Method for co-extracting protein and polysaccharide of cordyceps militaris

Abstract

The invention relates to a method for co-extracting protein and polysaccharide of cordyceps militaris. Through a process of combining high-pressure homogenization, helicase treatment and alkaline extraction, high-efficiency co-extraction of the protein and polysaccharide of the cordyceps militaris is realized. The method comprises the following specific steps: crushing cell walls of cordyceps militaris by using a high-pressure homogenization technology, then adding helicase to degrade polysaccharide in the cell walls, and finally promoting dissolution of protein by using an alkaline solution. The technology not only improves the extraction rates of protein and polysaccharide (respectively reaching 70% and 0.5 g / g dry powder), but also optimizes the resource utilization, reduces the production cost and reduces the environmental pollution. The method has a remarkable application prospect in the fields of health care products and functional foods.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a method for co-extracting Cordyceps militaris protein and polysaccharides. Background Technology

[0002] Cordyceps militaris, a fungus with significant medicinal value, has attracted widespread attention due to its rich array of active ingredients. It contains various bioactive substances beneficial to the human body, such as proteins, polysaccharides, nucleoside compounds, cordycepin, vitamins, and minerals. Among these, cordyceps proteins and polysaccharides are its two most important functional components, exhibiting significant antioxidant, anti-fatigue, immunomodulatory, and anti-tumor effects, and are widely used in health foods, pharmaceuticals, and cosmetics. Therefore, how to efficiently and economically extract proteins and polysaccharides from Cordyceps militaris has become a hot topic and a challenge in related research fields.

[0003] Cordyceps militaris protein is a crucial component of its functional activity, exhibiting significant antioxidant and immunomodulatory functions. Recent studies on Cordyceps militaris protein have revealed that its molecular structure and functional properties have numerous positive impacts on human health. Meanwhile, Cordyceps militaris polysaccharides, another important class of bioactive components, possess various biological activities, including enhancing immunity, anti-tumor activity, and lowering blood sugar.

[0004] Traditional Cordyceps militaris extraction techniques typically target a single component, lacking an effective method for simultaneously extracting both proteins and polysaccharides. This limitation of separate extraction not only leads to complex and costly processes but also risks the loss or destruction of some components. Therefore, researching and developing a technique capable of simultaneously extracting Cordyceps militaris proteins and polysaccharides has significant practical application value. Co-extraction simplifies the production process, reduces costs, improves resource utilization, and maximizes the preservation of the integrity and functionality of active substances in Cordyceps militaris. The co-extraction technology for Cordyceps militaris proteins and polysaccharides represents an optimization and innovation of traditional single-component extraction methods. In the future, with continuous advancements in extraction technology and the refinement of extraction processes, this co-extraction technology will demonstrate broad application prospects in the development of health products, pharmaceuticals, and functional foods. Through this technology, the medicinal value of Cordyceps militaris can be better utilized, and the sustainable development of related industries can be promoted.

[0005] Patent CN102690321A reports a protein extraction process from Cordyceps militaris that achieves a protein extraction rate of 31.2%.

[0006] Patent CN104292348A discloses a method for simultaneously extracting polysaccharides and proteins from processing by-products of king oyster mushrooms, with a polysaccharide yield of 10.69% and a protein yield of 11.23%. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to address the limitations of existing single-component extraction technology of Cordyceps militaris and to provide a method that combines high-pressure homogenization, snail enzyme extraction and alkaline extraction, which can simultaneously and efficiently extract Cordyceps militaris protein and polysaccharides.

[0008] To solve the above-mentioned technical problems, the present invention has the following approach:

[0009] Pretreatment of defatted Cordyceps militaris fruiting body freeze-dried powder using high-pressure homogenization effectively breaks down cell walls and dissolves some intracellular proteins, laying the foundation for subsequent extraction. To further efficiently release polysaccharides from the cell walls and promote the extraction of intracellular proteins, this invention employs snailase for enzymatic hydrolysis. This enzyme effectively degrades polysaccharides in the Cordyceps militaris cell walls, especially chitosan and other polysaccharides with complex structures, allowing them to fully dissolve in solution. Furthermore, the mild degradation characteristics of snailase help maintain the bioactivity of polysaccharides under lower temperatures and milder conditions, avoiding damage to active ingredients from high temperatures or chemical reagents. After the enzymatic hydrolysis reaction is complete, alkaline conditions are used to further increase protein solubility and promote efficient protein extraction.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0011] A method for co-extracting Cordyceps militaris protein and polysaccharides includes the following steps:

[0012] S1, High-pressure homogenization stage: Cordyceps militaris powder is mixed with water and its cell walls are broken using high-pressure homogenization technology to obtain a lysate.

[0013] S2, Snail enzyme treatment stage: Snail enzyme is added to the lysate, and the lysate is obtained by enzymatic hydrolysis.

[0014] S3, Alkaline Extraction Stage: Add NaOH solution to the enzymatic hydrolysate to adjust the pH to alkaline, let stand, centrifuge, and obtain Cordyceps militaris protein and polysaccharide.

[0015] In step S1, the Cordyceps militaris powder undergoes pretreatment, which involves freeze-drying and pulverizing the Cordyceps militaris, passing it through an 80-mesh sieve, decolorizing and degreasing it with ethanol, and then freeze-drying it to obtain Cordyceps militaris powder.

[0016] In step S1, the mass-to-volume ratio of the freeze-dried Cordyceps militaris to ethanol is 1 / 15 to 1 / 30 g / ml.

[0017] In step S1, the high-pressure homogenization technology uses a pressure of 500–1500 bar (preferably 900–1100 bar).

[0018] In step S1, the solid-liquid ratio of the Cordyceps militaris powder and water is 1 / 30 to 1 / 200 g / ml (preferably 1 / 75 to 1 / 100 g / ml).

[0019] In step S1, the high-pressure homogenization process is performed 1 to 5 times (preferably 3 to 5 times).

[0020] In step S2, the mass ratio of the snail enzyme to Cordyceps militaris powder is 50–250 mg / g (preferably 100–150 mg / g).

[0021] In step S2, the enzymatic hydrolysis is performed at a temperature of 25–75°C (preferably 45–55°C) and for a time of 30–180 min (preferably 90–120 min).

[0022] In step S3, the alkalinity is a pH value of 8 to 12 (preferably 10 to 12).

[0023] In step S3, the standing condition is room temperature and the time is 60 to 150 minutes.

[0024] Compared with the prior art, the present invention has the following significant advantages:

[0025] 1. Highly efficient resource utilization: Single extraction of edible fungi often leads to resource waste due to neglecting other components. Co-extraction methods can simultaneously recover two components in the same process, improving the utilization rate of raw materials, making the most of resources, and minimizing the generation of residue and waste.

[0026] 2. Reduced Costs: Single extraction typically requires designing independent processes for different components, increasing equipment investment and operating costs. Co-extraction integrates multiple steps, thereby simplifying the production process, reducing energy consumption and solvent usage, and significantly lowering overall costs.

[0027] 3. Reduced environmental impact: Single extraction typically requires multiple separation and purification steps, potentially generating more waste liquid and residue. Co-extraction technology reduces waste generation by minimizing process steps, contributing to green production.

[0028] 4. This invention uses a combined method of high-pressure homogenization, snail enzyme extraction, and alkaline extraction to simultaneously extract Cordyceps militaris protein and polysaccharides, and theoretically can achieve a protein extraction rate of 70% and a polysaccharide extraction rate of 0.5 g / g dry powder. Attached Figure Description

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0030] Figure 1Schematic diagram of the process for co-extraction of Cordyceps militaris protein and polysaccharides Detailed Implementation

[0031] Methods for measuring protein and polysaccharide content: Protein content was determined using the Coomassie protein assay, and total polysaccharide content was determined using the sulfuric acid-phenol method.

[0032]

[0033] Preparation of Cordyceps militaris powder: Fresh Cordyceps militaris fruiting bodies were freeze-dried and pulverized using a pulverizer to obtain 80-mesh powder. The powder was then decolorized and degreased by stirring with 80% ethanol (solid-liquid ratio 1:20 g / ml), and then freeze-dried for 12 hours before use.

[0034] The snail enzyme used in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number: S10083. In an isotonic sorbitol solution with pH 5.8-7.2, 30-40 mg of the enzyme per gram of cells was used to hydrate the cells at 37°C for 1 hour, which dissolved the cell walls and achieved a cell wall breakage rate of over 80-90%.

[0035] Example 1:

[0036] A method for co-extracting Cordyceps militaris protein and polysaccharides, comprising the following steps:

[0037] S1: High-pressure homogenization stage

[0038] After adding a certain amount of water to Cordyceps militaris powder, the cell walls are broken by high-pressure homogenization technology. The process is repeated 1 to 5 times to obtain the broken liquid.

[0039] S2: Snail enzyme treatment stage

[0040] Snail enzyme was added to the lysate obtained in step S1 for enzymatic hydrolysis to further extract proteins and polysaccharides.

[0041] S3: Alkali Extraction Stage

[0042] After the enzymatic hydrolysis in step S2 is completed, the reaction solution is adjusted to alkalinity by adding NaOH solution, and then allowed to stand at room temperature for a period of time before centrifugation to obtain Cordyceps militaris protein and polysaccharide.

[0043] The performance of high-pressure homogenization-enzyme-alkali combined treatment in improving the extraction rates of Cordyceps militaris protein and polysaccharides was compared with that of alkaline extraction, high-pressure homogenization-alkaline extraction, and snail enzyme-alkali extraction. Specific experimental parameters and results are shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047] Discussion: The experimental results show that the optimal extraction rates of protein and polysaccharides from Cordyceps militaris were 69.27% ​​and 0.5 g / g, respectively, achieved by high-pressure homogenization-enzyme-alkali combined treatment. It can also be seen that snail enzyme and high-pressure homogenization significantly promoted the extraction rates of protein and polysaccharides, respectively.

[0048] Example 2: Investigating the co-extraction performance of Cordyceps militaris protein and polysaccharide under different solid-liquid ratios in the high-pressure homogenization-enzyme-alkali combined method.

[0049] Experimental conditions: 1g of Cordyceps militaris powder was added to water, with solid-liquid ratios of 1 / 50, 1 / 75, 1 / 100, 1 / 125, 1 / 150, and 1 / 175g / ml, respectively.

[0050] Other conditions: High pressure homogenization pressure, enzyme addition amount, temperature, time, and pH of alkali extraction were 900 bar, 100 mg / g, 45℃, 90 min, and pH=10, respectively.

[0051] Experimental results:

[0052] Table 2

[0053] Solid-liquid ratio (g / ml) 1 / 50 1 / 75 1 / 100 1 / 125 1 / 150 1 / 175 Protein extraction rate (%) 56.58 70.00 69.61 63.75 57.12 60.00 Polysaccharide extraction yield (g / g) 0.40 0.49 0.50 0.47 0.47 0.43

[0054] Discussion: The preferred solid-liquid ratio range is 1 / 75 to 1 / 100 g / ml, and the extraction rates of protein and polysaccharides can be greater than 69% and greater than 0.49 g / g dry powder, respectively.

[0055] Example 3: Investigating the co-extraction performance of Cordyceps militaris protein and polysaccharide under different pressures in the high-pressure homogenization-enzyme-alkali combined method.

[0056] Experimental conditions: 1g of Cordyceps militaris powder, high pressure homogenization pressures of 500, 700, 900, 1100, 1300, and 1500 bar respectively;

[0057] Other conditions: solid-liquid ratio, enzyme addition amount, temperature, time, and pH of alkali extraction were 1 / 100g / ml, 100mg / g, 45℃, 90min, and pH=10, respectively.

[0058] Experimental results:

[0059] Table 3

[0060]

[0061] Discussion: The preferred pressure range is 900–1100 bar, where the extraction rates of protein and polysaccharides can exceed 68% and 0.49 g / g dry powder, respectively. Higher pressures have little impact on the extraction efficiency and result in higher energy costs.

[0062] Example 4: Investigating the co-extraction performance of Cordyceps militaris protein and polysaccharide under different enzyme addition amounts in the high-pressure homogenization-enzyme-alkali combined method.

[0063] Experimental conditions: 1g of Cordyceps militaris powder was added at concentrations of 0, 50, 100, 150, 200, and 250 mg / g relative to snail enzyme.

[0064] Other conditions: solid-liquid ratio, high-pressure homogenization pressure, temperature, time, and pH of alkali extraction were 1 / 100 g / ml, 900 bar, 45℃, 90 min, and pH=10, respectively.

[0065] Experimental results:

[0066] Table 4

[0067]

[0068] Discussion: The optimal snail enzyme dosage range is 100–150 mg / g dry powder, resulting in protein and polysaccharide extraction rates greater than 68% and greater than 0.49 g / g dry powder, respectively. Higher snail enzyme dosages lead to a decrease in protein extraction rate and higher reagent costs.

[0069] Example 5: Investigating the co-extraction performance of Cordyceps militaris protein and polysaccharide under different enzyme reaction times in the high-pressure homogenization-enzyme-alkali combined method.

[0070] Experimental conditions: 1g of Cordyceps militaris powder, reaction times of 30, 60, 90, 120, 150, and 180 min respectively;

[0071] Other conditions: solid-liquid ratio, high-pressure homogenization pressure, enzyme addition, temperature, and pH of alkaline extraction were 1 / 100g / ml, 900bar, 100mg / g, 45℃, and pH=10, respectively.

[0072] Experimental results:

[0073] Table 5

[0074]

[0075] Discussion: The optimal snail enzyme reaction time range is 90–120 min, achieving protein extraction rates greater than 67% and polysaccharide extraction rates greater than 0.49 g / g dry powder, respectively. Excessively long snail enzyme reaction times will decrease protein extraction rates and lead to higher energy costs.

[0076] Example 6: Investigating the co-extraction performance of Cordyceps militaris protein and polysaccharide at different enzyme reaction temperatures in the high-pressure homogenization-enzyme-alkali combined method.

[0077] Experimental conditions: 1g of Cordyceps militaris powder was used in experiments at temperatures of 25, 35, 45, 55, 65, and 75℃.

[0078] Other conditions: solid-liquid ratio, high-pressure homogenization pressure, enzyme addition amount, time, and pH of alkali extraction were 1 / 100g / ml, 900bar, 100mg / g, 90min, and pH=10, respectively.

[0079] Experimental results:

[0080] Table 6

[0081]

[0082] Discussion: The optimal snail enzyme reaction time range is 45–55℃, achieving protein extraction rates greater than 68% and polysaccharide extraction rates greater than 0.42 g / g dry powder, respectively. Excessively high snail enzyme reaction temperatures can cause a slight decrease in protein extraction rate and a sharp decrease in polysaccharide extraction rate, and also result in higher energy costs.

[0083] Example 7: Investigating the co-extraction performance of Cordyceps militaris protein and polysaccharide at different pH values ​​during the alkali treatment stage in the high-pressure homogenization-enzyme-alkali combined method.

[0084] Experimental conditions: 1g of Cordyceps militaris powder, pH of alkaline extraction at 8, 9, 10, 11, and 12;

[0085] Other conditions: solid-liquid ratio, high-pressure homogenization pressure, enzyme addition amount, temperature and time were 1 / 100g / ml, 900bar, 100mg / g, 45℃ and 90min, respectively.

[0086] Experimental results:

[0087] Table 7

[0088]

[0089] Discussion: The optimal pH range for the alkali treatment stage is 10–12, and the extraction rates of protein and polysaccharides can be greater than 69% and greater than 0.49 g / g dry powder, respectively.

[0090] Example 8: Investigating the performance of co-extraction of Cordyceps militaris protein and polysaccharide under optimal conditions in the high-pressure homogenization-enzyme-alkali combined method.

[0091] 1g of Cordyceps militaris powder, under optimal conditions: solid-liquid ratio (1 / 100g / ml), high-pressure homogenization pressure (900 bar), enzyme addition (100mg / g), temperature and time (45℃ and 90min), and alkaline extraction pH of 11, yielded protein and polysaccharide extraction rates of 70% and greater than 0.5g / g dry powder, respectively.

[0092] This invention provides a method for co-extracting Cordyceps militaris protein and polysaccharides. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for co-extracting Cordyceps militaris protein and polysaccharides, characterized in that, Includes the following steps: S1, High-pressure homogenization stage: Cordyceps militaris powder is mixed with water and its cell walls are broken using high-pressure homogenization technology to obtain a lysate. S2, Snail enzyme treatment stage: Snail enzyme is added to the lysate, and the lysate is obtained by enzymatic hydrolysis. S3, Alkaline Extraction Stage: Add NaOH solution to the enzymatic hydrolysate to adjust the pH to alkaline, let stand, centrifuge, and obtain Cordyceps militaris protein and polysaccharide.

2. The method according to claim 1, characterized in that, In step S1, the Cordyceps militaris powder undergoes pretreatment, which involves freeze-drying and pulverizing the Cordyceps militaris, passing it through an 80-mesh sieve, decolorizing and degreasing it with ethanol, and then freeze-drying it to obtain Cordyceps militaris powder.

3. The method according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of the freeze-dried Cordyceps militaris to ethanol is 1 / 15 to 1 / 30 g / ml.

4. The method according to claim 1, characterized in that, In step S1, the high-pressure homogenization technology uses a pressure of 500–1500 bar.

5. The method according to claim 1, characterized in that, In step S1, the solid-liquid ratio of the Cordyceps militaris powder and water is 1 / 30 to 1 / 200 g / ml.

6. The method according to claim 1, characterized in that, In step S1, the high-pressure homogenization technique is performed 1 to 5 times.

7. The method according to claim 1, characterized in that, In step S2, the mass ratio of the snail enzyme to Cordyceps militaris powder is 50–250 mg / g.

8. The method according to claim 1, characterized in that, In step S2, the enzymatic hydrolysis is performed at a temperature of 25–75°C for a time of 30–180 min.

9. The method according to claim 1, characterized in that, In step S3, the alkalinity is defined as a pH value of 8 to 12.

10. The method according to claim 1, characterized in that, In step S3, the standing condition is room temperature and the time is 60 to 150 minutes.

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