Extraction method and application of mushroom extract

Abstract

The invention provides an extraction method and application of a mushroom extract, and particularly belongs to the technical field of fungus extracts. The invention provides an extraction method of a shiitake mushroom extract, which comprises the following steps: mixing cell wall hydrolase, proteolytic enzyme and shiitake mushroom for enzymolysis to obtain the shiitake mushroom extract, the cell wall hydrolase comprises cellulase; the proteolytic enzyme comprises flavourzyme. The extraction method provided by the invention can improve the content of the shiitake mushroom extract, particularly the content of total reducing sugar, total polysaccharide, total amino acid and total protein, and has more advantages in the aspects of process feasibility and economy.

Description

Technical Field

[0001] The present invention belongs to the technical field of fungal extracts, and particularly relates to an extraction method and application of lentinula edodes extract. Background Art

[0002] Lentinula edodes extract is widely used in the food, health product and pharmaceutical industries because it is rich in polysaccharides, proteins and amino acids. However, traditional water extraction methods have problems such as low dissolution rate of active ingredients, poor flavor and low extraction efficiency. Ultrasonic extraction can improve the cell wall breakage rate, but it will increase energy consumption and equipment costs. High-temperature hydrolysis can promote the release of some components, but it will cause the degradation of some active ingredients. In recent years, enzymatic hydrolysis technology has been introduced into the field of lentinula edodes processing, but existing enzymatic hydrolysis methods often fail to accurately optimize the combination of enzyme species, resulting in poor extraction rates of components such as polysaccharides and proteins. Summary of the Invention

[0003] The purpose of the present invention is to provide an extraction method and application of lentinula edodes extract. The extraction method of the present invention can increase the content of lentinula edodes extract and has more advantages in terms of process feasibility and economy.

[0004] The present invention provides an extraction method of lentinula edodes extract, comprising the following steps:

[0005] Mix cell wall hydrolase, protease and lentinula edodes for enzymatic hydrolysis to obtain lentinula edodes extract;

[0006] The cell wall hydrolase includes cellulase; the protease includes flavourzyme.

[0007] Preferably, the cellulase includes the cellulase with the model of Cellulase T"Amano"4 from Amano Enzyme Company; the flavourzyme includes the flavourzyme with the model of Flavourzyme 1000L from Novozymes Company.

[0008] Preferably, the lentinula edodes includes lentinula edodes powder.

[0009] Preferably, the lentinula edodes is mixed with cell wall hydrolase and protease in the form of a lentinula edodes water mixture; the lentinula edodes water mixture is prepared by mixing lentinula edodes and water; the mass ratio of lentinula edodes to water for mixing is 1:30.

[0010] Preferably, the temperature of the enzymatic hydrolysis is 50°C.

[0011] Preferably, the time of the enzymatic hydrolysis is 6 - 9 h.

[0012] Preferably, after the enzymatic hydrolysis, it further includes terminating the enzymatic hydrolysis and solid-liquid separation.

[0013] Preferably, the conditions for terminating enzymatic hydrolysis are 80 °C for 10 min.

[0014] Preferably, the method for solid-liquid separation includes centrifuging to obtain the supernatant; the conditions for centrifugation include 5000 rpm, 20 °C, and 10 min.

[0015] The present invention also provides the application of the extraction method described in the above technical solution in increasing the content of any one or more of total reducing sugar, total polysaccharide, total amino acid, and total protein in the Lentinula edodes extract.

[0016] The present invention provides a method for extracting Lentinula edodes extract. Through enzyme species screening and enzymatic hydrolysis condition optimization, the present invention effectively improves the content and flavor of the Lentinula edodes extract, and provides a production plan with a higher extraction rate. The method for optimizing the content of Lentinula edodes extract by using a specific enzymatic hydrolysis combination provided by the present invention has the following beneficial effects: the content of active ingredients is significantly increased: through enzymatic hydrolysis, proteins and polysaccharides are degraded into small molecules, increasing the content of total reducing sugar (by 3 times), total polysaccharide (by 1 time), total amino acid (by 4 times), and total protein (by 1 time) in the Lentinula edodes extract; process optimization to improve enzymatic hydrolysis efficiency: by screening suitable cell wall hydrolases and proteolytic enzymes, and optimizing the enzymatic hydrolysis temperature and time, high-efficiency enzymatic hydrolysis is achieved in a short time, improving the extraction efficiency. Detailed implementation manners

[0017] The present invention provides a method for extracting Lentinula edodes extract, including the following steps:

[0018] Mix a cell wall hydrolase, a proteolytic enzyme, and Lentinula edodes for enzymatic hydrolysis to obtain a Lentinula edodes extract;

[0019] The cell wall hydrolase includes cellulase; the proteolytic enzyme includes flavor protease.

[0020] In the present invention, a cell wall hydrolase, a proteolytic enzyme, and Lentinula edodes are mixed for enzymatic hydrolysis to obtain a Lentinula edodes extract. In a specific embodiment, the Lentinula edodes includes Lentinula edodes powder. In a specific embodiment, the Lentinula edodes powder is derived from dried Lentinula edodes. In a specific embodiment, the source of the dried Lentinula edodes includes dried Lentinula edodes stalks. In a specific embodiment, the Lentinula edodes powder is obtained by crushing and sieving Lentinula edodes stalks. In a specific embodiment, the particle size of the Lentinula edodes powder is below 80 mesh. In a specific embodiment, the Lentinula edodes is mixed with the cell wall hydrolase and the proteolytic enzyme in the form of a Lentinula edodes aqueous mixture; the Lentinula edodes aqueous mixture is prepared by mixing Lentinula edodes and water; the mass ratio of Lentinula edodes to water for mixing is 1:30. After mixing Lentinula edodes and water in the present invention, it is stirred evenly.

[0021] In the present invention, the cell wall hydrolase includes cellulase; the proteolytic enzyme includes flavourzyme. The cell wall hydrolase of the present invention is used to degrade β-glucan and improve the water permeability of the cell wall; the protease of the present invention is used to degrade Lentinula edodes protein and increase the release of amino acids. Specifically, cellulase can deconstruct the cell wall, break the wall, and release saccharide substances. Flavourzyme is a type of compound proteolytic enzyme that decomposes proteins into flavour peptides and free amino acids. In a specific embodiment, the cellulase includes the cellulase of Amano Enzyme Co., Ltd. with the model of Cellulase T"Amano"4; the flavourzyme includes the flavourzyme of Novozymes Co., Ltd. with the model of Flavourzyme 1000L. The specific enzymatic hydrolysis combination of the present invention can increase the content of Lentinula edodes extract and significantly improve the content of active ingredients. In a specific embodiment, the addition amount of the cell wall hydrolase can be 0.1% w / w; in a specific embodiment, the addition amount of the proteolytic enzyme can be 0.1% w / w. The addition amount described in the present invention is the addition amount relative to the mass of the Lentinula edodes water mixture. In a specific embodiment, the temperature of the enzymatic hydrolysis is 50°C. In a specific embodiment, the time of the enzymatic hydrolysis is 6-9 h, specifically it can be 6 h, 8 h or 9 h. The settings of the enzymatic hydrolysis and time in the present invention are relatively mild and will not overly affect the quality of the raw materials. In a specific embodiment, after the enzymatic hydrolysis, it further includes terminating the enzymatic hydrolysis and solid-liquid separation. In a specific embodiment, the conditions for terminating the enzymatic hydrolysis are 80°C for 10 min. In a specific embodiment, the method of solid-liquid separation includes centrifuging to obtain the supernatant; the conditions of the centrifugation include 5000 rpm, 20°C for 10 min. The present invention screens and obtains suitable cell wall hydrolases and proteolytic enzymes (enzyme species screening), combined with the optimization of the enzymatic hydrolysis temperature and time, can achieve efficient enzymatic hydrolysis in a short time, improve the extraction efficiency, and increase the content of target components in the Lentinula edodes extract. The test results show that the extraction method of the present invention can, through enzymatic hydrolysis, degrade proteins and polysaccharides into small molecules, and increase the total reducing sugar content (increased by 3 times), total polysaccharide content (increased by 1 time), total amino acid content (increased by 4 times), and total protein content (increased by 1 time) in the Lentinula edodes extract.

[0022] The present invention also provides the application of the extraction method described in the above technical solution in increasing the content of any one or more of the total reducing sugar, total polysaccharide, total amino acid and total protein in the Lentinula edodes extract.

[0023] In order to further illustrate the present invention, the following describes in detail an extraction method and its application of a Lentinula edodes extract provided by the present invention in conjunction with embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0024] Detection methods for each component in the Lentinula edodes extract:

[0025] Total reducing sugar content (GB 5009.7-2016);

[0026] Total polysaccharide content (SN / T 4260-2015);

[0027] Total protein content (GB 5009.5-2016);

[0028] Total free amino acid content (GB 5009.124-2016).

[0029] Example 1

[0030] Lentinula edodes powder and water were mixed at a ratio of 1:30 (mass ratio). Specifically, 1.00 g of Lentinula edodes and 30.0 ml of water were taken and stirred evenly to obtain a Lentinula edodes-water mixture;

[0031] The mass percentage content of cellulase (model: Cellulase T"Amano"4) from Amano Enzyme Co., Ltd. added to the Lentinula edodes-water mixture was 0.1%, and the mass percentage content of flavour protease (model: Flavourzyme 1000L) from Novozymes Co., Ltd. added to the Lentinula edodes-water mixture was 0.1%;

[0032] The temperature was 50 °C and enzymatic hydrolysis extraction was carried out for 6 h;

[0033] Terminate enzymatic hydrolysis: Heat to 80 °C and maintain for 10 min;

[0034] Solid-liquid separation: By centrifugation (5000 rpm, 20 °C, 10 min), the supernatant was taken to obtain Lentinula edodes extract.

[0035] Example 2

[0036] Other conditions were the same as in Example 1, the difference was that:

[0037] The temperature was 40 °C and enzymatic hydrolysis extraction was carried out for 3 h.

[0038] Example 3

[0039] Other conditions were the same as in Example 1, the difference was that:

[0040] The temperature was 50 °C and enzymatic hydrolysis extraction was carried out for 3 h.

[0041] Example 4

[0042] Other conditions were the same as in Example 1, the difference was that:

[0043] The temperature was 60 °C and enzymatic hydrolysis extraction was carried out for 3 h.

[0044] Example 5

[0045] Other conditions are the same as those in Example 1, except that:

[0046] The temperature is 50°C, and enzymatic hydrolysis extraction is carried out for 9 h;

[0047] The measurement results of Examples 1 to 5 are shown in Table 1:

[0048] Table 1 Measurement Results

[0049] Total reducing sugar (mg / ml) Total polysaccharide (mg / ml) Total amino acid (mg / ml) Total protein (mg / ml) Example 1 2.88±0.28b 0.90±0.07c 1.72±0.05c 4.55±0.12c Example 2 1.57±0.43a 0.65±0.06def 0.89±0.24d 3.16±0.03b Example 3 2.35±0.28b 0.79±0.07cd 1.37±0.06e 4.14±0.12c Example 4 1.58±0.14a 0.66±0.09def 0.65±0.06bd 3.20±0.12b Example 5 2.83±0.28b 0.92±0.07c 1.48±0.22ce 4.67±0.35c

[0050] In the examples, the enzymatic hydrolysis temperature is 50°C, and the time is 6 - 9 h. The effect is better than that of other examples, and the comprehensive effect is the best when enzymatic hydrolysis is carried out at 50°C for 6 h.

[0051] Comparative Example 1

[0052] Other conditions are the same as those in Example 1, except that:

[0053] No enzyme is added;

[0054] The temperature is 50°C, and extraction is carried out for 3 h.

[0055] Comparative Example 2

[0056] Other conditions are the same as those in Example 1, except that:

[0057] No enzyme is added;

[0058] The temperature is 60°C, and extraction is carried out for 3 h.

[0059] Comparative Example 3

[0060] Other conditions are the same as those in Example 1, except that:

[0061] No enzyme is added;

[0062] The temperature is 60°C, and extraction is carried out for 6 h.

[0063] Comparative Example 4

[0064] Other conditions are the same as those in Example 1, except that:

[0065] No enzyme is added;

[0066] The temperature is 60°C, and extraction is carried out for 9 h;

[0067] The measurement results of Comparative Examples 1 to 4 are shown in Table 2:

[0068] Table 2 Measurement Results

[0069] Total reducing sugar (mg / ml) Total polysaccharide (mg / ml) Total amino acid (mg / ml) Total protein (mg / ml) Comparative Example 1 1.07±0.06a 0.39±0.01ab 0.24±0.01a 2.10±0.10a Comparative Example 2 1.09±0.05a 0.46±0.02bf 0.30±0.05a 2.16±0.12a Comparative Example 3 1.13±0.09a 0.52±0.02be 0.36±0.05ab 2.20±0.12a Comparative Example 4 1.21±0.06a 0.55±0.04be 0.42±0.06ab 2.23±0.14a

[0070] The results of the comparative examples show that without adding enzymes and only changing the temperature during water extraction, there are no significant differences in the extraction effects of the four indicators, and the extraction efficiency is significantly lower than that of Example 1.

[0071] Comparative Example 5

[0072] Other conditions are the same as those in Example 1, except that:

[0073] Replace 0.1% of the cellulase (model: Cellulase T"Amano"4) from Amano Enzyme with the pectinase (model: Pectionase PL"Amano") from Amano Enzyme.

[0074] Comparative Example 6

[0075] Other conditions are the same as those in Example 1, except that:

[0076] Replace the cellulase (model: Cellulase T"Amano"4) from Amano Enzyme with the pectinase (model: Pectionex XXL) from Novozymes.

[0077] Comparative Example 7

[0078] Other conditions are the same as those in Example 1, except that:

[0079] Replace 0.1% of the cellulase (model: Cellulase T"Amano"4) from Amano Enzyme with the plant hydrolase (model: Viscozyme L) from Novozymes.

[0080] Comparative Example 8

[0081] Other conditions are the same as those in Example 1, except that:

[0082] Replace 0.1% of the flavour protease (model: Flavourzyme 1000L) from Novozymes with the chymotrypsin (model: Formea Sol) from Novozymes.

[0083] Comparative Example 9

[0084] Other conditions are the same as those in Example 1, except that:

[0085] Replace 0.1% of Flavourzyme 1000L (produced by Novozymes) with Papain W-40 (produced by Amano Enzyme).

[0086] Comparative Example 10

[0087] Other conditions are the same as those in Example 1, except that:

[0088] Replace 0.1% of Flavourzyme 1000L (produced by Novozymes) with Protease HF "Amano" 150SD (produced by Amano Enzyme).

[0089] The measurement results of Comparative Examples 5 - 10 are shown in Table 3:

[0090] Table 3 Measurement Results

[0091] Total reducing sugar (mg / ml) Total polysaccharide (mg / ml) Total amino acid (mg / ml) Total protein (mg / ml) Comparative Example 5 2.42±0.24b 0.38±0.01ab 0.48±0.02ab 2.17±0.06a Comparative Example 6 2.73±0.35b 0.37±0.03b 0.33±0.02a 2.30±0.10a Comparative Example 7 2.73±0.50b 0.59±0.04adef 0.30±0.01a 2.13±0.12a Comparative Example 8 2.60±0.09b 0.66±0.04def 0.39±0.12ab 2.64±0.53ab Comparative Example 9 2.69±0.06b 0.79±0.23cd 0.39±0.13ab 2.69±0.56ab Comparative Example 10 2.56±0.13b 0.71±0.01ce 0.30±0.01a 2.32±0.06a

[0092] The results of the comparative examples show that improper combination of enzyme species will affect the extraction efficiency. Therefore, the correct selection of enzyme species combination has a direct impact on the content of active ingredients in the Lentinula edodes extract.

[0093] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting an extract of Lentinula edodes, characterized in that, It includes the following steps: Mix cell wall hydrolase, proteolytic enzyme and Lentinus edodes for enzymatic hydrolysis to obtain Lentinus edodes extract; The cell wall hydrolase includes cellulase; the proteolytic enzyme includes flavourzyme.

2. The extraction method according to claim 1, wherein The cellulase includes the cellulase with the model of Cellulase T"Amano"4 from Amano Enzyme Company; the flavourzyme includes the flavourzyme with the model of Flavourzyme 1000L from Novozymes Company.

3. The extraction method according to claim 1, wherein The Lentinus edodes includes Lentinus edodes powder.

4. The extraction method according to claim 1, wherein The Lentinus edodes is mixed with cell wall hydrolase and proteolytic enzyme in the form of a Lentinus edodes - water mixture; the Lentinus edodes - water mixture is prepared by mixing Lentinus edodes and water; the mass ratio of Lentinus edodes to water for mixing is 1:

30.

5. The extraction method according to claim 1, wherein The temperature of the enzymatic hydrolysis is 50°C.

6. The extraction method according to claim 1, wherein The time of the enzymatic hydrolysis is 6 - 9 h.

7. The extraction method according to claim 1, wherein After the enzymatic hydrolysis, it also includes terminating the enzymatic hydrolysis and solid - liquid separation.

8. The extraction method according to claim 7, characterized in that The conditions for terminating the enzymatic hydrolysis are 80°C and 10 min.

9. The extraction method according to claim 7, wherein The method for solid - liquid separation includes centrifuging to obtain the supernatant; the conditions for centrifuging include 5000 rpm, 20°C and 10 min.

10. Use of the extraction method according to any one of claims 1 - 9 in increasing the content of any one or more of total reducing sugar, total polysaccharide, total amino acid and total protein in Lentinus edodes extract.