CN112375799A - Method for improving oxidation resistance of abalone biological product - Google Patents
Method for improving oxidation resistance of abalone biological product Download PDFInfo
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- CN112375799A CN112375799A CN202011171977.1A CN202011171977A CN112375799A CN 112375799 A CN112375799 A CN 112375799A CN 202011171977 A CN202011171977 A CN 202011171977A CN 112375799 A CN112375799 A CN 112375799A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/16—Yeasts; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
Abstract
The invention discloses a method for improving the oxidation resistance of an abalone biological product, and belongs to the technical field of marine organisms. Abalone protein is used as a raw material, and a modern bioseparation technology, a biological enzymolysis technology and a mixed bacteria microbial fermentation technology are integrated to develop and develop an abalone polypeptide biological product with high in-vitro oxidation resistance, wherein mixed bacteria aerobic fermentation strains mainly comprise candida utilis and bacillus subtilis. The obtained abalone polypeptide has strong oxidation resistance, and the extract can be subjected to concentration, adsorption drying, alcohol precipitation or resin purification and other treatments according to application ways, so that the high-efficiency utilization in the fields of health care, food, medicine and the like is met. The process has the advantages of simple and efficient technology, strong oxidation resistance and free radical scavenging capacity of the obtained product, strong product stability, low production cost and easy industrial mass production.
Description
Technical Field
The invention relates to a method for improving the antioxidant capacity of an abalone biological product, in particular to an abalone polypeptide biological product with high antioxidant capacity prepared by utilizing synergistic fermentation of microorganisms, and belongs to the technical field of marine organisms.
Background
Abalone, ancient name abalone, mirror fish, nine-hole snail, eyesight improving fish and general cap, which are marine mollusks, belonging to the class gastropoda abalone family, single-shell marine shellfish, which is one of the traditional famous and precious food materials, ancient name abalone, mountain delicacy and seafood, and the saying "one bite abalone and one bite gold" is true. The record of the dietetic herbal medicine states that abalone enters liver to remove blood stasis and enters intestine to wash dirt without damaging primordial qi. Strengthening yang and promoting blood circulation, and can be used for treating upward reversal of liver heat, dizziness, bone steaming, fatigue heat, cataract, hypertension, and retinal hemorrhage; modern research results show that abalone is extremely rich in nutritive value and contains more than twenty kinds of amino acids, each hundred grams of fresh abalone meat contains 23.4 grams of protein, 3.4 grams of fat, 32 milligrams of inorganic salt calcium, 3.0 milligrams of iron, a considerable amount of iodine, zinc, phosphorus, vitamin A, B1 and the like, and when the abalone is eaten frequently, the abalone can nourish yin, calm the liver, strengthen the kidney, adjust adrenal secretion, regulate blood pressure, moisten dryness and promote intestines, and treat irregular menstruation, constipation and other diseases.
At present, abalone is deeply favored by consumers as a precious sea product, the eating mode mainly comprises instant and dry products, and certain disadvantages exist, and firstly, the eating mode is single, and active extracts with strong sea cucumber functionality and application thereof are lacked; secondly, a large amount of nutrient components are lost during cooking or extraction, so that the efficacy is influenced; thirdly, the food is inconvenient to eat. Therefore, the abalone nutrient health-care product can fully exert the food efficiency and drug effect functions of the abalone, is convenient for consumers to eat, conforms to the market demand, has better market prospect, and provides higher requirements for the extraction of functional active substances of the abalone.
In the prior art, abalone polypeptide and application thereof in food are rarely studied, for example, patent 200510068010.X discloses a preparation method and application of abalone peptide by degrading abalone protein by using compound plant protease (papain, ficin and bromelin), wherein the preparation method comprises the steps of freeze-drying fresh or foamed dry abalone to be crushed into powder, and degrading the abalone protein by using the above protease in a formula of 3: 2: 1.5 to obtain abalone polypeptide; patent 200410054770.0 discloses an abalone food and its preparation method, which is prepared by double enzymolysis of autolysis and As1.398 neutral protease and compound flavor protease or directly carrying out enzymolysis with As1.398 neutral protease and compound flavor protease. Comparing the above documents, it can be found that the research on the preparation and application of abalone polypeptide is less, and especially the lack of research on improving the nutrition of abalone polypeptide becomes a key bottleneck restricting the development of industry.
Disclosure of Invention
In order to overcome the defects of the prior art, the high-efficiency extraction and high-antioxidant-capacity application of abalone polypeptide are met, the application provides a method for improving the antioxidant capacity of abalone biological products, the modern bioseparation technology, the biological enzymolysis technology and the mixed-bacterium microbial fermentation technology are integrated, the abalone polypeptide biological products with high in-vitro antioxidant capacity are researched and developed, the application requirements of the fields of health care, medical treatment, food and the like on abalone polypeptide are met, the fermentation process of the process technology is simple and efficient, the obtained products are high in antioxidant capacity and free radical scavenging capacity, the production cost is low, and the industrial scale production is easy.
The invention realizes the technical effects through the following technical scheme:
a method for improving the antioxidant capacity of an abalone biological product is characterized in that the preparation method comprises the following steps: (1) dissolving: pulverizing abalone viscera protein dry powder, adding water, stirring to dissolve completely;
(2) enzymolysis: adding compound protease with the mass ratio of 2-3% of abalone protein into the dissolved abalone protein, and performing enzymolysis for 4-8 hours at the temperature of 40-60 ℃;
(3) aerobic fermentation: adding a compound microbial agent with the mass ratio of 2-3% of the enzymolysis liquid into the enzymolysis liquid after enzymolysis, and performing aerobic fermentation for 6-12 h at the temperature of 28-35 ℃;
(4) anaerobic fermentation: after the aerobic fermentation is finished, adding 1-2% of lactobacillus powder, and carrying out anaerobic fermentation for 8-16 h at the temperature of 28-35 ℃;
(5) and (3) filtering: performing coarse filtration to remove fermentation thalli;
(6) enzyme deactivation: inactivating enzyme at 100 deg.C for 10 min;
(7) centrifuging: centrifuging at 10000r/min, and collecting the supernatant as abalone polypeptide extractive solution.
The optimal water adding proportion in the step (1) is that the abalone visceral protein: water 1: 8.
Preferably, chitosan with the mass ratio of 2-3% of abalone protein is added in the abalone visceral protein dissolving process.
The compound protease in the step (2) is the combination of papain and neutral protease, and the mass ratio of the compound protease to the neutral protease is 2: 1.
The compound microbial agent in the step (3) is a mixed microbial agent of candida utilis and bacillus subtilis, and the mass ratio of the compound microbial agent to the mixed microbial agent is 2: 3.
The ventilation rate is 0.1-0.2 m when the aerobic fermentation process in the step (3) is carried out in a fermentation tank3/m3And min, and controlling the stirring speed to be 30-60 r/min.
The preparation method of the candida utilis seed solution comprises the following steps: under the aseptic operation condition, two rings of strains are picked from the inclined plane of the yeast YDP by using an inoculating ring, inoculated into a 500mL triangular flask containing 300mLPDA liquid culture medium, subjected to shaking culture at the temperature of between 28 and 32 ℃ and at the speed of 140r/min for 24 hours, and then inoculated into a seed tank for expanded culture according to the process to prepare the yeast seed liquid, wherein the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL。
The preparation method of the bacillus subtilis seed solution comprises the following steps: under the aseptic condition, LB culture medium is adopted to culture for 24 hours at the temperature of 28-32 ℃ to prepare liquid bacillus subtilis seed liquid, and the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL。
The invention provides a method for improving the antioxidant capacity of abalone biological products, the obtained abalone polypeptide has stronger antioxidant capacity, and the extract can be subjected to concentration, adsorption drying, alcohol precipitation, resin purification and other treatments according to application ways, so that the high-efficiency utilization in the fields of health care, food, medicine and the like is met.
The invention provides a method for improving the oxidation resistance of an abalone biological product, which has the following remarkable advantages compared with the prior art:
(1) the abalone visceral protein is used as a raw material, and the modern bioseparation technology, the biological enzymolysis technology and the mixed bacteria microbial fermentation technology are integrated, so that the oxidation resistance of the abalone visceral protein is remarkably improved; more importantly, the application creatively discovers that the antioxidant capacity of the abalone polypeptide prepared by the enzymolysis and fermentation method can be obviously improved by adding chitosan in a certain proportion into the abalone protein solution;
(2) this application utilizes good oxygen fungus (candida utilis, bacillus subtilis) to ferment abalone viscera protein jointly and draws abalone polypeptide, and the synergistic effect between the make full use of microorganism promotes abalone polypeptide's extraction rate and external antioxidant capacity, wherein: the candida utilis can generate various enzymes for degrading cell walls in the fermentation process, so that more functional active substances such as polypeptides and the like are released from the cell walls, and the yeast is an enzyme capable of releasing combined state phenolic acid, so that the in-vitro oxidation resistance of abalone polypeptides is improved; the bacillus subtilis can kill pathogenic bacteria in fermentation liquor in the high-temperature fermentation process, has strong capabilities of producing amylase, protease and the like, and can degrade macromolecular starch and protein substances into microorganisms for utilization;
(3) according to the invention, lactobacillus is adopted for anaerobic fermentation, so that the stability of abalone polypeptide is improved, and test results show that the influence of temperature and time on the oxidation resistance of abalone proteolytic peptide is small within the range of 20-80 ℃, the oxidation resistance begins to decrease along with the increase of temperature and time when the temperature exceeds 100 ℃, and the stability is higher than the technical level reported at present;
(4) by adopting liquid fermentation, the growth rate of the thalli is improved, the fermentation time is shortened, the production efficiency is improved, the fermentation process conditions are mild, the production cost is low, the large-scale production is easy, and the method plays an important role in improving the industrial economic benefit and large-scale popularization and application.
Detailed Description
In the present application, the activation method of candida utilis and bacillus subtilis is as follows:
(1) preparation of candida utilis seed liquidThe method comprises the following steps: under the aseptic operation condition, two rings of strains are picked from the inclined plane of the yeast YDP by using an inoculating ring, inoculated into a 500mL triangular flask containing 300mLPDA liquid culture medium, subjected to shaking culture at the temperature of between 28 and 32 ℃ and at the speed of 140r/min for 24 hours, and then inoculated into a seed tank for expanded culture according to the process to prepare the yeast seed liquid, wherein the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL；
(2) The preparation method of the bacillus subtilis seed solution comprises the following steps: under the aseptic condition, LB culture medium is adopted to culture for 24 hours at the temperature of 28-32 ℃ to prepare liquid bacillus subtilis seed liquid, and the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL。
Example 1
A method for improving the antioxidant capacity of an abalone biological product comprises the following specific process steps:
(1) dissolving: pulverizing abalone viscera protein dry powder, adding water, stirring to dissolve completely; the proportion of water added is abalone visceral protein: 1:8 of water;
(2) enzymolysis: adding compound protease with the mass ratio of 3% of abalone protein into the dissolved abalone protein, and performing enzymolysis for 6 hours at the temperature of 40-60 ℃; the compound protease is the combination of papain and neutral protease, and the mass ratio of the compound protease to the neutral protease is 2: 1;
(3) aerobic fermentation: adding a compound microbial agent with the mass ratio of 2% of the enzymolysis liquid into the enzymolysis liquid after enzymolysis, and carrying out aerobic fermentation for 12h at the temperature of 28-35 ℃; the compound microbial agent is a mixed microbial agent of candida utilis and bacillus subtilis, and the mass ratio of the compound microbial agent to the bacillus subtilis is 2: 3; the ventilation rate is 0.1-0.2 m when the aerobic fermentation process is carried out in a fermentation tank3/m3Min, controlling the stirring speed to be 30-60 r/min;
(4) anaerobic fermentation: after the aerobic fermentation is finished, 2% of lactobacillus powder is added, and anaerobic fermentation is carried out for 8 hours at the temperature of 28-35 ℃;
(5) and (3) filtering: performing coarse filtration to remove fermentation thalli;
(6) enzyme deactivation: inactivating enzyme at 100 deg.C for 10 min;
(7) centrifuging: centrifuging at 10000r/min, and collecting the supernatant as abalone polypeptide extractive solution.
The antioxidant capacity of the abalone polypeptide extract liquid in example 1 was measured, wherein the OH radical clearance was measured by the method described in "food chemistry test" of Shao Zhi et al; ABTS free radical clearance and DPPH free radical clearance are measured by referring to the method in the method of 'influence of reaction time on oxidation resistance results evaluated by DPPH method and ABTS method' of Linlian bamboo and the like; the results of the measurement were as follows:
in the experiment, the control group 1 is an abalone polypeptide product after the enzymatic inactivation and centrifugation of the enzymatic hydrolysate in the step (2) in the example 1; the control group 2 is the abalone polypeptide product fermented by adding only candida utilis in the aerobic fermentation in the example 1, and the rest is the same as the example 1; the control 3 group was the abalone polypeptide product fermented by adding only bacillus subtilis in the aerobic fermentation in example 1, and the rest was the same as example 1.
Example 2
A method for improving the antioxidant capacity of an abalone biological product comprises the following specific process steps:
(1) dissolving: crushing abalone viscera protein dry powder, adding water, stirring until the abalone viscera protein dry powder is completely dissolved, and adding chitosan with 2% of abalone protein by mass; the proportion of water added is abalone visceral protein: 1:8 of water;
(2) enzymolysis: adding compound protease with the mass ratio of 3% of abalone protein into the dissolved abalone protein, and performing enzymolysis for 6 hours at the temperature of 40-60 ℃; the compound protease is the combination of papain and neutral protease, and the mass ratio of the compound protease to the neutral protease is 2: 1;
(3) aerobic fermentation: adding a compound microbial agent with the mass ratio of 2% of the enzymolysis liquid into the enzymolysis liquid after enzymolysis, and carrying out aerobic fermentation for 12h at the temperature of 28-35 ℃; the compound microbial agent is a mixed microbial agent of candida utilis and bacillus subtilis, and the mass ratio of the compound microbial agent to the bacillus subtilis is 2: 3; the ventilation rate is 0.1-0.2 m when the aerobic fermentation process is carried out in a fermentation tank3/m3Min, controlling the stirring speed to be 30-60 r/min;
(4) anaerobic fermentation: after the aerobic fermentation is finished, 2% of lactobacillus powder is added, and anaerobic fermentation is carried out for 8 hours at the temperature of 28-35 ℃;
(5) and (3) filtering: performing coarse filtration to remove fermentation thalli;
(6) enzyme deactivation: inactivating enzyme at 100 deg.C for 10 min;
(7) centrifuging: centrifuging at 10000r/min, and collecting the supernatant as abalone polypeptide extractive solution.
The antioxidant activity of the abalone polypeptide extract liquid of example 2 was measured by the same method as that described in example 1. The test results are as follows:
the control group 1 in the experiment is the abalone polypeptide product after the enzymatic inactivation and centrifugation of the enzymatic hydrolysate in the step (2) in the example 2; the control group 2 is the abalone polypeptide product fermented by adding only candida utilis in the aerobic fermentation in the example 2, and the rest is the same as the example 2; the control 3 group is the abalone polypeptide product fermented by adding only bacillus subtilis in the aerobic fermentation in the example 2, and the rest is the same as the example 2.
Example 3
A method for improving the antioxidant capacity of an abalone biological product comprises the following specific process steps:
(1) dissolving: crushing abalone viscera protein dry powder, adding water, stirring until the abalone viscera protein dry powder is completely dissolved, and adding chitosan with the mass ratio of 3% of abalone protein; the proportion of water added is abalone visceral protein: 1:8 of water;
(2) enzymolysis: adding compound protease with the mass ratio of 3% of abalone protein into the dissolved abalone protein, and performing enzymolysis for 6 hours at the temperature of 40-60 ℃; the compound protease is the combination of papain and neutral protease, and the mass ratio of the compound protease to the neutral protease is 2: 1;
(3) aerobic fermentation: adding a compound microbial agent with the mass ratio of 2% of the enzymolysis liquid into the enzymolysis liquid after enzymolysis, and carrying out aerobic fermentation for 12h at the temperature of 28-35 ℃; the compound microbial agent is a mixed microbial agent of candida utilis and bacillus subtilis, and the mass ratio of the compound microbial agent to the bacillus subtilis is 2: 3; good tasteThe ventilation rate is 0.1-0.2 m when the oxygen fermentation process is carried out in a fermentation tank3/m3Min, controlling the stirring speed to be 30-60 r/min;
(4) anaerobic fermentation: after the aerobic fermentation is finished, 2% of lactobacillus powder is added, and anaerobic fermentation is carried out for 8 hours at the temperature of 28-35 ℃;
(5) and (3) filtering: performing coarse filtration to remove fermentation thalli;
(6) enzyme deactivation: inactivating enzyme at 100 deg.C for 10 min;
(7) centrifuging: centrifuging at 10000r/min, and collecting the supernatant as abalone polypeptide extractive solution.
The antioxidant activity of the abalone polypeptide extract liquid of example 3 was measured by the same method as that described in example 1. The test results are as follows:
in the experiment, the control group 1 is an abalone polypeptide product after the enzymatic inactivation and centrifugation of the enzymatic hydrolysate in the step (2) in the example 3; the control group 2 is the abalone polypeptide product fermented by adding only candida utilis in the aerobic fermentation in the example 3, and the rest is the same as the example 3; the control 3 group was the abalone polypeptide product fermented by adding only bacillus subtilis in the aerobic fermentation in example 3, and the rest was the same as in example 3.
The above embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the present invention as set forth in the appended claims.
Claims (10)
1. A method for improving the antioxidant capacity of an abalone biological product is characterized in that the preparation method comprises the following steps:
dissolving: pulverizing abalone viscera protein dry powder, adding water, stirring to dissolve completely;
enzymolysis: adding compound protease with the mass ratio of 2-3% of abalone protein into the dissolved abalone protein, and performing enzymolysis for 4-8 hours at the temperature of 40-60 ℃;
aerobic fermentation: adding a compound microbial agent with the mass ratio of 2-3% of the enzymolysis liquid into the enzymolysis liquid after enzymolysis, and performing aerobic fermentation for 6-12 h at the temperature of 28-35 ℃;
anaerobic fermentation: after the aerobic fermentation is finished, adding 1-2% of lactobacillus powder, and carrying out anaerobic fermentation for 8-16 h at the temperature of 28-35 ℃;
and (3) filtering: performing coarse filtration to remove fermentation thalli;
enzyme deactivation: inactivating enzyme at 100 deg.C for 10 min;
centrifuging: centrifuging at 10000r/min, and collecting the supernatant as abalone polypeptide extractive solution.
2. The method for improving the antioxidant capacity of abalone bioproducts according to claim 1, wherein the optimal water addition ratio in step (1) is abalone visceral protein: water =1: 8.
3. The method for improving the antioxidant capacity of the abalone biological product according to claim 1, wherein chitosan is added to the abalone viscera protein during the dissolution process, wherein the mass ratio of chitosan to abalone protein is 2-3%.
4. The method for improving the antioxidant capacity of the abalone biological product, according to claim 1, characterized in that in step (2), the compound protease is a combination of papain and neutral protease, and the mass ratio is 2: 1.
5. The method for improving the antioxidant capacity of the abalone biological product, according to claim 1, characterized in that the compound microbial agent in step (3) is a mixed microbial agent of candida utilis and bacillus subtilis, and the mass ratio of the compound microbial agent to the bacillus subtilis is 2: 3.
6. The method for improving the antioxidant capacity of the abalone biological product according to claim 1, wherein the ventilation rate of the aerobic fermentation process in the step (3) is 0.1-0.2 m when the aerobic fermentation process is performed in a fermentation tank3/m3And min, and controlling the stirring speed to be 30-60 r/min.
7. The method for improving the antioxidant capacity of abalone bioproducts as claimed in claim 1, wherein the candida utilis seed solution is prepared by the following steps: under the aseptic operation condition, two rings of strains are picked from the inclined plane of the yeast YDP by using an inoculating ring, inoculated into a 500mL triangular flask containing 300mLPDA liquid culture medium, subjected to shaking culture at the temperature of between 28 and 32 ℃ and at the speed of 140r/min for 24 hours, and then inoculated into a seed tank for expanded culture according to the process to prepare the yeast seed liquid, wherein the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL。
8. The method for improving the antioxidant capacity of an abalone biologic according to claim 1, characterized in that the bacillus subtilis seed solution is prepared by the following steps: under the aseptic condition, LB culture medium is adopted to culture for 24 hours at the temperature of 28-32 ℃ to prepare liquid bacillus subtilis seed liquid, and the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL。
9. An abalone polypeptide produced by the process of claims 1-8.
10. Use of an abalone polypeptide according to claim 9 in health care, food, medicine.
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| CN113331367A (en) * | 2021-06-30 | 2021-09-03 | 百珍堂生物科技(浙江)有限公司 | Abalone viscera treatment method, fermentation treatment method and preparation method thereof |
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| CN113331367A (en) * | 2021-06-30 | 2021-09-03 | 百珍堂生物科技(浙江)有限公司 | Abalone viscera treatment method, fermentation treatment method and preparation method thereof |
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