Preparation method and application of high-activity solid-state fermented ganoderma lucidum compound for repairing hepatocyte function

Abstract

The invention relates to the technical field of biological fermentation, in particular to a preparation method and application of a high-activity solid-state fermented ganoderma lucidum compound for repairing hepatocyte functions. According to the method, rice peptide, traditional Chinese medicines with homology of medicine and food and functional precursor substances are taken as substrates, and a three-stage composite process of enzymolysis pretreatment, synergistic pre-fermentation of probiotics and yeast and directional co-fermentation of lucid ganoderma is adopted. Through metabolism synergy and segmented temperature regulation and control of a specific strain, and in combination with an enzymolysis-water extraction-alcohol precipitation-ultrasonic composite post-treatment technology, oriented efficient enrichment of ganoderma lucidum polysaccharides and triterpenoids is realized. The content of active ingredients of the obtained compound is remarkably increased, and the obtained compound has excellent antioxidant, anti-inflammatory and hepatocyte repairing activity and can remarkably reduce ALT and AST levels in acute and chronic liver injury models. The method is good in process stability, low in production cost and suitable for development and production of liver protection drugs, functional foods and liver protection cosmetics.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a method for preparing and applying a highly active solid-state fermented Ganoderma lucidum complex for repairing liver cell function. Background Technology

[0002] Reishi mushroom, a traditional raw material listed in the "List of Food and Medicine of the Same Origin," is rich in polysaccharides and triterpenoids, which have clear effects on anti-oxidation, anti-inflammation, and liver protection. In industrial production, solid-state fermentation has become the mainstream preparation process due to its advantages such as high raw material utilization and low energy consumption.

[0003] However, existing solid-state fermentation technologies face significant technical bottlenecks in practical applications. Current single-fermentation systems often lack synergistic enzymatic hydrolysis and multi-strain metabolic complementarity mechanisms, leading to incomplete degradation of macromolecules in the substrate and insufficient supply of precursors for active ingredient synthesis. This limitation directly results in an increase in active ingredient content generally below 60%, failing to achieve exponential growth and making it difficult to meet the demands of modern pharmaceuticals and functional foods for high-purity, high-activity raw materials. Furthermore, existing process designs typically do not target the synthesis mechanisms of Ganoderma lucidum polysaccharides and triterpenoids, limiting their synthesis efficiency and resulting in weak product targeting.

[0004] In view of the above-mentioned technical pain points, developing a compound solid-state fermentation technology that can significantly improve the content of active ingredients and achieve process innovation is of great significance for breaking through the existing industrial bottlenecks. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function. This method, through the construction of a three-stage complex fermentation system combined with a specific matrix formulation and post-processing technique, significantly enhances the content and bioactivity of Ganoderma lucidum polysaccharides and triterpenoids.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function, comprising the following steps: S1 solid fermentation substrate preparation: Rice peptides, oat bran, Ophiopogon japonicus, Angelica sinensis, fennel, grape skin, licorice root, yeast extract, Cordyceps polysaccharide precursor and compound enzyme preparation are mixed, and water is added to adjust the humidity to obtain solid fermentation substrate. S2 Three-Stage Compound Fermentation: The solid fermentation substrate is subjected to enzymatic pretreatment, probiotic-yeast synergistic prefermentation, and Ganoderma lucidum directional co-fermentation in sequence; wherein, the enzymatic pretreatment aims to degrade the substrate macromolecules; the probiotic-yeast synergistic prefermentation system is inoculated with a compound bacterial solution containing Bifidobacterium longum, Lactobacillus plantarum, and Saccharomyces cerevisiae for cultivation; the Ganoderma lucidum directional co-fermentation system is based on the prefermentation product inoculated with Ganoderma lucidum strain spores for fermentation; S3 Post-processing: The fermentation end product is inactivated, and then the effective components are enriched by a composite extraction process including water extraction, enzymatic hydrolysis, alcohol precipitation and ultrasonic treatment. After drying, the Ganoderma lucidum complex is obtained.

[0007] Preferably, in step S1, the raw materials of the solid-state fermentation substrate consist of the following components by weight percentage: rice peptide 4.0%, oat bran 0.08%, Ophiopogon japonicus 0.06%, Angelica sinensis 0.07%, fennel 0.04%, grape skin 0.05%, licorice root 0.03%, yeast extract 1.0%, cordyceps polysaccharide precursor 0.06%, cellulase 0.02%, neutral protease 0.01%, with the remainder being deionized water with a moisture content adjusted to 55%-60%. This formula, through the scientific ratio of medicinal and edible raw materials and functional precursor substances, provides sufficient substrate for strain growth and active product synthesis.

[0008] Preferably, in step S2, the bacterial strains in the compound bacterial solution are specifically *Bifidobacterium longum* with accession number CICC 6193, *Lactobacillus plantarum* with accession number CICC 23941, and *Saccharomyces cerevisiae* with accession number CICC1339; and the *Ganoderma lucidum* strain is specifically *Ganoderma lucidum* with accession number CICC 14029.

[0009] Preferably, in step S2, the specific process parameters for each stage are controlled as follows: The enzymatic hydrolysis pretreatment was controlled at a temperature of 48℃-52℃, a pH of 5.3-5.7, and a hydrolysis time of 10-14 hours, with the reducing sugar content ≥3.2mg / g as the endpoint. The probiotic-yeast co-fermentation was carried out after enzymatic hydrolysis, substrate sterilization and cooling. The inoculation amount was 1.5%-2.5% of the dry weight of the substrate, the fermentation temperature was 36℃-38℃, the relative humidity was 85%-90%, the fermentation cycle was 32-40 hours, and the endpoint was when the substrate pH dropped to 4.2-4.4.

[0010] Preferably, in step S2, the Ganoderma lucidum directional co-fermentation stage employs a temperature gradient control strategy. Specifically, the pre-fermentation substrate is cooled to 28℃-30℃, and the inoculation concentration is ≥10... 8The inoculum consisted of Ganoderma lucidum spores at a concentration of 1 spore per mL, with an inoculum size of 6%-10% of the substrate dry weight. The fermentation process was carried out at the following temperatures: 27℃-29℃ for 20-28 hours, 29℃-31℃ for 18-22 hours, and 26℃-28℃ for 14-18 hours. The relative humidity was maintained at 90%-95% throughout the process, with an aeration rate of 0.5-0.7 m³ / mL. 3 / (m 3 ·h).

[0011] Preferably, in step S3, the specific steps of the composite extraction process are as follows: Water extraction: Add water at a material-to-liquid ratio of 1:22-1:28 and extract at 88℃-92℃ for 2.5-3.5 hours; Enzymatic extraction: Cool the aqueous extract to 53℃-57℃, add cellulase and neutral protease and hydrolyze for 1.5-2.5 hours; Alcohol precipitation and sonication: After inactivation of the enzymatic hydrolysate, add ethanol to achieve a final concentration of 72%-78%, refrigerate for 16-20 hours, and after redissolve the precipitate, perform ultrasonic treatment at a power of 380-420W for 40-50 minutes.

[0012] Preferably, in step S3, the drying method is vacuum freeze drying, with the cold trap temperature controlled at -58°C to -52°C, the vacuum degree at 0.008-0.01 MPa, and the composite moisture content ≤3.8%.

[0013] The present invention also claims protection for a composite solid-state fermented Ganoderma lucidum complex prepared by any of the above methods.

[0014] Furthermore, this invention also protects the application of the aforementioned composite solid-state fermented Ganoderma lucidum complex in the preparation of hepatocyte repair drugs, hepatoprotective functional foods, or hepatoprotective cosmetics. In specific applications, the drug can be used to treat or as an adjunct treatment for acute liver injury, alcoholic liver injury, and non-alcoholic fatty liver disease, exerting its therapeutic effect by reducing ALT and AST levels and inhibiting liver fibrosis markers.

[0015] This invention provides a method for preparing and applying a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function. It has the following beneficial effects: 1. This invention achieves deep utilization of the matrix and targeted enrichment of the product through a three-stage process of enzymatic pretreatment, probiotic-yeast synergy, and Ganoderma lucidum-directed co-fermentation. The content of Ganoderma lucidum polysaccharides in the resulting complex is increased by ≥2.83 times compared with the traditional process, and the content of triterpenoids is increased by ≥1.55 times.

[0016] 2. Through a triple mechanism of deep anti-oxidation, potent anti-inflammation, and targeted hepatocyte regeneration, the product of this invention achieves a repair effect of ALT and AST reduction of ≥85% in multiple models such as acute liver injury and alcoholic liver injury, and its liver protection efficacy is significantly better than that of single fermentation products.

[0017] 3. The process of this invention has good stability (RSD < 2.0%), and by optimizing fermentation parameters and improving raw material utilization, the overall cost is reduced by about 42%, which has good prospects for industrialization. Attached Figure Description

[0018] Figure 1 This is a graph showing the changes in the content of Ganoderma lucidum polysaccharides and triterpenoids during the three-stage fermentation process of this invention. Curve 1 represents Ganoderma lucidum polysaccharides, and curve 2 represents triterpenoids, demonstrating the rapid accumulation trend of active ingredients after enzymatic pretreatment.

[0019] Figure 2 The images show a comparison of HE staining of liver tissue from three different liver injury models. A represents the acute liver injury model group, A1 represents the acute liver injury model group of this invention, B represents the alcoholic liver injury model group, B1 represents the alcoholic liver injury model group of this invention, C represents the non-alcoholic fatty liver model group, and C1 represents the non-alcoholic fatty liver model group of this invention.

[0020] Figure 3 This is the HPLC-MS / MS chromatogram of the complex of the present invention. Peak 1 is oleanolic acid, and peak 2 is ferulic acid, indicating good separation of the target components.

[0021] Figure 4 This is a trend graph showing the effects of various factors on the content of Ganoderma lucidum polysaccharides in an orthogonal experiment. Curve 1 represents the amount of rice peptide, curve 2 represents the amount of yeast extract, and curve 3 represents the fermentation temperature. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0023] like Figure 1-4 As shown in the figure, this invention provides a method for preparing a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function, comprising the following steps: S1. Raw material preparation and matrix preparation Weigh the following raw materials according to the optimized formula (total mass 100kg): 4.0kg rice peptides (molecular weight 1000-3000Da, protein content ≥95%), 0.08kg oat bran, 0.06kg Ophiopogon japonicus, 0.07kg Angelica sinensis (from Minxian County, Gansu Province, 100 mesh, ferulic acid content ≥0.20%), 0.04kg fennel, 0.05kg grape skins, 0.03kg licorice root, 1.0kg yeast extract, 0.06kg cordyceps polysaccharide precursor, 0.02kg cellulase, and 0.01kg neutral protease. Add the above raw materials to 58kg deionized water, stir evenly, adjust the moisture content to 58%, and load into the fermentation equipment.

[0024] S2. Stage 1: Enzymatic pretreatment The substrate temperature was controlled at 50℃, the pH was adjusted to 5.5, and stirring was started (40 rpm). The enzymatic hydrolysis reaction continued for 12 hours. Testing showed that the reducing sugar content in the substrate reached 3.6 mg / g at the end of the enzymatic hydrolysis, indicating that large protein molecules were effectively degraded.

[0025] S3. Phase Two: Probiotic-Yeast Co-fermentation The enzymatically hydrolyzed matrix was autoclaved at 121°C for 35 minutes and then cooled to 37°C. 2.0 kg of a compound bacterial solution was then inoculated. This solution consisted of *Bifidobacterium longum* (CICC 6193), *Lactobacillus plantarum* (CICC 23941), and *Saccharomyces cerevisiae* (CICC1339) in a 1:1:0.5 ratio, with a bacterial concentration of 10. 10 CFU / mL.

[0026] The fermentation temperature was controlled at 37℃, the relative humidity at 88%, and the aeration rate at 0.4 m³ / h. 3 / (m 3 • h). During this period, stir once every 6 hours (60 rpm, for 6 minutes). After 36 hours of fermentation, the substrate pH drops to 4.3 and the lactic acid content is ≥1.8 mg / g, at which point the fermentation stage ends.

[0027] S4. Stage Three: Targeted Co-fermentation of Ganoderma lucidum Cool the pre-fermentation substrate to 29°C and inoculate it with 8 kg of Ganoderma lucidum strain (CICC 14029) spore suspension (concentration 10). 8 (number of cells / mL). Temperature gradient control was used: maintain 28℃ for the first 24 hours, increase to 30℃ for the middle 20 hours, and decrease to 27℃ for the last 16 hours. Relative humidity was maintained at 92% throughout the process, and ventilation rate was 0.6 m³ / mL. 3 / (m 3 Stir every 10 hours (45 rpm for 10 minutes). Total fermentation time: 60 hours. The final test showed the reducing sugar content had decreased to 0.7 mg / g.

[0028] S5. Post-processing technology The fermentation product was sterilized at 120℃ for 25 minutes, cooled, and then pulverized to 250 mesh. Water was added at a material-to-liquid ratio of 1:25 (w / v), and water extraction was performed at 90℃ for 3 hours. The temperature was then lowered to 55℃, and 0.05% cellulase and 0.03% neutral protease were added for enzymatic hydrolysis for 2 hours, followed by inactivation by heating.

[0029] After cooling, anhydrous ethanol was added to a volume fraction of 75%, and the mixture was precipitated at 4°C for 18 hours. The precipitate was then redissolved and subjected to ultrasonic treatment at 400W and 45kHz for 45 minutes. After filtration through a 0.45μm filter and secondary ethanol precipitation, the mixture was freeze-dried under vacuum at -55°C and 0.009MPa until the moisture content reached 3.2%, yielding a final product of 4.2 kg of the composite.

[0030] S6. Product Inspection HPLC-MS / MS analysis showed that the Ganoderma lucidum polysaccharide content in the obtained complex was 34.5 mg / g, the total triterpenoid content was 6.6 mg / g, and the oleanolic acid content was 2.15 mg / g. All indicators met the set standards. Example 2

[0031] This invention provides a method for preparing Ganoderma lucidum complex using traditional single solid-state fermentation. To verify the superiority of the process of this invention, a traditional single solid-state fermentation group was set up as a control.

[0032] Process: The basic raw material formula of Example 1 was used, but the enzyme preparation, yeast extract, and cordyceps polysaccharide precursor were removed. Without enzymatic pretreatment, 8 kg of Ganoderma lucidum spore suspension was directly sterilized and inoculated. Single-stage fermentation was carried out at a constant temperature of 28℃ for 108 hours. The post-processing was the same as in Example 1.

[0033] Results analysis: The test results showed that the content of Ganoderma lucidum polysaccharides in the control group was only 8.1 mg / g, and the total amount of triterpenoids was 3.1 mg / g, which were 76.5% and 53.0% lower than those in Example 1, respectively. Example 3

[0034] This invention provides the application of solid-state fermented Ganoderma lucidum complex in liver-protecting functional foods.

[0035] Formula: 30% of the complex prepared in Example 1 of this invention, 65% of maltodextrin, and 5% of magnesium stearate.

[0036] Preparation: Mix for 30 minutes (20 rpm) using a three-dimensional mixer, and compress to form tablets with a specification of 300 mg / tablet.

[0037] Results: Volunteers took 2 tablets daily (equivalent to an intake of 600mg of the complex) for 45 consecutive days. Results showed that the improvement rate of ALT and AST in the subjects was ≥85%, and no adverse reactions were observed. Example 4

[0038] This invention provides the application of solid-state fermented Ganoderma lucidum complex in hepatoprotective drugs.

[0039] Formulation: 45% of the complex prepared in Example 1 of this invention, 50% lactose, and 5% sodium carboxymethyl cellulose.

[0040] Preparation: After mixing evenly, fill into No. 0 capsules, with a specification of 200mg / capsule.

[0041] Effects: Clinical observations showed that after patients took one capsule three times a day for 28 consecutive days, ALT and AST levels decreased by ≥85%, and liver fibrosis indicators improved by more than 50%, demonstrating good medicinal potential. Example 5

[0042] This invention provides the application of solid-state fermented Ganoderma lucidum complex in liver-protecting cosmetics.

[0043] Formula: 8% of the complex prepared in Example 1 of this invention, 12% glycerol, 3% sodium hyaluronate, 2% panthenol, and 75% deionized water.

[0044] Preparation: After dissolving the aqueous components, the complex and panthenol were added at 40°C, and the serum was obtained by homogenization.

[0045] Results: Tests show that the serum has a transdermal absorption rate of 58%. After using it twice daily for four consecutive weeks, users experienced a 42% increase in skin brightness, effectively alleviating dull skin caused by liver damage.

[0046] Bioactivity and stability verification Cell and animal experiments In HepG2 cell experiments, the ROS clearance rate of the complex group of this invention was ≥98%, which was 55.6% higher than that of the traditional process group; the apoptosis rate of primary hepatocytes was reduced by 89%.

[0047] In a CCl4-induced acute liver injury model in mice, the high-dose group (100 mg / kg) reduced ALT by 87%, AST by 85%, and the area of ​​liver necrosis by 96%. In an alcoholic liver injury model, ALT and AST decreased by 83% and 81%, respectively. These data are significantly better than those of the conventional process group in Example 2 (e.g., the conventional group showed only a 20% decrease in ALT in the acute liver injury model).

[0048] Human clinical trials The results of a randomized, double-blind, placebo-controlled trial (100 patients with mild liver injury) showed that in the group taking the complex of the present invention (600 mg / day, 45 days), ALT decreased by an average of 86%, AST decreased by 84%, liver fibrosis markers (HA, LN, PCⅢ) decreased by an average of 58%, and blood routine and renal function indicators were normal.

[0049] Process stability The products were tested in 20 consecutive batches. The content of Ganoderma lucidum polysaccharides ranged from 34.0 to 35.2 mg / g (RSD=1.6%), and the total amount of triterpenoids ranged from 6.5 to 6.8 mg / g (RSD=1.4%), indicating that the process of the present invention has good reproducibility and industrial stability.

[0050] In summary, this invention, through an innovative three-stage compound fermentation process and specific post-processing techniques, successfully overcomes the shortcomings of traditional Ganoderma lucidum fermentation technology, such as low active ingredients and poor targeting, providing an efficient and stable technical path for the development of liver-protecting drugs and functional products.

[0051] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function, characterized in that, Includes the following steps: S1 solid fermentation substrate preparation: Rice peptides, oat bran, Ophiopogon japonicus, Angelica sinensis, fennel, grape skin, licorice root, yeast extract, Cordyceps polysaccharide precursor and compound enzyme preparation are mixed, and water is added to adjust the humidity to obtain solid fermentation substrate. S2 Three-Stage Compound Fermentation: The solid fermentation substrate is subjected to enzymatic pretreatment, probiotic-yeast synergistic prefermentation, and Ganoderma lucidum directional co-fermentation in sequence; wherein, the enzymatic pretreatment aims to degrade the substrate macromolecules; the probiotic-yeast synergistic prefermentation system is inoculated with a compound bacterial solution containing Bifidobacterium longum, Lactobacillus plantarum, and Saccharomyces cerevisiae for cultivation; the Ganoderma lucidum directional co-fermentation system is based on the prefermentation product inoculated with Ganoderma lucidum strain spores for fermentation; S3 Post-processing: The fermentation end product is inactivated, and then the effective components are enriched by a composite extraction process including water extraction, enzymatic hydrolysis, alcohol precipitation and ultrasonic treatment. After drying, the Ganoderma lucidum complex is obtained.

2. The method for preparing a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function according to claim 1, characterized in that, The raw materials of the solid fermentation substrate mentioned in step (1) are composed of the following components by weight percentage: rice peptide 4.0%, oat bran 0.08%, Ophiopogon japonicus 0.06%, Angelica sinensis 0.07%, fennel 0.04%, grape skin 0.05%, licorice root 0.03%, yeast extract 1.0%, cordyceps polysaccharide precursor 0.06%, cellulase 0.02%, neutral protease 0.01%, and the balance is deionized water with a water content adjusted to 55%-60%.

3. The method for preparing a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function according to claim 1, characterized in that, In step S2, the bacterial strains in the compound bacterial solution are specifically *Bifidobacterium longum* with accession number CICC 6193, *Lactobacillus plantarum* with accession number CICC 23941, and *Saccharomyces cerevisiae* with accession number CICC1339; the *Ganoderma lucidum* strain is specifically *Ganoderma lucidum* with accession number CICC 14029.

4. The method for preparing a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function according to claim 1, characterized in that, In step S2, the specific process parameters for each stage are controlled as follows: The enzymatic hydrolysis pretreatment was controlled at a temperature of 48℃-52℃, a pH of 5.3-5.7, and a hydrolysis time of 10-14 hours, with the reducing sugar content ≥3.2mg / g as the endpoint. The probiotic-yeast co-fermentation was carried out after enzymatic hydrolysis, substrate sterilization and cooling. The inoculation amount was 1.5%-2.5% of the dry weight of the substrate, the fermentation temperature was 36℃-38℃, the relative humidity was 85%-90%, the fermentation cycle was 32-40 hours, and the endpoint was when the substrate pH dropped to 4.2-4.

4.

5. The method for preparing a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function according to claim 1, characterized in that, In step S2, the Ganoderma lucidum directional co-fermentation stage employs a temperature gradient control strategy. Specifically, the pre-fermentation substrate is cooled to 28℃-30℃, and the inoculation concentration is ≥10... 8 The inoculum consisted of Ganoderma lucidum spores at a concentration of 1 spore per mL, with an inoculum size of 6%-10% of the substrate dry weight. The fermentation process was carried out at the following temperatures: 27℃-29℃ for 20-28 hours, 29℃-31℃ for 18-22 hours, and 26℃-28℃ for 14-18 hours. The relative humidity was maintained at 90%-95% throughout the process, with an aeration rate of 0.5-0.7 m³ / mL. 3 / (m 3 ·h).

6. The method for preparing a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function according to claim 1, characterized in that, In step S3, the specific steps of the composite extraction process are as follows: Water extraction: Add water at a material-to-liquid ratio of 1:22-1:28 and extract at 88℃-92℃ for 2.5-3.5 hours; Enzymatic extraction: Cool the aqueous extract to 53℃-57℃, add cellulase and neutral protease and hydrolyze for 1.5-2.5 hours; Alcohol precipitation and sonication: After inactivation of the enzymatic hydrolysate, add ethanol to achieve a final concentration of 72%-78%, refrigerate for 16-20 hours, and after redissolve the precipitate, perform ultrasonic treatment at a power of 380-420W for 40-50 minutes.

7. The preparation method and application of a highly active solid-state fermented Ganoderma lucidum complex for repairing hepatocyte function according to claim 1, characterized in that, In step S3, the drying method is vacuum freeze drying, with the cold trap temperature controlled at -58℃ to -52℃ and the vacuum degree at 0.008-0.01MPa, until the moisture content of the composite is ≤3.8%.

8. A composite solid-state fermented Ganoderma lucidum complex prepared by the method according to any one of claims 1 to 7.

9. The application of the composite solid-state fermented Ganoderma lucidum complex according to claim 8 in the preparation of hepatocyte repair drugs, liver-protecting functional foods, or liver-protecting cosmetics.

10. The application according to claim 9, characterized in that, The drug is used to treat or as an adjunct treatment for acute liver injury, alcoholic liver injury, and non-alcoholic fatty liver disease. It exerts its therapeutic effect by lowering ALT and AST levels and inhibiting liver fibrosis markers.

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