Lentinan and preparation method and application thereof

Abstract

The present application provides a kind of lentinan and its preparation method and application, lentinan preparation method includes: preparation method includes: after crushing lentinan raw material, auxiliary extraction is carried out by adding composite enzyme, decoction 3-5h, filter to obtain lentinan intermediate, then add alpha-glucosidase and beta-glucosidase to obtain reaction liquid, stir to 40-45 ℃, dry to obtain lentinan.The lentinan prepared by the lentinan preparation method of the present application has high activity and meets the quality requirements of lentinan characteristic atlas, the method does not remove protein and alcohol precipitation, can be mass-produced and reduces production cost.The lentinan is modified by using composite enzyme assisted extraction and alpha-glucosidase and beta-glucosidase, which improves the extraction efficiency, and the lentinan obtained has higher activity than ordinary lentinan, which can be used for the treatment of skin disease in pigs, reduces the use amount of hormone drugs, and reduces the drug residue in the body of pig.

Description

Technical Field

[0001] This invention relates to the technical field of lentinan processing, specifically to a lentinan, its preparation method, and its applications. Background Technology

[0002] In the prior art, the characteristic peaks of lentinan are tested by high performance liquid chromatography. The characteristic chromatogram requires that the peak area ratio of peak 1 is greater than 0.41, the peak area ratio of peak 2 is greater than 1.2, the retention time ratio of peak 1 is 0.483-0.533, and the retention time ratio of peak 2 is 0.587-0.649. Among them, the peak area ratio of peak 1 and peak 2 is the peak area ratio of characteristic peaks 1 and 2 relative to the reference (the reference is anhydrous glucose); the retention time ratio is the retention time ratio of each characteristic peak relative to the reference (the reference is anhydrous glucose).

[0003] Lentinan, a polysaccharide found in shiitake mushrooms, is typically obtained through alcohol precipitation, a process that requires alcohol treatment, resulting in high production costs and failing to meet the characteristic peak requirements of shiitake polysaccharides. Furthermore, the treatment of swine skin diseases primarily utilizes hormonal drugs, which leave residues in the pig's body. Currently, shiitake polysaccharides are not used in the treatment of swine skin diseases. Therefore, this technology requires further development. Summary of the Invention

[0004] To address the shortcomings of existing technologies and solve the aforementioned problems, a lentinan, its preparation method, and its applications are proposed, and the following technical solution is provided:

[0005] A method for preparing lentinan, comprising: pulverizing lentinan raw material, adding a compound enzyme for assisted extraction, decocting for 3-5 hours, filtering to obtain lentinan intermediate, then adding α-glucosidase and β-glucosidase to obtain a reaction solution, heating to 40-45℃ for stirring, and drying to obtain lentinan.

[0006] Furthermore, after the shiitake mushrooms are crushed, they are soaked in water for 20-40 minutes and then a compound enzyme is added.

[0007] Furthermore, the water used for soaking should be 10-20 times the weight of the shiitake mushroom raw material.

[0008] Furthermore, the amount of the compound enzyme used is 1%-2% of the shiitake mushroom raw material, based on a mass percentage.

[0009] Furthermore, the complex enzyme is a mixture of three enzymes: cellulase, hemicellulase, and pectinase.

[0010] Furthermore, by mass percentage, the complex enzyme consists of 30%-40% cellulase, 20%-30% hemicellulase, and 30%-50% pectinase.

[0011] Furthermore, the ratio of α-glucosidase and β-glucosidase to the mass of lentinan intermediate was 0.5:1.

[0012] Furthermore, by mass percentage, the ratio of α-glucosidase to β-glucosidase is: α-glucosidase 30-40%, β-glucosidase 60-70%.

[0013] Furthermore, the ratio of α-glucosidase and β-glucosidase to lentinan powder by mass is 0.5-0.7:1.

[0014] Furthermore, by mass percentage, the ratio of α-glucosidase to β-glucosidase is: α-glucosidase 30-50%, β-glucosidase 50-70%.

[0015] Furthermore, by mass percentage, the ratio of α-glucosidase to β-glucosidase is: α-glucosidase 40%, β-glucosidase 60%.

[0016] In addition, the present invention also provides lentinan prepared by the above method.

[0017] This invention also provides applications of the above-mentioned lentinan, which is used to treat swine skin diseases.

[0018] Beneficial effects:

[0019] 1. The lentinan prepared by the method of the present invention meets the quality requirements of the characteristic spectrum of lentinan. Ordinary lentinan has a large molecular weight and high viscosity, which affects its application. However, the lentinan that meets the requirements of the characteristic spectrum has higher activity than ordinary lentinan. Peak 1 and peak 2 are the main highly active components. It has antiviral, antitumor, immune regulation, hypoglycemic and antioxidant functions. It can also be used to treat swine skin diseases.

[0020] 2. The method for preparing lentinan of the present invention does not involve protein removal and alcohol precipitation, which enables large-scale production and reduces production costs.

[0021] 3. The method for preparing lentinan of the present invention uses a compound enzyme-assisted extraction, and then the lentinan is prepared by synergistic modification of lentinan by α-glucosidase and β-glucosidase. The lentinan obtained meets the quality requirements of the characteristic spectrum of lentinan. Attached Figure Description

[0022] Figure 1 This is a characteristic spectrum of lentinan from the present invention;

[0023] Figure 2 This is the characteristic chromatogram of the reference standard, anhydrous glucose;

[0024] The above figures include the following reference numerals:

[0025] 1. Peak 1; 2. Peak 2; 3. Peak 3; 4. Peak 4. Detailed Implementation

[0026] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0027] A method for preparing lentinan, comprising: pulverizing shiitake mushroom raw material, adding a compound enzyme for assisted extraction, decocting for 3-5 hours, filtering to obtain a lentinan intermediate, then adding α-glucosidase and β-glucosidase to obtain a reaction solution, heating to 40-45℃ and stirring, and drying to obtain lentinan. The method for preparing lentinan uses compound enzyme-assisted extraction, and the lentinan prepared by synergistic modification of lentinan with α-glucosidase and β-glucosidase meets the quality requirements of lentinan characteristic spectrum. This lentinan can be used to treat swine skin diseases.

[0028] After pulverizing shiitake mushrooms, soak them in water for 20-40 minutes, then add the compound enzyme. Soaking time less than 20 minutes results in a crude polysaccharide content of 13%; soaking time of 20-40 minutes results in 15%; and soaking time more than 40 minutes results in 15%. Compared to soaking for 20-40 minutes, the crude polysaccharide content is not significantly different. To save time and improve extraction efficiency, the soaking time should be controlled to 20-40 minutes.

[0029] The mass of water used for soaking should be 10-20 times the mass of the shiitake mushrooms. When the amount of water used for soaking is less than 10 times the mass of the shiitake mushrooms, the crude polysaccharide content of shiitake mushroom polysaccharides is 12%. When the amount of water used for soaking is 10-20 times the mass of the shiitake mushrooms, the crude polysaccharide content is 16%. When the amount of water used for soaking is more than 20 times the mass of the shiitake mushrooms, the crude polysaccharide content is 16.3%, which is not much different from the content when the amount of water used for soaking is 10-20 times the mass of the shiitake mushrooms. In order to save production costs, the mass of water used for soaking should be controlled to be 10-20 times the mass of the shiitake mushrooms.

[0030] The decoction time is 3-5 hours. When the decoction time is less than 3 hours, the crude polysaccharide content of shiitake mushroom polysaccharides is 10%. When the decoction time is 3-5 hours, the crude polysaccharide content is 16%. When the decoction time is greater than 5 hours, the crude polysaccharide content is 16.5%. Compared to a decoction time of 3-5 hours, the crude polysaccharide content of shiitake mushroom polysaccharides is not significantly different; therefore, to save production costs, the extraction time is controlled at 3-5 hours.

[0031] After spray drying, mannitol is added and mixed. Mannitol is an excipient and has no hygroscopic properties. Since lentinan has strong hygroscopic properties, adding mannitol can prevent lentinan from clumping.

[0032] The activity of lentinan intermediates is mainly determined by the macromolecular polysaccharides. The main chain of lentinan intermediates consists of glucans linked by β-1,3 glycosidic bonds, and there are three types of side chains: a single glucose molecule, oligosaccharides linked by β-1,6 glycosidic bonds, and oligosaccharides linked by β-1,3 glycosidic bonds. β-glucosidase can hydrolyze the non-reducing β-D-glucose bonds bound to the ends of oligosaccharides, releasing glucose and the corresponding ligands. Then, through the transglycosidic action of α-glucosidase, the free glucose residues can be transferred to another glucose substrate by α-1,6 glycosidic bonds, converting single glucose into oligosaccharides, increasing the content of macromolecular polysaccharides, and thus increasing the activity of lentinan.

[0033] First, a simple test was conducted to investigate the effect of the dosage of the compound enzyme. The shiitake mushroom raw material was pulverized and then extracted with the compound enzyme. The compound enzyme used for enzymatic hydrolysis was 1 wt% of the shiitake mushroom raw material. The compound enzyme consisted of the following enzyme composition by mass fraction: 30% cellulase, 20% hemicellulase, and the remainder being pectinase. The crude polysaccharide content of the unfiltered shiitake mushroom was 12%.

[0034] After pulverizing the shiitake mushroom raw material, a compound enzyme was added for extraction. The compound enzyme used for enzymatic hydrolysis was 1 wt% of the shiitake mushroom raw material. The compound enzyme consisted of the following enzyme composition by mass fraction: 35% cellulase, 25% hemicellulase, and the remainder being pectinase. The crude polysaccharide content of the unfiltered shiitake mushroom was 16.0%.

[0035] After pulverizing the shiitake mushroom raw material, a compound enzyme was added for extraction. The compound enzyme accounted for 1 wt% of the shiitake mushroom raw material and consisted of the following enzyme composition by mass fraction: 40% cellulase, 30% hemicellulase, and the remainder being pectinase. The crude polysaccharide content of the unfiltered shiitake mushroom was 16.5%.

[0036] Example 1

[0037] (1) Extraction: 350 kg of shiitake mushrooms were crushed, soaked in 15 times the amount of water for 30 min, and then heated to 45 degrees Celsius and 5.25 kg of compound enzyme was added. The compound enzyme consisted of the following enzymes in the indicated mass fractions: 40% cellulase, 30% hemicellulase, and 30% pectinase. After 1 h of enzyme-assisted extraction, the mixture was boiled for 3 h to obtain 4 t of extract. The extract was filtered into a filtrate using a disc centrifuge.

[0038] (2) Concentration: The filtrate is concentrated to a density of 1.10 kg / m³. 3 ;

[0039] (3) Spray drying: The concentrated liquid was spray dried and powdered to obtain 30 kg of shiitake mushroom polysaccharide intermediate with a polysaccharide content of 16%.

[0040] (4) Enzymatic hydrolysis: The lentinan intermediate obtained in step (3) was dissolved in 300 kg of water, and 15 kg of α-glucosidase and β-glucosidase were added to obtain a reaction solution, wherein α-glucosidase accounted for 40% and β-glucosidase accounted for 60%. The temperature was raised to 40℃ and stirred for 3 hours.

[0041] (5) Spray drying: The reaction solution was spray dried to collect 15 kg of powder with a shiitake mushroom polysaccharide content of 65%.

[0042] (6) Prepare 2.5% lentinan powder by adding mannitol as an excipient to prepare 2.5% lentinan powder.

[0043] Example 2

[0044] (1) Extraction: 350 kg of shiitake mushrooms were crushed, soaked in 20 times the amount of water for 20 min, and then heated to 45 degrees Celsius and 3.5 kg of compound enzyme was added. The compound enzyme was composed of the following enzymes in the following mass fractions: 35% cellulase, 25% hemicellulase, and 40% pectinase. After 1 h of compound enzyme-assisted extraction, the mixture was boiled and simmered for 5 h to obtain an extract with a mass of 4 t. The extract was filtered into a filtrate using a disc centrifuge.

[0045] (2) Concentration: The filtrate is concentrated to a density of 1.20 kg / m³. 3 ;

[0046] (3) Spray drying: The concentrated liquid is spray dried to collect 35 kg of shiitake mushroom polysaccharide intermediate, with a polysaccharide content of 15%.

[0047] (4) Enzymatic hydrolysis: The lentinan intermediate obtained in step (3) was dissolved in 350 kg of water, and 17.5 kg of α-glucosidase and β-glucosidase were added to obtain a reaction solution, wherein α-glucosidase accounted for 30% and β-glucosidase accounted for 70%. The temperature was raised to 45℃ and stirred for 3 hours.

[0048] (5) Spray drying: The reaction solution was spray dried, and 16 kg of powder was collected, with a shiitake mushroom polysaccharide content of 60%.

[0049] (6) Prepare 2.5% lentinan powder by adding mannitol as an excipient to prepare 2.5% lentinan powder.

[0050] Example 3

[0051] (1) Extraction: 350 kg of shiitake mushrooms were crushed, soaked in 10 times the amount of water for 40 min, and then heated to 45 degrees Celsius and 7 kg of compound enzyme was added. The compound enzyme consisted of the following enzyme composition by mass fraction: 30% cellulase, 20% hemicellulase, and 50% pectinase. After 1 h of enzyme-assisted extraction, the mixture was boiled and simmered for 4 h to obtain an extract of 3.0 t. The extract was then filtered into a filtrate using a disc centrifuge.

[0052] (2) Concentration: The filtrate is concentrated to a density of 1.30 kg / m³. 3 ;

[0053] (3) Spray drying: The concentrate is spray dried to collect 28 kg of shiitake mushroom polysaccharide intermediate, with a polysaccharide content of 12%.

[0054] (4) Enzymatic hydrolysis: The lentinan intermediate obtained in step (3) was dissolved in 280 kg of water, and 19.6 kg of α-glucosidase and β-glucosidase were added to obtain a reaction solution, wherein α-glucosidase accounted for 50% and β-glucosidase for 50%. The temperature was raised to 42℃ and stirred for 3 hours.

[0055] (5) Spray drying: The reaction solution was spray dried to collect 15 kg of powder with a shiitake mushroom polysaccharide content of 58%.

[0056] (6) Prepare 2.5% lentinan powder by adding mannitol as an excipient to prepare 2.5% lentinan powder.

[0057] Comparative Example 1

[0058] Compared with Example 1, Comparative Example 1 used only α-glucosidase for enzymatic hydrolysis, while the other steps were the same.

[0059] Comparative Example 2

[0060] Compared with Example 1, Comparative Example 2 used only β-glucosidase for enzymatic hydrolysis, while the other steps were the same.

[0061] Comparative Example 3

[0062] Compared with Example 1, Comparative Example 3 did not undergo enzymatic hydrolysis, but the other steps were the same.

[0063] Comparative Example 4

[0064] Compared to Example 1, Comparative Example 4 did not include cellulase, but all other steps were exactly the same.

[0065] Comparative Example 5

[0066] Compared with Example 1, Comparative Example 5 did not include pectinase, but all other steps were exactly the same.

[0067] Comparative Example 6

[0068] Compared to Example 1, Comparative Example 6 did not include hemicellulase, but all other steps were exactly the same.

[0069] The method for characteristic chromatographic testing using high-performance liquid chromatography is as follows:

[0070] The separation column was a TSK G4000PWx l (7.8 mm × 300 mm, 10 μm) gel chromatography column; the mobile phase was 0.02 mol / L potassium dihydrogen phosphate solution; the column temperature was 30℃ and the flow rate was 0.6 ml / min; the detection was performed.

[0071] Preparation of reference solution: Take an appropriate amount of anhydrous glucose reference standard, accurately weigh it, and add it to the mobile phase to prepare a solution containing 0.5 mg of reference standard per 1 mL.

[0072] Preparation of the test solution: Take 30.0 mg of the test sample, accurately add 10 mL of the mobile phase, seal, dissolve in a water bath, filter, and collect the filtrate.

[0073] Assay: Accurately pipette 20 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result. The chromatogram of the test sample should have four characteristic peaks. Calculate the retention time of each characteristic peak relative to the reference solution. The relative retention time should be within ±5% of the specified value, which is: 0.508 (peak 1), 0.618 (peak 2), 0.866 (peak 3), and 0.988 (peak 4). In the chromatogram of the test sample, the peak areas of peak 1 and peak 2 relative to the reference solution should be greater than 0.41 and 1.20, respectively.

[0074] The lentinan powder obtained from Example 1 and Comparative Examples 1-6 was subjected to characteristic spectrum detection, and the detection results are shown in Table 1 below.

[0075] Table 1 shows the characteristic spectral detection results of lentinan powder obtained in Example 1 and Comparative Examples 1-6.

[0076] Peak-to-peak area ratio Peak-to-peak area ratio Example 1 0.72 1.65 Comparative Example 1 0.33 1.05 Comparative Example 2 0.26 0.93 Comparative Example 3 0.40 1.24 Comparative Example 4 0.40 1.37 Comparative Example 5 0.25 0.96 Comparative Example 6 0.35 1.02

[0077] As shown in Table 1, the enzymatic hydrolysis in Example 1, using 40% α-glucosidase and 60% β-glucosidase, yielded peak area ratios for both peak 1 and peak 2 that met quality requirements. However, in Comparative Examples 1-6, both peak area ratios for peak 1 and peak 2 did not meet quality requirements. The final lentinan powder obtained in Example 1 was characterized by its characteristic spectral data, and the results are as follows: Figure 1 The above, Figure 1 The horizontal axis represents time, and the vertical axis represents peak height. In the figure, 1 represents peak 1; 2 represents peak 2; 3 represents peak 3; and 4 represents peak 4. Figure 1 The specific data is shown in Table 2 below.

[0078] Table 2 shows the characteristic spectral detection data of lentinan powder obtained in Example 1.

[0079]

[0080] The reference chromatogram of anhydrous glucose is as follows: Figure 2 As shown, Figure 2 The horizontal axis represents time, and the vertical axis represents peak height. Figure 2 The specific data is shown in Table 3 below.

[0081] Table 3. Detection data of characteristic chromatograms of reference standard anhydrous glucose.

[0082]

[0083] Analyzing the data in Tables 2 and 3, we obtained the retention time ratios of peak 1 and peak 2, the peak area ratio of peak 1, and the peak area ratio of peak 2 for the lentinan in Example 1. Specific values ​​are shown in Table 4.

[0084] Table 4 shows the detection data of the characteristic spectrum of lentinan powder obtained in Example 1.

[0085]

[0086]

[0087] As can be seen from Table 4, the lentinan prepared in Example 1 meets the quality requirements of the characteristic spectrum.

[0088] Clinical trials have verified that using 2.5% lentinan powder in combination with topical hydrocortisone acetate cream can treat swine eczema, reduce the dosage of glucocorticoids, and shorten the treatment time.

[0089] Clinical Trial 1

[0090] Table 5. Methods of adding groups in Clinical Trial 1

[0091]

[0092]

[0093] (1) Experimental subjects: 210 four-month-old pigs with red spots and erythema on their skin and ulceration were selected.

[0094] (2) Husbandry environment: During the experiment, all experiments were conducted in an isolated room, and the environment was kept dry and clean. Everything else remained the same.

[0095] (3) Experimental methods: The animals were randomly divided into 7 groups, with 30 animals in each group. They were fed according to Table 5. After 7 days of treatment, the animals were observed and recorded. See Table 6 for details. Another course of treatment was given after 7 days.

[0096] (4) Treatment standards and observation methods

[0097] Cured: The pig's skin disease was cured, and some symptoms, such as loss of appetite, were also cured.

[0098] Effective: Pig skin diseases were effectively alleviated.

[0099] Ineffective: The pig's skin disease was not effectively alleviated.

[0100] Table 6 Treatment results of each group in Clinical Trial 1

[0101] Cure rate after one course of treatment: % Cure rate after two courses of treatment: % Group 1 90 100 Group 2 95 100 Group 3 100 --- Group 4 80 85 Group 5 75 80 Group 6 70 75 Group 7 70 75

[0102] Table 6 shows that using 2.5% lentinan powder combined with topical hydrocortisone acetate cream can quickly improve the symptoms of skin diseases in pigs. After one course of treatment, the skin disease shows significant improvement, and most pigs recover completely after two courses of treatment, at which point the use of feed containing lentinan powder should be discontinued. No recurrence was observed for one month after recovery. The therapeutic effect of feeding pigs with skin diseases using lentinan powder mixed into feed is significantly better than using lentinan powder without this application. The therapeutic effect of feeding pigs with skin diseases using ordinary lentinan powder mixed into feed is not much different from that of using hydrocortisone acetate cream alone. Using highly active lentinan powder reduces the amount of hydrocortisone acetate cream required, thus reducing the impact of drug residues on human health.

[0103] Clinical Trial 2:

[0104] Table 7. Methods of administration for each group in Clinical Trial 2

[0105]

[0106] To demonstrate the feeding effect of this invention on pigs, multiple feeding trials were conducted at a pig farm. One hundred piglets of similar weight, size, and health status were randomly divided into five groups of 20 piglets each. They were fed according to the dosage in Table 7, twice daily, 1 kg each time, for seven consecutive days; a seven-day interval was then added to complete one treatment course. The feeding frequency and duration were identical for all five groups of piglets. The average performance indicators of the five groups are shown in Table 8. After one month of feeding, the condition of the five groups of piglets is shown in Table 8.

[0107] Table 8 Treatment results of each group in Clinical Trial 2

[0108]

[0109]

[0110] As can be clearly seen from Table 8, the daily weight gain of piglets in groups 1 to 3 was significantly higher than that of groups 4 and 5 after continuous feeding with feed containing lentinan powder. Furthermore, the piglets in groups 1 to 3 had a stronger appetite, greater vitality, better condition, shinier fur, and no skin diseases compared to groups 4 and 5.

[0111] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing lentinan, characterized in that, The preparation method includes: pulverizing shiitake mushroom raw materials, soaking them in water for 20-40 minutes, adding a compound enzyme for assisted extraction, decocting for 3-5 hours, and filtering to obtain shiitake polysaccharide intermediates; the amount of the compound enzyme is 1%-2% of the shiitake mushroom raw materials by mass percentage, and the compound enzyme is a mixture of cellulase, hemicellulase and pectinase; the compound enzyme is 30%-40% cellulase, 20%-30% hemicellulase and 30%-50% pectinase by mass percentage; then adding α-glucosidase and β-glucosidase to obtain a reaction solution, the mass ratio of α-glucosidase and β-glucosidase to shiitake polysaccharide intermediates is 0.5-0.7:1, and the mass percentage ratio of α-glucosidase to β-glucosidase is 30-50% α-glucosidase and 50-70% β-glucosidase; heating to 40-45℃ and stirring, then drying to obtain shiitake polysaccharide.

2. The method for preparing lentinan according to claim 1, characterized in that, The ratio of α-glucosidase to β-glucosidase by mass percentage is: α-glucosidase 40%, β-glucosidase 60%.

3. A lentinan, characterized in that, It is obtained by the method for preparing lentinan according to any one of claims 1-2.

4. An application of lentinan according to claim 3, characterized in that, The lentinan is used to prepare a medicine for treating swine eczema.

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