Lucid ganoderma melanin nano-selenium as well as preparation method and application thereof

Abstract

The invention discloses lucid ganoderma melanin nano-selenium as well as a preparation method and application thereof. The preparation method of the lucid ganoderma melanin nano-selenium comprises the following steps: preparing a lucid ganoderma melanin solution, uniformly mixing the lucid ganoderma melanin solution with Na2SeO3, SeO2 and H2SeO3 respectively, then adding a Vc solution, stirring to form an orange red solution, dialyzing, and freeze-drying to obtain the lucid ganoderma melanin nano-selenium. The lucid ganoderma melanin nano-selenium disclosed by the invention has anti-inflammatory, anti-oxidation and antibacterial properties, and has certain anti-oxidation capability in the aspects of DPPH., ABTS, hydroxyl free radicals and total reduction capability; the inhibited bacteria comprise escherichia coli, staphylococcus aureus and bacillus subtilis; the method can be used for keeping shiitake mushrooms fresh.

Description

Technical Field

[0001] The invention relates to ganoderma lucidum melanin nano-selenium and a preparation method and application thereof, belonging to the technical field of deep processing of edible fungi. Background Art

[0002] Selenium is an important micronutrient that is essential to human health. It plays a significant role in anti-oxidation, anti-cancer, and immune regulation. The human body cannot synthesize selenium on its own and can only be supplemented through food. However, the difference between the intake of selenium and the toxic dose is small. If it exceeds a certain safety limit, it will have a toxic effect on the human body. How to provide a safe and effective way to supplement selenium has become a difficult problem.

[0003] SeNPs are a new type of selenium particles. Studies have shown that the toxicity of SeNPs is much lower than that of inorganic selenium, and they have high biological activity and bioavailability, providing a new option for selenium supplementation. Supplementing SeNPs can not only solve the problem of selenium deficiency, but also regulate the physiological functions of the human body and improve the ability to resist diseases. However, SeNPs have a high surface free energy and are easy to aggregate and precipitate, which limits their application in the food field. Therefore, improving the stability of SeNPs is a current research hotspot. Commonly used stabilizers generally refer to macromolecular substances such as proteins, polysaccharides, and polyphenols, which can prevent the aggregation of nanoparticles. Melanin, as a natural polyphenolic substance, has rich functional groups and can bind to SeNPs through hydrogen bond interactions. It is an excellent stabilizer. Therefore, it is necessary to study melanin nanoselenium and its properties. Summary of the invention

[0004] The purpose of the present invention is to provide a ganoderma lucidum melanin nano-selenium and a preparation method thereof. The present invention uses GLM as a stabilizer, utilizes Vc to reduce sodium selenite, selenium dioxide and selenious acid, prepares ganoderma lucidum melanin nano-selenium S-GLM, D-GLM and A-GLM with different selenium sources, and conducts structural characterization and stability research on them.

[0005] The preparation method of ganoderma lucidum melanin nano-selenium provided by the present invention comprises the following steps:

[0006] The solution of Ganoderma lucidum melanin is prepared by mixing with Na2SeO3, SeO2 and H2SeO3 respectively, then adding Vc solution, stirring until an orange-red solution is formed, dialyzing and freeze-drying to obtain the solution.

[0007] Using water to prepare the solution of ganoderma lucidum melanin;

[0008] In the solution of the ganoderma lucidum melanin, the concentration of the ganoderma lucidum melanin is 1-2 mg / mL.

[0009] After stirring for 20-40 minutes with a magnetic stirrer, add the Vc solution;

[0010] The orange-red solution was formed by stirring at 50-70°C for 4-5 hours.

[0011] The preparation method of Ganoderma lucidum melanin comprises the following steps:

[0012] The fruiting body of Ganoderma lucidum was crushed, a NaOH solution was added, and the mixture was ultrasonically centrifuged, the pH value of the supernatant was adjusted to 1.5-2 with HCl, the mixture was centrifuged again after water bath, the precipitate was washed with distilled water until it was neutral, and then washed with chloroform and ethyl acetate, the precipitate was centrifuged, and then washed with 95% ethanol and 75% ethanol in sequence, and then washed with distilled water again after centrifugation until it was neutral to obtain a melanin precipitate;

[0013] The melanin precipitate is dissolved in NaOH solution 2, and then HCl solution of the same concentration is added to adjust the pH value to 7-7.2, dialyzed, and freeze-dried to obtain the Ganoderma lucidum melanin.

[0014] The ganoderma lucidum melanin nano-selenium of the invention has anti-inflammatory, anti-oxidation and antibacterial properties.

[0015] Specifically, the Ganoderma lucidum melanin nano-selenium reduces the content of NO, TNF-α, IL-1β, and IL-6 in inflammatory cells, and increases the content of IL-10 in inflammatory cells;

[0016] The ganoderma lucidum melanin nano-selenium reduces the mRNA expression of IL-1β, IL-6, NF-κB, iNOS and COX-2 in inflammatory cells, and increases the mRNA expression of IL-10 in inflammatory cells.

[0017] Specifically, the bacteria inhibited by the Ganoderma lucidum melanin nano-selenium include Escherichia coli, Staphylococcus aureus and Bacillus subtilis.

[0018] The ganoderma lucidum melanin nano-selenium of the present invention can be used for preserving shiitake mushrooms;

[0019] The ganoderma lucidum melanin nano-selenium delays the decrease of the weight loss rate, hardness and soluble solid content of shiitake mushrooms;

[0020] The ganoderma lucidum melanin nano-selenium inhibits the increase of the respiration rate, the opening rate, the color and the relative conductivity of the mushroom;

[0021] The ganoderma lucidum melanin nano-selenium reduces the activity of PPO, increases the activities of POD and CAT, reduces the content of MDA, protects the integrity of cell membranes, and delays aging.

[0022] The present invention establishes an LPS-induced RAW264.7 cell inflammatory injury model to evaluate its anti-inflammatory effect, antioxidant capacity and antibacterial effect; further explores the preservation effect of GLM, S-GLM, D-GLM and A-GLM on shiitake mushrooms. Through antioxidant tests, it is found that GLM, S-GLM, D-GLM and A-GLM have certain antioxidant capacity in terms of DPPH·, ABTS, hydroxyl free radicals and total reducing capacity. In terms of antibacterial effect, GLM, S-GLM, D-GLM and A-GLM have a certain degree of inhibitory effect on Escherichia coli, Staphylococcus aureus and Bacillus subtilis. By exploring the effects of GLM, S-GLM, D-GLM and A-GLM on the preservation of shiitake mushrooms, it was found that treatment with Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium can delay the decrease in weight loss rate, hardness and soluble solids content of shiitake mushrooms, inhibit the increase in respiration rate, opening rate, color and relative conductivity, effectively reduce PPO activity, increase POD and CAT activity, reduce MDA content, effectively inhibit the aging of shiitake mushrooms, and improve the quality of shiitake mushrooms during storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the measurement result of particle size and polydispersity index of S-GLM, D-GLM and A-GLM.

[0024] Figure 2 It is the UV-visible absorption spectra of S-GLM, D-GLM and A-GLM.

[0025] Figure 3 It is the infrared spectra of S-GLM, D-GLM and A-GLM.

[0026] Figure 4 These are scanning electron micrographs of GLM, S-GLM, D-GLM, and A-GLM (A, B, C, and D represent GLM, S-GLM, D-GLM, and A-GLM, respectively) (×30,000).

[0027] Figure 5 It is the thermogravimetric curve analysis of GLM, S-GLM, D-GLM and A-GLM.

[0028] Figure 6 It is the effect of pH value on the stability of S-GLM, D-GLM and A-GLM.

[0029] Figure 7 Yes + Effect of concentration on the stability of S-GLM, D-GLM and A-GLM.

[0030] Figure 8 It is Ca 2+Effect of concentration on the stability of S-GLM, D-GLM and A-GLM.

[0031] Fig. 9 It is the influence of S-GLM, D-GLM and A-GLM on storage stability.

[0032] Fig.10 It is the effect of GLM, S-GLM, D-GLM and A-GLM on the survival rate of RAW264.7 cells.

[0033] Fig.11 The figure shows the effect of different concentrations of LPS on the NO content in RAW264.7 cells.

[0034] Fig.12 The effects of GLM, S-GLM, D-GLM and A-GLM on the survival rate of RAW264.7 cells induced by LPS.

[0035] Fig.13 The effects of GLM, S-GLM, D-GLM and A-GLM on the changes in NO content in RAW264.7 cells induced by LPS.

[0036] Fig.14 The effects of GLM, S-GLM, D-GLM and A-GLM on the TNF-α content in RAW264.7 cells induced by LPS.

[0037] Fig.15 The effects of GLM, S-GLM, D-GLM and A-GLM on the IL-1β content in RAW264.7 cells induced by LPS.

[0038] Fig.16 The effects of GLM, S-GLM, D-GLM and A-GLM on the IL-6 content in RAW264.7 cells induced by LPS.

[0039] Fig.17 The figure shows the effects of GLM, S-GLM, D-GLM and A-GLM on the IL-10 content in RAW264.7 cells induced by LPS.

[0040] Fig.18 It is the effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of IL-1β mRNA in RAW264.7 cells induced by LPS.

[0041] Fig.19 It is the effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of IL-6 mRNA in RAW264.7 cells induced by LPS.

[0042] Fig. 20It is the effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of IL-10 mRNA in RAW264.7 cells induced by LPS.

[0043] Fig.21 It is the effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of NF-κB mRNA in RAW264.7 cells induced by LPS.

[0044] Fig. 22 The effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of iNOS mRNA in RAW264.7 cells induced by LPS.

[0045] Fig.23 It is the effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of COX-2 mRNA in RAW264.7 cells induced by LPS.

[0046] Fig.24 The effect of GLM, S-GLM, D-GLM and A-GLM on DPPH scavenging ability.

[0047] Fig.25 It is the effect of GLM, S-GLM, D-GLM and A-GLM on the ABTS scavenging ability.

[0048] Fig.26 It is the effect of GLM, S-GLM, D-GLM and A-GLM on the hydroxyl radical scavenging ability.

[0049] Fig. 27 It is the effect of GLM, S-GLM, D-GLM and A-GLM on the total reducing capacity.

[0050] Fig.28 The effect of different treatments on the color of shiitake mushrooms.

[0051] Fig.29 It is the apparent color change of shiitake mushrooms.

[0052] Fig.30 The effect of different treatments on the weight loss rate of shiitake mushrooms.

[0053] Fig.31 The effect of different treatments on the mushroom opening rate.

[0054] Fig.32 is the effect of different treatments on respiratory rate.

[0055] Fig.33 The effect of different treatments on the hardness of shiitake mushrooms.

[0056] Fig.34The effect of different treatments on the soluble solids content of shiitake mushrooms.

[0057] Fig.35 The effect of different treatments on the relative conductivity of shiitake mushrooms.

[0058] Fig.36 The effect of different treatments on the PPO activity of Lentinus edodes.

[0059] Fig.37 The effect of different treatments on the POD content of Lentinus edodes.

[0060] Fig.38 The effect of different treatments on CAT activity of Lentinus edodes.

[0061] Fig.39 The effect of different treatments on the MDA content of Lentinus edodes.

[0062] Different lowercase letters in the figure indicate significant differences (P<0.05). DETAILED DESCRIPTION

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0064] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0065] Example 1. Preparation and structural characterization of Ganoderma lucidum melanin nano-selenium

[0066] Experimental materials: Ganoderma lucidum fruiting bodies were purchased from the Edible Fungi Center of Shanxi Agricultural University.

[0067] Main reagents include: sodium hydroxide (Jinan Tianxiang Co., Ltd.), ethyl acetate (Jinan Shuangying Chemical Co., Ltd.), hydrochloric acid (Jiangsu Xuhong Environmental Protection Technology Co., Ltd.), chloroform (Hubei Shineng Chemical Technology Co., Ltd.), sodium selenite (Jiangsu Yuanzhiyuan Biotechnology Co., Ltd.), selenium dioxide (Jinan Mingxin Chemical Co., Ltd.), selenious acid (Beijing October New Materials Technology Co., Ltd.), and ascorbic acid (Nanjing Tongying Biotechnology Co., Ltd.).

[0068] Main instruments: nanoparticle size analyzer (939SZ, Shanghai Dachang Foreign Trade Co., Ltd.), electronic balance (DSC-500B, Shanghai Yi Yu Electronic Technology Co., Ltd.), heat-collecting constant temperature heating magnetic stirrer (DF-101S, Shanghai Meiyingpu Instrument Manufacturing Co., Ltd.), Fourier transform infrared spectrometer (Nicolet iS5, Tianjin Norei Xinda Technology Co., Ltd.), ultraviolet visible spectrophotometer (HD-UV90, Jiangsu Shengaohua Environmental Protection Technology Co., Ltd.), freeze dryer (FD-1C-50, Shanghai Jipu Electronic Technology Co., Ltd.), high-speed refrigerated centrifuge (AXDD5M, Changzhou Jintan Liangyou Instrument Co., Ltd.), inductively coupled plasma mass spectrometer (PlasmaMS300, Beijing Jingkerida Technology Co., Ltd.), scanning electron microscope (SU3800, Shanghai Xinu Optical Technology Co., Ltd.).

[0069] In this example, GLM was used as a stabilizer, Na3SeO3, SeO2, and H2SeO3 were used as selenium sources, and Vc was used as a reducing agent to prepare Ganoderma lucidum melanin nano-selenium, and its structural characterization, morphology, and stability were analyzed.

[0070] 1. Preparation of Ganoderma lucidum melanin nano-selenium

[0071] 1. Extraction of Ganoderma lucidum melanin

[0072] The fruiting bodies of Ganoderma lucidum were crushed, 1.25 mol / L NaOH solution was added, and then the mixture was ultrasonically centrifuged. The pH value of the supernatant was adjusted to 1.5 with HCl, and then centrifuged after being water bathed at 80°C. The precipitate was repeatedly washed with distilled water until it was neutral, and then chloroform and ethyl acetate were added, washed 3 times, and centrifuged to leave the precipitate. 95% ethanol and 75% ethanol were added in sequence, washed 3 times, centrifuged, and washed with distilled water until it was neutral. The melanin precipitate was dissolved in 0.1 mol / L NaOH solution, and then the pH value was adjusted to 7 with HCl solution of the same concentration, dialyzed, and freeze-dried to obtain Ganoderma lucidum melanin (GLM).

[0073] 2. Preparation of Ganoderma lucidum melanin nano-selenium

[0074] The Ganoderma lucidum melanin aqueous solution (concentration of 1 mg / mL, solvent is water) was mixed with Na2SeO3, SeO2 and H2SeO3 respectively, stirred with a magnetic stirrer for 20 minutes, Vc solution was added, stirred at 50°C for 4 hours until a stable orange-red solution was formed, dialyzed for 48 hours, and freeze-dried to obtain S-GLM, D-GLM and A-GLM.

[0075] 2. Characterization of Ganoderma lucidum melanin nanoselenium

[0076] SPSS software was used to analyze the data, and P < 0.05 indicated a significant difference.

[0077] 1. Determination of selenium content

[0078] Weigh a certain amount of S-GLM, D-GLM and A-GLM respectively and digest them in a 150mL digestion tube, add 9mL99.8% HNO3 and 2%1mL HClO4, heat it in a digestion furnace, and brown smoke will be produced after a period of time. After it disappears, adjust the temperature of the digestion furnace to 180℃. If the digestion solution turns brown-black, add a small amount of HNO3 appropriately, and terminate the reaction when the digestion solution becomes colorless and transparent, and then perform the measurement.

[0079] The results of the determination of the selenium content in the Ganoderma lucidum melanin nano-selenium of the present invention are shown in Table 1:

[0080] Table 1 Determination results of selenium content in S-GLM, D-GLM and A-GLM

[0081] Sample name Selenium content (mg / kg) Solution state S-GLM 2598.90 Colorless and transparent D-GLM 1771.41 Colorless and transparent A-GLM 1105.78 Colorless and transparent

[0082] As shown in Table 1, the selenium contents of S-GLM, D-GLM and A-GLM are 2598.90 mg / kg, 1771.41 mg / kg and 1105.78 mg / kg, respectively, which are higher than the selenium content in Forsythia polysaccharide nano-selenium (908 mg / kg).

[0083] 2. Particle size and polydispersity index determination

[0084] References (Yang XQ, Yang Fu, Zhang JB, et al. Preparation, characterization, and antioxidant and antiapoptotic activities of biosynthesized nano-selenium by yak-derived Bacillus cereus and chitosan-encapsulated chemically synthesized nano-selenium[J]. International Journal of Biological Macromolecules, 2023, 242(2):124708-124727.) was measured.

[0085] The results of the determination of the particle size and polydispersity index of the nano-selenium melanin in this example are as follows: Figure 1 shown.

[0086] Particle size and polydispersity index (PDI) are important indicators that affect the stability and dispersibility of nanoparticles. Larger nanoparticle sizes will cause aggregation on the one hand, and on the other hand, they will be more affected by gravity, with faster sedimentation and poorer stability. The larger the PDI, the worse the uniformity of the nanoparticles. Figure 1 It can be seen that the particle size of S-GLM is 54.81nm, the particle size of D-GLM is 74.74nm, and the particle size of A-GLM is 64.02nm, among which S-GLM has the smallest particle size (P<0.05). PDI below 0.3 indicates that the nanoparticles are more evenly distributed. The PDI of S-GLM, D-GLM and A-GLM are 0.11, 0.12, and 0.16, respectively, all within 0.3, indicating that the distribution of S-GLM, D-GLM and A-GLM is very uniform.

[0087] 3. UV-visible spectrum scanning determination

[0088] The determination was carried out according to reference (Jiao JS, Yu J, Ji HY, et al. Synthesis of macromolecular Astragalus polysaccharide-nano selenium complex and the inhibitory effects on HepG2 cells[J]. International Journal of Biological Macromolecules, 2022, 211(5): 481-489.).

[0089] Depend on Figure 2 It can be seen that the maximum absorption wavelength of GLM is 193nm, which is similar to the maximum characteristic absorption peaks of black fungus melanin (210nm) and black boletus melanin (209nm). The absorption is strong in the ultraviolet light region (10nm-380nm), and then the absorbance gradually decreases in the visible light region (400nm-700nm). 265nm is the characteristic absorption peak of SeNPs, the characteristic absorption peak of S-GLM is 232nm, and the characteristic absorption peak of D-GLM is 255nm. The reason may be that the preparation method causes different changes in the absorption peak. A-GLM has a characteristic absorption peak at 268nm, which is similar to the characteristic absorption peak of Forsythia polysaccharide nano-selenium (270nm). The above results show that S-GLM, D-GLM and A-GLM were successfully prepared.

[0090] 4. Infrared spectroscopy

[0091] The determination was carried out according to the method of reference (Luk KH, Chan CH, Liu ZW, et al. Selenium nanoparticlesfunctionalized by mushroom polysaccharide-protein complex: A novel nano-mineral for managing postmenopausal osteoporosis[J]. Journal of Functional Foods, 2023, 110(1): 105832-105849.).

[0092] Depend on Figure 3 It can be seen that the absorption peak heights of GLM, S-GLM, D-GLM and A-GLM are similar, indicating that no new covalent bond is formed between GLM and SeNPs. -1 The absorption peak at 2916 cm corresponds to OH stretching vibration. -1 The small peak at 1639 cm is the stretching vibration of aliphatic CH groups. -1 The strongest absorption peak at 3442 cm is the typical characteristic absorption peak of fungal melanin, which is caused by the stretching vibration of C=O group, C=C group or the bending vibration of NH group. -1 Move to 3543cm -1 The carbonyl absorption peak weakened, indicating that the carboxyl group and conjugated system in GLM reacted with SeO2. -1 Move to 3489cm -1 and 3479cm -1 , indicating that the OH groups of GLM interact with Se atoms in SeNPs through hydrogen bonding. -1 A sharp and narrow absorption peak appears at , corresponding to the C=O stretching vibration in the amide I bond.

[0093] 5. Scanning electron microscopy

[0094] The determination was carried out according to the method of Song (Song JY, Zhou JJ, Li X, et al. Nano-selenium stablilized by konjac glucommannan and its biological activity in vitro [J]. LWT, 2022, 161 (6): 113289-113298.)

[0095] Depend on Figure 4 As shown in Figure A, the fibrous connections or wrinkles with holes between GLMs are caused by the polysaccharides attached to the surface of GLMs. Figure 4 As shown in Figures B, C, and D, S-GLM, D-GLM, and A-GLM are all spherical particles.

[0096] 6. Thermogravimetric determination

[0097] A certain amount of GLM, S-GLM, D-GLM and A-GLM were weighed, and the temperature was gradually increased from 25°C to 600°C at a rate of 10°C / min for thermogravimetric measurement.

[0098] Thermal analysis is the mass loss caused by the increase of the temperature of the test sample, the change of its structure and composition, and further determines the thermal stability of the sample. Figure 5 It can be seen that GLM has two steps of weight loss, the first step of weight loss is at 100℃-210℃, which is caused by the evaporation of bound water in the sample. As the heating progresses, the second step of weight loss is mainly concentrated at 594℃, with a weight loss rate of 43.88%. The weight loss of S-GLM, D-GLM and A-GLM is concentrated around 590℃, with weight loss rates of 41.25%, 49.54% and 44.77% respectively.

[0099] 7. Effects of different factors on the stability of Ganoderma lucidum melanin nano-selenium

[0100] 1) Effect of pH on the stability of S-GLM, D-GLM and A-GLM

[0101] Ganoderma lucidum melanin nano-selenium solutions with different pH values ​​(2-10) were prepared, mixed, and the particle size and polydispersity index were measured by a nanoparticle size analyzer.

[0102] Depend on Figure 6 It can be seen that the pH value is relatively stable in the range of 5-10, and the particle size is below 72nm, among which S-GLM has the smallest particle size (P<0.05). Through PDI, it can be concluded that the PDI of S-GLM, D-GLM and A-GLM are all less than 0.3, indicating that S-GLM, D-GLM and A-GLM are evenly distributed. When the pH value is 2 or 3, the particle size is about 2000nm, and the PDI is greater than 0.6. At this time, S-GLM, D-GLM and A-GLM are extremely unstable. This is because the electrochemical properties of the SeNPs surface change. Secondly, by changing the pH value, new chemical bonds may be generated, further affecting the stability. In food production, processing, storage and other aspects, the pH value of food is changing all the time. Therefore, evaluating the pH stability of nanomaterials is very necessary for predicting food applications.

[0103] 2) Na+ Effect of concentration on the stability of S-GLM, D-GLM and A-GLM

[0104] Ganoderma lucidum melanin nano-selenium solutions with different NaCl concentrations (0.25-4 mg / mL) were prepared, mixed, and the particle size and polydispersity index were measured by a nanoparticle size analyzer.

[0105] Depend on Figure 7 It can be seen that when Na + When the concentration was 0.25-4 mg / mL, the particle size of S-GLM was stable at about 50 nm without large fluctuations, indicating that S-GLM + The particle sizes of D-GLM and A-GLM increase with the Na + The particle size gradually increases and then decreases with the increase of concentration. This is because in the high salt state, part of the NaCl is adsorbed on the surface of SeNPs to form large particles, which in turn increases the particle size. + In the concentration range of 0.25-4 mg / mL, the PDI of S-GLM was the lowest (P<0.05), and the PDI remained at around 0.11, which was significantly lower than that of D-GLM and A-GLM, indicating that S-GLM can be more evenly dispersed in NaCl solutions with higher concentrations. This may be because the covalent binding of polyphenols to proteins increases the steric hindrance effect. In food processing, the way to improve the taste of food and extend the shelf life of food is to add salt to the food. Therefore, exploring the effect of salt concentration on the stability of S-GLM, D-GLM and A-GLM is conducive to evaluating their potential application value.

[0106] 3) Ca 2+ Effect of concentration on the stability of S-GLM, D-GLM and A-GLM

[0107] Ganoderma lucidum melanin nano-selenium solutions with different CaCl2 concentrations (0.25-4 mg / mL) were prepared, mixed, and the particle size and polydispersity index were measured by a nanoparticle size analyzer.

[0108] Depend on Figure 8 It can be seen that when Ca 2+ When the concentration was 4 mg / mL, the particle sizes of S-GLM, D-GLM and A-GLM increased to several thousand nanometers, and the aggregation was serious, indicating that S-GLM, D-GLM and A-GLM were less stable in CaCl2 solution. It is speculated that the reason may be Ca 2 + The chelation with GLM leads to the destruction of the interaction between GLM and SeNPs. The PDI of S-GLM, D-GLM and A-GLM are all greater than 0.4, which also indicates that they are unstable in CaCl2 solution.

[0109] 4) Effects of S-GLM, D-GLM and A-GLM on Storage Days

[0110] The Ganoderma lucidum melanin nano-selenium was stored at 4°C for 30 days, and the particle size and polydispersity index were measured by nanoparticle size analyzer.

[0111] The changes of particle size and PDI of S-GLM, D-GLM and A-GLM during storage are shown in Figure 2. Fig. 9 As shown, with the extension of storage time, the particle size and PDI of S-GLM were the lowest (P<0.05), and the storage stability was the best, which was about 60nm.

[0112] In this embodiment, GLM is used as a stabilizer to prepare nano-selenium of ganoderma lucidum melanin from different selenium sources, and structural characterization and stability studies are performed on it. The results show that the selenium contents of S-GLM, D-GLM and A-GLM are 2598.90mg / kg, 1771.41mg / kg and 1105.78mg / kg respectively. The particle sizes of S-GLM, D-GLM and A-GLM are 54.81nm, 74.74nm and 64.02nm respectively. It is known by UV-visible absorption spectrum that the maximum characteristic absorption peaks of S-GLM, D-GLM and A-GLM are 232nm, 255nm and 268nm respectively, which are related to the characteristic absorption peaks, particle size and crystallinity of SeNPs. It can be concluded by FT-IR that the OH group of GLM acts on the Se atoms in SeNPs through similar hydrogen bonds. It is found by SEM that S-GLM, D-GLM and A-GLM are all spherical particles, which are formed under the GLM coating. The stability test showed that S-GLM, D-GLM and A-GLM could be + The stability of S-GLM, D-GLM and A-GLM was better. 2+ The results of storage stability showed that S-GLM had the best storage stability within 30 days. The above results laid a theoretical foundation for further research on Ganoderma lucidum melanin nano-selenium.

[0113] Example 2: Study on the anti-inflammatory, antioxidant and antibacterial activities of Ganoderma lucidum melanin nano-selenium

[0114] Under the stimulation of LPS, RAW264.7 macrophages can be activated into inflammatory macrophages and increase the NO content. If the NO content is high, it can inhibit the growth of T cells. The NO production pathway is through iNOS catalyzing the production of L-arginine. Therefore, inhibiting the expression of iNOS can effectively reduce the NO content. The expression of other inflammation-related cytokines such as TNF-α and COX-2 will also change with the changes in the inflammatory response. Detecting the expression of cellular inflammatory factors can clarify the relevant mechanisms of the inflammatory response.

[0115] In this example, an LPS-induced RAW264.7 cell inflammatory injury model was established to explore the effects of GLM, S-GLM, D-GLM and A-GLM on RAW264.7 cell survival rate, NO content and expression of related inflammatory factors under LPS stimulation, so as to evaluate their biological activity.

[0116] Main reagents: DMEM high glucose medium (Solarbio Co., Ltd.), fetal bovine serum (Thermo Fisher Scientific), PBS buffer (Shanghai Sigma-Aldrich Co., Ltd.), lipopolysaccharide (Shanghai Yuanxin Biotechnology Co., Ltd.), ABTS kit (Solarbio Co., Ltd.), MTT (Solarbio Co., Ltd.), dimethyl sulfoxide (Jiujiang Hengtong Chemical Co., Ltd.).

[0117] 1. Test methods

[0118] 1. Recovery, passaging and cryopreservation of RAW264.7 cells

[0119] 1) RAW264.7 cell recovery

[0120] Take out the cryotube containing RAW264.7 cell suspension from the liquid nitrogen tank, put it in a 37°C water bath, pour out the upper layer of cryofluid after centrifugation, add fresh culture medium, transfer it to a cell culture bottle after continuous pipetting several times, and culture it in a 37°C, 5% CO2 incubator, and observe the cell status regularly.

[0121] 2) RAW264.7 cell passaging

[0122] RAW264.7 cell culture medium is prepared in a ratio of 9:1 (DMEM high-glucose medium: fetal bovine serum). The culture medium is replaced after 24 hours and cultured in an incubator. When the cells grow to 80%, they are passaged. The cells in the culture bottle are carefully scraped off with a cell spatula, and the culture is centrifuged after repeated blowing and beating. The supernatant is poured out, fresh culture medium is added, and some of the cells are continued to be transferred to a new culture bottle and cultured in an incubator.

[0123] 3) Cryopreservation of RAW264.7 cells

[0124] Prepare an EP tube containing pre-frozen cells in advance, add 1 mL of freezing solution, put it into a freezing jar, then put the freezing jar into a -80℃ refrigerator, and after a period of time, transfer it to a liquid nitrogen tank for storage.

[0125] 2. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on the survival rate of RAW264.7 cells

[0126] The MTT method was used to evaluate the effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nanoselenium on the survival rate of RAW264.7 cells. RAW264.7 cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 1×10 6 100 μL of fresh culture medium was aspirated into a 96-well plate, and cultured in an incubator for 12 h. The culture medium was then aspirated. 100 μL of fresh culture medium was added to the blank group, and 100 μL of different concentrations of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium culture medium were added to the test group. After 24 h, the supernatant was discarded, 10 μL of 5 mg / mL MTT solution was added to each well, and cultured at 37 ° C for 4 h, the supernatant was discarded, 150 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured at a wavelength of 490 nm. The cell survival rate was calculated as follows:

[0127]

[0128] 3. Establishment of LPS-induced RAW264.7 cell inflammatory injury model

[0129] RAW264.7 cells were cultured at 1×10 6 Each well was connected to a 96-well plate and placed in an incubator overnight. The supernatant was discarded, and a blank group and a model group were set up. Fresh culture medium was added to the blank group, and fresh culture medium with different concentrations of LPS was added to the model group. After 24 hours, the supernatant was aspirated and the NO content was determined according to the instructions of the kit.

[0130] 4. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on the survival rate of LPS-induced RAW264.7 cells

[0131] The protective effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nanoselenium on LPS-induced inflammatory damage in RAW264.7 cells were studied. RAW264.7 cells were cultured at 1×10 6 The cells were connected to 96-well plates for overnight culture. The supernatant was discarded, and 100 μL of fresh culture medium was added to the blank group and model group, and 100 μL of different concentrations of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium culture medium were added to the sample group. After 24 hours, the supernatant was discarded, and LPS culture medium was added to the model group and sample group, and fresh culture medium was added to the blank group. After 4 hours, the cell survival rate was detected according to the method in 2 above.

[0132] 5. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on LPS-induced changes in NO levels in RAW264.7 cells

[0133] The RAW264.7 cell inflammatory injury model was established with reference to 3. Fresh culture medium was added to the blank group, LPS culture medium was added to the model group, and different concentrations of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium culture medium were added to the sample group. LPS culture medium was added 4 hours later. After further culture for 24 hours, the supernatant was taken and the NO content was determined.

[0134] 6. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on the secretion level of cellular inflammatory factors

[0135] A blank group, a model group, and a sample group (the cells were pretreated with 50, 100, 200, and 400 μg / mL of fresh culture medium for 4 h, followed by the addition of LPS culture medium) were set up. The supernatant was collected after 24 h, and the assay was performed according to the instructions in the kit.

[0136] 7. Effect of relative gene expression

[0137] 1) Extraction of total RNA from cells

[0138] After culturing the cells in the logarithmic growth phase in a 6-well plate for a period of time, the total RNA of RAW264.7 cells was extracted according to the instructions of the RNAiso Plus kit.

[0139] 2) RNA concentration and purity determination

[0140] 1 μL RNA sample was taken and measured using an ultra-micro volume high-precision spectrophotometer.

[0141] 3) Synthesis of cDNA

[0142] Table 2 cDNA synthesis system

[0143] <![CDATA[PrimeScript TM RT Master Mix]]> 1μL Total RNA 1μL DEPC water 8μL Total volume 10μL

[0144] 4) Operation method of real-time fluorescence quantitative PCR

[0145] (1) Primer design

[0146] The gene sequences of RAW264.7 cells were searched through the NCBI database: GAPDH, IL-6, IL-10, IL-1β, NF-κB, iNOS and COX-2. The primer sequences designed by Primer 5.0 are shown in Table 3.

[0147] Table 3 Primer sequences

[0148]

[0149]

[0150] (2) Reaction system and amplification conditions of real-time fluorescence quantitative PCR

[0151] Table 4 qRT-PCR reaction system

[0152]

[0153] qRT-PCR amplification conditions:

[0154]

[0155] 8. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on antioxidant activity

[0156] 1) Effects of GLM, S-GLM, D-GLM and A-GLM on DPPH clearance

[0157] Take 1mL of GLM, S-GLM, D-GLM, and A-GLM solutions in centrifuge tubes, add DPPH solution as test group A1, add anhydrous ethanol solution as control group A2, add distilled water and DPPH solution as blank group A0, react for 15 minutes, adjust distilled water and anhydrous ethanol to zero, and measure absorbance at 517nm. The DPPH clearance rate calculation formula is:

[0158]

[0159] 2) Effects of GLM, S-GLM, D-GLM and A-GLM on ABTS clearance

[0160] The ABTS free radical scavenging rate was determined using the ABTS free radical scavenging ability test kit. For specific operating procedures, please refer to the instructions.

[0161] 3) Effects of GLM, S-GLM, D-GLM and A-GLM on hydroxyl radical scavenging rate

[0162] Prepare GLM, S-GLM, D-GLM, and A-GLM concentration gradients of 0.25, 0.5, 1, 2, and 4 mg / mL, respectively. Add the prepared reagents in a certain proportion, mix well, react at 37°C for 30 min, and measure the absorbance at 510 nm.

[0163] 4) Effects of GLM, S-GLM, D-GLM and A-GLM on total reducing power

[0164] The total reducing power was determined using a total antioxidant capacity (T-AOC) detection kit. Please refer to the instructions for specific operating methods.

[0165] 9. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on antibacterial activity

[0166] The KB filter paper method was used to determine its antibacterial ability, and Escherichia coli, Staphylococcus aureus, and Bacillus subtilis were selected to prepare bacterial suspensions. 50 mg / mL of GLM, S-GLM, D-GLM, and A-GLM solutions were prepared, and sterile filter paper was immersed in the sample solution and taken out after 15 minutes. 90 μL of bacterial suspension was drawn onto LB culture medium, and evenly spread on the plate with a glass spatula. The infiltrated filter paper of GLM, S-GLM, D-GLM, and A-GLM was placed on the plate, and the filter paper of sterile water was used as the blank control group. The plate was then placed in a moderate incubator, and the inhibition zone was recorded every 24 hours until 72 hours. The minimum inhibitory concentration (MIC) of GLM, S-GLM, D-GLM, and A-GLM was determined, and the GLM, S-GLM, D-GLM, and A-GLM solutions were diluted by the two-fold dilution method, and the above operation was repeated.

[0167] 10. Data Processing

[0168] SPSS software was used for significance analysis, and P < 0.05 indicated a significant difference.

[0169] 2. Results and Analysis

[0170] 1. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on the survival rate of RAW264.7 cells

[0171] The MTT method was used to detect the effects of GLM, S-GLM, D-GLM and A-GLM on the survival rate of RAW264.7 cells. Fig.10 It can be seen that the effects of GLM, S-GLM, D-GLM and A-GLM on the survival rate of RAW264.7 cells are different. When the concentration is 400μg / mL, the survival rate of RAW264.7 cells is above 94.18%. When the concentration is 600μg / mL, the cell survival rate of GLM is 83.53%, the cell survival rate of S-GLM is 91.55%, the cell survival rate of D-GLM is 90.55%, and the cell survival rate of A-GLM is 90.34%. When the concentration is 1500μg / mL, the cell survival rate of S-GLM is 70.62%, and the cell survival rates of the remaining groups are all above 60.36%. It can be seen that different treatment groups can promote the growth of RAW264.7 cells within a certain concentration.

[0172] 2. Establishment of LPS-induced RAW264.7 cell inflammatory injury model

[0173] like Fig.11As shown in the figure, compared with the blank group, the addition of LPS can increase the NO release of RAW264.7 cells, and the NO release gradually increases with the increase of LPS concentration. When the LPS concentration is 1000ng / mL, the NO content of RAW264.7 cells is 63.52μmol, which is significantly higher than that of the blank group (P<0.05). When the LPS concentration is 2000ng / mL, the NO content of RAW264.7 cells is 62.04μmol, which is lower than the NO content of 1000ng / mL. Therefore, the LPS concentration of 1000ng / mL was selected to construct the RAW264.7 cell inflammation model.

[0174] 3. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on the survival rate of LPS-induced RAW264.7 cells

[0175] like Fig.12 As shown in the figure, compared with the blank group, the cell survival rate in the model group was significantly reduced. Compared with the model group, within the experimental concentration range, GLM, S-GLM, D-GLM and A-GLM all had a protective effect on the inflammatory injury of RAW264.7 cells. When the concentration was 600 μg / mL, the cell survival rate of GLM was 61.49%, the cell survival rate of S-GLM was 81.11%, the cell survival rate of D-GLM was 71.99%, and the cell survival rate of A-GLM was 71.78%. When the concentration was 50, 100, 200, and 400 μg / mL, the cell survival rate was above 75.82%. Therefore, concentrations of 50, 100, 200, and 400 μg / mL were selected for subsequent experiments.

[0176] 4. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on LPS-induced changes in NO levels in RAW264.7 cells

[0177] NO is a biologically active gas molecule that is closely related to inflammation. In the acute inflammation site, it can cause cell damage through cytotoxic effects, ultimately leading to inflammation and related diseases. Fig.13 It can be seen that compared with the blank group, the NO content of RAW264.7 cells in the model group after LPS induction was significantly increased, reaching 26.3% (P<0.05), indicating that the model was successfully constructed. Compared with the model group, the NO content produced by RAW264.7 cells after treatment with different concentrations of GLM, S-GLM, D-GLM and A-GLM decreased to varying degrees. When the concentration was 400μg / mL, the NO content of GLM, S-GLM, D-GLM, and A-GLM was 11.79μmol, 8.04μmol, 10.82μmol, and 9.12μmol, respectively. In summary, GLM, S-GLM, D-GLM and A-GLM all have a protective effect on RAW264.7 cells.

[0178] 5. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on the secretion of inflammatory factors in RAW264.7 cells induced by LPS

[0179] 1) Effects of GLM, S-GLM, D-GLM and A-GLM on TNF-α content

[0180] Tumor necrosis factor (TNF-α) is a cytokine produced by a variety of immune or non-immune cells. It can release IL-6 and IL-1β. It is one of the strongest inflammatory mediators and plays an important role in regulating the cytokine cascade reaction. If TNF-α is abnormal, it will lead to the occurrence of many diseases, such as cancer, rheumatoid arthritis, Crohn's disease, etc. Fig.14 It can be seen that compared with the blank group, the TNF-α content in the model group was 937.62pg / mL, which was significantly higher than that in the blank group. Under the stimulation of LPS, the TNF-α content decreased to varying degrees after treatment with samples of different concentrations. When the concentration was 400μg / mL, the TNF-α content of GLM, S-GLM, D-GLM and A-GLM were 479.38pg / mL, 420.53pg / mL, 465.42pg / mL and 467.09pg / mL, respectively, indicating that the TNF-α content in LPS-induced RAW264.7 cells could be reduced after treatment with GLM, S-GLM, D-GLM and A-GLM, among which S-GLM had the best inhibitory effect (P<0.05).

[0181] 2) Effects of GLM, S-GLM, D-GLM and A-GLM on IL-1β content

[0182] IL-1β is a pro-inflammatory cytokine that binds to immune cell surface receptors to activate NF-κB and MAPK signaling pathways. Fig.15 It can be seen that the IL-1β content in the blank group was 30.47pg / mL, and the IL-1β content in the model group was 106.57pg / mL, which was significantly higher than that in the blank group. After treatment with GLM, S-GLM, D-GLM and A-GLM, there was a decrease to varying degrees. When the concentration was 400μg / mL, the contents of GLM, S-GLM, D-GLM and A-GLM were 62.59pg / mL, 45.81pg / mL, 54.49pg / mL and 48.92pg / mL, respectively. Compared with other treatment groups, the S-GLM and A-GLM treatment groups had the best effect, which could significantly reduce the IL-1β content in LPS-induced RAW264.7 cells, and there was no significant difference between the two (P>0.05).

[0183] 3) Effects of GLM, S-GLM, D-GLM and A-GLM on IL-6 content

[0184] IL-6 is an important regulator of inflammatory cell differentiation and can maintain a stable internal environment. If the IL-6 content is too high, it may lead to the occurrence of a variety of diseases. Fig.16 It can be seen that the IL-6 content released by RAW264.7 cells in the model group was 245.23pg / mL, which was significantly higher than 44.43pg / mL in the blank group. When GLM, S-GLM, D-GLM and A-GLM were used to treat LPS-induced RAW264.7 cells, the IL-6 content in each treatment group showed a decreasing trend to varying degrees. When the concentration was 400μg / mL, the IL-6 content of GLM, S-GLM, D-GLM and A-GLM was 122.01pg / mL, 73.70pg / mL, 110.47pg / mL and 85.57pg / mL, respectively, among which the IL-6 content of S-GLM was the lowest (P<0.05), indicating that S-GLM can significantly reduce the release of IL-6 content in LPS-induced RAW264.7 cells.

[0185] 4) Effects of GLM, S-GLM, D-GLM and A-GLM on IL-10 content

[0186] The most important function of IL-10 is to inhibit the occurrence of inflammatory reactions and has an immunomodulatory effect on some inflammatory and autoimmune diseases. In addition, it can also reduce the production of other inflammatory factors, such as platelet activating factor. Fig.17 As shown in the figure, the IL-10 content of the blank group was 216.67pg / mL, and that of the model group was 85.50pg / mL. When the concentration was 400μg / mL, the IL-10 contents of GLM, S-GLM, D-GLM and A-GLM were 127.60pg / mL, 140.53pg / mL, 161.41pg / mL and 124.74pg / mL, respectively. Among them, the IL-10 content was the highest after D-GLM treatment (P<0.05), indicating that D-GLM can significantly increase the release of IL-10 content in LPS-induced RAW264.7 cells and reduce the occurrence of cellular inflammatory response.

[0187] 6. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on the relative expression of inflammatory mediators in LPS-induced RAW264.7 cells

[0188] 1) Effects of GLM, S-GLM, D-GLM and A-GLM on the relative expression of IL-1β mRNA

[0189] Fig.18The results show the effects of GLM, S-GLM, D-GLM and A-GLM on the relative expression of proinflammatory factor IL-1β in RAW264.7 cells induced by LPS. The results show that compared with the LPS model group, GLM, S-GLM, D-GLM and A-GLM can significantly inhibit the relative expression of IL-1β mRNA in RAW264.7 macrophages induced by LPS. When the concentration of S-GLM and A-GLM was 400 μg / mL, the inhibitory effects of the two were most obvious, and there was no significant difference between S-GLM and A-GLM (P>0.05).

[0190] 2) Effects of GLM, S-GLM, D-GLM and A-GLM on the relative expression of IL-6 mRNA

[0191] Depend on Fig.19 It can be seen that compared with the LPS model group, GLM, S-GLM, D-GLM and A-GLM can significantly inhibit the relative expression of IL-6 mRNA in RAW264.7 cells induced by LPS. When the concentration of S-GLM was 400 μg / mL, the inhibitory effect was most obvious (P<0.05), and the effect was the best.

[0192] 3) Effects of GLM, S-GLM, D-GLM and A-GLM on the relative expression of IL-10 mRNA

[0193] like Fig. 20 As shown in the figure, after treating LPS-induced RAW264.7 cells with GLM, S-GLM, D-GLM and A-GLM for a period of time, compared with the model group, GLM, S-GLM, D-GLM and A-GLM all had a significant promoting effect on the expression level of IL-10 in LPS-induced RAW264.7 cells. When the concentration of D-GLM was 400μg / mL, the promoting effect was the best. Wang et al. prepared Ganoderma lucidum polysaccharide nano-selenium through research and established an LPS-induced RAW264.7 cell inflammatory injury model. They found that after treatment with Ganoderma lucidum polysaccharide nano-selenium, the relative expression of IL-10mRNA was also significantly promoted.

[0194] 4) Effects of GLM, S-GLM, D-GLM and A-GLM on the relative expression of NF-κB mRNA

[0195] like Fig.21As shown in the figure, compared with the blank group, the relative expression of NF-κB mRNA in the model group increased significantly (P<0.05), and the relative expression of NF-κB mRNA could be reduced under the dual action of GLM, S-GLM, D-GLM, A-GLM and LPS. When the sample concentration was 400μg / mL, the relative expression of NF-κB mRNA by GLM, S-GLM, D-GLM and A-GLM decreased to varying degrees, and there was no significant difference between S-GLM, D-GLM and A-GLM (P>0.05). Tan Xiaoyan compared Schisandra polysaccharide and Schisandra polysaccharide nano-selenium and found that both could reduce the relative expression of NF-κB mRNA.

[0196] 5) Effects of GLM, S-GLM, D-GLM and A-GLM on the relative expression of iNOS mRNA

[0197] like Fig. 22 As shown in the figure, compared with the model group, after treatment with different concentrations of GLM, S-GLM, D-GLM and A-GLM for a period of time, the relative expression of iNOS mRNA was significantly reduced in a dose-dependent manner. When the concentration of S-GLM was 400 μg / mL, it can be intuitively seen that it had a more significant inhibitory effect on the expression of iNOS in cells, thereby further avoiding inflammatory damage to cells.

[0198] 6) Effects of GLM, S-GLM, D-GLM and A-GLM on the relative expression of COX-2 mRNA

[0199] like Fig.23 As shown in the figure, after LPS treatment, the relative expression of COX-2mRNA in the model group increased significantly, and decreased to varying degrees after GLM, S-GLM, D-GLM and A-GLM treatment. When the sample concentration of GLM, S-GLM, D-GLM and A-GLM was 400μg / mL, COX-2mRNA was significantly inhibited, and there was no significant difference among S-GLM, D-GLM and A-GLM (P>0.05).

[0200] 7. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on antioxidant activity

[0201] 1) Effects of GLM, S-GLM, D-GLM and A-GLM on DPPH free radical scavenging ability

[0202] The DPPH free radical scavenging principle is based on the electron-sharing reaction. When a free radical scavenging agent is added to the DPPH free radical solution, it will successfully pair with the lone pair of electrons, and the deep purple DPPH free radical will be reduced to the yellow DPPH-H non-free radical form. The degree of fading has a significant quantitative relationship with the number of electrons accepted, and its scavenging ability can be studied by measuring the absorbance of the solution. Fig.24 As shown in the figure, with Vc as the control group, within a certain concentration range, the scavenging rate of GLM, S-GLM, D-GLM and A-GLM on DPPH· showed a linear growth relationship with the concentration. When the concentration was 4 mg / mL, the scavenging ability of GLM, S-GLM, D-GLM and A-GLM for DPPH· free radicals was 75.44%, 80.31%, 86.8% and 94.34% respectively, and the order of their strength was A-GLM>D-GLM>S-GLM>GLM.

[0203] 2) Effects of GLM, S-GLM, D-GLM and A-GLM on ABTS free radical scavenging ability

[0204] ABTS can be oxidized to ABTS by potassium persulfate + , antioxidants scavenge ABTS by donating protons + , thus achieving the purpose of anti-oxidation. Fig.25 It can be seen that GLM, S-GLM, D-GLM and A-GLM all have different degrees of ABTS scavenging ability. With the increase of concentration, the ABTS scavenging ability of GLM, S-GLM, D-GLM and A-GLM also increased, and showed obvious dose-dependence. The ABTS scavenging ability of S-GLM was significantly higher than that of D-GLM, A-GLM and GLM. When the concentration was 4 mg / mL, the ABTS scavenging ability of GLM, S-GLM, D-GLM and A-GLM was 72.57%, 90.36%, 84.13% and 76.74%, respectively. Among them, S-GLM had the strongest ABTS scavenging ability (P<0.05), which may be due to size effect and surface effect.

[0205] 3) Effects of GLM, S-GLM, D-GLM and A-GLM on hydroxyl radical scavenging ability

[0206] Hydroxyl groups have strong antioxidant capacity. When reacting with metal ions such as iron ions and copper ions, lipid peroxidation will occur, generating ROS. There is a maximum characteristic absorption peak at a wavelength of 510nm. The darker the solution color, the higher the absorbance, indicating that more and more hydroxyl groups are combined with salicylic acid. Fig.26As shown in the figure, with the increase of concentration, the hydroxyl radical scavenging ability of GLM, S-GLM, D-GLM and A-GLM showed a gradual upward trend. When the concentration was 4 mg / mL, the hydroxyl radical scavenging ability of GLM, S-GLM, D-GLM and A-GLM was 60.48%, 67.69%, 75.18% and 82.78%, respectively, among which A-GLM had the highest hydroxyl radical scavenging ability (P<0.05).

[0207] 4) Effects of GLM, S-GLM, D-GLM and A-GLM on total reduction capacity

[0208] The total reducing capacity refers to the pH value that can produce blue Fe under acidic conditions. 2+ -TPTZ, the total reducing capacity of the sample can be obtained by measuring the sample absorbance at a wavelength of 593nm. The higher the total reducing capacity, the stronger the antioxidant capacity of the substance. Fig. 27 It can be seen that the total reducing capacity of Vc, GLM, S-GLM, D-GLM and A-GLM all increased with the increase of sample concentration. When the sample concentration was 4 mg / mL, the total reducing capacity of A-GLM was 0.73. Compared with GLM, S-GLM and D-GLM, A-GLM had the highest total reducing capacity. Therefore, the antioxidant capacity of A-GLM was stronger.

[0209] 8. Effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on antibacterial activity

[0210] Table 5 Effects of GLM, S-GLM, D-GLM and A-GLM on antibacterial activity

[0211]

[0212] Note: Different lowercase letters indicate significant differences (P<0.05)

[0213] As shown in Table 5, GLM, S-GLM, D-GLM and A-GLM have a certain degree of inhibitory effect on Escherichia coli, Staphylococcus aureus and Bacillus subtilis at a concentration of 50 mg / mL. The maximum diameter of the inhibition zone of GLM is 15.60±0.59 mm, the maximum diameter of the inhibition zone of S-GLM is 19.30±0.69 mm, the maximum diameter of the inhibition zone of D-GLM is 21.03±0.66 mm, and the maximum diameter of the inhibition zone of A-GLM is 23.68±0.35 mm. The diameters of the inhibition zones of S-GLM, D-GLM and A-GLM are all higher than that of GLM.

[0214] Table 6 Effects of GLM, S-GLM, D-GLM and A-GLM on the minimum inhibitory concentration of Escherichia coli

[0215]

[0216] Note: Different lowercase letters indicate significant differences (P<0.05)

[0217] The MIC values ​​of GLM, S-GLM, D-GLM and A-GLM were determined by the two-fold dilution method. As shown in Table 6, with the decrease of the concentrations of GLM, S-GLM, D-GLM and A-GLM, the diameter of the inhibition zone of Escherichia coli gradually decreased, and the antibacterial effect was gradually weakened. The MIC values ​​of GLM, S-GLM, D-GLM and A-GLM were all 12.5 mg / mL.

[0218] Table 7 Effects of GLM, S-GLM, D-GLM and A-GLM on the minimum inhibitory concentration of Staphylococcus aureus

[0219]

[0220] Note: Different lowercase letters indicate significant differences (P<0.05)

[0221] It can be seen from Table 7 that with the decrease in the concentrations of GLM, S-GLM, D-GLM and A-GLM, the diameter of the inhibition zone of Staphylococcus aureus gradually decreased, and the antibacterial effect gradually weakened. The MIC values ​​of GLM and S-GLM were both 3.13 mg / mL.

[0222] Table 8 Effects of GLM, S-GLM, D-GLM and A-GLM on the minimum inhibitory concentration of Bacillus subtilis

[0223]

[0224] Note: Different lowercase letters indicate significant differences (P<0.05)

[0225] As shown in Table 8, the diameter of the inhibition zone of Bacillus subtilis is positively correlated with the concentrations of GLM, S-GLM, D-GLM and A-GLM. When the concentrations of GLM, S-GLM, D-GLM and A-GLM decrease, the inhibitory effect gradually weakens. The MIC values ​​of GLM, S-GLM, D-GLM and A-GLM are all 6.25 mg / mL. When the concentration is 3.13 mg / mL, GLM, S-GLM, D-GLM and A-GLM have no inhibitory effect.

[0226] The present invention has found that GLM, S-GLM, D-GLM and A-GLM all have a protective effect on LPS-induced RAW264.7 cells, can reduce the content of cellular NO, TNF-α, IL-1β, IL-6, increase the content of IL-10, inhibit the mRNA expression of IL-1β, IL-6, NF-κB, iNOS and COX-2, and increase the mRNA expression of IL-10, thereby alleviating the inflammatory damage caused by LPS to RAW264.7 cells. It further illustrates that GLM, S-GLM, D-GLM and A-GLM can alleviate the inflammatory response of LPS-induced RAW264.7 cells by regulating the NF-κB signaling pathway.

[0227] Through antioxidant tests, it was found that GLM, S-GLM, D-GLM and A-GLM all had certain antioxidant capacity in terms of DPPH·, ABTS, hydroxyl radicals and total reducing capacity. In terms of antibacterial effect, GLM, S-GLM, D-GLM and A-GLM all had a certain degree of inhibitory effect on Escherichia coli, Staphylococcus aureus and Bacillus subtilis. On this basis, S-GLM, D-GLM and A-GLM all showed higher anti-inflammatory activity, antioxidant and antibacterial activity than GLM.

[0228] Example 3: Application of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium in the preservation of shiitake mushrooms

[0229] In this embodiment, ganoderma lucidum melanin and ganoderma lucidum melanin nano-selenium are used to preserve shiitake mushrooms, and the changes in various indicators are studied.

[0230] Experimental materials: Fresh shiitake mushrooms were purchased from the shiitake mushroom production base in Wanrong County, Shanxi Province. Rubber gloves were used during picking to prevent damage to the mushrooms. After completion, they were placed in foam boxes and transported back to the cold storage for pre-cooling for 24 hours. Then, shiitake mushrooms with uniform color and similar size were selected and grouped for subsequent index determination.

[0231] 1. Test methods

[0232] 1. Processing method

[0233] Select shiitake mushrooms of uniform size and without disease spots on the cap. Randomly divide them into 5 groups, 15 in each group. The blank group does not receive any treatment, and the other treatment groups are sprayed with Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium solution, respectively. After the treatment, samples are taken every 3 days for the determination of indicators.

[0234] 2. Determination of color difference

[0235] Use a colorimeter to measure the cap of Lentinus edodes, select three fixed points for recording, and use a white plate to calibrate the color difference before measurement.

[0236] 3. Determination of weight loss rate

[0237] The fresh weight of shiitake mushrooms was measured by weighing method, and the test period was 18 days, and the weight loss rate was calculated as follows:

[0238]

[0239] Where M1 is the mass before storage (g); M2 is the mass after storage (g).

[0240] 4. Determination of parachute opening rate

[0241] Wear rubber gloves when handling mushrooms to reduce direct contact with mushrooms. The calculation formula for the opening rate is:

[0242]

[0243] Where Q1 is the number of opened mushrooms (pieces); Q2 is the total number of mushrooms (pieces).

[0244] 5. Determination of respiratory rate

[0245] The determination was carried out according to the method of the literature (Ye JJ, Li JR, Han XX, et al. Effects of active modified atmosphere packaging on postharvest quality of shiitake mushrooms (Lentinulaedodes) stored at cold storage [J]. Journal of Integrative Agriculture, 2012, 11 (3): 474-482.).

[0246] 6. Determination of hardness

[0247] The determination was carried out with reference to Ye Jianbing's method (Ye Jianbing, Chen Fahe, Wu Guangbin. Effects of nitric oxide on postharvest physiology and quality of wax apple fruit [J]. Journal of Jimei University, 2012, 17(3): 180-185).

[0248] 7. Determination of soluble solids content

[0249] The determination was carried out according to the method of reference (Li HM, Li F, Wang L, et al. Effect of nano-packing on preservation quality of Chinese jujube (Ziziphus jujuba Mill. var. inermis (Bunge) Rehd) [J]. Food Chemistry, 2009, 114: 547-552.).

[0250] 8. Determination of relative conductivity

[0251] The determination was carried out according to the method of (Liu Pei. Screening of litchi composite preservatives and their preservation effects [D]. South China Agricultural University, 2019.).

[0252] 9. Determination of PPO, POD, CAT activity and MDA content

[0253] The Solarbio kit was used for determination according to the instructions.

[0254] 10. Data Processing

[0255] SPSS software was used to analyze the data, and P < 0.05 indicated a significant difference.

[0256] 2. Results and Analysis

[0257] 1. Effects of different treatments on the color of shiitake mushrooms

[0258] The color of shiitake mushrooms is an important indicator of their quality and maturity. If the color of shiitake mushrooms is too dark, it will reduce consumers' demand for purchase. The color change of shiitake mushrooms is often judged by the L value. The smaller the L value, the lower the degree of browning of the shiitake mushrooms. Fig.28 As shown in the figure, the L value of the blank group gradually decreased with the extension of storage time, indicating that the mushrooms had a certain degree of browning, which was mainly caused by the oxidation of enzymes and the massive reproduction of microorganisms. Fig.29 It can also be seen that compared with the blank group, GLM, S-GLM, D-GLM and A-GLM can delay the color deterioration of shiitake mushrooms to a certain extent.

[0259] 2. Effects of different treatments on the weight loss rate of shiitake mushrooms

[0260] The freshness of shiitake mushrooms is determined by the weight loss rate. If the weight loss rate is greater than 5%, the mushroom body becomes soft, resulting in a decrease in edible value. In serious cases, it will cause economic losses to mushroom farmers and enterprises. Weight loss is mainly caused by two reasons: on the one hand, it is due to water loss caused by transpiration; on the other hand, it is due to respiration. Among them, transpiration water loss is the main cause of quality loss. Fig.30 It can be seen that during the entire storage process, the weight loss rate of shiitake mushrooms gradually increased with the extension of storage time. This is because the water content in shiitake mushrooms is high. After harvesting, the water inside the mushroom body continues to evaporate, causing the weight of shiitake mushrooms to decrease and the quality to decline. Among them, the weight loss rate of the blank group was the most significant. On the 18th day, the weight loss rates of the blank group, GLM, S-GLM, D-GLM and A-GLM were 6.42%, 4.32%, 4.94%, 4.88% and 4.9%, respectively. The weight loss rates of S-GLM, D-GLM and A-GLM increased slightly compared with the early storage period, but were still within an acceptable range. It can be concluded that GLM, S-GLM, D-GLM and A-GLM can slow down the weight loss of shiitake mushrooms to a certain extent and better maintain the quality of shiitake mushrooms.

[0261] 3. Effects of different treatments on mushroom opening rate

[0262] like Fig.31 As shown in the data, during the storage from the 0th to the 18th day, the opening rates of GLM, S-GLM, D-GLM and A-GLM all showed a gradual upward trend. From the 6th to the 9th day, the opening rate remained basically unchanged after treatment with D-GLM and A-GLM, and gradually increased in the later stage of storage. However, during the entire storage period, the opening rates of GLM, S-GLM, D-GLM and A-GLM were lower than those of the blank group, indicating that the opening rate of shiitake mushrooms during storage can be inhibited after treatment with GLM, S-GLM, D-GLM and A-GLM.

[0263] 4. Effects of different treatments on the respiratory rate of Lentinus edodes

[0264] Depend on Fig.32 As shown in the figure, on the third day of storage, the respiration rate of each treatment group dropped rapidly. It is speculated that the reason may be that the storage temperature is too low, which leads to a sudden drop in the respiration rate of shiitake mushrooms. During the entire storage period, the respiration rates of S-GLM, D-GLM and A-GLM were lower than those of the blank group, indicating that the respiration rate of shiitake mushrooms can be effectively inhibited after treatment with S-GLM, D-GLM and A-GLM, thereby improving the storage resistance of shiitake mushrooms, and there was no significant difference among S-GLM, D-GLM and A-GLM (P>0.05).

[0265] 5. Effects of different treatments on the hardness of shiitake mushrooms

[0266] The most important indicator of the texture properties of shiitake mushrooms is hardness. A decrease in hardness will affect the quality and shelf life of shiitake mushrooms. Fig.33 It can be seen that with the extension of storage time, the hardness of different treatment groups gradually decreased, and the decrease was more obvious on the 12th day, which may be caused by excessive water loss, or the degradation of the cell wall of the mushroom by bacterial enzymes and the increase of endogenous autolysin activity, resulting in softening. Therefore, GLM, S-GLM, D-GLM and A-GLM can delay the decline of the hardness of the mushroom to a certain extent, but the difference with the blank group is not significant (P>0.05).

[0267] 6. Effects of different treatments on soluble solids content of shiitake mushrooms

[0268] Fig.34 The results reflect the changes in the soluble solids content of shiitake mushrooms in the blank group, GLM, S-GLM, D-GLM and A-GLM during the entire storage period. The soluble solids content of shiitake mushrooms in the five treatments showed a trend of gradual decrease and then increase. When the storage time was the third day, there was no significant difference in the soluble solids content of the blank group, GLM, S-GLM, D-GLM and A-GLM (P>0.05). In the later stage of storage, with the loss of water by transpiration, the soluble solids content gradually increased. On the 18th day, the soluble solids content after GLM, S-GLM, D-GLM and A-GLM treatments was higher than that of the blank group, which can improve the quality of shiitake mushrooms to a certain extent, and there was no significant difference among S-GLM, D-GLM and A-GLM (P>0.05).

[0269] 7. Effects of different treatments on relative conductivity of shiitake mushrooms

[0270] Relative conductivity can reflect the cell membrane permeability of shiitake mushrooms, which is related to the integrity of the cell membrane. When shiitake mushrooms are in a harsh living environment, the cell membrane will be damaged, the selective permeability of the cell will decrease or even disappear, and the water-soluble substances inside the shiitake mushrooms will gradually leak out, resulting in an increase in relative conductivity. The higher the relative conductivity, the greater the degree of damage to the cell membrane. Fig.35As shown in the figure, during the storage period, the relative conductivity of S-GLM, D-GLM and A-GLM of Lentinus edodes was lower than that of the blank group as time went on. On the 9th day, the relative conductivity of the blank group, GLM, S-GLM, D-GLM and A-GLM gradually increased. It is speculated that the reason may be that the surface of Lentinus edodes was damaged. As the storage time prolonged, the damage gradually intensified, and finally the cell membrane structure was destroyed, and the relative conductivity increased. At the end of storage, the relative conductivity of GLM, S-GLM, D-GLM and A-GLM were 26.35%, 24.65%, 24.67% and 21.56%, respectively. Among them, the relative conductivity of Lentinus edodes treated with A-GLM was the lowest (P<0.05), which can better maintain the integrity of Lentinus edodes cell membrane.

[0271] 8. Effects of different treatments on PPO activity of Lentinula edodes

[0272] Depend on Fig.36 As shown in the results, the PPO activity of the blank group gradually increased during storage and reached the highest value on the 18th day, close to 390U / g. The PPO activity of S-GLM, D-GLM and A-GLM during storage showed an overall trend of first increasing and then decreasing, which is speculated to be related to the relevant metabolism of Lentinus edodes. On the 3rd and 6th days of storage, the inhibitory effect of GLM, S-GLM, D-GLM and A-GLM on PPO activity was not obvious. At the end of storage, the PPO activity of the blank group was 385.40U / g, the PPO activity of GLM was 351.60U / g, the PPO activity of S-GLM was 231.39U / g, the PPO activity of D-GLM was 251.84U / g, and the PPO activity of A-GLM was 198.64U / g. In contrast, the inhibitory effect was more obvious after A-GLM treatment (P<0.05).

[0273] 9. Effects of different treatments on the activity of Lentinus edodes POD

[0274] The reduction of peroxidase (POD) activity will delay the oxidation of polyphenolic compounds, thereby slowing down the browning process of fruits and vegetables. Fig.37 It can be seen that the blank group, GLM, S-GLM, D-GLM and A-GLM all showed a trend of first increasing and then decreasing. The peak of the blank group appeared on the 6th day, while the peak of GLM, S-GLM, D-GLM and A-GLM appeared on the 9th day, which was later than the peak of the blank group. At the end of storage, the POD activity of the blank group was 294U / g, and the POD activity after treatment with GLM, S-GLM, D-GLM and A-GLM was significantly higher than that of the blank group.

[0275] 10. Effects of different treatments on CAT activity of Lentinus edodes

[0276] like Fig.38As shown in the figure, the CAT activity showed a trend of first decreasing and then increasing during storage. At the end of storage, the CAT activity of the blank group was 425.90 U / g, which was higher than that of the blank group after treatment with GLM, S-GLM, D-GLM and A-GLM. Among them, the effects after treatment with A-GLM and D-GLM were the best, and the differences were not significant (P>0.05).

[0277] 11. Effects of different treatments on MDA content of shiitake mushrooms

[0278] Depend on Fig.39 It can be seen that at the 9th day, the MDA content of the blank group reached the highest, rising from the initial 60.21nmlo / g to 100.75nmlo / g, which was significantly different from GLM, S-GLM, D-GLM and A-GLM (P<0.05). At the 18th day, the MDA content of GLM, S-GLM, D-GLM and A-GLM was 74.52nmlo / g, 67.96nmlo / g, 58.92nmlo / g, 58.02nmlo / g, respectively. Among them, D-GLM and A-GLM treatment can effectively delay the generation of lipid free radicals in shiitake mushroom cells, and the difference is not significant (P>0.05).

[0279] This example explores the effect of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium treatment on the preservation of shiitake mushrooms by measuring the weight loss rate, hardness, respiration rate, and umbrella opening rate of shiitake mushrooms. The test results show that compared with the blank group, Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium treatment can delay the decline in weight loss rate, hardness, and soluble solids content of shiitake mushrooms, inhibit the increase in respiration rate, umbrella opening rate, color, and relative conductivity, effectively reduce PPO activity, increase POD and CAT activity, reduce MDA content, effectively protect the integrity of cell membranes, delay aging of shiitake mushrooms, and improve the quality of shiitake mushrooms during storage.

Claims

1. A method for preparing nano-selenium melanin from Ganoderma lucidum, comprising the following steps: The solution of Ganoderma lucidum melanin is prepared by mixing with Na2SeO3, SeO2 and H2SeO3 respectively, then adding Vc solution, stirring until an orange-red solution is formed, dialyzing and freeze-drying to obtain the solution.

2. The preparation method according to claim 1, characterized in that: Using water to prepare the solution of ganoderma lucidum melanin; In the solution of the ganoderma lucidum melanin, the concentration of the ganoderma lucidum melanin is 1-2 mg / mL.

3. The preparation method according to claim 1 or 2, characterized in that: The mixture was stirred with a magnetic stirrer for 20-40 minutes and then the Vc solution was added; the mixture was stirred at 50-70° C. for 4-5 hours to form the orange-red solution.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The preparation method of ganoderma lucidum melanin comprises the following steps: The fruiting body of Ganoderma lucidum was crushed, a NaOH solution was added, and the mixture was ultrasonically centrifuged, the pH value of the supernatant was adjusted to 1.5-2 with HCl, the mixture was centrifuged again after water bath, the precipitate was washed with distilled water until it was neutral, and then washed with chloroform and ethyl acetate, the precipitate was centrifuged, and then washed with 95% ethanol and 75% ethanol in sequence, and then washed with distilled water again after centrifugation until it was neutral to obtain a melanin precipitate; The melanin precipitate is dissolved in NaOH solution 2, and then HCl solution of the same concentration is added to adjust the pH value to 7-7.2, dialyzed, and freeze-dried to obtain the Ganoderma lucidum melanin.

5. Ganoderma lucidum melanin nano-selenium prepared by the method according to any one of claims 1 to 4.

6. Use of the Ganoderma lucidum melanin nano-selenium as described in claim 5 in the preparation of anti-inflammatory products, antioxidant products and antibacterial products.

7. The use according to claim 6, characterized in that: The ganoderma lucidum melanin nano-selenium reduces the content of NO, TNF-α, IL-1β, and IL-6 in inflammatory cells, and increases the content of IL-10 in inflammatory cells; The ganoderma lucidum melanin nano-selenium reduces the mRNA expression of IL-1β, IL-6, NF-κB, iNOS and COX-2 in inflammatory cells, and increases the mRNA expression of IL-10 in inflammatory cells.

8. The use according to claim 6, characterized in that: The bacteria inhibited by the Ganoderma lucidum melanin nano-selenium include Escherichia coli, Staphylococcus aureus and Bacillus subtilis.

9. Use of the Ganoderma lucidum melanin nano-selenium as claimed in claim 5 in the preservation of shiitake mushrooms.

10. The use according to claim 9, characterized in that: The ganoderma lucidum melanin nano-selenium delays the decrease of the weight loss rate, hardness and soluble solid content of shiitake mushrooms; The ganoderma melanin nano-selenium inhibits the increase of the respiration rate, the opening rate, the color and the relative conductivity of the shiitake mushroom; the ganoderma melanin nano-selenium reduces the activity of PPO, increases the activities of POD and CAT, reduces the content of MDA, protects the integrity of the cell membrane, and delays aging.

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