CN110106219B - Method for recycling residue of cordyceps militaris solid culture medium - Google Patents
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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
Technical field:
the invention relates to a technology for recycling cordyceps militaris solid culture medium residues, in particular to a method for extracting blood pressure reducing functional factors from cordyceps militaris solid culture medium residues by adopting an ultrasonic-assisted enzymolysis method, and belongs to the technical field of biology.
The background technology is as follows:
the cordyceps militaris is also called as Cordyceps militaris, belongs to Cordyceps fungi, has no strict requirements on hosts and living environments, and is the only cordyceps militaris capable of realizing artificial cultivation at present. Because the cordyceps militaris has excellent medicine and nourishing effects, the cordyceps militaris is listed as a new resource food, and has very broad application prospect. Although the production of artificially cultured cordyceps militaris fruiting bodies is quite large-scale, when fruiting bodies are harvested, a large amount of residual cordyceps militaris culture medium residues are abandoned, so that environmental pollution is caused; the residue contains abundant Cordyceps polysaccharide, starch sugar and protein substances, and also causes resource waste. The method for recycling the cordyceps militaris culture medium residues becomes a current research hot spot, so that the environmental problem can be solved, and the economic profit of enterprises can be improved.
Related researches show that in order to better invade the host body and effectively utilize the nutrition components of the host body, cordyceps sinensis can secrete various biological enzymes into the culture medium, so that some macromolecular nutrition substances in the culture medium are subjected to enzymolysis. Yang Xue in the text of "influence of ultrasonic pretreatment on ACE inhibition rate of rice protein enzymolysis product", research finds that the enzymolysis product of rice protein has good blood pressure lowering activity; the invention patent with the Chinese patent number of 201810189732.8 discloses a separation and purification method of silkworm chrysalis protein polypeptide with good ACE inhibition activity; peng Lei et al, in the "research on garlic antihypertensive function factor by ultrasonic pretreatment enzymatic method", research has found that the antihypertensive factor (partial polysaccharide) of the incomplete polypeptide also has antihypertensive activity. At present, the research on the residue of a solid culture medium of cordyceps militaris is mostly remained in the extraction of single components such as cordyceps polysaccharide, cordycepin and the like, but the research on the blood pressure reducing activity of the residue of the culture medium developed by combining the metabolism of cordyceps sinensis with short-time enzymolysis has not been reported yet.
The ultrasonic auxiliary enzymolysis technology can overcome the defects of low reaction efficiency, low product activity, low product yield and the like in the traditional enzymolysis technology, and has the advantages of short acting time, simple operation, easy control, low temperature rise and the like, thereby being widely applied to enzymolysis strengthening research. Zhang Yanyan in the "preparation of gluten ACE inhibitory peptide based on ultrasonic pretreatment and in-situ monitoring technical research of the process thereof", it is pointed out that pretreatment of raw materials by adopting a proper ultrasonic working mode can improve enzymolysis characteristics and improve the antihypertensive activity of enzymolysis products. However, most of the current researches on ultrasonic technology are stopped in ultrasonic washing tanks, ultrasonic cell disruptors and other devices, and the working mode of the ultrasonic cannot be changed. In addition, the research related to the preparation of the cordyceps militaris solid medium residue by the ultrasonic-assisted enzymolysis technology is not reported.
According to the characteristics of the cordyceps militaris solid culture medium residues, the solid culture medium residues remained after fruiting bodies are harvested are used as raw materials by utilizing an ultrasonic auxiliary enzyme treatment technology to prepare the blood pressure reducing functional factors with high added value, so that the problem of waste generated in the cordyceps militaris cultivation industry can be effectively solved, and cordyceps militaris resources can be fully utilized.
Disclosure of Invention
The invention aims to overcome the defects of the traditional extraction or enzymolysis method in the prior art, fully utilizes the residue characteristics of the cordyceps militaris solid culture medium, and provides a novel method for preparing the cordyceps militaris solid culture medium residue blood pressure reducing functional factor by ultrasonic assistance. The invention uses the ultrasonic auxiliary enzymolysis technology, takes the residue of the solid culture medium remained after harvesting the fruiting bodies as the raw material, prepares the blood pressure reducing functional factors with high added value, can fully utilize the cordyceps militaris resources, and can effectively solve the problem of waste generated in the cordyceps militaris cultivation industry.
Aiming at the method of the invention, the technical scheme adopted specifically is as follows:
a method for recycling residue of Cordyceps militaris solid culture medium comprises the following steps:
step one, crushing the residues of the culture medium: pulverizing residue of Cordyceps militaris solid culture medium dried by hot air, sieving;
step two, pretreatment of the culture medium residues: adding water into the culture medium residue dry powder to obtain suspension, adjusting the temperature to 50 ℃ and the pH value to 10, and sequentially carrying out ultrasonic treatment through an ultrasonic equipment reaction cavity in a countercurrent circulation mode according to a set ultrasonic mode to obtain pretreatment liquid;
step three, amylase enzymolysis of the culture medium residue: adjusting the pH of the pretreatment solution to 6.5, and adding amylase to carry out enzymolysis by adding enzyme in an amount of 50.1U/g (mass of residual powder) to obtain amylase enzymolysis solution;
step four, protease enzymolysis of the culture medium residues: regulating pH of amylase enzymolysis liquid to 10, adding enzyme amount of 6000U/g (residue powder mass) and alkaline protease for enzymolysis, inactivating enzyme, centrifuging for 15min at 4000rad/min, and obtaining enzymolysis product with blood pressure lowering effect.
In the first step, the residue of the solid culture medium is the solid culture medium remained after harvesting the cordyceps militaris fruiting body, and is rich in hypha and cordyceps sinensis metabolites; the main raw materials of the solid culture medium are rice and silkworm chrysalis; the sieving treatment is that the waste residues and impurities can be effectively removed by sieving through a 60-mesh sieve.
In the second step, the concentration of the residue dry powder suspension is 10-50 g/L. .
In the second step, the ultrasonic equipment adopts an ultrasonic working mode of 20/28kHz sequential double-frequency countercurrent, the ultrasonic power density is 32-160W/L, and the pretreatment time in the reaction cavity is 30min.
Further, in step three, the amylase is an alpha-amylase; the amylase is used for enzymolysis, wherein the temperature is constant at 60 ℃, the pH is 6.5, and the enzymolysis is carried out for 20 minutes, so that the rapid and moderate enzymolysis of starch in the material can be ensured, and the protein and Cordyceps polysaccharide are fully released.
Further, in the fourth step, the protease is alkaline protease; the protease is subjected to enzymolysis, wherein the temperature is constant at 60 ℃, the pH is 10, and the enzymolysis is performed for 40 minutes, so that the protease in the material can be ensured to obtain the polypeptide with good ACE inhibition activity.
The invention adopts the ultrasonic-assisted enzymolysis method to prepare the blood pressure reducing functional factors of the cordyceps militaris solid medium residues, shortens the enzymolysis time, improves the enzymolysis effect and improves the activity and the yield of enzymolysis products by ultrasonic pretreatment.
Drawings
FIG. 1 is a schematic diagram of a 20/28kHz sequential dual-frequency counter-current ultrasonic device;
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 kettle.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to the accompanying drawings and the detailed description. The invention has universality for other culture mediums rich in polysaccharide and protein.
In the mechanism of hypertension formation, angiotensin Converting Enzyme (ACE) plays a key role in the blood pressure regulation process, and an evaluation system for in vitro detection index ACE inhibition rate is very mature, so that the ACE inhibition rate of an enzymolysis final product is selected to evaluate the blood pressure reduction activity of the final product. The enzymatic hydrolysis product inhibitory activity was measured according to the method of the literature "Huang L, ma H, li Y, et al, anti-hypersensitive activity of recombinant peptide IYPR expressed in Escherichia coli as inclusion bodies [ J ]. Protein Expression & Purification,2012,83 (1): 15-20 ], using N- [3- (2-furyl) acryloyl ] -L-phenylalanyl glycylglycine (FAPGG) as an ACE-catalyzed substrate, and the change in absorbance was studied and measured using an enzyme calibration instrument.
According to the figure 1, the structure of the 20/28kHz sequential double-frequency countercurrent ultrasonic equipment comprises an ultrasonic generator 1, a 28kHz ultrasonic reactor 2, a 20kHz ultrasonic reactor 3, a circulating pump 4 and a water bath kettle 5.
The ultrasonic generator 1 is respectively connected with the left bottom of the 28kHz ultrasonic reactor 2 and the left bottom of the 20kHz ultrasonic reactor 3 through wires; the top of the right side of the 28kHz ultrasonic reactor 2 is connected with the bottom of the left side of the 20kHz ultrasonic reactor 3 through a feed liquid pipe; the top of the right side (treatment fluid inlet) of the 20kHz ultrasonic reactor 3 is connected to the left side of the circulating pump 4 through a feed fluid pipe; the circulating pump 4 is connected with the water bath kettle 5 through a feed liquid pipe; the water bath 5 is respectively connected with the right side of the circulating pump 4 and the left side bottom (treatment fluid outlet) of the 28kHz ultrasonic reactor 2 through a material liquid pipe. The treatment fluid is sequentially circulated and pumped into the reaction chambers of the 20kHz ultrasonic reactor 3 and the 28kHz ultrasonic reactor 2 through the circulating pump 4 by the feed inlet for ultrasonic pretreatment, and the ultrasonic power and the total ultrasonic treatment time of the ultrasonic reactor can be controlled through the ultrasonic generator 1 in the whole process.
Comparative example:
(1) Pulverizing residue of Cordyceps militaris solid culture medium dried by hot air, and sieving with 60 mesh sieve to obtain residue dry powder;
(2) Conventional enzymatic hydrolysis (no ultrasonic pretreatment): preparing a cordyceps militaris solid culture medium residue suspension with the concentration of 30g/L, adjusting the temperature to 50 ℃ and maintaining the pH value to 10; placing into a constant temperature water bath with a rotor, stirring at 50deg.C for 30min, adjusting pH to 6.5, adjusting temperature to 60deg.C, adding alpha-amylase at 50.1U/g (residue powder mass), and rapidly adjusting pH to 10 after maintaining pH to 6.5 and temperature to 60deg.C during enzymolysis for 20 min.
Then adding enzyme amount of 6000U/g (mass of residue powder) and alkaline protease for enzymolysis, keeping pH at 10 and temperature at 60 ℃ in the enzymolysis process, inactivating enzyme at 100 ℃ for 10min after enzymolysis for 40min, centrifuging for 15min at 4000rad/min, and collecting supernatant to obtain Cordyceps militaris solid culture medium residue blood pressure lowering functional factor;
(3) Activity detection: 80mL of the supernatant was adjusted to pH 8.3 and then the volume was set to 100mL, and the ACE inhibitory activity was measured as shown in Table 1.
Example 1:
(1) Pulverizing residue of Cordyceps militaris solid culture medium dried by hot air, and sieving with 60 mesh sieve to obtain residue dry powder;
(2) Ultrasonic-assisted enzymolysis: preparing a cordyceps militaris solid culture medium residue suspension with the concentration of 30g/L, heating to 50 ℃, regulating the pH to 10, and performing ultrasonic treatment.
The treatment liquid passes through the reaction chamber in a countercurrent circulation mode, and the rotation speed of the circulation pump is 300rad/min. The ultrasonic treatment conditions are as follows: the initial temperature of the treatment liquid is 50 ℃, the pH is maintained at 10, the power density of ultrasonic treatment is 32W/L, the ultrasonic mode is 20/28kHz, and the ultrasonic treatment time is 30min.
After pretreatment, the mixture is put into a constant-temperature water bath with a rotor, the pH value is regulated to 6.5, the temperature is 60 ℃, the enzyme amount is added to 50.1U/g (the mass of residual powder), the alpha-amylase is added for enzymolysis, the pH value is kept to 6.5 in the enzymolysis process, the temperature is 60 ℃, and the pH value is regulated to 10 after 20 minutes of enzymolysis.
Then adding enzyme amount of 6000U/g (mass of residue powder) and alkaline protease for enzymolysis, keeping pH at 10 and temperature at 60 ℃ in the enzymolysis process, inactivating enzyme at 100 ℃ for 10min after enzymolysis for 40min, centrifuging for 15min at 4000rad/min, and collecting supernatant to obtain the Cordyceps militaris solid culture medium residue blood pressure lowering functional factor.
(3) Activity detection: 80mL of the supernatant was adjusted to pH 8.3 and then the volume was set to 100mL, and the ACE inhibitory activity was measured as shown in Table 1.
Example 2:
(4) Pulverizing residue of Cordyceps militaris solid culture medium dried by hot air, and sieving with 60 mesh sieve to obtain residue dry powder;
(5) Ultrasonic-assisted enzymolysis: preparing a cordyceps militaris solid culture medium residue suspension with the concentration of 50g/L, heating to 50 ℃, regulating the pH value to 10, and performing ultrasonic treatment.
The treatment liquid passes through the reaction chamber in a countercurrent circulation mode, and the rotation speed of the circulation pump is 300rad/min. The ultrasonic treatment conditions are as follows: the initial temperature of the treatment liquid is 50 ℃, the pH is maintained at 10, the power density of ultrasonic treatment is 128W/L, the ultrasonic mode is 20/28kHz, and the sequential double-frequency countercurrent ultrasonic treatment is carried out for 30min.
After pretreatment, the mixture is put into a constant-temperature water bath with a rotor, the pH value is regulated to 6.5, the temperature is 60 ℃, the enzyme amount is added to 50.1U/g (the mass of residual powder), the alpha-amylase is added for enzymolysis, the pH value is kept to 6.5 in the enzymolysis process, the temperature is 60 ℃, and the pH value is regulated to 10 after 20 minutes of enzymolysis.
Then adding enzyme amount of 6000U/g (mass of residue powder) and alkaline protease for enzymolysis, keeping pH at 10 and temperature at 60 ℃ in the enzymolysis process, inactivating enzyme at 100 ℃ for 10min after enzymolysis for 40min, centrifuging for 15min at 4000rad/min, and collecting supernatant to obtain the Cordyceps militaris solid culture medium residue blood pressure lowering functional factor.
(6) Activity detection: 80mL of the supernatant was adjusted to pH 8.3 and then the volume was set to 100mL, and the ACE inhibitory activity was measured as shown in Table 1.
Example 3:
(1) Pulverizing residue of Cordyceps militaris solid culture medium dried by hot air, and sieving with 60 mesh sieve to obtain residue dry powder;
(2) Ultrasonic-assisted enzymolysis: preparing a cordyceps militaris solid culture medium residue suspension with the concentration of 10g/L, heating to 50 ℃, regulating the pH value to 10, and performing ultrasonic treatment.
The treatment liquid passes through the reaction chamber in a countercurrent circulation mode, and the rotation speed of the circulation pump is 300rad/min. The ultrasonic treatment conditions are as follows: the initial temperature of the treatment liquid is 50 ℃, the pH is maintained at 10, the power density of ultrasonic treatment is 160W/L, the ultrasonic mode is 20/28kHz, and the ultrasonic treatment time is 30min.
After pretreatment, the mixture is put into a constant-temperature water bath with a rotor, the pH value is regulated to 6.5, the temperature is 60 ℃, the enzyme amount is added to 50.1U/g (the mass of residual powder), the alpha-amylase is added for enzymolysis, the pH value is kept to 6.5 in the enzymolysis process, the temperature is 60 ℃, and the pH value is regulated to 10 after 20 minutes of enzymolysis.
Then adding enzyme amount of 6000U/g (mass of residue powder) and alkaline protease for enzymolysis, keeping pH at 10 and temperature at 60 ℃ in the enzymolysis process, inactivating enzyme at 100 ℃ for 10min after enzymolysis for 40min, centrifuging for 15min at 4000rad/min, and collecting supernatant to obtain the Cordyceps militaris solid culture medium residue blood pressure lowering functional factor.
(3) Activity detection: 80mL of the supernatant was adjusted to pH 8.3 and then the volume was set to 100mL, and the ACE inhibitory activity was measured as shown in Table 1.
TABLE 1 results of ACE inhibitory Activity of different examples
| Sample numbering | ACE inhibition Rate (%) |
| Comparative example | 38.14 |
| Example 1 | 44.40 |
| Example 2 | 79.94 |
| Example 3 | 54.82 |
The results of the examples show that the preparation of the blood pressure reducing functional factor with high added value can be carried out by the method for recycling the residue of the cordyceps militaris solid culture medium, so that cordyceps militaris resources can be fully utilized, and the problem of waste generated in the cordyceps militaris cultivation industry can be effectively solved.
Claims (1)
1. A method for recycling residue of cordyceps militaris solid culture medium is characterized by comprising the following steps:
(1) Pulverizing residue of Cordyceps militaris solid culture medium dried by hot air, and sieving with 60 mesh sieve to obtain residue dry powder; the solid culture medium residue is the solid culture medium remained after harvesting the cordyceps militaris fruiting body, and is rich in hypha and cordyceps sinensis metabolites; the main raw materials of the solid culture medium are rice and silkworm chrysalis; the sieving treatment is that a 60-mesh sieve is adopted, so that useless residues and impurities can be effectively removed;
(2) Ultrasonic-assisted enzymolysis: preparing a cordyceps militaris solid culture medium residue suspension with the concentration of 50g/L from the residue dry powder obtained in the step (1), heating to 50 ℃, regulating the pH to 10, and performing ultrasonic treatment;
the treatment liquid passes through the reaction cavity in a countercurrent circulation mode, and the rotation speed of the circulation pump is 300rad/min; the ultrasonic treatment conditions are as follows: the initial temperature of the treatment liquid is 50 ℃, the pH is maintained at 10, the power density of ultrasonic treatment is 128W/L, the ultrasonic mode is 20/28kHz, and the sequential double-frequency countercurrent ultrasonic treatment is carried out for 30min;
after pretreatment, placing the mixture into a constant-temperature water bath kettle with a rotor, then adjusting the pH value to 6.5, adjusting the temperature to 60 ℃, adding the alpha-amylase with the enzyme amount of 50.1U/g (the mass of residual powder) for enzymolysis, keeping the pH value to 6.5 in the enzymolysis process, adjusting the pH value to 10 after 20 minutes of enzymolysis;
then adding enzyme amount of 6000U/g (mass of residue powder) and alkaline protease for enzymolysis, keeping pH at 10 and temperature at 60 ℃ in the enzymolysis process, inactivating enzyme at 100 ℃ for 10min after enzymolysis for 40min, centrifuging for 15min at 4000rad/min, and collecting supernatant to obtain the Cordyceps militaris solid culture medium residue blood pressure lowering functional factor.
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| CN114990183B (en) * | 2022-06-11 | 2023-11-21 | 江苏科技大学 | Method for preparing cordycepin and antihypertensive peptide based on rice cordyceps militaris fruiting body |
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