Preparation method and application of ganoderma lucidum and inonotus obliquus co-fermentation liquid extract

Abstract

The invention discloses a preparation method and application of a ganoderma lucidum and inonotus obliquus co-fermentation liquor extract. The ganoderma lucidum and inonotus obliquus co-fermentation liquor extract is prepared by co-culturing ganoderma lucidum and inonotus obliquus and extracting the fermentation liquor with ethanol and ethyl acetate. The ganoderma lucidum and inonotus obliquus co-fermentation liquid extract has the characteristics of remarkably inhibiting protein glycosylation and relieving human skin fibroblast senescence induced by advanced glycosylation end products.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to a preparation method of a ganoderma lucidum and inonotus obliquus co-fermentation liquid extract and application thereof. Background Art

[0002] "Glycation" is the process of attaching sugar molecules to proteins or lipid molecules without the control of enzymes. Studies have found that the continuous accumulation of glycation end products in the skin reduces skin elasticity and leads to skin aging, which is the result of a combination of various factors in the body. Advanced Glycation End Products (AGEs) are one of the key biomarkers of aging. Their accumulation in low metabolically active tissue proteins is closely related to aging. This accumulation can lead to damage to protein structure and function. Effectively inhibiting the level of glycation in the body can not only improve a variety of disease states, but also has the potential to delay biological aging. In recent years, as "anti-sugar" products have become more and more popular in the market, the research and development of AGEs has become a hot topic. The currently commonly used AGE inhibitor aminoguanidine (AG) has serious problems such as large side effects and limited applicable populations. It is urgent to develop safe and effective natural AGE inhibitors.

[0003] Inonotus obliquus Inonotus obliquus ), also known as Chaga, belongs to the genus Porites of the Polyporaceae family, and has been shown to have multiple effects such as hypoglycemic, lipid-lowering, anti-tumor, antiviral and antioxidant. The latest research shows that the content and bioavailability of active ingredients such as polysaccharides, triterpenes, steroids, polyphenols and flavonoids can be effectively improved through fungal co-culture fermentation technology. The applicant conducted a screening test on the activity of co-cultured fermentation broths of edible and medicinal fungi such as birch mushrooms, bright mushrooms, Ganoderma lucidum, mulberry linterus, cicada fungus and Cordyceps sinensis, and found that the extracts of the co-fermentation broth of Ganoderma lucidum and birch mushrooms have excellent anti-glycosylation and aging activity. There is currently no report on the anti-glycosylation effect of this extract. Summary of the invention

[0004] The present invention aims to provide a method for preparing an extract of a co-fermentation broth of Ganoderma lucidum and Inonotus obliquus and its application. The obtained IGE-e can significantly inhibit non-enzymatic glycosylation in vitro, effectively reduce the AGEs-induced senescent HFF (human skin fibroblast) β-galactosidase (SA-β-gal) staining positive cell rate, and can be used to prepare a new natural anti-glycation preparation.

[0005] The method for preparing the extract of the co-fermentation liquid of Ganoderma lucidum and Inonotus obliquus of the present invention comprises the following steps: Step 1: Inonotus obliquus mycelium culture 1-1 Activation of strains: The mother strain of Inonotus birchii was inoculated into PDA solid culture medium and cultured at 28°C for 7-10 days.

[0006] 1-2 Liquid fermentation culture: Cut 8-10 pieces of 0.5 × 0.5 cm from the activated Inonotus birchii solid culture medium. 2 The fungus block was inoculated into 100 mL of liquid culture medium of Inonotus birchii and cultured at 28°C and 120 r / min for 6-8 days.

[0007] The liquid culture medium of Inonotus obliquus consists of 1.0 L of potato boiling liquid, 20 g / L of glucose, 3 g / L of potassium dihydrogen phosphate, 1.5 g / L of anhydrous magnesium sulfate, and 0.03 g / L of vitamin B1. The specific preparation process of potato boiling liquid is as follows: 200 g of peeled potato pieces are boiled in boiling water for 30 min, and the filtrate is collected and fixed to 1.0 L.

[0008] Step 2: Ganoderma lucidum mycelium culture 2-1 Activation of strains: Ganoderma lucidum mother strains were inoculated into PDA solid culture medium and cultured at 28°C for 7-10 days.

[0009] 2-2 Liquid fermentation culture: Cut 8-10 pieces of 0.5 × 0.5 cm from the activated Ganoderma lucidum solid culture medium. 2 The fungus block was inoculated into 100 mL of Ganoderma lucidum mycelium liquid culture medium and cultured at 28°C and 120 r / min for 6-8 days.

[0010] The liquid culture medium for Ganoderma mycelium consists of 30 g / L glucose, 3 g / L yeast powder, 1.5 g / L potassium dihydrogen phosphate, 0.75 g / L anhydrous magnesium sulfate, and 0.03 g / L vitamin B1.

[0011] Step 3: Co-fermentation The bacterial solution obtained in step 2 was added to the bacterial solution in step 1 at an inoculation amount of 2-5% (w / w). The mixture was cultured at 28-30°C and 120 r / min for 14 days. The fermentation liquid was collected and centrifuged at 6000 rpm for 5-10 min. The supernatant was collected and freeze-dried to obtain co-fermentation liquid powder.

[0012] Step 4: Add 95% ethanol to the co-fermentation liquid powder obtained in step 3 at a solid-liquid ratio of 1:10-20 (g / mL), reflux extraction at 85°C for 60-120 min, and filter and collect the filtrate.

[0013] Step 5: The filtrate obtained in step 4 was concentrated to 1 / 9-1 / 10 of the original volume, centrifuged at 6000 rpm, the supernatant was collected, freeze-dried, and pure water was added at a solid-liquid ratio of 1:15-20 (g / mL) for ultrasonic dissolution, and graded extraction was performed by adding 2-3 times the volume of petroleum ether, ethyl acetate, and n-butanol in sequence. The extraction time for each extraction was 10-20 min. The ethyl acetate extract phase was concentrated and dried to obtain the ethyl acetate extract (IGE-e).

[0014] The invention discloses an application of the extract of the co-cultured fermentation liquid of Ganoderma lucidum and Inonotus obliquus in the preparation of an anti-glycosylation preparation.

[0015] The anti-glycosylation preparation has excellent anti-glycosylation aging efficacy, can effectively inhibit protein glycosylation, and significantly reduce the SA-β-gal staining positive cell rate of senescent HFF cells induced by advanced glycation end products (AGEs).

[0016] The beneficial effects of the present invention are embodied in: 1. A method for preparing an extract of a co-cultured fermentation broth of Ganoderma lucidum and Inonotus obliquus is provided, which has the characteristics of simple operation, low cost, high efficiency, etc.

[0017] 2. The extract of the co-fermentation broth of Ganoderma lucidum and Inonotus obliquus of the present invention is used to prepare an anti-glycosylation preparation. The preparation has an outstanding anti-glycosylation aging effect, can significantly inhibit protein glycosylation, and delay AGEs-induced HFF cell senescence. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the ITS species identification report of Ganoderma lucidum fungus.

[0019] Figure 2 The effects of co-culture fermentation broth and single culture fermentation broth on the formation of AGEs in the in vitro non-enzymatic glycosylation inhibition test. Figure 2 It can be seen that each co-culture fermentation broth has a certain inhibitory effect on the production of AGEs, among which the inhibitory effect of Inonotus obliquus-Ganoderma lucidum fermentation broth is the best, and the activity is significantly improved compared with the single culture of Inonotus obliquus and Ganoderma lucidum fermentation broth.

[0020] Figure 3 This is the effect of various extracts from the co-fermentation broth of Inonotus obliquus and Ganoderma lucidum on the formation of AGEs in the in vitro inhibition of non-enzymatic glycation test. Figure 3 The experimental results showed that the inhibitory activity of the ethyl acetate extract of Inonotus obliquus-Ganoderma lucidum fermentation broth (IGE-e) on AGEs was close to that of the positive control group.

[0021] Figure 4 , Figure 5 The anti-glycosylation senescence activity of the ethyl acetate extract of Inonotus obliquus-Ganoderma lucidum co-fermentation broth (IGE-e) on senescent HFF cells. Figure 4 , 5 The results showed that compared with the control group, IGE-e significantly reduced the rate of senescent blue-stained cells in a dose-dependent manner. DETAILED DESCRIPTION

[0022] The present invention is further described below with reference to specific embodiments. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

[0023] The Inonotus obliquus species used in the present invention was purchased from Beina Biological and is Inonotus obliquus (Ach.ex Pers.) Pilát, the strain bank number is BNCC117822.

[0024] The Lingzhi strain used was obtained from Hefei Lixin Strain Farm and tested by General Biotechnology (Anhui) Co., Ltd. to be Sichuan Lingzhi Ganoderma Sichuanense (Ganoderma lucidum ITS species identification report can be found at Figure 1 ). Example 1: Preparation of extract from co-fermentation of Inonotus obliquus and Ganoderma lucidum

[0025] Step 1: Inonotus obliquus mycelium culture Activation of the strain: inoculate the mother strain of Inonotus birchii on PDA solid culture medium and culture it at 28℃ for 7-10 days; Liquid fermentation culture: cut 8-10 pieces of 0.5 × 0.5 cm from the activated solid culture medium of Inonotus birchii 2 The fungus block was inoculated into 100 mL of Inonotus birchii liquid culture medium and cultured at 28°C and 120 r / min for 6-8 days; the composition of the Inonotus birchii liquid culture medium was: 1.0 L of potato boiling liquid, 20 g / L of glucose, 3 g / L of potassium dihydrogen phosphate, 1.5 g / L of anhydrous magnesium sulfate, and 0.03 g / L of vitamin B1. The specific preparation process of potato boiling liquid was as follows: 200 g of peeled potato blocks were boiled in boiling water for 30 min, and the filtrate was collected and fixed to 1.0 L.

[0026] Step 2: Ganoderma mycelium culture Activation of strains: inoculate the mother strain of Ganoderma lucidum on PDA solid culture medium and culture at 28℃ for 7-10 days; liquid fermentation culture: cut 8-10 pieces of 0.5 × 0.5 cm from the activated Ganoderma lucidum solid culture medium 2 The fungus block was inoculated into 100 mL of Ganoderma lucidum liquid culture medium and cultured at 28℃ and 120 r / min for 6-8 days; the Ganoderma lucidum mycelium liquid culture medium consisted of: 30 g / L glucose, 3 g / L yeast powder, 1.5 g / L potassium dihydrogen phosphate, 0.75 g / L anhydrous magnesium sulfate, and 0.03 g / L vitamin B1.

[0027] Step 3: Co-fermentation Add the bacterial solution obtained in step 2 to the bacterial solution in step 1, with an inoculation amount of 2-5% (w / w), and culture at 28-30°C and 120 r / min for 14 days; collect the fermentation liquid, centrifuge at 6000 rpm for 5-10 min, collect the supernatant, and freeze-dry to obtain the co-fermentation liquid powder; Step 4: Add 95% ethanol to the co-fermentation broth powder in step 3 at a solid-liquid ratio of 1:10-20 (g / mL), reflux extraction at 85°C for 60-120 min, and collect the filtrate; Step 5: The filtrate obtained in step 4 is concentrated to 1 / 9-1 / 10 of the original volume, centrifuged at 6000 rpm, the supernatant is collected, freeze-dried, and pure water is added at a solid-liquid ratio of 1:15-20 (g / mL) for ultrasonic dissolution, and 2-3 volumes of petroleum ether, ethyl acetate, and n-butanol are added in sequence for graded extraction. The specific operation is as follows: after adding petroleum ether and vigorous shaking, stand for stratification, remove the lower aqueous phase, repeat 3 times, combine the upper organic phase, and obtain a petroleum ether extract (IGE-p) after drying. The remaining aqueous phase is extracted with ethyl acetate to obtain an ethyl acetate extract (IGE-e), and the remaining aqueous phase is further extracted with n-butanol to obtain an n-butanol extract (IGE-n). Finally, the remaining phase is combined with the filter residue in step 4, and dried to obtain an alcohol extraction residue (IG-w). Comparative Example 1: Preparation of Inonotus obliquus-Argyrus fermentation liquid

[0028] Step 1: Cut a 2 × 1 cm piece from the solid culture medium 2 The bacterial block was inoculated into 500 mL of a liquid culture medium for bright bacteria, and cultured at 28° C. for 10-12 days; the composition of the liquid culture medium for bright bacteria was: 25 g / L glucose and 200 g / L potato.

[0029] Step 2: Cut 3-5 pieces of 0.5 × 0.5 cm from the activated Inonotus birchii solid culture medium. 2 The fungus block was inoculated into 100 mL of liquid culture medium of Inonotus birchii and cultured at 28°C and 120 r / min for 6-8 days.

[0030] Step 3: Add the inonotus birchii seed liquid obtained in step 2 to the sclerotium seed liquid obtained in step 1, the added amount being 3% of the volume of the sclerotium seed liquid, and culture at 28°C and 120r / min for 12-16 days. Collect the fermentation liquid, centrifuge at 6000 rpm for 5-10min, and freeze-dry the supernatant to obtain the co-fermentation liquid of inonotus birchii and sclerotium. Comparative Example 2: Preparation of Inonotus obliquus-Cordyceps sinensis fermentation liquid

[0031] Step 1: Cut 8-10 pieces of 0.5 × 0.5 cm from the activated Inonotus birchii solid culture medium. 2The fungus block was inoculated into 100 mL of liquid culture medium of Inonotus birchii and cultured at 28°C and 120 r / min for 6-8 days. Step 2: Cut 8-10 pieces of 0.5 × 0.5 cm from the activated Cordyceps sinensis solid culture medium 2 The fungus block was inoculated into 100 mL of liquid culture medium of Cordyceps sinensis and cultured at 28℃ and 120 r / min for 6-8 days; the liquid culture medium of Cordyceps sinensis mycelium consisted of 30 g / L glucose, 3 g / L yeast powder, 1.5 g / L potassium dihydrogen phosphate, 0.75 g / L anhydrous magnesium sulfate, and 0.03 g / L vitamin B1.

[0032] Step 3: Add the bacterial solution obtained in step 2 to the bacterial solution in step 1, with an inoculation amount of 2-5% (w / w), and culture at 28-30°C and 120 r / min for 14 days; collect the fermentation liquid, centrifuge at 6000 rpm for 5-10 min, and freeze-dry the supernatant to obtain the co-fermentation liquid of Inonotus obliquus and Cordyceps sinensis. Example 2: In vitro non-enzymatic glycosylation inhibition test of Example 1, Comparative Example 1 and Comparative Example 2

[0033] Experimental steps: The BSA-glucose in vitro non-enzymatic glycosylation reaction model was established. BSA (30 mg / mL) and glucose solution (300 mg / mL) were prepared in 0.2 mol / L PBS (pH 7.4, 0.2% proclin 300 as a preservative). The concentrations of aminoguanidine (AG) control group, IGE-p, IGE-e, IGE-n, and IG-w were all 0.5 mg / mL. In the clean bench, BSA, glucose, and IGE components were filtered through a 0.22 μm filter membrane and mixed in equal volumes. No IGE components or AG were added as negative controls. Glucose in the sample group and control group was replaced with PBS as the corresponding blank control for each group. The cells were cultured at 37°C for 10 days, and the culture medium was collected on days 1, 3, 5, 7, and 10. The fluorescence value in the culture medium was measured using a fluorescence spectrophotometer at excitation / emission wavelengths (ex / em) of 370 / 440 nm and 335 / 385 nm. The inhibition rate of each component of IGE or AG on fluorescent AGEs was calculated according to the following formula. = × 100% Where: A1 is the fluorescence value of the negative control; A0 is the fluorescence value of the group containing only BSA (bovine serum albumin); B1 is the fluorescence value of each component or AG group after adding IGE; B0 is the fluorescence value of the blank control group after adding each component of IGE or AG group. Example 3: Anti-glycosylation senescence activity on human skin fibroblasts (HFF)

[0034] Experimental method: Observe by staining of senescence-associated β-galactosidase (SA-β-gal) and detect by cell senescence β-galactosidase staining kit.

[0035] The specific operations are: AGEs co-reaction solution was prepared in 0.2 mol / L PBS: BSA 20 g / L, glucose 100 g / L. Filtered through a 0.22 μm membrane, incubated at 37°C under sealed conditions for 3 months. The brown reaction mixture was dialyzed against 0.01 mol / mL PBS overnight, and the unincorporated glucose was removed by the dialysis membrane to obtain AGEs. The AGEs-specific fluorescence was measured by a fluorescence spectrophotometer (excitation wavelength / emission wavelength of 370 / 440 nm), and the AGEs concentration was calculated based on the fluorescence value.

[0036] HFF cells were seeded in culture plates. After the cells adhered to the wall, the culture medium was aspirated and culture medium containing 200 μmol / mL D-gal was added for three passages. The cell concentration was diluted to 6 × 10 5 The cells were inoculated in a 6-well plate and culture medium containing 1 mg / mL AGEs was added. After the cells adhered to the wall, the culture medium was aspirated. The normal group (control), model group (AGEs 1 mg / mL) and IGE-e group (AGEs 1mg / mL + IGE-e 200, 400, 600 μg / mL) were set up, and the culture medium containing the corresponding concentration of IGE-e was added to each well for further culture for 24 hours. After the culture was completed, the cell culture medium was aspirated, the cells were washed twice with PBS, 1 mL of β-galactosidase staining fixative was added, and the cells were fixed at room temperature for 15 minutes. The fixative was discarded, and the cells were washed 3 times with PBS for 2 minutes each time. The PBS was aspirated, and 1 mL of β-galactosidase staining working solution was added to each well. The cells were incubated at 37℃ for 2 hours to overnight. The blue-stained cells were counted under an inverted microscope. The number of blue-stained cells / total cells was the positive rate.

Claims

1. A method for preparing an extract of a co-fermentation broth of Ganoderma lucidum and Inonotus obliquus, characterized in that The steps include: Step 1: Inonotus obliquus mycelium culture 1-1 Activation of strains: Inoculate the mother strain of Inonotus obliquus into PDA solid culture medium and culture it at 28°C for 7-10 days; 1-2 Liquid fermentation culture: Cut the bacterial blocks from the activated Inonotus birchii solid culture medium, inoculate them into the Inonotus birchii liquid culture medium, and culture them at 28°C and 120 r / min for 6-8 days; Step 2: Ganoderma mycelium culture 2-1 Activation of strains: Inoculate the mother strain of Ganoderma lucidum into PDA solid culture medium and culture it at 28℃ for 7-10 days; 2-2 Liquid fermentation culture: Cut the fungus blocks from the activated Ganoderma lucidum solid culture medium, inoculate them into the Ganoderma lucidum mycelium liquid culture medium, and culture them at 28°C and 120 r / min for 6-8 days; Step 3: Co-fermentation Add the bacterial solution obtained in step 2 to the bacterial solution in step 1, culture at 28-30°C and 120 r / min for 14 days, collect the fermentation liquid, collect the supernatant by centrifugation, and freeze-dry to obtain the co-fermentation liquid powder; Step 4: Add the co-fermentation liquid powder obtained in step 3 into 95% ethanol, reflux and extract at 85°C for 60-120 min, and filter and collect the filtrate; Step 5: The filtrate obtained in step 4 is concentrated to 1 / 9-1 / 10 of the original volume, the supernatant is collected by centrifugation, freeze-dried, added with pure water for ultrasonic dissolution, and then graded extraction is performed. The extract phase is concentrated and dried to obtain the extract.

2. The preparation method according to claim 1, characterized in that: In step 1, the liquid culture medium of Inonotus obliquus is composed of: 1.0 L of potato boiling liquid, 20 g / L of glucose, 3 g / L of potassium dihydrogen phosphate, 1.5 g / L of anhydrous magnesium sulfate, and 0.03 g / L of vitamin B1.

3. The preparation method according to claim 2, characterized in that: The preparation process of the potato cooking liquid is as follows: 200 g of peeled potato pieces are boiled in boiling water for 30 min, and the filtrate is collected and the volume is adjusted to 1.0 L.

4. The preparation method according to claim 1, characterized in that: In step 2, the liquid culture medium for Ganoderma lucidum mycelium is composed of: 30 g / L glucose, 3 g / L yeast powder, 1.5 g / L potassium dihydrogen phosphate, 0.75 g / L anhydrous magnesium sulfate, and 0.03 g / L vitamin B1.

5. The preparation method according to claim 1, characterized in that: In step 3, the bacterial solution obtained in step 2 is added to the bacterial solution in step 1, and the inoculation amount is 2-5%.

6. The preparation method according to claim 1, characterized in that: In step 4, the co-fermentation liquid powder obtained in step 3 is added to 95% ethanol at a material-liquid ratio of 1 g:10-20 mL.

7. The preparation method according to claim 1, characterized in that: In step 5, the graded extraction is to add 2-3 volumes of petroleum ether and ethyl acetate in sequence for extraction for 10-20 min, and the extract phase is concentrated and dried to obtain an extract.

8. Use of the extract of the co-fermentation broth of Ganoderma lucidum and Inonotus obliquus obtained by the preparation method according to any one of claims 1 to 7 in the preparation of an anti-glycosylation preparation.

9. The use according to claim 8, characterized in that: The ethyl acetate extract phase was concentrated and dried to obtain an extract for preparing an anti-glycosylation preparation.

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