CN110106219B - A method for reusing the residue of Cordyceps militaris solid medium - Google Patents

Abstract

The invention discloses a method for recycling cordyceps militaris solid culture medium residues, and relates to the technical field of recycling cordyceps militaris solid culture medium residues. Pulverizing residue of Cordyceps militaris solid culture medium dried by hot air, sieving; firstly adding water to dissolve residue dry powder, adjusting the temperature to 50 ℃ and the pH value to 10, and sequentially carrying out ultrasonic pretreatment through a reaction cavity of 20/28kHz sequential double-frequency countercurrent ultrasonic equipment in a circulating mode; then adjusting the pH value to 6.5, and adding alpha-amylase for enzymolysis; finally, the pH of the enzymolysis liquid is adjusted back to 10, and 6000U/g (mass of residual powder) of enzyme is added with alkaline protease for enzymolysis, so that the enzymolysis product with the blood pressure reducing function is obtained. By using an ultrasonic auxiliary enzyme treatment technology and taking the residue of a solid culture medium remained after harvesting fruiting bodies as a raw material, the preparation of the blood pressure reducing functional factors with high added value can fully utilize cordyceps militaris resources and can also effectively solve the problem of waste generated in the cordyceps militaris cultivation industry.

Description

A method for reusing the residue of Cordyceps militaris solid medium

Technical field:

The invention relates to a technology for reusing residues of a solid culture medium of Cordyceps militaris, in particular to extracting blood pressure-lowering functional factors from residues of a solid culture medium of Cordyceps militaris by using an ultrasonic-assisted enzymatic hydrolysis method, which belongs to the field of biotechnology.

Background technique:

Cordyceps militaris, also known as Cordyceps militaris, is a fungus of the genus Cordyceps. It has no strict requirements on the host and living environment, and is currently the only Cordyceps that can be cultivated artificially. Due to its excellent medicinal and nourishing effects, Cordyceps militaris has been listed as a new resource food, and its application prospects are very broad. Although the production of artificially cultured Cordyceps militaris sporocarps has reached a considerable scale, when the fruiting bodies are harvested, a large amount of residues of the Cordyceps militaris culture medium are discarded, which not only pollutes the environment; the residues contain rich Cordyceps polysaccharides, starch Sugar and protein substances also cause a waste of resources. The method of reusing the residues of Cordyceps militaris medium has become a current research hotspot, which can not only solve environmental problems, but also improve the economic profit of enterprises.

Relevant studies have found that in order to better invade the host and effectively utilize the host's nutrients, Cordyceps secretes a variety of biological enzymes into the medium to enzymolyze some macromolecular nutrients in the medium. In the article "Effect of Ultrasonic Pretreatment on the ACE Inhibition Rate of Rice Protein Enzymatic Hydrolyzate", Yang Xue found that the enzymatic hydrolyzate of rice protein has good blood pressure lowering activity; the invention patent with Chinese patent number 201810189732. Separation and purification method of silkworm chrysalis protein polypeptides with inhibitory activity; Peng Lei et al. in the article "Research on the Preparation of Garlic Antihypertensive Functional Factors by Ultrasonic Pretreatment Enzyme Method", found that the antihypertensive factors (partial polysaccharides) of incomplete polypeptides also have antihypertensive effects. active. At present, most of the studies on the residues of Cordyceps militaris solid medium remain on the extraction of single components such as Cordyceps polysaccharides and cordycepin, but there is no research on the use of the metabolism of Cordyceps fungus combined with short-term enzymatic hydrolysis to develop the antihypertensive activity of medium residues. reports.

Ultrasonic-assisted enzymatic hydrolysis technology is widely used because it can overcome the shortcomings of traditional enzymatic hydrolysis technology such as low reaction efficiency, product activity and product yield, and it has the advantages of short action time, simple operation, easy control, and low temperature rise. In enzymatic hydrolysis strengthening research. Zhang Yanyan pointed out in the article "Research on Preparation of Gluten Protein ACE Inhibiting Peptide Based on Ultrasonic Pretreatment and Its Process In-Situ Monitoring Technology" that using an appropriate ultrasonic working mode to pretreat raw materials can improve enzymatic hydrolysis characteristics and increase the yield of enzymatic hydrolysis products. Hypotensive activity. However, most of the current research on ultrasonic technology is limited to ultrasonic washing tanks, ultrasonic cell disruptors and other equipment, and none of them can change the working mode of ultrasonic. In addition, there is no report on the preparation of solid medium residues of Cordyceps militaris by ultrasonic-assisted enzymatic hydrolysis.

According to the characteristics of Cordyceps militaris solid medium residues, ultrasonic-assisted enzyme treatment technology is used to prepare high value-added hypotensive functional factors using the remaining solid medium residues after harvesting fruiting bodies as raw materials, which can not only effectively solve the problem of Cordyceps militaris The problem of waste produced by the cultivation industry can also make full use of the resources of Cordyceps militaris.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the traditional extraction or enzymatic hydrolysis method in the above-mentioned prior art, make full use of the residual characteristics of the solid medium of Cordyceps militaris, and provide a novel ultrasonic-assisted method for the preparation of the residue of the solid medium of Cordyceps militaris for reducing blood pressure. method. The invention utilizes ultrasonic-assisted enzymatic hydrolysis technology to prepare high value-added blood pressure-lowering functional factors by using the remaining solid medium residues after harvesting the fruiting bodies as raw materials. waste from industry.

For the method of the present invention, the technical scheme that specifically adopts is as follows:

A kind of method that Cordyceps militaris solid culture medium residue reuses, according to the following steps:

Step 1, crushing of medium residues: crushing the solid medium residues of Cordyceps militaris dried by hot air, and sieving;

Step 2, pretreatment of medium residues: add water to the dry medium residues to obtain a suspension, adjust the temperature to 50°C and pH 10, and then pass through the reaction chamber of the ultrasonic equipment in a countercurrent cycle according to the set ultrasonic mode Ultrasonic treatment is carried out to obtain a pretreatment solution;

Step 3, amylase enzymatic hydrolysis of culture medium residues: adjust the pH of the pretreatment solution to 6.5, add enzyme amount of 50.1U/g (residue powder mass) and add amylase for enzymolysis to obtain amylase enzymatic hydrolysis solution ;

Step 4, protease enzymatic hydrolysis of medium residues: adjust the pH of the amylase hydrolyzate back to 10, add alkaline protease to 6000U/g (residue powder mass) for enzymolysis, and the enzymatic hydrolyzate to inactivate the enzyme Finally, after centrifugation at 4000 rad/min for 15 min, the enzymatic hydrolyzate with hypotensive function was obtained.

Further, in step 1, the solid medium residue is the remaining solid medium after harvesting the fruiting bodies of Cordyceps militaris, which is rich in hyphae and metabolites of Cordyceps fungus; the main raw materials of the solid medium are rice and silkworm chrysalis; The sieving treatment is to pass through a 60-mesh sieve, which can effectively screen out useless residues and impurities.

Further, in step 2, the concentration of the residue dry powder suspension is 10-50 g/L. .

Further, in step 2, the ultrasonic equipment adopts the 20/28kHz sequential dual-frequency countercurrent ultrasonic working mode, the ultrasonic power density is 32-160W/L, and the pretreatment time in the reaction chamber is 30 minutes.

Further, in step 3, the amylase is α-amylase; the enzymolysis of the amylase is performed at a constant temperature of 60°C and a pH of 6.5 for 20 minutes, which can ensure rapid and moderate enzymolysis of starch in the material and full release Protein and Cordyceps polysaccharide.

Further, in step 4, the protease is alkaline protease; the enzymatic hydrolysis of the protease is carried out at a constant temperature of 60° C., pH 10, and 40 minutes of enzymatic hydrolysis, which can ensure that the proteolytic hydrolysis in the material obtains a polypeptide with good ACE inhibitory activity.

The invention adopts an ultrasonic-assisted enzymolysis method to prepare the antihypertensive functional factor of the residue of the Cordyceps militaris solid medium, and the ultrasonic pretreatment shortens the enzymolysis time, improves the enzymolysis effect, and increases the activity and yield of the enzymolysis product.

Description of drawings

Figure 1 is a schematic diagram of 20/28kHz sequential dual-frequency countercurrent ultrasonic equipment;

In the figure: 1 is an ultrasonic generator, 2 is a 28kHz ultrasonic reactor, 3 is a 20kHz ultrasonic reactor, 4 is a circulating pump, and 5 is a water bath.

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. The invention has universality to other culture media rich in polysaccharides and proteins.

In the formation mechanism of hypertension, angiotensin-converting enzyme (ACE) plays a key role in the process of blood pressure regulation, and the evaluation system of the in vitro detection index ACE inhibition rate is very mature, so the ACE inhibition rate of the end product of enzymatic hydrolysis is used to evaluate its Hypotensive activity. The determination of the inhibitory activity of the hydrolyzate was carried out according to the literature "Huang L, Ma H, Li Y, et al. Antihypertensive activity of recombinant peptide IYPR expressed in Escherichia coli as inclusion bodies. [J]. Protein Expression & Purification, 2012, 83 (1): 15- 20." method, using N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG) as the substrate catalyzed by ACE, to study the change of its absorbance, using Determination by enzyme-labeled quantitative instrument.

As shown in Figure 1, the 20/28kHz sequential dual-frequency countercurrent ultrasonic equipment in the present invention has a structure including an ultrasonic generator 1, a 28kHz ultrasonic reactor 2, a 20kHz ultrasonic reactor 3, a circulation pump 4, and a water bath 5.

Described sonotrode 1 connects the left bottom of 28kHz ultrasonic reactor 2, the left side bottom of 20kHz ultrasonic reactor 3 respectively through electric wire; The left bottom of device 3; the right top (treatment liquid inlet) of described 20kHz ultrasonic reactor 3 is connected to the left side of circulation pump 4 through feed liquid pipe; Described circulation pump 4 is connected to water bath pot 5 through feed liquid pipe ; The water bath 5 is respectively connected to the right side of the circulation pump 4 and the bottom left side of the 28kHz ultrasonic reactor 2 (treatment liquid outlet) through the feed liquid pipe. The treatment liquid is circulated and pumped into the reaction chambers of 20kHz ultrasonic reactor 3 and 28kHz ultrasonic reactor 2 sequentially through the circulation pump 4 from the feed port for ultrasonic pretreatment. The ultrasonic generator 1 can be used to control the ultrasonic power of the ultrasonic reactor, Total time for sonication.

Comparative example:

(1) pulverizing the residue of the solid culture medium of Cordyceps militaris dried by hot air, and passing through a 60-mesh sieve to obtain the residue dry powder;

(2) Conventional enzymatic hydrolysis (without ultrasonic pretreatment): prepare a suspension of Cordyceps militaris solid medium residue with a concentration of 30 g/L, adjust the temperature to 50°C, and maintain the pH at 10; put it into a constant temperature water bath with a rotor for 50 Stir at constant temperature at ℃ for 30 minutes, then adjust pH to 6.5, temperature at 60°C, add α-amylase at 50.1U/g (mass of residue powder) for enzymolysis, keep pH at 6.5, temperature at 60°C during enzymolysis, and enzymolysis After 20 minutes, quickly adjust the pH to 10.

Then add enzyme amount of 6000U/g (mass of residue powder) and alkaline protease for enzymolysis, keep pH 10 during enzymolysis, temperature 60°C, after 40min of enzymolysis, inactivate enzyme at 100°C for 10min, centrifuge at 4000rad/min for 15min , collecting the supernatant to obtain the functional factor for lowering blood pressure of the Cordyceps militaris solid medium residue;

(3) Activity detection: Take 80 mL of the supernatant to adjust the pH to 8.3, then adjust the volume to 100 mL, and measure the ACE inhibitory activity, see Table 1.

Example 1:

(1) pulverizing the residue of the solid culture medium of Cordyceps militaris dried by hot air, and passing through a 60-mesh sieve to obtain the residue dry powder;

(2) Ultrasound-assisted enzymatic hydrolysis: prepare a suspension of Cordyceps militaris solid medium residue at a concentration of 30 g/L, heat to 50° C., adjust the pH to 10, and perform ultrasonic treatment.

The treatment liquid passes through the reaction chamber in a countercurrent circulation mode, and the circulation pump speed is 300rad/min. The conditions of ultrasonic treatment were: the initial temperature of the treatment liquid was 50°C, the pH was maintained at 10, the power density of ultrasonic treatment was 32W/L, the ultrasonic mode was 20/28kHz sequential dual-frequency countercurrent ultrasonic, and the ultrasonic treatment time was 30min.

After the pretreatment, put it into a constant temperature water bath with a rotor, then adjust the pH to 6.5 and the temperature to 60°C, add α-amylase to 50.1U/g (residue powder mass) for enzymolysis, and enzymolysis During the process, the pH was kept at 6.5 and the temperature was 60°C, and the pH was quickly adjusted to 10 after 20 minutes of enzymatic hydrolysis.

Then add enzyme amount of 6000U/g (mass of residue powder) and alkaline protease for enzymolysis, keep pH 10 during enzymolysis, temperature 60°C, after 40min of enzymolysis, inactivate enzyme at 100°C for 10min, centrifuge at 4000rad/min for 15min and collecting the supernatant to obtain the functional factor for lowering blood pressure of the Cordyceps militaris solid medium residue.

(3) Activity detection: Take 80 mL of the supernatant to adjust the pH to 8.3, then adjust the volume to 100 mL, and measure the ACE inhibitory activity, see Table 1.

Example 2:

(4) pulverize the residue of the solid culture medium of Cordyceps militaris through hot air drying, and cross a 60-mesh sieve to obtain the residue dry powder;

(5) Ultrasound-assisted enzymatic hydrolysis: prepare a suspension of Cordyceps militaris solid medium residue at a concentration of 50 g/L, heat to 50° C., adjust the pH to 10, and perform ultrasonic treatment.

The treatment liquid passes through the reaction chamber in a countercurrent circulation mode, and the circulation pump speed is 300rad/min. The ultrasonic treatment conditions were as follows: the initial temperature of the treatment liquid was 50°C, the pH was maintained at 10, the power density of the ultrasonic treatment was 128W/L, the ultrasonic mode was 20/28kHz sequential dual-frequency countercurrent ultrasonic, and the ultrasonic treatment time was 30 minutes.

After the pretreatment, put it into a constant temperature water bath with a rotor, then adjust the pH to 6.5 and the temperature to 60°C, add α-amylase to 50.1U/g (residue powder mass) for enzymolysis, and enzymolysis During the process, the pH was kept at 6.5 and the temperature was 60°C, and the pH was quickly adjusted to 10 after 20 minutes of enzymatic hydrolysis.

Then add enzyme amount of 6000U/g (mass of residue powder) and alkaline protease for enzymolysis, keep pH 10 during enzymolysis, temperature 60°C, after 40min of enzymolysis, inactivate enzyme at 100°C for 10min, centrifuge at 4000rad/min for 15min and collecting the supernatant to obtain the functional factor for lowering blood pressure of the Cordyceps militaris solid medium residue.

(6) Activity detection: Take 80 mL of the supernatant to adjust the pH to 8.3, then adjust the volume to 100 mL, and measure the ACE inhibitory activity, see Table 1.

Example 3:

(1) pulverizing the residue of the solid culture medium of Cordyceps militaris dried by hot air, and passing through a 60-mesh sieve to obtain the residue dry powder;

(2) Ultrasound-assisted enzymatic hydrolysis: prepare a suspension of Cordyceps militaris solid medium residue at a concentration of 10 g/L, heat it to 50° C., adjust the pH to 10, and perform ultrasonic treatment.

The treatment liquid passes through the reaction chamber in a countercurrent circulation mode, and the circulation pump speed is 300rad/min. The ultrasonic treatment conditions were as follows: the initial temperature of the treatment liquid was 50°C, the pH was maintained at 10, the power density of the ultrasonic treatment was 160W/L, the ultrasonic mode was 20/28kHz sequential dual-frequency countercurrent ultrasonic, and the ultrasonic treatment time was 30min.

After the pretreatment, put it into a constant temperature water bath with a rotor, then adjust the pH to 6.5 and the temperature to 60°C, add α-amylase to 50.1U/g (residue powder mass) for enzymolysis, and enzymolysis During the process, the pH was kept at 6.5 and the temperature was 60°C, and the pH was quickly adjusted to 10 after 20 minutes of enzymatic hydrolysis.

Then add enzyme amount of 6000U/g (mass of residue powder) and alkaline protease for enzymolysis, keep pH 10 during enzymolysis, temperature 60°C, after 40min of enzymolysis, inactivate enzyme at 100°C for 10min, centrifuge at 4000rad/min for 15min and collecting the supernatant to obtain the functional factor for lowering blood pressure of the Cordyceps militaris solid medium residue.

(3) Activity detection: Take 80 mL of the supernatant to adjust the pH to 8.3, then adjust the volume to 100 mL, and measure the ACE inhibitory activity, see Table 1.

The result of table 1 different embodiment ACE inhibitory activity

Sample serial number ACE inhibition rate (%) comparative example 38.14 Example 1 44.40 Example 2 79.94 Example 3 54.82

As can be seen from the results of the examples, the method for reusing the residues of the Cordyceps militaris solid medium according to the present invention can be used to prepare high-value-added hypotensive functional factors, which can not only make full use of Cordyceps militaris resources, but also effectively solve the problem of Cordyceps militaris. Waste problems from the cultivation industry.

Claims (1)

1. a method for reusing the residue of Cordyceps militaris solid culture medium, is characterized in that according to the following steps: (1) Pulverize the residue of the solid culture medium of Cordyceps militaris through hot air drying, and cross a 60 mesh sieve to obtain dry powder of the residue; the residue of the solid culture medium is the remaining solid culture medium after harvesting the fruiting body of Cordyceps militaris, rich in mycelia and Cordyceps metabolites; the main raw materials of the solid medium are rice and silkworm chrysalis; the sieving process is to pass through a 60-mesh sieve, which can effectively screen out useless residues and impurities; (2) Ultrasound-assisted enzymatic hydrolysis: the residue dry powder obtained in step (1) is prepared into a suspension of Cordyceps militaris solid medium residue at a concentration of 50 g/L, heated to 50° C., adjusted to pH 10, and then ultrasonically treated; The treatment liquid passes through the reaction chamber in a countercurrent circulation mode, and the circulation pump speed is 300rad/min; the ultrasonic treatment conditions are: the initial temperature of the treatment liquid is 50°C, the pH is maintained at 10, the power density of the ultrasonic treatment is 128W/L, and the ultrasonic mode is 20/ 28kHz sequential dual-frequency countercurrent ultrasound, ultrasonic treatment time is 30min; After the pretreatment, put it into a constant temperature water bath with a rotor, then adjust the pH to 6.5 and the temperature to 60°C, add α-amylase to 50.1U/g (residue powder mass) for enzymolysis, and enzymolysis Keep the pH at 6.5 and the temperature at 60°C during the process, and quickly adjust the pH to 10 after 20 minutes of enzymatic hydrolysis; Then add enzyme amount of 6000U/g (mass of residue powder) and alkaline protease for enzymolysis, keep pH 10 during enzymolysis, temperature 60°C, after 40min of enzymolysis, inactivate enzyme at 100°C for 10min, centrifuge at 4000rad/min for 15min , collect the supernatant, and obtain the hypotensive function factor of the Cordyceps militaris solid medium residue.