CN108586581B - Soybean polypeptide and preparation method and application thereof - Google Patents
Soybean polypeptide and preparation method and application thereof Download PDFInfo
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- CN108586581B CN108586581B CN201810317023.3A CN201810317023A CN108586581B CN 108586581 B CN108586581 B CN 108586581B CN 201810317023 A CN201810317023 A CN 201810317023A CN 108586581 B CN108586581 B CN 108586581B
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
Abstract
The invention discloses a soybean polypeptide and a preparation method and application thereof. The method is characterized in that fermented bean curd is used as a raw material, and the soybean polypeptide is prepared by two-step extraction. Firstly, mixing fermented bean curd, a freeze-dried fermented bean curd product or defatted fermented bean curd according to the weight ratio of 1: 5-1: 10 adding water, extracting, centrifuging or filtering, collecting filtrate, evaporating, concentrating, and freeze drying; and then Sephadex G15 Sephadex gel chromatography filtration and fractional collection are carried out to obtain the water-soluble soybean polypeptide with the molecular weight of 500-. On the basis, extracting the residue left after water extraction with 60-90% ethanol, centrifuging or filtering, concentrating and drying to obtain the alcohol-soluble soybean polypeptide with molecular weight below 3000 Da. The soybean polypeptide purified and prepared by the method has the purity of more than 80 percent, the salt content of less than 0.5 percent, and the effects of antioxidation activity and inhibition of the growth of breast cancer cells MCF-7 and liver cancer cells HepG2 are obvious.
Description
Technical Field
The invention relates to the technical field of polypeptide preparation, in particular to a preparation technology for extracting, separating and purifying functional polypeptides for resisting oxidation and inhibiting the growth of cancer cells in fermented bean curd.
Background
China is a large producing country of soybeans and a large consuming country of soybean products. Soybeans contain abundant proteins, and the amino acid proportion of the soybeans is reasonable, so the soybeans are often used as substitutes of animal proteins. The soybean polypeptide is a compound generated by hydrolyzing soybean protein, the average peptide chain length is 2-10 amino acid residues, and the molecular weight distribution is usually below 3000 Da. The soybean polypeptide is easy to digest and absorb, has physiological functions of resisting oxidation, resisting aging, reducing blood pressure, reducing blood fat, resisting fatigue and the like, and is a functional raw material with wide application. The preparation of the soybean polypeptide mainly takes soybean protein isolate or soybean meal and the like as raw materials, and is obtained by applying protease to carry out enzymatic degradation under certain temperature and pH conditions. However, due to the fact that the commercial types are not many and the action sites are single, the polypeptide sequences generated by catalytic degradation are often homogenized, and some functions such as anticancer activity are reported less frequently. At present, soybean polypeptides are prepared by adopting a fermentation method more, namely, a bacterial strain with strong protease production capacity is screened to directly ferment soybean meal, so that the production cost can be reduced, the flavor of the polypeptides can be improved, and the polypeptides prepared by the method are usually low in purity and have unobvious efficacy.
The fermented bean curd is a traditional fermented soybean product in China, and because protease secreted by various microorganisms participates in the degradation of protein, polypeptide molecules in the fermented bean curd are quite possible to have more unique sequence and functional characteristics than those of the conventional enzyme preparation. However, the production conditions of fermented bean curd are extensive, and high-concentration salt is usually added to control the growth of harmful bacteria, so that the fermented bean curd is not suitable to be directly applied as functional food. In addition, a large amount of leftover materials and products with partial damaged appearance are generated in the production process of the fermented bean curd. Therefore, the comprehensive utilization of the fermented bean curd and the byproducts thereof is enhanced, and the method has important practical significance.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a soybean polypeptide product with simple operation, high purity and good efficacy and a production and preparation method thereof.
The invention also aims to provide the application of the functional soybean polypeptide in preparing health-care food or anti-cancer drugs.
The raw material for preparing the soybean polypeptide is finished fermented bean curd or fermented bean curd with broken appearance. The fermented bean curd is a seasoning prepared by taking soybeans as raw materials and performing soaking, pulping, separation, curding, squeezing, fermentation and after-ripening. Under the catalysis of microbial protease, protein in the fermented bean curd is usually hydrolyzed to generate polypeptide with molecular weight below 10000Da, and because the types of microbes participating in fermentation are many, the generated polypeptide sequence is rich, no bitter taste exists, and the defect of poor flavor of the hydrolyzed protein is avoided.
The preparation method of the invention takes the fermented bean curd as a raw material, and directly extracts, desalts and fractionates the polypeptide in the fermented bean curd to obtain the polypeptide product and the sequence which have obvious antioxidant activity and can inhibit the growth of cancer cells.
The invention uses water to extract water-soluble polypeptide, and then uses ethanol solution to extract alcohol-soluble polypeptide. The refining of the water-soluble soybean polypeptide adopts a gel filtration method, namely different molecular sieves are selected for desalting and purifying according to the molecular weight; the method has the advantages of good purification effect, high recovery rate, simple and convenient operation, and can be replaced by ultrafiltration in actual production. The alcohol-soluble soybean polypeptide does not need further purification and desalination treatment. The two-step extraction method can simultaneously obtain the water-soluble polypeptide and the alcohol-soluble polypeptide, and furthest utilizes the protein in the fermented bean curd raw material.
The purpose of the invention is realized by the following technical scheme:
a method for preparing functional soybean polypeptide comprises the following steps:
1) taking kilograms and liters as mass and volume units respectively, and mixing the fermented bean curd, the freeze-dried fermented bean curd product or the defatted fermented bean curd according to the weight ratio of 1: 5-1: 10, adding water, extracting by shaking at 20-30 ℃, collecting supernatant, repeating the operations, and combining the supernatants; evaporating, concentrating, and freeze drying to obtain water soluble polypeptide crude extract;
2) re-dissolving the crude water-soluble polypeptide extract prepared in the step 1), centrifuging and then loading the sample; using deionized water as eluent, detecting wavelength at 220nm, collecting chromatography liquid, collecting polypeptide with molecular weight distribution of 500-1000Da within 20-50min, and collecting polypeptide with molecular weight distribution of <500Da within 50-80 min; evaporating, concentrating, and freeze drying the two kinds of collected polypeptides to obtain water soluble soybean polypeptide powder.
To further achieve the object of the present invention, preferably, the shaking extraction time in step 1) is 30-90 min; the number of times of repeating the above operation in the step 1) is 1-2 times.
Preferably, the evaporation concentration in the step 1) is rotary evaporation concentration, the temperature is 50-60 ℃, and the time is 20-40 min; the evaporation concentration in the step 2) is rotary evaporation concentration, the temperature is 50-60 ℃, and the time is 15-30 min.
Preferably, the post-centrifugation sample loading in the step 2) is a post-centrifugation deionized water equilibrium Sephadex G-15 gel chromatographic column; the flow rate of the eluent is 0.8-1.2 mL/min.
A functional soybean polypeptide is prepared by the above preparation method; the functional soybean polypeptide is water-soluble soybean polypeptide powder, the protein content is 90-98% by mass, the sodium chloride content is not detected, the molecular weight distribution is below 1000Da, the main band in a mass spectrum is 943.5283, and the amino acid sequence is Phe-Asn-Lys-Thr-Ser-Ser-Phe-Asn.
The invention also discloses a preparation method of the functional soybean polypeptide, which comprises the following steps:
1) taking kilograms and liters as mass and volume units respectively, and mixing the fermented bean curd, the freeze-dried fermented bean curd product or the defatted fermented bean curd according to the weight ratio of 1: 5-1: 10, adding water, extracting by shaking at 20-30 ℃, collecting supernatant, repeating the operations, and combining the supernatants; evaporating, concentrating, and freeze drying to obtain water soluble polypeptide crude extract;
2) measured in kilograms and liters, respectively, as mass and volume units, as 1: 5-1: 10, adding 50-95% ethanol by volume concentration into the residue after the water-soluble polypeptide is extracted in the step 1) for further extraction, shaking and extracting at 20-30 ℃, collecting supernatant, repeating the above operation, and combining the supernatants; concentrating, and freeze drying to obtain alcohol soluble soybean polypeptide powder.
To further achieve the object of the present invention, preferably, the shaking extraction time in step 1) and step 2) is 30-90 min; the times of repeating the operations in the step 1) and the step 2) are 1-2 times.
Preferably, the evaporation concentration in the step 1) is rotary evaporation concentration, the temperature is 50-60 ℃, and the time is 20-40 min; the concentration temperature in the step 2) is 40-50 ℃, and the time is 15-30 min.
A functional soybean polypeptide is prepared by the above preparation method; the functional soybean polypeptide is alcohol-soluble polypeptide, wherein the content of the protein of the alcohol-soluble polypeptide is 80-98% by mass, the content of sodium chloride is 0-0.5%, the molecular weight distribution is below 3000Da, the main band in a mass spectrum is 2088.0157, and the amino acid sequence is Val-Ser-Ile-Ile-Asp-Thr-Asn-Ser-Leu-Gle-Asn-Gln-Leu-Asp-Gln-Met-Pro-Arg.
The functional soybean polypeptide is applied to the preparation of anti-cancer drugs. In-vitro experiments prove that all the prepared polypeptide samples have excellent antioxidant activity and the effect of inhibiting the growth of cancer cells, and the functional characteristics are obvious.
The water-soluble polypeptide and the alcohol-soluble polypeptide both have obvious antioxidant activity and inhibition effect on cancer cell growth. The ABTS free radical scavenging ability is 40-210 mu mol tocopherol/g polypeptide, the DPPH free radical scavenging ability is 60-130 mu mol tocopherol/g polypeptide, and the ferric iron reducing ability is 2.0-8.0mmol tocopherol/g polypeptide. The polypeptide sample with the concentration of 500 mug/ml is used for respectively treating the breast cancer cell MCF-7 and the liver cancer cell HepG2, the proliferation inhibition rates of the two cancer cells after 48 hours reach 26% -37% and 24% -32% respectively, and the proliferation inhibition rate of the normal liver cell LO2 is only 6% -14%.
The artificially synthesized high-purity polypeptides Phe-Asn-Lys-Thr-Ser-Ser-Phe-Asn and Val-Ser-Ile-Ile-Asp-Thr-Asn-Ser-Leu-Gle-Asn-Gln-Leu-Asp-Gln-Met-Pro-Arg are respectively used for treating the breast cancer cell MCF-7 and the liver cancer cell HepG2 to present an obvious dose-effect relationship, and further prove that the high-purity polypeptides have the effect of inhibiting the growth of the cancer cells.
When the functional fermented bean curd polypeptide is prepared, the raw material can also be defatted fermented bean curd. After the fermented bean curd is subjected to degreasing treatment, water and an alcohol solution are sequentially used for extraction, separation and purification according to the mode, so that the water extraction active polypeptides DP1 and DP2 of the degreased fermented bean curd and the alcohol extraction active polypeptide DEP of the degreased fermented bean curd are obtained.
The fermented bean curd polypeptide prepared by the method is proved to have remarkable efficacy through further in-vitro antioxidant and anticancer analysis.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention comprehensively utilizes the raw materials of the fermented bean curd, the damaged fermented bean curd and the like, adopts a two-step extraction method of extracting water and then extracting alcohol, can furthest extract the polypeptide components in the fermented bean curd, does not need further refining the obtained alcohol-soluble polypeptide, provides a brand-new and simple solution strategy for the preparation of the soybean polypeptide, and provides a possibility for the modern application of the traditional fermented food. Wherein, the extraction of the alcohol-soluble polypeptide is not reported.
(2) The soybean polypeptide prepared by the invention comprises water-soluble soybean polypeptide and alcohol-soluble soybean polypeptide, and has obvious antioxidant activity and activity for inhibiting the growth of cancer cells, wherein few reports of the soybean polypeptide for inhibiting the growth of the cancer cells exist, and the soybean polypeptide can be developed and applied as a functional food raw material. The sequences and functional properties of the water-soluble polypeptide FNKTSSFN (consisting of 8 amino acid residues) and the alcohol-soluble polypeptide VSIIDTNSLENQLDQMPR (consisting of 18 amino acid residues) have not been reported.
(3) The invention adopts a Sephadex G15 gel filtration chromatography method to separate and purify the water-soluble polypeptide of the preserved beancurd, and realizes the desalination treatment of the water-soluble polypeptide of the preserved beancurd; moreover, the method can be conveniently converted into an ultrafiltration method for desalination and fractional membrane separation in actual production, and has good industrial popularization and application prospects.
Drawings
FIG. 1 is a mass spectrum of a polypeptide P1 of fermented bean curd extracted with water in example 1 of the present invention;
FIG. 2 is a mass spectrum of a polypeptide P2 of fermented bean curd extracted with water in example 2 of the present invention;
FIG. 3 is a mass spectrum of an alcohol-extracted fermented bean curd polypeptide EP in example 3 of the present invention;
FIG. 4 is a mass spectrum of a polypeptide DP1 of water-extracted defatted fermented bean curd in example 4 of the present invention;
FIG. 5 is a mass spectrum of a polypeptide DP2 of water-extracted defatted fermented bean curd in example 5 of the present invention;
FIG. 6 is a mass spectrum of the alcohol-extracted defatted fermented bean curd polypeptide DEP in example 6 of the present invention.
Detailed Description
In order to better illustrate the present invention, the following description is given with reference to the accompanying drawings and examples, but the examples should not be construed as limiting the scope of the present invention.
Description of the test methods in the examples:
1) the method for measuring the contents of fat and NaCl comprises the following steps: the contents of fat and NaCl in the sample before and after degreasing and desalting respectively reflect the degreasing effect and the desalting effect of the fermented bean curd sample, which are respectively measured according to the methods described in the standards GB/T14772-2008 and GB/T12457-2008.
2) The method for measuring the protein content comprises the following steps: the total protein content, the water-soluble protein content and the amino nitrogen content in the preserved beancurd are measured according to the methods in GB5009.5-2010, NY/T1205-2006 and SB/T10170-2007, wherein the proportion of the protein and the water-soluble protein reflects the hydrolysis degree of the protein, the higher the water-soluble protein content is, the higher the proportion of the water-soluble protein content is, the larger the hydrolysis degree is, and the amino nitrogen content reflects the maturity of the preserved beancurd.
3) The method for analyzing and identifying the components of the polypeptide comprises the following steps: the determination is carried out by referring to the method described in Purification and identification of antibiotic peptides from the floor space (Misgurnus anguillatus) protein hydrostate by coherent chromatography and electrophoresis. RP-HPLC detection wavelength: 214 nm; column temperature: 300C; flow rate: 0.2 mL/min; sample introduction amount: 10 mu L of the solution; mobile phase A: water/TFA (1000: 0.25, v/v); mobile phase B: methanol. The gradient elution conditions were as follows: 0-10min, 0% -2% B (linear gradient); 10-15min, 2-10% B (linear gradient); 15-30min, 10-40% B (linear gradient); 30-35min, 40-80% B (linear gradient); 35-50min, 80% B (equivalent elution); 50-55min, 80-2% B (linear gradient); 55-60min, 2-0% B (linear gradient).
The flow of sample after RP-HPLC analysis entered the mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) through the electrospray interface at a rate of 100 μ L/min. The positive ion scanning mode is adopted, the atomizing gas and the drying gas are high-purity nitrogen, and the scanning range is that the mass-to-nucleus ratio is 0-3000 (m/z). The MS/MS peptide sequence analysis was calculated by means of Biotools (version 3.0, Bruker Daltonics Data analysis 4.1) software in combination with manual calculations. Finally, amino acid sequence combinations of the polypeptides were obtained by consulting a soybean polypeptide database at www.uniprot.org.
4) The method for measuring the in vitro antioxidant activity comprises the following steps:
determination of The ability of fermented bean curd polypeptide extract to reduce trivalent ironThe method comprises the following specific operations: preparing 10mmol/L TPTZ solution with 40mmol/L HCl solution, and adding 2.5mL TPTZ solution and 2.5mL 20mmol/L FeCl3Mixing the solution with 25mL of acetic acid buffer solution (300mmol/L, pH3.6) to obtain FRAP solution. Taking 100 μ L of sample and 3mL of FRAP to react at 37 deg.C for 10min, measuring absorbance value at 592nm, using tocopherol as standard substance, and obtaining standard curve equation of y-1.272 x-0.015 (R)20.998), and the antioxidant activity result of the fermented bean curd polypeptide extract is expressed as mmol/L tocopherol/mL sample solution.
Second, the Method described in the references of Kinetics and mechanics of antibiotic Activity using the DPPH.free radial Method for measuring the ability of fermented bean curd polypeptide extract to remove DPPH free radicals specifically comprises the following steps: diluting the polypeptide extract by 10 times, shaking 2mL diluted sample solution with 2mL DPPH ethanol solution (0.2mmol/L), standing at room temperature for 30min, and measuring absorbance value at 517 nm. Using tocopherol as standard curve, using different concentration (concentration gradient is 20 mu mol/L) of tocopherol solution as abscissa, and using correspondent light absorption value as ordinate to make plotting them to obtain standard curve whose y is-0.012 x +1.096, R is20.999. The determination result of the capability of the fermented bean curd polypeptide sample to eliminate DPPH free radicals is expressed as mu mol/L tocopherol/mL sample solution.
③ removing ABTS in polypeptide extract of fermented bean curd+The method for measuring the free radical capacity is described in the referential literature of effective application and improved ABTS radial location deconstruction assay, and the specific operation is as follows: ABTS-containing 7mmol/L ABTS solution and 2.45mmol/L potassium persulfate solution was prepared+The solution is used by diluting ABTS with ethanol in dark room temperature for 12-16h+The solution was allowed to absorb 0.70. + -. 0.02 at 734 nm. 30. mu.L of the sample solution was added with 3mL of the above diluted ABTS + solution, reacted at room temperature for 6min, and then the absorbance value was measured at 734 nm. Using tocopherol as a standard curve, the standard curve equation was found to be y ═ 0.009x +0.712, (R)20.992), the antioxidant activity result of the fermented bean curd polypeptide extract is expressed as μmol/L tocopherol/mL sample solution.
5) Determination of the anti-cancer activity of the fermented bean curd polypeptide on cells: the assay was carried out according to the methods described In the literature for In vitro antioxidant activities and anti-functional properties of the functional here abs cantoniensis and its main analog, as follows: the inoculation of breast cancer cells MCF-7 and liver cancer cells HepG2 cells and normal liver cells LO2 on a 96-well plate as a contrast, so that the cell density of each well reaches 3000-5000 cells under the condition of adding 100 mu L of culture solution, the culture solution is removed after the cells are cultured for 24h in an adherent manner, and each inoculation hole is washed by PBS. Then 100. mu.L of the prepared fermented bean curd active polypeptide solution with continuous concentration gradient is added into each well, 100. mu.L of DMEM medium is added into the control group, and the culture is continued for 24h, 48h and 72h under the conditions of 37 ℃ and 5% CO 2. After adding 100. mu.L (0.5mg/mL) of MTT solution to each well and further culturing for 4 hours, the supernatant was removed, washed with 150. mu.L of DMSO solution, shaken at room temperature for 10min, and the absorbance was read at 490nm, reference wavelength 655 nm.
The cellular inhibition rate calculation formula is as follows:
comparative example: water-soluble protein WP of fermented bean curd
Firstly, taking 5g of fermented bean curd freeze-dried powder in a 100mL centrifuge tube, sucking 50mL of distilled water in the centrifuge tube, oscillating in a water bath on a shaking table at 20 +/-2 ℃ for 60min, centrifuging for 10min at 8000r/min, pouring the supernatant into a 250mL round-bottom flask, adding 50mL of distilled water into the residue of the centrifuge tube again, repeating the process for 2 times, finally, completely merging the supernatant into the 250mL round-bottom flask, concentrating for 30min at 55 ℃ to obtain yellow water-soluble soybean polypeptide concentrated solution, freezing and drying, and preserving in a refrigerator at-20 ℃.
Secondly, the protein content of the water-soluble protein extracting solution is 38.4 percent, the fat content is 24.35 percent and the salt content is 24.91 percent through detection and analysis; the antioxidant activity of the compound is 62.08 mu mol of tocopherol/g sample, 68.69 mu mol of tocopherol/g sample and 1.19mmol of tocopherol/g sample respectively.
Example 1: water-extraction fermented bean curd polypeptide P1
Firstly, taking 5g of fermented bean curd freeze-dried powder in a 100mL centrifuge tube, sucking 50mL of distilled water in the centrifuge tube, oscillating in a water bath on a shaking table at 20 +/-2 ℃ for 60min, centrifuging for 10min at 8000r/min, pouring the supernatant into a 250mL round-bottom flask, adding 50mL of distilled water into the residue of the centrifuge tube again, repeating the process for 2 times, finally, completely merging the supernatant into the 250mL round-bottom flask, concentrating for 30min at 55 ℃ to obtain yellow water-soluble soybean polypeptide concentrated solution, freezing and drying, and preserving in a refrigerator at-20 ℃.
And secondly, balancing the Sephadex G-15 gel chromatographic column by using deionized water, re-dissolving the crude water-soluble polypeptide extract sample, and centrifuging and then loading the sample. Deionized water is used as eluent, the flow rate is 1mL/min, the detection wavelength is 220nm, and the eluent is collected by an automatic partial collector. Collecting the polypeptide eluate with molecular weight distribution of 500-1000Da for 20-50min, concentrating at 55 deg.C for 20min, and freeze drying to obtain polypeptide powder, which is designated as P1.
And thirdly, further analyzing the composition and molecular weight distribution of the P1, and finding that the protein content is up to 98.12 percent, and the fat content and the NaCl content are not detected, thereby indicating that the sample is purified. As can be seen from FIG. 1(HPLC-MS spectrum), its molecular weight distribution is between 500-1000Da, mainly composed of bands such as 943.5273 and 796.5724, wherein the band with the highest abundance is 943.5273, and its amino acid sequence is FNKTSSFN, i.e., Phe-Asn-Lys-Thr-Ser-Ser-Ser-Phe-Asn, obtained by searching soybean peptide database www.uniprot.org and combining manual calculation and analysis.
And fourthly, analyzing the antioxidant activity and the anticancer activity of P1 in one step, and finding that the antioxidant activity is greatly improved, the capability of eliminating ABTS free radicals is 196.17 mu mol of tocopherol/g sample, the capability of eliminating DPPH free radicals is 118.34 mu mol of tocopherol/g sample, and the reducing power of ferric iron is 6.86mmol of tocopherol/g sample. The anticancer activity of P1 is reflected in the inhibition effect on the proliferation of liver cancer cell HepG2 and breast cancer cell MCF-7, after the treatment of P1 with the concentration of 500 mug/mL for 48 hours, the inhibition efficiency on the proliferation of MCF-7 is 36.42 percent, the inhibition efficiency on the proliferation of HepG2 is 32.01 percent, and the inhibition efficiency on the proliferation of normal liver cell LO2 is only 13.84 percent, which shows that P1 has stronger selectivity on tumor cells and can inhibit the proliferation of the tumor cells.
And fifthly, verifying the experiment. Artificially synthesizing a high-purity polypeptide with the sequence of FNKTSSFN, testing the inhibition effect on the proliferation of liver cancer cells HepG2 and breast cancer cells MCF-7, and finding that the polypeptide has a dose-effect relation in the concentration range of 10-2000 ug/ml. Wherein, after being treated by the polypeptide with the concentration of 500 mug/mL for 24 hours, the proliferation inhibition efficiency of MCF-7 is 20.14 percent, the proliferation inhibition efficiency of HepG2 is 14.80 percent, and the proliferation inhibition efficiency of normal liver cell LO2 is-4.94 percent, which proves that the polypeptide in the sample P1 has the effect of inhibiting the growth of cancer cells.
At present, many antioxidant active peptides have been reported, but no antioxidant active peptide from fermented bean curd exists, and no research for efficiently preparing the antioxidant active peptide by taking fermented bean curd as a raw material exists. There are few reports of soybean polypeptides having an effect of inhibiting cancer cell proliferation.
Example 2: water-extraction fermented bean curd polypeptide P2
Firstly, taking 5g of fermented bean curd freeze-dried powder in a 100mL centrifuge tube, sucking 50mL of distilled water in the centrifuge tube, oscillating in a water bath on a shaking table at 20 +/-2 ℃ for 60min, centrifuging for 10min at 8000r/min, pouring the supernatant into a 250mL round-bottom flask, adding 50mL of distilled water into the residue of the centrifuge tube again, repeating the process for 2 times, finally, completely merging the supernatant into the 250mL round-bottom flask, concentrating for 30min at 55 ℃ to obtain yellow water-soluble soybean polypeptide concentrated solution, freezing and drying, and preserving in a refrigerator at-20 ℃.
And secondly, balancing a Sephadex G-15 gel chromatographic column with distilled water, desalting and performing fractional separation on the fermented bean curd soybean polypeptide concentrated solution through gel column chromatography, wherein the eluent is deionized water, the flow rate is 1mL/min, the detection wavelength is 220nm, and the eluent is collected by an automatic part collector. Collecting the polypeptide eluate with molecular weight distribution of <500Da for 50-80min, concentrating at 55 deg.C for 20min, and freeze drying to obtain polypeptide powder, which is designated as P2.
And thirdly, further analyzing the composition and molecular weight distribution of P2, and finding that the protein content is as high as 97.82%, and the fat content and the NaCl content are not detected, which indicates that the sample is purified. From the HPLC-MS spectrum (fig. 2), the molecular weight distribution was in the range <500Da, consisting mainly of bands 425.2166 and 301.1425, with the most abundant band being 425.2166.
And analyzing the antioxidant activity and the anticancer activity of P2 in one step, and finding that the antioxidant activity is obviously improved compared with that before purification, the ABTS free radical scavenging capacity is 38.49 mu mol of tocopherol/g of sample, the DPPH free radical scavenging capacity is 118.57 mu mol of tocopherol/g of sample, and the ferric iron reducing capacity is 2.97mmol of tocopherol/g of sample. The anticancer activity of P2 is reflected in the proliferation inhibition effect on liver cancer cells HepG2 and breast cancer cells MCF-7, after the treatment of P2 with the concentration of 500 mug/mL for 48 hours, the proliferation inhibition rate on MCF-7 is 26.29%, the proliferation inhibition rate on HepG2 is 23.48%, and the inhibition rate on normal liver cells LO2 is only 10.16%, which shows that the fermented bean curd water extract polypeptide P2 has stronger selectivity on tumor cells and can inhibit the proliferation of the tumor cells.
Example 3: alcohol extraction fermented bean curd polypeptide EP
The first step is to add 50mL 80% ethanol into the precipitate after the water-soluble polypeptide is extracted in example 1, carry out leaching, shake in water bath at 20 ℃ for 60min, centrifuge at 8000r/min for 10min, pour the supernatant into a 250mL round-bottom flask, add 50mL 80% ethanol into the residue of the centrifuge tube again, repeat the above process for 2 times, finally, incorporate the supernatant into the 250mL round-bottom flask completely, concentrate at 45 ℃ for 20min to obtain a yellow oily alcohol-soluble soybean polypeptide concentrate, freeze-dry to obtain a yellow oily freeze-dried substance, which is recorded as EP.
The second step, further analysis of the composition and molecular weight distribution of EP, found that it had a protein content of 81.13%, a fat content of 17.86% and a NaCl content of 0.44%, indicating that it had been enriched to a higher degree. FIG. 3 is a mass spectrogram of fermented bean curd alcohol extract polypeptide, the molecular weight distribution of which is below 3000Da, mainly comprising bands of 2088.0157, 943.5273 and 353.2306, wherein the band with the highest abundance is 2088.0157, and the amino acid sequence of which is VSIIDTNSLENQLDQMPR obtained by searching soybean polypeptide database www.uniprot.org and combining manual calculation and analysis, namely Val-Ser-Ile-Ile-Asp-Thr-Asn-Ser-Leu-Gle-Asn-Gln-Leu-Asp-Gln-Met-Pro-Arg.
The third step further analyzes the antioxidant activity and the anticancer activity of EP, and finds that the capability of eliminating ABTS free radicals is 43.80 mu mol tocopherol/g sample, the capability of eliminating DPPH free radicals is 62.57 mu mol tocopherol/g sample, and the ferric iron reducing power is 2.06mmol tocopherol/g sample. The anticancer activity of EP is reflected in the proliferation inhibition effect on liver cancer cells HepG2 and breast cancer cells MCF-7, after the EP with the concentration of 500 mug/mL is treated for 48 hours, the proliferation inhibition rate on MCF-7 is 26.00 percent, the proliferation inhibition rate on HepG2 is 24.15 percent, and the proliferation inhibition rate on normal liver cells LO2 is only 13.62 percent, which shows that the fermented bean curd alcohol extract polypeptide EP has stronger selectivity on tumor cells and can inhibit the proliferation of the tumor cells.
And step four, verifying the experiment. Artificially synthesizing a high-purity polypeptide with the sequence of VSIIDTNSLENQLDQMPR, testing the inhibition effect of the polypeptide on the proliferation of liver cancer cells HepG2 and breast cancer cells MCF-7, and finding that the polypeptide has a dose-effect relationship in the concentration range of 10-2000 ug/ml. Wherein, after being treated by the synthetic polypeptide with the concentration of 500 mug/mL for 48 hours, the proliferation inhibition efficiency of MCF-7 is 28.89%, the proliferation inhibition efficiency of HepG2 is 33.56%, and the proliferation inhibition efficiency of normal liver cell LO2 is-5.82%, which proves that the polypeptide in the sample EP has the effect of inhibiting the growth of cancer cells.
Example 4: defatted water-extracted fermented bean curd polypeptide DP1
In the first step, 100g of a mature fermented bean curd freeze-dried sample is taken and mixed with n-hexane according to the ratio of 1: mixing at a ratio of 2, extracting with shaking in 40 deg.C water bath for 30min, centrifuging at 10000g/min for 3min, discarding supernatant, adding n-hexane again, repeating for 2 times to remove fat, oven drying at 65 deg.C to constant weight, and sealing in bottle.
And secondly, taking 5g of defatted fermented bean curd powder into a 100mL centrifuge tube, sucking 50mL of water into the centrifuge tube, shaking in a water bath at 25 ℃ for 90min, centrifuging for 10min at 8000r/min, pouring the supernatant into a 250mL round-bottom flask, adding 50mL of distilled water into the residue of the centrifuge tube again, repeating the process for 1 time, finally, completely merging the supernatant into the 250mL round-bottom flask, concentrating at 50 ℃ for 40min to obtain a light yellow soybean polypeptide concentrated solution, freeze-drying, and preserving in a refrigerator at-20 ℃.
And thirdly, balancing the Sephadex G-15 gel chromatographic column with distilled water, performing desalination and fractionation on the fermented bean curd soybean polypeptide by gel column chromatography, wherein the eluent is deionized water, the flow rate is 1mL/min, the detection wavelength is 220nm, and the eluent is collected by an automatic part collector. Collecting the polypeptide eluate with molecular weight distribution of 500-1000Da for 20-50min, concentrating at 55 deg.C for 20min, and freeze drying to obtain polypeptide powder, which is designated as DP 1.
The composition and molecular weight distribution of DP1 were analyzed in one step, and the protein content was found to be as high as 98.55%, and no fat and NaCl were contained. FIG. 4 is a mass spectrum of DP1, and it can be seen that its molecular weight distribution is similar to P1, and between 500 and 1000Da, it is mainly composed of 943.5278 and 796.5724 bands, in which the most abundant band is also 943.5283.
The antioxidant activity and the anticancer activity of DP1 were analyzed in one step in the fifth step, and it was found that it had an ABTS radical scavenging ability of 196.50. mu. mol tocopherol/g sample, an DPPH radical scavenging ability of 116.92. mu. mol tocopherol/g sample, and a ferric iron reducing ability of 6.89mmol tocopherol/g sample. The anticancer activity of the polypeptide DP1 of the defatted fermented bean curd water extract is reflected in the proliferation inhibition effect on liver cancer cells HepG2 and breast cancer cells MCF-7, after the polypeptide DP1 with the concentration of 500 mug/mL is treated for 48 hours, the proliferation inhibition efficiency on the MCF-7 is 37.03 percent, the proliferation inhibition rate on HepG2 is 32.78 percent, and the inhibition rate on normal liver cells LO2 is only 14.21 percent, which shows that the polypeptide DP1 of the defatted fermented bean curd water extract has stronger selectivity on tumor cells and can inhibit the proliferation of the tumor cells.
Example 5: defatted water-extracted fermented bean curd polypeptide DP2
In the first step, 100g of a mature freeze-dried fermented bean curd sample is taken, and the ratio of 1: mixing 2 proportion with n-hexane, extracting with water bath oscillation at 40 deg.C for 30min, centrifuging at 10000g/min for 3min, discarding supernatant, adding n-hexane again, repeating for 2 times to remove fat, oven drying at 65 deg.C to constant weight, and sealing in bottle for storage.
And secondly, taking 5g of defatted fermented bean curd powder into a 100mL centrifuge tube, sucking 50mL of water into the centrifuge tube, shaking in a water bath at 25 ℃ for 90min, centrifuging for 10min at 8000r/min, pouring the supernatant into a 250mL round-bottom flask, adding 50mL of distilled water into the residue of the centrifuge tube again, repeating the process for 1 time, finally, completely merging the supernatant into the 250mL round-bottom flask, concentrating at 50 ℃ for 40min to obtain a light yellow soybean polypeptide concentrated solution, freeze-drying, and preserving in a refrigerator at-20 ℃.
And thirdly, balancing a Sephadex G-15 gel chromatographic column with distilled water, desalting and performing fractional separation on the defatted fermented bean curd soybean polypeptide by gel column chromatography, wherein the eluent is deionized water, the flow rate is 1mL/min, the detection wavelength is 220nm, and the eluent is collected by an automatic part collector for 10 mL/min. Collecting eluate between 50-80min, concentrating at 55 deg.C for 20min, and freeze drying to obtain defatted polypeptide powder, which is designated as DP 2.
The composition and molecular weight distribution of DP2 were analyzed in one step, and the protein content was found to be as high as 98.01%, and no fat and NaCl were contained. Fig. 5 is a mass spectrum of DP2, whose molecular weight distribution can be seen to be similar to P2, consisting mainly of 425.2166 and 301.1425 bands in the range <500 Da.
The antioxidant activity and the anticancer activity of DP2 were analyzed in one step in the fifth step, and it was found that the ability to scavenge ABTS free radicals was 38.87. mu. mol tocopherol/g sample, the ability to scavenge DPPH free radicals was 110.75. mu. mol tocopherol/g sample, and the ferric iron reducing power was 3.01mmol tocopherol/g sample. The anticancer activity of the aqueous extract polypeptide DP2 of the defatted fermented bean curd is reflected in the proliferation inhibition effect on liver cancer cells HepG2 and breast cancer cells MCF-7, after the polypeptide DP2 with the concentration of 500 mug/mL is treated for 48 hours, the proliferation inhibition rate on the MCF-7 is 26.71%, the proliferation inhibition rate on the HepG2 is 24.02%, and the inhibition rate on normal liver cells LO2 is only 11.35%, which shows that the aqueous extract polypeptide DP2 of the defatted fermented bean curd has stronger selectivity on tumor cells and can inhibit the proliferation of the tumor cells.
Example 6: active polypeptide DEP of defatted alcohol-extracted fermented bean curd
The first step is to obtain the defatted fermented bean curd as in example 4 and extract alcohol-soluble polypeptide from the precipitate after extracting water-soluble polypeptide. Namely: adding 50mL of 90% ethanol for extraction, shaking in a water bath at 25 ℃ for 50min, centrifuging at 8000r/min for 10min, pouring the supernatant into a 250mL round-bottom flask, adding 50mL of 90% ethanol into the residue of the centrifuge tube, repeating the above process for 2 times, finally adding all the ethanol extract into the 250mL round-bottom flask, concentrating at 45 ℃ for 20min to obtain a light yellow soybean polypeptide concentrate, and freeze-drying to obtain white powder which is recorded as DEP.
The second step, further analyzing the composition and molecular weight distribution of DEP, found that it had a protein content of 97.02%, a fat content of 2.11%, and a NaCl content of almost 0. FIG. 6 is a mass spectrum of the defatted fermented bean curd alcohol extract polypeptide, the molecular weight distribution thereof is similar to that of EP, and the most abundant band is 2088.0157.
The third step is to further analyze the anti-oxidation and anti-cancer activity of DEP, and the results show that the capability of eliminating ABTS free radicals is 62.81 mu mol of tocopherol/g sample, the capability of eliminating DPPH free radicals is 93.43 mu mol of tocopherol/g sample, and the ferric iron reducing power is 2.41mmol of tocopherol/g sample. The anticancer activity of the fermented bean curd alcohol extract polypeptide DEP is reflected in the proliferation inhibition effect on liver cancer cells HepG2 and breast cancer cells MCF-7, after the polypeptide DEP is treated by EP with the concentration of 500 mu g/mL for 48 hours, the proliferation inhibition efficiency on the MCF-7 is 30.33 percent, the proliferation inhibition rate on HepG2 is 27.54 percent, and the inhibition rate on normal liver cells LO2 is only 6.55 percent, which indicates that the defatted fermented bean curd alcohol extract polypeptide DEP has stronger selectivity on tumor cells and can inhibit the proliferation of the tumor cells.
As can be seen from the above examples, the water-soluble polypeptide in the humus milk has high purity after being filtered by the molecular sieve, and has significant antioxidant activity and activity of inhibiting the growth of cancer cells, wherein the polypeptide with the molecular weight distribution of 500-100Da has more prominent effect (example 1 and example 4). The alcohol-soluble polypeptides in the fermented bean curd also have good efficacy, wherein the polypeptide sample which is degreased firstly and then extracted with alcohol has higher purity and better functional activity (example 6). 2 polypeptides of which sequences were obtained all had potent inhibitory effects on cancer cells, wherein the sequence VSIIDTNSLENQLDQMPR, identified from the alcohol-soluble polypeptides, was more potent in inhibiting than the mixed peptides. Through a two-step extraction method and combined with molecular sieve gel filtration, the polypeptide in the fermented bean curd can be fully extracted and purified. In addition, the extraction and preparation method of the polypeptide can be conveniently converted into an ultrafiltration method for desalting and fractionation in the actual production, so that the production cost is reduced, and the production efficiency is improved.
Claims (2)
1. An application of a functional soybean polypeptide in preparing a medicament for resisting liver cancer or breast cancer is characterized in that the functional soybean polypeptide is water-soluble soybean polypeptide; the amino acid sequence of the water-soluble soybean polypeptide is Phe-Asn-Lys-Thr-Ser-Ser-Phe-Asn.
2. The application of the functional soybean polypeptide in preparing the medicine for resisting liver cancer or breast cancer is characterized in that the functional soybean polypeptide is alcohol-soluble polypeptide, and the amino acid sequence of the alcohol-soluble polypeptide is Val-Ser-Ile-Ile-Asp-Thr-Asn-Ser-Leu-Gle-Asn-Gln-Leu-Asp-Gln-Met-Pro-Arg.
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| KR20040013338A (en) * | 2002-08-05 | 2004-02-14 | 풀무원건강생활 주식회사 | Isolation and preparation of small functional peptides from tofu whey and It's application |
| CN1239711C (en) * | 2003-11-14 | 2006-02-01 | 华南理工大学 | Bitterless soybean polypeptide and its production method |
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2018
- 2018-04-10 CN CN201810317023.3A patent/CN108586581B/en active Active
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| CN102286584A (en) * | 2011-06-29 | 2011-12-21 | 刘长福 | Natural active water-soluble protein and polypeptide and preparation method thereof |
| WO2014188428A1 (en) * | 2013-05-22 | 2014-11-27 | Evogene Ltd. | Isolated polynucleotides and polypeptides, and methods of using same for increasing plant yield and/or agricultural characteristics |
Non-Patent Citations (2)
| Title |
|---|
| Ripening temperature affects the content and distribution of isoflavones in sufu, a fermented soybean curd;Yung-Hsin Huang 等;《International Journal of Food Science and Technology》;20110112;第46卷;第257-262页 * |
| 豆腐乳中的功能性成分;程丽娟 等;《中国调味品》;20051231(第12期);第10-13页 * |
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| CN108586581A (en) | 2018-09-28 |
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