Ganoderma lucidum fermentation lysate as well as preparation method and application thereof

Abstract

The invention provides a ganoderma lucidum fermentation lysate as well as a preparation method and application thereof in preparation of cosmetics. The preparation method of the ganoderma lucidum fermentation lysate comprises the steps of induced fermentation, catalytic degradation, membrane filtration, spray drying and the like. By inducing endogenous enzyme and metal ion regulation catalysis, the ganoderma lucidum fermentation lysate can be prepared in an environment-friendly, economical and efficient manner, the content of ganoderma lucidum polysaccharide in the prepared ganoderma lucidum fermentation lysate reaches 18% or above, and the content of beta-glucan in ganoderma lucidum polysaccharide is larger than 5%. Experiments prove that the ganoderma lucidum fermentation lysate has good antioxidant activity and an anti-aging effect, and has a wide market application prospect in the field of anti-aging cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological cosmetics, and in particular relates to a Ganoderma lucidum fermentation lysate and a preparation method and application thereof. Background Art

[0002] Lingzhi, also known as the "immortal grass," was first mentioned as "red ganoderma" in the Shennong Bencao Jing (Shen Nong's Herbal Classic). It was subsequently mentioned in Ming Yi Bie Lu (Famous Doctors' Records), Xin Xiu Ben Cao (Newly Revised Materia Medica), Zhong Hua Zi Ben (Chinese Classics), and Kai Yuan Ben Cao (Kai Yuan Materia Medica). The Compendium of Materia Medica states that Lingzhi "tonifies the middle qi, enhances wisdom, improves complexion, and, if consumed regularly, leads to a light and immortal body, prolonging life." Lingzhi, included in the 2020 edition of the Chinese Pharmacopoeia (Volume 1), is the dried fruiting body of Ganoderma lucidum (Leyss. ex Fr.) Karst. or Ganoderma sinense (Zhao, Xu et Zhang), a member of the Polyporaceae family. Red ganoderma is the representative species of Lingzhi, known for its qi-tonifying, calming, and relieving coughs and asthma. It is used to treat restlessness, insomnia, palpitations, lung deficiency, cough and asthma, consumptive fatigue, shortness of breath, and loss of appetite. Studies have shown that Ganoderma lucidum contains a variety of bioactive substances such as Ganoderma polysaccharides, Ganoderma triterpenes, sterols, nucleotides, peptidoglycan, etc., which have multiple biological activities such as immunomodulation, anti-tumor, anti-inflammatory, and regulation of intestinal flora.

[0003] Traditional Ganoderma production methods involve wild harvesting or artificial cultivation of fruiting bodies. These methods are subject to long production cycles, significant seasonal and climatic influences, unstable raw material quality, and pesticide residues, limiting the development and utilization of Ganoderma resources. Research has shown that Ganoderma mycelium produced through liquid fermentation has similar active ingredient content and physiological functions to Ganoderma fruiting bodies. Furthermore, Ganoderma liquid fermentation offers advantages such as a stable material source, a controllable and stable production process, immunity to environmental influences, a short production cycle, and the absence of pesticide residues. Consequently, research on Ganoderma liquid fermentation is gaining increasing attention.

[0004] Given the diverse bioactive substances and bioactivities found in Ganoderma lucidum, such as polysaccharides, triterpenes, and alkaloids, the research, application, and product development of Ganoderma lucidum wall-broken extracts in pharmaceuticals, foods, and cosmetics has attracted significant attention. Methods for breaking the wall of Ganoderma lucidum include physical, chemical, enzymatic, and biofermentation methods. For example, Patent 201810325349.0 discloses a method for extracting polysaccharides from Ganoderma lucidum mycelium through high-pressure wall-broken extraction. Patent ZL202111098884.5 discloses a process for preparing a Ganoderma lucidum extract with low color and high polysaccharide content using dynamic hot water extraction, microfiltration with a microfiltration membrane, and dynamic resin decolorization. Patent Application No. 202310938577.6 discloses a method for preparing polysaccharides from Ganoderma lucidum fruiting bodies for the prevention or treatment of neurodegenerative diseases, using Ganoderma lucidum fruiting bodies as raw materials through a series of steps: aqueous extraction, alkaline extraction, graded alcohol precipitation, deproteinization, ion exchange column chromatography, and molecular sieve column chromatography. Patent application number 202210082453.8 discloses a method for preparing a Ganoderma lucidum fruiting body extract for cosmetics using a combination of enzymatic hydrolysis, flash extraction, and microwave extraction. Patent ZL202210321892.X discloses a method for breaking the wall of Ganoderma lucidum using Trichoderma harzianum and extracting the broken Ganoderma lucidum. The method involves fermenting Ganoderma lucidum fruiting body powder with Trichoderma harzianum to break the wall, followed by water extraction of Ganoderma polysaccharides and ethanol extraction of Ganoderma triterpenes. Patent ZL202111602728.8 discloses a method for preparing a Ganoderma lucidum extract using yeast fermentation of a crude Ganoderma lucidum extract and combined enzymatic hydrolysis. Solvent extraction has a low extraction rate of active ingredients because the cell wall is not damaged, and there are problems such as organic solvent residues; ultrasonic extraction, microwave extraction, and high-pressure homogenization extraction have high equipment requirements and high energy consumption, which are not conducive to industrial production; bio-enzyme method and fermentation method have become the main methods for breaking the cell wall of Ganoderma lucidum due to their relatively mild reaction conditions and high product extraction rate, but there are problems such as exogenous addition of bio-enzymes and fermentation bacteria, complex extract composition, and unclear active functional substances. Summary of the Invention

[0005] In response to the aforementioned problems of the prior art, the present invention provides a Ganoderma lucidum fermentation lysate and a method for preparing the same. This method utilizes ultrafine oat beta-glucan powder and ultrafine Ganoderma lucidum mycelium powder in fed-batch fermentation to selectively enhance the endogenous enzyme activity of the Ganoderma lucidum fermentation mycelium. Furthermore, the endogenous enzymes are catalyzed by composite metal ions to degrade the Ganoderma lucidum fermentation mycelium, resulting in the economical and efficient preparation of a Ganoderma lucidum fermentation lysate with anti-aging properties.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: The present invention provides a Ganoderma lucidum CZDG10, which is classified as Ganoderma lucidum Ganoderma lucidum , deposited in the China Center for Type Culture Collection, and its deposit number is CCTCC NO: M20241696.

[0007] The present invention also provides a method for preparing a fermented lysate of Ganoderma lucidum, the method comprising the following steps: (1) Inoculate Ganoderma lucidum CZDG10 with a preservation number of CCTCC: M20241696 into a seed culture medium, and culture at 25-28°C and 140-160 rpm for 5-7 days to obtain a seed solution; (2) inoculating the seed solution of step (1) into the fermentation medium at a 10% (v / v) inoculation rate, and fermenting and culturing at 25-28°C and 140-160 rpm for 2 days, adding 0.2-0.8% (m / v) sterilization inducer to the Ganoderma lucidum fermentation culture, and continuing the fermentation and culturing for 5-8 days. When the glucose content of the Ganoderma lucidum fermentation supernatant is lower than 2 mg / L, the fermentation is terminated, the fermentation is centrifuged, and the fermented mycelium is collected; (3) adding 0.5-2 times the weight of purified water to the fermented mycelium of step (2), homogenizing at 8000 rpm for 5 min, and high-pressure homogenizing at 80-120 MPa twice to obtain a homogenized liquid of fermented mycelium of Ganoderma lucidum; (4) adding a metal ion catalyst with a final concentration of 1-10 mmol / L to the homogenized solution of the fermented mycelium of Ganoderma lucidum obtained in step (3), adjusting the temperature to 40-50°C and the pH to 4.5-5.5, and carrying out the reaction at this temperature for 1-3 hours. The mixture was then heated to 80-90°C and reacted for 1 hour. The mixture was then cooled to room temperature and centrifuged at 8000 rpm for 10 minutes. The supernatant was collected to obtain a lysate of the fermented mycelium of Ganoderma lucidum. (5) adding 0.5-1% (m / v) activated carbon to the lysate of the fermented mycelium of Ganoderma lucidum in step (4) for adsorption for 1 hour, filtering with a 0.45 μm filter membrane, collecting filtrate I, ultrafiltering filtrate I with an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, collecting filtrate II, and concentrating filtrate II with a nanofiltration membrane with a molecular weight cutoff of 200-300 Da to a soluble solids concentration of 8-30%, and collecting the retentate; (6) The retentate from step (5) is spray-dried to obtain a Ganoderma lucidum fermentation lysate.

[0008] Furthermore, the seed culture medium in step (1) is: 20 g / L glucose, 5 g / L yeast powder, 1.5 g / L potassium dihydrogen phosphate, 0.5 g / L MgSO4, and 5 mg / L thiamine.

[0009] Furthermore, the fermentation medium in step (2) is: 25 g / L glucose, 8 g / L yeast powder, 2 g / L potassium dihydrogen phosphate, 1 g / L MgSO4, and 8 mg / L thiamine.

[0010] Furthermore, in step (2), the inducers are oat beta glucan superfine powder and Ganoderma lucidum mycelium superfine powder, and the weight ratio of oat beta glucan superfine powder to Ganoderma lucidum mycelium superfine powder is 1:2.

[0011] Furthermore, the inducer concentration is selected to be 0.2%-0.8%, and the inducer particle size is selected to be 500 mesh-1000 mesh.

[0012] Furthermore, the metal ion catalyst in step (4) is K + 、Zn 2+ and Mn 2+ The mixture has a metal ion addition amount of 1mmol / L-10mmol / L.

[0013] Furthermore, the K ＋ :Zn 2+ :Mn 2+ The molar concentration ratio is 1:2:2.

[0014] Furthermore, K + It is one of KCl, K2SO4, K2CO4, Zn 2+ It is one of ZnCl2, ZnSO4, Mn 2+ It is one of MnCl2 and MnSO4.

[0015] The present invention also provides a Ganoderma lucidum fermentation lysate prepared by the preparation method. The Ganoderma lucidum fermentation lysate can significantly increase the activity of SOD enzyme, effectively inhibit H2O2-induced COL-I loss, significantly promote elastin production, significantly reduce MMP-1 gene expression, exert antioxidant function and delay cell aging function, and the Ganoderma lucidum polysaccharide content is greater than 18%, and the β-glucan content in the Ganoderma lucidum polysaccharide is greater than 5%.

[0016] The present invention also provides the use of the fermented lysate of Ganoderma lucidum in the preparation of cosmetics with anti-aging effects.

[0017] In one embodiment of the present invention, the anti-aging cosmetic is a facial mask, essence, lotion, cream, spray, aqueous solution or powder.

[0018] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: the present invention uses oat beta glucan superfine powder with a particle size of 500-1000 mesh and Ganoderma lucidum mycelium superfine powder as an inducer to induce Ganoderma lucidum Ganoderma lucidumCZDG10 (CCTCC:M 20241696) efficiently expresses intracellular cellulase and β-D-glucosidase. Compared with the absence of inducers, the enzymatic activities of cellulase and β-D-glucosidase were increased by more than 25 times. By further adding a composite metal ion catalyst to regulate the activity of the intracellular enzymes, the cellulase activity reached 1.8 times that of the control group, which can effectively degrade active substances such as the cell wall and intracellular polysaccharides of the fermented mycelium of Ganoderma lucidum. Combined with membrane filtration separation and spray drying, the fermented lysate of Ganoderma lucidum is prepared. The entire process does not require the participation of exogenous enzymes and / or organic solvents, making it economical, efficient, safe and environmentally friendly.

[0019] The Ganoderma lucidum fermentation lysate prepared by the present invention has a high content of active substances, the Ganoderma lucidum polysaccharide content is as high as 22.3%, and the beta-glucan content in the Ganoderma lucidum polysaccharide can reach 6.01%. At the cellular level, it can significantly reduce the accumulation of ROS caused by oxidative damage, significantly improve the activity of SOD enzyme, and exert antioxidant effect. At the cellular level, it can significantly increase the production of collagen and elastin in human dermal fibroblasts, reduce the activity of MMP-1 enzyme, inhibit collagen degradation, and exert anti-aging effect. Therefore, it has obvious advantages and broad application prospects in the preparation of anti-aging functional cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : The effect of the fermented lysate of Ganoderma lucidum on the viability of HaCaT cells; Figure 2 : The effect of the fermented lysate of Ganoderma lucidum on the ROS content of HaCaT cells damaged by H2O2.

[0021] Figure 3 : The effect of the fermented lysate of Ganoderma lucidum on the SOD content of HaCaT cells damaged by H2O2.

[0022] Figure 4 : The effect of the fermentation lysate of Ganoderma lucidum on the COL-I production of HSF cells.

[0023] Figure 5 : The effect of the fermentation lysate of Ganoderma lucidum on the ELN production of HSF cells.

[0024] Figure 6 : The effect of the fermented lysate of Ganoderma lucidum on the expression of MMP-1 gene in HSF cells.

[0025] * or ** indicates significant difference (p<0.05) or extremely significant difference (p<0.01) compared with the model group.

[0026] # or ## indicates that the model group had a significant difference (p<0.05) or an extremely significant difference (p<0.01) between the model group and the negative control group. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments, but the scope of protection claimed by the present invention is not limited to the following embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] 1. Culture medium PDA medium: potato 200 g / L, glucose 20 g / L, agar 20 g / L, natural pH.

[0029] Seed culture medium: glucose 20 g / L, yeast powder 5 g / L, potassium dihydrogen phosphate 1.5 g / L, MgSO4 0.5 g / L, thiamine 5 mg / L, sterilized at 121°C for 15 min.

[0030] Fermentation medium: glucose 25 g / L, yeast powder 8 g / L, potassium dihydrogen phosphate 2 g / L, MgSO4 1 g / L, thiamine 8 mg / L, sterilization at 121 °C for 15 min.

[0031] Inducers: oat beta-glucan ultrafine powder (commercially available), Ganoderma lucidum mycelium ultrafine powder (commercially available), sterilized at 121°C for 20 minutes.

[0032] 2. Enzyme Activity Assay Cellulase: Cellulase activity was determined using the carboxymethyl cellulose activity assay. A 0.5% sodium carboxymethyl cellulose solution (CMC-Na) was prepared using 0.1 mol / L acetate buffer (pH 4.5). 1.5 mL of sodium carboxymethyl cellulose solution was used as the substrate, and 0.5 mL of enzyme solution was added. The mixture was placed in a 50°C water bath for 30 minutes. 2 mL of DNS was added to terminate the reaction. The mixture was heated in a boiling water bath for 5 minutes to develop color. The mixture was cooled to room temperature and the volume was adjusted to 25 mL. A standard curve was drawn using glucose for determination. Absorbance at 540 nm (OD 540 ), using inactivated enzyme solution as blank control. A standard curve was drawn using glucose to calculate enzyme activity.

[0033] The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of reducing sugar per minute under experimental conditions, and is calculated in U / mL.

[0034] β-D-glucosidase: β-D-glucosidase was determined using the p-nitrophenol-β-D-glucoside (pNPG) method. A 5 mmol / L p-nitrophenol-β-D-glucoside (pNPG) solution was prepared in 0.1 mol / L acetate buffer (pH 4.5). 0.8 mL of p-nitrophenol-β-D-glucoside solution was used as substrate and the absorbance at 405 nm (OD405 ), use inactivated enzyme solution as blank control. Plot a standard curve with p-nitrophenol (pNP) to calculate enzyme activity.

[0035] The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze pNPG to produce 1 μmol pNP per minute under the experimental conditions, and is calculated in U / mL.

[0036] 3. Biomass Determination After the fermentation was completed, 50 mL of fermentation broth was centrifuged at 10,000 r / min for 10 min, the mycelium was washed three times with distilled water, dried at 80 °C for 48 h, weighed, and the biomass was calculated.

[0037] 4. Determination of polysaccharide content Ganoderma lucidum polysaccharide: The total sugar content was determined by the benzoic acid-sulfuric acid method, and the reducing sugar content was determined by the DNS method. Polysaccharide content = total sugar content - reducing sugar content.

[0038] β-glucan: β-glucanase assay kit was used to detect the β-glucan content.

[0039] β-glucan in Ganoderma lucidum polysaccharide content = β-glucan content / Ganoderma lucidum polysaccharide content × 100%.

[0040] Example 1: Screening and identification of Ganoderma lucidum CZDG10 Wild Ganoderma lucidum was harvested from Dabie Mountain. After surface disinfection, appropriate amount of solids were aseptically picked and inoculated onto PDA plate culture medium (10 plates) and cultured at 26°C for 10-15 days. Strain 2# had the largest colony diameter and was further screened.

[0041] Directed evolution of strains was performed by continuous plate rejuvenation and shake flask fermentation: (1) Plate culture: The strain from the strain library No. 2 slant was transferred to plate culture (20 plates), plate cultured at 26°C for 8 days (PDA medium), and the colony diameter was measured. (2) Shake flask culture: 5 dominant colonies were selected from the plate culture for shake flask culture, and 0.5 cm 2 Ten colonies from the plate were inoculated into a shake flask containing fermentation medium. The culture was incubated at 26°C, 150 rpm for 8 days as one generation, and three generations were subcultured. The biomass of the shake flask culture was measured. (3) Fine mycelia were selected from the dominant strain in the shake flask in step (2) and transferred to the plate culture (20 plates) in step (1). Five colonies with vigorous mycelial growth were selected for the shake flask culture in step (2).

[0042] After three cycles of directed evolution, strain #10, CZDG10, exhibited the highest colony diameter (3.6 ± 0.2 cm) and mycelial biomass (11.2 ± 0.3 g / L). The colonies were white, round, and adhered to the surface of the culture medium. The mycelium was dense in the center and sparse at the edges. Methylene blue staining revealed lock-like unions within the mycelium.

[0043] The universal primers ITS1 and ITS4 were used as primers, and the sequences of the primers were: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' ITS4: 5'-TCCTCCGCTTATTGATATGC-3' The ITS region sequence was amplified by PCR using total DNA from the mycelium of CZDG10 as a template.

[0044] The 50 μL PCR reaction system is as follows: 1 μL DNA template, 1 μL F (10 μM), 1 μL; R (10 μM), 1 μL; dNTP (2.5mM each), 4 μL; Taq (2U / μL), 1 μL; 10×Taq buffer, 5 μL; ddH2O, add to 50 μL.

[0045] The PCR amplification program was as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, 35 cycles of amplification, and final extension at 72°C for 5 min.

[0046] The PCR amplified products were sequenced in the ITS region, and the sequences were compared by BLAST on the website of the National Center for Bioinformation (NCBI). Ganoderma lucidum The sequence (ID: GU213479.1) has a similarity of 99.51%. Combining the results of morphological and molecular biological experiments, the strain was named Ganoderma lucidum CZDG10 ( Ganoderma lucidum CZDG10), and it was deposited in the China Center for Type Culture Collection.

[0047] The preservation information is: Ganoderma lucidum Ganoderma lucidum CZDG10 was deposited on July 26, 2024, in the China Center for Type Culture Collection, Ganoderma Lucidum, Wuhan University, Wuhan, China. Ganoderma lucidum The deposit number of CZDG10 is: CCTCC NO: M 20241696.

[0048] Example 2: Preparation of Ganoderma lucidum fermentation lysate Ganoderma lucidum Ganoderma lucidumCZDG10 (CCTCC NO:M20241696), take 8-10 0.5×0.5 cm mushroom blocks, inoculate them into 100 mL seed culture medium, and culture them at 26°C, 150 rpm for 7 days to obtain seed liquid; inoculate the seed liquid into 1000 mL fermentation medium at a 10% (v / v) inoculation rate, and ferment and culture at 26°C, 150 rpm for 2 days, add 0.5% (m / v) sterilization inducer to the Ganoderma lucidum fermentation culture (the inducer is prepared into a 10% (m / v) solution with purified water and sterilized at 121°C for 30 minutes), continue fermentation and culture for 5-8 days, terminate fermentation when the glucose content of the Ganoderma lucidum fermentation supernatant is lower than 2 mg / L, centrifuge at 8000 rpm for 10 minutes, and collect the fermentation mycelium.

[0049] 1 times the weight of purified water was added to the fermented mycelium, and the mixture was homogenized at 8000 rpm for 5 minutes. High-pressure homogenization was performed twice at 100 MPa to obtain a homogenized Ganoderma lucidum fermented mycelium solution. A metal ion catalyst (KCl:ZnCl2:MnCl2 molar ratio of 1:2:2) was added to the homogenized Ganoderma lucidum fermented mycelium solution at a concentration of 5 mmol / L. The reaction was adjusted to 45°C and pH 5.0. The mixture was kept warm for 2 hours, then heated to 85°C for 1 hour. The mixture was cooled to room temperature and centrifuged at 8000 rpm for 10 minutes. The supernatant was collected to obtain a Ganoderma lucidum fermented mycelium lysate.

[0050] 0.5% (m / v) activated carbon was added to the Ganoderma lucidum fermentation mycelium lysate for adsorption for 1 hour, and the mixture was filtered through a 0.45 μm filter membrane. Filtrate I was collected, and filtrate I was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. Filtrate II was collected, and filtrate II was concentrated through nanofiltration through a nanofiltration membrane with a molecular weight cutoff of 200-300 Da to a soluble solids concentration of approximately 15%. The retentate was collected, and the retentate was spray-dried to obtain the Ganoderma lucidum fermentation lysate.

[0051] Example 3: Effects of different inducers on Ganoderma lucidum fermentation lysate 1. Effect of inducer types on Ganoderma lucidum fermentation lysate On the basis of the basic process of Example 2, 0.5% (m / v) of oat β-glucan ultrafine powder (A), Ganoderma lucidum mycelium powder ultrafine powder (B), Ganoderma lucidum fruiting body ultrafine powder (C), Ganoderma lucidum spore ultrafine powder (D), and ultrafine powders of different mass ratios were added respectively. With no inducer added as the control, other process conditions remained unchanged, and the effects of different types of inducers on Ganoderma lucidum fermentation lysate were studied.

[0052] Table 1: Effects of different inducers on Ganoderma lucidum fermentation lysate

[0053] The results are shown in Table 1: Without the addition of inducers, the biomass of Ganoderma lucidum fermentation was low, the cellulase and β-D-glucosidase activities of Ganoderma lucidum mycelium were low, and the yield of fermentation lysate, Ganoderma lucidum polysaccharide, and β-glucan content were low. When 0.5% oat β-glucan ultrafine powder (A), Ganoderma lucidum mycelium powder ultrafine powder (B), and Ganoderma lucidum fruiting body powder ultrafine powder (C) were added to induce fermentation, the biomass of Ganoderma lucidum fermentation, the cellulase and β-D-glucosidase activities of Ganoderma lucidum fermentation mycelium, the yield of fermentation lysate, and the Ganoderma lucidum polysaccharide and β-glucan content of fermentation lysate were all increased. Among them, oat β-glucan ultrafine powder had a significant promoting effect on the improvement of β-D-glucosidase activity, and Ganoderma lucidum mycelium powder had a significant promoting effect on the improvement of cellulase activity.

[0054] Further research was conducted to investigate the effects of different mass ratios of oat β-glucan ultrafine powder and Ganoderma lucidum mycelium powder on Ganoderma lucidum fermentation lysate. The results showed that the addition of a composite powder with a mass ratio of 1:2 of oat β-glucan ultrafine powder to Ganoderma lucidum mycelium powder significantly increased Ganoderma lucidum fermentation biomass, Ganoderma lucidum fermentation mycelium cellulase and β-D-glucosidase enzyme activities, fermentation lysate yield, and Ganoderma lucidum polysaccharide and β-glucan contents in the fermentation lysate. The composite powder with a mass ratio of 1:2 of oat β-glucan ultrafine powder to Ganoderma lucidum mycelium powder was selected as the inducer.

[0055] 2. Effect of inducer concentration on Ganoderma lucidum fermentation lysate On the basis of the above basic process, 0.1%, 0.2%, 0.5%, 0.8%, 1.0%, 2.0% of oat beta glucan ultrafine powder and Ganoderma lucidum mycelium powder ultrafine powder in a weight ratio of 1:2 were added. No inducer was added as a control, and other process conditions remained unchanged. The effects of different inducer concentrations on Ganoderma lucidum fermentation lysate were studied.

[0056] Table 2: Effects of different inducer concentrations on Ganoderma lucidum fermentation lysate

[0057] The results are shown in Table 2: In the range of 0.1%-0.5%, with the increase of inducer concentration, the fermentation biomass of Ganoderma lucidum, the cellulase and β-D-glucosidase activities of Ganoderma lucidum fermentation mycelium, the yield of fermentation lysate, the Ganoderma lucidum polysaccharide and β-glucan content of fermentation lysate all increased. In the range of 0.5%-2.0%, with the increase of inducer concentration, all indicators decreased. This may be because the additive concentration was too high, resulting in excessive viscosity of the fermentation system, affecting Ganoderma lucidum fermentation and the extraction of fermentation lysate. The optimal inducer concentration was 0.5%. In the range of 0.2%-0.8%, the fermentation biomass of Ganoderma lucidum, the cellulase and β-D-glucosidase activities of Ganoderma lucidum fermentation mycelium, the yield of fermentation lysate, the Ganoderma lucidum polysaccharide and β-glucan content of fermentation lysate all remained at a high level. Therefore, the inducer concentration was selected to be 0.2%-0.8%.

[0058] 3. Effect of inducer particle size on Ganoderma lucidum fermentation lysate On the basis of the above basic process, oat beta-glucan ultrafine powder and Ganoderma lucidum mycelium ultrafine powder with particle sizes of 50 mesh, 200 mesh, 500 mesh, 800 mesh, 1000 mesh, 2000 mesh and 5000 mesh were selected, with a weight ratio of 1:2. No inducer was added as the control, and other process conditions remained unchanged. The effect of different inducer particle sizes on Ganoderma lucidum fermentation lysate was studied.

[0059] Table 3: Effects of different inducer particle sizes on Ganoderma lucidum fermentation lysate

[0060] The results are shown in Table 3. When the inducer particle size was between 500 mesh and 1000 mesh, the fermentation biomass of Ganoderma lucidum, the cellulase and β-D-glucosidase activities of the Ganoderma lucidum fermentation mycelium, the yield of the fermentation lysate, and the Ganoderma lucidum polysaccharide and β-glucan contents of the fermentation lysate all remained at high levels. When the inducer particle size was larger (100-200 mesh), the induction effect was poor, the biomass of Ganoderma lucidum fermentation mycelium was low, and the yield of the Ganoderma lucidum fermentation lysate was affected. When the inducer particle size was smaller (2000-5000 mesh), the induction effect was stronger, and the mycelial pellets were dense and filamentous, but the cellulase and β-D-glucosidase activities of the Ganoderma lucidum fermentation mycelium were reduced, which was not conducive to the preparation of the Ganoderma lucidum fermentation lysate. Therefore, the inducer particle size was selected to be between 500 mesh and 1000 mesh.

[0061] Example 4: Effects of different metal ions on the activity of cellulase in Ganoderma lucidum cells 1. Effect of metal ion types on the activity of intracellular cellulase in Ganoderma lucidum The homogenate of the fermented mycelium of Ganoderma lucidum in Example 2 was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. Na + , K＋ , Ca 2+ Mg 2+ 、Zn 2+ 、Cu 2+ 、Fe 2+ 、Mn 2+ ions, placed at room temperature for 1 hour, and the cellulase activity was determined. Different concentrations of metal ions were added to the crude enzyme solution inactivated in a boiling water bath as blank controls of the corresponding metal ions. The enzyme activity without added metal ions was 100%. The relative enzyme activity was used to compare the effects of metal ions on enzyme activity.

[0062] Table 4: Effects of different metal ions on the activity of cellulase in Ganoderma lucidum cells

[0063] The results are shown in Table 4: Mn 2+ 、Zn 2+ , K ＋ Ions have a significant activation effect on the cellulase activity in the mycelium of Ganoderma lucidum, among which potassium ions have the most obvious activation effect, and the enzyme activity is 162.1%±5.2% of the control group. + , Ca 2+ Mg ions had no significant effect on the cellulase activity in Ganoderma lucidum mycelium. 2+ 、Cu 2+ 、Fe 2+ Ions have a significant inhibitory effect on the activity of cellulase in the mycelium of Ganoderma lucidum, and the most obvious inhibitory effect is Cu 2+ , which is only 22.4%±2.5% of the control enzyme activity. Therefore, during the use of Ganoderma lucidum mycelium intracellular cellulase, it should be avoided as much as possible from contact with Mg. 2+ 、Cu 2+ 、Fe 2+ ions, Mn can be added appropriately 2+ 、Zn 2+ , K ＋ Ions increase enzyme activity.

[0064] 2. Effects of different metal ion molar concentration ratios on the activity of intracellular cellulase in Ganoderma lucidum Take the homogenate of Ganoderma lucidum fermentation mycelium in Example 2, centrifuge at 8000rpm for 10min, and collect the supernatant. Add K ＋ 、Zn 2+ 、Mn 2+ ions, placed at room temperature for 1 hour, and the cellulase activity was determined. Different concentrations of metal ions were added to the crude enzyme solution inactivated in a boiling water bath as blank controls of the corresponding metal ions. The enzyme activity without added metal ions was 100%. The relative enzyme activity was used to compare the effects of metal ions on enzyme activity.

[0065] Different metal ions have different binding sites for the activation of cellulase. Further research was conducted to study the effects of different metal ion molar concentration ratios on the cellulase activity in Ganoderma lucidum mycelium. The results are shown in Table 5. The results showed that K ＋ :Zn 2+ :Mn 2+ The molar concentration ratio of 1:2:2 had the most significant effect on activating the intracellular cellulase activity of Ganoderma lucidum mycelium, reaching as high as 178.5%±6.6%.

[0066] Table 5: Effects of different metal ion ratios on the activity of cellulase in Ganoderma lucidum cells

[0067] 3. Effects of different metal ion additions on Ganoderma lucidum fermentation lysate The homogenate of the fermented mycelium of Ganoderma lucidum in Example 2 was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. K was added to the supernatant at final concentrations of 0.5 mmol / L, 1 mmol / L, 5 mmol / L, 10 mmol / L, 20 mmol / L, and 50 mmol / L, respectively. ＋ :Zn 2 + :Mn 2+ The molar concentration ratio of metal ions was 1:2:2, and the cellulase activity was determined at room temperature for 1 hour. Different concentrations of metal ions were added to the crude enzyme solution inactivated in a boiling water bath as blank controls of the corresponding metal ions. The enzyme activity without added metal ions was 100%, and the relative enzyme activity was used to compare the effects of metal ions on enzyme activity.

[0068] Table 6: Effects of different metal ion additions on the activity of intracellular enzymes in Ganoderma lucidum

[0069] The results are shown in Table 6: The results show that within the range of 0.5mmol / L-5mmol / L of metal ion addition, as the metal ion concentration increases, the relative activity of cellulase activity gradually increases. Within the range of 5mmol / L-50mmol / L of metal ion addition, as the metal ion concentration increases, the relative activity of cellulase activity gradually decreases. In particular, after the concentration exceeds 10mmol / L, the relative activity of cellulase activity decreases significantly. At 50mmol / L, the relative activity of cellulase activity is only 34.7%±1.9% of the enzyme activity of the control group. The optimal metal ion addition amount is 5mmol / L. Within the range of 1mmol / L-10mmol / L, the relative activity of cellulase activity remains at a high level. Therefore, the metal ion addition amount is 1mmol / L-10mmol / L.

[0070] Example 5: Preparation of Ganoderma lucidum fermentation lysate Ganoderma lucidum Ganoderma lucidum CZDG10 (CCTCC:M20241696), take 8-10 0.5×0.5cm mushroom blocks, inoculate them into 100mL seed culture medium, and culture them at 26℃, 150rpm for 7 days to obtain seed liquid; inoculate the seed liquid into 1000mL fermentation medium at a 10% (v / v) inoculation rate, and ferment and culture at 26℃, 150rpm for 2 days, add 5g inducer to the Ganoderma lucidum fermentation culture, the inducer is a sterilized compound powder with a particle size of 800 mesh and a weight ratio of oat β-glucan ultrafine powder and Ganoderma lucidum mycelium ultrafine powder of 1:2 (the compound powder is prepared into a 10% (m / v) solution with purified water and sterilized at 121℃ for 30 minutes), continue fermentation and culture for 6 days, terminate the fermentation when the glucose content of the Ganoderma lucidum fermentation supernatant is lower than 2mg / L, centrifuge at 8000rpm for 10min, and collect 565g of fermented mycelium.

[0071] 565 g of purified water was added to the fermented mycelium, and the mixture was homogenized at 8000 rpm for 5 minutes and then high-pressure homogenized twice at 100 MPa to obtain a homogenized Ganoderma lucidum fermented mycelium liquid. The supernatant of the homogenized Ganoderma lucidum fermented mycelium liquid had a cellulase activity of 3.96 U / mL and a β-D-glucosidase activity of 2.79 U / mL.

[0072] A metal ion catalyst (K) with a concentration of 5 mmol / L was added to the homogenate of Ganoderma lucidum fermentation mycelium. ＋ :Zn 2+ :Mn 2 The molar concentration ratio was 1:2:2), the temperature was adjusted to 45°C, the pH was 5.0, the reaction was kept warm for 2 hours, the temperature was further raised to 85°C for 1 hour, the mixture was cooled to room temperature, and the mixture was centrifuged at 8000 rpm for 10 minutes. The supernatant was collected to obtain the lysate of the fermented mycelium of Ganoderma lucidum.

[0073] 0.5% (m / v) activated carbon was added to the fermented mycelial lysate of Ganoderma lucidum for adsorption for 1 hour. The mixture was then filtered through a 0.45 μm filter membrane, and filtrate I was collected. Filtrate I was then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, and filtrate II was collected. Filtrate II was then concentrated through nanofiltration using a nanofiltration membrane with a molecular weight cutoff of 200-300 Da to a soluble solids concentration of approximately 15%, and the retentate was collected. The retentate was spray-dried to obtain 14.6 g of Ganoderma lucidum fermentation lysate. The Ganoderma lucidum fermentation lysate contained 21.5% Ganoderma lucidum polysaccharide and 1.25% β-glucan, with β-glucan accounting for 5.81% of the total Ganoderma lucidum polysaccharide content.

[0074] Example 6: Preparation of Ganoderma lucidum fermentation lysate Ganoderma lucidum Ganoderma lucidumCZDG10 (CCTCC:M20241696), take 8-10 0.5×0.5cm mushroom blocks, inoculate them into 100mL seed culture medium, and culture them at 28℃, 140rpm for 5 days to obtain seed liquid; inoculate the seed liquid into 1000mL fermentation medium at a 10% (v / v) inoculation rate, and ferment and culture at 28℃, 140rpm for 2 days, add 10g inducer to the Ganoderma lucidum fermentation culture, the inducer is a sterilized compound powder with a particle size of 500 mesh, oat β-glucan ultrafine powder and Ganoderma lucidum mycelium ultrafine powder in a weight ratio of 1:2 (the compound powder is prepared into a 10% (m / v) solution with purified water, and sterilized at 121℃ for 20 minutes), continue fermentation and culture for 8 days, terminate the fermentation when the glucose content of the Ganoderma lucidum fermentation supernatant is lower than 2mg / L, centrifuge at 8000rpm for 10min, and collect 602g of fermented mycelium.

[0075] 1204 g of purified water was added to the fermented mycelium, and the mixture was homogenized at 8000 rpm for 5 min. High-pressure homogenization was performed twice at 80 MPa to obtain a homogenized Ganoderma lucidum fermented mycelium solution. The supernatant of the homogenized Ganoderma lucidum fermented mycelium solution had a cellulase activity of 2.45 U / mL and a β-D-glucosidase activity of 1.85 U / mL.

[0076] A metal ion catalyst (K ＋ :Zn 2+ :Mn 2 The molar concentration ratio was 1:2:2), the temperature was adjusted to 50°C and the pH was 4.5, the reaction was kept warm for 3 hours, the temperature was further raised to 90°C for 1 hour, the mixture was cooled to room temperature, and the mixture was centrifuged at 8000 rpm for 10 minutes. The supernatant was collected to obtain the lysate of the fermented mycelium of Ganoderma lucidum.

[0077] 0.5% (m / v) activated carbon was added to the fermented mycelial lysate of Ganoderma lucidum for adsorption for 1 hour. The mixture was then filtered through a 0.45 μm filter membrane, and filtrate I was collected. Filtrate I was then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, and filtrate II was collected. Filtrate II was then concentrated through a nanofiltration membrane with a molecular weight cutoff of 200-300 Da to a soluble solids concentration of approximately 8%, and the retentate was collected. The retentate was spray-dried to obtain 14.1 g of Ganoderma lucidum fermentation lysate. The Ganoderma lucidum fermentation lysate contained 22.3% Ganoderma lucidum polysaccharide and 1.34% β-glucan, with β-glucan accounting for 6.01% of the total Ganoderma lucidum polysaccharide content.

[0078] Example 7: Preparation of Ganoderma lucidum fermentation lysate Ganoderma lucidum Ganoderma lucidumCZDG10 (CCTCC:M20241696), take 8-10 0.5×0.5cm mushroom blocks, inoculate them into 100mL seed culture medium, and culture them at 25℃, 160rpm for 6 days to obtain seed liquid; inoculate the seed liquid into 1000mL fermentation medium at a 10% (v / v) inoculation rate, and ferment and culture at 25℃, 160rpm for 2 days. Add 2g of inducer to the Ganoderma lucidum fermentation culture. The inducer is a sterilized compound powder with a particle size of 1000 mesh and a weight ratio of oat β-glucan ultrafine powder and Ganoderma lucidum mycelium ultrafine powder of 1:2 (the compound powder is prepared into a 10% (m / v) solution with purified water and sterilized at 121℃ for 60 minutes). Continue fermentation and culture for 5 days. When the glucose content of the Ganoderma lucidum fermentation supernatant is lower than 2mg / L, terminate the fermentation, centrifuge at 8000rpm for 10min, and collect 480g of fermented mycelium.

[0079] 240 g of purified water was added to the fermented mycelium, and the mixture was homogenized at 8000 rpm for 5 min. The mixture was then homogenized twice at 120 MPa to obtain a homogenized Ganoderma lucidum fermented mycelium solution. The supernatant of the homogenized Ganoderma lucidum fermented mycelium solution had a cellulase activity of 5.05 U / mL and a β-D-glucosidase activity of 3.78 U / mL.

[0080] A metal ion catalyst (K ＋ :Zn 2+ :Mn 2 The molar concentration ratio was 1:2:2), the temperature was adjusted to 40°C and the pH was 5.5, the reaction was kept warm for 1 hour, the temperature was further raised to 80°C for 1 hour, the mixture was cooled to room temperature, and the mixture was centrifuged at 8000 rpm for 10 minutes. The supernatant was collected to obtain the lysate of the fermented mycelium of Ganoderma lucidum.

[0081] 1% (m / v) activated carbon was added to the fermented mycelial lysate of Ganoderma lucidum for adsorption for 1 hour. The mixture was then filtered through a 0.45 μm filter membrane, and filtrate I was collected. Filtrate I was then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, and filtrate II was collected. Filtrate II was then concentrated through a nanofiltration membrane with a molecular weight cutoff of 200-300 Da to a soluble solids concentration of approximately 30%, and the retentate was collected. The retentate was spray-dried to obtain 12.5 g of Ganoderma lucidum fermentation lysate. The Ganoderma lucidum fermentation lysate contained 18.2% Ganoderma lucidum polysaccharide and 0.92% β-glucan, with β-glucan accounting for 5.05% of the total Ganoderma lucidum polysaccharide content.

[0082] Example 8: Preparation of Ganoderma lucidum fermentation lysate Ganoderma lucidum Ganoderma lucidumCZDG10 (CCTCC:M20241696) was inoculated into the seed culture medium and cultured at 26°C, 150 rpm for 7 days to obtain the first-level seed solution. The first-level seed solution was inoculated into the seed culture medium and cultured at 26°C, 150 rpm for 7 days to obtain the second-level seed solution.

[0083] A 300L fermentation tank was filled with 200L fermentation medium and incubated at 121°C for 30 minutes. When the temperature dropped to about 26°C, the secondary seed liquid was inoculated into the fermentation tank at a 10% (v / v) inoculation rate. The temperature was controlled at 26°C, the rotation speed was 200rpm, and the ventilation volume was 100L / min. The fermentation culture was carried out for 2 days. 1kg of inducer was added to the Ganoderma lucidum fermentation culture. The inducer was a sterilized compound powder with a particle size of 800 mesh and a weight ratio of oat beta-glucan ultrafine powder and Ganoderma lucidum mycelium ultrafine powder of 1:2 (the compound powder was prepared into a 10% (m / v) solution with purified water and sterilized at 121°C for 30 minutes). The fermentation culture was continued for 6 days. The fermentation was terminated when the glucose content of the Ganoderma lucidum fermentation supernatant was lower than 2mg / L. The culture was centrifuged at 8000rpm for 10min, and 11.2kg of fermented mycelium was collected.

[0084] 11.2 kg of purified water was added to the fermented mycelium, and the mixture was homogenized at 8000 rpm for 5 minutes. The mixture was then homogenized twice at 100 MPa to obtain a homogenized Ganoderma lucidum fermentation mycelium solution. The supernatant of the homogenized Ganoderma lucidum fermentation mycelium solution had a cellulase activity of 3.85 U / mL and a β-D-glucosidase activity of 2.72 U / mL.

[0085] A metal ion catalyst (K) with a concentration of 5 mmol / L was added to the homogenate of Ganoderma lucidum fermentation mycelium. ＋ :Zn 2+ :Mn 2 The molar concentration ratio was 1:2:2), the temperature was adjusted to 45°C, the pH was 5.0, the reaction was kept warm for 2 hours, the temperature was further raised to 85°C for 1 hour, the mixture was cooled to room temperature, and the mixture was centrifuged at 8000 rpm for 10 minutes. The supernatant was collected to obtain the lysate of the fermented mycelium of Ganoderma lucidum.

[0086] 0.5% (m / v) activated carbon was added to the fermented mycelial lysate of Ganoderma lucidum for adsorption for 1 hour. The mixture was then filtered through a 0.45 μm filter membrane, and filtrate I was collected. Filtrate I was then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, and filtrate II was collected. Filtrate II was then concentrated through a nanofiltration membrane with a molecular weight cutoff of 200-300 Da to a soluble solids concentration of approximately 15%, and the retentate was collected. The retentate was spray-dried to obtain 2.85 kg of Ganoderma lucidum fermentation lysate. The Ganoderma lucidum fermentation lysate contained 20.7% Ganoderma lucidum polysaccharide and 1.13% β-glucan, with β-glucan accounting for 5.46% of the total Ganoderma lucidum polysaccharide content.

[0087] Example 9: Safety of Ganoderma lucidum fermentation lysate The MTT assay was used to investigate the cytotoxicity of Ganoderma lucidum fermentation lysate on HaCaT cells. Generally speaking, when cell viability is above 80%, it is considered that the sample has no significant toxicity to the cells. After reviving, HaCaT cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double-antibody to a confluence of 80-90%. After digestion, the cells were seeded in 96-well plates at a seeding density of 5×10 4 Cells were inoculated at 100 μL per well and incubated at 37°C in a 5% CO2 incubator for 12-24 hours. The culture medium was then discarded and various concentrations of the sample prepared in Example 3 were added to each well. The culture was continued for 24 hours. Then, 20 μL of a 5 mg / mL MTT solution was added to each well. After 4 hours, the solution was removed and 150 μL of dimethyl sulfoxide was added to each well. After thorough mixing, the absorbance at 490 nm was measured using a microplate reader.

[0088] See the results Figure 1 ,Depend on Figure 1 It can be seen that when the concentration of Ganoderma lucidum fermentation lysate samples is in the range of 50-1000μg / mL, the cell viability of HaCaT cells is higher than 80%, and when the sample concentration is lower than 600μg / mL, the cell viability is above 90%. Therefore, it can be considered that within the concentration range of 50-1000μg / mL, Ganoderma lucidum extract has no obvious toxicity to HaCaT cells.

[0089] Example 10: Antioxidant activity of Ganoderma lucidum fermentation lysate (1) Reactive oxygen species (ROS) scavenging HaCaT cells were plated at 1.2×10 6 Cells were seeded into 6-well plates at 1000 cells / well. After culturing with complete medium for 12-24 hours, all cells were divided into four groups according to the following table:

[0090] Incubation was continued for 24 hours according to the experimental groupings described above. The concentrations of Ganoderma lucidum fermentation lysate in the sample groups were 100, 200, and 500 μg / mL. After the incubation and stimulation process, the culture medium was aspirated and the cells were washed with PBS. The cells were then treated with 10 μM DCFH-DA fluorescent probe at 37°C for 30 minutes, washed twice with PBS, digested, and harvested. The DCF fluorescence intensity was measured using a multi-functional microplate reader at an emission wavelength of 525 nm (excitation wavelength of 488 nm).

[0091] See the results Figure 2 ,Depend on Figure 2It can be seen that after HaCaT cells were treated with H2O2, the ROS level increased significantly, about 160% of the control group. After being treated with different concentrations of Ganoderma lucidum fermentation lysate, the fluorescence intensity decreased significantly with the increase of sample concentration (P<0.01). When the sample concentration was 500μg / mL, the fluorescence intensity was 80.17% of the model group, indicating that Ganoderma lucidum extract can effectively remove ROS caused by H2O2, prevent the accumulation of ROS in cells, and help resist oxidative damage.

[0092] (2) Superoxide dismutase (SOD) activity HaCaT cells were plated at 1.2×10 6 Cells were seeded at 100 cells / well in a 6-well plate and cultured in complete medium for 12-24 hours. All cells were then divided into four groups according to the ROS scavenging protocol. After the culture and stimulation phases, the cells were harvested, lysed, and total protein was extracted and measured for concentration. Superoxide dismutase (SOD) activity was then assayed according to the kit instructions.

[0093] See the results Figure 3 ,Depend on Figure 3 It can be seen that after H2O2 treatment, the SOD activity of the model group decreased to 67.11% of that of the control group (P<0.01). With the increase of the concentration of Ganoderma lucidum fermentation lysate sample, the SOD enzyme activity gradually increased. When the concentration of Ganoderma lucidum extract was 200 μg / mL, the SOD enzyme activity increased significantly (P<0.05), which was 116.82% of that of the model group, indicating that Ganoderma lucidum fermentation lysate can significantly increase the activity of SOD enzyme and exert an antioxidant effect.

[0094] Example 11: Effect of Ganoderma lucidum fermentation lysate on delaying cell aging (1) Effects of Ganoderma lucidum fermentation lysate on human dermal fibroblasts (HSF) under oxidative stress Elastin and collagen are two important and ubiquitous proteins in the body. They play a vital role in our skin and overall health. Fibroblasts, the primary cells in the dermis, are responsible for the synthesis of the dermal extracellular matrix and the remodeling of collagen fiber structure, playing a crucial role in maintaining skin condition. Type I collagen (COL-I), secreted by fibroblasts, accounts for over 80% of young skin and is the predominant collagen in the skin, determining its appearance. Elastin (ELN), the primary component of elastic fibers, plays a key role in maintaining the elasticity and flexibility of skin tissue, maintaining its elasticity, plumpness, and firmness, and preventing wrinkles. Decreased levels of COL-I and ELN can lead to sagging and wrinkling, and are key indicators of skin aging. This study used H2O2 to induce a damaged cell model in human dermal fibroblasts (HSFs) and used collagen and elastin as indicators to investigate the anti-aging effects of Ganoderma lucidum fermentation lysate.

[0095] HSF cells were cultured at a rate of 5 × 10 5 Cells were seeded into 12-well plates at 1000 cells / well and cultured in complete medium for 12-24 hours to allow the cells to adhere to the wall. All cells were then divided into four groups according to the following table:

[0096] Incubate for another 24 hours according to the experimental grouping described above. The concentrations of Ganoderma lucidum fermentation lysate in the sample groups were 100 μg / mL, 200 μg / mL, and 500 μg / mL. After the incubation and stimulation process, carefully collect the culture supernatant into a centrifuge tube and centrifuge at 3000 rpm for 10 minutes to remove cell debris and other impurities. The supernatant was then assayed for COL-I and ELN content according to the ELISA kit instructions.

[0097] The results of the effect of Ganoderma lucidum fermentation lysate on COL-I production in HSF cells under oxidative stress are shown in Figure 4 ,Depend on Figure 4 It can be seen that, with VC as the positive control, H2O2 treatment of cells resulted in a significant decrease in COL-I production, which was 33.56% lower than that of the blank control group ( p <0.01), after treatment with different concentrations of Ganoderma lucidum fermentation lysate, the COL-I production was significantly increased compared with the model group, indicating that Ganoderma lucidum fermentation lysate can effectively inhibit the loss of COL-I induced by H2O2, thereby playing an anti-aging effect.

[0098] The results of the effect of Ganoderma lucidum fermentation lysate on ELN production in HSF cells under oxidative stress are shown in Figure 5 ,Depend on Figure 5 It can be seen that under oxidative stress, the ELN content of cells decreased significantly, falling to 84.23% of the blank control group ( P<0.01), with the increase of the concentration of Ganoderma lucidum fermentation lysate sample, the ELN production gradually increased. When the concentration of Ganoderma lucidum extract was 200 μg / mL, the ELN production increased to 121.45% of that in the model group ( P <0.01), and the ELN content exceeded that of the positive control group (VC), indicating that Ganoderma lucidum extract can significantly promote the production of elastin under oxidative stress, thereby playing an anti-aging effect.

[0099] (3) RT-PCR detection of matrix metalloproteinase-1 (MMP-1) gene expression in HSF cells After oxidative stress treatment of HSF, total RNA was extracted using the Trizol method. RNA concentration and purity were determined by measuring absorbance at 260 nm and 280 nm using a UV spectrophotometer. A 20 μL qRT-PCR reaction system was established, and the PCR amplification conditions were: initial denaturation at 95°C for 15 min; denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 20 s, for 40 cycles. Melting curve analysis was performed using a temperature control reaction at 95°C for 1 min, followed by a temperature control reaction at 55°C for 30 s, with fluorescence signals collected every 5°C increase.

[0100] Effects of Ganoderma lucidum fermentation lysate on MMP-1 expression in HSF cells damaged by H2O2 Figure 6 ,Depend on Figure 6 It can be seen that under oxidative stress, the MMP-1 mRNA level in HSF cells of the model group increased significantly. After treatment with different concentrations of Ganoderma lucidum fermentation lysate, the MMP-1 mRNA level was significantly reduced. This shows that Ganoderma lucidum fermentation lysate can significantly reduce the gene expression of MMP-1, thereby inhibiting the collagen degradation caused by MMP-1 and exerting its anti-aging effect.

[0101] In summary, the Ganoderma lucidum fermentation lysate prepared by the present invention has high contents of Ganoderma lucidum polysaccharides and β-glucan. At the cellular level, it can significantly reduce ROS accumulation caused by oxidative damage, significantly increase SOD enzyme activity, and exert antioxidant efficacy. At the cellular level, it can significantly increase collagen and elastin production in human dermal fibroblasts, reduce MMP-1 enzyme activity, inhibit collagen degradation, and exert anti-aging efficacy. The Ganoderma lucidum fermentation lysate prepared by the present invention has a high content of active polysaccharides and exhibits excellent antioxidant and anti-aging efficacy. It has significant advantages and broad application prospects in the preparation of anti-aging cosmetics (such as facial masks, essences, lotions, creams, sprays, aqueous solutions, or powders).

[0102] The above factual cases only represent the technical solutions of the present invention, and do not limit the experiments. Although we have improved the experimental solutions, researchers in the same field can still make further improvements to the experimental solutions described above or make scientific equivalent replacements for the experimental steps. These changes do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A strain of Ganoderma lucidum CZDG10, characterized by: Ganoderma lucidum Ganoderma lucidum , deposited in China Center for Type Culture Collection, and its deposit number is CCTCC NO: M 20241696.

2. A method for preparing a fermented lysate of Ganoderma lucidum, characterized in that: The preparation method comprises the following steps: (1) inoculating Ganoderma lucidum CZDG10 with a deposit number of CCTCC: M 20241696 into a seed culture medium for culturing to obtain a seed solution; (2) inoculating the seed liquid of step (1) into a fermentation medium for fermentation culture, adding an inducer to the fermentation culture, continuing the fermentation culture, terminating the fermentation when the glucose content of the Ganoderma lucidum fermentation supernatant is lower than 2 mg / L, and collecting the fermentation mycelium by centrifugation; (3) adding purified water to the fermented mycelium of step (2), homogenizing, and high-pressure homogenizing to obtain a homogenized liquid of fermented mycelium of Ganoderma lucidum; (4) adding a metal ion catalyst to the homogenized solution of the fermented mycelium of Ganoderma lucidum obtained in step (3), adjusting the temperature to 40-50° C. and the pH value to 4.5-5.5, keeping the temperature for reaction for 1-3 hours, further raising the temperature to 80-90° C. for reaction for 1 hour, cooling to room temperature, and collecting the supernatant by centrifugation to obtain a lysate of the fermented mycelium of Ganoderma lucidum; (5) adding activated carbon adsorption to the lysate of the fermented mycelium of Ganoderma lucidum in step (4), collecting the filtrate I by filtering through a filter membrane, ultrafiltering the filtrate I through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, collecting the filtrate II, and concentrating the filtrate II through a nanofiltration membrane with a molecular weight cutoff of 200-300 Da to a soluble solid concentration of 8-30%, and collecting the retentate; (6) spray drying the retentate from step (5) to obtain the Ganoderma lucidum fermentation lysate.

3. The method for preparing the fermented lysate of Ganoderma lucidum according to claim 2, characterized in that: In the step (2), the inducer is oat beta glucan superfine powder and Ganoderma lucidum mycelium superfine powder, and the weight ratio of oat beta glucan superfine powder to Ganoderma lucidum mycelium superfine powder is 1:

2.

4. The method for preparing the fermented lysate of Ganoderma lucidum according to claim 3, characterized in that: The inducer concentration is selected to be 0.2%-0.8%, and the inducer particle size is selected to be 500 mesh-1000 mesh.

5. The method for preparing the fermented lysate of Ganoderma lucidum according to claim 2, characterized in that: The metal ion catalyst in step (4) is K + 、Zn 2+ and Mn 2+ The mixture has a metal ion addition amount of 1mmol / L-10mmol / L.

6. The method for preparing the fermented lysate of Ganoderma lucidum according to claim 5, characterized in that: The K ＋ :Zn 2+ :Mn 2+ The molar concentration ratio is 1:2:

2.

7. The Ganoderma lucidum fermentation lysate produced by fermenting Ganoderma lucidum CZDG10 according to claim 1 or the Ganoderma lucidum fermentation lysate prepared by the preparation method according to any one of claims 2 to 6.

8. The fermented lysate of Ganoderma lucidum according to claim 7, characterized in that: The fermented lysate of Ganoderma lucidum can significantly increase the activity of SOD enzyme, effectively inhibit the loss of COL-I induced by H2O2, significantly promote the production of elastin, significantly reduce the gene expression of MMP-1, exert antioxidant function and delay cell aging function, the content of Ganoderma lucidum polysaccharide is greater than 18%, and the content of β-glucan in Ganoderma lucidum polysaccharide is greater than 5%.

9. Use of the fermented lysate of Ganoderma lucidum according to claim 5 in the preparation of cosmetics with anti-aging effects.

10. The use according to claim 9, characterized in that: The cosmetics are facial masks, essences, lotions, creams, sprays, aqueous solutions or powders.

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