Method for extracting grease in walnut shells by using biological enzyme method

Abstract

The invention relates to the field of comprehensive utilization of biological resources, and discloses a method for extracting grease in walnut shells by using a biological enzyme method, and the method comprises the following steps: raw material pretreatment, first-stage enzymolysis, second-stage enzymolysis, extraction and post-treatment. According to the grease extraction method, waste utilization of the walnut shells is achieved, the grease in the walnut shells is extracted in combination with a biological enzyme method, and the influence of high-temperature and high-pressure conditions on the quality of the extracted grease is avoided; meanwhile, the oil extraction method disclosed by the invention is low in energy consumption, the use amount of an organic solvent is greatly reduced, and the environmental protection property is strong; moreover, the oil extraction method disclosed by the invention is mild in condition and suitable for industrial production, the final extraction rate of the oil in the walnut shells can reach 4.08-4.4%, and efficient, environment-friendly and low-cost industrial production of the oil is realized.

Description

Technical Field

[0001] This invention relates to the field of comprehensive utilization of biological resources, specifically to a method for extracting oil from walnut shells using a biological enzymatic method. Background Technology

[0002] Walnut( Royal walnut Walnuts (L.) belong to the genus Juglans of the family Juglandaceae, and are also known as "walnuts" or "Qiang walnuts." Walnuts are rich in nutrients and are considered one of the world's "four major dried fruits." my country has abundant walnut cultivation resources, with annual walnut production reaching several million tons, resulting in a large amount of byproducts—walnut shells. Walnut shells are a byproduct of kernel extraction; a very small amount is used to extract walnut brown pigment and produce activated carbon, while the majority is discarded or incinerated as waste. The waste gas from incineration and the soil pollution from landfilling are becoming increasingly serious problems. At the same time, the usable components contained in walnut shells have not been effectively developed, resulting in resource waste. Summary of the Invention

[0003] This invention provides a method for extracting oil from walnut shells using a bio-enzymatic method, thereby solving the problem of efficient extraction and utilization of oil from walnut shells.

[0004] In a first aspect, the present invention provides a method for extracting oil from walnut shells using a biological enzymatic method, the method comprising the following steps: Raw material pretreatment steps: The walnut shells are crushed and then extracted to obtain walnut shell extract; One enzymatic hydrolysis step: Adjust the pH of the walnut shell extract obtained from the raw material pretreatment step to 4.5-5.5, adjust the temperature to 40℃-50℃, add the first group of enzymes, the amount of the first group of enzymes added to the walnut shell extract is 200U / g-1000U / g, and obtain the first enzymatic hydrolysis product after 0.5-3 hours of enzymatic hydrolysis. Two-stage enzymatic hydrolysis step: Adjust the pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step to 8.0-9.0, adjust the temperature to 45℃-55℃, add the second group of enzymes, the amount of the second group of enzymes added to the first enzymatic hydrolysis product is 800U / g-4000U / g, and obtain the second enzymatic hydrolysis product after enzymatic hydrolysis for 0.5-3 hours; Extraction steps: Extractant is added to the second enzymatic hydrolysis product obtained in the two-stage enzymatic hydrolysis step and then extracted. After extraction, the supernatant is collected. Post-processing step: Remove the extractant from the supernatant obtained in the extraction step to obtain the oil.

[0005] In one alternative embodiment, the amount of enzyme added refers to the enzyme activity units added per gram of substrate.

[0006] In one optional embodiment, in the first enzymatic hydrolysis step, the amount of the first group of enzymes added to the walnut shell extract is 1000 U / g; in the second enzymatic hydrolysis step, the amount of the second group of enzymes added to the first enzymatic hydrolysis product is 4000 U / g.

[0007] In one alternative embodiment, the amount of enzyme added refers to the enzyme activity units added per gram of substrate.

[0008] In one optional embodiment, the enzymatic hydrolysis time is 1 hour in the first enzymatic hydrolysis step and 2 hours in the second enzymatic hydrolysis step.

[0009] In one optional embodiment, in the first enzymatic hydrolysis step, the first group of enzymes includes cellulase; in the second enzymatic hydrolysis step, the second group of enzymes includes alkaline protease.

[0010] In one optional embodiment, the cellulase has an enzyme activity of 50,000 U / g, and the alkaline protease has an enzyme activity of 200,000 U / g.

[0011] In one optional embodiment, the raw material pretreatment step includes: crushing the walnut shells, soaking them in water, and then subjecting them to ultrasonic treatment to obtain a walnut shell extract.

[0012] In one optional embodiment, in the raw material pretreatment step, the ratio of walnut shells to water is 1:6 (g:mL), and the soaking time is 20-24 hours.

[0013] In one optional embodiment, in the raw material pretreatment step, the ultrasonic power is 500W and the time is 25-30 minutes.

[0014] In one optional embodiment, the extractant in the extraction step includes n-hexane; the ratio of the second enzymatic hydrolysis product to n-hexane is 1:5 to 1:9 (g:mL).

[0015] In one optional embodiment, the ratio of the second enzymatic hydrolysis product to n-hexane is 1:7 (g:mL).

[0016] In one alternative implementation, the extraction step utilizes ultrasound-assisted processing.

[0017] In one optional embodiment, the ultrasonic-assisted treatment has a power of 500W and a duration of 35-45 minutes.

[0018] In one alternative implementation, the ultrasound-assisted treatment lasts for 40 minutes.

[0019] In one alternative embodiment, the post-processing step includes: rotary evaporation to remove the extractant.

[0020] In one optional embodiment, in the post-processing step, the rotary evaporation temperature is 55-60°C, the rotation speed is 80-120 r / s, and the time is 60-120 min.

[0021] The technical solution of this invention has the following advantages: This invention provides a method for extracting oil from walnut shells using a bio-enzymatic method. The method includes the following steps: raw material pretreatment, primary enzymatic hydrolysis, secondary enzymatic hydrolysis, extraction, and post-treatment. This oil extraction method utilizes walnut shells as waste material and, by combining it with a bio-enzymatic method, achieves an oil extraction rate of 4.08-4.4%, enabling efficient, environmentally friendly, and low-cost industrial production.

[0022] Furthermore, the bio-enzymatic method is an emerging oil extraction method that applies enzyme preparations to oil separation. Through the mechanical disruption of oilseed cell walls and enzymatic degradation, the oil extraction rate is improved. Because it avoids the use of organic solvents and high-temperature, high-pressure conditions, the processing conditions are milder compared to traditional oil extraction processes. The degradation products in the system generally do not react with the extract, effectively protecting usable components such as oils, proteins, and gums, thus ensuring product quality and environmental safety. The oil extraction method described in this invention, by using a bio-enzymatic method to extract oil from walnut shells, avoids the impact of high-temperature, high-pressure conditions on the quality of the extracted oil. It also features low energy consumption, significantly reduced organic solvent usage, strong environmental friendliness, and mild process conditions, making it suitable for industrial production.

[0023] Furthermore, walnut shells, as a byproduct of walnut production, represent a significant waste of resources. This invention, however, discovers that walnut shells contain a certain amount of oil and provides a bio-enzymatic method for extracting this oil. Using the oil extraction method described in this invention, the oil extraction rate from walnut shells can reach 4.08% to 4.4%, achieving the green reuse of walnut shells. Detailed Implementation

[0024] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0025] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0026] The reagent and material information used in the embodiments and comparative examples of this invention is as follows: HCl (1 mol / L) solution: Beijing Chemical Plant NaOH (1 mol / L) solution: Beijing Chemical Plant Cellulase: Purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with an enzyme activity of 50,000 U / g; Alkaline protease: purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with an enzyme activity of 200,000 U / g.

[0027] Example 1: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0028] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 50℃. Cellulase was added at a concentration of 1000 U / g, that is, 1000 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 1 hour of enzymatic hydrolysis.

[0029] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 50℃. Alkaline protease was added at a concentration of 4000 U / g, that is, 4000 U of enzyme activity units were added per g of substrate (first enzymatic hydrolysis product). After 2 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0030] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 40 minutes. After ultrasonic treatment, the supernatant was collected.

[0031] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 120r / s for 120min to remove n-hexane from the supernatant, yielding oil.

[0032] Example 2: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0033] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.5 using HCl (1 mol / L) solution, and the temperature was adjusted to 40℃. Cellulase was added at a concentration of 200 U / g, that is, 200 U of enzyme activity units were added per gram of substrate (walnut shell extract). After enzymatic hydrolysis for 0.5 hours, the first enzymatic hydrolysis product was obtained.

[0034] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 45℃. Alkaline protease was added at a concentration of 800 U / g, that is, 800 U of enzyme activity units were added per g of substrate (first enzymatic hydrolysis product). After enzymatic hydrolysis for 0.5 hours, the second enzymatic hydrolysis product was obtained.

[0035] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:5 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 35 minutes. After ultrasonic treatment, the supernatant was collected.

[0036] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 55℃ and 80r / s for 60min to remove n-hexane from the supernatant, yielding oil.

[0037] Example 3: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0038] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 5.5 with HCl (1 mol / L) solution and the temperature was adjusted to 50℃. Cellulase was added at a concentration of 1000 U / g, that is, 1000 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 3 hours of enzymatic hydrolysis.

[0039] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 9.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 55℃. Alkaline protease was added at a concentration of 4000 U / g, that is, 4000 U of enzyme activity units were added per gram of substrate (first enzymatic hydrolysis product). After 3 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0040] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:9 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 45 minutes. After ultrasonic treatment, the supernatant was collected.

[0041] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 120r / s for 120min to remove n-hexane from the supernatant, yielding oil.

[0042] Example 4: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0043] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 5.0 with HCl (1 mol / L) solution and the temperature was adjusted to 45℃. Cellulase was added at a concentration of 400 U / g, that is, 400 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 2 hours of enzymatic hydrolysis.

[0044] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.5 using NaOH (1 mol / L) solution, and the temperature was adjusted to 55℃. Alkaline protease was added at a concentration of 1600 U / g, that is, 1600 U of enzyme activity units were added per gram of substrate (first enzymatic hydrolysis product). After 2 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0045] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:8 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 40 minutes. After ultrasonic treatment, the supernatant was collected.

[0046] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 55℃ and 100r / s for 80min to remove n-hexane from the supernatant, yielding oil.

[0047] Example 5: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0048] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 5.0 with HCl (1 mol / L) solution and the temperature was adjusted to 40℃. Cellulase was added at a concentration of 600 U / g, that is, 600 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 1 hour of enzymatic hydrolysis.

[0049] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.5 using NaOH (1 mol / L) solution, and the temperature was adjusted to 45℃. Alkaline protease was added at a concentration of 2400 U / g, that is, 2400 U of enzyme activity units were added per g of substrate (first enzymatic hydrolysis product). After 1 hour of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0050] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:6 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 40 minutes. After ultrasonic treatment, the supernatant was collected.

[0051] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 80r / s for 100min to remove n-hexane from the supernatant, yielding oil.

[0052] Example 6: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0053] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 40℃. Cellulase was added at a concentration of 800 U / g, that is, 800 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 1 hour of enzymatic hydrolysis.

[0054] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 50℃. Alkaline protease was added at a concentration of 3200 U / g, that is, 3200 U of enzyme activity units were added per gram of substrate (first enzymatic hydrolysis product). After 2 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0055] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 35 minutes. After ultrasonic treatment, the supernatant was collected.

[0056] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 120r / s for 120min to remove n-hexane from the supernatant, yielding oil.

[0057] Example 7: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0058] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 50℃. Cellulase was added at a concentration of 600 U / g, that is, 600 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 1.5 hours of enzymatic hydrolysis.

[0059] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 40℃. Alkaline protease was added at a concentration of 2400 U / g, that is, 2400 U of enzyme activity units were added per g of substrate (first enzymatic hydrolysis product). After 1.5 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0060] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 40 minutes. After ultrasonic treatment, the supernatant was collected.

[0061] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 100r / s for 120min to remove n-hexane from the supernatant, yielding oil.

[0062] Example 8: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0063] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 50℃. Cellulase was added at a concentration of 1000 U / g, that is, 1000 U of enzyme activity units were added per gram of substrate (walnut shell extract). After enzymatic hydrolysis for 2.5 hours, the first enzymatic hydrolysis product was obtained.

[0064] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 50℃. Alkaline protease was added at a concentration of 4000 U / g, that is, 4000 U of enzyme activity units were added per gram of substrate (first enzymatic hydrolysis product). After 2.5 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0065] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 40 minutes. After ultrasonic treatment, the supernatant was collected.

[0066] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 120r / s for 120min to remove n-hexane from the supernatant, yielding oil.

[0067] Comparative Example 1: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0068] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 40℃. Cellulase was added at a concentration of 1000 U / g, that is, 1000 U of enzyme activity units were added per gram of substrate (walnut shell extract). After 1 hour of enzymatic hydrolysis, the enzymatic hydrolysis product was obtained.

[0069] 3. Extraction After cooling the enzymatic hydrolysis product obtained from the enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 30 minutes. After ultrasonic treatment, the supernatant was collected.

[0070] 4. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 120r / s for 120min to remove n-hexane from the supernatant, yielding oil.

[0071] Comparative Example 2: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0072] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 40℃. Alkaline protease was added at a concentration of 4000 U / g, that is, 4000 U of enzyme activity units were added per gram of substrate (walnut shell extract). After 2 hours of enzymatic hydrolysis, the enzymatic hydrolysis product was obtained.

[0073] 3. Extraction After cooling the enzymatic hydrolysis product obtained from the enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 30 minutes. After ultrasonic treatment, the supernatant was collected.

[0074] 4. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 120r / s for 120min to remove n-hexane from the supernatant, yielding oil.

[0075] Comparative Example 3: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0076] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 7.0 using HCl (1 mol / L) and NaOH (1 mol / L) solutions, and the temperature was adjusted to 50℃. Cellulase and alkaline protease were added. The amount of cellulase added was 1000 U / g, that is, 1000 U of enzyme activity units were added per g of substrate (walnut shell extract); the amount of alkaline protease added was 4000 U / g, that is, 4000 U of enzyme activity units were added per g of substrate (walnut shell extract). After 2 hours of enzymatic hydrolysis, the enzymatic hydrolysis product was obtained.

[0077] 3. Extraction After cooling the enzymatic hydrolysis product obtained from the enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 30 minutes. After ultrasonic treatment, the supernatant was collected.

[0078] 4. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 80r / s for 100min to remove n-hexane from the supernatant, yielding oil.

[0079] Comparative Example 4: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0080] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 50℃. Alkaline protease was added at a concentration of 4000 U / g, that is, 4000 U of enzyme activity units were added per gram of substrate (walnut shell extract). After 2 hours of enzymatic hydrolysis, the first enzymatic hydrolysis product was obtained.

[0081] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 50℃. Cellulase was added at a concentration of 1000 U / g, that is, 1000 U of enzyme activity units were added per gram of substrate (first enzymatic hydrolysis product). After 1 hour of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0082] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 30 minutes. After ultrasonic treatment, the supernatant was collected.

[0083] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 80r / s for 100min to remove n-hexane from the supernatant, yielding oil.

[0084] Comparative Example 5: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0085] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 50℃. Cellulase was added at a concentration of 1000 U / g, that is, 1000 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 1 hour of enzymatic hydrolysis.

[0086] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 50℃. Alkaline protease was added at a concentration of 4000 U / g, that is, 4000 U of enzyme activity units were added per g of substrate (first enzymatic hydrolysis product). After 2 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0087] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 30 minutes. After ultrasonic treatment, the supernatant was collected.

[0088] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 80r / s for 100min to remove n-hexane from the supernatant, yielding oil.

[0089] Comparative Example 6: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0090] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 40℃. Cellulase was added at a concentration of 1000 U / g, that is, 1000 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 1 hour of enzymatic hydrolysis.

[0091] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 40℃. Alkaline protease was added at a concentration of 4000 U / g, that is, 4000 U of enzyme activity units were added per gram of substrate (first enzymatic hydrolysis product). After 2 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0092] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 10 minutes. After ultrasonic treatment, the supernatant was collected.

[0093] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 80r / s for 100min to remove n-hexane from the supernatant, yielding oil.

[0094] Comparative Example 7: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0095] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 40℃. Cellulase was added at a concentration of 1000 U / g, that is, 1000 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 1 hour of enzymatic hydrolysis.

[0096] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 40℃. Alkaline protease was added at a concentration of 4000 U / g, that is, 4000 U of enzyme activity units were added per gram of substrate (first enzymatic hydrolysis product). After 2 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0097] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 20 minutes. After ultrasonic treatment, the supernatant was collected.

[0098] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 80r / s for 100min to remove n-hexane from the supernatant, yielding oil.

[0099] Comparative Example 8: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0100] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 40℃. Cellulase was added at a concentration of 1000 U / g, that is, 1000 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 1 hour of enzymatic hydrolysis.

[0101] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 40℃. Alkaline protease was added at a concentration of 4000 U / g, that is, 4000 U of enzyme activity units were added per gram of substrate (first enzymatic hydrolysis product). After 2 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0102] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 30 minutes. After ultrasonic treatment, the supernatant was collected.

[0103] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 80r / s for 100min to remove n-hexane from the supernatant, yielding oil.

[0104] Comparative Example 9: A method for extracting oil from walnut shells using a bio-enzymatic method 1. Raw material pretreatment After crushing the walnut shells, add water at a ratio of 1:6 (g:mL) and soak for 24 hours. After soaking, sonicate at 500 W for 30 minutes to obtain walnut shell extract.

[0105] 2. Enzymatic hydrolysis The walnut shell extract obtained from the raw material pretreatment step was adjusted to pH 4.8 using HCl (1 mol / L) solution, and the temperature was adjusted to 40℃. Cellulase was added at a concentration of 1000 U / g, that is, 1000 U of enzyme activity units were added per gram of substrate (walnut shell extract). The first enzymatic hydrolysis product was obtained after 1 hour of enzymatic hydrolysis.

[0106] 3. Two-stage enzymatic hydrolysis The pH of the first enzymatic hydrolysis product obtained from the first enzymatic hydrolysis step was adjusted to 8.0 using NaOH (1 mol / L) solution, and the temperature was adjusted to 40℃. Alkaline protease was added at a concentration of 4000 U / g, that is, 4000 U of enzyme activity units were added per gram of substrate (first enzymatic hydrolysis product). After 2 hours of enzymatic hydrolysis, the second enzymatic hydrolysis product was obtained.

[0107] 4. Extraction After cooling the second enzymatic hydrolysis product obtained from the two-stage enzymatic hydrolysis step to 25°C, n-hexane was added at a material-to-liquid ratio of 1:7 (g:mL). After the addition was completed, the mixture was extracted by ultrasonic treatment at 500 W for 50 minutes. After ultrasonic treatment, the supernatant was collected.

[0108] 5. Post-processing The supernatant obtained from the extraction step was rotary evaporated at 60℃ and 80r / s for 100min to remove n-hexane from the supernatant, yielding oil.

[0109] Experimental Example 1: Calculating the oil extraction rate This experimental example provides a method for calculating the oil extraction rate of Examples 1-8 and Comparative Examples 1-9 above by weighing. The specific experimental steps are as follows: After rotary evaporation, the clear oil portion is removed, weighed, and the oil extraction rate is calculated using the following formula.

[0110]

[0111] In the formula: X is the oil extraction rate (%); m1 is the mass of the receiving bottle and oil after constant weight (g); m0 is the mass of the receiving bottle (g); m2 is the mass of the walnut shell powder (g).

[0112] The results are shown in Table 1. The extraction rates of walnut shell oil by the methods in Examples 1-8 were 4.08-4.4%, which were significantly higher than those in Comparative Examples 1-9. This indicates that the methods in Examples 1-8 have achieved the utilization of walnut shell waste and are suitable for efficient, environmentally friendly and low-cost industrial production of oil.

[0113] Table 1 Results of Walnut Shell Oil Extraction Rate Determination

[0114] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for extracting oil from walnut shell by using a biological enzyme, characterized in that, The method comprises the following steps: A raw material pretreatment step: walnut shell is crushed and extracted to obtain a walnut shell extract; A first enzymolysis step: the walnut shell extract obtained in the raw material pretreatment step is adjusted to a pH of 4.5-5.5 and a temperature of 40-50°C, a first group of enzymes is added, the addition amount of the first group of enzymes in the walnut shell extract is 200-1000 U / g, and a first enzymolysis product is obtained after enzymolysis for 0.5-3 hours; A second enzymolysis step: the first enzymolysis product obtained in the first enzymolysis step is adjusted to a pH of 8.0-9.0 and a temperature of 45-55°C, a second group of enzymes is added, the addition amount of the second group of enzymes in the first enzymolysis product is 800-4000 U / g, and a second enzymolysis product is obtained after enzymolysis for 0.5-3 hours; An extraction step: an extractant is added to the second enzymolysis product obtained in the second enzymolysis step, and after the extraction is completed, the supernatant is collected; A post-treatment step: the extractant in the supernatant obtained in the extraction step is removed to obtain oil.

2. The method of claim 1, wherein, In the first enzymolysis step, the addition amount of the first group of enzymes in the walnut shell extract is 1000 U / g; in the second enzymolysis step, the addition amount of the second group of enzymes in the first enzymolysis product is 4000 U / g.

3. The method of claim 1, wherein, In the first enzymolysis step, the enzymolysis time is 1 hour; in the second enzymolysis step, the enzymolysis time is 2 hours.

4. The method according to any one of claims 1 to 3, characterized in that, In the first enzymolysis step, the first group of enzymes comprises cellulase; in the second enzymolysis step, the second group of enzymes comprises alkaline protease.

5. The method according to any one of claims 1 to 3, characterized in that, The raw material pretreatment step comprises: after the walnut shell is crushed, it is first soaked in water and then ultrasonically treated to obtain a walnut shell extract; Preferably, in the raw material pretreatment step, the solid-liquid ratio of the walnut shell and water is 1:6 (g:mL), and the soaking time is 20-24 hours.

6. The method of claim 5, wherein, In the raw material pretreatment step, the ultrasonic power is 500 W, and the time is 25-30 minutes.

7. The method of claim 1, wherein, In the extraction step, the extractant comprises n-hexane; the solid-liquid ratio of the second enzymolysis product and n-hexane is 1:5-1:9 (g:mL); Preferably, the solid-liquid ratio of the second enzymolysis product and n-hexane is 1:7 (g:mL).

8. The method of claim 1, wherein, In the extraction step, the extraction process uses ultrasonic auxiliary treatment; Preferably, the ultrasonic auxiliary treatment has a power of 500 W and a time of 35-45 minutes; More preferably, the ultrasonic auxiliary treatment has a time of 40 minutes.

9. The method of claim 1, wherein, The post-treatment step comprises: removing the extractant by rotary evaporation.

10. The method of claim 9, wherein, In the post-treatment step, the rotary evaporation has a temperature of 55-60°C, a rotation speed of 80-120 r / s, and a time of 60-120 min.

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