared with the normal group, the Oil Red O staining in zebrafish of the model group deepens, indicating an increase in the relative fat level in zebrafish of the model group; meanwhile, compared with the normal group (100.00 ± 11.46%), the relative fat level in zebrafish of the model group (225.41 ± 1.46%) increases significantly (p < 0.001), indicating that the high-fat zebrafish model is successfully constructed. The Oil Red O staining in zebrafish of the positive control group becomes lighter, and compared with the model group (225.41 ± 1.46%), the relative fat level in zebrafish of the positive control group (102.77 ± 6.44%) decreases significantly (p < 0.001). Therefore, atorvastatin calcium has the effect of reducing body fat, which is consistent with the clinical results, indicating that the efficacy evaluation experiment for reducing body fat this time is effective.

[0123] The Oil Red O staining in zebrafish of the Polygonatum sibiricum fermented liquid group, the processed Polygonatum sibiricum group, the raw Polygonatum sibiricum group, the Ginseng, Notoginseng, Ginkgo biloba and Polygonatum sibiricum tea group, and the Tongren Qingzhi tea group all become lighter, and the measured relative fat levels in zebrafish are 96.0 ± 4.74%, 151.23 ± 4.83%, 180.36 ± 7.13%, 162.54 ± 9.34% and 165.34 ± 8.47% respectively, which are lower compared with the model control group (225.41 ± 1.46%) and show significant differences (p < 0.001). Among them, the relative fat level in zebrafish of the Polygonatum sibiricum fermented liquid group is the lowest compared with other test groups, proving that its lipid-lowering effect is the best.

[0124] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of probiotic fermentation liquid of Polygonatum sibiricum in the preparation of lipid-lowering products, characterized in that: The probiotic fermented polygonatum sibiricum liquid is obtained by fermenting polygonatum sibiricum with Lactobacillus plantarum NX-1, and the preservation number of the Lactobacillus plantarum NX-1 is CGMCC No.20109; The preparation method of the polygonatum probiotic fermentation liquid is as follows: (1) Seed solution preparation: Lactobacillus plantarum NX-1 was cultured to OD 600 The pH value is 1.8-2.2, and the Lactobacillus plantarum NX-1 seed solution is obtained and placed in a 4°C refrigerator for later use; (2) Preparation of Polygonatum sibiricum aqueous solution: Dry the raw polygonatum to constant weight, grind it to obtain polygonatum powder, prepare the polygonatum powder into a 5%-15% polygonatum aqueous solution with pure water, sterilize it and set it aside; (3) Fermentation: The Lactobacillus plantarum NX-1 seed liquid obtained in step (1) is inoculated into the sterilized polygonatum sibiricum aqueous solution in step (2) at an inoculation amount of 2%, and the mixture is allowed to ferment at 32° C.-42° C. for 2-4 days to obtain a fermentation liquid; the fermentation liquid is centrifuged, and the supernatant is taken to obtain a polygonatum sibiricum probiotic fermentation liquid.

2. The use according to claim 1, characterized in that: The specific steps of preparing the seed solution in step (1) are as follows: inoculate Lactobacillus plantarum NX-1 onto an MRS plate using the plate streak method, place it in a 37°C constant temperature incubator and culture it for 24 hours; then pick a single colony from the plate and inoculate it into an MRS liquid culture medium, and culture it at 37°C until OD 600 The concentration of Lactobacillus plantarum NX-1 was 1.8-2.2, and the seed solution was obtained and placed in a 4°C refrigerator for later use.

3. The use according to claim 1, characterized in that: The specific steps of preparing the polygonatum sibiricum aqueous solution in step (2) are as follows: take raw polygonatum sibiricum, place it in a 60°C forced air drying oven and dry it to constant weight, grind the dried raw polygonatum sibiricum into powder with uniform particles through a grinder, use pure water to prepare the polygonatum sibiricum powder into a 5%-15% polygonatum sibiricum aqueous solution, sterilize it at 118°C for 15 minutes and set it aside.

4. The use according to claim 1, characterized in that: The centrifugation conditions in step (3) are: 4°C, 4000 rpm / min for 20 min.

5. Use of the probiotic fermentation broth of Polygonatum sibiricum according to any one of claims 1 to 4 in the preparation of a product that improves cholesterol binding rate and pancreatic lipase inhibition rate.

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