阅读说明：本技术 一种发酵酶解提高河豚鱼皮抗氧化能力的工艺 (Process for improving oxidation resistance of globefish skin through fermentation and enzymolysis ) 是由 杨庆利 丁加玲 王冠丹 于 2019-10-11 设计创作，主要内容包括：本发明公开了一种发酵酶解提高河豚鱼皮抗氧化能力的工艺,属于海洋生物技术领域。本发明以河豚鱼皮为原料,集成现代生物分离技术、生物酶解技术、混菌微生物发酵技术,研制开发出高体外抗氧化能力的河豚鱼皮多肽生物制品,其中混菌好氧发酵菌种主要包括黑曲霉和枯草芽孢杆菌。所得河豚鱼皮多肽具有较强的抗氧化能力,提取液可根据应用途径做浓缩、吸附干燥、醇析或树脂纯化等处理,满足在保健、食品、医药等领域的高效利用。该工艺技术简单高效、所得产品抗氧化能力和自由基清除能力强,产品稳定性强,生产成本低,易于工业化规模生产。(The invention discloses a process for improving the oxidation resistance of globefish skin through fermentation and enzymolysis, and belongs to the technical field of marine organisms. The invention takes globefish skin as a raw material, integrates the modern bioseparation technology, the biological enzymolysis technology and the mixed bacteria microbial fermentation technology, and develops the globefish skin polypeptide biological product with high in-vitro oxidation resistance, wherein the mixed bacteria aerobic fermentation strain mainly comprises aspergillus niger and bacillus subtilis. The obtained globefish skin 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.)

1. A process for improving the oxidation resistance of globefish skin by fermentation and enzymolysis is characterized by comprising the following steps:

(1) homogenizing: pulverizing globefish skin, adding water, and homogenizing;

(2) enzymolysis: adding 3000-5000U/g of compound protease into the homogenate, and performing enzymolysis for 4 hours at the temperature of 40-60 ℃;

(3) aerobic fermentation: adding a compound microbial agent with the mass ratio of 3-5% of the enzymolysis liquid into the enzymolysis liquid after enzymolysis, and performing aerobic fermentation for 12-24 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 to obtain supernatant as Fugu ocellatus skin polypeptide extractive solution.

2. The process for improving the oxidation resistance of the globefish skin by fermentation and enzymolysis according to claim 1, wherein the optimal water adding proportion in the step (1) is that the globefish skin: water =1: 8.

3. The process for improving the oxidation resistance of globefish skin through fermentation and enzymolysis according to claim 1, wherein honeysuckle flower with the globefish skin protein mass ratio of 1-2% is added in the homogenizing process.

4. The process for improving the oxidation resistance of the globefish skin through fermentation and enzymolysis according to claim 1, wherein the compound protease is a combination of neutral protease, papain, pepsin, trypsin and flavourzyme, and the mass ratio of the compound protease to the flavourzyme is 2: 1.

5. The process for improving the oxidation resistance of the globefish skin by fermentation and enzymolysis as claimed in claim 4, wherein the compound protease is a combination of neutral protease and flavourzyme, and the mass ratio of the neutral protease to the flavourzyme is 2: 1.

6. The process for improving the oxidation resistance of the globefish skin through fermentation and enzymolysis according to claim 1, wherein the compound microbial agent is a mixed microbial agent of aspergillus niger and bacillus subtilis, and the mass ratio of the compound microbial agent to the bacillus subtilis is 3: 1.

7. The process for improving the oxidation resistance of globefish skin through fermentation and enzymolysis as claimed in claim 1, wherein the ventilation rate is 0.1-0.2 m when the aerobic fermentation process is carried out in a fermentation tank³/m³Min, and the stirring speed is controlled at 300 r/min.

8. The process for improving the oxidation resistance of globefish skin by fermentation and enzymolysis according to claim 1, wherein the preparation method of the aspergillus niger seed liquid comprises the following steps: culturing for 24h at 35-40 deg.C in potato culture medium under aseptic condition to obtain Aspergillus niger seed liquid with effective viable count not less than 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。

9. A puffer fish skin polypeptide prepared according to the process of claims 1-8.

10. The puffer fish skin polypeptide of claim 9 for use in health care, food, and medicine.

Technical Field

The invention relates to a process for improving the oxidation resistance of globefish skin by fermentation and enzymolysis, in particular to a globefish skin polypeptide biological product with high oxidation resistance prepared by combining microbial synergistic fermentation and enzymolysis processes, belonging to the technical field of marine organisms.

Background

According to the history, the eating of globefish in China has been recorded for more than four thousand years, but ancient people are commendable and dispute on the eating of globefish, and not only are "sweet and far-win west milk", Wuwang has not known "praise on the delicious taste of globefish meat in the current year, but also" boil is prairie and disappointed, and enters the throat is "a tom 38104". Because the globefish contains tetrodotoxin, people who have lost their lives because of eating the globefish every year, so that the globefish is fasted in China. In 2016, the nation forbids the red-fin eastern globefish and the dark-stripe eastern globefish, and forbids the globefish with the age of 26 years to be served on the dining table of the consumer, which undoubtedly promotes the development of globefish breeding industry and enables people to be full of luck. With the opening of globefish market and the development of the industry of artificially culturing nontoxic globefish, the demand of the globefish in China will gradually increase, and the research on the nutritive value of the globefish and the comprehensive utilization of the by-products thereof has important significance for the development of the culture and processing industry of the globefish in the future

Globefish skin contains abundant collagen, and the collagen has the effects of resisting oxidation, resisting tumor, reducing blood pressure, promoting wound healing, beautifying and the like, and is widely used in the industries of food, cosmetics and medicines. At present, collagen derived from mammals such as pigs and cows is threatened by various infectious diseases such as foot-and-mouth disease and mad cow disease, and fish skin and fish scale collagen are better sources for replacing the same. The globefish skin has higher collagen content and higher extraction rate than other mammal collagen. The content of protein in the skin of Takifugu rubripes determined by Junfeng is about 97.30% of the dry weight of the skin, and the content of collagen is 90% of the total protein content. Extracting collagen peptide from globefish skin, and measuring its free radicals OH, DPPH, and O₂ ^—Discovery of the scavenging ability ofThe globefish skin collagen peptide has strong oxidation resistance.

At present, the research and utilization of the globefish in China are still in the primary stage, and with the gradual release of the national limitation on the globefish, the research on the globefish is more comprehensive. In the prior art, the research on the application of the globefish skin polypeptide in food is less, for example, patent 201510951376.5 discloses a production process of a compound fermentation type globefish skin health beverage, wherein the globefish skin, sea cucumber, medlar, hawthorn, yam, rhodiola rosea, stevia rebaudiana, polygonum multiflorum, acanthopanax, ginseng, cistanche salsa, kudzu vine root and liquorice are selected as raw materials to produce the globefish skin health beverage through the compound fermentation production process, but the patent does not disclose how to prepare the high-purity globefish skin polypeptide; patent 201410078719.7 discloses a chitosan oligosaccharide-based modified fish skin protein polypeptide compound with anti-freezing function, which is mainly prepared by grinding fish skin into slurry and centrifuging to remove fat; then, carrying out ultrasonic treatment and enzymolysis by using compound enzyme, centrifuging an enzymolysis product, and taking supernatant; and (3) ultrafiltering the supernatant in two steps to obtain the fish skin protein polypeptide with the molecular weight of 5000-8000 daltons, and reacting with chitosan oligosaccharide to obtain the fish skin protein polypeptide compound. Comparing the above documents, it can be found that the preparation of the globefish skin polypeptide mostly adopts a complex enzymolysis method, the application of a key technology of complex microbial fermentation is lacked, and especially the research of lack of improvement of the trophism of the globefish skin polypeptide becomes a key bottleneck restricting the industrial development.

Disclosure of Invention

In order to overcome the defects of the prior art and meet the requirements of high-efficiency extraction and high oxidation resistance application of the globefish skin polypeptide, the application provides a process for improving the oxidation resistance of the globefish skin by fermentation and enzymolysis, integrates a modern bioseparation technology, a biological enzymolysis technology and a mixed bacteria microbial fermentation technology, develops and develops a globefish skin polypeptide biological product with high in-vitro oxidation resistance, meets the application requirements of fields such as health care, medical treatment, food and the like on the globefish skin polypeptide, and has the advantages of simple and high-efficiency process, strong oxidation resistance and free radical scavenging capacity of the obtained product, low production cost and easiness in industrial scale production.

The invention realizes the technical effects through the following technical scheme:

a process for improving the oxidation resistance of globefish skin by fermentation and enzymolysis is characterized by comprising the following steps:

(1) homogenizing: pulverizing globefish skin, adding water, and homogenizing;

(2) enzymolysis: adding 3000-5000U/g of compound protease into the homogenate, and performing enzymolysis for 4 hours at the temperature of 40-60 ℃;

(3) aerobic fermentation: adding a compound microbial agent with the mass ratio of 3-5% of the enzymolysis liquid into the enzymolysis liquid after enzymolysis, and performing aerobic fermentation for 12-24 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 to obtain supernatant as Fugu ocellatus skin polypeptide extractive solution.

The optimal water adding proportion in the step (1) is that the globefish skin: water =1: 8.

Preferably, honeysuckle with the globefish skin protein mass ratio of 1-2% is added in the globefish skin homogenizing process.

The compound protease in the step (2) is a combination of one of neutral protease, papain, pepsin and trypsin and flavourzyme, and the mass ratio of the compound protease to the flavourzyme is 2: 1.

Most preferably, the compound protease is a combination of neutral protease and flavourzyme, and the mass ratio of the neutral protease to the flavourzyme is 2: 1.

The compound microbial agent in the step (3) is a mixed microbial agent of aspergillus niger and bacillus subtilis, and the mass ratio of the compound microbial agent to the bacillus subtilis is 3: 1.

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/m³Min, and the stirring speed is controlled at 300 r/min.

Preparation method of aspergillus niger seed liquidThe method comprises the following steps: culturing for 24h at 35-40 deg.C in potato culture medium under aseptic condition to obtain Aspergillus niger seed liquid with effective viable count not less than 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 process for improving the oxidation resistance of globefish skin by fermentation and enzymolysis, the obtained globefish skin polypeptide has stronger 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 invention provides a process for improving the oxidation resistance of globefish skin by fermentation and enzymolysis, which has the following remarkable advantages compared with the prior art:

(1) according to the method, the globefish skin is used as a raw material, and a modern bioseparation technology, a biological enzymolysis technology and a mixed-bacterium microbial fermentation technology are integrated, so that the oxidation resistance of the globefish skin is remarkably improved; more importantly, the application creatively discovers that the oxidation resistance of the globefish skin polypeptide prepared by the enzymolysis and fermentation method can be obviously improved by adding a certain proportion of honeysuckle into the globefish skin protein solution, and the reason is analyzed, although the heat-clearing and detoxifying Chinese herbal medicine honeysuckle has a certain inhibition effect on microorganisms, the active substances in the honeysuckle and the globefish skin polypeptide generate complex chemical reaction, so that the inhibition effect of an inhibition factor in the enzymolysis and combined fermentation process in the globefish skin is broken through, and a little inhibition effect still exists on a single enzymolysis or fermentation process; the test result also shows that the tonifying traditional Chinese medicines (such as codonopsis pilosula and ginseng) and the like can not improve the oxidation resistance of the globefish skin polypeptide;

(2) the method utilizes aerobic bacteria (Aspergillus niger and Bacillus subtilis) to jointly ferment the globefish skin to extract the globefish skin polypeptide, makes full use of the synergistic effect among microorganisms, and improves the extraction rate and the in-vitro oxidation resistance of the globefish skin polypeptide, wherein: aspergillus niger can generate various enzymes for degrading cell walls in the fermentation process, so that more functional active substances such as polypeptide and the like are released from the cell walls, and the in-vitro antioxidant capacity of the globefish skin polypeptide 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; the synergistic effect of the aspergillus niger and the bacillus subtilis obviously improves the oxidation resistance of the product;

(3) according to the invention, lactobacillus is adopted for anaerobic fermentation, so that the stability of the globefish skin polypeptide is improved, test results show that the temperature and time have little influence on the oxidation resistance of the globefish skin protein hydrolysis peptide within the range of 20-80 ℃, the oxidation resistance begins to decrease along with the increase of the temperature and time when the temperature exceeds 100 ℃, and the stability is higher than the technical level reported at present;

(4) according to the invention, the oxidation resistance of the globefish skin polypeptide is improved by adopting a fermentation method, and the globefish skin is directly used as a raw material under the process condition of the application, so that non-collagen is removed without extracting collagen by water washing, and the production water is saved; the invention has mild fermentation process conditions, low production cost and easy large-scale production.

Detailed Description

In the present application, the activation method of Aspergillus niger and Bacillus subtilis is as follows:

(1) the preparation method of the aspergillus niger seed liquid comprises the following steps: culturing for 24h at 35-40 deg.C in potato culture medium under aseptic condition to obtain Aspergillus niger seed liquid with effective viable count not less than 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。