Preparation method and application of probiotic fermented black fungus extract

Abstract

The invention discloses a preparation method and application of a probiotic fermented black fungus extract and belongs to the technical field of biological medicine, and the probiotic fermented black fungus extract is prepared by fermenting black fungus through mixed bacteria of casei casei and lactobacillus plantarum. The extract can effectively improve oxidative damage of H9c2 myocardial cells induced by high glucose, and compared with an unfermented black fungus extract, the probiotic fermented black fungus extract shows better cell protection activity under the same concentration, the action mechanism is more comprehensive and systematic, and the probiotic fermented black fungus extract can be used as a probiotic fermented black fungus extract. A new natural medicine candidate and an effective solution are provided for preventing and treating diabetic cardiomyopathy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a method for preparing probiotic fermented black fungus extract and its application. Background Technology

[0002] Currently, heart disease has become one of the leading causes of death worldwide. The occurrence of diabetic cardiomyopathy is closely related to oxidative damage to myocardial cells under hyperglycemic conditions. This damage not only affects the normal function of myocardial cells, but may also lead to long-term damage to the structure and function of the heart. Diabetic patients are often accompanied by hyperglycemia, and a hyperglycemic environment is one of the important pathophysiological mechanisms of diabetic cardiomyopathy.

[0003] Black fungus, a common edible and medicinal fungus, has been used to prevent various diseases since ancient times. Modern research shows that the polysaccharides, proteins, trace elements and other components in black fungus have a variety of biological activities such as anticoagulation, lipid reduction, anti-inflammation and anti-oxidation, which have a protective effect on the cardiovascular system. In particular, in terms of anti-oxidation, the melanin in black fungus can effectively scavenge free radicals, reduce oxidative stress and protect myocardial cells from damage.

[0004] The metabolites produced by probiotics during the fermentation process can improve their active ingredients, thereby enhancing their pharmacological effects. In addition, probiotics can also regulate the balance of intestinal flora and enhance the body's immunity, providing a new perspective for improving heart disease. Summary of the Invention

[0005] To address the above problems, the purpose of this invention is to provide a method for preparing probiotic fermented black fungus extract and its application.

[0006] To achieve the above objectives, the present invention employs the following technical solution: A method for preparing a probiotic fermented black fungus extract, wherein the probiotic fermented black fungus extract is prepared by fermenting black fungus with a mixed bacteria of Lactobacillus casei and Lactobacillus plantarum.

[0007] The probiotic fermented black fungus extract was prepared according to the following method: 1) After cleaning and drying the black fungus, add sterile water and sterilize at 121℃ for 30 minutes to obtain a mixture; 2) After cooling the mixture obtained in step 1), add white sugar to it in a clean bench to obtain a fermentation base liquid. Activate the mixed bacterial powder of Lactobacillus casei and Lactobacillus plantarum in 0.9% physiological saline to obtain a mixed bacterial solution. Add the mixed bacterial solution to the fermentation base liquid. 3) Seal fermentation. After fermentation, centrifuge at 4℃ and 8000rpm for 15min, take the supernatant, filter it with a 0.22μm microporous membrane for sterilization, collect the filtrate, and obtain probiotic fermented black fungus extract.

[0008] The mass-to-volume ratio of black fungus to sterile water in step 1) is 1:10.

[0009] The mass ratio of *Lactobacillus casei* to *Lactobacillus plantarum* in step 2) is 1:1.

[0010] The amount of white sugar added in step 2) is 2% of the total mass of black fungus and sterile water in step 1); the inoculation amount of the mixed bacterial solution is 2% of the fermentation base liquid volume, and the concentration of the mixed bacterial solution is 1×10⁻⁶. 7 CFU / mL.

[0011] The sealed fermentation temperature in step 3) is 28℃, and the fermentation time is 3 days.

[0012] The probiotic fermented black fungus extract described in step 3) is frozen and stored at -20°C.

[0013] The present invention also includes the application of probiotic fermented black fungus extract in the preparation of drugs to improve myocardial cell oxidative damage.

[0014] The oxidative damage to the cardiomyocytes was high glucose-induced oxidative damage to H9c2 cardiomyocytes.

[0015] The probiotic fermented black fungus extract enhances the activity of intracellular SOD, GSH-Px, and GSH antioxidant enzymes, reduces MDA content, inhibits the release of LDH, CK, and troponin I, markers of myocardial injury, and downregulates the expression of IL-1β, IL-6, and TNF-α inflammatory factors.

[0016] The effective concentration of the probiotic fermented black fungus extract in the preparation of drugs to improve myocardial cell oxidative damage is 160 μg / mL to 640 μg / mL.

[0017] The present invention has the following advantages over the prior art: This invention relates to a probiotic-fermented black fungus extract, which innovatively employs a mixed fermentation of *Lactobacillus casei* and *Lactobacillus plantarum* to successfully prepare a fermented extract with significant cardioprotective effects. Verification has shown that this extract effectively improves high-glucose-induced oxidative damage to H9c2 cardiomyocytes. Its mechanism of action is achieved through multi-target synergy: on the one hand, it significantly enhances the activity of intracellular antioxidant enzymes such as SOD and GSH-Px, and reduces MDA content; on the other hand, it effectively inhibits the release of myocardial injury markers such as LDH, CK, and troponin I, while downregulating the levels of key inflammatory factors such as IL-1β, IL-6, and TNF-α. Compared with unfermented black fungus extract, this probiotic-fermented black fungus extract exhibits superior cell-protective activity at the same concentration, and its mechanism of action is more comprehensive and systematic, providing a new natural drug candidate and effective solution for the prevention and treatment of diabetic cardiomyopathy. Attached Figure Description

[0018] Figure 1 The effect of different concentrations of high glucose on the survival rate of H9c2 cardiomyocytes (n=6); Figure 2 To study the effects of different concentrations of black fungus extract (BF) and probiotic-fermented black fungus extract (FBF) on... Effect of H9c2 on cardiomyocyte viability (n=6); Figure 3 The effect of different concentrations of black fungus extract (BF) and probiotic-fermented black fungus extract (FBF) on the viability of high glucose-induced H9c2 cardiomyocytes (n=6). Figure 4 The effect of different concentrations of black fungus extract (BF) and probiotic-fermented black fungus extract (FBF) on the levels of SOD, MDA, GSH and GSH-PX in high glucose-induced H9c2 cardiomyocytes (n=6). Figure 5 Figure 1 shows the effects of different concentrations of black fungus extract (BF) and probiotic-fermented black fungus extract (FBF) on the levels of LDH, CK and troponin I in high glucose-induced H9c2 cardiomyocytes (n=6). Figure 6 The effect of different concentrations of black fungus extract (BF) and probiotic-fermented black fungus extract (FBF) on the levels of IL-6, IL-1β and TNF-α in high glucose-induced H9c2 cardiomyocytes (n=6). Detailed Implementation

[0019] To better understand the technical solution of the present invention, the following detailed embodiments further illustrate the above-mentioned content of the present invention. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention.

[0020] In the following examples, black fungus was purchased from Jilin Heizun Food Co., Ltd.; *Lactobacillus casei* and *Lactobacillus plantarum* were purchased from Zhongke Jiayi; rat cardiomyocyte H9c2 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; MTT assay was purchased from McEly Biotech, Inc. (USA); CK, troponin I, and LDH kits were purchased from Shanghai Yaji Biotechnology Co., Ltd.; GSH-Px, GSH, SOD, and MDA kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.; inflammatory factor detection kits were purchased from Shanghai Enzyme Immunosorbent Assay Biotechnology Co., Ltd.; trypsin and PBS buffer were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; DMEM, high glucose culture medium, and fetal bovine serum were purchased from Gibco; and six-well plates were purchased from Thermo Fisher Scientific.

[0021] Preparation of black fungus extract (BF): Select black fungus with intact appearance, dark black color and uniform texture, wash and dry it. Add sterile water to the dried black fungus at a ratio of 1:10 (w / v) and soak it in an environment of 20℃ for 3 days. Filter the black fungus extract, collect the filtrate and store it in sterile containers.

[0022] Example 1: Preparation of Probiotic Fermented Black Fungus Extract (FBF) 1) After washing and drying 60g of black fungus, add 600ml of sterile water and sterilize at 121℃ for 30min to obtain a mixture; 2) After cooling the mixture obtained in step 1), add 2% by weight of white sugar in a clean bench to obtain the fermentation base liquid. Mix *Lactobacillus casei* and *Lactobacillus plantarum* at a mass ratio of 1:1 to obtain 0.1g of mixed bacterial powder. Activate and revive the mixed bacterial powder with 0.9% sterile physiological saline, and dilute and adjust the bacterial concentration to 1×10⁻⁶. 7 CFU / mL was used to obtain a mixed bacterial solution, which was then added to the fermentation base liquid at a volume ratio of 2%. 3) Seal and ferment at 28℃ for 3 days. After fermentation, centrifuge at 4℃ and 8000rpm for 15min, take the supernatant, filter and sterilize using a 0.22μm microporous membrane, collect the filtrate, and obtain probiotic fermented black fungus extract. After aliquoting, freeze and store at -20℃.

[0023] Example 2: Establishment of a high-glucose-induced oxidative damage model in H9c2 cardiomyocytes H9c2 cardiomyocytes were passaged and seeded in new culture dishes for high glucose treatment. A gradient concentration of glucose solution was added to DMEM medium to simulate a hyperglycemic state. Cells were cultured in the high glucose medium for 24 hours. Cell viability was then assessed using the MTT assay to evaluate the survival of cardiomyocytes under high glucose conditions. The results are shown below. Figure 1 As shown, by Figure 1 It can be seen that as glucose concentration increases, cell viability decreases significantly, and its IC50 value also decreases. 50 The value was 33.14 ± 0.94 mmol / L. Therefore, subsequent experiments selected a glucose concentration of 33 mmol / L for 24 hours to establish a stable H9c2 cardiomyocyte oxidative damage model.

[0024] Example 3: Determination of the safe concentration range of probiotic-fermented black fungus extract for normal H9c2 cells H9c2 cardiomyocytes were used at 5×10 3 Cells were seeded at a density of [number] cells / well in 96-well plates and treated with different concentrations of FBF and BF for 48 hours after cell adhesion. Subsequently, 180 μL of serum-free culture medium and 20 μL of MTT solution were added to each well, and the cells were incubated at 37°C in the dark for 4 hours. After incubation, the supernatant was removed, and 150 μL of DMSO was added to each well to dissolve the formed metronidazole crystals. The absorbance (A value) of each well was measured at 570 nm using a microplate reader. Cell viability was calculated using the following formula: (A experimental group - A blank group) / (A control group - A blank group) × 100% to evaluate the effect of FBF and BF treatment on the viability of H9c2 cardiomyocytes. The results are shown below. Figure 2 As shown, from Figure 2 As can be seen, neither extract showed significant cytotoxicity within the tested concentration range. Among them, the probiotic fermented black fungus extract had a slight promoting effect on cell viability at concentrations of 160 μg / mL and 640 μg / mL. Therefore, 160 μg / mL (low dose) and 640 μg / mL (high dose) were selected as the effective concentrations for subsequent experiments.

[0025] Example 4: Verification of the protective effect of probiotic-fermented black fungus extract on model cells H9c2 cells were randomly divided into the following groups: Control group (CN): cultured in normal culture medium containing 5.5 mmol / L glucose.

[0026] High glucose group (MN): cultured in a high glucose medium containing 33 mmol / L glucose.

[0027] BF low-dose group (BF-L): 33 mmol / L high glucose culture medium + 160 μg / mL black fungus extract.

[0028] BF high-dose group (BF-H): 33 mmol / L high glucose culture medium + 640 μg / mL black fungus extract.

[0029] Low-dose FBF group (FBF-L): 33 mmol / L high-glucose medium + 160 μg / mL probiotic fermentation Black fungus extract.

[0030] High-dose FBF group (FBF-H): 33 mmol / L high-glucose medium + 640 μg / mL probiotic fermentation Black fungus extract.

[0031] After 24 hours of treatment, each group underwent the following tests: 1) Cell viability: The detection method was the same as in Example 2. The detection results are as follows: Figure 3 As shown, by Figure 3 It can be seen that, compared with the high glucose group, both the high and low dose FBF groups can significantly improve cell viability (P<0.05), and the effect is better than that of the corresponding concentration of BF group.

[0032] 2) Oxidative stress indicators: Cell supernatant or lysate was collected, and the activities or contents of superoxide dismutase (SOD), malondialdehyde (MDA), reduced glutathione (GSH), and glutathione peroxidase (GSH-Px) were measured strictly according to the instructions of the kits. The results are as follows: Figure 4 As shown, by Figure 4 It can be seen that FBF can significantly reverse the decrease in SOD, GSH-Px activity and GSH content and the increase in MDA content caused by high sugar (P<0.05).

[0033] 3) Markers of myocardial injury: Cell supernatant was collected, and the activities or concentrations of lactate dehydrogenase (LDH), creatine kinase (CK), and troponin I (cTnI) were detected using a kit. The results are as follows: Figure 5 As shown, by Figure 5 It can be seen that FBF can significantly inhibit the release of LDH, CK and troponin I induced by high glucose (P<0.05).

[0034] 4) Inflammatory factor levels: Cell supernatant was collected, and the concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were detected using an ELISA kit. The results are as follows: Figure 6 As shown, by Figure 6As shown, FBF can significantly reduce the levels of these inflammatory factors in a high-glucose environment (P<0.05).

[0035] The results above indicate that FBF can effectively improve cell survival, reduce oxidative stress indicators, and decrease the levels of myocardial damage and inflammatory factors based on behavioral indicators and the use of aura-boosting marker genes. Compared with the control group, FBF treated with probiotics significantly improved the survival rate of H9c2 cardiomyocytes under high glucose conditions and reduced intracellular reactive oxygen species levels, thus reducing oxidative stress damage. Furthermore, probiotic-fermented black fungus extract significantly reduced MDA and GSH levels, suggesting that it may alleviate oxidative damage by reducing lipid peroxidation and increasing glutathione levels. Therefore, this invention provides a scientific basis for developing new strategies for the prevention and treatment of heart disease and opens up new avenues for the modern application of traditional Chinese medicinal materials that are both food and medicine.

[0036] While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A method for preparing a fermented black fungus extract with probiotics, characterized by: The probiotic fermented Auricularia auricula extract is prepared by fermenting Auricularia auricula with mixed bacteria of Lactobacillus casei and Lactobacillus plantarum.

2. The method of claim 1, wherein the preparation of the probiotic fermented black fungus extract is characterized by: The probiotic fermented Auricularia auricula extract is prepared according to the following method: 1) After Auricularia auricula is washed and dried, sterile water is added, and sterilization is carried out at 121 DEG C for 30 min to obtain a mixture; 2) After the mixture obtained in step 1) is cooled, white granulated sugar is added in an ultra-clean bench to obtain a fermentation base liquid, Lactobacillus casei and Lactobacillus plantarum mixed bacteria powder are activated in 0.9% physiological saline to obtain a mixed bacteria liquid, and the mixed bacteria liquid is added to the fermentation base liquid; 3) Sealed fermentation, after the fermentation is completed, centrifugation is carried out at 4 DEG C and 8000 rpm for 15 min, the supernatant is taken, 0.22 mu m microporous filter membrane is used for filtration and sterilization, the filtrate is collected, and the probiotic fermented Auricularia auricula extract is obtained.

3. The method of claim 2, wherein the fermented black fungus extract is prepared by fermenting the black fungus extract with the probiotic bacteria. In step 1), the mass-volume ratio of Auricularia auricula to sterile water is 1:

10.

4. The preparation method of probiotic fermented black fungus extract as described in claim 2, characterized in that: In step 2), the mass ratio of Lactobacillus casei to Lactobacillus plantarum is 1:

1.

5. The method for preparing probiotic fermented black fungus extract as described in claim 2, characterized in that: The amount of white sugar added in step 2) is 2% of the total mass of black fungus and sterile water in step 1); the inoculation amount of the mixed bacteria solution is 2% of the volume of the fermentation base liquid, and the concentration of the mixed bacteria solution is 1 x 10 7 CFU / mL.

6. The method of claim 2, wherein the preparation of the probiotic fermented Auricularia auricula-judae extract is characterized by: In step 3), the sealed fermentation temperature is 28 DEG C, and the fermentation time is 3 days; in step 3), the probiotic fermented Auricularia auricula extract is stored at -20 DEG C.

7. Use of the probiotic fermented Auricularia auricula extract according to claim 1, characterized in that: In the preparation of a drug for improving oxidative damage of myocardial cells.

8. The use of a probiotic fermented Auricularia auricula extract according to claim 7, characterized in that: The oxidative damage of myocardial cells is high-glucose-induced oxidative damage of H9c2 myocardial cells.

9. The use of a probiotic fermented Auricularia auricula extract according to claim 8, characterized in that: The probiotic fermented Auricularia auricula extract is an extract for improving the antioxidant enzyme activities of intracellular SOD, GSH-Px and GSH, reducing the content of MDA, inhibiting the release of LDH, CK and troponin I myocardial injury markers, and down-regulating the expression of IL-1beta, IL-6 and TNF-alpha inflammatory factors.

10. The use of the probiotic fermented Auricularia auricula extract according to claim 7, characterized in that: The effective concentration of the probiotic fermented Auricularia auricula extract in the preparation of a drug for improving oxidative damage of myocardial cells is 160 mu g / mL ~ 640 mu g / mL.

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