阅读说明：本技术 一种兔血抗氧化肽的发酵制备方法 (Fermentation preparation method of rabbit blood antioxidant peptide ) 是由 肖岚 辛松林 董平 李燮昕 安攀宇 于 2019-09-20 设计创作，主要内容包括：本发明提供了一种兔血抗氧化肽的发酵制备方法,其包括步骤：(1)、分别制备枯草芽孢杆菌种子培养液、凝结芽孢杆菌种子培养液和植物乳杆菌种子培养液；按体积比3:1:1的比例,将枯草芽孢杆菌种子培养液、凝结芽孢杆菌种子培养液和植物乳杆菌种子培养液混合,得到混合种子液；(2)、以灭菌后的兔血作为培养底物,将混合种子液接种至培养底物中,接种量为2.5～6.5％；(3)、在30～40℃、120-150r/min转速下,培养2-4天,对所得培养液进行离心、抽滤和超滤之后,即得抗氧化肽。本发明所得抗氧化肽对ABTS+、·OH和·O<Sub>2</Sub>-自由基具有优异的清除能力,本发明方法所得多肽含量可达到3.42mg/ml。(The invention provides a fermentation preparation method of rabbit blood antioxidant peptide, which comprises the following steps: (1) respectively preparing a bacillus subtilis seed culture solution, a bacillus coagulans seed culture solution and a lactobacillus plantarum seed culture solution; mixing a bacillus subtilis seed culture solution, a bacillus coagulans seed culture solution and a lactobacillus plantarum seed culture solution according to the volume ratio of 3:1:1 to obtain a mixed seed solution; (2) inoculating the mixed seed liquid to culture medium by taking sterilized rabbit blood as culture substrateIn the substrate, the inoculation amount is 2.5-6.5%; (3) culturing for 2-4 days at 30-40 ℃ and 120-150r/min, and centrifuging, filtering and ultrafiltering the obtained culture solution to obtain the antioxidant peptide. The antioxidant peptide pair ABTS +,. OH and. O obtained by the invention 2 The free radicals have excellent scavenging capacity, and the content of the polypeptide obtained by the method can reach 3.42 mg/ml.)

1. a fermentation preparation method of rabbit blood antioxidant peptide is characterized by comprising the following steps:

(1) Respectively preparing a bacillus subtilis seed culture solution, a bacillus coagulans seed culture solution and a lactobacillus plantarum seed culture solution; mixing a bacillus subtilis seed culture solution, a bacillus coagulans seed culture solution and a lactobacillus plantarum seed culture solution according to the volume ratio of 3:1:1 to obtain a mixed seed solution;

(2) Inoculating the mixed seed solution into a culture substrate by taking the sterilized rabbit blood as the culture substrate, wherein the inoculation amount is 2.5-6.5%;

(3) And (3) culturing the product obtained in the step (2) for 2-4 days at the temperature of 30-40 ℃ and the rotating speed of 120-150r/min, and then centrifuging, filtering and ultrafiltering the obtained culture solution to obtain the antioxidant peptide.

2. The preparation method of claim 1, wherein the bacillus subtilis seed culture solution is prepared by inoculating activated single colony of bacillus subtilis slant strain into sterilized seed culture solution, and culturing for 12h in a constant temperature shaking incubator at 35 ℃ and 135r/min, wherein the formula of the seed culture solution is as follows: 10g/L of glucose, 10g/L of beef extract, 10g/L of peptone and 5g/L of sodium chloride, and the pH value is 7.0.

3. The preparation method of claim 1, wherein the bacillus coagulans seed culture solution is prepared by inoculating activated bacillus coagulans slant strain single colony into sterilized seed culture solution, and culturing for 12h in a constant temperature shaking incubator with 35 ℃ and 135r/min of rotation speed, wherein the formula of the seed culture solution is as follows: 6g/L glucose, 10g/L beef extract, 10g/L peptone, 2g/L potassium monohydrogen phosphate and 1g/L manganese sulfate heptahydrate, and the pH value is 7.0.

4. The preparation method of claim 1, wherein the lactobacillus plantarum seed culture solution is prepared by inoculating activated lactobacillus plantarum slant strain single colony into a sterilized seed liquid culture medium, and culturing for 12h in a constant temperature shaking incubator with 35 ℃ and 135r/min of rotation speed, wherein the formula of the seed liquid culture medium is as follows: 20g/L of glucose, 10g/L of beef extract, 10g/L of peptone, 2g/L of potassium monohydrogen phosphate, 0.25g/L of manganese sulfate heptahydrate and 3g/L of trisodium citrate, wherein the pH value is 7.0.

5. The method of claim 1, wherein the concentration of rabbit blood is 6-14 g/mL.

6. The method of claim 1, wherein the concentration of rabbit blood is 10 g/mL.

7. The method according to claim 6, wherein the amount of the mixed seed solution inoculated is 4.5%.

8. The process according to claim 1, wherein in the step (3), the product obtained in the step (2) is cultured at 37 ℃ and 135r/min for 3.5 days.

9. The method according to claim 1, wherein in the step (3), the centrifugation is performed by a high-speed refrigerated centrifuge, and the centrifugation parameters are as follows: the temperature is 4 deg.C, the rotation speed is 6000r/min, and the time is 20 min.

10. The production method according to claim 1, wherein in the step (3), the filtration is performed with a 0.45 μm filter; when the ultrafiltration is carried out, a 5KD filter membrane is used in a centrifuge, and the centrifugation parameters are as follows: the temperature is 4 ℃, the rotating speed is 4000r/min, and the time is 10 min.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a fermentation preparation method of rabbit blood antioxidant peptide.

Background

China is a rabbit producing country and the first rabbit product export country [9] in the world, and rabbit blood resources are very rich. The blood volume of healthy rabbits is about 55-65ml/kg, but because of the heavy fishy smell, poor palatability, difficult digestion and the like, a large amount of rabbit blood is directly discarded and is not fully utilized.

Rabbits are divided into three directions according to the economic use subject: meat, hair and skin, but not relates to the recycling of rabbit blood, and are all discarded in the slaughtering process. However, various studies have proved that rabbit blood has a very large effect, and researchers have separated substances for treating insomnia from rabbit blood. However, the research on extracting antioxidant peptide from rabbit blood has not been reported so far.

The peptide is a substance which is composed of two or more than two amino acids, has simple structure and strong physiological activity, and has the functions of regulating the metabolism of a human body, enhancing the immunity of the organism, preventing diseases and the like. An antioxidant peptide is one of the bioactive peptides, which can be defined by its main functional properties as: biological active peptides, which are collectively called antioxidant peptides, have the functions of maintaining the balance of free radicals of the body, improving the aging resistance of the body, inhibiting lipid peroxidation, eliminating free radicals and resisting diseases. In addition, the antioxidant peptide has anticancer, anti-stress, and anti-blood pressure effects. At present, antioxidant peptides can be classified into various types, such as animal-derived antioxidant peptides, plant-derived antioxidant peptides, microbial antioxidant peptides, endogenous and exogenous antioxidant peptides.

The animal-derived antioxidant peptide is derived from animal protein, can be classified into endogenous and exogenous ones, and is obtained from animal tissue organ or in vivo protein hydrolysate and has effects of inhibiting macromolecule peroxidation or scavenging in vivo free radical. At present, the sources for preparing animal-derived antioxidant peptides mainly include various sea sources, milk sources, blood sources, poultry egg sources and the like. The animal protein-derived antioxidant peptide has effective antioxidant activity (such as scavenging free radicals, inhibiting oxidation of oil and fat, and chelating metal ions) in food and organism.

At present, a problem to be solved in the art is how to efficiently extract antioxidant peptides from rabbit blood by fermentation.

Disclosure of Invention

aiming at the defects and the needs of the prior art, the invention aims to provide an antioxidant peptide extracted from rabbit blood by a fermentation method with high efficiency, so that the obtained antioxidant peptide has excellent ABTS free radical clearance rate.

In order to achieve the above object, the present invention provides the following solutions:

A fermentation preparation method of rabbit blood antioxidant peptide comprises the following steps:

(1) Respectively preparing a bacillus subtilis seed culture solution, a bacillus coagulans seed culture solution and a lactobacillus plantarum seed culture solution; mixing a bacillus subtilis seed culture solution, a bacillus coagulans seed culture solution and a lactobacillus plantarum seed culture solution according to the volume ratio of 3:1:1 to obtain a mixed seed solution;

(2) inoculating the mixed seed solution into a culture substrate by taking the sterilized rabbit blood as the culture substrate, wherein the inoculation amount is 2.5-6.5%;

(3) And (3) culturing the product obtained in the step (2) for 2-4 days at the temperature of 30-40 ℃ and the rotating speed of 120-150r/min, and then centrifuging, filtering and ultrafiltering the obtained culture solution to obtain the antioxidant peptide.

As an optional embodiment of the invention, the Bacillus subtilis seed culture solution is prepared by inoculating activated single colony of Bacillus subtilis slant strain into sterilized seed liquid culture medium, and culturing for 12h in a constant temperature shaking incubator at 35 deg.C and 135r/min, wherein the seed liquid culture medium has a formula: 10g/L of glucose, 10g/L of beef extract, 10g/L of peptone and 5g/L of sodium chloride, and the pH value is 7.0.

As an optional embodiment of the invention, the Bacillus coagulans seed culture solution is prepared by inoculating activated single colony of Bacillus coagulans slant strain into sterilized seed liquid culture medium, and culturing for 12h in a constant temperature shaking incubator at 35 ℃ and 135r/min, wherein the seed liquid culture medium has the formula: 6g/L glucose, 10g/L beef extract, 10g/L peptone, 2g/L potassium monohydrogen phosphate and 1g/L manganese sulfate heptahydrate, and the pH value is 7.0.

as an optional embodiment of the invention, the lactobacillus plantarum seed culture solution is prepared by inoculating activated lactobacillus plantarum slant strain single colony into a sterilized seed liquid culture medium, and culturing for 12 hours in a constant-temperature shaking incubator at 35 ℃ and 135r/min, wherein the formula of the seed liquid culture medium is as follows: 20g/L of glucose, 10g/L of beef extract, 10g/L of peptone, 2g/L of potassium monohydrogen phosphate, 0.25g/L of manganese sulfate heptahydrate and 3g/L of trisodium citrate, wherein the pH value is 7.0.

The concentration of the rabbit blood is 6-14 g/mL.

in a preferred embodiment of the invention, the concentration of the rabbit blood is 10 g/mL.

in a preferred embodiment of the present invention, the inoculation amount of the mixed seed solution is 4.5%.

In a preferred embodiment of the present invention, in the step (3), the product obtained in the step (2) is cultured at 37 ℃ and 135r/min for 3.5 days.

In a preferred embodiment of the present invention, in the step (3), the centrifugation is performed by a high-speed refrigerated centrifuge, and the centrifugation parameters are as follows: the temperature is 4 deg.C, the rotation speed is 6000r/min, and the time is 20 min.

As a preferable scheme of the invention, in the step (3), when the suction filtration is carried out, a 0.45-micron filter membrane is used for filtration; when the ultrafiltration is carried out, a 5KD filter membrane is used in a centrifuge, and the centrifugation parameters are as follows: the temperature is 4 ℃, the rotating speed is 4000r/min, and the time is 10 min.

Before the invention, the process optimization of preparing antioxidant peptide by fermenting yak blood with bacillus subtilis and the research on comparing and separating and purifying the antioxidant peptide in the yak blood utilize the bacillus subtilis to extract the antioxidant peptide from the yak blood, and good results are obtained. On the basis of the reported technology, the rabbit blood is subjected to corresponding experiments, and a certain effect is obtained, but in general, the clearance rate of the obtained antioxidant peptide on ABTS + is still unsatisfactory, and the content of the polypeptide is low.

On the basis of using bacillus subtilis as zymocyte, the inventor performs mixed fermentation on bacillus coagulans and bacillus subtilis, but the ABTS + clearance and polypeptide content of the obtained antioxidant peptide are not obviously improved. Through a large amount of grope, the inventor finds that the mixed fermentation of the bacillus subtilis, the bacillus coagulans and the lactobacillus plantarum can greatly improve the clearance rate of the antioxidant peptide obtained by fermentation to ABTS + and the polypeptide content. In addition, the inventor also finds that the antioxidant peptide obtained by mixed fermentation of the invention can also greatly improve the resistance to OH and O₂-scavenging of free radicals. Therefore, the present invention provides a method for preparing antioxidant peptides with high antioxidant activity from rabbit blood. On the basis of the obtained experimental results, the inventor of the invention utilizes bacillus subtilis and lactobacillus plantarum to carry out mixed fermentation, and finds that the ABTS clearance rate of the obtained antioxidant peptide is not improved compared with that of the antioxidant peptide obtained by utilizing bacillus subtilis to carry out single-strain fermentation. Therefore, it can be determined that the bacillus subtilis, the bacillus coagulans and the lactobacillus plantarum play a role in coordination and synergism on the inoxidizability and the polypeptide content of the obtained antioxidant peptide when the bacillus subtilis, the bacillus coagulans and the lactobacillus plantarum are fermented.

the invention has the beneficial effects that:

1. The clearance rate of the antioxidant peptide on ABTS free radicals can reach 94%, and the antioxidant peptide has an excellent ABTS free radical clearance effect.

2. The polypeptide prepared by the preparation method has high content, and the content of the polypeptide can reach 3.42 mg/ml.

3. The clearance rate of the antioxidant peptide on OH free radicals can reach 89.31%, and on O₂the clearance rate of free radicals can reach 91.27%.

Detailed Description

The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.