CN109180788B

CN109180788B - Preparation method and application of camel milk antioxidant polypeptide

Info

Publication number: CN109180788B
Application number: CN201811122673.9A
Authority: CN
Inventors: 阿布力米提·伊力; 艾合米丁·外力; 阿吉艾克拜尔·艾萨; 高彦华
Original assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Current assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Priority date: 2018-09-26
Filing date: 2018-09-26
Publication date: 2021-07-02
Anticipated expiration: 2038-09-26
Also published as: CN109180788A

Abstract

The invention relates to a preparation method and application of camel milk antioxidant polypeptide, wherein fresh camel milk is used as a raw material, butter is removed by centrifugation at 4 ℃, total casein is obtained by adopting the traditional isoelectric precipitation, dialysis bag, concentration and freeze drying, trypsin is used for hydrolysis, and the antioxidant polypeptide is prepared by freeze drying and refining, and then three camel milk antioxidant polypeptides with stronger antioxidant activity are obtained by ultrafiltration membrane, gel column and high performance liquid phase separation and purification. The primary deconstruction of amino acid is determined to be three polypeptides by combining high-resolution mass spectrum and liquid chromatography-mass spectrometry: RLDGQGRPRVWLGR (1665.94Da), TPDNIDIWLGGIAEPQVKR (2122.13Da) and VAYSDDGENWTEYRDQGAVEGK (2489.09 Da). The polypeptide has the functions of enhancing humoral immunity and systemic immunity, is rich in pure natural protein and 18 amino acids which are easily digested and absorbed by human bodies, and is more suitable for being used as an ideal dairy product for old people and infants.

Description

Preparation method and application of camel milk antioxidant polypeptide

Technical Field

The invention relates to a preparation method and application of camel milk antioxidant polypeptide.

Background

Through the development of recent 20 years, the yield of dairy products in China in 2016 is 2782.5 ten thousand tons, and 1466.3 thousand tons in the first half of 2017, so that the dairy product industry becomes an important industry related to the national civilization. But meanwhile, the unreasonable product structure, the serious homogenization phenomenon, the downward profit slip of enterprises and the like also become obstacles for the industry to move forward continuously.

Camels play an important role in the lives of people in many countries around the world, particularly in those arid regions. Besides the functions of riding, carrying, pulling, plowing and the like, camels can provide milk and meat, and make contributions to economic life and food sources of people. According to food and agricultural organization statistics of the united nations, about 1900 million camels exist all over the world, about 1500 million camels are produced in Africa, and about 400 million camels are produced in Asia. It is also estimated that a dromedary, of which about 1700 ten thousand peaks, is mainly used as milk; the hump is about 200 ten thousand humps and is mainly used for service and wool. The lactation period of Bactrian camels such as China, Mongolia and the like is 14-16 months, the average daily milk yield of mother camels is about 5kg, the individual body is as high as 20kg, usually 2kg is used by people, and the rest is eaten by young camels. The camel milk is rich in nutrition and has good health care function. Through comparison of a large amount of domestic and foreign literature data, the functions of camel milk in the aspects of nutrition and health care are comprehensively reviewed, and some theoretical bases are provided for popularization and deep processing of camel milk.

Camel milk is called desert platinum in Arabic area, has slightly sweet taste and higher nutritive value than milk, and is a common beverage in local areas. The camel milk contains 3 times and 10 times of vitamin C and iron respectively, the content of unsaturated fatty acid, protein, magnesium, calcium and the like is higher than that of the milk, 1L of camel milk contains 52 units of insulin and far higher than 0.016 unit of the milk, the camel milk is rich in lactotransferrin of milk transferrin which is lacked by the milk and has the functions of sterilization and human immunity enhancement, and the fat content is lower than that of the milk. Therefore, the camel milk has the effects of building body, improving human immunity, resisting cancer, treating diabetes and the like. The camel milk has long been used by the Kazak population to treat pulmonary tuberculosis. Clinical research in the second subsidiary hospital of Xinjiang medical university in 2009 shows that under the condition of not accepting formal treatment, part of hepatitis B patients achieve hepatitis B negative conversion after drinking camel milk for a period of time. The camel milk has the effects of nourishing yin and tonifying yang, Chinese doctors such as Russia, Hassakestan and India use the camel milk as prescription drugs for helping patients to recover, and drinking the camel milk helps patients suffering from edema, jaundice, spleen diseases, tuberculosis, bobble, anemia, hemorrhoids and the like to recover. AIDS patients in some countries of Africa are advised to take camel milk, which is believed by Somari to have therapeutic effects. Comparative physiological studies in israel and germany show that camel milk has a pronounced anti-diabetic profile. The camel milk can help patients with type I diabetes to greatly reduce the insulin injection amount.

From China, the camel milk is originally a beverage which is self-squeezed and drunk by farmers and herds and can serve guests at home, is rarely sold to the outside, and the camel milk yield is kept about 4 ten thousand t throughout the year and only accounts for 0.74 percent of the total world production. However, the problems of the dairy industry occur frequently in this year, and the camel milk becomes a new high-end drink due to the unique components and efficacies of the camel milk, so that the camel milk is becoming a new fashion. Camel milk in domestic market is mainly produced in Xinjiang, and the consumers are mainly Kazak people, rehabilitation people, old people, medical workers, teachers and the like. At present, camel milk is sold in places such as Beijing, Shanghai, Fujian, Zhejiang, Jiangsu, Hebei, Heilongjiang, Chongqing, hong Kong, Taiwan and the like, and has a certain degree of popularity. The camel milk has a high price, one bottle of 180g of the camel milk has a sale price of 48 yuan, one can of 300g of camel milk has a sale price of 588 yuan, but the camel milk still has good sale and is in short supply, and the sale space of the camel milk in the inland cities is initially opened, so that the prospect is bright.

The camel milk is a nutrient substance deduced by the life of the camel, and is purely natural and green. Besides high nutritive value, camel milk can be used for adjuvant treatment of diabetes and iron supplement, and belongs to natural multifunctional milk product. Recent studies have shown that a daily intake of about 500 ml camel milk is effective in lowering blood glucose levels and reducing the human insulin demand. The relevant people indicate that the camel milk contains insulin-like factors, can assist in reducing blood sugar and can be used for treating diabetes.

Disclosure of Invention

The invention aims to provide a preparation method and application of camel milk antioxidant polypeptide, wherein fresh camel milk is used as a raw material, butter is removed by centrifugation at 4 ℃, total casein is obtained by traditional isoelectric precipitation, dialysis bag, concentration and freeze drying, trypsin is used for hydrolysis, antioxidant polypeptide is prepared by freeze drying and refining, and three camel milk antioxidant polypeptides with stronger antioxidant activity are obtained by ultrafiltration membrane, gel column and high performance liquid phase separation and purification. The primary deconstruction of amino acid is determined to be three polypeptides by combining high-resolution mass spectrum and liquid chromatography-mass spectrometry: RLDGQGRPRVWLGR (1665.94Da), TPDNIDIWLGGIAEPQVKR (2122.13Da) and VAYSDDGENWTEYRDQGAVEGK (2489.09 Da). The polypeptide has the functions of enhancing humoral immunity and systemic immunity, is rich in natural proteins which are easily digested and absorbed by human bodies, 18 amino acids (containing all 8 amino acids which are necessary for human bodies but can not be synthesized), iron, vitamin B, vitamin C and other nutrient elements, and is more suitable for being used as ideal dairy products for old people and infants.

The preparation method of the camel milk antioxidant polypeptide comprises the following steps:

a. centrifuging fresh camel milk at 4 deg.C and 10000r/min for 20min, repeating for 2 times, removing butter, and mixing the supernatants;

b. b, regulating the pH of the supernatant obtained in the step a to be 4.0-4.5 by using 1mol/L HCl solution, and standing in a refrigerator at the temperature of 4 ℃ for 12 hours to precipitate camel cheese protein;

c. c, centrifuging the precipitate in the step b at the temperature of 4 ℃ and the rotating speed of 10000r/min for 20min, adding the precipitate into distilled water for dissolving, adjusting the pH value to 7.0 by using 1mol/l HCl, and stirring by using a magnetic stirrer for complete dissolution;

d. dialyzing the solution in the step c with distilled water for 48h for desalination, concentrating the desalted casein, and freeze-drying for 24h at the temperature of-80 ℃ and the pressure of 9.8Pa by using a freeze dryer, wherein the specification of a dialysis bag used for dialysis is 1000 Da;

e. d, measuring the antioxidant activity of the casein in the step d through electrophoresis, and measuring the protein content, wherein the electrophoresis separation gel is acrylamide with the volume of 15%;

f. e, adding distilled water into the casein obtained in the step e to dissolve the protein, adjusting the mass concentration of the solution to be 50g/L, adjusting the pH value of the solution to be 8.0 by using NaOH with the concentration of 1mol/L, dissolving the solution in a water bath kettle at the temperature of 37 ℃, adding trypsin with the mass ratio of 1.0 percent when the solution has no obvious particles and the pH value has no obvious change, maintaining the pH value of the casein hydrolysate to be 8.0 by using NaOH with the concentration of 1mol/L in the constant-temperature water bath kettle, slowly stirring for enzymolysis for 4 hours, inactivating the enzyme in a boiling water bath at the temperature of 100 ℃ for 10 minutes, and naturally cooling to the room temperature;

g. adding ethanol into the total polypeptide in the step f according to the volume ratio of 1:3, and standing in a refrigerator at the temperature of 4 ℃ for 12 hours to precipitate macromolecular protein;

h. centrifuging the solution obtained in the step g for 20min at the temperature of 4 ℃ and the rotating speed of 10000r/min, removing proteins with large molecular weight, concentrating by using a rotary evaporator, and freeze-drying to obtain total polypeptides with small molecular weight;

i. separating the total polypeptide in the step h by an ultrafiltration membrane of 3000Da to obtain a marker I with the molecular weight of less than 3000Da and a marker II with the molecular weight of more than 3000 Da;

j. separating the part I with the molecular weight of less than 3000Da obtained in the step i by using a gel column to obtain two parts Ia and Ib, and comparing the antioxidant activity of the two parts, wherein the buffer solution used by the gel column is trifluoroacetic acid with the volume of 0.1%;

k. and (d) separating the part Ib obtained in the step j again by using high performance liquid chromatography with the gradient of 25-70% acetonitrile for 0-40min and the flow rate of 2.5mL/min to obtain three camel milk antioxidant polypeptides Ib-1, Ib-2 and Ib-3.

The camel milk antioxidant polypeptide obtained by the method is used for preparing health care products.

The camel milk antioxidant polypeptide obtained by the method is used for preparing food.

The camel milk antioxidant polypeptide obtained by the method is used for preparing food additives.

The invention relates to a preparation method and application of camel milk antioxidant polypeptide, wherein the molecular weight of camel milk antioxidant polypeptide obtained by the method is greatly different due to different amino acid sequences. The camel cheese has high protein content, can improve the immunity and disease resistance of infants, and can meet the nutritional requirements of the growth and development of the infants. The camel milk has higher nucleotide content than milk and human milk, and is beneficial to the development and intelligence enhancement of infant brain. Camel cheese protein is used as an important source of protein due to its balanced amino acid composition, high bioavailability and low anti-nutritional factor, and is also a good resource for obtaining bioactive peptides. The method takes fresh camel milk as a raw material, degreases the fresh camel milk, adopts the traditional isoelectric point method to precipitate and extract total casein, then obtains total polypeptide through trypsin hydrolysis, and finally obtains three camel milk antioxidant polypeptides with different molecular weights and different amino acid sequences through ultrafiltration membrane, gel chromatography and high performance liquid chromatography separation and purification. Then, each part of polypeptide solution is dried in vacuum, the yield of the polypeptides with different molecular weights is measured, a 5800MALDI-TOF/TOF (AB SCIEX) mass spectrometer is used for carrying out MS/MS mass spectrum analysis on the protein enzymolysis products, and then database software matching is carried out to obtain the qualitative information of the proteins. The amino acid sequences and molecular weights of three antioxidant polypeptides obtained by subjecting the part Ib to high performance liquid chromatography are respectively as follows: RLDGQGRPRVWLGR (1665.94Da), TPDNIDIWLGGIAEPQVKR (2122.13Da) and VAYSDDGENWTEYRDQGAVEGK (2489.09 Da). When the protease is hydrolyzed properly, the activity of the protease is released, the protease is easy to digest and absorb, and has various functions of regulating the metabolism and physiological functions of human bodies, so that the protease is a natural resource treasure house for developing functional foods and screening medicines. The biological characteristics of casein are studied more, and a plurality of active peptides are gradually developed, so that hydrolyzed peptide fragments are easier to be absorbed by human bodies, and the allergic reaction of infants is reduced. It is safe to eat, has high nutritive value, and has strong physiological health promotion function. The method has positive significance for promoting the progress of the Xinjiang-style animal husbandry industrialization development, optimizing the industrial structure, reducing the production cost of enterprises, increasing the economic benefit of the enterprises, preventing the environmental pollution, saving energy and reducing emission.

The antioxidant polypeptide obtained by the method has the functions of enhancing humoral immunity and systemic immunity, is rich in natural high-quality protein which is easily digested and absorbed by human bodies, has complete amino acid types, 18 amino acids (containing all 8 amino acids which are necessary for human bodies but can not be synthesized), iron, vitamin B, vitamin C and other nutrient elements, and is more suitable for the old and infants. The effect of adding the antioxidant polypeptide into daily skin care products can improve the skin care more upwards. Generally speaking, it can prevent skin from relaxing and aging, and further achieve the effect of anti-oxidation.

The antioxidant polypeptide has the characteristics that the original nutritional value of camel milk is kept, and meanwhile, the bioactive functional components of camel milk are analyzed, so that the camel milk is beneficial to scientific eating of camel milk resources, and the healthy, orderly and sustainable development of the camel milk industry in our district is better promoted. The camel milk polypeptide also has the effects of promoting digestion, reducing blood sugar, reducing blood pressure, preventing and resisting cancer and the like. The product provides scientific basis for becoming a functional health-care product for preventing and assisting in treating diabetes, also provides technical support for popularization and application of camel milk and related products, and lays a foundation for promoting economic development of camel breeding industry and camel milk industry. Along with the deep knowledge of camel milk, the market demand of camel milk polypeptide is larger and larger.

Drawings

FIG. 1 is a 15% SDS-PAGE Coomassie Brilliant blue electrophoresis chart of camel milk casein and camel milk casein trypsin hydrolysate of the invention: wherein M is a standard molecular weight protein; BCM is camel milk casein; TF is camel milk casein trypsin hydrolysate;

FIG. 2 is a gel chromatogram of a tryptic hydrolysate of the present invention;

FIG. 3 is a high performance liquid chromatogram of three polypeptides of the invention;

FIG. 4 is a high performance liquid chromatography chromatogram for the purity analysis of the Ib-1 polypeptide of the invention;

FIG. 5 is a high performance liquid chromatography chromatogram for the purity analysis of the Ib-2 polypeptide of the invention;

FIG. 6 is a high performance liquid chromatography chromatogram for the purity analysis of the Ib-3 polypeptide of the invention;

FIG. 7 is a mass spectrometry (LC/MS) profile of the present invention, wherein A is an Ib-1 polypeptide; b is Ib-2 polypeptide; c is Ib-3 polypeptide.

Detailed Description

Example 1

a. Centrifuging fresh camel milk 100ml at 4 deg.C and rotation speed of 10000r/min for 20min, repeating for 2 times, removing butter, and mixing the supernatants;

b. b, regulating the pH value of the supernatant obtained in the step a to 4.0 by using a HCL solution with the concentration of 1mol/l, and standing in a refrigerator at the temperature of 4 ℃ for 12 hours to precipitate camel cheese protein;

c. c, centrifuging the precipitate in the step b at the temperature of 4 ℃ and the rotating speed of 10000r/min for 20min, adding the precipitate into distilled water for dissolving, adjusting the pH value to 7.0 by using 1mol/l NaOH, and stirring by using a magnetic stirrer for completely dissolving;

d. d, dialyzing the solution obtained in the step c with distilled water for 48 hours for desalination, wherein the specification of a dialysis bag is 1000Da, concentrating the desalted casein, and freeze-drying the casein for 24 hours at the temperature of minus 80 ℃ and under the pressure of 9.8Pa by using a freeze dryer, wherein the polypeptide content and the antioxidant activity are shown in Table 2;

e. d, measuring the antioxidant activity and the protein content of the casein in the step d through electrophoresis to obtain 3.2g of total casein, and calculating the yield (mg/ml) shown in table 1, wherein the electrophoretic separation gel is acrylamide with the volume of 15%;

f. e, adding 200ml of distilled water into 10g of casein obtained in the step e to dissolve the protein, adjusting the mass concentration of the solution to 50g/L, adjusting the pH value of the solution to 8.0 by using NaOH with the concentration of 1mol/L, dissolving the solution in a water bath kettle at the temperature of 37 ℃, adding 1.0% of trypsin by mass when the solution has no obvious particles and the pH value has no obvious change, maintaining the pH value of the casein hydrolysate to 8.0 by using NaOH with the concentration of 1mol/L in the constant-temperature water bath kettle, slowly stirring for enzymolysis for 4 hours, inactivating the enzyme in a boiling water bath at the temperature of 100 ℃ for 10 minutes, and naturally cooling to room temperature;

g. adding ethanol into the hydrolysate obtained in the step f according to the volume ratio of 1:3, and standing in a refrigerator at the temperature of 4 ℃ for 12 hours to precipitate macromolecular protein;

h. centrifuging the solution in the step g for 20min at the rotation speed of 10000r/min and the temperature of 4 ℃, removing proteins with large molecular weight, concentrating by using a rotary evaporator, and freeze-drying to obtain total polypeptide;

k. and (3) separating the part Ib obtained in the step j by high performance liquid chromatography with the gradient of 25-70% acetonitrile for 0-40min and the flow rate of 2.5mL/min to obtain three camel milk antioxidant polypeptides Ib-1, Ib-2 and Ib-3 (figure 3).

TABLE 1 Effect of different pH values on Casein yield and protein content

TABLE 2 analysis of antioxidant Activity of camel cheese protein Trypsin hydrolysate

Example 2

b. b, regulating the pH value of the supernatant obtained in the step a to 4.3 by using 1mol/l HCl solution, and standing in a refrigerator at the temperature of 4 ℃ for 12 hours to precipitate camel cheese protein;

e. d, measuring the antioxidant activity and the protein content of the casein in the step d through electrophoresis to obtain 4.1g of total casein, wherein the calculation yield (mg/ml) is shown in table 1, and the volume of the electrophoretic separation gel is 15% of acrylamide;

h. centrifuging the solution in the step g for 20min at the rotation speed of 10000r/min and the temperature of 4 ℃, removing protein with large molecular weight, concentrating by using a rotary evaporator, and freeze-drying to obtain total polypeptide with small molecular weight;

Example 3

b. b, regulating the pH value of the supernatant obtained in the step a to 4.5 by using 1mol/l HCl solution, and standing in a refrigerator at the temperature of 4 ℃ for 12 hours to precipitate camel cheese protein;

e. d, measuring the antioxidant activity and the protein content of the casein in the step d through electrophoresis to obtain 2.9g of total casein, wherein the calculation yield (mg/ml) is shown in table 1, and the volume of the electrophoretic separation gel is 15% of acrylamide;

Example 4

d. d, dialyzing the solution obtained in the step c with distilled water for 48 hours for desalination, wherein the specification of a dialysis bag is 1000Da, concentrating the desalted casein, and freeze-drying the concentrated solution for 24 hours at the temperature of 80 ℃ below zero and under the pressure of 9.8Pa by using a freeze dryer to obtain 3.8g of hydrolyzed polypeptide, wherein the extraction rate of the polypeptide is 3.8g/10g which is 38%, the hydrolysis degree is calculated to be 17.6%, and the content and the antioxidant activity of the polypeptide are shown in Table 2;

h. centrifuging the ethanol aqueous solution obtained in the step g at the temperature of 4 ℃ and the rotating speed of 10000r/min for 10min to remove proteins with large molecular weight, concentrating by using a rotary evaporator, and freeze-drying to obtain total polypeptides with small molecular weight;

Example 5

Determining the molecular weight and the amino acid sequence of the obtained three antioxidant polypeptides Ib-1, Ib-2 and Ib-3 by combining a high-resolution mass spectrum and a liquid chromatography-mass spectrometry technology: RLDGQGRPRVWLGR (1665.94Da), TPDNIDIWLGGIAEPQVKR (2122.13Da) and VAYSDDGENWTEYRDQGAVEGK (2489.09Da) (FIGS. 4-7).

Claims

1. A preparation method of camel milk antioxidant polypeptide is characterized by comprising the following steps:

2. Use of camel milk antioxidant polypeptides obtained according to the method of claim 1 in the preparation of a health product.

3. Use of camel milk antioxidant polypeptides obtained according to the method of claim 1 in the preparation of a food product.

4. Use of camel milk antioxidant polypeptides obtained according to the method of claim 1 in the preparation of a food additive.