CN112552371A - Method for preparing olive kernel extract based on extraction and purification technology - Google Patents
Method for preparing olive kernel extract based on extraction and purification technology Download PDFInfo
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- 240000007817 Olea europaea Species 0.000 title claims abstract description 83
- 238000000605 extraction Methods 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000005516 engineering process Methods 0.000 title abstract description 9
- 238000000746 purification Methods 0.000 title abstract description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 39
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000012074 organic phase Substances 0.000 claims abstract description 31
- 239000000872 buffer Substances 0.000 claims abstract description 18
- 238000001035 drying Methods 0.000 claims abstract description 13
- 239000012065 filter cake Substances 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 12
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 12
- 238000003756 stirring Methods 0.000 claims abstract description 10
- 238000000498 ball milling Methods 0.000 claims abstract description 7
- 238000004821 distillation Methods 0.000 claims abstract description 7
- 238000010298 pulverizing process Methods 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 11
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- 239000007983 Tris buffer Substances 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 8
- 229940125532 enzyme inhibitor Drugs 0.000 claims description 8
- 239000002532 enzyme inhibitor Substances 0.000 claims description 8
- RYYKJJJTJZKILX-UHFFFAOYSA-M sodium octadecanoate Chemical compound [Na+].CCCCCCCCCCCCCCCCCC([O-])=O RYYKJJJTJZKILX-UHFFFAOYSA-M 0.000 claims description 8
- 238000001694 spray drying Methods 0.000 claims description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 8
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 8
- 235000019270 ammonium chloride Nutrition 0.000 claims description 7
- 229960001484 edetic acid Drugs 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 238000005485 electric heating Methods 0.000 claims description 2
- 238000007602 hot air drying Methods 0.000 claims description 2
- 239000012071 phase Substances 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 13
- 108090000623 proteins and genes Proteins 0.000 abstract description 13
- 239000012535 impurity Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 239000000428 dust Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000007873 sieving Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 241000208229 Burseraceae Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Extraction Or Liquid Replacement (AREA)
- Fats And Perfumes (AREA)
Abstract
The invention provides a method for preparing an olive kernel extract based on an extraction and purification technology, wherein the protein content of the extract prepared by the method is more than 60%. The method comprises the following steps: pulverizing fructus Canarii albi; adding ethanol with the mass ratio of 5-10 times, stirring for 1-3 hours, and filtering to obtain a filter cake; adding a buffer extracting solution into the filter cake, ball-milling for 2-3 hours by using a ball mill, and filtering by using an ultrafiltration device to obtain an extracting solution; concentrating the extract; sequentially carrying out gradient extraction on the extracting solution by using ethanol/n-hexane, ethanol/n-hexane and n-hexane with the volume ratio of 1:1, 1:5, carrying out reduced pressure distillation on an organic phase, and drying to obtain the extract. The invention adopts the buffer extract, increases the solubility of protein, and simultaneously improves the extraction efficiency of the olive kernel extract by combining the ball milling technology; in the post-treatment process of the extract, the ultrafiltration technology and the extraction technology are combined, so that the extraction degree of the olive kernel extract is improved, and the purity of the extract is increased.
Description
Technical Field
The invention relates to the field of deep processing of agricultural products, in particular to an extraction process of an olive kernel extract.
Background
The olive kernel is the kernel of olive of family Burseraceae, and accounts for about 6% of fresh olive fruit weight and 25% of kernel weight. The olive kernel is rich in protein besides oil and unsaturated fatty acid, the protein content is about 40%, and the protein covers 8 essential amino acids needed by human body. The olive kernel has the effects of moistening dryness, sobering up and detoxifying, and has good nutritional value and medicinal value. Therefore, an extraction method capable of efficiently extracting the olive kernel is developed and utilized in an olive industry chain, the deep processing of the olive can be realized, the additional value of the olive is improved, and the method has important economic and social significance for increasing the economic income of farmers. At present, the development of olive kernels in China is very little, for example, in patent CN201710483962.0, olive kernel protein is prepared by an alkali extraction and acid precipitation method, and the protein extraction amount is not high because water-insoluble protein cannot be extracted. Therefore, there is a need to develop a method for extracting water-soluble protein and organic solvent-soluble protein from olive kernel simultaneously without increasing the production cost.
Disclosure of Invention
The invention aims to solve the problem of low protein content in the olive kernel extract in the prior art so as to further improve the added value of the olive.
The invention provides a preparation method of an olive kernel extract, which is characterized in that the olive kernel extract with the protein content of more than 60 percent is obtained by a gradient extraction method.
Further, in order to obtain an olive kernel extract with a protein content of more than 60%, the extraction method comprises the following steps:
step (1): pulverizing fructus Canarii albi to below 100 mesh to obtain fructus Canarii albi powder;
step (2): adding 5-10 times of ethanol into the olive kernel powder in the step (1) according to the mass ratio, stirring for 1-3 hours, filtering, and taking a filter cake to obtain a degreased olive kernel filter cake;
and (3): adding 5-10 times of low-temperature mixed buffer extracting solution into the degreased olive kernel filter cake in the step (2), ball-milling for 2-3 hours by using a ball mill, and filtering by using an ultrafiltration device to obtain extracting solution;
and (4): concentrating the extract of step (3);
and (5): and (3) sequentially carrying out gradient extraction on the extracting solution in the step (4) by using ethanol/n-hexane, ethanol/n-hexane and n-hexane in a volume ratio of 1:1, separating a water phase and an organic phase, carrying out pressure distillation on the combined organic phases, and drying to obtain the olive kernel extract.
Further, in order to improve the extraction rate of the protein in the olive kernel extract, the mixed buffer extract in the step (3) comprises the following components: beta-mercaptoethanol, sodium stearate, ethylene diamine tetraacetic acid, Tris acid, enzyme inhibitor and ammonium chloride; preferably, the mixed buffer extract comprises the following components: 500mmol of beta-mercaptoethanol, 300mmol of sodium stearate, 50mmol of ethylenediamine tetraacetic acid, 500mmol of Tris acid, 50mmol of enzyme inhibitor and 100mmol of ammonium chloride.
Furthermore, in order to improve the extraction rate of protein in the olive kernel extract, the mixed buffer extract is prepared by firstly mixing beta-mercaptoethanol, sodium stearate, ethylene diamine tetraacetic acid and ammonium chloride, then storing at low temperature, and dispersing Tris acid and an enzyme inhibitor into the mixed buffer extract when in use.
Furthermore, in order to reduce the denaturation probability of the protein in the olive kernel extract, the low temperature range related to the mixed buffer extract is 0-5oC。
Further, in order to improve the extraction purity of the olive kernel extract, the ultrafiltration device in the step (3) is composed of filters with different filter diameters which are connected in series; preferably, the ultrafiltration device consists of a 0.45 μm filter in series with a 0.1 μm filter.
Further, the drying method of the olive kernel extract comprises any one or a combination of electric heating drying, spray drying, hot air drying and freeze drying; in order to better maintain the properties of the proteins in the olive kernel extract, preferably the drying method of the olive kernel extract is one of freeze drying or spray drying.
The invention has the beneficial effects that:
according to the invention, the mixed buffer extracting solution with a specific formula is adopted, so that the solubility of protein in the extracting solution is increased, and meanwhile, the olive kernel powder is fully contacted and mixed with the extracting solution by combining a ball milling technology, so that the extraction efficiency of the olive kernel extract is greatly improved; meanwhile, in the post-treatment process of the olive kernel extract, the ultrafiltration technology and the extraction technology are combined, mechanical micro-impurities in the extracting solution are removed through an ultrafiltration series device, and then the olive kernel extract is extracted by adopting a gradient extraction process, so that the extraction degree of the olive kernel extract is improved, and the purity of the extract is increased.
Detailed Description
The present invention is described in further detail below with reference to specific examples, which are not to be construed as limiting the scope of the invention as claimed.
Example one
< preparation of Mixed buffer extract >
Mixing ethylene diamine tetraacetic acid, ammonium chloride, sodium stearate and beta-mercaptoethanol in a molar ratio of 1:2:6:10, stirring, uniformly mixing, and adding 0oAnd C, storing in an environment.
When in use, the additive is added into the mixture, and the mixture is also 0oC, 1 stoichiometric amount of enzyme inhibitor and 10 stoichiometric amounts of Tris acid, and mixing them uniformly.
< preparation of Olive seed extract >
A method for preparing olive kernel extract based on extraction and purification technology comprises the following steps:
washing the collected olive kernels with clear water to remove impurities such as dust and mud, filtering, washing with clear water for 3 times, and standing the washed olive kernels at 35 deg.CoC, drying in an oven, crushing the olive kernel to the particle size of below 100 meshes after drying, and sieving to obtain olive kernel powder;
weighing 500g of olive kernel powder, adding 4000g of ethanol in batches under the condition of stirring, continuously stirring for 3 hours, and filtering to obtain a degreased olive kernel filter cake;
adding 5000g of low-temperature mixed buffer extract into the defatted olive kernel filter cake in batches under the stirring condition, ball-milling for 3 hours by using a ball mill, sequentially passing through a 0.45-micrometer filter and a 0.1-micrometer filter, and filtering to obtain an extract;
concentrating the extract by reduced pressure distillation; extracting the extract with ethanol/n-hexane at a volume ratio of 1:1 for the first time, separating the water layer from the organic phase layer, and collecting the organic phase layer; extracting the water layer after the first extraction with ethanol/n-hexane at a volume ratio of 1:5 for the second time, separating the water layer from the organic phase layer, and collecting the organic phase layer; extracting the water layer after the second extraction with n-hexane for the third time, separating the water layer from the organic phase layer, and collecting the organic phase layer; mixing the organic phase layers, distilling under reduced pressure, and spray drying to obtain white or off-white olive kernel extract.
< protein content test of Olive Kernel extract >
The protein content of the olive kernel extract is measured by a Coomassie brilliant blue method, and the protein content of the olive kernel extract is 62.62%.
Example two
The difference from example one is that the gradient extraction process is different.
Concentrating the extract by reduced pressure distillation; extracting the extract with ethanol/n-hexane at a volume ratio of 1:1 for the first time, separating the water layer from the organic phase layer, and collecting the organic phase layer; extracting the water layer after the first extraction with ethanol/n-hexane at a volume ratio of 1:8 for the second time, separating the water layer from the organic phase layer, and collecting the organic phase layer; extracting the water layer after the second extraction with n-hexane for the third time, separating the water layer from the organic phase layer, and collecting the organic phase layer; mixing the organic phase layers after the three-time extraction, distilling under reduced pressure, and spray drying to obtain the olive kernel extract. The olive kernel extract is white to off-white powder, wherein the protein content is 60.07%.
EXAMPLE III
The difference from example one is that the gradient extraction process is different.
The gradient extraction process comprises the following steps: concentrating the extract by reduced pressure distillation; extracting the extract with ethanol/n-hexane at a volume ratio of 1:1 for the first time, separating the water layer from the organic phase layer, and collecting the organic phase layer; extracting the water layer after the first extraction for the second time by using normal hexane, separating the water layer from the organic phase layer, and collecting the organic phase layer; mixing the organic phase layers, distilling under reduced pressure, and spray drying to obtain fructus Canarii albi extract. The olive kernel extract is white to off-white powder, wherein the protein content is 52.13%.
Example four
The difference from the first embodiment is that the mixed buffer extract is prepared in a different manner.
< preparation of Mixed buffer extract >
Mixing EDTA, enzyme inhibitor, and chlorineAmmonium chloride, sodium stearate, beta-mercaptoethanol and Tris acid are stirred and mixed according to the molar ratio of 1:1:2:6:10:10, and are added with 0 after being mixed evenlyoAnd C, storing in an environment.
The olive kernel extract was extracted in the same manner as in example one. The olive kernel extract is prepared as white to off-white powder with a protein content of 38.77%.
EXAMPLE five
The difference from the first example is that the extract is not filtered by an ultrafiltration device.
< preparation of Olive seed extract >
A method for preparing olive kernel extract based on extraction and purification technology comprises the following steps:
washing the collected olive kernels with clear water to remove impurities such as dust and mud, filtering, washing with clear water for 3 times, and standing the washed olive kernels at 35 deg.CoC, drying in an oven, crushing the olive kernel to the particle size of below 100 meshes after drying, and sieving to obtain olive kernel powder;
weighing 500g of olive kernel powder, adding 4000g of ethanol in batches under the condition of stirring, continuously stirring for 3 hours, and filtering to obtain a degreased olive kernel filter cake;
adding 5000g of low-temperature mixed buffer extracting solution into the degreased olive kernel filter cake in batches under the stirring condition, ball-milling for 3 hours by using a ball mill, and filtering by using common filter paper to obtain extracting solution;
concentrating the extract by reduced pressure distillation; extracting the extract with ethanol/n-hexane at a volume ratio of 1:1 for the first time, separating the water layer from the organic phase layer, and collecting the organic phase layer; extracting the water layer after the first extraction with ethanol/n-hexane at a volume ratio of 1:5 for the second time, separating the water layer from the organic phase layer, and collecting the organic phase layer; extracting the water layer after the second extraction with n-hexane for the third time, separating the water layer from the organic phase layer, and collecting the organic phase layer; mixing the organic phase layers after the three-time extraction, distilling under reduced pressure, and spray drying to obtain the olive kernel extract. The olive kernel extract is a yellowish to yellow powder with a protein content of 54.97%.
Based on the experimental data, the scheme of the embodiment of the invention combines the protein mixed buffer extract, adopts the gradient extraction process, can obviously improve the protein content in the olive kernel extract, can remove micromechanical impurities in the extract after passing through an ultrafiltration device, improves the purity of the extract, and ensures the color of the olive kernel extract.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for preparing fructus Canarii albi extract is characterized by using gradient extraction method to obtain extract with protein content of more than 60%
The olive kernel extract of (1).
2. The method of preparing an olive kernel extract according to claim 1, comprising the steps of:
step (1): pulverizing fructus Canarii albi to below 100 mesh to obtain fructus Canarii albi powder;
step (2): adding 5-10 times of ethanol into the olive kernel powder in the step (1) according to the mass ratio, stirring for 1-3 hours,
filtering to obtain a filter cake, and obtaining a degreased olive kernel filter cake;
and (3): adding 5-10 times of low-temperature mixed buffer extracting solution into the degreased olive kernel filter cake in the step (2) to facilitate
Ball-milling for 2-3 hours by using a ball mill, and filtering by using an ultrafiltration device to obtain an extracting solution;
and (4): concentrating the extract of step (3);
and (5): sequentially adding ethanol/n-hexane with the volume ratio of 1:1, ethanol/n-hexane with the volume ratio of 1:5 and n-hexane to the mixture obtained in the step (4)
Performing gradient extraction on the extracting solution, separating a water phase and an organic phase, performing pressure distillation on the combined organic phase, and drying to obtain the olive kernel extract.
3. The method for preparing olive kernel extract according to claim 2, wherein the mixing in the step (3) is slow
The extracting solution comprises the following components: beta-mercaptoethanol, sodium stearate, ethylene diamine tetraacetic acid, Tris acid, an enzyme inhibitor and ammonium chloride.
4. The method of preparing the olive kernel extract according to claim 3, wherein the mixed buffer extract is prepared by mixing beta-mercaptoethanol, sodium stearate, ethylenediamine tetraacetic acid and ammonium chloride, storing at low temperature, and dispersing Tris acid and an enzyme inhibitor therein when in use.
5. A method of preparing an olive kernel extract according to claim 3 or claim 4, wherein the mixed buffered extract comprises: 500mmol of beta-mercaptoethanol, 300mmol of sodium stearate, 50mmol of ethylenediamine tetraacetic acid, 500mmol of Tris acid, 50mmol of enzyme inhibitor and 100mmol of ammonium chloride.
6. The method for preparing olive kernel extract according to claim 2 or claim 3, wherein the low temperature range of the mixed buffer extract is 0 to 5%oC。
7. The method for preparing olive kernel extract according to claim 2, wherein the ultrafiltration package in the step (3)
The device is composed of filters with different filter diameters connected in series.
8. The method for the preparation of olive kernel extract according to claim 7, wherein the ultrafiltration device consists of a 0.45 μm filter in series with a 0.1 μm filter.
9. The method for preparing olive kernel extract according to claim 2, wherein the drying method is any one or combination of electric heating drying, spray drying, hot air drying and freeze drying.
10. The method of preparing an olive kernel extract according to claim 9, wherein the drying method is one of freeze drying or spray drying.
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