CN114592024A - Sheep placenta polypeptide and preparation method and application thereof - Google Patents
Sheep placenta polypeptide and preparation method and application thereof Download PDFInfo
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- 230000007365 immunoregulation Effects 0.000 description 1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- 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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Abstract
The invention discloses a sheep placenta polypeptide and a preparation method and application thereof, belonging to the technical field of protein engineering. It comprises the following steps: (1) processing sheep placenta; (2) preparing sheep placenta polypeptide by an enzymolysis method; (3) removing fishy smell of placenta Caprae Seu Ovis enzymolysis solution. Experiments show that the optimal enzymolysis process determined by screening pepsin and papain is as follows: papain 5 per mill, pH6.40, enzymolysis at 55 deg.C for 6 hr, centrifuging at 10000rpm for 20 min. The best conditions for fermentation and deodorization of saccharomyces cerevisiae are as follows: 5% of yeast liquid, and culturing for 24 hours in a shaking culture box at 28 ℃, wherein enzymolysis liquid under different extraction conditions has antioxidant activity of different degrees.
Description
Technical Field
The invention belongs to the technical field of protein engineering, and particularly relates to an improved sheep placenta polypeptide.
Background
The sheep placenta is a tissue responsible for exchanging maternal and fetal blood and nutrients when a ewe breeds a fetus, can generate more active substances, and has a nutritional composition close to that of a human placenta. The sheep placenta contains rich protein, vitamins, lipopolysaccharide, active polypeptide, growth factor, amino acids, trace elements, etc. and antibody, etc. relevant to immunity and maintaining normal running of body, and may be absorbed well. According to the records of the compendium of materia medica, the placenta is also called as placenta or human placenta, has sweet, salty and warm taste, enters the lung, liver and kidney meridians, has various effects of warming the middle-jiao and replenishing qi, nourishing yin and enriching the blood, beautifying, preventing cancer, resisting aging, regulating endocrine, improving the immunity of the organism and the like, and is widely applied to the fields of medical care, food, cosmetics and the like. In the production of cosmetics, the extract of sheep placenta from Swiss is added, and at the same time, some domestic researchers also apply sheep placenta antioxidant peptide to the cosmetics with partial anti-aging effect. Because the antioxidant peptide has the advantages of good antioxidant activity, high stability, easy absorption, safety, nutrition and the like, the antioxidant peptide becomes a hot spot of domestic and foreign research in recent years.
The placenta peptide is also called placenta immune live cell, placenta immune regulating peptide, placenta transfer factor and placenta immune regulating factor, and the placenta immune regulating peptide is placenta extract with relatively deep research and has components and stability related to the technological process. The placenta immunoregulation peptide belongs to small molecular substance, is a mixture of small molecular weight nucleotide and peptide substance, has a molecular weight less than 5000Da, and contains multiple amino acids. Zhoushanhua et al report that placental peptide is colorless or yellowish transparent liquid, contains various amino acids, has various characteristics such as permeability, ultrafiltration, negative protein reaction, no antigenicity, etc., and the freeze-dried product is usually white loose powder and is very soluble in water.
The enzymatic hydrolysis method is a common method for preparing bioactive peptides on a large scale at present, and the moderate hydrolysis of proteins is achieved by adding protease. The principle of enzymolysis is to utilize the specificity, high efficiency, selectivity and other features of protease. The enzymolysis process is generally carried out in a relatively mild environment, and although the enzymolysis process is difficult to control, the enzymolysis method is also widely regarded due to the advantages of high safety, mild conditions, low cost and the like.
The sheep placenta polypeptide is prepared from sheep placenta as raw material by enzymolysis, and is subjected to protein content determination and deodorization treatment, and simultaneously, the antioxidant activity of the sheep placenta peptide is extracted under different conditions.
Disclosure of Invention
1. Problems to be solved
Aiming at the problems in the prior art, the invention provides a preparation method and application of sheep placenta polypeptide, and specifically comprises the following steps: the purpose of the invention is as follows: comparing the yields of the sheep placenta polypeptides extracted by two different proteases, screening out an optimal process, and performing deodorization treatment, protein content determination and antioxidant activity comparison on the extracts under different conditions; the invention method comprises the following steps: carrying out enzymolysis on sheep placenta by adopting protease, carrying out deodorization treatment on enzymolysis liquid by using saccharomyces cerevisiae, carrying out rating and scoring on the deodorized enzymolysis liquid by randomly searching 34 persons, then carrying out ultrafiltration to obtain enzymolysis liquid with different molecular weights, determining the protein content by using a Kjeldahl apparatus, and finally determining the activity by using an antioxidant activity determination kit; and (4) conclusion: the optimum enzymolysis process is determined by screening pepsin and papain: papain 5 ‰, pH6.40, enzymolysis at 55 deg.C for 6 hr, and centrifuging at 10000rpm for 20 min. The best conditions for fermentation and deodorization of saccharomyces cerevisiae are as follows: 5% of yeast liquid, and culturing for 24 hours in a shaking culture box at 28 ℃, wherein enzymolysis liquid under different extraction conditions has antioxidant activity of different degrees.
2. Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
A preparation method of sheep placenta polypeptide comprises the following steps:
(1) treatment of sheep placenta
Thawing sheep placenta at room temperature, washing with clear water, soaking in 75% ethanol for 30min, shearing, draining, weighing, pouring into a wall breaking machine, adding normal saline, and mashing to obtain sheep placenta homogenate;
(2) method for preparing sheep placenta polypeptide by enzymolysis
Adding pepsin or papain into the sheep placenta homogenate obtained in the step (1), treating the sheep placenta homogenate, placing the treated sheep placenta homogenate in a shaking culture box for carrying out first enzymolysis for 6 hours, centrifuging the treated sheep placenta homogenate to obtain supernatant and a precipitate part, adding pepsin or papain into the precipitate part again for carrying out second enzymolysis for 4 hours, centrifuging the precipitate part, combining the supernatants for the second time, and refrigerating the combined supernatant to obtain an enzymolysis liquid;
(3) fishy smell removing method for sheep placenta enzymolysis liquid
And (3) adding saccharomyces cerevisiae into the enzymatic hydrolysate obtained in the step (2), and transferring to a shaking incubator for culture.
In the preparation method of the sheep placenta polypeptide,
in the step (1), the mass ratio of the physiological saline to the sheep is 2: 1.
in the preparation method of the sheep placenta polypeptide,
in the step (2), the added mass percent of the pepsin or the papain is 5 per mill.
In the preparation method of the sheep placenta polypeptide,
the centrifugation parameters in the step (2) are 10000rpm and 2 h.
In the preparation method of the sheep placenta polypeptide,
and (2) adding pepsin into the sheep placenta homogenate obtained in the step (1), adjusting the pH value to 2.0, treating the sheep placenta homogenate, placing the treated sheep placenta homogenate in a shaking incubator for carrying out first enzymolysis for 6 hours at the temperature of 37 ℃, centrifuging the sheep placenta homogenate to obtain a supernatant and a precipitate, adding pepsin into the precipitate again for carrying out second enzymolysis for 4 hours at the temperature of 55 ℃, and refrigerating the supernatant and the precipitate after centrifuging the sheep placenta homogenate.
In the preparation method of the sheep placenta polypeptide,
and (2) adding papain into the sheep placenta homogenate obtained in the step (1), adjusting the pH value to 6.4, treating the mixture, placing the treated mixture in a shaking incubator for carrying out first enzymolysis for 6 hours at the temperature of 37 ℃, centrifuging the treated mixture to obtain a supernatant and a precipitate, adding papain into the precipitate again for carrying out second enzymolysis for 4 hours at the temperature of 55 ℃, and refrigerating the supernatant and the precipitate after centrifuging the treatment.
In the preparation method of the sheep placenta polypeptide,
and (4) adding the saccharomyces cerevisiae with the mass percentage of 5 per mill in the step (3).
In the preparation method of the sheep placenta polypeptide,
the temperature for culturing in the step (3) is 28 ℃;
the culture time in the step (3) is 24 h.
A sheep placenta polypeptide obtained by the preparation method.
An application of the sheep placenta polypeptide in antioxidation.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
the invention takes sheep placenta as raw material, adopts enzymolysis method to prepare sheep placenta polypeptide, takes enzymolysis rate as index, determines that papain can be adopted for hydrolysis in the process production, and the optimal process conditions are as follows: papain 5 ‰, pH6.40, enzymolysis at 55 deg.C for 6 hr, and centrifuging at 10000rpm for 20 min. The enzymolysis rate of the sheep placenta under the condition is 95.02 percent. The method adopts saccharomyces cerevisiae to screen the fishy smell removing condition of the sheep placenta enzymatic hydrolysate, and determines the optimal condition as follows: 5 percent of yeast liquid and 24 hours of shaking culture box culture at 28 ℃. And separating the deodorized enzymolysis liquid by using an ultrafiltration centrifugal tube, and determining the protein content and activity of the enzymolysis liquid with three different molecular weights, wherein the highest protein content is the enzymolysis liquid with the molecular weight of 3-10kDa pepsin and papain at 37 ℃. The best DPPH free radical clearance rate in activity determination is enzymolysis liquid with the molecular weight of 3-10kDa pepsin + papain at 37 ℃, among three enzymolysis liquid with different molecular weights, the best DPPH free radical clearance rate of the enzymolysis liquid with the molecular weight of 3-10kDa is the enzymolysis liquid with the molecular weight of 3-10kDa, the best test effect of inhibiting and generating superoxide anion free radicals is the raw liquid after deodorizing by papain + pepsin at 37 ℃, the highest hydroxyl free radical clearance rate is the enzymolysis liquid with the molecular weight of 3-10kDa papain + pepsin at 37 ℃, and the best total oxidation resistance is the enzymolysis liquid with the molecular weight of less than 3kDa pepsin + papain at 37 ℃.
Drawings
FIG. 1 is a graph showing a standard curve of DPPH radical scavenging ability according to the present invention;
FIG. 2 is a graph of a vitamin C standard curve according to the present invention;
FIG. 3 is a standard curve diagram of the total antioxidant capacity of the present invention.
Detailed Description
The invention is further described with reference to specific examples.
1. Experimental Material
1.1 Experimental materials
Frozen sheep placenta (provided by Huaxi Biotech limited of Zhuhai city) 1.2 medicine and reagent
As shown in table 1.
TABLE 1
| Methyl Red | TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd. |
| Sulfuric acid (analytically pure) | GUANGDONG GUANGSHI REAGENTS TECHNOLOGY Co.,Ltd. |
| Copper sulfate (analytically pure) | TIANJIN DAMAO CHEMICAL REAGENT FACTORY |
| Potassium sulphate (super pure) | TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd. |
| Sodium chloride (analytically pure) | SHANGHAI MACKLIN BIOCHEMICAL Co.,Ltd. |
| Papain | Guangzhou Liyuan food additives Co Ltd |
| Boric acid (analytically pure) | TIANJIN DAMAO CHEMICAL REAGENT FACTORY |
| Sodium hydroxide (NaOH) | TIANJIN DAMAO CHEMICAL REAGENT FACTORY |
| Anhydrous ethanol | TIANJIN FUYU FINE CHEMICAL Co.,Ltd. |
| Pepsin | Guangzhou Liyuan food additives Co Ltd |
| Bromocresol green | TIANJIN KERMEL CHEMICAL REAGENT Co.,Ltd. |
| Hydrochloric acid | Guangzhou Chemical Reagent Factory |
| DPPH free radical scavenging ability kit | Nanjing institute of biological engineering |
| Hydroxy radical testing box | Nanjing institute of biological engineering |
| Test box for inhibiting and generating superoxide anion free radical | Nanjing institute of biological engineering |
| Total antioxidant capacity (T-AOC) test box | Nanjing institute of biological engineering |
1.3 Experimental instruments
As shown in table 2.
TABLE 2
2. Experimental method
2.1 treatment of sheep placenta
Thawing sheep placenta at room temperature, washing with clear water, soaking in 75% ethanol for about 30min, shearing, draining, weighing (m is 179.677g), pouring into a wall breaking machine, adding physiological saline (sheep placenta: physiological saline: 1:2), and mashing to obtain sheep placenta homogenate.
2.2 preparation of sheep placenta polypeptide by enzymolysis
Adding pepsin or 5 per mill of papain into the sheep placenta homogenate, treating the sheep placenta homogenate according to the following conditions, then placing the processed sheep placenta homogenate into an oscillation incubator for enzymolysis for 6 hours, adding papain or 5 per mill of pepsin into a precipitate part obtained after centrifugation at 10000rpm for 20 minutes for secondary enzymolysis for 4 hours, and obtaining a supernatant and storing the precipitate in a chromatographic experiment refrigerator after centrifugation at 10000rpm for 20 minutes so as to carry out the next experiment. By comparing the enzymatic hydrolysis rates, the best extraction process was selected, as shown in table 3.
TABLE 3 conditions of enzymatic hydrolysis
| Protease enzyme | pH | Temperature of first enzymolysis (. degree.C.) | Second enzymolysis temperature (. degree.C.) |
| Pepsin | 2.0 | 37 | 55 |
| Papain | 6.4 | 37 | 55 |
2.3 fermentation of Saccharomyces cerevisiae to remove fishy smell
2.3.1 preparation of the culture Medium
2% of each protein powder and glucose, sterilizing at 121 deg.C for 20min, adding Saccharomyces cerevisiae 5 μ L, and culturing in 28 deg.C shaking incubator.
2.3.2 deodorization of sheep placenta enzymatic hydrolysate
Adding 5% of saccharomyces cerevisiae into the sheep placenta enzymatic hydrolysate, culturing in a shaking incubator at 28 ℃, randomly searching 34 persons for rating and scoring the enzymatic hydrolysate subjected to deodorization treatment, and performing comparative fermentation culture for 0 hour; 2 hours; 4 hours; 8 hours; the fishy smell removing effect of the enzymolysis liquid is 24 hours.
2.4 protein content determination
And (3) carrying out ultrafiltration and centrifugation on the deodorized sheep placenta enzymatic hydrolysate to obtain samples with different molecular weights, and then determining the protein content by using a Kjeldahl apparatus.
2.5 determination of antioxidant Activity
2.5.1 measurement of DPPH radical scavenging ability
The operation is carried out according to the instruction of the DPPH free radical scavenging kit, the absorbance of A517nm is measured by an enzyme labeling instrument, 80% methanol is used as a blank control, and vitamin C ethyl ether is used as a positive control.
DPPH free radical clearance (%) {1- (assay-a control)/a blank } × 100%
2.5.2 scavenging superoxide anion free radical test
The operation is carried out according to the instruction steps of the test box for inhibiting and generating superoxide anion free radicals, the absorbance of A550 nm of the test box is measured by an enzyme-labeling instrument, ultrapure water is used as a blank control, and vitamin C ethyl ether is used as a positive control.
Clearance (%) { (control a-assay tube)/control a } × 100%
2.5.3 hydroxy radical scavenging experiments
The operation is carried out according to the steps of the specification of the hydroxyl radical determination kit, the absorbance at A550 nm is measured by an enzyme-labeling instrument, ultrapure water is used as a blank control, and vitamin C ethyl ether is used as a positive control.
Clearance (%) { (a control-a assay)/a control } × 100%
2.5.4 Total Oxidation resistance test
The operation is carried out according to the steps of the instruction of the total antioxidant capacity determination kit, the absorbance of A593 nm of the total antioxidant capacity determination kit is measured by an enzyme-labeling instrument, distilled water is used as a blank control, and vitamin C ethyl ether is used as a positive control.
Total antioxidant capacity (mmol/g) ═ (3.654x-0.4109) x protein concentration (mg/mL)
Wherein x represents the absorbance of A593 nm of the sample measured by a microplate reader.
2.6 statistical analysis
All test data herein are triplicate experiments, and the results are expressed as mean plus minus standard deviation (x ± S), and statistical analysis of the results was performed using the LSD method using software SPSS 22.0.
As a result:
1. preparation of sheep placenta polypeptide by enzymolysis
As shown in table 4, when the single enzyme enzymolysis rate is highest at 55 ℃, the enzymolysis rate is 95.02%, the combined enzyme enzymolysis rate is highest at 98.39% when the papain is pepsin at 55 ℃, the enzymolysis rate of the papain is only as high as 95.02%, and the papain can be used for enzymolysis and extraction of the sheep placenta only in consideration of both economy and enzymolysis rate.
TABLE 4 enzymatic hydrolysis ratio% of different proteases (x. + -. S, n ═ 3)
Note: the different lower case letters on the right shoulder of the same column number represent significant differences (P <0.05), and the same letter represents no significant difference (P > 0.05); gastric juice at 37 ℃: adding pepsin for the first time, and adding papain for the second time at 37 ℃ for enzymolysis; gastrowood at 55 ℃: adding pepsin for the first time, and adding papain for the second time at 55 ℃ for enzymolysis; wood stomach 37 ℃: adding papain for the first time, and adding pepsin for the second time at 37 ℃ for enzymolysis; wood stomach 55 ℃: adding papain for the first time, adding pepsin for the second time at 55 ℃ for enzymolysis, and the same below.
2. Saccharomyces cerevisiae fermentation deodorization score
As can be seen from Table 5, from the scores of the fishy smell, the off-flavor and the overall evaluation, the score was the highest in 24 hours of fermentation for any sample, which indicates that the time for the sheep placenta enzymatic hydrolysate to deodorize was 24 hours at 28 ℃ and 5% of the yeast solution.
Table 5 fishy smell elimination scoring experimental results (x ± S, n ═ 3)
Note: the different lower case letters on the right shoulder of the same column number represent significant differences (P <0.05), and the same letter represents no significant difference (P > 0.05); the different capital letters of the right shoulder of the same row number represent significant differences (P <0.05), and the same letter represents no significant difference (P > 0.05); no. 1, stomach wood 37 ℃, No. 2, stomach wood 55 ℃, No. 3, stomach wood 37 ℃, No. 4 and stomach wood 55 ℃; the score is 1-9 points and 1-3 points, which are unacceptable; 4-6 points, still acceptable; and 7-9 points, which are completely accepted, wherein the higher the value, the better the fishy smell removing effect is.
3. Method for determining protein content by Kjeldahl determination apparatus
As is clear from tables 6, 7 and 8, the percentage of the samples of three fractions having different molecular weights after the ultrafiltration treatment was not 100%, and it is possible that some of the samples were stuck to the filter during the ultrafiltration to cause a small amount of loss.
TABLE 6 volume percent of samples of different molecular weights
| Sample name | <3kDa(％) | 3-10kDa(％) | >10kDa(％) |
| Stomach → mu 37 deg.C | 62.45 | 32.65 | 4.50 |
| Stomach → mu 55 deg.C | 43.47 | 28.57 | 21.02 |
| Mu → stomach 37 deg.C | 49.80 | 37.55 | 6.73 |
| Mu → stomach 55 deg.C | 61.23 | 24.90 | 8.16 |
TABLE 7 protein content
TABLE 8 protein quality before and after deodorization
| Sample name | Sample name | Concentration (mg/mL) | Mass (mg) |
| Stomach → mu 37 deg.C | Raw liquid without fishy smell | 1.7267 | 84.6067 |
| Stomach → mu 55 deg.C | 4.8657 | 238.4177 | |
| Mu → stomach 37 deg.C | 2.7600 | 135.2400 | |
| Mu → stomach 55 deg.C | 4.2440 | 207.9560 | |
| Stomach → mu 37 deg.C | Raw liquid after deodorization | 1.1303 | 55.3863 |
| Stomach → mu 55 deg.C | 1.7533 | 85.9133 | |
| Mu → stomach 37 deg.C | 1.1897 | 58.2937 | |
| Mu → stomach 55 deg.C | 3.4440 | 168.7560 |
4. Determination of antioxidant Activity
4.1DPPH radical scavenging Capacity determination
As shown in Table 9 and FIG. 1, the pepsin papain with the molecular weight of 3-10kDa at 37 ℃ has the highest clearance rate with the non-fishy smell stock solution, which is 42.95% and 42.20% respectively, and the enzymolysis liquid under other extraction conditions also has DPPH free radical clearance capacity, which is about 30% basically. Compared with the original liquid without fishy smell removal by the same protease, the clearance rate of the original liquid without fishy smell removal is higher than that of the original liquid after fishy smell removal; the clearance rate of the molecular weight of 3-10kDa is higher than that of the other two groups of molecular weights, which shows that the difference of temperature, deodorization treatment and molecular weight can influence the DPPH free radical clearance capability.
TABLE 9% DPPH radical scavenging ratio of sheep placenta polypeptide hydrolysate
n＝3)
Note: the different lower case letters on the right shoulder of the same column number represent significant differences (P <0.05), and the same letter represents no significant difference (P > 0.05); the different capital letters in the right shoulder of the same row number represent significant differences (P <0.05), and the inclusion of the same letter represents no significant difference (P > 0.05).
4.2 superoxide anion radical scavenging assay
As shown in Table 10 and FIG. 2, the best clearance rate measured by superoxide anion scavenging ability was papain → pepsin, and the stock solution after deodorization under 37 ℃ enzymolysis condition was 20.34%. The removal rate of the stock solution after fishy smell removal is higher than that of the stock solution without fishy smell removal, which indicates that the fishy smell removal treatment may have influence on the antioxidation activity.
TABLE 10 percentage of removal of superoxide anion radical by sheep placenta polypeptide enzymolysis solution%
n＝3)
| Stomach → mu 37 deg.C | Stomach → mu 55 deg.C | Mu → stomach 37 deg.C | Mu → stomach 55 deg.C | |
| <3kDa | -2.28±0.05bB | 16.97±2.22aA | -33.29±3.91cB | -0.64±4.18aBC |
| 3-10kDa | 3.01±2.09bA | 8.35±1.31bA | -2.32±7.73bA | 3.97±2.81aA |
| >10kDa | 1.51±2.28bA | -13.64±3.19dC | -7.87±5.26bB | -3.65±3.52aAB |
| Raw liquid without fishy smell | 4.67±6.58bA | -3.13±0.18cA | -6.34±2.51bA | 6.73±9.46aA |
| Raw liquid after deodorization | 13.25±4.85aA | 13.23±2.47aA | 20.34±7.85aA | 11.25±10.25aA |
Note: the different lower case letters on the right shoulder of the same column number represent significant differences (P <0.05), and the same letter represents no significant difference (P > 0.05); the different capital letters in the right shoulder of the same row number represent significant differences (P <0.05), and the inclusion of the same letter represents no significant difference (P > 0.05).
4.3 hydroxy radical scavenging experiments
As can be seen from Table 11, the enzyme solution without deodorization except the papain pepsin at 55 deg.C has a clearance rate of only 6.66%. The removal rate of the stock solution after fishy smell removal is higher than that of the stock solution without fishy smell removal, which indicates that the fishy smell removal treatment can not reduce the inhibition rate of the sample and can also increase the removal rate.
TABLE 11% hydroxy radical scavenging rate of sheep placenta polypeptidase hydrolysate%
n＝3)
| Stomach → mu 37 deg.C | Stomach → mu 55 deg.C | Mu → stomach 37 deg.C | Mu → stomach 55 deg.C | |
| <3kDa | 66.18±2.44acA | 62.78±0.60bA | 58.55±1.19cB | 37.02±2.38bC |
| 3-10kDa | 65.92±2.61adA | 61.09±2.96bB | 72.60±0.68aB | 47.45±3.53aC |
| >10kDa | 56.14±1.80bC | 69.00±1.44aA | 64.51±1.48bB | 35.96±3.78bD |
| Raw liquid without fishy smell | -30.31±1.26eA | -32.67±5.38cB | -30.54±6.91dB | 6.66±3.74cB |
| Raw liquid after deodorization | 67.70±2.38aA | 59.46±1.80bB | 60.58±0.53bcB | 43.46±0.82aC |
Note: the different lower case letters on the right shoulder of the same column number represent significant differences (P <0.05), and the same letter represents no significant difference (P > 0.05); the different capital letters in the right shoulder of the same row number represent significant differences (P <0.05), and the inclusion of the same letter represents no significant difference (P > 0.05).
4.4 Total Oxidation resistance test
As shown in Table 12, pepsin → papain with a molecular weight of less than 3kDa at 37 ℃ has the best total antioxidant capacity, and the difference between the total antioxidant capacity of the sample before and after deodorization is not large, which indicates that the influence of deodorization treatment on the total antioxidant capacity is small.
TABLE 12 Total antioxidant capacity mmol/g of sheep placenta polypeptide hydrolysate: (
n＝3)
| Stomach → mu 37 deg.C | Stomach → mu 55 deg.C | Mu → stomach 37 deg.C | Mu → stomach 55 deg.C | |
| <3kDa | 0.16±0.01aA | 0.13±0.01aA | 0.12±0.01aA | 0.14±0.02aB |
| 3-10kDa | 0.11±0.01cA | 0.10±0.01bA | 0.12±0.03aA | 0.13±0.02aA |
| >10kDa | 0.11±0.00bC | 0.10±0.01bB | 0.10±0.01aB | 0.12±0.01abA |
| Raw liquid without fishy smell | 0.11±0.00cA | 0.08±0.01bC | 0.09±0.01aB | 0.09±0.01bB |
| Raw liquid after deodorization | 0.11±0.01bA | 0.07±0.00cB | 0.11±0.02aB | 0.10±0.01bA |
Note: the different lower case letters on the right shoulder of the same column number represent significant differences (P <0.05), and the same letter represents no significant difference (P > 0.05); the different capital letters in the right shoulder of the same row number represent significant differences (P <0.05), and the inclusion of the same letter represents no significant difference (P > 0.05).
Discussion:
the invention takes sheep placenta as raw material, prepares sheep placenta polypeptide by an enzymolysis method, determines that papain can be adopted for hydrolysis in the process production by taking the enzymolysis rate as an index, and has the optimal process conditions as follows: papain 5 ‰, pH6.40, enzymolysis at 55 deg.C for 6 hr, and centrifuging at 10000rpm for 20 min. Under the condition, the enzymolysis rate of the sheep placenta is 95.02 percent. The method adopts saccharomyces cerevisiae to screen the fishy smell removing condition of the sheep placenta enzymatic hydrolysate, and determines the optimal condition as follows: 5 percent of yeast liquid and 24 hours of shaking culture box culture at 28 ℃. And separating the deodorized enzymolysis liquid by using an ultrafiltration centrifugal tube, and determining the protein content and activity of the enzymolysis liquid with three different molecular weights, wherein the highest protein content is the enzymolysis liquid with the molecular weight of 3-10kDa pepsin and papain at 37 ℃. The best DPPH free radical clearance rate in activity determination is enzymolysis liquid with the molecular weight of 3-10kDa pepsin + papain at 37 ℃, among three enzymolysis liquid with different molecular weights, the best DPPH free radical clearance rate of the enzymolysis liquid with the molecular weight of 3-10kDa is the enzymolysis liquid with the molecular weight of 3-10kDa, the best test effect of inhibiting and generating superoxide anion free radicals is the raw liquid after deodorizing by papain + pepsin at 37 ℃, the highest hydroxyl free radical clearance rate is the enzymolysis liquid with the molecular weight of 3-10kDa papain + pepsin at 37 ℃, and the best total oxidation resistance is the enzymolysis liquid with the molecular weight of less than 3kDa pepsin + papain at 37 ℃. The sample obtained by ultrafiltration in the experiment is yellowish transparent liquid, and possible reasons are as follows: the actual reasons of the different extraction methods and conditions, the species of sheep, the number of fetuses of sheep placenta, and the like are still to be researched.
The sheep placenta has extremely low development and utilization cost and utilization rate, causes resource waste and environmental pollution to a great extent, but has wide sources, rich bioactive substances and extremely high edible and medicinal values, and meanwhile, the sheep placenta and human placenta have great similarity in nutritional ingredients and efficacies and relatively high safety guarantee, so that the active substances in the placenta are further refined, the current processing technology is improved, the effective ingredients are fully utilized, the stability of the active substances is improved, and the product is favorable for quality control and industrial production.
And (4) conclusion: the optimal technological conditions for extracting the sheep placenta polypeptide by the enzymolysis method are as follows: papain 5 per mill, pH6.40, enzymolysis at 55 deg.C for 6 hr, and sheep placenta polypeptide enzymolysis rate of 95.02%. The best condition of the deodorization treatment of the saccharomyces cerevisiae is as follows: 5 percent of yeast liquid and 24 hours of shaking culture box culture at 28 ℃. By comparing the enzymolysis liquid with different extraction conditions, the enzymolysis liquid has antioxidant activity with different degrees.
While the invention has been described in further detail in connection with specific embodiments thereof, it will be understood that the invention is not limited thereto, and that various other modifications and substitutions may be made by those skilled in the art without departing from the spirit of the invention, which should be considered to be within the scope of the invention as defined by the appended claims.
Claims (10)
1. A preparation method of sheep placenta polypeptide is characterized by comprising the following steps:
the method comprises the following steps:
(1) treatment of sheep placenta
Thawing sheep placenta at room temperature, washing with clear water, soaking in 75% ethanol for 30min, shearing, draining, weighing, pouring into a wall breaking machine, adding normal saline, and mashing to obtain sheep placenta homogenate;
(2) method for preparing sheep placenta polypeptide by enzymolysis
Adding pepsin or papain into the sheep placenta homogenate obtained in the step (1), treating the sheep placenta homogenate, placing the treated sheep placenta homogenate in a shaking culture box for carrying out first enzymolysis for 6 hours, centrifuging the treated sheep placenta homogenate to obtain supernatant and a precipitate part, adding pepsin or papain into the precipitate part again for carrying out second enzymolysis for 4 hours, centrifuging the precipitate part, combining the supernatants, and refrigerating the mixed supernatants to obtain enzymatic hydrolysate;
(3) fishy smell removing method for sheep placenta enzymolysis liquid
And (3) adding saccharomyces cerevisiae into the enzymatic hydrolysate obtained in the step (2), and transferring to a shaking incubator for culture.
2. The method for preparing the sheep placenta polypeptide according to claim 1, wherein the method comprises the following steps:
in the step (1), the mass ratio of the physiological saline to the sheep is 2: 1.
3. the method for preparing sheep placenta polypeptide according to claim 1, wherein:
in the step (2), the added mass percent of the pepsin or the papain is 5 per mill.
4. The method for preparing sheep placenta polypeptide according to claim 1, wherein:
the centrifugation parameters in the step (2) are 10000rpm and 2 h.
5. The method for preparing sheep placenta polypeptide according to claim 1, wherein:
and (2) adding pepsin into the sheep placenta homogenate obtained in the step (1), adjusting the pH value to 2.0, treating the sheep placenta homogenate, placing the treated sheep placenta homogenate in a shaking incubator for carrying out first enzymolysis for 6 hours at the temperature of 37 ℃, centrifuging the sheep placenta homogenate to obtain a supernatant and a precipitate, adding pepsin into the precipitate again for carrying out second enzymolysis for 4 hours at the temperature of 55 ℃, and refrigerating the supernatant and the precipitate after centrifuging the sheep placenta homogenate.
6. The method for preparing sheep placenta polypeptide according to claim 1, wherein:
and (2) adding papain into the sheep placenta homogenate obtained in the step (1), adjusting the pH value to 6.4, treating the mixture, placing the treated mixture in a shaking incubator for carrying out first enzymolysis for 6 hours at the temperature of 37 ℃, centrifuging the treated mixture to obtain a supernatant and a precipitate, adding papain into the precipitate again for carrying out second enzymolysis for 4 hours at the temperature of 55 ℃, and refrigerating the supernatant and the precipitate after centrifuging the treatment.
7. The method for preparing sheep placenta polypeptide according to claim 1, wherein:
and (4) adding the saccharomyces cerevisiae with the mass percentage of 5 per mill in the step (3).
8. The method for preparing sheep placenta polypeptide according to claim 1, wherein:
the temperature for culturing in the step (3) is 28 ℃;
the culture time in the step (3) is 24 h.
9. A sheep placenta polypeptide obtained by the method of claim 1.
10. Use of the sheep placenta polypeptide of claim 9 for anti-oxidant.
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