CN111202163A - Potato-derived protease inhibitor mixture, and extraction method and application thereof - Google Patents
Potato-derived protease inhibitor mixture, and extraction method and application thereof Download PDFInfo
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- CN111202163A CN111202163A CN202010082104.7A CN202010082104A CN111202163A CN 111202163 A CN111202163 A CN 111202163A CN 202010082104 A CN202010082104 A CN 202010082104A CN 111202163 A CN111202163 A CN 111202163A
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/16—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste water of starch-manufacturing plant or like wastes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
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- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Gerontology & Geriatric Medicine (AREA)
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- Dermatology (AREA)
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Abstract
The invention relates to the technical field of natural product extraction and application, and provides a potato-derived protease inhibitor mixture, and an extraction method and application thereof. Adding ammonium sulfate into organic waste liquid from potato starch production, stirring, centrifuging, precipitating, redissolving, adjusting pH, standing, centrifuging, extracting supernatant, dialyzing, freeze drying, and extracting to obtain potato sourceA mixture of protease inhibitors. The extracted protease inhibitor mixture is composed of proteins with molecular weight of 5-25kDa, has good scavenging effect on ABTS free radicals, has outstanding antioxidant capacity in a grease system, and shows excellent high pressure resistance and stability. The product of the invention also has good oxidation resistance in cells, and protects HUVEC cells from H2O2The induced oxidative stress damaged and inhibited the growth of GIST882 tumor cells. The invention widens the application range of the existing potato source protease inhibitor, improves the comprehensive utilization of byproducts, reduces the sewage treatment cost of enterprises, protects the environment and has good application prospect.
Description
Technical Field
The invention relates to the technical field of natural product extraction and application, and particularly provides a potato-derived protease inhibitor mixture, and an extraction method and application thereof.
Background
In the production process of potato starch, a large amount of organic waste liquid is generated, wherein the organic waste liquid mainly contains protein and other components, such as saccharides, micro-fibers, a small amount of starch and the like. Although these organic compounds are not toxic, the discharge of high-concentration protein waste liquid into the environment is a great waste of resources, and the imbalance of water resource ecological environment is caused, which affects the life of fish and other aquatic animals and plants. It has adverse effect on human production and life. Therefore, how to efficiently extract, separate and purify the potato protein and how to obtain beneficial application is one of important ways for solving the problems of organic waste liquid treatment, resource recycling and economic benefit improvement of starch enterprises. However, the current related research reports are scarce, and the sustainable development of the potato industry is restricted.
Studies have shown that potato protein is mainly composed of three parts, of which protease inhibitor (a mixture of proteins with molecular weights ranging from 5 to 25 kDa) is the main component, accounting for about 50% of the total protein, patatin for 40%, and high molecular weight protein for 10%. The premise of researching the functions and the applications of the proteins is to realize the extraction and the separation of the proteins of each component. Salting out and isoelectric point separation are common methods for the extraction of large amounts of plant proteins. The salting-out can ensure that the protein is precipitated from the solution after high-concentration neutral salt is added, has reversibility, can keep the biological activity of the protein, is simple and convenient to operate, has low cost, is suitable for crude extraction of the protein, and has low purity of the obtained protein. The pH value of the solution is adjusted to be acidic, so that the protein can be redissolved after being precipitated at the isoelectric point of the protein, the protein is separated from other soluble components, and the isoelectric point separation method has low product cost, higher purity and low extraction rate. Other methods such as chromatographic column separation have complex production engineering and high cost, although the product has high purity and good separation effect, and are not suitable for large-scale separation and extraction.
Disclosure of Invention
In order to solve the technical problems, the invention provides a potato-derived protease inhibitor mixture, an extraction method and application thereof, the potato-derived protease inhibitor mixture is prepared by a low-cost and high-efficiency method, the in-vitro antioxidant capacity and the processing stability of the potato-derived protease inhibitor mixture are found, and the potato-derived protease inhibitor mixture can effectively protect HUVEC cells from H2O2The induced oxidative stress damaged and inhibited the growth of GIST882 tumor cells.
The invention is realized in such a way that the method for extracting the potato-derived protease inhibitor mixture comprises the following steps:
1) adding ammonium sulfate into the industrial waste liquid of the potato starch at room temperature to ensure that the saturation degree of the industrial waste liquid reaches 30% -80%, oscillating and extracting the obtained system for 90-120 min at 80-150 rpm under the condition of 4-6 ℃, and carrying out solid-liquid separation for 20-30 min at 8000-10000 r/min to obtain a precipitate which is a crude potato protein extract;
2) re-dissolving the crude potato protein extract prepared in the step 1) in distilled water at a feed-to-liquid ratio of 1: 5-1: 10(g/mL) to obtain a crude potato protein extract;
3) adjusting the pH value of the crude potato protein extracting solution prepared in the step 2) to 2.5-5.0, standing, performing solid-liquid separation at 8000-10000 r/min for 20-30 min, dialyzing the supernatant for 36-48 h, and performing freeze drying treatment to obtain the potato-derived protease inhibitor mixture.
The invention also provides a potato-derived protease inhibitor mixture extracted by the method for extracting the potato-derived protease inhibitor mixture.
The invention also provides the application of the potato-derived protease inhibitor mixture in the direction of antioxidant protection.
Further, the applications of the above potato-derived protease inhibitor mixture in the direction of antioxidant protection include antioxidant capacity and processing stability in vitro, intracellular and extracellular antioxidant ability, and protection of cells from H2O2Use of an induced oxidative stress injury direction.
The invention also provides application of the potato-derived protease inhibitor mixture in an anti-tumor direction.
Further, the above potato-derived protease inhibitor mixture can be used in the anti-tumor direction, including the use in inhibiting the growth direction of GIST882 tumor cells.
Compared with the prior art, the invention has the advantages that:
the potato source protease inhibitor mixture is extracted through the synergistic effect of salting out and isoelectric point, the extraction rate of the protease inhibitor is improved to 80.18% at most, and the method has the advantages of simple operation, low cost and suitability for large-scale production;
the preparation process of the protease inhibitor mixture simultaneously discloses that the protease inhibitor mixture has a good scavenging effect on ABTS free radicals, has outstanding oxidation resistance in a grease system, shows excellent high-pressure-resistant stability and has good industrial prospects in a cold sterilization system. Meanwhile, the compound has the functional activities of eliminating free radicals, protecting cells from oxidative damage, inhibiting the growth of tumor cells and the like. Has good application potential in the fields of functional foods, medicaments and cosmetics, and has important economic value and market value.
Drawings
The invention is described in further detail below with reference to the following figures and embodiments:
FIG. 1 is a diagram showing the results of gel electrophoresis of a potato-derived protease inhibitor;
FIG. 2-A is a graph showing the in vitro antioxidant activity-total reducing power test results of potato-derived protease inhibitors;
FIG. 2-B is a graph showing the results of in vitro antioxidant-hydroxyl radical scavenging ability assay of potato-derived protease inhibitor;
FIG. 2-C is a graph showing the results of in vitro antioxidant-DPPH free radical scavenging ability assay of potato-derived protease inhibitor;
FIG. 2D is a graph showing the in vitro antioxidant-ABTS free radical scavenging ability test results of potato-derived protease inhibitors;
FIG. 2-E is a graph showing the results of in vitro antioxidant-lipid peroxidation inhibition assays of potato-derived protease inhibitors;
FIG. 3-A is a graph showing the effect of processing conditions (temperature) on the antioxidant capacity of potato derived protease inhibitors;
FIG. 3-B is a graph showing the effect of processing conditions (pH) on the antioxidant capacity of potato derived protease inhibitors;
FIG. 3-C is a graph of the results of the effect of processing conditions (high pressure) on the antioxidant capacity of potato derived protease inhibitors;
FIG. 4 is a graph of potato derived protease inhibitor vs. H2O2Results of the effect of induced ROS in HUVEC cells.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
The present invention will be further described with reference to specific embodiments, but the scope of the invention as claimed is not limited to the following embodiments.
Early researches find that the potato protease inhibitor has good solubility in the whole pH range, and patatin and high molecular weight protein precipitate at isoelectric points. Meanwhile, the potato protease inhibitor has new applications of resisting oxidation, inhibiting tumor cells and the like besides inhibiting the protease activity.
EXAMPLE 1 preparation of Potato derived protease inhibitor cocktail
Adding ammonium sulfate into the industrial waste liquid at room temperature to reach 40% saturation, shaking the obtained system at 4 deg.C and 100rpm for 90min, and performing solid-liquid separation at 4 deg.C and 8000rpm for 20min to obtain crude potato protein extract. Redissolving the mixture in distilled water according to the ratio of 1:10 to obtain potato protein extracting solution. Adjusting pH of the crude potato protein solution to 3.4, standing at 4 deg.C for 3min, performing solid-liquid separation at 4 deg.C and 8000rpm for 20min, dialyzing the supernatant, and freeze drying to obtain potato source protease inhibitor mixture. The electrophoresis results are shown in FIG. 1.
Example 2 in vitro antioxidant Activity
(1) Total reducing power
1mL each of sample solutions (0.1, 0.5, 1, 2, 3, 4, 5mg/M L), PBS (0.2M, pH 6.6) and potassium ferricyanide solution (1%) at different concentrations were mixed, 1mL of trichloroacetic acid (10%) was added after reaction at 50 ℃ for 20min, 1mL of supernatant was mixed with 1mL each of distilled water and ferric trichloride (0.1%) after centrifugation at 4000r/min for 20min, and the mixture was reacted at room temperature for 10 min. While replacing the sample with 1ml PBS, a blank was prepared. Each sample was plated with 3 replicate wells and the assay was repeated at least 3 times. And taking an average value, and calculating the total reducing power according to the following formula.
Total reducing power (A)x-A0)/A0×100％
AxDenotes the absorbance of the sample set, A0The absorbance of the blank control group was represented by using PBS instead of the sample. IC (integrated circuit)50For measuring the total reducing power level.
As shown in FIG. 2-A, the total reducing power of potato-derived protease inhibitors was significantly higher than that of ovalbumin, but much lower than that of vitamin C. IC of potato-derived protease inhibitors50The value was 20.018 + -0.212 mg/mL, which is about 9-fold higher than ovalbumin (180.039 + -0.68 mg/mL). The oxidation resistance is as follows: vitamin C>Potato derived protease inhibitors>Ovalbumin (FIG. 2-A).
(2) Hydroxyl radical scavenging ability
Sample solutions of different concentrations (0.1, 0.5, 1, 2, 3, 4, 5mg/m L), salicylic acid (9mM), ferrous sulfate solution (9mM) and H2O2(8.8mM) 1mL of each was mixed and reacted at 37 ℃ for 30 min. While replacing the sample and H with 2mL of distilled water2O2Solution, prepare blank control. . Repeat 3 times, take the average value, according to the following formula to calculate the clearance.
Clearance (%) ═ a0-(Ax-AX0)]/A0×100％
AxDenotes the absorbance of the sample set, AX0Denotes the absorbance of the sample blank, A0The absorbance of the blank control group is shown. IC (integrated circuit)50For measuring the level of hydroxyl radical scavenging activity.
As shown in FIG. 2-B, potato-derived protease inhibitors have a radical scavenging ability similar to ovalbumin in the hydroxyl radical assay, IC502.395 +/-0.822 g/mL and 23.203 +/-0.181 mg/mL respectively are far lower than vitamin C (0.137 +/-0.238 mg/mL). The hydroxyl radical scavenging capacity is as follows: vitamin C>Ovalbumin>Potato derived protease inhibitors (fig. 2B).
(3) DPPH radical scavenging ability
Sample solutions of different concentrations (0.1, 0.5, 1, 2, 3, 4, 5mg/m L) and 0.04g/L of ethanol DPPH solution were each added to a tube at 2mL and mixed rapidly, and the reaction was carried out at room temperature in the dark for 20min while replacing the sample and DPPH solution with 2mL of ethanol to prepare a blank. Repeat 3 times, take the average value, according to the following formula to calculate the clearance.
Clearance (%) ═ a0-(Ax-AX0)]/A0×100％
AxDenotes the absorbance of the sample set, AX0Denotes the absorbance of the sample blank, A0The absorbance of the blank control group is shown. IC (integrated circuit)50For measuring DPPH scavenging activity levels.
As shown in FIG. 2-C, the potato-derived protease inhibitors have a higher radical scavenging capacity than ovalbumin, but a lower capacity than vitamin C in the DPPH assay. The DPPH free radical scavenging capacity is as follows: vitamin C > potato-derived protease inhibitor > ovalbumin.
(4) ABTS free radical scavenging ability
0.2mL of sample solutions of different concentrations (0.1, 0.5, 1, 2, 3, 4, 5mg/m L) and 0.8mL of ABTS working solution were added to the tube and mixed rapidly, and reacted at room temperature in the dark for 6min while replacing the sample with 0.2mL of ethanol to prepare a blank. Repeat 3 times, take the average value, according to the following formula to calculate the clearance.
Clearance (%) ═ a0-A)/A0×100％
A represents the absorbance of the sample group, A0The absorbance of the blank control group is shown. IC (integrated circuit)50For measuring the level of ABTS scavenging activity.
As shown in FIG. 2-D, potato-derived protease inhibitors have a radical scavenging capacity similar to ovalbumin and lower than vitamin C in the ABTS assay. Potato derived protease inhibitionIC of agent50The value was 0.018. + -. 0.118mg/mL, which is 24.7-fold that of ovalbumin (0.445. + -. 0.332 mg/mL). The ABTS free radical scavenging capacity is as follows: vitamin C>Potato derived protease inhibitors>Egg white protein.
(5) Oil and fat peroxidation inhibiting ability
Adding 1mL of sample solutions (0.1, 0.5, 1, 2, 3, 4, 5mg/m L) with different concentrations, a lipid dispersion system and ferrous sulfate (0.4mM) into a test tube respectively, quickly mixing, carrying out a light-shielding reaction at 37 ℃ for 60min, adding 2mL of TCA-TBA-HCl mixed solution, carrying out water bath at 90-100 ℃ for 15min, quickly cooling, and centrifuging at 3000r/min for 10 min. The supernatant was collected and absorbance was measured at 525 nm. While the sample was replaced with distilled water, a blank control was prepared. Repeat 3 times, take the average value, according to the following formula calculate the inhibition rate.
Inhibition ratio (%) ═ a0-A)/A0×100％
A represents the absorbance of the sample group, A0The absorbance of the blank control group is shown. IC (integrated circuit)50Used to measure the level of lipid peroxidation inhibition.
As shown in the accompanying FIG. 2-E, the fat peroxidation inhibition ability of potato-derived protease inhibitor was higher than that of vitamin C and ovalbumin. IC of potato-derived protease inhibitors50The value was 0.296. + -. 0.522mg/mL, about 1.8 times more potent than vitamin C (0.54. + -. 0.71mg/mL), and about 2.8 times more potent than ovalbumin (0.822. + -. 0.83 mg/mL). The lipid peroxidation inhibition capacity is as follows in sequence: potato derived protease inhibitors>Vitamin C>Ovalbumin (FIG. 2-E).
Example 3 Effect of different processing factors on Oxidation resistance
(1) Temperature stability
Preparing potato source protease inhibitor into solution (1mg/mL), heating at 4-100 deg.C for 1 hr, and rapidly cooling. Measuring the change curve of the in vitro antioxidant capacity with the temperature.
As shown in FIG. 3-A, the in vitro antioxidant capacity of potato-derived protease inhibitors is inversely related to temperature. The total reducing power, DPPH removing capacity, hydroxyl radical removing capacity and lipid peroxidation inhibition rate of the composition slowly decrease with the increase of temperature. The scavenging capacity of ABTS free radicals has two gradient descending stages, and the ABTS free radicals still have higher antioxidant capacity before 50 ℃. The results show that the potato-derived protease inhibitor can maintain stable antioxidant activity in a certain temperature range.
(2) Stability of pH
Dissolving potato source protease inhibitor in different pH solutions to prepare protein solution with concentration of 1mg/mL, standing at 4 deg.C for 1h, and determining the variation curve of its in vitro antioxidant ability with pH.
As shown in FIG. 3-B, there are different effects of pH on the in vitro antioxidant capacity of potato-derived protease inhibitors. In a hydroxyl free radical system, the potato-derived protease inhibitor has better scavenging activity under acidic or alkaline conditions. The DPPH free radical scavenging ability is inversely related to pH, and the DPPH free radical scavenging ability is better under acidic conditions. ABTS is contrary to DPPH trend. In a grease system, the potato-derived protease inhibitor has a good inhibition rate on lipid peroxidation under an acidic weakly alkaline condition. The result shows that the potato-derived protease inhibitor has good oxidation resistance in a wider pH range and has wide application prospect.
(3) Stability against high pressure
The potato-derived protease inhibitor was prepared as a solution (1mg/mL) and treated at 25 ℃ under 100-600MPa for 10 min. Measuring the change curve of the in vitro antioxidant capacity of the strain along with the pressure.
As shown in FIG. 3-C, the potato-derived protease inhibitor showed no significant change in oxidation resistance after high pressure treatment. The results show that the potato-derived protease inhibitor can maintain good structure and antioxidant activity in a short time and high pressure treatment. Therefore, the potato-derived protease inhibitor can be used as a potential functional factor capable of resisting ultra-high pressure sterilization and applied to food, medicines and cosmetics.
Example 4 intracellular and extracellular Oxidation resistance
Human liver cancer HepG2 cells in logarithmic growth phase according to 6X 105one/mL was inoculated into a black clear flat-bottomed 96-well plate and cultured overnight. The cell culture medium was aspirated and washed with PBS buffer. Sample and DCFH-DA prepared with cell culture fluid at a 1:1 ratioFor example, the cells were incubated in a cell incubator for 1 h. DCFH-DA was aspirated, and the first 3 wells of each sample were washed with PBS and the last 3 wells were not washed with PBS. ABAP dissolved in HBSS is quickly added by a discharging gun and is quickly put into a multifunctional fluorescence microplate reader for determination, and the molecular weight ratio of ABAP to HBSS is determined according to the excitation wavelength: 485nm, emission wavelength: the change in fluorescence intensity was measured every 5min at 538nm, and the change in fluorescence intensity was measured over 60 min. And calculating the area under the curve, and bringing the area into a standard curve to obtain the equivalent weight of the quercetin of the sample. As shown in Table 1, the potato-derived protease inhibitor has a concentration dependency on the intracellular and extracellular antioxidant ability, and provides scientific basic data and possibility for further application of antioxidant activity in human body in the future.
TABLE 1 antioxidant action of potato protease inhibitors in and out of cells
Example 5 protective action against oxidative Damage
HUVEC cells with good growth state in logarithmic growth phase are taken and cultured at 2X 105Inoculating in one hole, culturing overnight, discarding culture solution, adding potato protease inhibitor, pre-incubating with cells for 1 hr, and adding H2O2Incubate for 1 h. The kit is used for detecting the change of ROS, LDH, SOD, NO and MDA before and after damage, and 6 parallel reagents are arranged in each group. The results are shown in Table 2 and FIG. 4, that potato-derived protease inhibitors can effectively protect HUVEC cells from H2O2Oxidative stress damage.
Comparative example 1: normal growth cell
Comparative example 2: h2O2Model group:
TABLE 2 Potato protease inhibitor vs. H2O2Induced effects of LDH, SOD, MDA, NO in HUVEC cells
Note: lower case letters in the table indicate statistically significant differences at P < 0.05.
Example 6 antitumor Effect
GIST882 in logarithmic growth phase at 1X 105one/mL of the cells were inoculated into a 96-well plate and placed in an incubator overnight. Discarding supernatant, adding sample, 37 deg.C, 5% CO2The culture box is used for culturing for 48 hours. Adding MTT solution, keeping out of the sun, incubating at 37 ℃ for 4h, discarding the supernatant, adding DMSO, measuring the absorbance at 490nm, and calculating the survival rate. Each set of samples was set in 6 replicates. The potato protease inhibitor has certain inhibiting effect on GIST882 tumor cells, IC50The content of the active ingredients in the product is 10.532 +/-0.87 mg/mL, so that the product can be used in health-care food for inhibiting tumor growth.
The foregoing embodiments illustrate and describe the principles and general features of the present invention and its advantages. It will be understood by those skilled in the art that the present invention is not limited by the embodiments described above, which are given by way of illustration of the principles of the invention and are not to be taken as limiting the scope of the invention in any way, and that various changes and modifications may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (6)
1. A method for extracting a potato-derived protease inhibitor mixture, which is characterized by comprising the following steps:
1) adding ammonium sulfate into the industrial waste liquid of the potato starch at room temperature to ensure that the saturation degree of the industrial waste liquid reaches 30% -80%, oscillating and extracting the obtained system for 90-120 min at 80-150 rpm under the condition of 4-6 ℃, and carrying out solid-liquid separation for 20-30 min at 8000-10000 r/min to obtain a precipitate which is a crude potato protein extract;
2) re-dissolving the crude potato protein extract prepared in the step 1) in distilled water at a feed-to-liquid ratio of 1: 5-1: 10(g/mL) to obtain a crude potato protein extract;
3) adjusting the pH value of the crude potato protein extracting solution prepared in the step 2) to 2.5-5.0, standing, performing solid-liquid separation at 8000-10000 r/min for 20-30 min, dialyzing the supernatant for 36-48 h, and performing freeze drying treatment to obtain the potato-derived protease inhibitor mixture.
2. A potato-derived protease inhibitor blend extracted by the method of claim 1.
3. Use of the potato-derived protease inhibitor cocktail of claim 2 for protecting against oxidative stress.
4. The use of the potato-derived protease inhibitor cocktail of claim 3 for antioxidant protection in an in vitro antioxidant capacity and process stability, intracellular and extracellular antioxidant activity, and for protecting cells from H2O2Use of an induced oxidative stress injury direction.
5. Use of the potato-derived protease inhibitor cocktail of claim 2 for combating tumor.
6. The use of the potato derived protease inhibitor cocktail of claim 5 for inhibiting the growth of GIST882 tumor cells in an anti-tumor orientation.
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| CN101597332A (en) * | 2009-07-14 | 2009-12-09 | 江南大学 | A kind of preparation method of potato asparaginic acid protease inhibitors |
| CN104387443A (en) * | 2014-11-26 | 2015-03-04 | 江南大学 | Method for efficiently recycling protein and free amino acid from potato starch processing wastewater |
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