CN111850075A - Cervus elaphus linnaeus soluble protein extract and extraction method and application thereof - Google Patents
Cervus elaphus linnaeus soluble protein extract and extraction method and application thereof Download PDFInfo
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Abstract
The invention discloses a cervus elaphus linnaeus soluble protein extract and an extraction method and application thereof. The invention adopts colloid mill grinding combined with enzyme method to extract soluble protein in pilose antler, takes the pilose antler protein extraction rate as an evaluation index, inspects the influence of enzymolysis time, temperature, enzyme adding amount and the like on the protein extraction process, obtains the optimal process for extracting the pilose antler soluble protein by colloid mill auxiliary enzyme, further researches the nonspecific immunity activity of the protein extract, and determines the phagocytosis index of mononuclear macrophage, serum lysozyme, acid phosphatase and other indexes through carbon clearance test, thereby showing that the pilose antler soluble protein has obvious immunity regulation effect and good immunity activity.
Description
Technical Field
The invention relates to the technical field of protein extraction, in particular to a cervus elaphus linnaeus soluble protein extract and an extraction method and application thereof.
Background
The cornu Cervi Pantotrichum is young horn of male deer (Cervus nippon Temminck) or Cervus Elaphus Temminck) of Cervus nippon Temminck, which is not ossified and dense cornu Cervi Pantotrichum, and is a rare Chinese medicinal material. The cornu Cervi Pantotrichum contains phospholipid, glycolipid, gum lipid, hormone, fatty acid, amino acid, protein, and calcium, phosphorus, magnesium, and sodium, wherein the amino acid component accounts for more than half of the total components. Ancient physicians thought that the essence of deer is in the horn, antler is the tender shoot of the horn, and the qi is full without excretion, so the most powerful of tonifying yang and nourishing blood. Ming Dynasty Li Shizhen called pilose antler in Ben Cao gang mu, which is good at tonifying kidney and strengthening yang, producing essence and benefiting blood, replenishing marrow and strengthening bone.
At present, the major protein extraction processes mainly comprise a hot water extraction method, an alkaline method, an ultrasonic method, a microwave method and the like, but the methods are all deficient in the aspects of protein extraction rate and activity of an extract.
Disclosure of Invention
The invention aims to provide a cervus elaphus linnaeus soluble protein extract and an extraction method and application thereof.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention firstly provides an extraction method of a cervus elaphus linnaeus soluble protein extract, which comprises the steps of crushing cervus elaphus linnaeus slices into fine powder, adding 8-10 times of volume of water, uniformly mixing, adjusting tooth grinding gaps of a colloid mill to be less than or equal to 90 mu m, adding papain with the enzyme amount of 20-60 mg/g when the pH value is 6.5, carrying out enzymolysis for 30-70 min, carrying out enzymolysis at the temperature of 45-65 ℃, homogenizing for 20-40 min by the colloid mill, collecting homogenate, centrifuging for 10min at 4000r/min, and taking supernatant to obtain the cervus elaphus linnaeus soluble protein extract.
Preferably, the grinding tooth gap of the colloid mill is 10-70 μm.
Preferably, the tooth clearance of the colloid mill is 50 μm.
Preferably, the grinding time of the colloid mill is 25-35 min.
Preferably, the colloid mill is used for grinding for 30 min.
Preferably, the enzyme adding amount is 30-50 mg/g.
Preferably, the enzymolysis time is 40-60 min.
Preferably, the enzyme adding amount is 40mg/g, the enzymolysis time is 40min, and the enzymolysis temperature is 50 ℃.
The invention also aims to provide the cervus elaphus linnaeus soluble protein extract, which is prepared by the extraction method of the cervus elaphus linnaeus soluble protein extract.
The invention further aims to provide application of the cervus elaphus linnaeus soluble protein extract in preparing a medicine for treating low immunocompetence caused by cyclophosphamide.
Compared with the prior art, the invention has the beneficial effects that:
the invention adopts colloid mill grinding combined with enzyme method to extract soluble protein in pilose antler, takes the pilose antler protein extraction rate as an evaluation index, inspects the influence of enzymolysis time, temperature, enzyme adding amount and the like on the protein extraction process, obtains the optimal process for extracting the pilose antler soluble protein by colloid mill auxiliary enzyme, further researches the nonspecific immunity activity of the protein extract, and determines the phagocytosis index of mononuclear macrophage, serum lysozyme, acid phosphatase and other indexes through carbon clearance test, thereby showing that the pilose antler soluble protein has obvious immunity regulation effect and good immunity activity.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
FIG. 1 is a graph showing the effect of the grinding gap of a colloid mill on the extraction rate of deer antler protein according to example 1 of the present invention.
FIG. 2 is a graph showing the effect of colloid milling time on the extraction rate of deer antler protein according to example 2 of the present invention.
Fig. 3 shows the effect of the added amount of papain on the extraction rate of deer antler protein provided in example 3 of the present invention.
Fig. 4 is a graph showing the influence of enzymolysis time on the extraction rate of deer antler protein according to embodiment 3 of the present invention.
Fig. 5 shows the influence of the enzymolysis temperature on the extraction rate of the deer antler protein provided in embodiment 3 of the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments. Cornu cervi pantotrichum (purchased from deer village in double yang areas of Jilin), bromelain (original leaf biotechnology limited), bovine serum albumin (Beijing solibao technology limited), phosphoric acid (AR, Beijing chemical plant), Coomassie brilliant blue G-250 (Beijing solibao technology limited), cyclophosphamide for injection (Jiangsu Shengdi medicine limited), Indian ink (Shanghai Kayon biotechnology limited), Lysozyme (LZM) test box (Shanghai Lanton biotechnology limited) and acid phosphatase (ACP) test box (Biyunshi biotechnology limited), Kunming mouse (Changchun responsibility Yice laboratory animal technology limited), Zhenqi righting granules (correction pharmaceutical industry) all adopt commercially available products.
EXAMPLE 1 selection of tooth gap (degree of pulverization of raw material slurry) of colloid mill
Adding 10g of cornu Cervi Pantotrichum fine powder into one group, 7 groups in total, adding 10 times of water, mixing, homogenizing for 30min with colloid mill, adjusting tooth clearance of colloid mill to 10, 30, 50, 70, 90, 110, 130 μm, centrifuging at 4000r/min for 10min to obtain extractive solution, and determining protein extraction rate. Each set of experiments was repeated three times and the mean was calculated.
The calculation method of the extraction rate of the pilose antler protein comprises the following steps: referring to the Bradford method, 1mg/ml Bovine Serum Albumin (BSA) standard protein solutions of 0, 10, 20, 40, 60, 80 and 100. mu.L were precisely measured, and distilled water was added to 100. mu.L, followed by shaking to prepare BSA control solutions having mass concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mg/ml, respectively. 5mL of Coomassie brilliant blue G-250 staining solution was added to the reaction mixture, and after 5min of reaction, absorbance was measured at 595 nm. Taking BSA content (μ g) as abscissa and A595 value as ordinate to obtain a standard curve, where R is 0.0059X +0.094820.9993 (linear range of 10-100. mu.g/100. mu.L). Accurately transferring 20 μ L of cornu Cervi Pantotrichum enzymolysis extractive solution, supplementing ultrapure water to 100 μ L, measuring absorbance at 595nm, and calculating cornu Cervi Pantotrichum protein extraction rate according to linear equation.
The influence of the grinding gap of the colloid mill on the extraction rate of the pilose antler protein is shown in fig. 1, and the pilose antler powder is ground by the colloid mill with different tooth grinding gaps, so that the smaller the gap is, the finer the raw material is crushed. As can be seen from FIG. 1, the protein extraction rate of the colloid mill grinding treatment group tends to increase rapidly with the decrease of the tooth clearance, and the protein extraction rate peaks at a tooth clearance of 50 μm, so that 50 μm is selected as the optimum tooth clearance of the colloid mill.
EXAMPLE 2 selection of grinding time of colloid mill
Adding 10 times of water into 7 groups of 10g of cornu Cervi Pantotrichum fine powder, mixing, adjusting tooth gap to 50 μm, homogenizing for 10, 15, 20, 25, 30, 35, and 40min, centrifuging at 4000r/min for 10min to obtain extractive solution, and determining protein extraction rate. The method for calculating the extraction rate of the pilose antler protein is also shown in the example 1. Each set of experiments was repeated three times and the mean was calculated.
The gap between the grinding teeth of the colloid mill is 50 μm, and the results of examining the influence of different grinding times on the extraction rate of the deer antler protein are shown in fig. 2. As can be seen from FIG. 2, the extraction rate of protein was the highest when the milling time was 30min, and the increase in extraction rate was moderate when the time continued to increase, so 30min was selected as the optimum colloid milling time.
Example 3 Single factor test of Process for extracting deer antler protein by enzyme method
Taking 10ml of each group of extract, adjusting the pH value of the solution to 6.5, adding a certain amount or different amounts of papain, carrying out water bath at different temperatures or the optimum temperature of enzyme of 55 ℃ for certain time or different times, centrifuging at 4000r/min for 10min to obtain an enzymolysis extract, measuring the protein content and calculating the protein extraction rate. The method for calculating the extraction rate of the pilose antler protein is also shown in the example 1. And performing a single-factor variable test each time, taking the optimal parameter obtained by the previous single-factor verification as the quantitative parameter of the next single-factor test, and determining different levels of all factors of the orthogonal test.
Taking 10ml of each group, adjusting pH to 6.5, adding papain 10, 20, 30, 40, 50 and 60mg/g respectively, performing water bath at 55 deg.C for 60min at optimum enzyme temperature, centrifuging at 4000r/min for 10min to obtain enzymolysis extractive solution, measuring protein content and calculating extraction rate, the result is shown in FIG. 3. As can be seen from FIG. 3, the protein extraction rate was significantly increased with the increase of the amount of papain, and when the amount of papain added was 30mg/g, the protein extraction rate was the highest, followed by a tendency to decrease, so that the addition of papain at 20, 30, and 40mg/g was selected as 3 levels for the orthogonal test.
Taking 10ml of each group, adjusting pH to 6.5, adding papain 30mg/g, centrifuging at 4000r/min for 10min at 55 deg.C water bath temperature for 20, 30, 40, 50, 60, and 70min to obtain enzymolysis extractive solution, measuring protein content, and calculating extraction rate, the result is shown in FIG. 4. As can be seen from fig. 4, the protein extraction rate increases with the longer enzymolysis time, and the protein extraction rate is the highest at 50min and slowly decreases after 50min, so the enzymolysis times 40, 50 and 60min are selected as 3 levels of the orthogonal test.
Taking 10ml of each group, adjusting pH to 6.5, adding papain 30mg/g, performing enzymolysis for 50min at 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 4000r/min, centrifuging for 10min to obtain enzymolysis extractive solution, measuring protein content and calculating extraction rate, the result is shown in FIG. 5. As can be seen from FIG. 5, the protein extraction rate increased with the increase of the enzymatic hydrolysis temperature, and slightly decreased after 55 ℃, so that the enzymatic hydrolysis temperatures of 50, 55 and 60 ℃ were selected as 3 levels of the orthogonal test.
Example 4 orthogonal test for screening of optimal extraction Process of deer antler protein
And (3) crushing by a colloid mill and performing protein extraction by combining enzymolysis. According to the single-factor test result, 3 factors of enzyme addition amount, enzymolysis temperature and enzymolysis time are selected as L 9(34) And (4) performing orthogonal test. The test adopts the process of firstly grinding by a colloid mill and then carrying out enzymolysis, centrifuging for 10min at 4000r/min after the enzymolysis is finished to obtain supernatant enzymolysis extracting solution, measuring the protein content and calculating the protein extraction rate. The levels of the design orthogonal test factors are shown in table 1, the protein extraction rate is used as a survey index, and the orthogonal test results are shown in table 2. The analysis of variance is shown in Table 3.
TABLE 1 orthogonal test factor horizon
TABLE 2 orthogonal test design and results table
TABLE 3 ANOVA TABLE
As can be seen from the results of the analysis of variance, the order of the influencing factors from large to small is as follows: a is more than C and B, and A factor (enzyme adding amount) is the main factor of extraction, and C factor (enzymolysis time) and B factor (colloid mill grinding time) are secondary factors. The optimal process is considered by combining the visual analysis table and the significance result of the variance analysis, and comprises the following steps: the A3B1C1, namely the enzyme adding amount is 40mg/g, the enzymolysis time is 40min, the enzymolysis temperature is 50 ℃, because the A factor is the third level, the A factor is increased by one level (the enzyme adding amount is 50mg/g) according to the orthogonal test conditions, the test result of the A4B1C1 is compared with the three verification tests, and the result is shown in Table 4.
TABLE 4 additional test results
The test result shows that each index component of the additional test result has no significant difference with the verification test result, so the enzyme adding amount is 40mg/g, the enzymolysis time is 40min, the enzymolysis temperature is 50 ℃ as the test condition, and the protein extraction rate under the process condition is verified to be the highest.
Example 5 test of the immunological Activity of velvet antler protein extract
Selecting 60 healthy Kunming small animals, 10 animals per group, and male and female halves, and randomly dividing into blank control group, model control group, positive control group (Zhenqi Fuzheng granule), cornu Cervi Pantotrichum protein extract low dose group (low dose group), cornu Cervi Pantotrichum protein extract medium dose group (medium dose group), and cornu Cervi Pantotrichum protein extract high dose group (high dose group). Preparing cornu Cervi Pantotrichum protein extract by optimum process, dissolving in normal saline, and performing intragastric administration on mice with low, medium and high doses at 200, 400 and 800mg/kg respectively; 1.52g/kg of glossy privet fruit and astragalus root strengthening particles are taken for the positive medicine by intragastric administration; the model control group and the blank control group are given with 10mL/kg of physiological saline; 1 time per day for 10 days. On the 2 nd and 3 rd days of gastric lavage, cyclophosphamide aqueous solution was injected into the abdominal cavity of each group of mice except the blank control group at a dose of 40mg/kg, resulting in low immune function of the mice. On day 11, collecting blood from eyeball, centrifuging at 3500r/min for 10min, collecting serum, removing excessive tissues from liver, spleen and thymus after cervical dislocation, cleaning with normal saline, drying with filter paper, and calculating viscera index; performing a carbon clearance test to determine the phagocytic index of the mononuclear macrophage; and (3) measuring the content of serum lysozyme, serum acid phosphatase and liver tissue acid phosphatase activity. Statistical analysis of the data was performed using IBM SPSS20.0 statistical software and the results are expressed as mean ± standard deviation.
Measurement results of mouse organ index: the spleen is an important peripheral immune organ of an organism, a large number of immune cells exist in the spleen, the spleen is also a place for generating immune response, the size of the spleen index is one of important indexes reflecting the immune regulation capability of the organism, and the increase of the immunity of the organism can be shown from the change of the spleen index. The thymus is an important central immune organ of an organism, T lymphocytes are differentiated and matured in the thymus, and immune response of the T lymphocytes and B lymphocytes after being stimulated by antigens also occurs in the thymus, so that the thymus index can effectively judge whether the immune function is high or low, and is another important index for reflecting the immune function of the organism. The calculation formula is as follows:
spleen index ═ 10 (spleen weight mg/mouse body weight g) ×
Thymus index (weight mg of thymus/g of mouse body weight) × 10
TABLE 5 measurement results of organ index of mouse
Note: p <0.05 compared to the blank control group is denoted by a and P <0.05 compared to the model control group is denoted by b.
As can be seen from Table 5, the low, medium and high dose groups and the positive control group of the deer antler protein extract have different increases compared with the blank control group and the model control group, the medium and high dose groups have significant differences compared with the blank control group and the model control group, the average spleen index of the high dose group is 134.07g/100g, the average thymus index of the high dose group is 15.22g/100g, and the increase is obvious compared with the model control group, which indicates that the deer antler water-soluble protein has obvious efficacy of improving immunity, and the immune effect has an enhancement trend along with the increase of the dose.
Results of nonspecific immune function assay: serum lysozyme, serum acid phosphatase, and liver tissue acid phosphatase activities of the mice were measured according to the kit instructions and the results are shown in table 6.
TABLE 6 Effect of deer antler protein extract on non-specific immune function in mice
Note: p <0.05 compared to the blank control group is denoted by a and P <0.05 compared to the model control group is denoted by b.
As can be seen from Table 6, compared with the model control group, the deer antler protein extract has very significant differences in the phagocytic index of mononuclear macrophages, the concentration of serum lysozyme, the activity of serum acid phosphatase and the activity of liver tissue acid phosphatase in the low, medium and high dose groups, and compared with the blank control group, each index in the high dose group has very significant difference, the average phagocytic index of mononuclear macrophages in the low and medium dose groups, which have significant difference in the activity of serum acid phosphatase and the activity of liver tissue acid phosphatase, in the high dose group is 5.98, and the average mass concentration of serum lysozyme is 23.92. mu.g.L-1The average value of the activity of the serum acid phosphatase is 93.24 U.L-1The average value of the activity of the liver tissue acid phosphatase is 91.60 U.g-1. The data show that the pilose antler protein extract can enhance the macrophage phagocytosis index, the concentration of serum lysozyme and the activity of acid phosphatase in serum and liver tissues of mice with low immunity caused by cyclophosphamide, and has obvious nonspecific immune activity regulation effect.
While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that the described embodiments may be modified in various different ways without departing from the spirit and scope of the invention. Accordingly, the foregoing description is illustrative in nature and is not to be construed as limiting the scope of the invention as claimed.
Claims (10)
1. A method for extracting a cervus elaphus linnaeus soluble protein extract is characterized by comprising the steps of crushing cervus elaphus linnaeus slices into fine powder, adding 8-10 times of volume of water, uniformly mixing, adjusting tooth grinding gaps of a colloid mill to be less than or equal to 90 mu m, adding papain with the enzyme amount of 20-60 mg/g when the pH value is 6.5, carrying out enzymolysis for 30-70 min, carrying out enzymolysis at the temperature of 45-65 ℃, homogenizing for 20-40 min by the colloid mill, collecting homogenate, centrifuging for 10min at 4000r/min, and taking supernatant to obtain the cervus elaphus linnaeus soluble protein extract.
2. The method for extracting soluble protein extract from cervus elaphus linnaeus as claimed in claim 1, wherein the gap between the grinding teeth of the colloid mill is 10-70 μm.
3. The method for extracting soluble protein extract from cornu Cervi Pantotrichum according to claim 1, wherein the tooth clearance of colloid mill is 50 μm.
4. The method for extracting the cervus elaphus linnaeus soluble protein extract as claimed in claim 1, wherein the colloid mill is used for 25-35 min.
5. The method for extracting soluble protein extract from cervus elaphus linnaeus as claimed in claim 1, wherein the colloid mill is used for 30 min.
6. The method for extracting the cervus elaphus linnaeus soluble protein extract as claimed in claim 1, wherein the enzyme is added in an amount of 30-50 mg/g.
7. The method for extracting the cervus elaphus linnaeus soluble protein extract as claimed in claim 1, wherein the enzymolysis time is 40-60 min.
8. The method for extracting soluble protein extract from cervus elaphus linnaeus as claimed in claim 1, wherein the amount of enzyme added is 40mg/g, the enzymolysis time is 40min, and the enzymolysis temperature is 50 ℃.
9. A cervus elaphus linnaeus soluble protein extract, which is prepared by the extraction method of the cervus elaphus linnaeus soluble protein extract as claimed in any one of claims 1 to 8.
10. The use of the soluble protein extract of cervus elaphus linnaeus as claimed in claim 9 for preparing a medicine for treating immunocompromised deficiency caused by cyclophosphamide.
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