Process for the enrichment of water-soluble active factors and associated equipment

Abstract

The application provides a water-soluble active factor enrichment process and related equipment, and relates to the technical field of water-soluble active factor enrichment. The specific steps of the water-soluble active factor enrichment process are as follows: washing Ganoderma lucidum, drying and crushing, sieving, and obtaining Ganoderma lucidum powder; adding the Ganoderma lucidum powder into a buffer solution and uniformly mixing to form a Ganoderma lucidum powder solution, inoculating a composite enzyme, adjusting the temperature, performing enzymolysis, and inactivating the enzyme to obtain an extraction liquid A; the composite enzyme is obtained by configuring cellulase and beta-glucanase at a mass ratio of 2:0.59-0.67. The application uses Ganoderma lucidum as the main extraction material, uses the composite enzyme for the early-stage treatment of Ganoderma lucidum, performs system cleaning after enzymolysis, inactivates the composite enzyme, uses pectinase for the late-stage treatment of the reaction system, makes the pectinase participate in the reaction system, improves the yield of flavones, and the improvement rate of the flavone enrichment process is about 23%-67% compared to other enzymolysis enrichment processes, and the antioxidant capacity of the enriched flavones is improved.

Description

Technical Field

[0001] This invention relates to the field of water-soluble active factor enrichment technology, specifically to water-soluble active factor enrichment process and related equipment. Background Technology

[0002] Flavonoids are a class of bioactive substances with unique health benefits and therapeutic effects. Various extraction methods exist, including hot water extraction, alkaline extraction, acetone extraction, ethanol extraction, microwave extraction, ultrasonic extraction, enzymatic hydrolysis, macroporous resin adsorption, and ultrafiltration. Currently, traditional methods such as solvent extraction and hot water extraction still dominate flavonoid extraction. However, compared to enzymatic hydrolysis, ultrafiltration, and microwave extraction techniques, these methods have revealed several problems, such as high extraction temperatures, long extraction times, high energy consumption, poor protection of the flavonoid nucleus activity, low flavonoid yield in the final product, and, in particular, the presence of solvent residue. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a process and related equipment for enriching water-soluble active factors, the specific solution of which is as follows: The specific steps of a water-soluble active factor enrichment process are as follows: S1. Wash the Ganoderma lucidum from the linden wood, dry it, pulverize it, and sieve it to obtain Ganoderma lucidum powder; S2. Add Ganoderma lucidum powder to buffer solution and mix well to form Ganoderma lucidum powder solution. Inoculate with compound enzyme, adjust temperature, perform enzymatic hydrolysis, and inactivate enzyme to obtain extract A. The composite enzyme is prepared by mixing cellulase and β-glucanase at a mass ratio of 2:0.59-0.67. S3. Adjust the temperature, inoculate pectinase into extract A, hydrolyze the enzyme, and inactivate the enzyme to obtain extract B; S4. Centrifuge extract B, take the supernatant, remove impurities, and make up to volume to obtain the active factor enrichment solution.

[0004] Preferably, the sieving in step S1 specifically involves passing the material through a 20-mesh sieve.

[0005] Preferably, in step S2, the pH value of the buffer solution is 5.0-5.5, the mass ratio of the buffer solution to Ganoderma lucidum powder is 2:1, the temperature is adjusted to 50-55℃, the enzymatic hydrolysis is carried out for 60-62 minutes, the enzyme is inactivated, and the solution is placed in a boiling water bath at 100℃ for 10-12 minutes. The mass ratio of Ganoderma lucidum powder liquid to compound enzyme is 100:1-2.

[0006] Preferably, in step S3, the temperature is adjusted to 50-52℃, the mass ratio of pectinase to cellulase is 0.43-0.69:2, the enzymatic hydrolysis is carried out for 20-30 minutes, and the enzyme is inactivated by a water bath in boiling water at 100℃ for 5-10 minutes.

[0007] Preferably, the centrifugation speed is 4300-4500 r / min and the treatment time is 12-15 min.

[0008] A water-soluble active factor enrichment device includes: The pretreatment module includes a washing device, a drying device, a crushing device, and a sieving device connected in sequence. The enzymatic hydrolysis module includes a first enzymatic hydrolysis chamber for inoculating a compound enzyme and a second enzymatic hydrolysis chamber for inoculating a pectinase. The post-processing module includes a centrifuge for centrifuging extract B and collecting the supernatant, the centrifuge being connected to a purification device for removing impurities, and the purification device being connected to a volume-fixing device for volume adjustment.

[0009] This invention provides a process and related equipment for enriching water-soluble active factors, which has the following advantages compared with the prior art: This invention uses Ganoderma lucidum from linden wood as the main extraction material. It employs a compound enzyme to pre-treat the Ganoderma lucidum from linden wood, and cleans the system after enzymatic hydrolysis to inactivate the compound enzyme. Then, it uses pectinase to inoculate the reaction system in the later stage, allowing it to participate in the reaction system and improve the yield of flavonoids. Compared with other enzymatic hydrolysis enrichment processes, the improvement rate is about 23%-67%, and the antioxidant capacity of the enriched flavonoids is also improved. Attached Figure Description

[0010] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a schematic diagram of the enrichment process of the present invention.

[0011] In the diagram: 1. Pretreatment module; 101. Cleaning device; 102. Drying device; 103. Crushing device; 104. Sieving device; 2. Enzymatic hydrolysis module; 201. First enzymatic hydrolysis chamber; 202. Second enzymatic hydrolysis chamber; 3. Posttreatment module; 301. Centrifuge; 302. Impurity removal device; 303. Volume control device. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 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.

[0013] Unless otherwise specified, all reagents used in the following schemes are commercially available products.

[0014] Cellulase (purchased from Acmec, enzyme activity 3U / mg); β-glucanase (purchased from Beijing Ita Biotechnology Co., Ltd., enzyme activity 3.5 million u / ml); pectinase (purchased from Kramar, enzyme activity 30 u / mg).

[0015] Example 1 S1. Wash the linden wood Ganoderma lucidum in the cleaning device 101, dry it in the drying device 102, and then pulverize it through the pulverizing device 103 until it passes through the 20-mesh sieve 104 to obtain Ganoderma lucidum powder. S2. Add Ganoderma lucidum powder to the first enzymatic hydrolysis chamber 201. First, add an acetate-sodium acetate buffer solution (pH=5.5) with a mass ratio of 2:1 to Ganoderma lucidum powder, mix well to form Ganoderma lucidum powder solution, and then inoculate with a compound enzyme (the compound enzyme is cellulase and β-glucanase in a mass ratio of 2:0.67). The mass ratio of Ganoderma lucidum powder solution to compound enzyme is 100:2. Adjust the temperature to 53℃, enzymatically hydrolyze for 61 min, and then bathe in boiling water at 100℃ for 11 min to obtain extract A. S3. Add extract A to the second enzymatic hydrolysis chamber 202. First, adjust the temperature to 51℃. Inoculate extract A with pectinase. The mass ratio of pectinase to cellulase is 0.43-0.69:2. Enzymatically hydrolyze for 25 minutes, and then bathe in boiling water at 100℃ for 7 minutes to obtain extract B. S4. Add extract B to centrifuge 301 and centrifuge at 4400 r / min for 13 min. Take the supernatant and put it into impurity removal device 302. Add petroleum ether to remove impurities. After impurity removal, put it into volume making device 303 and make up to 10 mL to obtain active factor enrichment solution.

[0016] Example 2 This embodiment refers to the enrichment process of Example 1, only the mixing ratio of the complex enzymes is different, specifically: The complex enzyme is composed of cellulase and β-glucanase in a mass ratio of 2:0.59.

[0017] Comparative Example 1 This comparative example refers to the enrichment process of Example 1, only adjusting step S2, specifically: S2. Add Ganoderma lucidum powder to the first enzymatic hydrolysis chamber 201. First, add acetate-sodium acetate buffer (pH=5.5) and mix well to form Ganoderma lucidum powder solution. Then, inoculate with cellulase. The mass ratio of Ganoderma lucidum powder solution to cellulase is 100:1. Adjust the temperature to 53℃ and enzymatically hydrolyze for 61 min. Then, bathe in boiling water at 100℃ for 11 min to obtain extract A.

[0018] Comparative Example 2 This comparative example refers to the enrichment process of Example 1, only adjusting step S2, specifically: S2. Add Ganoderma lucidum powder to the first enzymatic hydrolysis chamber 201. First, add an acetate-sodium acetate buffer solution (pH=5.5) with a mass ratio of 2:1 to Ganoderma lucidum powder, mix well to form Ganoderma lucidum powder solution, and then inoculate with β-glucanase. The mass ratio of Ganoderma lucidum powder solution to β-glucanase is 100:1. Adjust the temperature to 55℃, enzymatically hydrolyze for 50 min, and then water bath in boiling water at 100℃ for 11 min to obtain extract A.

[0019] Comparative Example 3 This comparative example refers to the enrichment process of Example 1, only adjusting steps S2 and S3, specifically: S2. Add Ganoderma lucidum powder to the first enzymatic hydrolysis chamber 201. First, add an acetate-sodium acetate buffer solution (pH=5.5) with a mass ratio of 2:1 to Ganoderma lucidum powder, mix well to form Ganoderma lucidum powder solution, and then inoculate with a compound enzyme (the compound enzyme is cellulase and pectinase in a mass ratio of 2:0.67). The mass ratio of Ganoderma lucidum powder solution to compound enzyme is 100:1. Adjust the temperature to 53℃, enzymatically hydrolyze for 61 min, and then bathe in boiling water at 100℃ for 11 min to obtain extract A. S3. Add extract A to the second enzymatic hydrolysis chamber 202. First, adjust the temperature to 51℃. Inoculate β-glucanase into extract A. The mass ratio of β-glucanase to cellulase is 0.43-0.69:2. Enzymatically hydrolyze for 25 minutes, and then bathe in boiling water at 100℃ for 7 minutes to obtain extract B.

[0020] Comparative Example 4 This comparative example refers to the enrichment process of Example 1, only adjusting steps S2 and S3, specifically: S2. Add Ganoderma lucidum powder to the first enzymatic hydrolysis chamber 201. First, add an acetate-sodium acetate buffer solution (pH=5.5) with a mass ratio of 2:1 to Ganoderma lucidum powder, mix well to form Ganoderma lucidum powder solution, and then inoculate with a compound enzyme (the compound enzyme is β-glucanase and pectinase in a mass ratio of 0.55:0.67). The mass ratio of Ganoderma lucidum powder solution to compound enzyme is 100:1. Adjust the temperature to 53℃, enzymatically hydrolyze for 61 min, and then water bath in boiling water at 100℃ for 11 min to obtain extract A. S3. Add extract A to the second enzymatic hydrolysis chamber 202. First, adjust the temperature to 51℃. Inoculate cellulase into extract A. The mass ratio of cellulase to pectinase is 2:0.55. Hydrolyze for 25 minutes. Then, bathe in boiling water at 100℃ for 7 minutes to obtain extract B.

[0021] Comparative Example 5 This comparative example refers to the enrichment process of Example 1, only adjusting steps S2 and S3, specifically: S2. Add Ganoderma lucidum powder to the first enzymatic hydrolysis chamber 201. First, add an acetate-sodium acetate buffer solution (pH=5.5) with a mass ratio of 2:1 to Ganoderma lucidum powder, mix well to form Ganoderma lucidum powder solution, adjust the temperature to 51℃, inoculate with pectinase, the mass ratio of pectinase to cellulase is 0.43-0.69:2, enzymatic hydrolysis for 25 min, and then water bath in boiling water at 100℃ for 7 min to obtain extract A. S3. Add extract A to the second enzymatic hydrolysis chamber 202, inoculate extract A with a compound enzyme (the compound enzyme is cellulase and β-glucanase in a mass ratio of 2:0.67), the mass ratio of Ganoderma lucidum powder liquid to compound enzyme is 100:2, adjust the temperature to 53℃, enzymatically hydrolyze for 61 min, and then water bath in boiling water at 100℃ for 11 min to obtain extract B.

[0022] Comparative Example 6 This comparative example refers to the enrichment process of Example 1, only adjusting steps S2 and S4, and deleting S3, specifically: S2. Add Ganoderma lucidum powder to the first enzymatic hydrolysis chamber 201. First, add an acetate-sodium acetate buffer solution (pH=5.5) with a mass ratio of 2:1 to Ganoderma lucidum powder, mix well to form Ganoderma lucidum powder solution, and then inoculate with a compound enzyme (the compound enzyme is cellulase, β-glucanase and pectinase in a mass ratio of 2:0.67:0.55). The mass ratio of Ganoderma lucidum powder solution to compound enzyme is 100:2. Adjust the temperature to 53℃, enzymatically hydrolyze for 86 min, and then water bath in boiling water at 100℃ for 11 min to obtain the extract. S4. Add the extract to a centrifuge and centrifuge at 4400 r / min for 13 min. Take the supernatant and put it into a purification device. Add petroleum ether to remove impurities. After purification, put it into a volume-adjusting device to make up to 10 mL to obtain the active factor enrichment solution.

[0023] Detection: Active factor enrichment solutions were prepared as test samples according to the enrichment processes of Examples 1-2 and Comparative Examples 1-6.

[0024] 1. Extraction rate determination: Take 1 mL of the test sample into a 25 mL volumetric flask, and determine the total flavonoids by the NaNO2-Al(NO3)3-NaOH colorimetric method. According to the standard curve regression equation, the mass concentration of flavonoids in the extract is calculated from the absorbance. The flavonoid yield is calculated as shown in formula (1): Flavonoid yield (mg / g) = C × V / m (1) In the formula: C - flavonoid mass concentration, mg / mL; V - final volume, mL; m - raw material mass (3g), g.

[0025] The results are shown in Table 1 below: Table 1 As shown in Table 1 above, the highest flavonoid yield was 0.95 mg / g in Example 1. The specific process of Example 1 involved first inoculating the compound enzyme onto *Ganoderma lucidum* wood and then inoculating it with pectinase after enzymatic hydrolysis, achieving stepwise synergistic decomposition. Observing Table 1, the flavonoid yield of Comparative Example 4 was relatively higher than the other comparative examples. In Comparative Example 4, the compound enzyme was replaced with pectinase and cellulase, followed by inoculation with β-glucanase. Although the flavonoid yield increased somewhat, it was still lower than that of Example 1.

[0026] 2. Determination of DPPH free radical scavenging rate: Prepare a 0.04 mg / mL solution of DPPH using anhydrous ethanol. Add 2 mL of DPPH to 1 mL of test samples of different concentrations (0.2, 0.8, 1.0, 2.0, 3.0 mg / mL), mix thoroughly, and react at room temperature in the dark for 30 min. Measure the absorbance at 517 nm. Use vitamin C as a positive control. Plot the IC50 of DPPH scavenging rate. 50 The DPPH removal rate is calculated as described in formula (2): FPPH clearance rate (%) = {[A1-(A2-A3)]} / A1×100% (2) In the formula: A1 - absorbance value of the sample replaced by ethanol; A2 - absorbance value of the sample being tested; A3 - absorbance value of the sample replaced by ethanol.

[0027] The results are shown in Table 2 below: Table 2 As shown in Table 3 above, Example 1 had the lowest concentration at which a 50% clearance rate was achieved, indicating that the sample prepared in Example 1 had stronger antioxidant capacity. Comparative Example 5, compared to Example 1, had a different inoculation order of pectinase and the complex enzyme. While it showed better results than other comparative examples, its concentration was still higher than that of Example 1. Therefore, using the complex enzyme to first decompose the Ganoderma lucidum before introducing pectinase can have beneficial effects.

[0028] 3. Determination of hydroxyl radical scavenging rate: DPPH was prepared into a 0.04 mg / mL solution using anhydrous ethanol. FeSO4 (9 mmol / L, 1 mL), salicylic acid-ethanol solution (9 mmol / L, 1 mL), and H2O2 (9 mmol / L, 1 mL) were added to 1 mL of test samples of different concentrations (0.2, 0.8, 1.0, 2.0, 3.0 mg / mL), respectively. The reaction was carried out in a water bath at 37℃ for 30 min, and the absorbance was measured at 510 nm. The IC50 of DPPH scavenging rate was plotted. 50Vitamin C was used as a positive control. The hydroxyl radical scavenging rate was calculated as described in formula (3): Hydroxyl radical scavenging rate (%) = {[A1-(A2-A3)]} / A1×100% (3) In the formula: A1 - absorbance value when distilled water is used instead of the sample; A2 - absorbance value of the sample being tested; A3 - absorbance value when distilled water is used instead of H2O2.

[0029] The results are shown in Table 3 below: Table 3 As shown in Table 3, the concentration of Example 1 was the lowest when a 50% clearance rate was achieved, indicating that the test sample prepared in Example 1 had stronger antioxidant capacity than the test samples prepared in Comparative Examples 1-6.

[0030] In summary, based on Tables 1, 2, and 3, the sample prepared in Example 1 showed better yield and antioxidant capacity. Although Comparative Example 5 also had a relatively low IC50 concentration for FPPH scavenging, its flavonoid yield was also low, and its IC50 concentration for hydroxyl radical scavenging was also low. 50 The measured concentrations showed no advantage, and other comparative examples also showed no advantage over Example 1. Based on the above three tables, when enriching water-soluble flavonoids in *Ganoderma lucidum*, inoculating the *Ganoderma lucidum* with a compound enzyme, completing enzymatic hydrolysis, and then inoculating with pectinase achieves stepwise synergistic decomposition, which can improve the flavonoid yield.

[0031] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A process for enriching water-soluble active factors, characterized in that, The specific steps of the enrichment process are as follows: S1. Wash the Ganoderma lucidum from the linden wood, dry it, pulverize it, and sieve it to obtain Ganoderma lucidum powder; S2. Add Ganoderma lucidum powder to buffer solution and mix well to form Ganoderma lucidum powder solution. Inoculate with compound enzyme, adjust temperature, perform enzymatic hydrolysis, and inactivate enzyme to obtain extract A. The composite enzyme is prepared by mixing cellulase and β-glucanase at a mass ratio of 2:0.59-0.67; S3. Adjust the temperature, inoculate pectinase into extract A, hydrolyze the enzyme, and inactivate the enzyme to obtain extract B; S4. Centrifuge extract B, take the supernatant, remove impurities, and make up to volume to obtain the active factor enrichment solution.

2. The enrichment process according to claim 1, characterized in that, The sieving process in step S1 specifically involves passing the material through a 20-mesh sieve.

3. The enrichment process according to claim 1, characterized in that, In step S2, the pH of the buffer solution is 5.0-5.5, the mass ratio of the buffer solution to Ganoderma lucidum powder is 2:1, the temperature is adjusted to 50-55℃, the enzymatic hydrolysis is carried out for 60-62 minutes, the enzyme is inactivated, and the solution is placed in a boiling water bath at 100℃ for 10-12 minutes. The mass ratio of Ganoderma lucidum powder liquid to compound enzyme is 100:1-2.

4. The enrichment process according to claim 1, characterized in that, In step S3, the temperature is adjusted to 50-52℃, the mass ratio of pectinase to cellulase is 0.43-0.69:2, the enzymatic hydrolysis is carried out for 20-30 minutes, and the enzyme is inactivated by a water bath in boiling water at 100℃ for 5-10 minutes.

5. The enrichment process according to claim 1, characterized in that, The centrifugation speed is 4300-4500 r / min, and the treatment time is 12-15 min.

6. A water-soluble active factor enrichment device, implementing the water-soluble active factor enrichment process as described in any one of claims 1-5, characterized in that, include: The pretreatment module (1) includes a washing device (101), a drying device (102), a crushing device (103), and a sieving device (104) connected in sequence. The enzymatic hydrolysis module (2) includes a first enzymatic hydrolysis chamber (201) for inoculating a compound enzyme and a second enzymatic hydrolysis chamber (202) for inoculating a pectinase. The post-processing module (3) includes a centrifuge (301) for centrifuging extract B and collecting supernatant, the centrifuge being connected to a purification device (302) for removing impurities, and the purification device being connected to a volume-fixing device (303) for volume-fixing.

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