CN105624242A - Method for preparing active peptide through high-temperature fermentation of aquatic protein - Google Patents
Method for preparing active peptide through high-temperature fermentation of aquatic protein Download PDFInfo
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Abstract
The invention discloses a method for preparing active peptide through high-temperature fermentation of aquatic protein. The method mainly includes the steps that the fresh aquatic protein is minced; the minced aquatic protein is added to a fermentation tank, nutrients and water are added, and the mixture is mixed to be uniform to serve as a fermentation culture medium; the fermentation tank is inoculated with Bacillus licheniformis HL-3 capable of generating high-temperature-resistant protease for aerobic fermentation for 42 h-48 h at the temperature of 55 DEG C-60 DEG C; after fermentation is completed, fermentation liquor is subjected to filtration, ultra-filtration and nano-filtration in sequence, concentrated liquor is obtained, and the finished active peptide is obtained after freeze drying of the concentrated liquor. According to the technology, high-temperature treatment is not needed, biological activity of functional ingredients in the prepared functional peptide and the aquatic protein is better kept, preparation procedures of the active peptide are simplified, the production cost of the active peptide is reduced, and good application prospects are achieved.
Description
Technical field
The present invention relates to a kind of method utilizing aquatic product protein thermophilic fermentation to prepare bioactive peptide, belong to the field of processing of aquatic products and utilization.
Background technology
The little peptide of product after protein digestion not only readily digested, absorb, but also there is hypertension, anti-cholesterol, antithrombus formation, improve lipid metabolism, strengthen human body physical agility, help to recover tired, promote the absorption of calcium phosphorus and other trace element, promote brain development, improve memory, strengthen scavenger cell and B cell vigor, strengthen immunologic function, protect epidermic cell, prevent melanin deposition, eliminate the effects such as interior free yl.
About the preparation of aquatic product protein bioactive peptide, domestic carry out big quantifier elimination. At present, the preparation method of aquatic product protein bioactive peptide mainly contains the method these four kinds that self-dissolving enzymolysis, enzymolysis process, liquid fermentation method and solid state fermentation combine with liquid enzymolysis. A kind of method that patent application 200810195646.4 technique of bioactive micro peptide " endogenous enzyme prepare " discloses endogenous enzyme self-dissolving enzymolysis utilized in marine low-value fish body prepares the method for aquatic product protein bioactive micro peptide. Patent application 201410145196.3 " a kind of method preparing scallop body bioactive peptide " discloses a kind of method that enzymolysis process prepares aquatic product protein bioactive peptide. Patent application CN102028091A " a kind of method of preparing low molecular weight fish peptide through Bacillus natto fermentation method " discloses a kind of method that solution fermentation prepares fish protein peptide, and patent application 201210539941.3 " a kind of by fish protein peptide and the production method of protein peptide that forms with soybean peptides " discloses a kind of combining with liquid enzymolysis by solid state fermentation and prepares the method for aquatic product protein bioactive peptide.
There is following defect in existing aquatic product protein bioactive peptide technology of preparing. First, existing aquatic product protein bioactive peptide technology of preparing has the process of pyroprocessing. Above technology of preparing there is high temperature go out this operation of enzyme; The method raw material of fermentable needs sterilizing. These pyroprocessing above, can in the biologic activity of the contained functional ingredient of functional peptides or aquatic product protein itself to a certain degree weakening preparation; Pyroprocessing can significantly strengthen Maillard reaction in addition so that enzymolysis solution color becomes dark, for the finished product course of processing adds burden. Secondly, the method for additional enzyme enzymolysis needs added proteins enzyme, adds cost; Self-dissolving enzymolysis, due to the deficiency of endogenous enzyme, causes enzymolysis not thorough. In addition, although microbe fermentation method is without added proteins enzyme, but raw material needs sterilizing to cause endogenous enzyme inactivation, can not fully effectively utilize endogenous enzyme.
Summary of the invention
For the problem existing for existing aquatic product protein bioactive peptide technology of preparing. The main purpose of the present invention is to provide a kind of method preparing aquatic product protein bioactive peptide of low temperature, to retain the functional peptides of preparation and the biologic activity of the contained functional ingredient of aquatic product protein itself better. It is a further object of the present invention to provide a kind of method that fermentation method prepares bioactive peptide so that fermentable and endogenous enzyme enzymolysis process can organically combine. The present invention also has an object to be the preparation section simplifying bioactive peptide, reduces its production cost.
In order to solve the problem, the technical solution adopted in the present invention is as follows: a kind of method utilizing aquatic product protein thermophilic fermentation to prepare bioactive peptide, comprises the steps:
(1) in the LB liquid nutrient medium cooled after Bacillus licheniformis (Bacilluslicheniformis) HL-3 that can produce thermostable proteinase of slant preservation is linked into sterilizing, in 55 DEG C, 150r/min cultivate 12h, be prepared into Bacillus licheniformis (Bacilluslicheniformis) HL-3 seed liquor for subsequent use;
(2) by for subsequent use after the rubbing of fresh aquatic product protein;
(3) aquatic product protein of rubbing step (2) obtained is put in fermentor tank, and adds water and nutrition in fermentor tank, obtains fermention medium after mixed even;
(4) fermentation culture obtained toward step (3) accesses Bacillus licheniformis (Bacilluslicheniformis) the HL-3 seed liquor prepared by step (1);
(5) control of liquid state fermentation process: leavening temperature is 55 DEG C��60 DEG C, and sterile air intake is 0.5vvm��2.5vvm, and the rotating speed of fermentor tank is 150rpm��350rpm, and fermentation time is 42h��48h;
(6) product handling system: undertaken the fermented liquid that step (5) obtains filtering and remove precipitation and obtain supernatant liquor; The ultrafiltration removal macro-molecular protein that supernatant liquor is the ultra-filtration membrane of 5000 dalton by molecular weight cut-off obtains permeate; The nanofiltration removal amino acid that then permeate is the nanofiltration membrane of 200 dalton by molecular weight cut-off obtains concentrated solution; Concentrated solution prepares bioactive peptide finished product through lyophilize.
Bacillus licheniformis (Bacilluslicheniformis) HL-3 that can produce thermostable proteinase used in described step (1) is preserved in Guangdong Province's Culture Collection on January 18th, 2016, deposit number is GDMCCNo:60003, and preservation address is Guangdong Microbes Inst, compound 5th floors, No. 59 building, No. 100, Xianlie Middle Road, Guangzhou City.
Aquatic product protein in described step (2) is the one in leftovers of tilapia, krill, shrimp head, scallop body, cod tankage, squid tankage, Harengulazunasi (Bleeker) tankage, black carp tankage, silver carp tankage, grass carp tankage and stripped tuna tankage.
In described step (3), the add-on of aquatic product protein accounts for fermention medium total mass per-cent in the quality of the aquatic product protein added, and is 10%��30%. Described step (3) Middle nutrition thing is the mixture of glucose, food-grade calcium chloride, food grade iron protochloride and food grade phosphoric acid hydrogen two potassium composition. Nutraceutical add-on in described step (3) accounts for fermention medium total mass per-cent in the nutraceutical quality added, it is respectively: glucose 0.01%��5%, food-grade calcium chloride 0.01%��5%, food grade iron protochloride 0.01%��5%, food grade phosphoric acid hydrogen two potassium 0.01%��5%.
In described step (4), the inoculum size of Bacillus licheniformis (Bacilluslicheniformis) HL-3 seed liquor accounts for fermention medium total mass per-cent in the quality of the seed liquor accessed, and is 1%��15%.
Compared with prior art, the present invention has the following advantages.
(1) whole preparation process does not have pyroprocessing, and this not only can retain the biologic activity of the contained functional ingredient of aquatic product protein itself better, such as, astaxanthin in shrimp head and krill; And the biologic activity of prepared functional peptides can also be retained better. The present patent application adopts high temperature resistant the lichen bacillus ferments, the growth of pathogenic bacterium, spoilage organism and other miscellaneous bacteria can be suppressed under the high temperature conditions, therefore in this technology substratum without the need to sterilizing, this also existing employing normal temperature bacterial classification carry out fermentation method and prepare bioactive peptide and cannot accomplish. In addition, this technology adopts ultra-filtration membrane to remove the proteolytic enzyme in enzymolysis solution, it is not necessary to adopt the mode of high temperature to carry out going out enzyme.
(2) simplify operation, reduce cost. Owing to the present patent application does not have high temperature to go out the process of enzyme, this not only reduces energy consumption, but also can greatly reduce the Maillard reaction of enzymolysis solution, so the color of fermented liquid is more shallow. And follow-up membrane sepn has certain decolorizing effect, therefore this technology without the need to this program of decolouring, can simplify operation, reduce cost.
(3) perfect adaptation of endogenous enzyme enzymolysis and fermentable. In existing fermentation technique, raw material all needs sterilizing, and this not only adds energy consumption, and makes again the endogenous enzyme inactivation of aquatic product protein. And raw material is without the need to sterilizing in this technology, remain the activity of endogenous enzyme in aquatic product protein. Further, the temperature of fermentable is consistent with the peak enzymolysis-ability temperature of endogenous enzyme so that endogenous enzyme enzymolysis is together with fermentable energy perfect adaptation. Just owing to the present invention making full use of the enzymolysis of endogenous enzyme so that the present invention is without the need to separately adding proteolytic enzyme.
(4) preparation cost not only will lower than existing fermentation technique, and ferment effect is also better than existing fermentation technique. Compared with the technology that existing employing fermentation method prepares bioactive peptide, the present patent application raw material without the need to extinction, capable of reducing energy consumption and cost. The technical scheme provided due to the present patent application is become extinct without the need to high temperature, the enzyme remaining aquatic product protein endogenous proteinase is lived, this is that existing employing normal temperature fermentation bacterial classification is prepared bioactive peptide and cannot be accomplished, in addition, at high temperature the activity of enzyme is stronger, therefore, this technology adopts high temperature resistant bacterial classification to ferment, and the degree of hydrolysis of aquatic product protein is higher than existing normal temperature strain fermentation technology.
The present invention provides a kind of method utilizing aquatic product protein thermophilic fermentation to prepare bioactive peptide. The technology of the present invention action condition is gentle, remains the functional peptides of preparation and the biologic activity of the contained functional ingredient of aquatic product protein itself more, and simplifies operation prepared by bioactive peptide and the production cost reducing bioactive peptide, has good application prospect.
Embodiment
In order to understand the present invention better, with regard to the selection of the kind of aquatic product protein, fermentation parameter and fermentation mode, the invention will be further described in conjunction with the embodiments below, but protection scope of the present invention is not limited to this.
Embodiment 1: in the LB liquid nutrient medium cooled after Bacillus licheniformis (Bacilluslicheniformis) HL-3 that can produce thermostable proteinase of slant preservation is linked into sterilizing, in 55 DEG C, 150r/min cultivate 12h, be prepared into Bacillus licheniformis (Bacilluslicheniformis) HL-3 seed liquor for subsequent use. Fresh krill is rubbed rear for subsequent use. 20 kilograms of krills rubbed and 100 kg of water are joined in fermentor tank, and in fermentor tank, adds 300 grams of glucose, 300 grams of food-grade calcium chlorides, 300 grams of food grade iron protochlorides and 500 grams of food grade phosphoric acid hydrogen two potassium, obtain fermention medium; 8 kilograms of Bacillus licheniformis (Bacilluslicheniformis) HL-3 seed liquor are added in the fermention medium in fermentor tank; Leavening temperature is 55 DEG C, and sterile air intake is 1.5vvm��2.5vvm, and the rotating speed of fermentor tank is 250rpm��350rpm, and fermentation time is 42h; Fermentation ends secondary fermentation liquid is removed precipitation by filtering and is obtained supernatant liquor; The ultrafiltration removal macro-molecular protein that supernatant liquor is the ultra-filtration membrane of 5000 dalton by molecular weight cut-off obtains permeate; The nanofiltration removal amino acid that then permeate is the nanofiltration membrane of 200 dalton by molecular weight cut-off obtains concentrated solution; Concentrated solution obtains bioactive peptide finished product through lyophilize.
Embodiment 2: in the LB liquid nutrient medium cooled after Bacillus licheniformis (Bacilluslicheniformis) HL-3 that can produce thermostable proteinase of slant preservation is linked into sterilizing, in 55 DEG C, 150r/min cultivate 12h, be prepared into Bacillus licheniformis (Bacilluslicheniformis) HL-3 seed liquor for subsequent use. Fresh shrimp head is rubbed rear for subsequent use. 22 kilograms of shrimp heads rubbed and 100 kg of water are joined in fermentor tank, and in fermentor tank, adds 200 grams of glucose, 400 grams of food-grade calcium chlorides, 400 grams of food grade iron protochlorides and 600 grams of food grade phosphoric acid hydrogen two potassium, obtain fermention medium; 9 kilograms of Bacillus licheniformis (Bacilluslicheniformis) HL-3 seed liquor are added in the fermention medium in fermentor tank; Leavening temperature is 60 DEG C, and sterile air intake is 1.5vvm��2.5vvm, and the rotating speed of fermentor tank is 250rpm��350rpm, and fermentation time is 48h; Fermentation ends secondary fermentation liquid is removed precipitation by filtering and is obtained supernatant liquor; The ultrafiltration removal macro-molecular protein that supernatant liquor is the ultra-filtration membrane of 5000 dalton by molecular weight cut-off obtains permeate; The nanofiltration removal amino acid that then permeate is the nanofiltration membrane of 200 dalton by molecular weight cut-off obtains concentrated solution; Concentrated solution obtains bioactive peptide finished product through lyophilize.
Embodiment 3: in the LB liquid nutrient medium cooled after Bacillus licheniformis (Bacilluslicheniformis) HL-3 that can produce thermostable proteinase of slant preservation is linked into sterilizing, in 55 DEG C, 150r/min cultivate 12h, be prepared into Bacillus licheniformis (Bacilluslicheniformis) HL-3 seed liquor for subsequent use. Fresh leftovers of tilapia is rubbed rear for subsequent use. 25 kilograms of leftovers of tilapia rubbed and 100 kg of water are joined in fermentor tank, and in fermentor tank, adds 450 grams of glucose, 450 grams of food-grade calcium chlorides, 350 grams of food grade iron protochlorides and 580 grams of food grade phosphoric acid hydrogen two potassium, obtain fermention medium; 10 kilograms of Bacillus licheniformis (Bacilluslicheniformis) HL-3 seed liquor are added in the fermention medium in fermentor tank; Leavening temperature is 57 DEG C, and sterile air intake is 1.5vvm��2.5vvm, and the rotating speed of fermentor tank is 250rpm��350rpm, and fermentation time is 45h; Fermentation ends secondary fermentation liquid is removed precipitation by filtering and is obtained supernatant liquor; The ultrafiltration removal macro-molecular protein that supernatant liquor is the ultra-filtration membrane of 5000 dalton by molecular weight cut-off obtains permeate; The nanofiltration removal amino acid that then permeate is the nanofiltration membrane of 200 dalton by molecular weight cut-off obtains concentrated solution; Concentrated solution obtains bioactive peptide finished product through lyophilize.
Claims (7)
1. one kind utilizes the method that aquatic product protein thermophilic fermentation prepares bioactive peptide, it is characterised in that: the method comprises the steps:
(1) in the LB liquid nutrient medium cooled after Bacillus licheniformis (Bacilluslicheniformis) HL-3 that can produce thermostable proteinase of slant preservation is linked into sterilizing, in 55 DEG C, 150r/min cultivate 12h, be prepared into Bacillus licheniformis (Bacilluslicheniformis) HL-3 seed liquor for subsequent use;
(2) by for subsequent use after the rubbing of fresh aquatic product protein;
(3) aquatic product protein of rubbing step (2) obtained is put in fermentor tank, and adds water and nutrition in fermentor tank, obtains fermention medium after mixed even;
(4) fermentation culture obtained toward step (3) accesses Bacillus licheniformis (Bacilluslicheniformis) the HL-3 seed liquor prepared by step (1);
(5) control of liquid state fermentation process: leavening temperature is 55 DEG C��60 DEG C, and sterile air intake is 0.5vvm��2.5vvm, and the rotating speed of fermentor tank is 150rpm��350rpm, and fermentation time is 42h��48h;
(6) product handling system: undertaken the fermented liquid that step (5) obtains filtering and remove precipitation and obtain supernatant liquor; The ultrafiltration removal macro-molecular protein that supernatant liquor is the ultra-filtration membrane of 5000 dalton by molecular weight cut-off obtains permeate; The nanofiltration removal amino acid that then permeate is the nanofiltration membrane of 200 dalton by molecular weight cut-off obtains concentrated solution; Concentrated solution prepares bioactive peptide finished product through lyophilize.
2. a kind of method utilizing aquatic product protein thermophilic fermentation to prepare bioactive peptide according to claim 1, it is characterised in that: Bacillus licheniformis (Bacilluslicheniformis) HL-3 that can produce thermostable proteinase used in described step (1) is GDMCCNo:60003 at the deposit number of Guangdong Province's Culture Collection.
3. a kind of method utilizing aquatic product protein thermophilic fermentation to prepare bioactive peptide according to claim 1, it is characterised in that: the aquatic product protein in described step (2) is the one in leftovers of tilapia, krill, shrimp head, scallop body, cod tankage, squid tankage, Harengulazunasi (Bleeker) tankage, black carp tankage, silver carp tankage, grass carp tankage and stripped tuna tankage.
4. a kind of method utilizing aquatic product protein thermophilic fermentation to prepare bioactive peptide according to claim 1, it is characterized in that: in described step (3), the add-on of aquatic product protein accounts for fermention medium total mass per-cent in the quality of the aquatic product protein added, it is 10%��30%.
5. a kind of method utilizing aquatic product protein thermophilic fermentation to prepare bioactive peptide according to claim 1, it is characterised in that: described step (3) Middle nutrition thing is the mixture of glucose, food-grade calcium chloride, food grade iron protochloride and food grade phosphoric acid hydrogen two potassium composition.
6. a kind of method utilizing aquatic product protein thermophilic fermentation to prepare bioactive peptide according to claim 1, it is characterized in that: the nutraceutical add-on in described step (3) accounts for fermention medium total mass per-cent in the nutraceutical quality added, it is respectively: glucose 0.01%��5%, food-grade calcium chloride 0.01%��5%, food grade iron protochloride 0.01%��5%, food grade phosphoric acid hydrogen two potassium 0.01%��5%.
7. a kind of method utilizing aquatic product protein thermophilic fermentation to prepare bioactive peptide according to claim 1, it is characterized in that: in described step (4), the inoculum size of Bacillus licheniformis (Bacilluslicheniformis) HL-3 seed liquor accounts for fermention medium total mass per-cent in the quality of the seed liquor accessed, it is 1%��15%.
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Cited By (2)
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| CN108771194A (en) * | 2018-07-02 | 2018-11-09 | 广东海洋大学 | A method of assist enzymolysis aquatic product protein to prepare seafood seasoning packet using high temperature resistant bacillus licheniformis |
| CN112852909A (en) * | 2021-01-25 | 2021-05-28 | 韩山师范学院 | Method for preparing ACE inhibitory peptide by solid-state fermentation of shrimp heads with mixed strains |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108771194A (en) * | 2018-07-02 | 2018-11-09 | 广东海洋大学 | A method of assist enzymolysis aquatic product protein to prepare seafood seasoning packet using high temperature resistant bacillus licheniformis |
| CN112852909A (en) * | 2021-01-25 | 2021-05-28 | 韩山师范学院 | Method for preparing ACE inhibitory peptide by solid-state fermentation of shrimp heads with mixed strains |
| CN112852909B (en) * | 2021-01-25 | 2023-05-09 | 韩山师范学院 | Method for preparing ACE (angiotensin converting enzyme) inhibitory peptide by solid-state fermentation of shrimp heads by mixed strains |
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