CN114214118A - Process for extracting wood frog egg oil by enzyme method - Google Patents
Process for extracting wood frog egg oil by enzyme method Download PDFInfo
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- 241000191896 Rana sylvatica Species 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 50
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 36
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 36
- 230000008569 process Effects 0.000 title claims description 30
- 235000013601 eggs Nutrition 0.000 claims abstract description 67
- 238000000605 extraction Methods 0.000 claims abstract description 55
- 239000007857 degradation product Substances 0.000 claims abstract description 43
- 238000000498 ball milling Methods 0.000 claims abstract description 41
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000002156 mixing Methods 0.000 claims abstract description 24
- 239000000047 product Substances 0.000 claims abstract description 23
- 238000003756 stirring Methods 0.000 claims abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000002002 slurry Substances 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000000843 powder Substances 0.000 claims abstract description 16
- 238000006731 degradation reaction Methods 0.000 claims abstract description 12
- 238000001556 precipitation Methods 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 241001336827 Rana chensinensis Species 0.000 claims abstract description 10
- 238000003825 pressing Methods 0.000 claims abstract description 8
- 230000015556 catabolic process Effects 0.000 claims abstract description 7
- 230000004048 modification Effects 0.000 claims abstract description 7
- 238000012986 modification Methods 0.000 claims abstract description 7
- 230000000415 inactivating effect Effects 0.000 claims abstract description 4
- 229940088598 enzyme Drugs 0.000 claims description 32
- 239000002904 solvent Substances 0.000 claims description 15
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 claims description 14
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 claims description 14
- 210000003101 oviduct Anatomy 0.000 claims description 14
- 235000019441 ethanol Nutrition 0.000 claims description 11
- 238000000265 homogenisation Methods 0.000 claims description 8
- 238000001704 evaporation Methods 0.000 claims description 7
- 108090000145 Bacillolysin Proteins 0.000 claims description 6
- 102000035092 Neutral proteases Human genes 0.000 claims description 6
- 108091005507 Neutral proteases Proteins 0.000 claims description 6
- 108090000526 Papain Proteins 0.000 claims description 6
- 239000004365 Protease Substances 0.000 claims description 6
- 102000004142 Trypsin Human genes 0.000 claims description 6
- 108090000631 Trypsin Proteins 0.000 claims description 6
- 229940055729 papain Drugs 0.000 claims description 6
- 235000019834 papain Nutrition 0.000 claims description 6
- 239000012588 trypsin Substances 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 239000012752 auxiliary agent Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical group [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- MCMNRKCIXSYSNV-UHFFFAOYSA-N Zirconium dioxide Chemical compound O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 description 12
- 239000011324 bead Substances 0.000 description 12
- 150000004676 glycans Chemical class 0.000 description 8
- 229920001282 polysaccharide Polymers 0.000 description 8
- 239000005017 polysaccharide Substances 0.000 description 8
- 239000002245 particle Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 230000009849 deactivation Effects 0.000 description 4
- 238000000227 grinding Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 150000002978 peroxides Chemical class 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- 206010010904 Convulsion Diseases 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
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- 230000036541 health Effects 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
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- 229940118019 malondialdehyde Drugs 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- 229930182833 estradiol Natural products 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
- C11B9/025—Recovery by solvent extraction
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Fats And Perfumes (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides an extraction process of wood frog egg oil by an enzyme method, which comprises the steps of firstly, ball-milling dried wood frog eggs into wood frog egg powder, pressing the wood frog egg powder into blank pieces, and performing subcritical extraction to obtain extraction oil and residues; adding the residue into water, stirring and mixing uniformly to obtain slurry, adding mixed enzyme into the slurry, performing enzymolysis, and inactivating enzyme to obtain an enzymolysis product; then performing microwave degradation on the enzymolysis product, centrifuging to obtain supernatant, performing alcohol precipitation to obtain a degradation product, and performing modification treatment on the degradation product by using acetic anhydride to obtain a modified degradation product; and finally, adding the modified degradation product into the extracted oil, uniformly mixing, and homogenizing to obtain the rana chensinensis egg oil. The woodfrog egg oil obtained by the invention has high content of effective components and high stability.
Description
Technical Field
The invention relates to the technical field of processing of wood frog eggs, in particular to an extraction process for processing wood frog egg oil by an enzyme method.
Background
The wood frog egg is a common byproduct after wood frog oil extraction, has complex components, and contains basic components such as water, polysaccharide, protein, phospholipids, amino acid, fatty acid, various trace elements and the like. In recent years, through research, the wood frog eggs have the potential of being developed in the fields of medicines and health care products, are novel raw materials, and can fully improve the utilization rate of wood frog resources. The wood frog egg has obvious effects of resisting tumor, convulsion, fatigue, oxidation, blood fat and liver injury.
The wood frog egg oil is egg oil obtained by extracting female adult wood frog, and is dark brown liquid oil. Through research, the wood frog egg oil has the effects of resisting convulsion, resisting anxiety, regulating blood fat and the like, the effective components of the wood frog egg oil comprise protein, amino acid, unsaturated fatty acid, vitamin E, trace elements and the like, the basic category of the wood frog oil is the same as that of the wood frog oil, but the female hormone components such as estradiol and the like are far higher than the wood frog oil, the content of the female hormone components is more than 1000 times of that of the wood frog oil, and the wood frog egg oil is an important raw material source of health-care food and cosmetics.
The existing extraction method of woodfrog egg oil comprises solvent extraction method and supercritical CO2Extraction method, etc. all need to use a large amount of organic solvent petroleum ether or cyclohexane, the production process has great harm to human body, and the residual amount of solvent in the product is large.
Patent application CN101690732A discloses a method for preparing a health product of wood frog oil, wherein, the wood frog egg oil adopts supercritical CO2The extraction method is adopted. However, supercritical CO2The extraction method has expensive production equipment, large energy consumption and high production cost, is not suitable for industrial production, and currently still stays in the laboratory production scale. In addition, the acid value and the peroxide value of the woodfrog egg oil obtained by the current method are high, and the peroxide value exceeds the sanitary standard, which indicates that the woodfrog egg oil is not fresh oil and begins to be rancid; the acid value is beyond the hygienic standard, indicating that the grease is and has been rancid.
Disclosure of Invention
The invention aims to provide an extraction process for the enzymatic processing of wood frog egg oil, and the obtained wood frog egg oil has high content of effective components and high stability.
In order to achieve the purpose, the invention is realized by the following scheme:
the process for extracting the wood frog egg oil by the enzyme method comprises the following specific steps:
(1) ball-milling dried oviductus Ranae into oviductus Ranae powder, pressing into blank, and performing subcritical extraction to obtain extract oil and residue;
(2) adding the residue into water, stirring and mixing uniformly to obtain slurry, adding mixed enzyme into the slurry, performing enzymolysis, and inactivating enzyme to obtain an enzymolysis product;
(3) then performing microwave degradation on the enzymolysis product, centrifuging to obtain supernatant, performing alcohol precipitation to obtain a degradation product, and performing modification treatment on the degradation product by using acetic anhydride to obtain a modified degradation product;
(4) and (3) finally, adding the modified degradation product obtained in the step (3) into the extracted oil obtained in the step (1), uniformly mixing, and homogenizing to obtain the rana chensinensis egg oil.
Preferably, in the step (1), the preparation method of the rana chensinensis egg powder comprises the following steps: and mixing the wood frog eggs with a ball milling auxiliary agent, and carrying out ball milling for 2-3 hours to obtain wood frog egg powder.
Further preferably, the ball-milling auxiliary agent is sodium hydroxide, the using amount of the ball-milling auxiliary agent is 25-35% of the weight of the wood frog eggs, zirconia ball grinding beads with the particle size of 10mm are used during ball milling, and the mass ratio of the ball grinding beads to the materials is 6-8: 1, the ball milling process conditions are as follows: ball milling is carried out for 30-40 minutes at 700-800 rpm.
Preferably, in the step (1), the pressed blank is made into a blank sheet with the diameter of 5-8 mm.
Preferably, in the step (1), the solvent used for subcritical extraction is a mixed gas of ethane and n-butane, and the volume ratio of the ethane to the n-butane is 1: the specific extraction method comprises the following steps: extracting for 2-3 times at the extraction pressure of 5-6 MPa and the extraction temperature of 45-55 ℃ for 50-60 minutes each time, combining the extracted liquid, and evaporating the solvent at the temperature of 30-40 ℃ to obtain the extracted oil.
Preferably, in the step (2), the mass ratio of the residue, the water and the mixed enzyme is 5-7: 30-35: 0.05 to 0.07.
Preferably, in the step (2), the enzyme mixture comprises the following components in parts by weight: 3-5 parts of papain, 0.5-0.8 part of trypsin and 8-10 parts of neutral protease.
Preferably, in the step (2), the enzymolysis process conditions are as follows: carrying out enzymolysis for 50-60 minutes at 50-55 ℃.
Preferably, in the step (3), the microwave degradation process conditions are as follows: and (3) performing microwave irradiation for 8-10 minutes at 300-400W.
Preferably, in the step (3), the alcohol precipitation method comprises the following specific steps in parts by volume: and taking 5-7 parts of supernatant, adding 15-20 parts of absolute ethyl alcohol, standing for 12-15 hours at 0-4 ℃, centrifuging and taking precipitate to obtain a degradation product.
Preferably, in the step (3), the specific method of modification treatment is as follows: adding 3-5 parts of degradation product into 15-18 parts of water, stirring until the degradation product is completely dissolved, adjusting the pH value to 7.5-8.5, dropwise adding 0.8-1 part of acetic anhydride while stirring, keeping the pH value unchanged in the process, continuing stirring for reacting for 4-5 hours after the dropwise adding is finished, adjusting the pH value to 7, and freeze-drying to obtain the modified degradation product.
Preferably, in the step (4), the mass ratio of the modified degradation product to the extracted oil is 13-15: 100.
preferably, in the step (4), the process conditions of the homogenization treatment are as follows: homogenizing for 3-4 times at 20-30 MPa.
Compared with the prior art, the invention has the beneficial effects that:
ball milling dried wood frog eggs into wood frog egg powder, compacting the wood frog egg powder into blank pieces, and performing subcritical extraction to obtain extract oil and residues; adding the residue into water, stirring and mixing uniformly to obtain slurry, adding mixed enzyme into the slurry, performing enzymolysis, and inactivating enzyme to obtain an enzymolysis product; then performing microwave degradation on the enzymolysis product, centrifuging to obtain supernatant, performing alcohol precipitation to obtain a degradation product, and performing modification treatment on the degradation product by using acetic anhydride to obtain a modified degradation product; and finally, adding the modified degradation product into the extracted oil, uniformly mixing, and homogenizing to obtain the rana chensinensis egg oil. The woodfrog egg oil obtained by the invention has high content of effective components and high stability.
The polysaccharide and the like contained in the wood frog egg are almost completely discarded in the existing wood frog egg oil extraction process, which is undoubtedly great nutrition loss, and the technical key point of the invention is to retain the polysaccharide and improve the performance of the wood frog egg oil. The invention adopts subcritical extraction to obtain the extraction oil, thereby reducing the loss of effective components. Adding the residual residue of subcritical extraction into water to prepare slurry, performing enzyme processing, and performing enzymolysis of mixed enzyme to obtain enzymolysis product and release polysaccharide component therein.
However, the polysaccharides have larger molecular weight, which is not beneficial to the dispersion in the wood frog egg oil, and the improvement effect on the stability of the wood frog egg oil is not good enough, so the application can further degrade the enzymolysis product by microwave, reduce the molecular weight of the polysaccharides, and utilize acetic anhydride to carry out modification treatment, so that the polysaccharides can be uniformly dispersed in the wood frog egg oil, and the low molecular weight polysaccharides play a role in protecting the wood frog egg oil, thereby greatly improving the stability of the wood frog egg oil.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The process for extracting the wood frog egg oil by the enzyme method comprises the following specific steps:
(1) mixing 1kg of dried oviductus Ranae with 2.5g of sodium hydroxide, ball-milling for 3 hours to obtain oviductus Ranae powder, pressing into blanks with diameter of 5mm, and performing subcritical extraction to obtain extract oil and residue;
(2) adding 70g of residues into 300g of water, uniformly stirring to obtain slurry, adding 0.7g of mixed enzyme into the slurry, performing enzymolysis, and performing enzyme deactivation to obtain an enzymolysis product;
(3) then performing microwave degradation on the enzymolysis product, centrifuging to obtain supernatant, performing alcohol precipitation to obtain a degradation product, adding 30g of the degradation product into 180g of water, stirring until the degradation product is completely dissolved, adjusting the pH to 7.5, dropwise adding 10g of acetic anhydride while stirring, keeping the pH unchanged in the process, continuously stirring for reacting for 4 hours after the dropwise addition is finished, adjusting the pH to 7, and performing freeze drying to obtain a modified degradation product;
(4) and finally, adding 15g of modified degradation products into 100g of extraction oil, uniformly mixing, and homogenizing to obtain the rana chensinensis egg oil.
In the step (1), zirconia ball milling beads with the particle size of 10mm are used during ball milling, the weight of the added ball milling beads is 6kg, and the ball milling process conditions are as follows: ball milling was carried out at 800rpm for 30 minutes.
In the step (1), the solvent adopted by the subcritical extraction is mixed gas of ethane and n-butane, and the volume ratio of the ethane to the n-butane is 1: the specific extraction method comprises the following steps: extracting at 45 deg.C under 6MPa for 3 times, each for 50 min, mixing extractive solutions, and evaporating solvent at 40 deg.C to obtain extract oil.
In the step (2), the mixed enzyme comprises: 3g of papain, 0.8g of trypsin and 8g of neutral protease.
In the step (2), the enzymolysis process conditions are as follows: enzymolysis is carried out for 50 minutes at 55 ℃.
In the step (3), the microwave degradation process conditions are as follows: the 400W microwave irradiation is carried out for 8 minutes.
In the step (3), the alcohol precipitation method specifically comprises the following steps: taking 7L of supernatant, adding 15L of absolute ethyl alcohol, standing for 12 hours at 4 ℃, centrifuging and taking precipitate to obtain the degradation product.
In the step (4), the process conditions of the homogenization treatment are as follows: homogenizing at 30MPa for 3 times.
Example 2
The process for extracting the wood frog egg oil by the enzyme method comprises the following specific steps:
(1) mixing 1kg of dried wood frog egg with 3.5g of sodium hydroxide, ball milling for 2 hours to prepare wood frog egg powder, pressing into a blank with the diameter of 8mm, and performing subcritical extraction to obtain extract oil and residue;
(2) adding 50g of residues into 350g of water, uniformly stirring to obtain slurry, adding 0.5g of mixed enzyme into the slurry, performing enzymolysis, and performing enzyme deactivation to obtain an enzymolysis product;
(3) then performing microwave degradation on the enzymolysis product, centrifuging to obtain a supernatant, performing alcohol precipitation to obtain a degradation product, adding 50g of the degradation product into 150g of water, stirring until the degradation product is completely dissolved, adjusting the pH value to 8.5, dropwise adding 8g of acetic anhydride while stirring, keeping the pH value unchanged in the process, continuously stirring for reacting for 5 hours after the dropwise adding is finished, adjusting the pH value to 7, and performing freeze drying to obtain a modified degradation product;
(4) and finally, adding 13g of modified degradation products into 100g of extraction oil, uniformly mixing, and homogenizing to obtain the rana chensinensis egg oil.
In the step (1), zirconia ball milling beads with the particle size of 10mm are used during ball milling, the weight of the added ball milling beads is 8kg, and the ball milling process conditions are as follows: ball milling was carried out at 700rpm for 40 minutes.
In the step (1), the solvent adopted by the subcritical extraction is mixed gas of ethane and n-butane, and the volume ratio of the ethane to the n-butane is 1: the specific extraction method comprises the following steps: extracting at 55 deg.C and 5MPa for 2 times (60 min each time), mixing extractive solutions, and evaporating solvent at 30 deg.C to obtain extract oil.
In the step (2), the mixed enzyme comprises: 5g of papain, 0.5g of trypsin and 10g of neutral protease.
In the step (2), the enzymolysis process conditions are as follows: enzymolysis is carried out for 60 minutes at 50 ℃.
In the step (3), the microwave degradation process conditions are as follows: 300W microwave irradiation for 10 minutes.
In the step (3), the alcohol precipitation method specifically comprises the following steps: and taking 5L of supernatant, adding 20L of absolute ethyl alcohol, standing for 15 hours at 0 ℃, centrifuging and taking precipitate to obtain the degradation product.
In the step (4), the process conditions of the homogenization treatment are as follows: homogenization at 20MPa was performed 4 times.
Example 3
The process for extracting the wood frog egg oil by the enzyme method comprises the following specific steps:
(1) firstly, mixing 1kg of dried wood frog eggs with 3g of sodium hydroxide, ball-milling for 2.5 hours to prepare wood frog egg powder, then pressing the wood frog egg powder into blank sheets with the diameter of 7mm, and performing subcritical extraction to obtain extract oil and residues;
(2) adding 60g of residues into 320g of water, uniformly stirring to obtain slurry, adding 0.6g of mixed enzyme into the slurry, performing enzymolysis, and performing enzyme deactivation to obtain an enzymolysis product;
(3) then performing microwave degradation on the enzymolysis product, centrifuging to obtain supernatant, performing alcohol precipitation to obtain a degradation product, adding 40g of the degradation product into 160g of water, stirring until the degradation product is completely dissolved, adjusting the pH value to 8, dropwise adding 9g of acetic anhydride while stirring, keeping the pH value unchanged in the process, continuously stirring and reacting for 4.5 hours after the dropwise addition is finished, adjusting the pH value to 7, and performing freeze drying to obtain a modified degradation product;
(4) and finally, adding 14g of the modified degradation product into 100g of the extraction oil, uniformly mixing, and homogenizing to obtain the rana chensinensis egg oil.
In the step (1), zirconia ball milling beads with the particle size of 10mm are used during ball milling, the weight of the added ball milling beads is 7kg, and the ball milling process conditions are as follows: ball milling was carried out at 800rpm for 35 minutes.
In the step (1), the solvent adopted by the subcritical extraction is mixed gas of ethane and n-butane, and the volume ratio of the ethane to the n-butane is 1: the specific extraction method comprises the following steps: extracting at 50 deg.C under 6MPa for 3 times (55 min each time), mixing extractive solutions, and evaporating solvent at 35 deg.C to obtain extract oil.
In the step (2), the mixed enzyme comprises: 4g of papain, 0.6g of trypsin and 9g of neutral protease.
In the step (2), the enzymolysis process conditions are as follows: enzymolysis was carried out at 52 ℃ for 55 minutes.
In the step (3), the microwave degradation process conditions are as follows: the 400W microwave irradiation was carried out for 9 minutes.
In the step (3), the alcohol precipitation method specifically comprises the following steps: taking 6L of supernatant, adding 18L of absolute ethyl alcohol, standing for 13 hours at 2 ℃, centrifuging and taking precipitate to obtain the degradation product.
In the step (4), the process conditions of the homogenization treatment are as follows: homogenization at 25MPa was performed 4 times.
Comparative example 1
The process for extracting the wood frog egg oil comprises the following specific steps: mixing 1kg of dried oviductus Ranae with 2.5g of sodium hydroxide, ball milling for 3 hours to obtain oviductus Ranae powder, pressing into blank with diameter of 5mm, and performing subcritical extraction to obtain oviductus Ranae oil.
Wherein, zirconia ball grinding beads with the particle size of 10mm are used during ball milling, the weight of the added ball grinding beads is 6kg, and the technical conditions of the ball milling are as follows: ball milling was carried out at 800rpm for 30 minutes.
The subcritical extraction adopts a mixed gas of ethane and n-butane as a solvent, and the volume ratio of the ethane to the n-butane is 1: the specific extraction method comprises the following steps: extracting at 45 deg.C under 6MPa for 3 times, each for 50 min, mixing extractive solutions, and evaporating solvent at 40 deg.C to obtain extract oil.
Comparative example 2
The process for extracting the wood frog egg oil by the enzyme method comprises the following specific steps:
(1) mixing 1kg of dried oviductus Ranae with 2.5g of sodium hydroxide, ball-milling for 3 hours to obtain oviductus Ranae powder, pressing into blanks with diameter of 5mm, and performing subcritical extraction to obtain extract oil and residue;
(2) adding 70g of residues into 300g of water, uniformly stirring to obtain slurry, adding 0.7g of mixed enzyme into the slurry, performing enzymolysis, and performing enzyme deactivation to obtain an enzymolysis product;
(3) and finally, adding 15g of the enzymolysis product into 100g of the extraction oil, uniformly mixing, and homogenizing to obtain the rana chensinensis egg oil.
In the step (1), zirconia ball milling beads with the particle size of 10mm are used during ball milling, the weight of the added ball milling beads is 6kg, and the ball milling process conditions are as follows: ball milling was carried out at 800rpm for 30 minutes.
In the step (1), the solvent adopted by the subcritical extraction is mixed gas of ethane and n-butane, and the volume ratio of the ethane to the n-butane is 1: the specific extraction method comprises the following steps: extracting at 45 deg.C under 6MPa for 3 times, each for 50 min, mixing extractive solutions, and evaporating solvent at 40 deg.C to obtain extract oil.
In the step (2), the mixed enzyme comprises: 3g of papain, 0.8g of trypsin and 8g of neutral protease.
In the step (2), the enzymolysis process conditions are as follows: enzymolysis is carried out for 50 minutes at 55 ℃.
In the step (3), the process conditions of the homogenization treatment are as follows: homogenizing at 30MPa for 3 times.
The stability of the woodfrog egg oil obtained in the examples 1-3 and the comparative examples 1 and 2 is respectively checked, the stability comprises acid value (GB5009.229-2016), peroxide value (GB5009.227-2016) and malondialdehyde (GB5009.181-2016), and the results are shown in the table 1.
TABLE 1 stability study
As can be seen from Table 1, the oviductus ranae obtained in examples 1-3 has low acid value, low peroxide value and low malondialdehyde, which indicates that the oviductus ranae has better stability (no relevant standard exists in the oviductus ranae at present, and reference is made to GB10146-2015, the content of the invention is far lower than the relevant index value specified therein, which indicates that the stability meets the edible requirement).
The comparative example 1 directly performs subcritical extraction on the wood frog eggs to obtain wood frog egg oil, the enzymolysis products of the comparative example 2 are not degraded, and the stability of the obtained wood frog egg oil is obviously poor, which indicates that the extract in the residue after the wood frog eggs are extracted plays a role in protecting the wood frog egg oil and improves the stability of the product.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. The extraction process of the wood frog egg oil by the enzyme method is characterized by comprising the following specific steps:
(1) ball-milling dried oviductus Ranae into oviductus Ranae powder, pressing into blank, and performing subcritical extraction to obtain extract oil and residue;
(2) adding the residue into water, stirring and mixing uniformly to obtain slurry, adding mixed enzyme into the slurry, performing enzymolysis, and inactivating enzyme to obtain an enzymolysis product;
(3) then performing microwave degradation on the enzymolysis product, centrifuging to obtain supernatant, performing alcohol precipitation to obtain a degradation product, and performing modification treatment on the degradation product by using acetic anhydride to obtain a modified degradation product;
(4) and (3) finally, adding the modified degradation product obtained in the step (3) into the extracted oil obtained in the step (1), uniformly mixing, and homogenizing to obtain the rana chensinensis egg oil.
2. The extraction process according to claim 1, wherein the preparation method of the rana chensinensis egg powder in the step (1) is as follows: and mixing the wood frog eggs with a ball milling auxiliary agent, and carrying out ball milling for 2-3 hours to obtain wood frog egg powder.
3. The extraction process according to claim 1, wherein in the step (1), the solvent used for subcritical extraction is a mixed gas of ethane and n-butane, and the volume ratio of the ethane to the n-butane is 1: the specific extraction method comprises the following steps: extracting for 2-3 times at the extraction pressure of 5-6 MPa and the extraction temperature of 45-55 ℃ for 50-60 minutes each time, combining the extracted liquid, and evaporating the solvent at the temperature of 30-40 ℃ to obtain the extracted oil.
4. The extraction process according to claim 1, wherein in the step (2), the mass ratio of the residue, the water and the mixed enzyme is 5-7: 30-35: 0.05 to 0.07.
5. The extraction process according to claim 1, wherein in the step (2), the mixed enzyme comprises the following components in parts by weight: 3-5 parts of papain, 0.5-0.8 part of trypsin and 8-10 parts of neutral protease.
6. The extraction process according to claim 1, wherein in the step (2), the enzymolysis process conditions are as follows: carrying out enzymolysis for 50-60 minutes at 50-55 ℃.
7. The extraction process according to claim 1, wherein in the step (3), the microwave degradation process conditions are as follows: and (3) performing microwave irradiation for 8-10 minutes at 300-400W.
8. The extraction process according to claim 1, wherein in the step (3), the alcohol precipitation is carried out by the following specific method in parts by volume: and taking 5-7 parts of supernatant, adding 15-20 parts of absolute ethyl alcohol, standing for 12-15 hours at 0-4 ℃, centrifuging and taking precipitate to obtain a degradation product.
9. The extraction process according to claim 1, wherein in the step (3), the specific method of modification treatment comprises the following steps in parts by weight: adding 3-5 parts of degradation product into 15-18 parts of water, stirring until the degradation product is completely dissolved, adjusting the pH value to 7.5-8.5, dropwise adding 0.8-1 part of acetic anhydride while stirring, keeping the pH value unchanged in the process, continuing stirring for reacting for 4-5 hours after the dropwise adding is finished, adjusting the pH value to 7, and freeze-drying to obtain the modified degradation product.
10. The extraction process according to claim 1, wherein in step (4), the process conditions of the homogenization treatment are as follows: homogenizing for 3-4 times at 20-30 MPa.
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