Method for improving liver-protecting and lipid-lowering effects by fermenting pine pollen with mucor racemosus
By fermenting pine pollen by mucor racemax, releasing and transforming its active ingredients, the problem of limited effect in the treatment of alcoholic fatty liver in the prior art is solved, and a stronger liver protection and lipid reduction effect is achieved.
Patent Information
- Application Number
- CN202510021558.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art has limited effect in the treatment of alcoholic fatty liver and lacks effective means of protecting the liver and reducing lipids.
By fermenting pine pollen with mucor racemos, it releases and transforms active ingredients such as polyphenols and flavonoids in pine pollen, and improves its liver protection and lipid-lowering activity.
The pine pollen after fermentation significantly increases its polyphenols and flavonoid content, enhances its liver protection and lipid reduction effect on alcoholic fatty liver, can significantly reduce fat accumulation and liver enzyme content in cells, and improve oxidative stress.
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Figure CN119632234A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of pine pollen fermentation, and in particular relates to a method for improving the liver-protecting and lipid-lowering effects by utilizing pine pollen fermented by Mucor racemosus. Background Art
[0002] Pine pollen is the male pollen of plants in the pine family. It is a traditional material that can be used as both medicine and food, and has always been hailed as a natural "treasure trove of nutrition". Pine pollen is rich in a variety of essential nutrients for the human body, including amino acids, minerals, vitamins, enzymes, and polyphenol compounds, and has high nutritional value and biological activity. It not only contains rich plant protein and dietary fiber, but also contains antioxidant ingredients that are beneficial to the human body, such as flavonoids and total phenols, which help regulate the immune system, resist aging, and improve intestinal health. Therefore, pine pollen is widely used in many fields such as medicine, health care, food, and cosmetics.
[0003] Alcoholic fatty liver is a liver fat deposition disease caused by long-term excessive drinking and is the early stage of alcoholic liver disease. Its main feature is fat accumulation in liver cells. As the disease progresses, it may develop into alcoholic hepatitis, liver fibrosis and even cirrhosis. The occurrence of alcoholic fatty liver is closely related to abnormal liver fat metabolism, increased oxidative stress and immune response disorders caused by excessive alcohol intake. With the increase in global alcohol consumption, the incidence of alcoholic fatty liver is on the rise and has become a global public health problem. The harm of alcoholic fatty liver is not only limited to the liver, but is also related to chronic diseases such as diabetes and cardiovascular disease, which seriously affects the health and quality of life of patients. At present, treatment is based on limiting alcohol intake, changing diet and increasing exercise. Drug treatment mainly includes antioxidants, lipid-lowering drugs and liver protectors, but the effect is limited and there is no specific drug. In recent years, with the in-depth research on liver metabolism and other related issues, new treatment methods such as natural plant extracts have gradually gained attention. These natural ingredients may provide new ideas for the treatment of alcoholic fatty liver by improving intestinal flora, reducing oxidative stress, regulating lipid metabolism and other pathways.
[0004] In recent years, fermentation technology has been widely used in the development and utilization of plant resources, especially in improving the bioavailability of substrates. Many plant raw materials are rich in active substances such as polyphenols, flavonoids, polysaccharides and amino acids. The fermentation process not only helps to degrade plant cell walls and promote the release of these active ingredients, but also improves their biological activity through transformation and enhances their liver-protecting effect. At present, research on fermenting pine pollen to enhance its liver-protecting function is relatively limited. Summary of the invention
[0005] The present invention aims to provide a method for improving the liver protection and lipid-lowering effects by fermenting pine pollen with Mucor racemosus. The specific technical scheme is as follows:
[0006] A method for improving the liver protection and lipid-lowering effects by fermenting pine pollen with Mucor racemosus, comprising the following steps:
[0007] (1) taking broken pine pollen and placing it in deionized water, stirring it evenly to prepare a pine pollen liquid culture medium;
[0008] (2) adding glucose to the pine pollen liquid culture medium in step (1), sterilizing the culture medium, and cooling the culture medium to room temperature after sterilization;
[0009] (3) adding a spore suspension of Mucor racemosus (10 7 CFU / mL), and fermented at 26°C and 180r / min for 5 days to obtain the fermentation product.
[0010] Furthermore, the racemosus is Mucor racemosus 40481. 40481).
[0011] Furthermore, the weight ratio of the pine pollen to deionized water is 1:20.
[0012] Furthermore, in the step (2), the mass fraction of glucose added is 2%, which means that glucose accounts for 2% of the mass of the pine pollen liquid culture medium.
[0013] Furthermore, the sterilization treatment in step (2) comprises the following steps: heating the pine pollen liquid culture medium with glucose added in a boiling water bath until the central temperature reaches 85° C., and placing it in a 85° C. water bath for 30 minutes for pasteurization.
[0014] Furthermore, the volume ratio of the Mucor racemosus to the pine pollen liquid culture medium is 1:10.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] The present invention utilizes Mucor racemosus to ferment pine pollen. Mucor racemosus is a filamentous fungus that can be used for food and contains a rich enzyme system. The present invention utilizes Mucor racemosus to ferment pine pollen. The fermentation can further release the cell active substances of the raw material substrate, free the small molecular active ingredients such as phenols and flavonoids that exist in a state of binding with macromolecules, further increase the polyphenol and flavonoid content of the pine pollen, and use an ethanol-induced HepG2 cell in vitro alcoholic fatty liver model to verify that its fermentation product has stronger liver-protecting and lipid-lowering activity, thereby providing theoretical guidance for the development of pine pollen products with higher liver-protecting activity. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1This is a graph showing the effect of pine pollen on the levels of TG, ALT and AST in HepG2 cells before and after fermentation in Example 2;
[0018] Figure 2 This is a graph showing the effect of pine pollen on the content of MDA, SOD, GPx and CAT in HepG2 cells before and after fermentation in Example 3. DETAILED DESCRIPTION
[0019] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.
[0020] Example 1
[0021] (1) placing broken pine pollen in deionized water and stirring evenly to prepare a pine pollen liquid culture medium, wherein the weight ratio of pine pollen to deionized water is 1:20;
[0022] (2) adding 2% by mass of glucose to the pine pollen liquid culture medium in step (1), heating the pine pollen liquid culture medium with glucose in a boiling water bath until the center temperature reaches 85° C., placing it in a 85° C. water bath for 30 minutes for pasteurization, and cooling it to room temperature after completion;
[0023] (3) Add Mucor racemosus to the sterilized pine pollen liquid culture medium in step (2) at a ratio of 10% of the inoculum. Spore suspension (10 7 CFU / mL), and fermented at 26°C and 180r / min for 5 days to obtain the fermentation product.
[0024] The contents of total phenols and total flavonoids before and after fermentation were determined and compared with the unfermented pine pollen water extract as control.
[0025] The total phenol content of the unfermented pine pollen water extract was 6.41±0.14mg / g, and the total flavonoid content was 14.14±1.26mg / g. The total phenol content of the pine pollen fermented by Mucor racemosus was 9.25±0.10mg / g, and the total flavonoid content was 28.46±0.19mg / g, which were 1.44 and 2.01 times of the unfermented ones (based on the dry weight of freeze-dried powder). The results showed that after the pine pollen was fermented by Mucor racemosus, polyphenols and flavonoids, these small molecular active substances, were further released, and their content was significantly increased.
[0026] Example 2: Effects of Pine Pollen Fermentation by Mucor racemosus on Fat and Liver Enzyme Content in Alcoholic Fatty Liver Cells
[0027] (1) placing broken pine pollen in deionized water and stirring evenly to prepare a pine pollen liquid culture medium, wherein the weight ratio of pine pollen to deionized water is 1:20;
[0028] (2) adding 2% by mass of glucose to the pine pollen liquid culture medium in step (1), heating the pine pollen liquid culture medium with glucose in a boiling water bath until the center temperature reaches 85° C., placing it in a 85° C. water bath for 30 minutes for pasteurization, and cooling it to room temperature after completion;
[0029] (3) Add Mucor racemosus to the sterilized pine pollen liquid culture medium in step (2) at a ratio of 10% of the inoculum. Spore suspension (10 7 CFU / mL), and fermented at 26°C and 180r / min for 5 days to obtain the fermentation product, which was freeze-dried for later use.
[0030] (4) HepG2 human liver cancer cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixture at 37°C and 5% CO2. When the cell density reached 80%, different grouping treatments were performed. Normal group: cultured in ordinary DMEM medium; Model group: cultured in medium containing 250mM anhydrous ethanol for 48h; Positive control: first prevented and intervened with normal medium containing 25μM resveratrol for 48h, and then cultured with medium containing 250mM anhydrous ethanol for 48h; Low-dose fermentation broth group: first prevented and intervened with normal medium containing 100μg / mL fermentation broth for 48h, and then cultured with medium containing 250mM anhydrous ethanol for 48h; High-dose fermentation broth group: first prevented and intervened with 200μg / mL fermentation broth mL concentration of fermentation liquid in normal culture medium for 48 hours, and then cultured in medium containing 250mM anhydrous ethanol for 48 hours; unfermented low-dose group: first used normal culture medium with 100μg / mL concentration of unfermented water extract for 48 hours, and then cultured in medium containing 250mM anhydrous ethanol for 48 hours; unfermented high-dose group: first used normal culture medium with 200μg / mL concentration of unfermented water extract for 48 hours, and then cultured in medium containing 250mM anhydrous ethanol for 48 hours.
[0031] (5) Cells were cultured at 2×10 5 The cells were inoculated into a six-well plate at a density of 100 cells / well and grouped according to (4). After the cells were lysed with IP lysis buffer, the supernatant was obtained and the intracellular TG, ALT and AST contents were determined according to the instructions of the BCA protein concentration assay kit, triglyceride (TG) test kit, alanine aminotransferase (ALT) test kit and aspartate aminotransferase (AST) test kit.
[0032] The results are as follows Figure 1As shown in the results, compared with the model group, both the low- and high-concentration fermentation groups could significantly reduce the TG, ALT, and AST content of cells (p<0.05), and the effect was better than that of the non-fermentation group; after the intervention of fermented pine pollen, the TG, ALT, and AST content could be restored to a level close to that of normal cells. The results showed that fermentation could significantly increase the liver-protecting and lipid-lowering effects of pine pollen on alcoholic fatty liver.
[0033] Example 3: Effects of pine pollen fermented by Mucor racemosus on lipid peroxidation and oxidoreductase content in alcoholic fatty liver cells
[0034] (1) placing broken pine pollen in deionized water and stirring evenly to prepare a pine pollen liquid culture medium, wherein the weight ratio of pine pollen to deionized water is 1:20;
[0035] (2) adding 2% by mass of glucose to the pine pollen liquid culture medium in step (1), heating the pine pollen liquid culture medium with glucose in a boiling water bath until the center temperature reaches 85° C., placing it in a 85° C. water bath for 30 minutes for pasteurization, and cooling it to room temperature after completion;
[0036] (3) Add Mucor racemosus to the pine pollen liquid culture medium in step (2) at a ratio of 10% of the inoculum. Spore suspension (10 7 CFU / mL), and fermented at 26°C and 180r / min for 5 days to obtain the fermentation product, which was freeze-dried for later use.
[0037] (4) HepG2 human liver cancer cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixture at 37°C and 5% CO2. When the cell density reached 80%, different grouping treatments were performed. Normal group: cultured in ordinary DMEM medium; Model group: cultured in medium containing 250mM anhydrous ethanol for 48h; Positive control: first prevented and intervened with normal medium containing 25μM resveratrol for 48h, and then cultured with medium containing 250mM anhydrous ethanol for 48h; Low-dose fermentation broth group: first prevented and intervened with normal medium containing 100μg / mL fermentation broth for 48h, and then cultured with medium containing 250mM anhydrous ethanol for 48h; High-dose fermentation broth group: first prevented and intervened with 200μg / mL fermentation broth mL concentration of fermentation liquid in normal culture medium for 48 hours, and then cultured in medium containing 250mM anhydrous ethanol for 48 hours; unfermented low-dose group: first used normal culture medium with 100μg / mL concentration of unfermented water extract for 48 hours, and then cultured in medium containing 250mM anhydrous ethanol for 48 hours; unfermented high-dose group: first used normal culture medium with 200μg / mL concentration of unfermented water extract for 48 hours, and then cultured in medium containing 250mM anhydrous ethanol for 48 hours.
[0038] (5) Cells were cultured at 2×10 5 The cells were inoculated into a six-well plate at a density of 100 cells / well. After grouping and culturing according to (4), the cells were lysed using IP lysis buffer and the supernatant was obtained. The cell MDA, SOD, GPx and CAT contents were determined according to the instructions of the BCA protein concentration assay kit, malondialdehyde (MDA) test kit, superoxide dismutase (SOD) test kit, glutathione peroxidase (GPx) and peroxidase (CAT) test kit.
[0039] The results are as follows Figure 2 As shown in the results, fermented pine pollen can significantly improve alcoholic fatty liver fat peroxidation and oxidative stress, increase the level of antioxidant enzymes, and the effect of fermented pine pollen is better than that of unfermented pine pollen. After the cells were intervened by fermented pine pollen, the MDA content was significantly reduced (p<0.05), and the SOD, GPx and CAT contents were significantly increased (p<0.05), and they were dose-dependent.
[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for improving the liver protection and lipid-lowering effects by fermenting pine pollen with Mucor racemosus, characterized in that: The steps include: (1) taking broken pine pollen and placing it in deionized water, stirring it evenly to prepare a pine pollen liquid culture medium; (2) adding glucose to the pine pollen liquid culture medium in step (1), sterilizing the culture medium, and cooling the culture medium to room temperature after sterilization; (3) adding a spore suspension of Mucor racemosus (10 7 CFU / mL), and fermented at 26°C and 180r / min for 5 days to obtain the fermentation product.
2. The method for improving the liver protection and lipid-lowering effect by fermenting pine pollen with Mucor racemosus according to claim 1, characterized in that: The Mucor racemosus is Mucor racemosus 40481.
3. The method for improving the liver protection and lipid-lowering effect by fermenting pine pollen with Mucor racemosus according to claim 2, characterized in that: The weight ratio of the pine pollen to deionized water is 1:
20.
4. The method for improving the liver protection and lipid-lowering effect by fermenting pine pollen with Mucor racemosus according to claim 1, characterized in that: In the step (2), the mass fraction of glucose added is 2%.
5. The method for improving the liver protection and lipid-lowering effects by fermenting pine pollen with Mucor racemosus according to claim 4, characterized in that: The sterilization treatment in step (2) comprises the following steps: heating the pine pollen liquid culture medium with glucose added in a boiling water bath until the center temperature reaches 85° C., and placing it in a water bath at 85° C. for 30 minutes for pasteurization.
6. The method for improving the liver protection and lipid-lowering effects by fermenting pine pollen with Mucor racemosus according to any one of claims 1 to 5, characterized in that: The volume ratio of the Mucor racemosus to the pine pollen liquid culture medium is 1:10.