Method for extracting effective components of edible mushrooms and determination method thereof

Abstract

According to the method for extracting the effective components of the edible mushrooms and the determination method thereof, the edible mushrooms are freeze-dried, cell walls of the edible mushrooms are damaged in the freeze-drying process, and the effective components are extracted more easily, so that the content of the effective components is increased, and the effective components in the edible mushrooms can be well protected; the freeze-dried product is more convenient to transport and store; the edible mushrooms are crushed and subpackaged in the containing bags, the contact area of the edible mushrooms and water is increased, sufficient extraction is facilitated, the nutritional value and biological activity of effective components are protected through the water extraction mode, and then the concentration of the effective components is further improved through concentration of membrane separation equipment.

Description

Technical Field

[0001] The invention relates to the technical field of edible fungus processing, and in particular to a method for extracting effective components of edible fungi and a determination method thereof. Background Art

[0002] Edible fungi are rich in a variety of biologically active ingredients, such as polysaccharides, terpenes, nucleosides, proteins and amino acids, and have extremely high application value in the fields of medicine, health products, cosmetics, etc. Edible fungi extracts contain a mixture of melanin precursors and a variety of amino acids necessary for hair synthesis and other active ingredients. Edible fungi extracts can be used as raw materials to prepare black hair products. With the continuous deepening of research on edible fungi, it is particularly important to develop extraction methods that retain active ingredients to the greatest extent.

[0003] Traditional extraction processes for active ingredients from edible fungi mainly include solvent extraction, hot water extraction, etc. Although the organic solvents commonly used in solvent extraction can dissolve the target active ingredients to a certain extent, they have many defects. For example, organic solvents can easily destroy the structure and activity of certain environmentally sensitive active ingredients in edible fungi, resulting in a decrease in the content and purity of the extracted active ingredients; moreover, most organic solvents are volatile and toxic, which will not only harm the health of operators during the extraction process, but also pollute the environment.

[0004] Hot water extraction generally uses water at high temperature to extract polysaccharides and other ingredients from edible fungi. However, this method takes a long time to extract, usually several hours or even longer, which not only consumes a lot of energy, but also long-term high-temperature treatment easily causes some effective ingredients to undergo chemical reactions such as oxidation and hydrolysis, reducing their quality. At the same time, the extract obtained by hot water extraction has a relatively high impurity content, and the subsequent purification steps are relatively cumbersome, increasing production costs.

[0005] In addition, there are some emerging extraction technologies such as supercritical fluid extraction. Although they have the advantages of high extraction efficiency and good selectivity, the equipment is expensive, the operation is complicated, and it requires high technical personnel. It is not suitable for the general extraction and production of active ingredients of edible fungi.

[0006] In summary, the existing extraction processes for active ingredients of edible fungi have certain limitations and are difficult to meet the current demand for high-content and high-purity extraction. Summary of the invention

[0007] The main purpose of the present invention is to provide a method for extracting effective components of edible fungi and a method for determining the same, aiming to solve the technical problem that the existing edible fungi effective components are extracted in small amounts and with low purity.

[0008] In order to achieve the above object, the present invention provides a method for extracting effective components of edible fungi in a first aspect, comprising the following steps:

[0009] S1: crushing and freeze-drying the edible fungi;

[0010] S2: Packing the freeze-dried edible fungi into holding bags, adding the holding bags into an extraction tank for water extraction for 30 minutes to 1 hour;

[0011] S3: The extract obtained in S2 is concentrated by a membrane separation device to obtain.

[0012] Furthermore, the step S1 includes:

[0013] S1.1: crushing and sieving the edible fungi;

[0014] S1.2: Place the sieved edible fungi into a freeze-drying chamber, freeze for 4 to 6 hours, and sublimate and dry for 7 to 8 hours.

[0015] Furthermore, the crushing method includes slicing and crushing, and the edible fungi are in the form of flakes, blocks or powder after being crushed.

[0016] Furthermore, the containing bag is a 60-count cotton bag.

[0017] Furthermore, the membrane separation equipment is a RO reverse osmosis membrane.

[0018] Furthermore, the edible fungi include one or more of Volvariella volvacea, Flammulina velutipes, Pleurotus ostreatus, Agaricus bisporus and Hericium erinaceus.

[0019] Another aspect of the present invention provides a method for determining the effective components of edible fungi, which is used to determine the effective components of edible fungi. The steps of the determination method include:

[0020] Take the concentrated solution obtained in S3 and add 5 times the volume fraction of 95% alcohol, shake well and centrifuge, take the supernatant and put it into liquid chromatography for detection;

[0021] The detection parameters of the liquid chromatograph are:

[0022] (1) The chromatographic column is a C18 column;

[0023] (2) The detection wavelength is 300nm;

[0024] (3) The flow rate of the mobile phase is 1 mL / min;

[0025] (4) Column temperature is 35°C ± 1°C;

[0026] (5) Injection volume: 5 μL to 7.5 μL;

[0027] (6) Mobile phase A was a methanol solution containing 0.5% by mass formic acid, and mobile phase B was a 50% by mass acetonitrile aqueous solution. A gradient elution procedure was used.

[0028] Further, the gradient elution procedure is:

[0029] 0-2min, volume fraction 30%-50% mobile phase A, 50%-70 mobile phase B;

[0030] 2-4 min, volume fraction 50%-80% mobile phase A, 20%-50 mobile phase B;

[0031] 4 to 6 min, mobile phase A by volume: 100%.

[0032] Beneficial effects:

[0033] The invention discloses a method for extracting effective components of edible fungi and a method for determining the same. The edible fungi are freeze-dried, and the cell walls of the edible fungi are destroyed during the freeze-drying process, so that the effective components are more easily extracted, thereby increasing the content of the effective components, and the effective components therein can be well protected, and the freeze-dried edible fungi are more convenient to transport and store. The edible fungi are crushed and divided into a containing bag, so that the contact area between the edible fungi and water is increased, which is conducive to full extraction. The water extraction method also protects the nutritional value and biological activity of the effective components, and then a membrane separation device is used for concentration to further increase the concentration of the effective components. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The liquid chromatogram of the effective ingredient extracted from Example 1 of the present invention;

[0035] Figure 2 This is the standard spectrum of melanin precursor 1 of the present invention;

[0036] Figure 3 This is the standard spectrum of melanin precursor 2 of the present invention.

[0037] Figure 4 The liquid chromatogram of the effective ingredient extracted from Example 2 of the present invention;

[0038] Figure 5 The liquid chromatogram of the effective ingredient extracted from Example 3 of the present invention;

[0039] Figure 6 The liquid chromatogram of the effective ingredient extracted from Example 4 of the present invention;

[0040] Figure 7 This is a liquid chromatogram of the effective ingredients extracted from Comparative Example 5 of the present invention;

[0041] Figure 8This is a liquid chromatogram of the effective ingredients extracted in Comparative Example 6 of the present invention.

[0042] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0043] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0044] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0045] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0046] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0047] The present invention is further described in the following examples, but the examples are not intended to limit the scope of protection of the present invention.

[0048] An embodiment of the present invention provides a method for extracting effective components of edible fungi, comprising the following steps:

[0049] S1: crushing and freeze-drying the edible fungi;

[0050] S2: Packing the freeze-dried edible fungi into holding bags, adding the holding bags into an extraction tank for water extraction for 30 minutes to 1 hour;

[0051] S3: The extract obtained in S2 is concentrated by a membrane separation device to obtain.

[0052] In the above embodiment, by freeze-drying the edible fungi, the cell walls of the edible fungi are destroyed during the freeze-drying process, making it easier to extract the effective ingredients, thereby increasing the content of the effective ingredients, and the effective ingredients therein can be well protected, making transportation and storage more convenient; the edible fungi are crushed and packaged in a container bag, which increases their contact area with water and is conducive to full extraction. The water extraction method also protects the nutritional value and biological activity of the effective ingredients, which are then concentrated using a membrane separation device to further increase the concentration of the effective ingredients.

[0053] In the above embodiment, the material of the container bag does not react chemically with water or edible fungi, and the pores of the container bag are small enough to ensure that water and effective ingredients can freely enter and exit, so as to prevent fragments or particles of edible fungi from mixing into the extract, thereby avoiding increasing the difficulty of subsequent processing.

[0054] In one embodiment, the step S1 includes:

[0055] S1.1: crushing the edible fungi and sieving;

[0056] S1.2: Place the sieved edible fungi into a freeze-drying chamber, freeze for 4 to 6 hours, and sublimate and dry for 7 to 8 hours.

[0057] In the above embodiment, the edible fungi are broken, and the breaking methods include slicing or crushing, etc., which can break down the whole edible fungi into flakes, blocks or powdered edible fungi. The broken edible fungi can be fully in contact with water so that effective substances can be fully extracted. The sieved edible fungi after breaking are freeze-dried, so that the problem of being difficult to filter and remove due to leakage from the containing bag during subsequent water extraction due to being too broken will not occur.

[0058] In the above embodiment, the crushed edible fungi are placed in a freeze-drying device, and the freeze-drying conditions are as follows: the average freezing rate should be 1°C / min, the freezing time is 4 to 6 hours, the freezing end temperature is -30°C, and then sublimation drying is performed. The temperature of the edible fungi rises from (-25 to -20)°C to 45°C, the sublimation drying time is (7 to 8) hours, and the pressure is 10Pa. When the temperature of the edible fungi tends to be consistent with the environment inside the equipment, the drying process can be ended, and the total duration is (45 to 55) hours.

[0059] In one embodiment, the containing bag is a 60-count cotton bag. During the water extraction process, water molecules can penetrate through the pores of the cotton cloth into the cotton cloth bag, contact with the effective ingredients in the edible fungus cells and dissolve them, and the dissolved effective ingredients can also diffuse into the external extract through the pores of the cotton cloth. In addition, the cotton cloth bag can be reused in industrial-scale edible fungus extraction production, reducing the consumption of packaging materials, reducing costs, and improving resource utilization.

[0060] The inventors found that using a low-count cotton fiber with a coarser count, such as a 40-count cotton cloth bag, would result in excessive impurities in the extract and reduced purity; using a high-count cotton fiber with a finer count, such as a 100-count cotton cloth bag, would result in reduced extraction efficiency and content, and using a 60-count cotton cloth bag would have the best extraction effect.

[0061] In one embodiment, the membrane separation device is a RO reverse osmosis membrane. The RO reverse osmosis membrane can separate water and solutes under pressure, allowing only water molecules to pass through, thereby concentrating the effective components.

[0062] In one embodiment, the edible fungi include one or more of Volvariella volvacea, Flammulina velutipes, Oyster mushroom, Agaricus bisporus and Hericium erinaceus. It can be reasonably inferred that other edible fungi can also achieve the same effect using this method.

[0063] Another aspect of the present invention provides a method for determining the effective components of edible fungi, which is used to determine the effective components of edible fungi. The steps of the determination method include:

[0064] Take the concentrated solution obtained in S3 and add 5 times the volume fraction of 95% alcohol, shake well and centrifuge, take the supernatant and put it into liquid chromatography for detection;

[0065] The detection parameters of the liquid chromatograph are:

[0066] (1) The chromatographic column is a C18 column;

[0067] (2) The detection wavelength is 300nm;

[0068] (3) The flow rate of the mobile phase is 1 mL / min;

[0069] (4) Column temperature is 35°C ± 1°C;

[0070] (5) Injection volume: 5 μL to 7.5 μL;

[0071] (6) Mobile phase A was a methanol solution containing 0.5% by mass formic acid, and mobile phase B was a 50% by mass acetonitrile aqueous solution. A gradient elution procedure was used.

[0072] In the above embodiment, the gradient elution procedure is:

[0073] 0-2min, volume fraction 30%-50% mobile phase A, 50%-70% mobile phase B;

[0074] 2-4 min, volume fraction 50%-80% mobile phase A, 20%-50% mobile phase B;

[0075] 4 to 6 min, mobile phase A by volume: 100%.

[0076] The liquid phase detection equipment used in the present invention is a liquid chromatograph LC-100 produced by Shanghai Wufeng Scientific Instrument Co., Ltd. The inventors investigated and analyzed the chromatographic conditions. Under the above chromatographic conditions, the separation of the chromatographic peaks of the effective ingredients is the best and the baseline is stable. The inventors used liquid chromatography to detect the content of the effective ingredients finally concentrated, thereby optimizing the various conditions in the freeze-drying and extraction processes to achieve the best extraction effect.

[0077] The different types of fresh edible fungi used in Examples 1 to 4 are first crushed, placed in a freeze-drying device for freeze-drying, and packaged in cotton bags of different specifications. The cotton bags are added to an extraction tank for water extraction for 30 minutes to 1 hour. The obtained extract is then concentrated by a RO reverse osmosis membrane at an operating pressure of about 10 to 20 MPa to obtain a concentrated solution of the active ingredient.

[0078] Specific embodiments and comparative examples are as follows:

[0079] Example 1

[0080] Slice the straw mushrooms, sieve them, put them into a freeze-drying device for freeze-drying, and pack them into 60 cotton bags. Add the cotton bags into an extraction tank and extract them with water for 45 minutes. The obtained extract is then concentrated by a RO reverse osmosis membrane to obtain a concentrated solution of active ingredients.

[0081] Example 2

[0082] The only difference from Example 1 is that the edible fungus species is changed to Flammulina velutipes, the cotton cloth bag specification is 40 pieces, and the extraction time is 30 minutes.

[0083] Example 3

[0084] The only difference from Example 1 is that the edible fungus species is changed to Pleurotus ostreatus, the cotton cloth bag specification is 100 pieces, and the extraction time is 40 minutes.

[0085] Example 4

[0086] The only difference from Example 1 is that the edible fungus species is changed to Hericium erinaceus, the cotton cloth bag specification is 40 pieces, and the extraction time is 1 hour.

[0087] Comparative Example 1

[0088] The only difference from Example 1 is that freeze-drying is not adopted.

[0089] Comparative Example 2

[0090] The only difference from Example 2 is that freeze-drying is not adopted.

[0091] Comparative Example 3

[0092] The only difference from Example 3 is that freeze-drying is not adopted.

[0093] Comparative Example 4

[0094] The only difference from Example 4 is that freeze-drying is not adopted.

[0095] Comparative Example 5

[0096] The only difference from Example 1 is that freeze drying is replaced by drying, the drying temperature is 65° C., and the drying time is 10 h.

[0097] Comparative Example 6

[0098] The only difference from Example 1 is that the extract is directly extracted with water without being crushed or freeze-dried.

[0099] Preparation of sample solution: Add 5 times the volume fraction of 95% alcohol to the concentrated solution obtained in Examples 1 to 4, shake well and centrifuge, take the supernatant to be tested, draw the supernatant with a 1 ml syringe, filter it with a 0.45 μm filter into a 1.5 ml centrifuge tube, and obtain the sample solution to be tested.

[0100] Preparation of reference solution: Add 5 times the volume fraction of 95% alcohol to the concentrated solution obtained in Comparative Examples 1 to 6, shake well and centrifuge, take the supernatant to be tested, draw the supernatant with a 1 ml syringe, filter it with a 0.45 μm filter into a 1.5 ml centrifuge tube, and obtain the reference solution to be tested.

[0101] Preparation of standard solution: 5,6-dihydroxyindole (DHI) standard and 5,6-dihydroxyindole carboxylic acid (DHICA) standard were respectively added into 95% by volume alcohol to dissolve and prepare standard solutions.

[0102] Pipette appropriate amounts of standard solution, sample solution and reference solution into liquid chromatography bottles, place the bottles in the edited sequence and perform injection testing according to the sequence. The corresponding HPLC chromatogram is as follows: Figures 1 to 8 shown.

[0103] The content of melanin precursor can be estimated based on the peak area detected in the liquid chromatogram, and the purity of melanin precursor can be estimated based on the detected concentration.

[0104] Calculation of melanin precursor content: According to the time when the characteristic peak of the standard appears, find the peak area A around 3.5 minutes 3.5min Calculate the content of melanin precursor substance 1, the specific formula is melanin precursor substance 1 content = A 3.5min / 1800(g / L), where 1800g / L is the amount of melanin precursor substance 1 (DHICA) corresponding to the unit peak area; similarly, the peak area A4.1min around 4.1min is found to calculate the content of melanin precursor substance 2 (DHI), and the specific formula is melanin precursor substance 2 content = A 4.5min / 2000 (g / L), wherein 2000 g / L is the amount of melanin precursor substance 2 corresponding to unit peak area.

[0105] Melanin precursor substance content = melanin precursor substance 1 content + melanin precursor substance 2 content;

[0106] The purity of the melanin precursor substance = the concentration of the melanin precursor substance 1 + the concentration of the melanin precursor substance 2.

[0107] According to the above formula, the content of melanin precursor in the extracted mushroom of Example 1 is: (1699 / 1800+7865 / 2000) g / L≈4.8 g / L; the purity is: (14.67+67.90)%=82.5%. The calculation methods of other examples and comparative examples are similar.

[0108] The specific extraction methods and extraction results of Examples 1 to 4 and Comparative Examples 1 to 6 are referred to Tables 1 to 2 below:

[0109] Table 1:

[0110] Example Project Example 1 Example 2 Example 3 Example 4 Types of Edible Fungi straw mushroom Flammulina velutipes Oyster Mushroom Hericium erinaceus Extraction time 45min 30min 40min 1h Cotton bag specifications 60 pieces 40 sticks 100 pieces 40 sticks Active ingredient content 4.8g / L 2.9g / L 4.7g / L 3.8g / L Purity of active ingredients 82.5％ 75.8％ 80.7％ 73.7％

[0111] Table 2:

[0112]

[0113] Referring to Examples 1 to 3 and Comparative Examples 1 to 6 in Tables 1 to 2, it can be seen that, compared with the traditional extraction method, the method of first crushing the edible fungi and then freeze-drying them before extraction can increase the content of the melanin precursor mixture in the extract by 10 to 15%, and the purity can also be increased by 5 to 10%, indicating that freeze-drying the edible fungi can well protect the active ingredients therein, and because the cell wall of the edible fungi is destroyed during the freeze-drying process, it is easier to extract the active ingredients.

[0114] In Comparative Example 5, freeze-drying was replaced by drying. Although the content of the active ingredient was improved, the purity of the active ingredient decreased significantly. This may be because the drying process usually involves a higher temperature, which causes some active ingredients to decompose and produce impurities, affecting their purity.

[0115] In Comparative Example 6, the edible fungi were not crushed or freeze-dried, which greatly reduced the efficiency of water extraction and made it difficult for the active ingredients to be completely dissolved in the extract, thereby reducing the content of the active ingredients.

[0116] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for extracting effective ingredients from edible fungi, characterized in that: The following steps are involved: S1: crushing and freeze-drying the edible fungi; S2: Packing the freeze-dried edible fungi into holding bags, adding the holding bags into an extraction tank for water extraction for 30 minutes to 1 hour; S3: The extract obtained in S2 is concentrated by a membrane separation device to obtain.

2. The method for extracting effective ingredients from edible fungi according to claim 1, characterized in that: The steps of S1 include: S1.1: crushing and sieving the edible fungi; S1.2: Place the sieved edible fungi into a freeze-drying chamber, freeze for 4 to 6 hours, and sublimate and dry for 7 to 8 hours.

3. The method for extracting effective ingredients from edible fungi according to claim 1 or 2, characterized in that: The crushing method includes slicing and crushing, and the edible fungi are in the form of flakes, blocks or powder after being crushed.

4. The method for extracting effective ingredients from edible fungi according to claim 1, characterized in that: The containing bag is a 60-count cotton bag.

5. The method for extracting effective ingredients from edible fungi according to claim 1, characterized in that: The membrane separation equipment is a RO reverse osmosis membrane.

6. The method for extracting effective ingredients from edible fungi according to claim 1, characterized in that: The edible fungi include one or more of straw mushroom, enoki mushroom, oyster mushroom, button mushroom and hericium erinaceus.

7. A method for determining the effective ingredients of edible fungi, characterized in that: For determining the effective ingredients of edible fungi according to any one of claims 1 to 6, the determination method comprises the following steps: Take the concentrated solution obtained in S3 and add 5 times the volume fraction of 95% alcohol, shake well and centrifuge, take the supernatant and put it into liquid chromatography for detection; The detection parameters of the liquid chromatograph are: (1) The chromatographic column is a C18 column; (2) The detection wavelength is 300nm; (3) The flow rate of the mobile phase is 1 mL / min; (4) Column temperature is 35°C ± 1°C; (5) Injection volume: 5 μL to 7.5 μL; (6) Mobile phase A was a methanol solution containing 0.5% by mass formic acid, and mobile phase B was a 50% by mass acetonitrile aqueous solution. A gradient elution procedure was used.

8. The method for determining the effective components of edible fungi according to claim 7, characterized in that: The gradient elution procedure is: 0-2min, volume fraction 30%-50% mobile phase A, 50%-70% mobile phase B; 2-4 min, volume fraction 50%-80% mobile phase A, 20%-50% mobile phase B; 4 to 6 min, mobile phase A by volume: 100%.

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