Ganoderma lucidum melanin nano selenium, and preparation method and application thereof

Abstract

The application discloses ganoderma lucidum melanin nano selenium as well as a preparation method and application thereof. The preparation method of the ganoderma lucidum melanin nano selenium comprises the following steps: preparing a solution of ganoderma lucidum melanin, mixing the 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 ganoderma lucidum melanin nano selenium. The ganoderma lucidum melanin nano selenium has the performances of anti-inflammation, anti-oxidation and antibiosis, has certain anti-oxidation capacity in terms of DPPH·, ABTS, hydroxyl radical and total reduction capacity, can inhibit escherichia coli, staphylococcus aureus and bacillus subtilis, and can be used for fresh-keeping of lentinus edodes.

Description

TECHNICAL FIELD

[0001] The present application relates to ganoderma melanin nano selenium and its preparation method and application, and belongs to the technical field of deep processing of edible fungi. BACKGROUND

[0002] Selenium is an important micronutrient, which is essential for human health, and plays a significant role in antioxidant, anticancer, immune regulation, etc. Human body cannot synthesize selenium itself, and can only supplement it through food. However, the difference between the intake of selenium and the toxic dose is small, and exceeding a certain safety limit will be toxic to the human body. Therefore, how to provide a safe and effective way to supplement selenium has become a difficult problem.

[0003] SeNPs is a new type of selenium particles. Studies have shown that the toxicity of SeNPs is much lower than that of inorganic selenium, and it has high biological activity and bioavailability, providing a new choice 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, the surface free energy of SeNPs is high, which is easy to aggregate and precipitate, and its application in the field of food is limited. Therefore, improving the stability of SeNPs is the current research hotspot. Common stabilizers are generally large molecules such as proteins, polysaccharides, and polyphenols, which can prevent the aggregation of nanoparticles. Melanin, as a natural polyphenol, has rich functional groups and can be combined with SeNPs through hydrogen bond interaction, which is an excellent stabilizer. Therefore, it is necessary to study melanin nano selenium and its properties. SUMMARY

[0004] The purpose of the present application is to provide a kind of ganoderma melanin nano selenium and its preparation method, and the present application uses GLM as stabilizer, utilizes Vc to reduce sodium selenite, selenium dioxide and selenious acid, and prepares ganoderma melanin nano selenium S-GLM, D-GLM and A-GLM of different selenium sources, and carries out structure characterization and stability research on it.

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

[0006] Prepare a solution of ganoderma melanin, mix with Na2SeO3, SeO2 and H2SeO3 respectively, then add Vc solution, stir to form an orange-red solution, dialyze, freeze-dry to obtain.

[0007] The solution of ganoderma melanin is prepared with water;

[0008] The concentration of ganoderma melanin in the solution of ganoderma melanin is 1-2 mg / mL.

[0009] After stirring with a magnetic stirrer for 20-40 min, the Vc solution is added;

[0010] Stirring at 50-70 DEG C for 4-5h forms the orange red solution.

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

[0012] The ganoderma lucidum fruiting body is crushed, NaOH solution is added and ultrasonic is used, then centrifugation is carried out, the supernatant is adjusted to pH 1.5-2 by using HCl, water bath is used again, centrifugation is carried out again, the precipitate is washed to neutral by using distilled water, then the precipitate is washed by using chloroform and ethyl acetate, centrifugation is carried out, the precipitate is washed by using 95% ethanol and 75% ethanol in sequence, centrifugation is carried out again, and the precipitate is washed to neutral by using distilled water again, so that the melanin precipitate is obtained.

[0013] The melanin precipitate is dissolved in NaOH solution II, then the same concentration of HCl solution is added to adjust the pH value to 7-7.2, dialysis is carried out, and freeze-drying is carried out, so that the ganoderma lucidum melanin is obtained.

[0014] The ganoderma lucidum melanin nano selenium has the performances of anti-inflammation, anti-oxidation and antibacterial.

[0015] Specifically, the ganoderma lucidum melanin nano selenium reduces the content of NO, TNF-alpha, IL-1beta, IL-6 in inflammatory cells, and improves the content of IL-10 in inflammatory cells.

[0016] The ganoderma lucidum melanin nano selenium reduces the mRNA expression of IL-1beta, IL-6, NF-kappa B, iNOS and COX-2 in inflammatory cells, and improves 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 can be used for lentinus edodes preservation.

[0019] The ganoderma lucidum melanin nano selenium delays the decrease of weight loss rate, hardness and soluble solid content of lentinus edodes.

[0020] The ganoderma lucidum melanin nano selenium inhibits the increase of respiration rate, umbrella opening rate, color and relative conductivity of lentinus edodes.

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

[0022] The application establishes LPS-induced RAW264.7 cell inflammatory injury model, evaluates the anti-inflammatory effect, antioxidant capacity and antibacterial effect; further explores the preservation effect of GLM, S-GLM, D-GLM and A-GLM on shiitake mushroom. Through the antioxidant test, it is found that GLM, S-GLM, D-GLM and A-GLM have certain antioxidant capacity in DPPH·, ABTS, hydroxyl radical and total reducing capacity. In terms of antibacterial effect, GLM, S-GLM, D-GLM and A-GLM have a certain degree of inhibition effect on escherichia coli, staphylococcus aureus and bacillus subtilis. Through the exploration of the influence of GLM, S-GLM, D-GLM and A-GLM on the preservation effect of shiitake mushroom, it is concluded that after the treatment of ganoderma lucidum melanin and ganoderma lucidum melanin nano selenium, the weight loss rate, hardness and soluble solid content of shiitake mushroom can be delayed, the increase of respiration rate, umbrella opening rate, color and relative conductivity can be inhibited, the PPO activity can be effectively reduced, the POD and CAT activities can be improved, the MDA content can be reduced, the aging of shiitake mushroom can be effectively inhibited, and the quality of shiitake mushroom during storage can be improved. BRIEF DESCRIPTION OF DRAWINGS

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

[0024] Figure 2 is the ultraviolet-visible absorption spectrum of S-GLM, D-GLM and A-GLM.

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

[0026] Figure 4 is the scanning electron microscope graph of GLM, S-GLM, D-GLM and A-GLM (A, B, C, D graphs represent GLM, S-GLM, D-GLM and A-GLM respectively) (×30000).

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

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

[0029] Figure 7 is the influence of Na + concentration on the stability of S-GLM, D-GLM and A-GLM.

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

[0031] Figure 9 Effect of S-GLM, D-GLM and A-GLM on the storage stability.

[0032] Figure 10 Effect of GLM, S-GLM, D-GLM and A-GLM on the survival rate of RAW264.7 cells.

[0033] Figure 11 Effect of different concentrations of LPS on the content of NO in RAW264.7 cells.

[0034] Figure 12 Effect of GLM, S-GLM, D-GLM and A-GLM on the survival rate of LPS-induced RAW264.7 cells.

[0035] Figure 13 Effect of GLM, S-GLM, D-GLM and A-GLM on the content of NO in LPS-induced RAW264.7 cells.

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

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

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

[0039] Figure 17 Effect of GLM, S-GLM, D-GLM and A-GLM on the content of IL-10 in LPS-induced RAW264.7 cells.

[0040] Figure 18 Effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of IL-1β mRNA in LPS-induced RAW264.7 cells.

[0041] Figure 19 Effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of IL-6 mRNA in LPS-induced RAW264.7 cells.

[0042] Figure 20Effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of IL-10 mRNA in LPS-induced RAW264.7 cells.

[0043] Figure 21 Effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of NF-κB mRNA in LPS-induced RAW264.7 cells.

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

[0045] Figure 23 Effect of GLM, S-GLM, D-GLM and A-GLM on the relative expression of COX-2 mRNA in LPS-induced RAW264.7 cells.

[0046] Figure 24 Effect of GLM, S-GLM, D-GLM and A-GLM on DPPH· scavenging capacity.

[0047] Figure 25 Effect of GLM, S-GLM, D-GLM and A-GLM on ABTS scavenging capacity.

[0048] Figure 26 Effect of GLM, S-GLM, D-GLM and A-GLM on hydroxyl radical scavenging capacity.

[0049] Figure 27 Effect of GLM, S-GLM, D-GLM and A-GLM on total reducing capacity.

[0050] Figure 28 Effect of different treatments on the color of shiitake mushroom.

[0051] Figure 29 Effect of different treatments on the apparent color change of shiitake mushroom.

[0052] Figure 30 Effect of different treatments on the weight loss rate of shiitake mushroom.

[0053] Figure 31 Effect of different treatments on the opening rate of shiitake mushroom.

[0054] Figure 32 Effect of different treatments on the respiration rate.

[0055] Figure 33 Effect of different treatments on the hardness of shiitake mushroom.

[0056] Figure 34Figure 7 is the effect of different treatments on the soluble solid content of Lentinula edodes.

[0057] Figure 35 Figure 8 is the effect of different treatments on the relative electrical conductivity of Lentinula edodes.

[0058] Figure 36 Figure 9 is the effect of different treatments on the PPO activity of Lentinula edodes.

[0059] Figure 37 Figure 10 is the effect of different treatments on the POD content of Lentinula edodes.

[0060] Figure 3 Figure 11 is the effect of different treatments on the CAT activity of Lentinula edodes.

[0061] Figure 39 Figure 12 is the effect of different treatments on the MDA content of Lentinula edodes.

[0062] In the figure, different lowercase letters represent significant differences (P < 0.05). DETAILED DESCRIPTION

[0063] In the following examples, the experimental methods used are conventional methods unless otherwise specified.

[0064] In the following examples, the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0065] Example 1, preparation and structure characterization of ganoderma melanin nano selenium

[0066] Test material: Ganoderma lucidum fruiting body was purchased from Shanxi Agricultural University Edible Mushroom Center.

[0067] Main reagents such as sodium hydroxide (Jinan Tianxiang Co., Ltd.), ethyl acetate (Jinan Shuangying Chemical Co., Ltd.), hydrochloric acid (Jiangsu Xuhong Environmental Protection Technology Co., Ltd.), chloroform (Hubei Shennei Chemical Technology Co., Ltd.), sodium selenite (Jiangsu Yuanzhiyuan Biological Technology Co., Ltd.), selenium dioxide (Jinan Mingsheng Chemical Co., Ltd.), sodium selenite (Beijing October New Material Technology Co., Ltd.), ascorbic acid (Nanjing Tongying Biological Technology Co., Ltd.).

[0068] Main instruments: Nanoparticle size analyzer (939SZ, Shanghai Dachang Yhang Co., Ltd.), electronic balance (DSC-500B, Shanghai Yiyu Electronics Technology Co., Ltd.), heat collecting constant temperature heating magnetic stirrer (DF-101S, Shanghai Meiyangpu Instrument and Meter Manufacturing Co., Ltd.), Fourier infrared spectrometer (Nicolet iS5, Tianjin Norelco Science and Technology Co., Ltd.), ultraviolet visible spectrophotometer (HD-UV90, Jiangsu Shengao Hua Environmental Protection Technology Co., Ltd.), freeze dryer (FD-1C-50, Shanghai Jisupu Electronics Technology Co., Ltd.), high-speed refrigerated centrifuge (AXDD5M, Changzhou Jintan Liangyou Instrument Co., Ltd.), inductively coupled plasma mass spectrometer (Plasma MS300, Beijing Jingke Ruida Technology Co., Ltd.), scanning electron microscope (SU3800, Shanghai Xiniu Optics Technology Co., Ltd.).

[0069] In this embodiment, 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 melanin nanometer selenium. The structure, morphology and stability of the prepared product were analyzed.

[0070] I. Preparation of ganoderma melanin nanometer selenium

[0071] 1. Extraction of ganoderma melanin

[0072] The fruiting body of ganoderma was crushed and ultrasonicated in 1.25 mol / L NaOH solution, and then centrifuged. The pH value of the supernatant was adjusted to 1.5 with HCl, and then centrifuged after water bath at 80℃. The precipitate was washed with distilled water until neutral, and then chloroform and ethyl acetate were added and washed for 3 times. The precipitate was centrifuged and washed with 95% ethanol and 75% ethanol for 3 times, and then centrifuged and washed with distilled water until 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. After dialysis and freeze-drying, ganoderma melanin (GLM) was obtained.

[0073] 2. Preparation of ganoderma melanin nanometer selenium

[0074] The aqueous solution of ganoderma melanin (concentration of 1 mg / mL, solvent of water) was mixed with Na2SeO3, SeO2 and H2SeO3 respectively, and stirred with a magnetic stirrer for 20 min. Vc solution was added, and stirred at 50℃ for 4 h until a stable orange-red solution was formed. After dialysis for 48 h, S-GLM, D-GLM and A-GLM were obtained by freeze-drying.

[0075] II. Characterization of ganoderma melanin nanometer selenium

[0076] The data results were analyzed for significance using SPSS software, and P<0.05 represented significant difference.

[0077] 1. Selenium content determination

[0078] A certain amount of S-GLM, D-GLM and A-GLM was weighed into a 150 mL digestion tube for digestion, 9 mL of 99.8% HNO3 and 2% 1 mL of HClO4 were added, and the digestion furnace was heated. After a period of time, brown smoke was generated, and after it disappeared, the temperature of the digestion furnace was adjusted to 180℃. If the digestion solution becomes brown-black, a small amount of HNO3 needs to be added appropriately. When the digestion solution is colorless and transparent, the reaction is terminated, and then the determination is performed.

[0079] The determination results of the selenium content in the ganoderma melanin nano-selenium of the present application 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] Reference (Yang X Q, Yang Fu, Zhang J B, 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 used for determination.

[0085] The determination results of the particle size and polydispersity index of the ganoderma melanin nano-selenium of the present example are shown in Table 2: Figure 1

[0086] ​Particle size and polydispersity index (PDI) are important indicators affecting the stability and dispersibility of nanoparticles. Larger nanoparticle sizes can lead to aggregation and increased susceptibility to gravity, resulting in faster sedimentation and decreased stability. A higher PDI indicates poorer uniformity of the nanoparticles. Figure 1 The particle size of S-GLM is 54.81 nm, that of D-GLM is 74.74 nm, and that of A-GLM is 64.02 nm, with S-GLM having the smallest particle size (P<0.05). A PDI below 0.3 indicates a more uniform distribution of nanoparticles. The PDIs of S-GLM, D-GLM, and A-GLM are 0.11, 0.12, and 0.16, respectively, all below 0.3, indicating that S-GLM, D-GLM, and A-GLM have very uniform distributions.

[0087] 3. Ultraviolet-Vis Spectroscopy Scanning Measurement

[0088] The results were obtained from the 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 is known that the maximum absorption wavelength of GLM is 193 nm, similar to the maximum characteristic absorption peaks of melanin from black fungus (210 nm) and black boletus (209 nm). Absorption is strong in the ultraviolet region (10 nm-380 nm), then gradually decreases in the visible region (400 nm-700 nm). 265 nm is the characteristic absorption peak for SeNPs, while the characteristic absorption peak for S-GLM is 232 nm, and for D-GLM it is 255 nm. This difference in absorption peaks may be due to variations in the preparation methods. A-GLM exhibits a characteristic absorption peak at 268 nm, similar to the characteristic absorption peak of selenium nanoparticles in forsythia polysaccharide (270 nm). These results indicate that S-GLM, D-GLM, and A-GLM were successfully prepared.

[0090] 4. Infrared spectroscopy determination

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

[0092] Depend on Figure 3 It can be seen that the absorption peaks of GLM are highly similar to those of S-GLM, D-GLM, and A-GLM, indicating that no new covalent bonds are formed between GLM and SeNPs. In the infrared spectrum of GLM, the peak value is 3442 cm⁻¹. -1 The absorption peak at 2916 cm⁻¹ corresponds to the OH stretching vibration. -1 The small peak at 1639 cm⁻¹ represents the stretching vibration of aliphatic CH groups. -1 The strongest absorption peak is a typical characteristic absorption peak of fungal melanin, caused by the stretching vibration of C=O or C=C groups or the bending vibration of NH groups. When GLM binds to SeNPs, D-GLM reaches a peak at 3442 cm⁻¹. -1 The location was moved to 3543cm. -1 The carbonyl absorption peak weakens, indicating that the carboxyl group and conjugated system in GLM react with SeO2. The S-GLM and A-GLM peaks decrease from 3442 cm⁻¹. -1 Moved to 3489cm -1 and 3479cm -1 This indicates that the OH group of GLM interacts with the Se atom in SeNPs via a hydrogen-like bond. S-GLM, D-GLM, and A-GLM show a similar interaction at 1626 cm⁻¹. -1 A sharp and narrow absorption peak appears at this point, corresponding to the C=O stretching vibration in the amide I bond.

[0093] 5. Scanning electron microscopy determination

[0094] The assay was performed according to the method described in Song (Song JY, Zhou JJ, Li X, et al. Nano-selenium stablilized bykonjac 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 porous wrinkles between GLMs are caused by polysaccharides attached to the surface of the GLMs. Figure 4 As shown in Figures B, C, and D, S-GLM, D-GLM, and A-GLM all exhibit spherical particles.

[0096] 6. Thermogravimetric determination

[0097] Weigh out a certain amount of GLM, S-GLM, D-GLM and A-GLM, and gradually increase the temperature from 25℃ to 600℃ at a rate of 10℃ / min to perform thermogravimetric analysis.

[0098] Thermal analysis assesses the thermal stability of a sample by examining the mass loss caused by changes in its structure and composition as the temperature rises. Figure 5 It can be seen that GLM undergoes two-step weight loss. The first step, occurring between 100℃ and 210℃, is due to the evaporation of bound water in the sample. As heating continues, the second step of weight loss mainly occurs 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 influencing 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 then the particle size and polydispersity index were determined by a nanoparticle size analyzer.

[0102] Depend on Figure 6 It can be seen that the particles are relatively stable within the pH range of 5-10, with particle sizes all below 72 nm, among which S-GLM has the smallest particle size (P<0.05). The PDI (partial density index) shows 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 uniformly distributed. When the pH is 2 or 3, the particle size is around 2000 nm, and the PDI is greater than 0.6. At this point, S-GLM, D-GLM, and A-GLM are extremely unstable. This is because the electrochemical properties of the SeNPs surface change, and secondly, changing the pH may generate new chemical bonds, further affecting stability. In food production, processing, and storage, the pH value of food is constantly changing. Therefore, evaluating the pH stability of nanomaterials is essential for predicting their applications in food.

[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 well, and then the particle size and polydispersity index were determined by a nanoparticle size analyzer.

[0105] Depend on Figure 7 It can be seen that when Na + At concentrations of 0.25-4 mg / mL, the particle size of S-GLM remained stable at around 50 nm without significant fluctuations, indicating that S-GLM in Na+... + The particle size of D-GLM and A-GLM is relatively stable. + The particle size gradually increases and then decreases with increasing concentration because, under high salt conditions, some NaCl adsorbs onto the surface of SeNPs, forming large particles, which in turn increases the particle size. When Na... + Within the concentration range of 0.25-4 mg / mL, S-GLM exhibited the lowest PDI (P<0.05), maintaining a PDI of approximately 0.11, significantly lower than D-GLM and A-GLM. This indicates that S-GLM can be more uniformly dispersed in higher concentration NaCl solutions, possibly due to the increased steric hindrance effect after polyphenols covalently bind to proteins. In food processing, adding salt is a common method to improve food flavor and extend shelf life. Therefore, investigating the effect of salt concentration on the stability of S-GLM, D-GLM, and A-GLM is beneficial for assessing 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 then the particle size and polydispersity index were determined by a nanoparticle size analyzer.

[0108] Depend on Figure 8 It can be seen that when Ca 2+ At a concentration of 4 mg / mL, the particle size of S-GLM, D-GLM, and A-GLM increased to several thousand nanometers, indicating severe aggregation and suggesting poor stability of S-GLM, D-GLM, and A-GLM in CaCl2 solution. The reason for this is speculated to be that Ca... 2 + The chelation with GLM disrupts the interaction between GLM and SeNPs. The PDI of S-GLM, D-GLM, and A-GLM are all greater than 0.4, indicating that they are all unstable in CaCl2 solution.

[0109] 4) The effects of S-GLM, D-GLM and A-GLM on storage days

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

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

[0112] In this embodiment, GLM was used as a stabilizer to prepare Ganoderma lucidum melanin nanoparticles with different selenium sources, and their structure was characterized and stability was studied. The results showed that the selenium contents of S-GLM, D-GLM, and A-GLM were 2598.90 mg / kg, 1771.41 mg / kg, and 1105.78 mg / kg, respectively. The particle sizes of S-GLM, D-GLM, and A-GLM were 54.81 nm, 74.74 nm, and 64.02 nm, respectively. UV-Vis absorption spectroscopy revealed that the maximum characteristic absorption peaks of S-GLM, D-GLM, and A-GLM were 232 nm, 255 nm, and 268 nm, respectively, which are related to the characteristic absorption peaks, particle size, and crystallinity of SeNPs. FT-IR analysis showed that the OH groups of GLM interact with the Se atoms in SeNPs through hydrogen-like bonds. SEM analysis revealed that S-GLM, D-GLM, and A-GLM all exhibited spherical particles, which is attributed to their formation under GLM coating. Stability tests showed that S-GLM, D-GLM, and A-GLM can be used in Na... + The stability of the samples remained relatively stable, with S-GLM showing better stability. S-GLM, D-GLM, and A-GLM showed stability against Ca2+. 2+ All were unstable. Storage stability analysis showed that S-GLM exhibited the best storage stability within 30 days. These results lay 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] LPS stimulation can activate RAW264.7 macrophages into inflammatory macrophages, increasing NO levels. High NO levels can inhibit T cell growth. NO production occurs through the catalysis of L-arginine by iNOS. Therefore, inhibiting iNOS expression can effectively reduce NO levels. The expression of other inflammation-related cytokines, such as TNF-α and COX-2, also changes with the inflammatory response. Detecting the expression of cellular inflammatory factors can clarify the relevant mechanisms of the inflammatory response.

[0115] This embodiment establishes an LPS-induced inflammatory damage model of RAW264.7 cells and investigates the effects of GLM, S-GLM, D-GLM, and A-GLM on the survival rate, NO content, and expression of related inflammatory factors of RAW264.7 cells under LPS stimulation, thereby evaluating their biological activity.

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

[0117] I. Experimental Methods

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

[0119] 1) RAW264.7 cell resuscitation

[0120] Remove the cryovial containing the RAW264.7 cell suspension from the liquid nitrogen tank, place it in a 37°C water bath, centrifuge, discard the supernatant, add fresh culture medium, repeatedly pipette and transfer to a cell culture flask, and incubate at 37°C in a 5% CO2 incubator. Observe the cell status regularly.

[0121] 2) Passaging of RAW264.7 cells

[0122] RAW264.7 cell culture medium was prepared at a ratio of 9:1 (DMEM high glucose medium: fetal bovine serum). The medium was changed after 24 hours and cultured in an incubator. When the cells reached 80% confluence, they were passaged. Cells were carefully scraped from the culture flask with a cell scraper, repeatedly pipetted, centrifuged, the supernatant was discarded, fresh medium was added, and a portion of the cells were transferred to a new culture flask and placed in an incubator for further culture.

[0123] 3) Cryopreservation of RAW264.7 cells

[0124] Prepare EP tubes containing pre-frozen cells, add 1 mL of cryopreservation solution, place them in a cryopreservation container, and then place the cryopreservation container in a -80°C freezer. After a period of time, transfer them to a liquid nitrogen container 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 effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium on the survival rate of RAW264.7 cells were evaluated using the MTT assay. RAW264.7 cells in the logarithmic growth phase were harvested and their concentration adjusted to 1×10⁻⁶ cells / cells. 6 100 μL of fresh culture medium was transferred to each well of a 96-well plate and incubated for 12 h. The culture medium was then aspirated. 100 μL of fresh culture medium was added to the control group, and 100 μL of different concentrations of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium culture medium were added to the experimental groups. After 24 h, the supernatant was discarded, and 10 μL of 5 mg / mL MTT solution was added to each well. The plates were incubated at 37°C for 4 h, and the supernatant was discarded. 150 μL of dimethyl sulfoxide was added to each well, and the absorbance was measured at 490 nm. The cell viability was calculated using the following formula:

[0127]

[0128] 3. Establishment of an LPS-induced inflammatory damage model in RAW264.7 cells

[0129] RAW264.7 cells were divided into groups of 1×10⁻⁶. 6 Connect each sample to a 96-well plate and incubate overnight. Discard the supernatant, and set up a blank control group and a model group. Add fresh culture medium to the blank control group and add fresh culture medium of different concentrations of LPS to the model group. After 24 hours, aspirate the supernatant and determine the NO content according to the kit instructions.

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

[0131] This study investigated the protective effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium against LPS-induced inflammatory damage in RAW264.7 cells. RAW264.7 cells were cultured at a concentration of 1×10⁻⁶ cells / cells. 6 Cells were inoculated into 96-well plates and cultured overnight. The supernatant was discarded, and 100 μL of fresh culture medium was added to the blank group and the model group. 100 μL of different concentrations of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium culture medium was added to the sample group. After 24 h, the supernatant was discarded, and LPS culture medium was added to the model group and the sample group, while fresh culture medium was added to the blank group. After 4 h, the cell viability was detected according to the method in section 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] A RAW264.7 cell inflammatory injury model was established according to reference 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. After 4 hours, LPS culture medium was added, and the cells were cultured for another 24 hours. The supernatant was then collected, and the NO content was measured.

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

[0135] Set up a blank group, a model group, and a sample group (pretreated cells with 50, 100, 200, and 400 μg / mL fresh culture medium for 4 h, followed by the addition of LPS culture medium). After 24 h, collect the supernatant and perform the assay according to the instructions in the kit.

[0136] 7. Effects of relative gene expression levels

[0137] 1) Extraction of total RNA from cells

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

[0139] 2) RNA concentration and purity determination

[0140] Take 1 μL of RNA sample and measure it using an ultra-micro high-precision spectrophotometer.

[0141] 3) cDNA synthesis

[0142] Table 2 cDNA synthesis system

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

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

[0145] (1) Primer design

[0146] Gene sequences of RAW264.7 cells were retrieved from the NCBI database: GAPDH, IL-6, IL-10, IL-1β, NF-κB, iNOS, and COX-2. Primer sequences designed using Primer 5.0 are shown in Table 3.

[0147] Table 3 Primer sequences

[0148]

[0149]

[0150] (2) Reaction system and amplification conditions for real-time 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· removal rate

[0157] Take 1 mL of GLM, S-GLM, D-GLM, and A-GLM solutions respectively into centrifuge tubes. Add DPPH solution and label it as experimental group A1. Add anhydrous ethanol solution and label it as control group A2. Add distilled water and DPPH solution and label it as blank group A0. React for 15 min. Zero the instrument with distilled water and anhydrous ethanol, and measure the absorbance at 517 nm. The DPPH scavenging rate is calculated as follows:

[0158]

[0159] 2) The effects of GLM, S-GLM, D-GLM and A-GLM on ABTS removal rate

[0160] The ABTS radical scavenging rate was measured using the ABTS radical scavenging ability test kit. Refer to the instruction manual for specific operating procedures.

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

[0162] GLM, S-GLM, D-GLM, and A-GLM were prepared with concentration gradients of 0.25, 0.5, 1, 2, and 4 mg / mL, respectively. The prepared reagents were added in a certain proportion, mixed well, and reacted at 37℃ for 30 min. The absorbance was measured at 510 nm.

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

[0164] Total reducing power was determined using the Total Antioxidant Capacity (T-AOC) assay kit. For specific operating procedures, please refer to the instruction manual.

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

[0166] The antibacterial activity of GLM, S-GLM, D-GLM, and A-GLM was determined using the KB filter paper disc method. Bacterial suspensions of *Escherichia coli*, *Staphylococcus aureus*, and *Bacillus subtilis* were prepared. 50 mg / mL solutions of GLM, S-GLM, D-GLM, and A-GLM were prepared. Sterile filter paper discs were immersed in the sample solutions for 15 min and then removed. 90 μL of the bacterial suspension was transferred to LB agar plates and evenly spread using a glass spatula. Filter paper discs containing GLM, S-GLM, D-GLM, and A-GLM were placed on the plates, with sterile water filter paper discs serving as a blank control. The plates were then placed in a humidification incubator, and inhibition zones were recorded at 24-hour intervals, up to 72 hours. The minimum inhibitory concentration (MIC) of GLM, S-GLM, D-GLM, and A-GLM was determined. The GLM, S-GLM, D-GLM, and A-GLM solutions were diluted using the two-fold dilution method, and the above procedure was repeated.

[0167] 10. Data Processing

[0168] Significance analysis was performed using SPSS software; P < 0.05 indicates a significant difference.

[0169] II. 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 effects of GLM, S-GLM, D-GLM, and A-GLM on the viability of RAW264.7 cells were detected using the MTT assay. Figure 10 It was found that GLM, S-GLM, D-GLM, and A-GLM had different effects on the survival rate of RAW264.7 cells. At a concentration of 400 μg / mL, the survival rate of RAW264.7 cells was above 94.18%. At a concentration of 600 μg / mL, the cell survival rate was 83.53% for GLM, 91.55% for S-GLM, 90.55% for D-GLM, and 90.34% for A-GLM. At a concentration of 1500 μg / mL, the cell survival rate was 70.62% for S-GLM, while the survival rates of the other groups were all above 60.36%. Therefore, different treatments can promote the growth of RAW264.7 cells within a certain concentration range.

[0172] 2. Establishment of an LPS-induced inflammatory damage model in RAW264.7 cells

[0173] like Figure 11As shown, compared with the control group, the addition of LPS increased the NO release from RAW264.7 cells, and the NO release gradually increased with increasing LPS concentration. When the LPS concentration was 1000 ng / mL, the NO content in RAW264.7 cells was 63.52 μmol, significantly higher than that in the control group (P<0.05). When the LPS concentration was 2000 ng / mL, the NO content in RAW264.7 cells was 62.04 μmol, lower than that at 1000 ng / mL. Therefore, an LPS concentration of 1000 ng / 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 LPS-induced survival rate of RAW264.7 cells

[0175] like Figure 12 As shown, the cell viability in the model group was significantly lower than that in the control group. Compared with the model group, GLM, S-GLM, D-GLM, and A-GLM all had a protective effect against inflammatory damage to RAW264.7 cells within the experimental concentration range. At a concentration of 600 μg / mL, the cell viability of GLM was 61.49%, S-GLM was 81.11%, D-GLM was 71.99%, and A-GLM was 71.78%. At concentrations of 50, 100, 200, and 400 μg / mL, the cell viability was all 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 gaseous molecule closely associated with inflammation. At sites of acute inflammation, it can cause cell damage through cytotoxic effects, ultimately leading to inflammation and related diseases. Figure 13 The results showed that, compared with the control group, the NO content in RAW264.7 cells in the model group was significantly increased after LPS induction, reaching 26.3% (P<0.05), indicating that the model was successfully established. Compared with the model group, the NO content produced by RAW264.7 cells decreased to varying degrees after treatment with different concentrations of GLM, S-GLM, D-GLM, and A-GLM. At a concentration of 400 μg / mL, the NO contents of GLM, S-GLM, D-GLM, and A-GLM were 11.79 μmol, 8.04 μmol, 10.82 μmol, and 9.12 μmol, respectively. In conclusion, 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 LPS-induced secretion of inflammatory factors in RAW264.7 cells

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

[0180] Tumor necrosis factor (TNF-α) is a cytokine produced by various immune and non-immune cells. It releases IL-6 and IL-1β and is one of the most potent inflammatory mediators, playing a crucial role in regulating the cytokine cascade. Abnormalities in TNF-α can lead to numerous diseases, such as cancer, rheumatoid arthritis, and Crohn's disease. Figure 14 It was found that the TNF-α content in the model group was 937.62 pg / mL, significantly higher than that in the control group, compared to the blank group. Under LPS stimulation, the TNF-α content decreased to varying degrees after treatment with different concentrations of samples. When the concentration was 400 μg / mL, the TNF-α contents of GLM, S-GLM, D-GLM, and A-GLM were 479.38 pg / mL, 420.53 pg / mL, 465.42 pg / mL, and 467.09 pg / mL, respectively, indicating that treatment with GLM, S-GLM, D-GLM, and A-GLM could reduce the LPS-induced TNF-α content in RAW264.7 cells, with S-GLM showing the best inhibitory effect (P<0.05).

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

[0182] IL-1β is a pro-inflammatory cytokine that binds to receptors on the surface of immune cells, thereby activating the NF-κB and MAPK signaling pathways. Figure 15 The results showed that the IL-1β content in the control group was 30.47 pg / mL, while the IL-1β content in the model group was 106.57 pg / mL, both significantly higher than the control group. Treatment with GLM, S-GLM, D-GLM, and A-GLM all resulted in varying degrees of decrease. At a concentration of 400 μg / mL, the contents of GLM, S-GLM, D-GLM, and A-GLM were 62.59 pg / mL, 45.81 pg / mL, 54.49 pg / mL, and 48.92 pg / mL, respectively. Compared to the other treatment groups, the S-GLM and A-GLM treatment groups showed the best effects, significantly reducing LPS-induced IL-1β levels in RAW264.7 cells, with no significant difference between the two groups (P>0.05).

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

[0184] IL-6 is an important regulator of inflammatory cell differentiation and helps maintain homeostasis. Excessive IL-6 levels may lead to various diseases. Figure 16 The results showed that the IL-6 content released by RAW264.7 cells in the model group was 245.23 pg / mL, significantly higher than that in the control group (44.43 pg / mL). Treatment of LPS-induced RAW264.7 cells with GLM, S-GLM, D-GLM, and A-GLM resulted in varying degrees of decrease in IL-6 content. At a concentration of 400 μg / mL, the IL-6 content of GLM, S-GLM, D-GLM, and A-GLM were 122.01 pg / mL, 73.70 pg / mL, 110.47 pg / mL, and 85.57 pg / mL, respectively, with S-GLM showing the lowest IL-6 content (P<0.05), indicating that S-GLM significantly reduced the release of IL-6 from LPS-induced RAW264.7 cells.

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

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

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

[0189] Figure 18This study investigated the effects of GLM, S-GLM, D-GLM, and A-GLM on the relative expression of the pro-inflammatory cytokine IL-1β induced by LPS in RAW264.7 cells. Analysis showed that, compared to the LPS model group, GLM, S-GLM, D-GLM, and A-GLM all significantly inhibited the relative expression of IL-1β mRNA in RAW264.7 macrophages induced by LPS. The inhibitory effects of S-GLM and A-GLM were most pronounced at a concentration of 400 μg / mL, 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 level of IL-6 mRNA

[0191] Depend on Figure 19 It was found that, compared with the LPS model group, GLM, S-GLM, D-GLM, and A-GLM could all significantly inhibit the relative expression of IL-6 mRNA in RAW264.7 cells induced by LPS. The inhibitory effect was most significant (P<0.05) when the concentration of S-GLM was 400 μg / mL, showing the best effect.

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

[0193] like Figure 20 As shown, after treatment of 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 significantly promoted the expression level of IL-10 in LPS-induced RAW264.7 cells. The promoting effect was optimal when the concentration of D-GLM was 400 μg / mL. Wang et al. prepared Ganoderma lucidum polysaccharide nano-selenium and established an LPS-induced RAW264.7 cell inflammatory injury model. They found that treatment with Ganoderma lucidum polysaccharide nano-selenium also significantly promoted the relative expression level of IL-10 mRNA.

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

[0195] like Figure 21As shown, compared with the blank group, the relative expression level of NF-κB mRNA in the model group was significantly increased (P<0.05). The relative expression level of NF-κB mRNA could be reduced under the combined effects of GLM, S-GLM, D-GLM, and A-GLM with LPS. When the sample concentration was 400 μg / mL, GLM, S-GLM, D-GLM, and A-GLM all reduced the relative expression level of NF-κB mRNA to varying degrees, with no significant difference among S-GLM, D-GLM, and A-GLM (P>0.05). Tan Xiaoyan found that both Schisandra chinensis polysaccharide and Schisandra chinensis polysaccharide nano-selenium could reduce the relative expression level of NF-κB mRNA by comparing them.

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

[0197] like Figure 22 As shown, compared to the model group, the relative expression levels of iNOS mRNA were significantly reduced after treatment with different concentrations of GLM, S-GLM, D-GLM, and A-GLM for a period of time, and this reduction was dose-dependent. Specifically, when the concentration of S-GLM was 400 μg / mL, it was clearly observed to have a more significant inhibitory effect on iNOS expression in cells, thus further preventing cellular inflammatory damage.

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

[0199] like Figure 23 As shown, LPS treatment significantly increased the relative expression level of COX-2 mRNA in the model group, while treatment with GLM, S-GLM, D-GLM, and A-GLM resulted in varying degrees of decrease. When the sample concentration of GLM, S-GLM, D-GLM, and A-GLM was 400 μg / mL, they all significantly inhibited COX-2 mRNA expression, and there were no significant differences 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 the DPPH· radical scavenging ability

[0202] The DPPH· radical scavenging principle is based on a common-electron reaction. When a radical scavenging agent is added to a DPPH· radical solution, it successfully pairs with lone pairs of electrons, reducing the deep purple DPPH· radical to the yellow DPPH-H non-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. Figure 24 As shown, with vitamin C as the control group, within a certain concentration range, the DPPH· scavenging rates of GLM, S-GLM, D-GLM, and A-GLM showed a linear increasing relationship with concentration. At a concentration of 4 mg / mL, the DPPH· free radical scavenging abilities of GLM, S-GLM, D-GLM, and A-GLM were 75.44%, 80.31%, 86.8%, and 94.34%, respectively, and their ranking from strongest to weakest was A-GLM > D-GLM > S-GLM > GLM.

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

[0204] ABTS can be oxidized to ABTS by potassium persulfate. + Antioxidants scavenge ABTS by providing protons. + This achieves the purpose of anti-oxidation. Figure 25 It was found that GLM, S-GLM, D-GLM, and A-GLM all exhibited varying degrees of ABTS scavenging ability. With increasing concentration, the ABTS scavenging ability of GLM, S-GLM, D-GLM, and A-GLM also increased, showing a clear dose-dependent effect. S-GLM showed significantly higher ABTS scavenging ability than D-GLM, A-GLM, and GLM. At a concentration of 4 mg / mL, the ABTS scavenging abilities of GLM, S-GLM, D-GLM, and A-GLM were 72.57%, 90.36%, 84.13%, and 76.74%, respectively, with S-GLM showing the strongest ABTS scavenging ability (P<0.05). This is presumably due to size and surface effects.

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

[0206] Hydroxyl groups possess strong antioxidant capabilities. They react with metal ions such as iron and copper ions to undergo lipid peroxidation, producing reactive oxygen species (ROS). A maximum characteristic absorption peak is observed at 510 nm; the deeper the solution color, the higher the absorbance, indicating that more and more hydroxyl groups are binding with salicylic acid. For example... Figure 26As shown, the hydroxyl radical scavenging abilities of GLM, S-GLM, D-GLM, and A-GLM gradually increased with increasing concentration. At a concentration of 4 mg / mL, the hydroxyl radical scavenging abilities of GLM, S-GLM, D-GLM, and A-GLM were 60.48%, 67.69%, 75.18%, and 82.78%, respectively, with A-GLM exhibiting the highest hydroxyl radical scavenging ability (P<0.05).

[0207] 4) The effect of GLM, S-GLM, D-GLM and A-GLM on total reducing power

[0208] Total reducing power refers to the ability of a substance to produce blue Fe under acidic conditions. 2+ -TPTZ, by measuring the absorbance of the sample at a wavelength of 593 nm, yields the total reducing power of the sample. A higher total reducing power indicates a stronger antioxidant capacity. Figure 27 It can be seen that the total reducing power of Vc, GLM, S-GLM, D-GLM, and A-GLM all increases with increasing sample concentration. When the sample concentration is 4 mg / mL, the total reducing power of A-GLM is 0.73, which is the highest compared with GLM, S-GLM, and D-GLM. Therefore, A-GLM has the stronger antioxidant capacity.

[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] Table 5 shows that GLM, S-GLM, D-GLM, and A-GLM all exhibited certain inhibitory effects on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis at a concentration of 50 mg / mL. The largest inhibition zone diameter was 15.60 ± 0.59 mm for GLM, 19.30 ± 0.69 mm for S-GLM, 21.03 ± 0.66 mm for D-GLM, and 23.68 ± 0.35 mm for A-GLM. The inhibition zone diameters of S-GLM, D-GLM, and A-GLM were all larger than those 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, as the concentrations of GLM, S-GLM, D-GLM and A-GLM decreased, the diameter of the inhibition zone of Escherichia coli gradually decreased, and the antibacterial effect 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] As shown in Table 7, as the concentrations of GLM, S-GLM, D-GLM and A-GLM decreased, 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] Table 8 shows that the diameter of the inhibition zone of Bacillus subtilis is positively correlated with the concentrations of GLM, S-GLM, D-GLM, and A-GLM. The inhibitory effect gradually weakens as the concentrations of GLM, S-GLM, D-GLM, and A-GLM decrease. The MIC values ​​of GLM, S-GLM, D-GLM, and A-GLM are all 6.25 mg / mL. At a concentration of 3.13 mg / mL, GLM, S-GLM, D-GLM, and A-GLM show no inhibitory effect.

[0226] This invention has revealed that GLM, S-GLM, D-GLM, and A-GLM all have protective effects against LPS-induced RAW264.7 cells. They can reduce the levels of NO, TNF-α, IL-1β, and IL-6 in cells, increase the level 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. This further demonstrates that GLM, S-GLM, D-GLM, and A-GLM can mitigate the inflammatory response of LPS-induced RAW264.7 cells by regulating the NF-κB signaling pathway.

[0227] Antioxidant assays revealed that GLM, S-GLM, D-GLM, and A-GLM all exhibited certain antioxidant capabilities in terms of DPPH·, ABTS, hydroxyl radicals, and total reducing power. Regarding antibacterial effects, GLM, S-GLM, D-GLM, and A-GLM all showed inhibitory effects against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. Furthermore, S-GLM, D-GLM, and A-GLM all demonstrated higher anti-inflammatory, antioxidant, and antibacterial activities 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 were used to preserve shiitake mushrooms, and the changes in various indicators were studied.

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

[0231] I. Experimental Methods

[0232] 1. Handling method

[0233] Select shiitake mushrooms of uniform size with no disease spots on the cap. Randomly divide them into 5 groups of 15 mushrooms each. The control group received no treatment, while the other treatment groups were sprayed with Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium solution, respectively. After treatment, samples were taken every 3 days to measure the indicators.

[0234] 2. Measurement of color difference

[0235] The colorimeter was used to measure the color difference of shiitake mushroom caps. Three fixed sites were selected for recording. A white plate was used to calibrate the color difference before the measurement.

[0236] 3. Determination of weight loss rate

[0237] The fresh weight of shiitake mushrooms was measured using a weighing method over an 18-day experiment, and the weight changes were recorded. The formula for calculating the weight loss rate is:

[0238]

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

[0240] 4. Determination of umbrella opening rate

[0241] Wear rubber gloves when handling shiitake mushrooms to minimize direct contact. The formula for calculating the cap opening rate is:

[0242]

[0243] In the formula, Q1 represents the number of mushrooms that have opened; Q2 represents the total number of shiitake mushrooms.

[0244] 5. Measurement of respiratory rate

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

[0246] 6. Hardness Measurement

[0247] The method of Ye Jianbing was used for determination (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 method described in the 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.) was used for determination.

[0250] 8. Determination of relative conductivity

[0251] The method was followed in (Liu Pei. Screening of compound preservatives for litchi and their preservation effect [D]. South China Agricultural University, 2019.).

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

[0253] The Solarbio kit was used, and the assay was performed according to the instruction manual.

[0254] 10. Data Processing

[0255] The data results were analyzed for significance using SPSS software. P < 0.05 was considered statistically significant.

[0256] II. 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 is too dark, it will reduce consumer demand. The L value is commonly used to assess the color change of shiitake mushrooms. The smaller the L value, the lower the degree of browning. Figure 28 As shown, the L value of the blank group gradually decreased with the extension of storage time, indicating that the shiitake mushrooms underwent a certain degree of browning. This browning was mainly caused by enzymatic oxidation and the rapid proliferation of microorganisms. Combined with... Figure 29 It can also be seen that, compared with the blank group, GLM, S-GLM, D-GLM and A-GLM can all 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 assessed based on their weight loss rate. If the weight loss rate exceeds 5%, the mushrooms soften, leading to a decrease in edible value and, in severe cases, economic losses for mushroom farmers and businesses. Weight loss is primarily caused by two factors: water loss through transpiration and respiration. Transpiration is the main cause of weight loss. Figure 30 It can be seen that the weight loss rate of shiitake mushrooms gradually increases with the extension of storage time throughout the entire storage process. This is because shiitake mushrooms have a high water content, and after harvesting, the internal water of the mushroom body continuously evaporates outward, resulting in a decrease in the weight and quality of the shiitake mushrooms. Among them, the weight loss rate of the control group is the most significant. On day 18, the weight loss rates of the control group, GLM, S-GLM, D-GLM, and A-GLM are 6.42%, 4.32%, 4.94%, 4.88%, and 4.9%, respectively. The weight loss rates of S-GLM, D-GLM, and A-GLM are slightly higher than those in the early stage of storage, but still within an acceptable range. Therefore, it can be concluded that GLM, S-GLM, D-GLM, and A-GLM can all 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 the cap opening rate of shiitake mushrooms

[0262] like Figure 31 As shown, during the storage period from day 0 to day 18, the cap opening rates of GLM, S-GLM, D-GLM, and A-GLM all showed a gradual upward trend. From day 6 to day 9, the cap opening rates remained basically unchanged after treatment with D-GLM and A-GLM. The cap opening rate gradually increased in the later stage of storage, but the cap opening rates of GLM, S-GLM, D-GLM, and A-GLM were all lower than those of the control group throughout the entire storage period. This indicates that treatment with GLM, S-GLM, D-GLM, and A-GLM can inhibit the cap opening rate of shiitake mushrooms during storage.

[0263] 4. Effects of different treatments on the respiration rate of shiitake mushrooms

[0264] Depend on Figure 32 As shown, the respiration rate of all treatment groups decreased rapidly on the 3rd day of storage, presumably due to the low storage temperature causing a sharp drop in the respiration rate of shiitake mushrooms. Throughout the storage period, the respiration rates of S-GLM, D-GLM, and A-GLM were all lower than those of the control group, indicating that treatment with S-GLM, D-GLM, and A-GLM can effectively inhibit the respiration rate of shiitake mushrooms, thereby improving their storage tolerance. Furthermore, there were no significant differences between S-GLM, D-GLM, and A-GLM treatments (P>0.05).

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

[0266] The most important textural property of shiitake mushrooms is firmness; decreased firmness affects the quality and shelf life of the mushrooms. Figure 33 It was found that the hardness of all treatment groups gradually decreased with prolonged storage time, with a more significant decrease on day 12. This may be due to excessive water loss, or it may be due to the degradation of the cell wall of shiitake mushrooms by bacterial enzymes and the increase in endogenous autolysin activity, resulting in softening. Therefore, GLM, S-GLM, D-GLM, and A-GLM can all delay the decrease in shiitake mushroom hardness to some extent, but the difference from the control group was not significant (P>0.05).

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

[0268] Figure 34 This study reflects the changes in soluble solids content of shiitake mushrooms during storage using the control group, GLM, S-GLM, D-GLM, and A-GLM treatments. All five treatments showed a trend of gradually decreasing followed by increasing soluble solids content. At day 3 of storage, there was no significant difference in soluble solids content among the control group, GLM, S-GLM, D-GLM, and A-GLM treatments (P>0.05). In the later stages of storage, the soluble solids content gradually increased with transpiration and water loss. At day 18, the soluble solids content after GLM, S-GLM, D-GLM, and A-GLM treatments was higher than that after the control group, indicating that these treatments could improve the quality of shiitake mushrooms to some extent. Furthermore, there was no significant difference between S-GLM, D-GLM, and A-GLM treatments (P>0.05).

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

[0270] Relative conductivity reflects the permeability of the cell membrane in shiitake mushrooms, which is related to the integrity of the cell membrane. When shiitake mushrooms are in harsh environments, the cell membrane is damaged, the selective permeability of the cell decreases or even disappears, and water-soluble substances inside the mushroom gradually leak out, leading to an increase in relative conductivity. The higher the relative conductivity, the greater the degree of cell membrane damage. Figure 35As shown, during the storage period of shiitake mushrooms, the relative conductivity of S-GLM, D-GLM, and A-GLM was lower than that of the control group with increasing storage time. On day 9, the relative conductivity of the control group, GLM, S-GLM, D-GLM, and A-GLM gradually increased. It is speculated that this is because the surface of the shiitake mushroom was damaged, and the damage gradually intensified with the extension of storage time, eventually destroying the cell membrane structure and increasing the relative conductivity. 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 shiitake mushrooms treated with A-GLM was the lowest (P<0.05), which can better maintain the integrity of the shiitake mushroom cell membrane.

[0271] 8. Effects of different treatments on PPO activity in shiitake mushrooms

[0272] Depend on Figure 36 As shown, the PPO activity in the control group gradually increased during storage, reaching a peak of approximately 390 U / g on day 18. The PPO activities of S-GLM, D-GLM, and A-GLM generally showed an initial increase followed by a decrease during storage, which is presumably related to the metabolism of shiitake mushrooms. On days 3 and 6 of storage, the inhibitory effects of GLM, S-GLM, D-GLM, and A-GLM on PPO activity were not very significant. At the end of storage, the PPO activity in the control group was 385.40 U / g, the PPO activity in GLM was 351.60 U / g, the PPO activity in S-GLM was 231.39 U / g, the PPO activity in D-GLM was 251.84 U / g, and the PPO activity in A-GLM was 198.64 U / g. In comparison, the inhibitory effect after treatment with A-GLM was more significant (P<0.05).

[0273] 9. Effects of different treatments on POD activity of shiitake mushrooms

[0274] Reduced peroxidase (POD) activity slows the oxidation of polyphenolic compounds, thus mitigating the browning process in fruits and vegetables. Figure 37 It can be seen that the blank group, GLM, S-GLM, D-GLM, and A-GLM all showed an overall trend of first increasing and then decreasing. The peak value of the blank group appeared on day 6, while the peak values ​​of GLM, S-GLM, D-GLM, and A-GLM appeared on day 9, later than the peak value of the blank group. At the end of storage, the POD activity of the blank group was 294 U / g, and the POD activities of the groups treated with GLM, S-GLM, D-GLM, and A-GLM were significantly higher than those of the blank group.

[0275] 10. Effects of different treatments on CAT activity in shiitake mushrooms

[0276] like Figure 38As shown, CAT activity generally 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. The CAT activities of the blank group were higher after treatment with GLM, S-GLM, D-GLM and A-GLM. Among them, the effects of A-GLM and D-GLM treatment were the best, and the differences were not significant (P>0.05).

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

[0278] Depend on Figure 39 It was found that the MDA content in the blank group reached its highest level on day 9, increasing from 60.21 nmol / g to 100.75 nmol / g, which was significantly different from that of GLM, S-GLM, D-GLM, and A-GLM (P<0.05). On day 18, the MDA contents of GLM, S-GLM, D-GLM, and A-GLM were 74.52 nmol / g, 67.96 nmol / g, 58.92 nmol / g, and 58.02 nmol / g, respectively. Among them, treatment with D-GLM and A-GLM could effectively delay the production of lipid free radicals in shiitake mushroom cells, and the difference was not significant (P>0.05).

[0279] This embodiment investigates the effects of Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium treatment on the preservation effect of shiitake mushrooms by measuring indicators such as weight loss rate, firmness, respiration rate, and cap opening rate. The results show that, compared with the control group, treatment with Ganoderma lucidum melanin and Ganoderma lucidum melanin nano-selenium treatment can delay the decrease in weight loss rate, firmness, and soluble solids content of shiitake mushrooms, inhibit the increase in respiration rate, cap opening rate, color, and relative conductivity, effectively reduce PPO activity, increase POD and CAT activities, reduce MDA content, effectively protect cell membrane integrity, delay senescence, and improve the quality of shiitake mushrooms during storage.

Claims

1. A method for preparing Ganoderma lucidum melanin nano-selenium, comprising the following steps: To prepare a solution of Ganoderma lucidum melanin, mix it with Na2SeO3, SeO2 and H2SeO3 respectively, then add vitamin C solution and stir until an orange-red solution is formed. Dialyze the solution and freeze-dry it to obtain the final product. A solution of the Ganoderma lucidum melanin was prepared using water; In the solution of Ganoderma lucidum melanin, the concentration of Ganoderma lucidum melanin is 1-2 mg / mL; The vitamin C solution was added after stirring with a magnetic stirrer for 20-40 minutes; the solution was then stirred at 50-70°C for 4-5 hours to form the orange-red solution. The method for preparing Ganoderma lucidum melanin includes the following steps: The fruiting bodies of Ganoderma lucidum were crushed, NaOH solution was added, and the mixture was sonicated and centrifuged. The pH of the supernatant was adjusted to 1.5-2 with HCl, and the mixture was centrifuged again after a water bath. The precipitate was washed with distilled water until neutral, and then washed with chloroform and ethyl acetate. The precipitate was centrifuged and washed with 95% ethanol and 75% ethanol in sequence. After centrifugation, the precipitate was washed again with distilled water until neutral to obtain melanin precipitate. The melanin precipitate was dissolved in NaOH solution II, and then HCl solution of the same concentration was added to adjust the pH value to 7-7.

2. After dialyzing and freeze-drying, the Ganoderma lucidum melanin was obtained.

2. The Ganoderma lucidum melanin nano-selenium prepared by the method of claim 1.

3. The application of the Ganoderma lucidum melanin nano-selenium as described in claim 2 in the preparation of anti-inflammatory products, antioxidant products and antibacterial products.

4. The application according to claim 3, characterized in that: The Ganoderma lucidum melanin nano-selenium reduces the levels of NO, TNF-α, IL-1β, and IL-6 in inflammatory cells and increases the level 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.

5. The application according to claim 3, characterized in that: The bacteria inhibited by the Ganoderma lucidum melanin nano-selenium include Escherichia coli, Staphylococcus aureus, and Bacillus subtilis.

6. The application of the Ganoderma lucidum melanin nano-selenium as described in claim 2 in the preservation of shiitake mushrooms.

7. The application according to claim 6, characterized in that: The Ganoderma lucidum melanin nano-selenium delays the decrease in weight loss rate, hardness, and soluble solids content of shiitake mushrooms. The Ganoderma lucidum melanin nano-selenium inhibits the increase in respiration rate, cap opening rate, color and relative conductivity of shiitake mushrooms; The Ganoderma lucidum melanin nano-selenium reduces PPO activity, increases POD and CAT activity, reduces MDA content, protects cell membrane integrity, and delays aging.

Images