CN110527705B - Method for preparing antioxidant oligopeptide by enzymolysis of rabbit blood fermentation broth - Google Patents
Method for preparing antioxidant oligopeptide by enzymolysis of rabbit blood fermentation broth Download PDFInfo
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Abstract
The application provides a method for preparing antioxidant oligopeptide by enzymolysis of rabbit blood fermentation broth, which comprises the following steps: firstly, respectively preparing a bacillus subtilis seed culture solution, a bacillus coagulans seed culture solution and a lactobacillus plantarum seed culture solution; mixing the bacillus subtilis seed culture solution, the bacillus coagulans seed culture solution and the lactobacillus plantarum seed culture solution according to the volume ratio of 3:1:1 to obtain mixed seed solution; then, taking sterilized rabbit blood as a culture substrate, inoculating the mixed seed liquid into the culture substrate, culturing for 2-4 days at the temperature of 30-40 ℃ and the rotating speed of 120-150r/min, and taking the supernatant of the obtained culture liquid; and then carrying out enzymolysis by utilizing alkaline protease and papain compound enzyme liquid, and then carrying out centrifugation, suction filtration and ultrafiltration to obtain the enzyme-activated protein. The application can obtain antioxidant oligopeptide with excellent oxidation resistance, and meanwhile, the content of the obtained polypeptide is high.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a method for preparing antioxidant oligopeptide by enzymolysis of rabbit blood fermentation broth.
Background
The China is the first big rabbit raising country in the world, and is the big country for producing and processing the rabbit meat in the world. The rabbit industry is an dominant industry with development characteristics, and plays an important part in the comprehensive development of animal husbandry in China. It is found that the protein and lysine contents in rabbit meat are up to 21.21%, 10.6%, respectively, the digestibility is up to 85%, the fat content is only 3.89%, and the cholesterol content is lower than 63%, 41%, 8% and 15% respectively compared with pig, cow, sheep and chicken. The rabbit meat is rich in various minerals required by human body, wherein the calcium content is up to 12.8mg/100g, and the rabbit meat is low in pollution degree caused by toxic and harmful substances such as environment, agricultural and veterinary drugs and the like due to the growth characteristics of the rabbit meat, and has extremely high nutritive value. The long-term intake of the Chinese herbal medicine can improve the immunity of a human body, promote the growth and development of teenagers, reduce the incidence rate of cardiovascular diseases and cerebrovascular diseases, improve the metabolism of the human body and the like.
The main uses of the rabbit industry according to economic development are three aspects: meat, wool and skin, wherein rabbit blood is not recycled. The blood is largely discarded in the slaughtering process, which is not only a waste of protein resources, but also causes environmental pollution. The current research reports on rabbit blood are relatively few, the research direction is mainly focused on biomedicine, yang Wenrun and the like discuss the feasibility of the rabbit blood to replace human blood in the blood compatibility evaluation of medical instruments (Yang Wenrun, tian Shenghui, ke Jun. The feasibility research of the rabbit blood applied to the blood compatibility evaluation of medical instruments [ J ]. Chinese medical instrument information, 2016,22 (13): 41-45); zeng Li the extraction and application of superoxide dismutase in rabbit blood (Zeng Li. Extraction of superoxide dismutase from rabbit blood and its application in yogurt [ D ]. Sichuan university of agriculture, 2014); the medicine for treating insomnia is separated from the rabbit blood (the medicine for treating insomnia is separated from the rabbit blood [ J ]. Medical information communication, 1985 (01): 8). However, no studies on the preparation of antioxidant oligopeptides using rabbit blood fermentation broth have been reported so far.
The bioactive peptide is an amphoteric compound which is formed by dehydrating two or more amino acids according to different compositions and arrangement modes, contains carboxyl and amino and has special physiological regulation function. The antioxidant oligopeptide is one of various bioactive peptides, and has the functions of scavenging free radicals, maintaining free radical balance, inhibiting or slowing down oxidation reaction, improving organism immunity, resisting aging and the like, and can be absorbed more and digested faster than other free amino acids. Because of the strong antioxidant activity, diversity and high safety, the compound has been paid attention to in recent years, and has wide application prospect in the fields of health care products, medicines, cosmetics, food additives and the like. The antioxidant peptides can be divided into two types of chemical synthetic antioxidant peptides and natural antioxidant peptides according to different products prepared by the antioxidant peptides. The chemical synthesis of antioxidant peptide has obvious advantages and disadvantages, such as oil oxidation inhibition, relatively low price, and strict control of usage amount and obvious toxic and side effects. However, natural antioxidant peptides between proteins and amino acids have extremely strong biological activity and diversity, high safety and more remarkable oxidation resistance. Thus, various natural antioxidant peptides are paid attention to in recent years, and have wide application prospects in the fields of health care products, medicines, cosmetics, food additives and the like.
There are many methods for preparing antioxidant oligopeptides. At present, three methods are mainly adopted at home and abroad, namely a microbial fermentation method, an enzymolysis method for controlling hydrolysis parameters, and a most reasonable and most commonly used preparation method by combining bacteria and enzymes.
Microbial fermentation refers to the production of large amounts of proteases by certain microorganisms under suitable metabolic conditions. The protease produced by these bacteria can hydrolyze the fermentation substrate to produce bioactive peptides, which can promote the metabolism and enzyme production of the microorganism. The two are mutually promoted, thereby greatly improving the production rate of the polypeptide. At present, microorganisms such as bacillus subtilis, aspergillus niger, lactobacillus, microzyme and the like are widely applied in a microbial fermentation method, wherein mould and bacillus subtilis are respectively involved in solid state fermentation and liquid state fermentation. The research of Qihong soldiers and the like shows that the in-vitro free radical scavenging capability index of the fermented supernatant liquid obtained by liquid fermentation of rice bran by a microbial fermentation method is obviously improved (Qihong soldiers, chen Jun, he Jia and the like; the antioxidant function research of the fermented rice bran supernatant liquid of bacillus natto [ J ]. Food science, 2008 (03): 293-297). Li Feng the low molecular weight silkworm chrysalis protein peptide is obtained by fermenting silkworm chrysalis protein with natto bacillus, and the yield is up to 53.75% (Li Feng, li Li, zong Xuyan. The process for preparing the low molecular weight silkworm chrysalis protein by fermenting silkworm chrysalis protein with natto bacillus is optimized [ J ]. Food industry, 2014 (04): 14-17).
The enzymolysis method is widely applied to the preparation of antioxidant peptides, and protein is decomposed by protease to obtain peptide fragments, so as to generate bioactive peptides. The enzymolysis method has obvious advantages and disadvantages, and the advantages are mainly that the nutrition value of the polypeptide can be better preserved, the reaction condition is milder, no toxic byproducts are generated, and the maximized target peptide can be obtained. The disadvantages are mainly that the enzymolysis method can produce bitter peptide to make the flavor of the product worsen, and the commercial enzyme has few varieties, high price and small enzymolysis scale. Rainbow uses papain to hydrolyze soybean to prepare antioxidant active peptide, and the product is found to have strong antioxidant activity (Rainbow, soybean polypeptide antioxidant research [ D ]. Lanzhou, lanzhou university of science, 2007). When the antioxidant capacity of soybean enzymolysis products is studied by Yan Jiu Fang et al under certain conditions, the hydrolysate of papain has higher antioxidant activity (Yan Jiu, zhang Lili, wang Tian. The antioxidant property of soybean enzymolysis products is studied [ J ]. Grain and oil processing, 2006 (03): 45-46).
The bacterial-enzyme combination method combines the microbial fermentation method and the enzymolysis method, and combines the advantages of the microbial fermentation method and the enzymolysis method to degrade the protein more thoroughly and obtain smaller peptide fragments. Meanwhile, in the degradation process of the protein, the bacterial enzyme modifies the tail end of the small peptide, so that the generation of bitter peptide is avoided, the taste and flavor of a polypeptide product are improved, and the nutritional value and quality of the polypeptide are improved. Sun Hong the method of combining bacteria and enzymes is used for cooperatively treating cottonseed meal, and the bacillus subtilis is used for fermentation and enzymolysis by papain, so that the in vitro digestibility and DPPH clearance capacity of cottonseed protein are both obviously enhanced (Sun Hong. The method of cooperatively treating the nutrition characteristics of cottonseed meal, the preparation of cottonseed peptide and the research on the antioxidant activity of cottonseed peptide [ D ]. Hangzhou, zhejiang university, 2013).
In order to obtain high-efficiency antioxidant peptide from rabbit blood through fermentation and enzymolysis, the method comprises the steps of preparing crude peptide liquid with relatively high oxidation resistance by fermenting rabbit blood, selecting a proper enzymolysis mode, and improving corresponding indexes of oxidation resistance and polypeptide content. However, no relevant report exists in the prior art.
Disclosure of Invention
Aiming at the defects and demands of the prior art, the application aims to provide a preparation method which can obtain high oxidation resistance and oxidation resistance oligopeptide and high polypeptide content and takes rabbit blood as a raw material.
In order to achieve the above purpose, the present application adopts the following technical scheme:
a method for preparing antioxidant oligopeptide by enzymolysis of rabbit blood fermentation broth, which comprises the following steps:
(1) Respectively preparing bacillus subtilis seed culture solution, bacillus coagulans seed culture solution and lactobacillus plantarum seed culture solution; mixing the bacillus subtilis seed culture solution, the bacillus coagulans seed culture solution and the lactobacillus plantarum seed culture solution according to the volume ratio of 3:1:1 to obtain mixed seed solution;
(2) Taking sterilized rabbit blood as a culture substrate, and inoculating the mixed seed liquid into the culture substrate, wherein the inoculation amount is 2.5-6.5%;
(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 taking the supernatant of the obtained culture solution;
(4) Performing enzymolysis on the supernatant obtained in the step (3) by utilizing an alkaline protease and papain mixed enzyme solution, wherein the enzyme-to-enzyme ratio is 100-500U/g, the enzymolysis time is 2-4 h, the pH value is 8.0-8.5, and the enzymolysis temperature is 50-60 ℃; wherein, the ratio of the activity units of the alkaline protease to the papain is 1.5-4:1;
(5) And (3) centrifuging, suction filtering and ultrafiltration are carried out on the enzymolysis liquid obtained in the step (4) to obtain the antioxidant peptide.
As an alternative embodiment of the application, the bacillus subtilis seed culture solution is prepared by inoculating activated bacillus subtilis inclined plane strain single colony into a sterilized seed liquid culture medium, and culturing for 12 hours in a constant-temperature shaking incubator at 35 ℃ and the rotating speed of 135r/min, wherein the seed liquid culture medium comprises the following formula: glucose 10g/L, beef extract 10g/L, peptone 10g/L, sodium chloride 5g/L, pH=7.0.
As an alternative embodiment of the application, the bacillus coagulans seed culture solution is prepared by inoculating activated bacillus coagulans inclined plane strain single colony into a sterilized seed liquid culture medium, and culturing for 12h in a constant-temperature shaking incubator at 35 ℃ and the rotating speed of 135r/min, wherein the seed culture liquid culture medium comprises the following formula: glucose 6g/L, beef extract 10g/L, peptone 10g/L, potassium dihydrogen phosphate 2g/L, manganese sulfate heptahydrate 1g/L, and pH=7.0.
As an alternative embodiment of the application, the lactobacillus plantarum seed culture solution is prepared by inoculating activated lactobacillus plantarum inclined plane strain single colony into a sterilized seed liquid culture medium, and culturing the seed liquid culture medium in a constant-temperature shaking incubator at 35 ℃ and the rotating speed of 135r/min for 12 hours, wherein the seed liquid culture medium comprises the following formula: glucose 20g/L, beef extract 10g/L, peptone 10g/L, potassium dihydrogen phosphate 2g/L, manganese sulfate heptahydrate 0.25g/L, trisodium citrate 3g/L, and pH=7.0.
As a preferred embodiment of the present application, the concentration of the rabbit blood is 10g/mL. In the step (4), the enzyme-to-enzyme ratio is 200U/g, the enzymolysis time is 2.5h, the pH value is 8.5, and the enzymolysis temperature is 60 ℃; wherein, the ratio of the activity units of the alkaline protease to the papain is 2:1
As a preferred embodiment of the present application, in the step (3), the resultant of the step (2) is cultured at 37℃and a rotation speed of 135r/min for 3.5 days.
As an alternative embodiment of the present application, in the step (5), centrifugation is performed by using a high-speed refrigerated centrifuge, and the centrifugation parameters are: the temperature is 4 ℃, the rotating speed is 6000r/min, and the time is 20min.
As an alternative embodiment of the application, in the step (5), filtering is performed by using a 0.45 μm filter membrane; during ultrafiltration, a 5KD filter membrane is utilized for carrying out the ultrafiltration in a centrifuge, and the centrifugation parameters are as follows: the temperature is 4 ℃, the rotating speed is 4000r/min, and the time is 10min.
Prior to the application, the preparation process optimization of the antioxidant peptide by fermenting the yak blood by using bacillus subtilis and the comparison, separation and purification research of the antioxidant peptide by using the yak blood have utilized the bacillus subtilis to extract the antioxidant peptide from the yak blood, and good results are obtained. Based on the report technology, the application carries out corresponding experiments on rabbit blood and obtains a certain effect, but the clearance rate of the obtained antioxidant peptide to ABTS+ is still not satisfactory, and the polypeptide content is lower.
On the basis of using bacillus subtilis as zymogenThe inventor carries out mixed fermentation on bacillus coagulans and bacillus subtilis, but the ABTS+ clearance rate and the polypeptide content of the obtained antioxidant peptide are not obviously improved. Through a great deal of fumbling, the inventor discovers that the clearance rate and the polypeptide content of the antioxidant peptide obtained by fermentation on ABTS+ can be greatly improved by mixing bacillus subtilis, bacillus coagulans and lactobacillus plantarum for mixed fermentation. In addition, the inventors have found that the antioxidant peptides obtained by the mixed fermentation of the present application can also significantly improve the oxidation resistance to OH and O 2 -scavenging of free radicals. Accordingly, the present application has been made in view of the above-described problems, and an object of the present application is to provide a method for preparing an antioxidant peptide having high antioxidant activity using rabbit blood. Based on the experimental results, the inventor of the application utilizes bacillus subtilis and lactobacillus plantarum to perform mixed fermentation, and discovers that the clearance rate of the obtained antioxidant peptide to ABTS is not improved compared with that of the antioxidant peptide obtained by single-bacteria fermentation by using the bacillus subtilis. Therefore, it can be determined that bacillus subtilis, bacillus coagulans viable bacteria and lactobacillus plantarum play a role in coordinating and enhancing the oxidation resistance and the polypeptide content of the obtained antioxidant peptide when fermentation is performed.
Based on the experimental results, the inventors of the present application examined the selection of the enzymatic hydrolysis method. The process optimization of preparing the antioxidant peptide by the enzymolysis of the yak blood fermentation liquid examines the influence of the combined fermentation of several enzymes on the obtained antioxidant peptide. However, the inventors of the present application found that for the present application, only single enzyme combination fermentation was used, neither the oxidation resistance nor the increase in polypeptide content was significantly affected. After extensive investigation, the inventors found that, when the antioxidative peptide is prepared by fermenting with a complex enzyme of alkaline protease and papain, the antioxidative peptide prepared by the fermentation process has significantly improved oxidation resistance and polypeptide content compared with those prepared by the fermentation process alone.
The application has the beneficial effects that:
1. the antioxidant oligopeptide obtained by the application has the clearance rate of 96.20% to the ABTS free radical and has 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 4.77mg/ml.
3. The antioxidant oligopeptide obtained by the application has the scavenging rate of 93.22% for OH free radical and the scavenging rate of 93.22% for O 2 The radical scavenging rate can reach 94.10%.
Detailed Description
The present application is described in detail below by way of examples, which are necessary to be pointed out herein for further illustration of the application and are not to be construed as limiting the scope of the application, since numerous insubstantial modifications and adaptations of the application will occur to those skilled in the art in light of the foregoing disclosure.
Example 1
1.1 Experimental materials
Bacillus subtilis: SICC1.197. Bacillus coagulans from the platform bacterial collection of microbial resources, inc. Of Sichuan province: lactobacillus plantarum of China general microbiological culture collection center (CGMCC) No. 6729: CGMCC No.5105 China general microbiological culture Collection center alkaline protease (enzyme activity 85.61U/mg): shanghai Kayon Co Ltd
Papain (enzyme activity 800U/mg): shanghai Kayon Co Ltd
1.2 Experimental reagents
lowry protein concentration kit: chemical analysis purity of domestic products of Beijing Soy Bao technology Co., ltd.): beef extract, peptone, yeast extract, sodium chloride, glucose, agar, ABTS, K2S2O8, trichloroacetic acid (TCA), potassium dihydrogen phosphate, manganese sulfate heptahydrate, sodium dihydrogen phosphate, and trisodium citrate.
1.3 laboratory apparatus
HHS-8S type electronic constant temperature stainless steel water bath pot: shanghai optical land instruments, inc.;
H2050R bench type high-speed refrigerated centrifuge: a long sand xiang instrument centrifuge limited;
QYC-2102C type constant temperature shaking incubator: shanghai Fuma laboratory facility Co., ltd;
XW-80A vortex mixer: shanghai Chi electronic Co., ltd;
FALL04N type electronic balance: positron instruments, inc;
UV Power type ultraviolet-visible spectrophotometer: beijing Laibaitake instruments Co., ltd;
STARTER3100 PH meter: shanghai Orhaus Co., ltd;
XHF-D high speed disperser (internal refiner): ningbo Xinzhi biotechnology Co., ltd;
GM-0.33 type diaphragm vacuum pump: the experimental facilities of the Jiuteng, tianjin, inc.;
WP-UP-UV-20 type ultra-pure water machine: sichuan Wo Teer technology development Co., ltd; JJ-CJ-2FD type clean bench: suzhou city gold purification equipment technology development limited;
gZ-150-S biochemical incubator: shaoguan city Guangzhi scientific and technological equipment limited company;
pipetting gun: brand, germany;
ultrafiltration tube (5 kDa, 1 kDa): sartorius company, germany, pall company, usa.
1.4 preparation of Mixed seed solution
Respectively picking appropriate amount of activated Bacillus subtilis, bacillus coagulans and Lactobacillus plantarum, inoculating into conical flask containing 50mL of each liquid culture medium, culturing in constant temperature shaking incubator at 35deg.C and rotation speed of 135r/min for 12 hr to obtain respective seed culture solutions (1×10) 9 cfu/mL). The formulation parameters of the test media are shown in Table 1 below:
TABLE 1 preparation of seed culture Medium
The seed culture medium 1 is a bacillus subtilis seed culture liquid culture medium, the seed culture medium 2 is a bacillus coagulans seed culture liquid culture medium, and the seed culture medium 3 is a lactobacillus plantarum seed culture liquid culture medium; after adjusting to proper pH, sterilizing in a high-pressure steam sterilizing pot for 20 minutes, and cooling for standby.
1.5 preparation of antioxidant oligopeptides by fermentation and enzymolysis
Preparing fermentation liquid: mixing the bacillus subtilis seed culture solution, the bacillus coagulans seed culture solution and the lactobacillus plantarum seed culture solution according to the volume ratio of 3:1:1 to obtain mixed seed solution; taking sterilized rabbit blood as a culture substrate, and inoculating the mixed seed liquid into the culture substrate, wherein the inoculation amount is 4.5%; culturing at 37deg.C and 135r/min for 3.5 days, and collecting supernatant.
Enzymolysis: performing enzymolysis on the supernatant of the fermented culture solution by using an alkaline protease and papain mixed enzyme solution, wherein the enzyme-to-enzyme ratio is 200U/g, the enzymolysis time is 2.5h, the pH value is 8.5, and the enzymolysis temperature is 60 ℃; wherein, the ratio of the activity units of the alkaline protease to the papain is 2:1.
And (3) centrifuging: weighing the fermented liquid after culture into a centrifuge tube with the same weight, symmetrically placing the fermented liquid into a high-speed refrigerated centrifuge, and setting centrifugal parameters as follows: the temperature is 4 ℃, the rotating speed is 6000r/min, and the time is 20min. After the completion, the supernatant was aspirated by a pipette, and stored at low temperature.
And (3) suction filtration: filtering the centrifuged rabbit blood crude extract with 0.45 μm filter membrane in a suction filtration device, removing thallus and residues to obtain filtered liquid, and preserving at low temperature.
Ultrafiltration: selecting a 5KD filter membrane, weighing equal amount of filtrate, symmetrically placing in a centrifugal machine, and setting the parameters as follows: the temperature is 4 ℃, the rotating speed is 4000r/min, the time is 10min, and a liquid in the centrifuge tube is sucked by a liquid-transferring gun, so that the rabbit blood antioxidant oligopeptide with higher purity is obtained.
1.6 measurement of antioxidant Activity
1.6.1 determination of ABTS+ clearance of antioxidant oligopeptides
Dissolving a proper amount of ABTS powder in a certain amount of distilled water to prepare an ABTS solution with the concentration of 7mmol/L, adding an equal volume of 2.45mmol/L K2S2O8 solution, uniformly mixing, standing the mixed solution for 12-16h at room temperature and in a dark condition, diluting with a phosphate buffer solution (0.2 mol/L, pH 7.4) to ensure that the absorbance reaches 0.70+/-0.02 at 734nm to form a dark green ABTS+ measuring solution.
And (3) adding 100 mu L of a rabbit blood antioxidant active peptide sample solution into 4.0mL of an ABTS+ measuring solution, accurately oscillating for 30s, and measuring the absorbance value of a reaction system at 734 nm. The blank tube was replaced with phosphate buffer for the sample and the control tube was replaced with phosphate buffer for abts+ assay. The abts+ clearance calculation formula is as follows:
ABTS radical clearance (%) = [ a0- (A1-A2) ]/a0×100%
Wherein: a0-absorbance of blank tube; a1-measuring the absorbance of the sample; a2-absorbance of control tube.
1.6.2 determination of OH clearance of antioxidant oligopeptides
The method in the thesis of the academic paper Zhang Fengying, namely the enzymolysis of pig blood hemoglobin and the research on the antioxidant activity of the product thereof, is specifically as follows: 1mL of the sample solution to be measured is added into each test tube, and 1mL of 9mmol/L salicylic acid-ethanol solution and 1mL of 9 mmol/LFASO are respectively added 4 Mixing well, adding 1mL of 8.8 mmol/L H 2 O 2 Mixing, standing in a water bath at 37deg.C, maintaining the temperature for 30min, and measuring OD510nm with ultraviolet-visible spectrophotometer. The blank group replaced the sample solution with ultrapure water, and the control group replaced 3 solutions except the sample with ultrapure water. The sample clearance to hydroxyl radicals was calculated as follows:
ABTS radical clearance (%) = [1- (A1-A2) ]/a0×100%
Wherein: a0-absorbance of blank tube; a1-measuring the absorbance of the sample; a2-absorbance of control tube.
1.6.3 antioxidant oligopeptides O 2 Clearance determination
200. Mu.L of soybean lecithin solution (20 mg lecithin, dissolved in 5mL of 0.05mol/L pH7.4 phosphate buffer) and 1mL of sample solution of different concentrations were uniformly mixed, 50. Mu.L of 50mmol/L FeSO4 was added, and the total volume of the system was made up to 3mL by 0.1mmol/L pH7.4 phosphate buffer, water bath was 37℃for 40min, 0.8mL of 15g/LTCA and 1mL of 0.8g/LTBA were added respectively, water bath was carried out for 15min, cooling was carried out, 6000r/min was centrifuged for 10min, and absorbance of the supernatant was measured at 532nm wavelength by an ultraviolet-visible spectrophotometer. The blank group replaced the sample solution with phosphate buffer. The lipid peroxidation clearance was calculated as follows:
lipid peroxidation clearance (%) = (1-A1/A0) ×100%
Wherein A0 is absorbance of a blank group; a1 is the absorbance of the experimental group.
1.7 determination of polypeptide content
Adding trichloroacetic acid (TCA) with the same volume of 15% (w/v) into 4mL of polypeptide sample, uniformly mixing, standing to precipitate macromolecular protein, centrifuging in a high-speed centrifuge for 15min with the setting parameter of 6000r/min, and measuring the protein content of the supernatant by using a Lowry method to obtain the polypeptide content of the supernatant.
Namely, a proper amount of Folin phenol A reagent A and a proper amount of Folin phenol A reagent B are taken according to a weight ratio of 50:1 (expiration date 24 hours after mixing, expiration date) and according to the required amount, diluting the BSA standard 10 times to a concentration of 0.5mg/ml with PBS, taking a 5ml centrifuge tube, labeling, adding 200ul of properly diluted polypeptide, adding 2ml of Folin phenol A reagent, mixing uniformly, and standing at room temperature for 10 minutes. And adding 200ul of Folin phenol B reagent, uniformly mixing, standing at a constant temperature of 37 ℃ for 30 minutes, measuring by an ultraviolet-visible spectrophotometer, setting the wavelength to 650nm, and calculating the protein concentration of the supernatant by the obtained absorbance to obtain the polypeptide content in the supernatant.
1.8 experimental results
The antioxidant oligopeptide obtained by the application has the following characteristics of ABTS+,. O 2 The results of the clearance of the sum OH and the results of the polypeptide content obtained by the preparation method of the application are shown in Table 2:
TABLE 2
| ABTS + clearance (%) | ·O 2 Clearance (%) | OH clearance (%) | Polypeptide content (mg/ml) |
| 96.20 | 93.22 | 94.10 | 4.77 |
Comparative example 1
The procedure of example 1 was followed except that the enzymatic hydrolysis step was omitted, and the resulting antioxidant peptide had an ABTS+ clearance of 94.00%, an O2-clearance of 89.31%, an OH clearance of 91.27% and a polypeptide content of 3.42mg/ml.
Comparative example 2
The procedure was as in comparative example 1, except that the bacteria in the seed culture contained only Bacillus subtilis. The ABTS+ clearance rate of the obtained antioxidant peptide is 84.64%, the O2 clearance rate is 77.35%, the OH clearance rate is 79.66%, and the polypeptide content is 2.84mg/ml.
Comparative example 3
Comparative example 1 was conducted in the same manner as in comparative example 1 except that the mixed seed liquid was obtained by mixing the seed culture liquid of Bacillus subtilis and the seed culture liquid of Bacillus coagulans in a ratio of 3:1. The ABTS+ clearance rate of the obtained antioxidant peptide is 86.13%, the O2 clearance rate is 78.22%, the OH clearance rate is 80.17%, and the polypeptide content is 2.80mg/ml.
Comparative example 4
The procedure is as in example 1, except that the complex enzyme is changed to alkaline protease alone. The ABTS+ clearance rate of the obtained antioxidant oligopeptide is 94.35%, the O2 clearance rate is 90.65%, the OH clearance rate is 92.36%, and the polypeptide content is 3.51mg/ml.
Claims (7)
1. A method for preparing antioxidant oligopeptide by enzymolysis of rabbit blood fermentation broth, which is characterized by comprising the following steps:
(1) Respectively preparing bacillus subtilis seed culture solution, bacillus coagulans seed culture solution and lactobacillus plantarum seed culture solution; mixing the bacillus subtilis seed culture solution, the bacillus coagulans seed culture solution and the lactobacillus plantarum seed culture solution according to the volume ratio of 3:1:1 to obtain mixed seed solution;
(2) Taking sterilized rabbit blood as a culture substrate, and inoculating the mixed seed liquid into the culture substrate, wherein the inoculation amount is 4.5%;
(3) Culturing the product obtained in the step (2) for 3.5 days at 37 ℃ and a rotating speed of 135r/min, and taking the supernatant of the obtained culture solution;
(4) Performing enzymolysis on the supernatant obtained in the step (3) by utilizing an alkaline protease and papain mixed enzyme solution, wherein the enzyme-to-enzyme ratio is 200U/g, the enzymolysis time is 2.5h, the pH value is 8.5, and the enzymolysis temperature is 60 ℃; wherein, the ratio of the activity units of the alkaline protease to the papain is 2:1;
(5) Centrifuging, suction filtering and ultrafiltration are carried out on the enzymolysis liquid obtained in the step (4), so that the antioxidant peptide is obtained;
the bacillus subtilis is as follows: SICC1.197 is deposited in the microorganism resource platform bacterial collection of Sichuan province; the bacillus coagulans is: CGMCC No.6729, which is preserved in China general microbiological culture collection center; the lactobacillus plantarum is: CGMCC No.5105, which is preserved in China general microbiological culture collection center;
the bacterial concentration of the bacillus subtilis seed culture solution, the bacillus coagulans seed culture solution and the lactobacillus plantarum seed culture solution is 1 multiplied by 10 9 cfu/mL；
And 5KD filter membrane is selected for ultrafiltration during the ultrafiltration.
2. The method according to claim 1, wherein the bacillus subtilis seed culture solution is prepared by inoculating activated bacillus subtilis slant strain single colony into sterilized seed liquid culture medium, and culturing the seed liquid culture medium in a constant-temperature shaking incubator at 35 ℃ and a rotation speed of 135r/min for 12 hours, wherein the seed liquid culture medium comprises the following formula: glucose 10g/L, beef extract 10g/L, peptone 10g/L, sodium chloride 5g/L, pH=7.0.
3. The method according to claim 1, wherein the bacillus coagulans seed culture solution is prepared by inoculating activated bacillus coagulans slant strain single colonies into a sterilized seed liquid culture medium, and culturing the seed liquid culture medium in a constant-temperature shaking incubator at 35 ℃ and a rotation speed of 135r/min for 12 hours, wherein the seed liquid culture medium comprises the following formula: glucose 6g/L, beef extract 10g/L, peptone 10g/L, potassium dihydrogen phosphate 2g/L, manganese sulfate heptahydrate 1g/L, and pH=7.0.
4. The method according to claim 1, wherein the lactobacillus plantarum seed culture solution is prepared by inoculating activated lactobacillus plantarum bevel strain single colonies into a sterilized seed liquid culture medium, and culturing the seed liquid culture medium in a constant-temperature shaking incubator at 35 ℃ and a rotating speed of 135r/min for 12 hours, wherein the seed liquid culture medium comprises the following formula: glucose 20g/L, beef extract 10g/L, peptone 10g/L, potassium dihydrogen phosphate 2g/L, manganese sulfate heptahydrate 0.25g/L, trisodium citrate 3g/L, and pH=7.0.
5. The method of claim 1, wherein the concentration of rabbit blood is 10g/mL.
6. The method according to claim 1, wherein in the step (5), centrifugation is performed by a high-speed refrigerated centrifuge, and the centrifugation parameters are: the temperature is 4 ℃, the rotating speed is 6000r/min, and the time is 20min.
7. The method according to claim 1, wherein in the step (5), filtration is performed by using a 0.45 μm filter membrane; during ultrafiltration, a 5KD filter membrane is utilized for carrying out the ultrafiltration in a centrifuge, and the centrifugation parameters are as follows: the temperature is 4 ℃, the rotating speed is 4000r/min, and the time is 10min.
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