A ganoderma lucidum fermentation lysate, a preparation method and application thereof

Abstract

The present application provides a Ganoderma lucidum fermentation lysate, a preparation method thereof and application thereof in preparing cosmetics. The preparation method of the Ganoderma lucidum fermentation lysate comprises the steps of inducing fermentation, catalytic degradation, membrane filtration, spray drying and the like. The present application can prepare the Ganoderma lucidum fermentation lysate in an environmentally friendly, economical and efficient manner through inducing endogenous enzyme and metal ion catalytic regulation. The Ganoderma lucidum fermentation lysate prepared has a Ganoderma lucidum polysaccharide content of more than 18%, and the content of beta glucan in the Ganoderma lucidum polysaccharide is greater than 5%. The present application is proved by experiments that the Ganoderma lucidum fermentation lysate has good antioxidant activity and anti-aging effect, and has a broad market application prospect in the field of anti-aging cosmetics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological cosmetics, and particularly relates to a Ganoderma lucidum fermentation lysate as well as a preparation method and application thereof. BACKGROUND

[0002] Ganoderma lucidum, also known as Xiancao, was first recorded in the “Red Ganoderma” in Shennong's Herbal Classic, and was also recorded in the Name Medical Records, New Revised Herbal, Chinese Herbal, and Kaiyuan Herbal. The Compendium of Materia Medica recorded that Ganoderma lucidum “tonifies the middle and benefits qi, increases wisdom, good color, long-term consumption lightens the body, does not age, and prolongs life and immortality”. Ganoderma lucidum has been included in the Chinese Pharmacopoeia 2020 Edition, Part I, as the dried fruiting body of the Polyporaceae fungus Ganoderma lucidum (Leyss. ex Fr.) Karst. or Ganoderma sinense Zhao, Xu et Zhang, wherein Ganoderma lucidum is the representative variety of Ganoderma lucidum, has the effects of tonifying qi and soothing the nerves, relieving cough and asthma, and is used for restlessness of the heart and spirit, insomnia and palpitation, lung deficiency with cough and asthma, debilitation with shortness of breath, and loss of appetite. Studies have shown that Ganoderma lucidum contains many bioactive substances such as Ganoderma lucidum polysaccharides, Ganoderma lucidum triterpenes, sterols, nucleotides, and peptidoglycans, and has many biological activities such as immune regulation, anti-tumor, anti-inflammatory, and regulation of intestinal flora.

[0003] The traditional production method of Ganoderma lucidum is wild picking or artificial cultivation of Ganoderma lucidum fruiting bodies, which has the problems of long production cycle, great influence by seasons and climate, unstable quality of raw materials, pesticide residues, and the like, which limits the development and utilization of Ganoderma lucidum resources. Studies have shown that Ganoderma lucidum mycelium produced by liquid fermentation has similar active substance content and physiological functions as Ganoderma lucidum fruiting bodies, and has the advantages of stable material source, stable and controllable production process, no influence by environment, short production cycle, and no pesticide residues, and the like, and the research on Ganoderma lucidum liquid fermentation has gradually attracted attention.

[0004] Based on the bioactive substances and bioactivities of Ganoderma lucidum, such as Ganoderma lucidum polysaccharides, Ganoderma lucidum triterpenes and alkaloids, the research and application of Ganoderma lucidum broken cell extract in medicines, foods and cosmetics and the product development thereof have attracted much attention. The methods for breaking the cell wall of Ganoderma lucidum include physical method, chemical method, enzymatic method and biological fermentation method. For example, patent 201810325349.0 discloses a method for extracting Ganoderma lucidum mycelium polysaccharides by high-pressure breaking. Patent ZL202111098884.5 discloses a process for preparing Ganoderma lucidum extract with low color and high polysaccharide content by hot water dynamic extraction, microfiltration membrane precision filtration and resin dynamic decolorization. Patent with application number 202310938577.6 discloses a method for preparing Ganoderma lucidum sporocarp polysaccharides with the function of preventing or treating neurodegenerative diseases by using Ganoderma lucidum sporocarp as raw material, through water extraction and alkali extraction, fractional alcohol precipitation, deproteinization, ion exchange column chromatography and molecular sieve column chromatography. Patent with application number 202210082453.8 discloses a method for preparing Ganoderma lucidum sporocarp extract for cosmetics by using enzymatic hydrolysis, flash extraction and microwave extraction in combination. Patent ZL202210321892.X discloses a method for breaking the cell wall of Ganoderma lucidum and extracting broken Ganoderma lucidum by using Trichoderma harzianum. The cell wall of Ganoderma lucidum is broken by using Trichoderma harzianum to ferment Ganoderma lucidum sporocarp powder, and then Ganoderma lucidum polysaccharides are extracted by water, and Ganoderma lucidum triterpenes are extracted by ethanol. Patent ZL202111602728.8 discloses a method for preparing yeast fermented Ganoderma lucidum extract by yeast fermentation of Ganoderma lucidum crude extract and compound enzymatic hydrolysis. The solvent extraction has the problems of low extraction rate of active ingredients and organic solvent residue due to the fact that the cell wall is not broken. The ultrasonic extraction, microwave extraction and high-pressure homogenization extraction have the problems of high equipment requirement and high energy consumption, which are not conducive to industrial production. The biological enzyme method and the fermentation method have the problems of exogenous addition of biological enzymes and fermentation bacteria, complex composition of the extract, and unclear active functional substances. SUMMARY

[0005] In view of the above problems of the prior art, the present application aims to provide a Ganoderma lucidum fermentation lysate and a preparation method thereof. The present application uses oat beta-glucan ultrafine powder and Ganoderma lucidum mycelium ultrafine powder for fed-batch fermentation, directionally induces the endogenous enzyme activity of Ganoderma lucidum fermentation mycelium, and then degrades the Ganoderma lucidum fermentation mycelium by using compound metal ions to catalyze the endogenous enzyme, so as to economically and efficiently prepare Ganoderma lucidum fermentation lysate with the function of delaying aging.

[0006] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

[0007] The present application provides a Ganoderma lucidum strain CZDG10, which is classified and named as Ganoderma lucidum Ganoderma lucidum , and is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M20241696.

[0008] The present application also provides a preparation method of Ganoderma lucidum fermentation lysate, which comprises the following steps:

[0009] (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 liquid;

[0010] (2) inoculate the seed liquid of step (1) into a fermentation culture medium at an inoculation amount of 10% (v / v), and ferment at 25-28°C and 140-160 rpm for 2 days; add 0.2-0.8% (m / v) of sterilized inducer to the Ganoderma lucidum fermentation culture, and continue to culture for 5-8 days; terminate the fermentation when the glucose content of the Ganoderma lucidum fermentation supernatant is lower than 2 mg / L, centrifuge, and collect the fermentation mycelium;

[0011] (3) add 0.5-2 times weight of purified water to the fermentation mycelium of step (2), homogenize at 8000 rpm for 5 min, and high-pressure homogenize twice at 80-120 MPa to obtain Ganoderma lucidum fermentation mycelium homogenate;

[0012] (4) add a metal ion catalyst with a final concentration of 1-10 mmol / L to the Ganoderma lucidum fermentation mycelium homogenate of step (3), adjust the temperature to 40-50°C and the pH to 4.5-5.5, and incubate for 1-3 hours; continue to heat to 80-90°C for 1 hour, cool to room temperature, centrifuge at 8000 rpm for 10 min, collect the supernatant, and obtain Ganoderma lucidum fermentation mycelium lysate;

[0013] (5) add 0.5-1% (m / v) of activated carbon to the Ganoderma lucidum fermentation mycelium lysate of step (4) for adsorption for 1 hour, filter through a 0.45 μm filter membrane, collect the filtrate I, ultrafilter the filtrate I through an ultrafiltration membrane with a molecular weight cut-off of 100 kDa, collect the filtrate II, and concentrate the filtrate II through a nanofiltration membrane with a molecular weight cut-off of 200-300 Da, and collect the retentate;

[0014] (6) spray dry the retentate of step (5) to obtain Ganoderma lucidum fermentation lysate.

[0015] Further, the seed culture medium in step (1) is: glucose 20 g / L, yeast powder 5 g / L, potassium dihydrogen phosphate 1.5 g / L, MgSO4 0.5 g / L, and thiamine 5 mg / L.

[0016] Further, the fermentation culture medium in step (2) is: glucose 25 g / L, yeast powder 8 g / L, potassium dihydrogen phosphate 2 g / L, MgSO4 1 g / L, and thiamine 8 mg / L.

[0017] Further, the inducer in the step (2) is oat beta glucan ultrafine powder and Ganoderma lucidum mycelium ultrafine powder, and the weight ratio of the oat beta glucan ultrafine powder to the Ganoderma lucidum mycelium ultrafine powder is 1:2.

[0018] Further, the concentration of the inducer is selected from 0.2%-0.8%, and the particle size of the inducer is selected from 500-1000 mesh.

[0019] Further, the metal ion catalyst in the step (4) is a mixture of K + , Zn 2+ and Mn 2+ , and the addition amount of the metal ion is 1mmol / L-10mmol / L.

[0020] Further, the molar concentration ratio of K ＋ :Zn 2+ :Mn 2+ is 1:2:2.

[0021] Further, K + is one of KCl, K2SO4 and K2CO4, Zn 2+ is one of ZnCl2 and ZnSO4, and Mn 2+ is one of MnCl2 and MnSO4.

[0022] The present application also provides the Ganoderma lucidum fermentation lysate prepared by the preparation method, and the Ganoderma lucidum fermentation lysate can significantly improve the activity of SOD enzyme, effectively inhibit the H2O2-induced COL-I loss, significantly promote the production of elastin, significantly reduce the gene expression of MMP-1, play the antioxidant function and the cell aging delay function, the content of Ganoderma lucidum polysaccharide is greater than 18%, and the content of beta glucan in the Ganoderma lucidum polysaccharide is greater than 5%.

[0023] The present application also provides the application of the Ganoderma lucidum fermentation lysate in preparing the anti-aging cosmetic.

[0024] In an embodiment of the present application, the anti-aging cosmetic is a mask, an essence, a lotion, a cream, a spray, a water agent or a powder.

[0025] Compared with the prior art, the present application has the following advantages and beneficial effects: the present application uses the oat beta glucan ultrafine powder and the Ganoderma lucidum mycelium ultrafine powder with a particle size of 500-1000 mesh as the inducer to induce the Ganoderma lucidum to produce the polysaccharide, and the polysaccharide has the functions of delaying aging, resisting oxidation and resisting cell aging. Ganoderma lucidumCZDG10 (CCTCC:M 20241696) can highly express intracellular cellulase and β-D-glucosidase, and the cellulase and β-D-glucosidase enzyme activities are increased by more than 25 times compared with no inducer. Further, by adding a composite metal ion catalyst, the intracellular enzyme activity is adjusted, the cellulase enzyme activity is 1.8 times that of the control group, and the active substances such as the cell wall and intracellular polysaccharides of Ganoderma lucidum fermentation mycelium can be efficiently degraded. The Ganoderma lucidum fermentation lysate is prepared by combining membrane filtration separation and spray drying, and the whole process is free of exogenous enzymes and / or organic solvents, which is economical, efficient, safe and environmentally friendly.

[0026] The Ganoderma lucidum fermentation lysate prepared by the method has high active substance content, the content of Ganoderma lucidum polysaccharide is as high as 22.3%, and the content of β-glucan in Ganoderma lucidum polysaccharide can reach 6.01%. At the cellular level, the ROS accumulation caused by oxidative damage can be significantly reduced, the SOD enzyme activity can be significantly improved, the antioxidant effect can be played, the collagen and elastin production in human dermal fibroblasts can be significantly improved, the MMP-1 enzyme activity can be reduced, the degradation of collagen can be inhibited, the anti-aging effect can be played, and the Ganoderma lucidum fermentation lysate has obvious advantages and broad application prospects in the preparation of anti-aging cosmetic. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 : Effect of the Ganoderma lucidum fermentation lysate on the activity of HaCaT cells;

[0028] Figure 2 : Effect of the Ganoderma lucidum fermentation lysate on the ROS content of H2O2-induced damaged HaCaT cells.

[0029] Figure 3 : Effect of the Ganoderma lucidum fermentation lysate on the SOD content of H2O2-induced damaged HaCaT cells.

[0030] Figure 4 : Effect of the Ganoderma lucidum fermentation lysate on the COL-I production of HSF cells.

[0031] Figure 5 : Effect of the Ganoderma lucidum fermentation lysate on the ELN production of HSF cells.

[0032] Figure 6 : Effect of the Ganoderma lucidum fermentation lysate on the MMP-1 gene expression of HSF cells.

[0033] * or ** indicates that there is a significant difference (p<0.05) or a very significant difference (p<0.01) compared with the model group.

[0034] # or ## indicates that there is a significant difference (p<0.05) or a very significant difference (p<0.01) between the model group and the negative control group. DETAILED DESCRIPTION

[0035] The application will be further described in conjunction with the specific embodiments. However, the scope of protection of the application is not limited to the following embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the application without creative labor fall within the scope of protection of the application.

[0036] 1. Culture medium

[0037] PDA culture medium: potato 200 g / L, glucose 20 g / L, agar 20 g / L, pH value natural.

[0038] 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 minutes.

[0039] Fermentation culture medium: glucose 25 g / L, yeast powder 8 g / L, potassium dihydrogen phosphate 2 g / L, MgSO4 1 g / L, thiamine 8 mg / L, sterilized at 121°C for 15 minutes.

[0040] Inducer: oat beta-glucan ultrafine powder (commercially available), Ganoderma lucidum mycelium ultrafine powder (commercially available), sterilized at 121°C for 20 minutes.

[0041] 2. Enzyme activity determination

[0042] Cellulase: cellulase was determined by carboxymethyl cellulase activity determination method. A 0.5% carboxymethyl cellulose sodium solution (CMC-Na) was prepared using 0.1 mol / L acetate buffer (pH 4.5). 1.5 mL of the carboxymethyl cellulose sodium solution was taken as a substrate, 0.5 mL of enzyme solution was added, and the mixture was placed in a 50°C water bath for incubation for 30 min. Then, 2 mL of DNS was added to terminate the reaction, and the mixture was heated in a boiling water bath for 5 min for color development. After cooling to room temperature, the mixture was diluted to 25 mL. A standard curve was drawn using glucose, and the enzyme activity was determined.

[0043] 540 nm wavelength absorbance (OD 540 ), and the inactivated enzyme solution was used as a blank control. A standard curve was drawn using glucose, and the enzyme activity was calculated.

[0044] The enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the production of 1 μmol of reducing sugar per minute under the experimental conditions, which was 1 enzyme activity unit, measured in U / mL.

[0045] β-D-glucosidase: β-D-glucosidase was determined by p-nitrophenyl-β-D-glucoside (pNPG) method. A 5 mmol / L p-nitrophenyl-β-D-glucoside (pNPG) solution was prepared using 0.1 mol / L acetate buffer (pH 4.5). 0.8 mL of the p-nitrophenyl-β-D-glucoside solution was taken as the substrate, and the absorbance value (OD 405 ) at 405 nm was measured, with inactivated enzyme solution as a blank control. The standard curve was drawn using p-nitrophenol (pNP), and the enzyme activity was calculated.

[0046] The enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the production of 1 μmol of pNP from pNPG per minute under experimental conditions, and was expressed in U / mL.

[0047] 3. Biomass determination

[0048] After fermentation, 50 mL of the fermentation broth was centrifuged at 10,000 r / min for 10 min, the mycelium was washed with distilled water for 3 times, and dried at 80 ℃ for 48 h. The biomass was weighed and calculated.

[0049] 4. Determination of polysaccharide content

[0050] Ganoderma lucidum polysaccharide: The total sugar content was determined by the benzenesulfonic acid-sulfuric acid method, and the reducing sugar content was determined by the DNS method. The polysaccharide content = total sugar content - reducing sugar content.

[0051] β-glucan: The β-glucan content was detected by a β-glucanase kit.

[0052] The content of β-glucan in Ganoderma lucidum polysaccharide = β-glucan content / Ganoderma lucidum polysaccharide content × 100%.

[0053] Example 1: Screening and identification of Ganoderma lucidum CZDG10

[0054] Wild Ganoderma lucidum was picked from Dabie Mountain, and after surface disinfection, a suitable amount of fruiting body was aseptically picked and inoculated into PDA plate medium (10 plates). After 10-15 days of culture at 26 ℃, the 2# strain colony diameter was the largest, and further screening was carried out.

[0055] Strain directed evolution was carried out by continuous plate rejuvenation-shaking flask fermentation: (1) Plate culture: the 2# slope strain was transferred to plate culture (20 plates), and the plate culture was carried out at 26 ℃ for 8 days (PDA medium). The colony diameter was measured. (2) Shaking flask culture: 5 dominant colonies were picked from the plate culture mycelium for shaking flask culture, and 0.5 cm 2Ten plate colonies were inoculated into shake flasks containing fermentation medium and cultured at 26℃ and 150 rpm for 8 days as one generation. The culture was continuously subcultured for 3 generations. The biomass of the shake flask culture was measured. (3) Fine mycelia were picked from the dominant strains in the shake flasks in step (2) and transferred to the plate culture in step (1) (20 plates). Five colonies with vigorous mycelial growth were selected for the shake flask culture in step (2).

[0056] After three consecutive 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, with dense hyphae in the center and sparse hyphae at the edges. Methylene blue staining revealed clamp connections within the mycelium.

[0057] Using universal primers ITS1 and ITS4 as primers, the primer sequences are as follows:

[0058] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'

[0059] ITS4: 5'-TCCTCCGCTTATTGATATGC-3'

[0060] Using total DNA from CZDG10 mycelium as a template, ITS region sequence was amplified by PCR.

[0061] The 50 μL PCR reaction system is as follows: 1 μL of DNA template and 10 μM F are added to a 50 μL reaction system.

[0062] 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.

[0063] The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 2 min, 35 cycles of amplification, and 72℃ final extension for 5 min.

[0064] The PCR amplification products were sequenced for the ITS region, and the sequences were aligned using BLAST on the National Center for Biotechnology Information (NCBI) website. The alignment results showed that the sequences were similar to the original sequences. Ganoderma lucidum The sequence similarity (ID: GU213479.1) was 99.51%. Based on the morphological and molecular biological experimental results, the strain was named Ganoderma lucidum CZDG10 (…). Ganoderma lucidum CZDG10), which is deposited at the China Center for Type Culture Collection.

[0065] The preservation information is: Red Ganoderma Ganoderma lucidum CZDG10 was preserved in China Center for Type Culture Collection on July 26, 2024, located at Wuhan University, Wuhan, China. Ganoderma lucidum The preservation number of CZDG10 is CCTCC NO: M 20241696.

[0066] Example 2: Preparation of Red Ganoderma fermentation lysate

[0067] Fresh culture strain Red Ganoderma Ganoderma lucidum CZDG10 (CCTCC NO: M 20241696) was taken 8-10 pieces of 0.5x0.5cm mycelium blocks, inoculated into 100mL seed culture medium, cultured at 26℃, 150rpm for 7 days to obtain seed liquid; the seed liquid was inoculated into 1000mL fermentation medium at an inoculation amount of 10% (v / v), and fermented at 26℃, 150rpm for 2 days. 0.5% (m / v) of sterilized inducer was added to the Red Ganoderma fermentation culture, and the fermentation culture was continued for 5-8 days. When the glucose content of the Red Ganoderma fermentation supernatant was less than 2mg / L, the fermentation was terminated, and the fermentation mycelium was collected by centrifugation at 8000rpm for 10min.

[0068] 1 times weight of purified water was added to the fermentation mycelium, homogenized at 8000rpm for 5min, and homogenized twice at 100MPa high pressure to obtain Red Ganoderma fermentation mycelium homogenate. A metal ion catalyst with a concentration of 5mmol / L (molar concentration ratio of KCl:ZnCl2:MnCl2 is 1:2:2) was added to the Red Ganoderma fermentation mycelium homogenate, the temperature was adjusted to 45℃, the pH was adjusted to 5.0, and the reaction was incubated for 2 hours. The temperature was then increased to 85℃ for 1 hour, and then cooled to room temperature. The supernatant was collected by centrifugation at 8000rpm for 10min to obtain the Red Ganoderma fermentation mycelium lysate.

[0069] 0.5% (m / v) of activated carbon was added to the Red Ganoderma fermentation mycelium lysate and adsorbed for 1 hour, then filtered through a 0.45μm filter membrane to collect the filtrate I. The filtrate I was ultrafiltered using an ultrafiltration membrane with a molecular weight cut-off of 100kDa, and the filtrate II was collected. The filtrate II was concentrated by nanofiltration using a nanofiltration membrane with a molecular weight cut-off of 200-300Da, and the concentration of soluble solids was adjusted to about 15%. The retentate was collected and spray dried to obtain the Red Ganoderma fermentation lysate.

[0070] Example 3: Effect of different inducers on Red Ganoderma fermentation lysate

[0071] 1. Effect of inducer type on Red Ganoderma fermentation lysate

[0072] 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 different mass ratios of ultrafine powder were added respectively, with no inducer added as the control, and other process conditions unchanged, to study the effects of different inducer types on Ganoderma lucidum fermentation lysate.

[0073] Table 1: Effects of different inducer types on Ganoderma lucidum fermentation lysate

[0074]

[0075] The results are shown in Table 1: The biomass of Ganoderma lucidum fermentation mycelium, the cellulase and β-D-glucosidase enzyme activities of Ganoderma lucidum mycelium, the yield of fermentation lysate, and the contents of Ganoderma lucidum polysaccharide and β-glucan were low without adding inducer. The biomass of Ganoderma lucidum fermentation mycelium, the cellulase and β-D-glucosidase enzyme activities of Ganoderma lucidum mycelium, the yield of fermentation lysate, and the contents of Ganoderma lucidum polysaccharide and β-glucan in fermentation lysate were all improved by adding 0.5% of oat β-glucan ultrafine powder (A), Ganoderma lucidum mycelium powder ultrafine powder (B), and Ganoderma lucidum fruiting body ultrafine powder (C) for induction fermentation. Among them, oat β-glucan ultrafine powder had a significant promoting effect on the improvement of β-D-glucosidase enzyme activity, and Ganoderma lucidum mycelium powder ultrafine powder had a significant promoting effect on the improvement of cellulase enzyme activity.

[0076] Further study on the effects of different mass ratios of oat β-glucan ultrafine powder and Ganoderma lucidum mycelium powder ultrafine powder on Ganoderma lucidum fermentation lysate showed that the biomass of Ganoderma lucidum fermentation, the cellulase and β-D-glucosidase enzyme activities of Ganoderma lucidum mycelium, the yield of fermentation lysate, and the contents of Ganoderma lucidum polysaccharide and β-glucan in fermentation lysate were all significantly improved by adding a compound powder of oat β-glucan ultrafine powder and Ganoderma lucidum mycelium powder ultrafine powder with a mass ratio of 1:2. The compound powder of oat β-glucan ultrafine powder and Ganoderma lucidum mycelium powder ultrafine powder with a mass ratio of 1:2 was selected as the inducer.

[0077] 2. Effects of inducer concentration on Ganoderma lucidum fermentation lysate

[0078] On the basis of the above basic process, 0.1%, 0.2%, 0.5%, 0.8%, 1.0%, and 2.0% of a compound powder of oat β-glucan ultrafine powder and Ganoderma lucidum mycelium powder ultrafine powder with a weight ratio of 1:2 were added, with no inducer added as the control, and other process conditions unchanged, to study the effects of different inducer concentrations on Ganoderma lucidum fermentation lysate.

[0079] Table 2: Effects of different inducer concentrations on Ganoderma lucidum fermentation lysate

[0080]

[0081] The results are shown in Table 2: in the range of 0.1%-0.5%, with the increase of the concentration of the inducing agent, the fermentation biomass of Ganoderma lucidum, the cellulase and β-D-glucosidase enzyme activity of Ganoderma lucidum fermentation mycelium, the yield of fermentation lysate, the Ganoderma lucidum polysaccharide and β-glucan content of fermentation lysate are all increased; in the range of 0.5%-2.0%, with the increase of the concentration of the inducing agent, all the indexes are decreased, which is probably due to the fact that the high concentration of the additive causes the high viscosity of the fermentation system, which affects the fermentation of Ganoderma lucidum and the extraction of fermentation lysate; the optimal concentration of the inducing agent is 0.5%; in the range of 0.2%-0.8%, the fermentation biomass of Ganoderma lucidum, the cellulase and β-D-glucosidase enzyme activity of Ganoderma lucidum fermentation mycelium, the yield of fermentation lysate, the Ganoderma lucidum polysaccharide and β-glucan content of fermentation lysate are all kept at a high level; therefore, the concentration of the inducing agent is selected to be 0.2%-0.8%.

[0082] 3. Effect of the particle size of the inducing agent on the fermentation lysate of Ganoderma lucidum

[0083] On the basis of the above basic process, the oat β-glucan ultrafine powder with a particle size of 50 mesh, 200 mesh, 500 mesh, 800 mesh, 1000 mesh, 2000 mesh and 5000 mesh is respectively mixed with the Ganoderma lucidum mycelium powder ultrafine powder at a weight ratio of 1:2, and the process conditions are the same as those in the above basic process except that no inducing agent is added, so as to study the effect of different particle sizes of the inducing agent on the fermentation lysate of Ganoderma lucidum.

[0084] Table 3: Effect of different particle sizes of the inducing agent on the fermentation lysate of Ganoderma lucidum

[0085]

[0086] The results are shown in Table 3: when the particle size of the inducing agent is between 500 mesh and 1000 mesh, the fermentation biomass of Ganoderma lucidum, the cellulase and β-D-glucosidase enzyme activity of Ganoderma lucidum fermentation mycelium, the yield of fermentation lysate, the Ganoderma lucidum polysaccharide and β-glucan content of fermentation lysate are all kept at a high level; when the particle size of the inducing agent is large (100-200 mesh), the inducing effect is poor, the fermentation biomass of Ganoderma lucidum is low, and the yield of Ganoderma lucidum fermentation lysate is affected; when the particle size of the inducing agent is small (2000-5000 mesh), the inducing effect is strong, the mycelium is fine and in a filamentous shape, but the cellulase and β-D-glucosidase enzyme activity of Ganoderma lucidum fermentation mycelium is reduced, which is not conducive to the preparation of Ganoderma lucidum fermentation lysate. Therefore, the particle size of the inducing agent is selected to be 500 mesh-1000 mesh.

[0087] Example 4: Effect of different metal ions on the cellulase enzyme activity of Ganoderma lucidum

[0088] 1. Effect of the type of metal ion on the cellulase enzyme activity of Ganoderma lucidum

[0089] The homogenate of Ganoderma lucidum fermentation mycelium 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+ , and Mn 2+ ions were added to the supernatant to a final concentration of 5 mmol / L, respectively, and the mixture was placed at room temperature for 1 hour. The cellulase enzyme activity was determined. The crude enzyme solution inactivated by a boiling water bath was added with different concentrations of metal ions as the blank control of the corresponding metal ions. The enzyme activity without the addition of metal ions was 100%. The relative enzyme activity was used to compare the effect of metal ions on enzyme activity.

[0090] Table 4: Effect of different metal ions on the enzyme activity of Ganoderma lucidum intracellular cellulase

[0091]

[0092] The results are shown in Table 4: Mn 2+ , Zn 2+ , and K ＋ ions had a significant activating effect on the enzyme activity of Ganoderma lucidum mycelium intracellular cellulase, among which the activating effect of potassium ion was the most obvious, and the enzyme activity was 162.1% ± 5.2% of the control group. Na + and Ca 2+ ions had no significant effect on the enzyme activity of Ganoderma lucidum mycelium intracellular cellulase. Mg 2+ , Cu 2+ , and Fe 2+ ions had a significant inhibitory effect on the enzyme activity of Ganoderma lucidum mycelium intracellular cellulase, among which Cu 2+ had the most obvious inhibitory effect, and the enzyme activity was only 22.4% ± 2.5% of the control. Therefore, during the use of Ganoderma lucidum mycelium intracellular cellulase, Mg 2+ , Cu 2+ , and Fe 2+ ions should be avoided as much as possible, and Mn 2+ , Zn 2+ , and K ＋ ions can be appropriately added to improve the enzyme activity.

[0093] 2. Effect of different molar concentration ratios of metal ions on the enzyme activity of Ganoderma lucidum intracellular cellulase

[0094] The homogenate of Ganoderma lucidum fermentation mycelium in Example 2 was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. K ＋ , Zn 2+ , and Mn2+ The crude enzyme solution inactivated by boiling water bath was added with different concentrations of metal ions to serve as blank controls of the corresponding metal ions. The enzyme activity without the addition of metal ions was 100%. The effects of metal ions on enzyme activity were compared in terms of relative enzyme activity.

[0095] The binding sites of different metal ions for the activation of cellulase were different. Further study was made on the effects of different molar concentration ratios of metal ions on the enzyme activity of intracellular cellulase of Ganoderma lucidum mycelium. The results, as shown in Table 5, indicated that K ＋ : Zn 2+ : Mn 2+ The activation of intracellular cellulase of Ganoderma lucidum mycelium was most obvious when the molar concentration ratio was 1:2:2, reaching as high as 178.5%±6.6%.

[0096] Table 5: Effects of different metal ion ratios on the enzyme activity of intracellular cellulase of Ganoderma lucidum

[0097]

[0098] 3. Effects of different amounts of metal ions on the fermentation and lysis of Ganoderma lucidum

[0099] The homogenate of Ganoderma lucidum mycelium in Example 2 was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The supernatant was added with K ＋ : Zn 2 + : Mn 2+ The crude enzyme solution inactivated by boiling water bath was added with different concentrations of metal ions to serve as blank controls of the corresponding metal ions. The enzyme activity without the addition of metal ions was 100%. The effects of metal ions on enzyme activity were compared in terms of relative enzyme activity.

[0100] Table 6: Effects of different amounts of metal ions on the enzyme activity of Ganoderma lucidum

[0101]

[0102] The results are shown in Table 6: The results show that in the range of 0.5 mmol / L-5 mmol / L of metal ion addition amount, with the increase of metal ion concentration, the relative enzyme activity of cellulase activity gradually increases, in the range of 5 mmol / L-50 mmol / L of metal ion addition amount, with the increase of metal ion concentration, the relative enzyme activity of cellulase activity gradually decreases, especially when the concentration is more than 10 mmol / L, the relative enzyme activity of cellulase activity significantly decreases, and 50 mmol / L is only 34.7%±1.9% of the enzyme activity of the control group. The optimal metal ion addition amount is 5 mmol / L, and in the range of 1 mmol / L-10 mmol / L, the relative enzyme activity of cellulase activity remains at a high level, therefore, the metal ion addition amount is 1 mmol / L-10 mmol / L.

[0103] Example 5: Preparation of Ganoderma lucidum fermentation lysate

[0104] Freshly cultured strain Ganoderma lucidum Ganoderma lucidum CZDG10 (CCTCC:M20241696), 8-10 pieces of 0.5×0.5 cm mycelial blocks were taken and inoculated into 100 mL of seed culture medium, cultured at 26°C, 150 rpm for 7 days to obtain a seed liquid; the seed liquid was inoculated into 1000 mL of fermentation medium at an inoculation amount of 10% (v / v), and fermented at 26°C, 150 rpm for 2 days. 5 g of inducer was added to the Ganoderma lucidum fermentation culture, the inducer was a sterilized compound powder with a particle size of 800 meshes, and the weight ratio of oat β-glucan ultrafine powder and Ganoderma lucidum mycelium ultrafine powder was 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, and the fermentation was terminated when the glucose content of the Ganoderma lucidum fermentation supernatant was less than 2 mg / L. The fermentation mycelium was collected by centrifugation at 8000 rpm for 10 min, and the weight of the fermentation mycelium was 565 g.

[0105] 565 g of purified water was added to the fermentation mycelium, homogenized at 8000 rpm for 5 min, and homogenized twice at 100 MPa high pressure to obtain Ganoderma lucidum fermentation mycelium homogenate. The cellulase enzyme activity in the supernatant of the Ganoderma lucidum fermentation mycelium homogenate was 3.96 U / mL, and the β-D-glucosidase enzyme activity was 2.79 U / mL.

[0106] 5 mmol / L of metal ion catalyst (K ＋ :Zn 2+ :Mn 2 with a molar concentration ratio of 1:2:2) was added to the Ganoderma lucidum fermentation mycelium homogenate, the temperature was adjusted to 45°C, the pH was adjusted to 5.0, and the reaction was incubated for 2 hours. The temperature was then increased to 85°C for 1 hour, and then cooled to room temperature. The supernatant was collected by centrifugation at 8000 rpm for 10 min to obtain Ganoderma lucidum fermentation mycelium lysate.

[0107] The 0.5% (m / v) activated carbon was added to the Ganoderma lucidum fermentation mycelium lysate, and adsorbed for 1 hour, and then filtered through a 0.45 μm filter membrane to collect the filtrate I. The filtrate I was ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 100 kDa, and the filtrate II was collected. The filtrate II was concentrated by nanofiltration through a nanofiltration membrane with a molecular weight cut-off of 200-300 Da, and the concentration of soluble solids was about 15% after concentration, and the retentate was collected. The retentate was spray dried to obtain 14.6 g of Ganoderma lucidum fermentation lysate. The content of Ganoderma lucidum polysaccharide in the Ganoderma lucidum fermentation lysate was 21.5%, and the content of β-glucan was 1.25%, and the content of β-glucan accounted for 5.81% of the Ganoderma lucidum polysaccharide.

[0108] Example 6: Preparation of Ganoderma lucidum fermentation lysate

[0109] Freshly cultured strain Ganoderma lucidum Ganoderma lucidum CZDG10 (CCTCC:M20241696), 8-10 pieces of 0.5×0.5 cm mycelium were taken and inoculated into 100 mL of seed culture medium, and cultured at 28°C and 140 rpm for 5 days to obtain a seed liquid; the seed liquid was inoculated into 1000 mL of fermentation medium at an inoculation amount of 10% (v / v), and fermented at 28°C and 140 rpm for 2 days. 10 g of inducer was added to the Ganoderma lucidum fermentation culture, and the inducer was a sterilized compound powder with a particle size of 500 mesh, and the weight ratio of oat β-glucan ultrafine powder to Ganoderma lucidum mycelium ultrafine powder was 1:2 (the compound powder was prepared into a 10% (m / v) solution with purified water, and sterilized at 121°C for 20 minutes). The fermentation culture was continued for 8 days, and the fermentation was terminated when the glucose content of the Ganoderma lucidum fermentation supernatant was less than 2 mg / L. The fermentation mycelium was collected by centrifugation at 8000 rpm for 10 min, and the weight of the fermentation mycelium was 602 g.

[0110] 1204 g of purified water was added to the fermentation mycelium, and homogenized at 8000 rpm for 5 min, and homogenized twice at 80 MPa to obtain Ganoderma lucidum fermentation mycelium homogenate. The cellulase enzyme activity in the supernatant of the Ganoderma lucidum fermentation mycelium homogenate was 2.45 U / mL, and the β-D-glucosidase enzyme activity was 1.85 U / mL.

[0111] A metal ion catalyst (K ＋ :Zn 2+ :Mn 2 ) with a concentration of 10 mmol / L was added to the Ganoderma lucidum fermentation mycelium homogenate, the temperature was adjusted to 50°C, the pH was adjusted to 4.5, and the reaction was incubated for 3 hours. The temperature was continued to be raised to 90°C for 1 hour, and then cooled to room temperature. The supernatant was collected by centrifugation at 8000 rpm for 10 min to obtain Ganoderma lucidum fermentation mycelium lysate.

[0112] The 0.5% (m / v) activated carbon was added to the Ganoderma lucidum fermentation mycelium lysate, and adsorbed for 1 hour, and then filtered through a 0.45 μm filter membrane to collect the filtrate I. The filtrate I was ultrafiltered by using an ultrafiltration membrane with a molecular weight cut-off of 100 kDa, and the filtrate II was collected. The filtrate II was concentrated by using a nanofiltration membrane with a molecular weight cut-off of 200-300 Da, and the concentration of soluble solids was about 8%. The retentate was collected. The retentate was spray dried to obtain 14.1 g of Ganoderma lucidum fermentation lysate. The content of Ganoderma lucidum polysaccharide in the Ganoderma lucidum fermentation lysate was 22.3%, and the content of β-glucan was 1.34%, and the content of β-glucan accounted for 6.01% of the Ganoderma lucidum polysaccharide.

[0113] Example 7: Preparation of Ganoderma lucidum fermentation lysate

[0114] Freshly cultured strain Ganoderma lucidum Ganoderma lucidum CZDG10 (CCTCC: M20241696), 8-10 pieces of 0.5x0.5 cm mycelium were taken, inoculated into 100 mL seed culture medium, and cultured at 25°C, 160 rpm for 6 days to obtain seed liquid; the seed liquid was inoculated into 1000 mL of fermentation medium at an inoculation amount of 10% (v / v), and fermented at 25°C, 160 rpm for 2 days. 2 g of inducer was added to the Ganoderma lucidum fermentation culture, and the inducer was a sterilized compound powder with a particle size of 1000 mesh, and the weight ratio of oat β-glucan ultrafine powder to Ganoderma lucidum mycelium ultrafine powder was 1:2. The compound powder was prepared into a 10% (m / v) solution with purified water, and sterilized at 121°C for 60 minutes. The fermentation was continued for 5 days. When the glucose content of the Ganoderma lucidum fermentation supernatant was less than 2 mg / L, the fermentation was terminated, and the fermentation mycelium was collected by centrifugation at 8000 rpm for 10 min, and the weight of the fermentation mycelium was 480 g.

[0115] 240 g of purified water was added to the fermentation mycelium, and homogenized at 8000 rpm for 5 min, and homogenized twice at 120 MPa high pressure to obtain Ganoderma lucidum fermentation mycelium homogenate. The cellulase enzyme activity in the supernatant of the Ganoderma lucidum fermentation mycelium homogenate was 5.05 U / mL, and the β-D-glucosidase enzyme activity was 3.78 U / mL.

[0116] A metal ion catalyst (K ＋ :Zn 2+ :Mn 2 ) with a concentration of 1 mmol / L was added to the Ganoderma lucidum fermentation mycelium homogenate, the temperature was adjusted to 40°C, the pH was adjusted to 5.5, and the reaction was incubated for 1 hour. The temperature was then increased to 80°C for 1 hour, and then cooled to room temperature. The supernatant was collected by centrifugation at 8000 rpm for 10 min to obtain Ganoderma lucidum fermentation mycelium lysate.

[0117] The 1% (m / v) activated carbon was added to the Ganoderma lucidum fermentation mycelium lysate, and adsorbed for 1 hour. The filtrate I was collected by 0.45 μm filter membrane filtration. The filtrate I was ultrafiltered by using an ultrafiltration membrane with a molecular weight cut-off of 100 kDa. The filtrate II was collected. The filtrate II was concentrated by using a nanofiltration membrane with a molecular weight cut-off of 200-300 Da, and concentrated to a soluble solid concentration of about 30%. The retentate was collected. The retentate was spray dried to obtain 12.5 g of Ganoderma lucidum fermentation lysate. The content of Ganoderma lucidum polysaccharide in the Ganoderma lucidum fermentation lysate was 18.2%, and the content of β-glucan was 0.92%. The content of β-glucan accounted for 5.05% of the content of Ganoderma lucidum polysaccharide.

[0118] Example 8: Preparation of Ganoderma lucidum fermentation lysate

[0119] The fresh culture strain Ganoderma lucidum was taken Ganoderma lucidum CZDG10 (CCTCC: M20241696) was inoculated into a seed culture medium, and cultured at 26°C and 150 rpm for 7 days to obtain a first-stage seed liquid. The first-stage seed liquid was inoculated into a seed culture medium, and cultured at 26°C and 150 rpm for 7 days to obtain a second-stage seed liquid.

[0120] The 200 L fermentation medium was loaded into a 300 L fermentation tank, and sterilized at 121°C for 30 minutes. When the temperature decreased to about 26°C, the second-stage seed liquid was inoculated into the fermentation tank at an inoculation amount of 10% (v / v). The temperature was controlled at 26°C, the rotation speed was 200 rpm, and the aeration amount was 100 L / min. The fermentation culture was carried out for 2 days. 1 kg of inducer was added to the Ganoderma lucidum fermentation culture. The inducer was a sterilized compound powder with a particle size of 800 meshes, and the weight ratio of oat β-glucan ultrafine powder to Ganoderma lucidum mycelium ultrafine powder was 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. When the glucose content of the Ganoderma lucidum fermentation supernatant was less than 2 mg / L, the fermentation was terminated. The fermentation mycelium was collected by centrifugation at 8000 rpm for 10 min, and the weight of the fermentation mycelium was 11.2 kg.

[0121] The 11.2 kg of purified water was added to the fermentation mycelium, and homogenized at 8000 rpm for 5 min. The Ganoderma lucidum fermentation mycelium homogenate was obtained by high-pressure homogenization twice at 100 MPa. The cellulase enzyme activity in the supernatant of the Ganoderma lucidum fermentation mycelium homogenate was 3.85 U / mL, and the β-D-glucosidase enzyme activity was 2.72 U / mL.

[0122] The metal ion catalyst (K ＋ :Zn 2+ :Mn 2The molar concentration ratio was 1:2:2. The temperature was adjusted to 45℃ and the pH to 5.0. The reaction was maintained at this temperature for 2 hours. The temperature was then increased to 85℃ and the reaction was continued for 1 hour. The mixture was cooled to room temperature, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected to obtain the Ganoderma lucidum fermentation mycelium lysate.

[0123] Add 0.5% (m / v) activated carbon to the lysate of Ganoderma lucidum fermentation mycelium for 1 hour for adsorption, filter through a 0.45 μm filter membrane, and collect filtrate I. Filtrate I is then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, and filtrate II is collected. Filtrate II is then concentrated using a nanofiltration membrane with a molecular weight cutoff of 200-300 Da, until the soluble solids concentration is approximately 15%, and the retentate is collected. The retentate is spray-dried to obtain 2.85 kg of Ganoderma lucidum fermentation lysate. The Ganoderma lucidum fermentation lysate contains 20.7% Ganoderma lucidum polysaccharides and 1.13% β-glucan, with β-glucan accounting for 5.46% of the total Ganoderma lucidum polysaccharides.

[0124] Example 9: Safety of Ganoderma lucidum fermentation lysate

[0125] The MTT assay was used to investigate the cytotoxicity of Ganoderma lucidum fermentation lysate to HaCaT cells. Generally, when cell viability is above 80%, the sample is considered to have no significant toxicity to cells. HaCaT cells were resuscitated and cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics until confluence reached 80-90%. After digestion, the cells were seeded in 96-well plates at a density of 5 × 10⁶ cells / well. 4 Inoculation was performed at 100 μL per cell / mL, and the cells were incubated at 37°C with 5% CO2 for 12–24 h. The culture medium was then discarded, and samples prepared using Experiment 3 at different concentrations were added to each well. Incubation continued for 24 h. Then, 20 μL of 5 mg / mL MTT solution was added to each well. After 4 h, the culture medium was discarded, and 150 μL of dimethyl sulfoxide was added to each well. The mixture was thoroughly mixed, and the absorbance was measured at 490 nm using a microplate reader.

[0126] See 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 50-1000 μg / mL, the cell viability of HaCaT cells is higher than 80%, and when the sample concentration is lower than 600 μg / mL, the cell viability is above 90%. Therefore, it can be considered that Ganoderma lucidum extract has no obvious toxicity to HaCaT cells in the concentration range of 50-1000 μg / mL.

[0127] Example 10: Antioxidant activity of Ganoderma lucidum fermentation lysate

[0128] (1) Reactive oxygen species (ROS) scavenging

[0129] HaCaT cells were 1.2 × 10⁻⁶ 6Cells were seeded per well in 6-well plates and cultured in complete culture medium for 12-24 hours. All cells were then divided into four groups according to the table below:

[0130]

[0131] The cells were incubated for another 24 hours according to the experimental groups described above, with the concentrations of the fermented Ganoderma lucidum lysate in the sample groups being 100, 200, and 500 μg / mL. After the culture and stimulation process was completed, the culture medium was aspirated and the cells were washed with PBS. The cells were then treated with a 10 μM DCFH-DA fluorescent probe at 37°C for 30 min, followed by washing with PBS twice, digestion, and cell collection. The intensity of the fluorescent DCF was detected using a multi-functional microplate reader at an emission wavelength of 525 nm (excitation wavelength of 488 nm).

[0132] See results Figure 2 ,Depend on Figure 2 It was found that after H2O2 treatment, the ROS level of HaCaT cells increased significantly, reaching approximately 160% of that of the control group. After treatment with different concentrations of Ganoderma lucidum fermentation lysate, the fluorescence intensity decreased significantly with increasing sample concentration (P<0.01). When the sample concentration was 500 μg / mL, the fluorescence intensity was 80.17% of that of the model group, indicating that Ganoderma lucidum extract can effectively remove ROS caused by H2O2, prevent the accumulation of intracellular ROS, and help resist oxidative damage.

[0133] (2) Superoxide dismutase (SOD) activity

[0134] HaCaT cells were 1.2 × 10⁻⁶ 6 Cells were seeded per well in 6-well plates and cultured in complete medium for 12-24 hours. All cells were then divided into four groups according to the ROS scavenging assay protocol. After culture and stimulation, cells were collected, lysed, and total protein was extracted and its concentration determined. Superoxide dismutase (SOD) activity was then measured according to the kit instructions.

[0135] See results Figure 3 ,Depend on Figure 3 It was found that after H2O2 treatment, the SOD activity in the model group decreased to 67.11% of that in the control group (P<0.01). As the concentration of Ganoderma lucidum fermentation lysate increased, the SOD activity gradually increased. When the concentration of Ganoderma lucidum extract was 200 μg / mL, the SOD activity increased significantly (P<0.05), which was 116.82% of that in the model group. This indicates that Ganoderma lucidum fermentation lysate can significantly increase the activity of SOD enzyme and exert an antioxidant effect.

[0136] Example 11: The Anti-Cellular Senescence Effect of Ganoderma Lucidum Fermentation Lysate

[0137] (1) Effects of Ganoderma lucidum fermentation lysate on human dermal fibroblasts (HSF) under oxidative stress

[0138] In vivo, elastin and collagen are two important and widely existing proteins, which play a crucial role in our skin and even body health. Fibroblasts are the main cells in the dermis of the skin, responsible for the synthesis of the extracellular matrix of the dermis 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 more than 80% in young skin, which is the most important collagen in the skin and determines the appearance of the skin. Elastin (ELN) is the main component of elastic fibers, which plays a major role in maintaining skin tissue elasticity and flexibility, maintaining skin elasticity and firmness, and preventing wrinkle formation. The decrease of COL-I and ELN content will lead to skin relaxation and wrinkle, which is an important indicator of skin aging. The present application uses H2O2 to induce human dermal fibroblasts (HSF) to construct a damaged cell model, and uses collagen and elastin as indicators to study the anti-aging effect of Ganoderma lucidum fermentation lysate.

[0139] HSF cells were inoculated in 12-well plates at 5×10 5 cells / well, and cultured in complete medium for 12-24 h to adhere the cells, and then all the cells were divided into four groups according to the following table:

[0140]

[0141] The samples were incubated for 24 h according to the above experimental grouping, and the concentration of Ganoderma lucidum fermentation lysate in the sample group was 100 μg / mL, 200 μg / mL and 500 μg / mL. After the culture and stimulation process was completed, the culture supernatant was carefully collected into a centrifuge tube, centrifuged at 3000 rpm for 10 min to remove cell debris and other impurities, and the supernatant was detected for COL-I and ELN content according to the ELISA kit instructions.

[0142] The results of the effect of Ganoderma lucidum fermentation lysate on the production of COL-I in HSF cells under oxidative stress are shown in Figure 4 , and Figure 4 it can be seen that, taking VC as a positive control, H2O2 treatment of cells leads to a significant decrease in COL-I production, which is 33.56% lower than the blank control group (P<0.01). p The results of the effect of Ganoderma lucidum fermentation lysate on the production of COL-I in HSF cells under oxidative stress are shown in

[0143] The results of the effect of Ganoderma lucidum fermentation lysate on the production of ELN in HSF cells under oxidative stress are shown inFigure 5 Figure 5 P <0.01), with the increase of the concentration of Ganoderma lucidum fermentation lysate sample, the ELN yield gradually increased, when the concentration of Ganoderma lucidum extract was 200 μg / mL, the ELN yield increased to 121.45% of the model group (P<0.01), and the ELN content was higher than that of the positive control group (VC), indicating that the Ganoderma lucidum extract can significantly promote the production of elastin under oxidative stress, thereby playing a role in delaying aging. P

[0144] (3) RT-PCR detection of HSF cell matrix metalloproteinase-1 (MMP-1) gene expression

[0145] After HSF oxidative stress treatment, total RNA was extracted by Trizol method, and the absorbance values of samples at 260 nm and 280 nm were detected by ultraviolet spectrophotometer to detect the concentration and purity of RNA. A 20 μL qRT-PCR reaction system was established, and the PCR amplification conditions were as follows: pre-denaturation 95℃, 15 min; denaturation 95℃, 15 s, annealing 60℃, 30 s, extension 72℃, 20 s, reaction cycle 40 times. The melting curve reaction conditions were as follows: 95℃ for 1 min, 55℃ for 30 s, and fluorescence signal was collected once every 5℃ increase.

[0146] The effect of Ganoderma lucidum fermentation lysate on MMP-1 expression of H2O2-induced damaged HSF cells is shown in Table 2. Figure 6 Figure 6 It can be seen that under oxidative stress, the MMP-1 mRNA level of the model group HSF cells was significantly increased, and after treatment with different concentrations of Ganoderma lucidum fermentation lysate, the MMP-1 mRNA level was significantly reduced. It is shown that the Ganoderma lucidum fermentation lysate can significantly reduce the gene expression of MMP-1, thereby inhibiting the collagen decomposition caused by MMP-1, and playing a role in delaying aging.

[0147] In summary, the Ganoderma lucidum fermentation lysate prepared in the present application has high content of ganoderma polysaccharide and beta glucan, can significantly reduce the accumulation of ROS caused by oxidative damage at the cellular level, and can significantly improve the SOD enzyme activity, thereby playing an antioxidant effect. At the cellular level, it can significantly improve the production of collagen and elastin of human dermal fibroblasts, reduce the activity of MMP-1 enzyme, inhibit the degradation of collagen, and play a role in delaying aging. The Ganoderma lucidum fermentation lysate prepared in the present application has high content of active polysaccharide, has good antioxidant and anti-aging effects, and has obvious advantages and broad application prospects in the preparation of anti-aging cosmetic products (such as masks, essences, lotions, creams, sprays, water or powder).

[0148] ​​​​The above fact cases only represent the technical solutions of the present application, and are not limited to the experiments. Although the experimental schemes have been improved, researchers in the same field can still further improve the experimental schemes described above or make scientific equivalent replacements to the experimental steps, and these changes do not make the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A strain of Ganoderma lucidum CZDG10, characterized in that: The classification name is Ganoderma lucidum Ganoderma lucidum , and is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: M 20241696.

2. A method for preparing Ganoderma lucidum fermentation lysate, characterized in that, The preparation method comprises the following steps: (1) inoculating Ganoderma lucidum CZDG10 with a preservation number of CCTCC:M 20241696 into a seed culture medium for culture to obtain a seed liquid; (2) inoculating the seed liquid in step (1) into a fermentation culture medium for fermentation culture, adding an inducing agent to the fermentation culture, continuing the fermentation culture, terminating the fermentation when the glucose content in the Ganoderma lucidum fermentation supernatant is lower than 2 mg / L, and centrifuging to collect the fermentation mycelium; the inducing agent is oat beta glucan ultrafine powder and Ganoderma lucidum mycelium ultrafine powder, the weight ratio of the oat beta glucan ultrafine powder to the Ganoderma lucidum mycelium ultrafine powder is 1:2, the concentration of the inducing agent is selected from 0.2% to 0.8%, and the particle size of the inducing agent is selected from 500 meshes to 1000 meshes; (3) adding purified water to the fermentation mycelium in step (2), homogenizing, and high-pressure homogenizing to obtain Ganoderma lucidum fermentation mycelium homogenate; (4) Add a metal ion catalyst to the homogenized liquid of Ganoderma lucidum fermentation mycelium from step (3), adjust the temperature to 40-50℃ and pH to 4.5-5.5, keep the reaction at this temperature for 1-3 hours, continue to raise the temperature to 80-90℃ and react for 1 hour, cool to room temperature, centrifuge and collect the supernatant to obtain the Ganoderma lucidum fermentation mycelium lysate; the metal ion catalyst is K + Zn 2+ and Mn 2+ The mixture contains metal ions added at concentrations of 1 mmol / L to 10 mmol / L, wherein the K ＋ Zn 2+ Mn 2+ The molar concentration ratio is 1:2:2; (5) adding activated carbon adsorption to the Ganoderma lucidum fermentation mycelium homogenate in step (4), filtering to collect filtrate I, using an ultrafiltration membrane with a molecular weight cut-off of 100 kDa to ultrafilter the filtrate I, collecting filtrate II, using a nanofiltration membrane with a molecular weight cut-off of 200-300 Da to nanofiltration concentrate the filtrate II, concentrating to a soluble solid concentration of 8-30%, and collecting the retentate; (6) spray drying the retentate in step (5) to obtain the Ganoderma lucidum fermentation lysate.

3. The Ganoderma lucidum fermentation lysate prepared by the preparation method in claim 2.

4. The Ganoderma lucidum fermentation lysate of claim 3, wherein the Ganoderma lucidum is Ganoderma lucidum (Leyss. ex Fr.) Karst. The Ganoderma lucidum fermentation lysate can significantly improve the activity of SOD enzyme, effectively inhibit the H2O2-induced COL-I loss, significantly promote the production of elastin, significantly reduce the gene expression of MMP-1, play an antioxidant function and a cell aging delaying function, the content of Ganoderma lucidum polysaccharide is greater than 18%, and the content of beta glucan in the Ganoderma lucidum polysaccharide is greater than 5%.

5. The application of the Ganoderma lucidum fermentation lysate in claim 3 in the preparation of a cosmetic with a cell aging delaying function.

6. Use according to claim 5, characterized in that, The cosmetic is a mask, an essence, a lotion, a cream, a spray, a water agent or a powder.

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