Method for extracting glutamic acid from hemp

Abstract

The invention provides a method for extracting glutamic acid from cannabis sativa. The method comprises the following steps: a) pretreating a cannabis sativa sample; b) carrying out composite enzymatic hydrolysis on the extract obtained after pretreatment in the step a); c) fermenting by taking the enzymolysis product obtained in the step b) as a substrate; d) concentrating and crystallizing the fermentation liquor obtained in the step c) to obtain glutamic acid crystals; and e) recrystallizing and purifying the glutamic acid crystal, and drying. According to the method, the hemp hydrolysis sugar liquid is used as a carbon source, technologies such as concentration isoelectric point are adopted, operation is simple, and the obtained glutamic acid is good in condition.

Description

Technical Field

[0001] The present invention belongs to the technical field of amino acid production, and specifically relates to a method for extracting glutamic acid from cannabis. Background Art

[0002] Glutamic acid plays an important role in the process of protein metabolism in organisms and is involved in many important chemical reactions in animals, plants, and microorganisms. It is also the most abundant excitatory neurotransmitter in the vertebrate nervous system. Glutamic acid plays an important role in the central nervous system. It can not only regulate synaptic activity and maintain the stability of the neural network, but also is the hub of nerve cell damage and inflammatory reactions.

[0003] Glutamic acid has a wide range of uses and can be used in the pharmaceutical, food industry, and agriculture. In medicine, glutamic acid is mainly used to treat hepatic coma and also to improve children's intellectual development. In the food industry, monosodium glutamate is a commonly used food flavor enhancer, and its main component is sodium glutamate. In agriculture, glutamic acid can be made into a citrus sweetener or used as a carrier for micronutrient fertilizers. Under the condition that the basic requirements of nitrogen, phosphorus, and potassium are basically met, the micronutrient fertilizer sprayed on the leaf surface has the characteristics of low input and high efficiency.

[0004] The preparation processes of glutamic acid mainly include the fermentation method and the extraction method. The fermentation method is to ferment through specific production strains, such as Corynebacterium glutamicum, under suitable culture media and fermentation conditions to produce a large amount of glutamic acid. However, there are problems such as phage contamination and difficulty in controlling fermentation conditions during the fermentation process, which will affect the yield and quality of glutamic acid. The extraction method is to extract and purify the glutamic acid in the fermentation broth by methods such as the isoelectric point method, ion exchange method, and metal salt method. Among them, the isoelectric point method has a simple process, but there is more residual glutamic acid in the mother liquor; the ion exchange method has a high recovery rate, but a large amount of acid and alkali is used, and the wastewater discharge is large.

[0005] Generally speaking, the existing glutamic acid preparation processes still need to be improved in terms of efficiency and environmental protection. It is necessary to continuously optimize the fermentation conditions and improve the extraction methods to reduce pollution, increase the yield and quality, and reduce the impact on the environment. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for extracting glutamic acid from cannabis.

[0007] To achieve the purpose of the present invention, the present invention provides a method for extracting glutamic acid from cannabis, including the following steps: a) Pretreat the cannabis sample; b) Perform complex enzymatic hydrolysis on the extract obtained in step a) after pretreatment; c) Ferment using the enzymatic hydrolysis product obtained in step b) as a substrate; d) The fermentation broth obtained in step c) is concentrated and crystallized to obtain glutamic acid crystals; e) The glutamic acid crystals are recrystallized and purified, and then dried to obtain the product.

[0008] Furthermore, step a) includes: a1) Mix the dried hemp powder with a NaOH solution with a concentration of 0.5 - 1.0 mol / L in a ratio of 1 g:(20 - 25) mL, and let it stand at room temperature for 24 - 26 hours to ensure sufficient infiltration; a2) Place the mixture in a magnetic stirrer and extract at a temperature of 90°C - 100°C for 1 - 2 hours each time; after the extraction is completed, centrifuge at a speed of 6000 - 7000 rpm for 15 - 20 minutes, collect the residue after centrifugation, and extract the residue again under the above conditions, that is, add the residue to sodium hydroxide again for extraction according to the method in a1); a3) Wash the residue with distilled water until the washed residue is neutral (pH value within the range of 6.0 - 7.0); a4) Finally, dry the washed residue (extract) for later use.

[0009] Furthermore, step b) includes: b1) Preparation of the enzyme solution: Weigh each enzyme according to the amount of 10 - 30 U of cellulase, 5 - 15 U of hemicellulase, and 0.5 - 2 U of β - glucosidase per gram of residue, and dissolve them in a citrate - phosphate buffer solution with a concentration of 0.05 M and a pH value of 4.8 to prepare the enzyme solution; b2) Mix the residue obtained in step a) with the enzyme solution in a ratio of 1 g:5 mL - 10 mL evenly, and then carry out enzymatic hydrolysis at the optimal temperature of 50°C of cellulase, control the pH range of the reaction system to be 4.8 - 5.5, and during the enzymatic hydrolysis process, stir at a low speed of 100 - 200 rpm for 24 - 72 hours; b3) After the enzymatic hydrolysis is completed, separate the sugar solution obtained by enzymatic hydrolysis from the undegraded solid substances by filtration or centrifugation to obtain the required sugar solution (enzymatic hydrolysis product).

[0010] Furthermore, step c) includes: c1) Using the sugar solution obtained in step b) as a carbon source, prepare the following fermentation medium: total sugar concentration 50 - 100 g / L, ammonium sulfate 10 - 15 g / L, KH 2 PO 4 1 - 2 g / L, MgSO 4 ·7H 2 O 0.5 - 1.0 g / L, MnSO 40.01 - 0.1 g / L, biotin 1 µg / L, corn steep liquor 20 - 30 g / L, adjust the pH to 6.8 - 7.5 with NH 4 OH; c2) Inoculate 5% - 15% v / v of Corynebacterium glutamicum ( Corynebacterium glutamicum ) seed liquor into the fermentation medium, and ferment in a shaker at 150 rpm - 200 rpm for 48 - 72 hours at 30°C - 35°C, maintaining the saturated dissolved oxygen concentration at 20% - 50%.

[0011] The Corynebacterium glutamicum used in the present invention is preferably ATCC13032, purchased from the Shanghai Center for Microbial Culture Collection.

[0012] Furthermore, the bacterial content of Corynebacterium glutamicum in the seed liquor is about 10 6 CFU / mL.

[0013] Furthermore, step d) includes: d1) Take out the fermentation broth from the shaker, centrifuge at 8000 - 10000 rpm for 10 - 15 minutes; then, transfer the obtained supernatant to a rotary evaporator for vacuum concentration treatment; d2) Add 1 mol / L hydrochloric acid to the concentrated supernatant, stir at 40 - 50°C, adjust the pH value of the supernatant to the isoelectric point of glutamic acid 3.22, and then let it stand in the refrigerator to wait for the formation of glutamic acid crystals.

[0014] Furthermore, step e) includes: Dissolve the obtained glutamic acid crystals in hot water at 60 - 80°C, adjust the pH to 3.2 - 3.5 again until glutamic acid crystals precipitate again, and repeat the filtration and washing operations to obtain purified crystals.

[0015] Furthermore, the purified crystals are vacuum - dried at 50 - 60°C to obtain pure glutamic acid (with a moisture content of 0.5% - 1%).

[0016] By the above - mentioned technical solution, the present invention has at least the following advantages and beneficial effects: The present invention uses hemp hydrolyzed sugar solution as a carbon source, and adopts techniques such as concentration and isoelectric point. The operation is simple, and the obtained glutamic acid has good quality. The purity is 79.5%. Brief Description of the Drawings

[0017] Figure 1 It is the result of a single - factor experiment on ultrasonic extraction of hemp polysaccharide in a preferred embodiment of the present invention.

[0018] Figure 2This is the response surface 3D map and contour map of the interaction of enzymolysis time, enzymolysis temperature, and liquid-to-solid ratio on the yield of cannabis polysaccharide in the preferred embodiment of the present invention. Detailed implementation mode

[0019] The present invention provides a method for extracting glutamic acid from cannabis, which includes the following steps: Step 1) Pretreat the cannabis sample, Step 2) Perform complex enzymolysis on the extract, Step 3) Fermentation process, Step 4) Glutamic acid crystallization, Step 5) Recrystallization purification, and Step 6) Dry to obtain glutamic acid crystals.

[0020] Further, Step 1) includes the following steps: First, prepare a NaOH solution with a concentration of 0.5 - 1.0 mol / L and set it aside.

[0021] Next, mix the dried cannabis powder with the prepared NaOH solution at a ratio of 1:20 g / mL, and let it stand at room temperature for 24 hours to ensure sufficient infiltration.

[0022] After that, place the mixture in a magnetic stirrer and perform two extractions at a temperature of 90°C - 100°C, with each extraction time being 1 - 2 hours. After the extraction is completed, centrifuge at a speed of 6000 rpm for 15 minutes, collect the centrifuged residue, and set it aside.

[0023] To remove the excess alkali and dissolved lignin in the residue, wash the residue with distilled water until the washed residue is neutral (pH value within the range of 6.0 - 7.0). Finally, dry the washed residue and set it aside.

[0024] Further, Step 2) includes the following steps: The volume of the enzyme solution needs to be determined according to the actual situation of the residue. In the final reaction system, the content of each enzyme should be controlled within the following range: cellulase 10 - 30 U / g, hemicellulase 5 - 15 U / g, β-glucosidase 0.5 - 2 U / g. These enzymes need to be dissolved in 0.05 M citric acid-phosphate buffer with a pH value of 4.8.

[0025] Mix the residue and the enzyme solution evenly at a ratio of 1 g:10 mL, and then perform enzymolysis at the optimal temperature of 50°C for cellulase. Since the enzyme activity is best within the optimal pH range of 4.8 - 5.5, this pH value needs to be maintained. According to the sugar production requirement, the enzymolysis time can be 24 - 72 hours, and it can be appropriately extended if necessary.

[0026] During the enzymolysis process, stir at a low speed of 100 - 200 rpm to ensure sufficient contact between the substrate and the enzyme. At the same time, attention should be paid to avoiding the influence of mechanical damage on the enzyme activity.

[0027] After enzymatic hydrolysis is completed, the sugar solution is separated from the undegraded solid substances by filtration or centrifugation to obtain the required sugar solution.

[0028] Further, step 3) includes the following steps: Using the hydrolyzed sugar solution of industrial hemp as a carbon source, controlling its total sugar concentration within the range of 50 to 100 g / L. Using ammonium sulfate at 10 to 15 g / L as a nitrogen source, and at the same time using 1 to 2 g / L of KH 2 PO 4 to provide phosphorus element. MgSO 4 ·7H 2 O as an important cofactor, its concentration is maintained at 0.5 to 1.0 g / L. In order to induce the secretion of glutamic acid, 0.01 to 0.1 g / L of MnSO 4 needs to be added to the culture medium. In addition, 1 µg / L of biotin as a vitamin essential for fermentation, and 20 to 30 g / L of corn steep liquor need to be added to provide additional nitrogen source and trace elements. During the fermentation process, NH 4 OH is used to adjust the pH value to keep it within the appropriate range of 6.8 to 7.5. Add 5% - 15% Corynebacterium glutamicum (v / v) to the culture medium, and ferment in a shaker at 150 rpm at 30°C for 48 - 72 hours, maintaining a saturated dissolved oxygen concentration of 20% - 50%.

[0029] Further, step 4) includes the following steps: Take out the fermented broth from the shaker, place it in a centrifuge, and centrifuge at a speed of 10000 rpm for 10 minutes. Subsequently, transfer the obtained supernatant to a rotary evaporator for vacuum concentration treatment.

[0030] Add 1 mol / L hydrochloric acid to the concentrated supernatant, while controlling the temperature within the range of 40 - 50°C and slowly stirring. By adding hydrochloric acid, gradually adjust the pH value of the supernatant to the isoelectric point of glutamic acid, which is 3.22. After the pH adjustment is completed, place the solution in the refrigerator and let it stand to wait for the formation of glutamic acid crystals.

[0031] Further, step 5) includes: Dissolve the obtained glutamic acid crystals in hot water at 60 - 80°C, adjust the pH to 3.2 - 3.5 again until glutamic acid crystals precipitate again, and repeat the filtration and washing operations. Use the method of slow cooling to promote the uniform growth of glutamic acid crystals and improve the crystal quality.

[0032] Further, step 6) includes: The moisture content of the dry glutamic acid product is usually controlled at 0.5% - 1%, and vacuum drying is carried out at 50 - 60°C to prevent thermal degradation of glutamic acid.

[0033] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0034] In the following examples, the CAS numbers of cellulase, hemicellulase and β-glucosidase used are 9012-54-8, 9025-56-3 and 9001-22-3 respectively, and they are purchased from Shanghai Hushi Laboratory Equipment Co., Ltd.

[0035] Example 1 Method for Extracting Glutamic Acid from Cannabis 1. Pretreatment of Cannabis Samples After freeze-drying the cannabis with a freeze dryer, it is pulverized with a pulverizer, and 1 mol / L NaOH is added at a liquid-to-solid ratio of 20 mL:1 g, and it is stirred in a magnetic stirrer at room temperature for 24 h, and then refluxed and extracted at 90 °C - 100 °C for 1 - 2 h. The above operations are repeated twice, and centrifuged at a speed of 6000 rpm for 15 min. The residue is washed with distilled water to remove excess alkali and dissolved lignin until the residue is neutral (pH 6.0 - 7.0), and the residue is dried in an oven at 50 °C.

[0036] 2. Complex Enzymatic Hydrolysis Weigh 200 g of the filter residue and mix it with the enzyme solution at a ratio of 1 g:10 mL. For the preparation of the enzyme solution, a total of 4000 U of cellulase, 2000 U of hemicellulase and 250 U of β-glucosidase are weighed and placed in 1 L of 0.05 M, pH 4.8 citrate-phosphate buffer solution. The mixture is placed on a shaker at 100 - 200 rpm for enzymatic hydrolysis for 24 h. Then it is heated to 95 °C to inactivate the enzyme for 3 min, and the undegraded solids are separated by centrifugation. The obtained sugar solution is evaporated and concentrated for the next fermentation step.

[0037] 3. Fermentation Process (1) Take the raw materials of the fermentation medium and configure them according to the following concentrations: 80 g / L of cannabis hydrolyzed sugar solution, (NH 4 ) 2 SO 4 10 g / L, 2 g / L KH 2 PO 4 , 0.5 g / L MgSO 4 ·7H 2 O, 0.01 g / L MnSO 4 , 1 μg / L biotin, 20 g / L corn steep liquor. After stirring evenly, adjust the pH to 6.8 with NH 4 OH, sterilize at 121 °C for 15 min, and cool naturally.

[0038] (2) The seed solution of Corynebacterium glutamicum ATCC13032 (purchased from Shanghai Center for Microbial Preservation) had a bacterial content of 10 6 CFU / mL and was inoculated into the sterilized fermentation medium at an inoculation amount of 10%. The initial inoculation concentration of the bacteria was OD 600nm = 0.8. Fermentation was carried out under constant temperature at 30 °C and on a shaker at 150 rpm, maintaining the dissolved oxygen concentration in the fermentation broth at 30% saturation level. After 48 hours of fermentation, the fermentation broth was collected. Throughout the process, ensure stable fermentation conditions to obtain the best fermentation effect.

[0039] 4. Glutamic acid crystallization (1) The fermentation broth was centrifuged at 10000 rpm for 10 minutes in a centrifuge, and the supernatant was collected. Then, at a temperature of 40 °C, 1 mol / L hydrochloric acid was used to adjust its pH value to the isoelectric point of glutamic acid, 3.22. Then it was placed in the refrigerator and left standing until glutamic acid crystals were formed.

[0040] (2) The obtained glutamic acid crystals were dissolved in hot water at 60 °C, and the pH was adjusted again to 3.2 - 3.5 until glutamic acid crystals were precipitated again. The filtration and washing operations were repeated. The slow cooling method was used to promote uniform growth of glutamic acid crystals and improve crystal quality.

[0041] 5. Drying The moisture content of the dry glutamic acid product was usually controlled at 0.5% - 1%, and vacuum drying was carried out at 50 - 60 °C to prevent thermal degradation of glutamic acid.

[0042] The purity of the dry glutamic acid product was 79.5%.

[0043] Example 2 Method for extracting glutamic acid from cannabis 1. Pretreatment of cannabis samples After freeze-drying the cannabis with a freeze-dryer, it was crushed with a crusher. According to the liquid-solid ratio of 20 mL:1 g, 1 mol / L NaOH was added, and it was stirred in a magnetic stirrer at room temperature for 24 h, and then refluxed and extracted at 90 °C - 100 °C for 1 - 2 h. The above operations were repeated twice, and centrifuged at a speed of 6000 rpm for 15 min. The residue was washed with distilled water to remove excess alkali and dissolved lignin until the residue was neutral (pH 6.0 - 7.0), and the residue was dried in an oven at 50 °C.

[0044] 2. Composite enzymatic hydrolysis Weigh 200 g of the filter residue and mix it with the enzyme solution at a ratio of 1 g:10 mL. For the preparation of the enzyme solution, a total of 1000 U of cellulase, 800 U of hemicellulase, and 50 U of β-glucosidase are weighed and placed in 1 L of 0.05 M, pH 4.8 citrate-phosphate buffer. The mixture is placed on a shaker at 100 - 200 rpm for enzymatic hydrolysis for 24 h. Then, the temperature is raised to 95 °C to inactivate the enzyme for 3 min, and the undegraded solids are separated by centrifugation. The obtained sugar solution is evaporated and concentrated for the next fermentation step.

[0045] 3. Fermentation process (1) Take the raw materials of the fermentation medium and prepare them at the following concentrations: 80 g / L of hemp hydrolysate sugar solution, 10 g / L of (NH 4 ) 2 SO 4 10, 2 g / L of KH 2 PO 4 , 0.5 g / L of MgSO 4 ·7H 2 O, 0.01 g / L of MnSO 4 , 1 μg / L of biotin, 20 g / L of corn steep liquor. After stirring evenly, adjust the pH to 6.8 with NH 4 OH, sterilize at 121 °C for 15 min, and cool naturally.

[0046] (2) Use Corynebacterium glutamicum ATCC13032 (purchased from the Shanghai Center for Microbial Culture Collection) as the seed liquid with a bacterial content of 10 6 CFU / mL, inoculate it into the sterilized fermentation medium at an inoculation amount of 10%, and the initial inoculation concentration of the bacteria is OD 600nm = 0.8. Ferment under the conditions of constant temperature at 30 °C and a shaker at 150 rpm, maintaining the dissolved oxygen concentration in the fermentation broth at 30% of the saturation level. After 48 hours of fermentation, collect the fermentation broth. Throughout the process, ensure that the fermentation conditions are stable to obtain the best fermentation effect.

[0047] 4. Glutamic acid crystallization (1) Centrifuge the fermentation broth at 10000 rpm for 10 minutes in a centrifuge, collect the supernatant, and then adjust its pH value to the isoelectric point of glutamic acid, 3.22, with 1 mol / L hydrochloric acid at a temperature of 40 °C. Then place it in the refrigerator and let it stand until glutamic acid crystals are formed.

[0048] (2) Dissolve the obtained glutamic acid crystals in hot water at 60 °C, adjust the pH to 3.2 - 3.5 again until glutamic acid crystals precipitate again, and repeat the filtration and washing operations. Use the method of slow cooling to promote the uniform growth of glutamic acid crystals and improve the crystal quality.

[0049] 5. Drying The moisture content of the dry glutamate product is usually controlled at 0.5% - 1%, and vacuum drying is carried out at 50 - 60 °C to prevent thermal degradation of glutamate.

[0050] The purity of the dry glutamate product is 63.2%.

[0051] Example 3 Method for Extracting Glutamate from Cannabis 1. Pretreatment of Cannabis Samples After freeze - drying the cannabis with a freeze - dryer, it is crushed using a pulverizer. Add 1 mol / L NaOH according to a liquid - to - solid ratio of 20 mL:1 g, and stir in a magnetic stirrer at room temperature for 24 h. Then reflux and extract at 90 - 100 °C for 1 - 2 h. The above operations are repeated twice, and centrifuged at 6000 rpm for 15 min. Wash the residue with distilled water to remove excess alkali and dissolved lignin until the residue is neutral (pH 6.0 - 7.0), and dry the residue in an oven at 50 °C.

[0052] 2. Composite Enzymolysis Weigh 200 g of the filter residue and mix it with the enzyme solution according to a ratio of 1 g:10 mL. For the preparation of the enzyme solution, a total of 6000 U of cellulase, 4000 U of hemicellulase, and 500 U of β - glucosidase are weighed and placed in 1 L of 0.05 M, pH 4.8 citrate - phosphate buffer. Place the mixture on a shaker at 100 - 200 rpm for enzymatic hydrolysis for 24 h. Then raise the temperature to 95 °C to inactivate the enzyme for 3 min, and separate the undegraded solids by centrifugation. The obtained sugar solution is evaporated and concentrated for the next fermentation step.

[0053] 3. Fermentation Process (1) Take the raw materials of the fermentation medium and configure them according to the following concentrations: 80 g / L of cannabis hydrolysate, 10 g / L of (NH 4 ) 2 SO 4 10 g / L, 2 g / L of KH 2 PO 4 , 0.5 g / L of MgSO 4 ·7H 2 O, 0.01 g / L of MnSO 4 , 1 μg / L of biotin, 20 g / L of corn steep liquor. Stir evenly, adjust the pH to 6.8 with NH 4 OH, sterilize at 121 °C for 15 min, and cool naturally.

[0054] (2) The seed liquid of Corynebacterium glutamicum ATCC13032 (purchased from Shanghai Center for Microbial Culture Collection) has a bacterial content of 10 6 CFU / mL and is inoculated into the sterilized fermentation medium at an inoculation amount of 10%. The initial inoculation concentration of the bacteria is OD 600nm= 0.8, and fermentation was carried out under the conditions of constant temperature at 30 °C and a shaker speed of 150 rpm, maintaining the dissolved oxygen concentration in the fermentation broth at 30% of the saturation level. After 48 hours of fermentation, the fermentation broth was collected. Throughout the process, ensure stable fermentation conditions to obtain the best fermentation effect.

[0055] 4. Glutamic acid crystallization (1) Centrifuge the fermentation broth at 10000 rpm for 10 minutes in a centrifuge, collect the supernatant, and then adjust its pH value to the isoelectric point of glutamic acid, 3.22, with 1 mol / L hydrochloric acid at a temperature of 40 °C. Then place it in the refrigerator and let it stand until glutamic acid crystals are formed.

[0056] (2) Dissolve the obtained glutamic acid crystals in hot water at 60 °C, adjust the pH to 3.2 - 3.5 again until glutamic acid crystals precipitate again, and repeat the filtration and washing operations. Use the method of slow cooling to promote uniform growth of glutamic acid crystals and improve crystal quality.

[0057] 5. Drying The moisture content of the dry glutamic acid product is usually controlled at 0.5% - 1%, and vacuum drying is carried out at 50 - 60 °C to prevent thermal degradation of glutamic acid.

[0058] The purity of the dry glutamic acid product is 75.4%.

[0059] Example 4 Method for extracting glutamic acid from cannabis 1. Pretreatment of cannabis samples After freeze-drying the cannabis with a freeze-dryer, it was pulverized with a pulverizer, and 1 mol / L NaOH was added at a liquid-to-solid ratio of 20 mL:1 g. It was stirred in a magnetic stirrer at room temperature for 24 h, and then refluxed and extracted at 90 °C - 100 °C for 1 - 2 h. The above operations were repeated twice, and centrifuged at a speed of 6000 rpm for 15 min. The residue was washed with distilled water to remove excess alkali and dissolved lignin until the residue was neutral (pH 6.0 - 7.0), and the residue was dried in an oven at 50 °C.

[0060] 2. Composite enzymatic hydrolysis Weigh 200 g of the filter residue and mix it with the enzyme solution at a ratio of 1 g:10 mL. For the preparation of the enzyme solution, a total of 4000 U of cellulase, 2000 U of hemicellulase, and 250 U of β-glucosidase were weighed and placed in 1 L of 0.05 M, pH 4.8 citrate-phosphate buffer. The mixture was placed on a shaker at 100 - 200 rpm for enzymatic hydrolysis for 12 h. Then the temperature was raised to 95 °C to inactivate the enzyme for 3 min, and the undegraded solids were separated by centrifugation. The obtained sugar solution was evaporated and concentrated for the next fermentation step.

[0061] 3. Fermentation process (1) Take the raw materials of the fermentation medium and configure them according to the following concentrations: 80 g / L of hydrolyzed hemp sugar solution, (NH 4 ) 2 SO 4 10 g / L, 2 g / L of KH 2 PO 4 , 0.5 g / L of MgSO 4 ·7H 2 O, 0.01 g / L of MnSO 4 , 1 μg / L of biotin, 20 g / L of corn steep liquor. After stirring evenly, adjust the pH to 6.8 with NH 4 OH, sterilize at 121 °C for 15 min, and cool naturally.

[0062] (2) The seed liquid of Corynebacterium glutamicum ATCC13032 (purchased from Shanghai Center for Microbial Culture Collection) has a bacterial content of 10 6 CFU / mL. Inoculate it into the sterilized fermentation medium at an inoculation amount of 10%, and the initial inoculation concentration of the bacteria is OD 600nm = 0.8. Ferment under the conditions of constant temperature at 30 °C and a shaker at 150 rpm, and maintain the dissolved oxygen concentration in the fermentation broth at 30% of the saturation level. After 48 hours of fermentation, collect the fermentation broth. During the whole process, ensure that the fermentation conditions are stable to obtain the best fermentation effect.

[0063] 4. Glutamic acid crystallization (1) Centrifuge the fermentation broth at 10000 rpm for 10 minutes in a centrifuge, collect the supernatant, and then adjust its pH value to the isoelectric point of glutamic acid, 3.22, with 1 mol / L hydrochloric acid at a temperature of 40 °C. Then place it in the refrigerator and let it stand until glutamic acid crystals are formed.

[0064] (2) Dissolve the obtained glutamic acid crystals in hot water at 60 °C, and adjust the pH to 3.2 - 3.5 again until glutamic acid crystals precipitate again. Repeat the filtration and washing operations. Use the method of slow cooling to promote the uniform growth of glutamic acid crystals and improve the crystal quality.

[0065] 5. Drying The moisture content of the dried glutamic acid product is usually controlled at 0.5% - 1%, and vacuum drying is carried out at 50 - 60 °C to prevent thermal degradation of glutamic acid.

[0066] The purity of the dried glutamic acid product is 69.7%.

[0067] Example 5 Optimization of process conditions Enzymolysis temperature: Take 5 g of pretreated hemp powder, with a liquid-to-solid ratio of 10 mL:1 g and an extraction time of 24 h. Investigate the effect of enzymolysis temperature (40, 45, 50, 55, 60 °C) on sugar content.

[0068] Enzymolysis time: Take 5 g of pretreated hemp powder, with an enzymolysis temperature of 50 °C and a liquid-to-solid ratio of 15 mL:1 g. Investigate the effect of enzyme extraction time (18, 20, 22, 24, 26 h) on sugar content.

[0069] Liquid-to-solid ratio: Take 5 g of pretreated hemp powder, with an enzymolysis temperature of 55 °C and an extraction time of 24 h. Investigate the effect of liquid-to-solid ratio (5 mL:1 g, 10 mL:1 g, 15 mL:1 g, 20 mL:1 g, 25 mL:1 g) on sugar content.

[0070] According to the results of single-factor experiments, three factors were selected for response surface design (Table 1): Table 1 Response surface level table It can be seen from Figure 1 that the yield of polysaccharides increases with the increase of temperature between 40 and 50 °C. However, when the extraction temperature exceeds 50 °C, the polysaccharide yield decreases rapidly. This may be because the composite enzyme has the strongest activity at 50 °C and the best effect on decomposing the cell wall. When the temperature continues to rise, the enzyme activity decreases due to the heat denaturation of the enzyme protein, resulting in a decrease in the polysaccharide yield. Therefore, 45 - 55 °C was selected for the response surface experiment.

[0071] Enzymolysis time is also an important factor in polysaccharide extraction. Longer enzyme treatment is beneficial to the production of polysaccharides. As can be seen from the figure, the polysaccharide yield gradually increases with the increase of time, but the growth trend flattens out later. Therefore, 22 - 24 h was selected for the subsequent response surface experiment.

[0072] The polysaccharide yield first increases and then decreases with the increase of the liquid-to-solid ratio. It reaches the maximum at 15 mL:1 g, probably because the increase in volume expands the contact area between polysaccharides and the composite enzyme in the reaction system. As the liquid-to-solid ratio increases, the viscosity of the extraction solution decreases, and more polysaccharide molecules are dissolved. When the solvent dosage exceeds a certain range, the mass concentration of the composite enzyme will be significantly reduced, thereby affecting the catalytic efficiency of the enzyme. This is the reason why the polysaccharide yield decreases instead as the solvent dosage increases continuously. Therefore, 5:1 - 15:1 (mL:g) was selected for the response surface experiment (Table 2).

[0073] Table 2 Response surface design and experimental results Table 3 Results of regression statistical analysis Using software for multiple regression fitting and calculating the regression coefficients, the multiple regression equation of the polysaccharide yield with respect to the enzymolysis temperature (C), enzymolysis time (B), and liquid-to-solid ratio (C) can be obtained as: Y = 13.26 - 0.2875A - 0.2B - 0.1625C - 0.35AB + 0.125AC - 1.27A 2 -0.8425B 2 -0.5675C 2 。This model has a significant variance, while the lack-of-fit term is not significant, indicating that this model can better predict the polysaccharide yield. It can be seen from the software that the optimal extraction process is a liquid-to-solid ratio of 9.22 mL:1 g, at 49.56 °C, and extracting for 23.84 h, with a yield of 13.30% at this time. In actual operation, a liquid-to-solid ratio of 10 mL:1 g is selected, extracting at 50 °C for 24 h, with a yield of 13.05% and an error of 0.25% (Table 3).

[0074] The response surface 3D diagrams and contour diagrams of the interaction between enzymolysis time, enzymolysis temperature, and liquid-to-solid ratio on the yield of cannabis polysaccharide are shown in Figure 2 。

[0075] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for extracting glutamic acid from cannabis, characterized in that: The steps include: a) Pre-treatment of cannabis samples; b) performing complex enzymatic hydrolysis on the extract obtained after the pre-treatment in step a); c) fermenting the enzymatic hydrolysis product obtained in step b) as a substrate; d) the fermentation broth obtained in step c) is concentrated and crystallized to obtain glutamate crystals; e) The glutamic acid crystals are recrystallized, purified, and dried.

2. The method according to claim 1, characterized in that Step a) comprises: a1) Mix the dried cannabis powder with a 0.5-1.0 mol / L NaOH solution at a ratio of 1 g: (20-25) mL and let stand at room temperature for 24-26 hours; a2) placing the mixture in a magnetic stirrer and extracting at 90°C-100°C for 1-2 hours; after the extraction is completed, centrifuging at 6000-7000 rpm for 15-20 minutes, collecting the residue after centrifugation, and extracting the residue again under the above conditions; a3) washing the residue with distilled water until the residue becomes neutral; a4) Finally, dry the washed residue for later use.

3. The method according to claim 2, characterized in that Step b) comprises: b1) Preparation of enzyme solution: weigh 10-30 U of cellulase, 5-15 U of hemicellulase, and 0.5-2 U of β-glucosidase per gram of residual residue, and dissolve them in 0.05 M citric acid-phosphate buffer with a pH value of 4.8 to prepare enzyme solution; b2) the residue obtained in step a) is mixed evenly with the enzyme solution at a ratio of 1 g: (5-10) mL, and then enzymolysis is performed at the optimum temperature of cellulase, 50°C, and the pH range of the reaction system is controlled to be 4.8-5.

5. During the enzymolysis process, stirring is performed at a low speed of 100-200 rpm, and the enzymolysis is performed for 24-72 hours; b3) After the enzymatic hydrolysis is completed, the sugar solution obtained by the enzymatic hydrolysis is separated from the undegraded solid matter by filtering or centrifuging to obtain the desired sugar solution.

4. The method according to claim 3, characterized in that Step c) comprises: c1) using the sugar solution obtained in step b) as a carbon source, preparing the following fermentation medium: total sugar concentration 50-100 g / L, ammonium sulfate 10-15 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 0.5-1.0 g / L, MnSO4 0.01-0.1 g / L, biotin 1 µg / L, corn steep liquor 20-30 g / L, and adjusting the pH to 6.8-7.5 with NH4OH; c2) Inoculate 5% to 15% v / v Corynebacterium glutamicum into the fermentation medium ( Corynebacterium glutamicum ) Seed solution, ferment at 30-35℃ in a shaker at 150-200rpm for 48-72 hours, maintaining a saturated dissolved oxygen concentration of 20%-50%.

5. The method according to claim 4, characterized in that The Corynebacterium glutamicum is ATCC13032.

6. The method according to claim 4, characterized in that The bacterial content of Corynebacterium glutamicum in the seed solution is 10 6 CFU / mL.

7. The method according to any one of claims 4 to 6, characterized in that: Step d) comprises: d1) taking out the fermentation broth from the shaker and centrifuging it at 8000-10000 rpm for 10-15 minutes; then, transferring the obtained supernatant to a rotary evaporator for vacuum concentration; d2) Add 1 mol / L hydrochloric acid to the concentrated supernatant, stir at 40-50°C, adjust the pH value of the supernatant to the isoelectric point of glutamic acid 3.22, and then let it stand in a refrigerator to form glutamic acid crystals.

8. The method according to claim 7, characterized in that Step e) comprises: dissolving the obtained glutamic acid crystals in hot water at 60-80° C., adjusting the pH to 3.2-3.5 again until glutamic acid crystals are precipitated again, and repeating the filtering and washing operations to obtain purified crystals.

9. The method according to claim 8, characterized in that The purified crystals were vacuum dried at 50-60°C to obtain pure glutamic acid.

10. The method according to claim 9, characterized in that The water content of pure glutamic acid is 0.5%-1%.

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