CN105624242A - Method for preparing active peptide through high-temperature fermentation of aquatic protein - Google Patents

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

A method for preparing active peptides by high-temperature fermentation of aquatic proteins

technical field

The invention relates to a method for preparing active peptides through high-temperature fermentation of aquatic proteins, belonging to the field of processing and utilization of aquatic products.

Background technique

The small peptides produced by protein enzymatic hydrolysis are not only easy to digest and absorb, but also have anti-hypertensive, anti-cholesterol, anti-thrombosis, improve lipid metabolism, enhance human physical fitness, help restore fatigue, promote calcium, phosphorus and other trace elements. Absorption, promote brain development, improve memory, enhance the vitality of macrophages and B cells, enhance immune function, protect epidermal cells, prevent melanin precipitation, eliminate free radicals in the body, etc.

A lot of research has been done in China on the preparation of aquatic protein active peptides. At present, the preparation methods of aquatic protein active peptides mainly include autolytic enzymatic hydrolysis, enzymatic hydrolysis, liquid fermentation and solid-state fermentation combined with liquid enzymatic hydrolysis. Patent application 200810195646.4 "Process of preparing small biologically active peptides with endogenous enzymes" discloses a method for preparing small biologically active peptides from aquatic proteins by using endogenous enzymes in marine low-value fish to autolyze and enzymolyze. Patent application 201410145196.3 "A method for preparing scallop skirt active peptide" discloses a method for preparing aquatic protein active peptide by enzymatic hydrolysis. Patent application CN102028091A "A Method for Preparing Low Molecular Fish Peptides by Bacteria Natto Fermentation" discloses a method for preparing fish protein peptides by liquid fermentation. Patent application 201210539941.3 "A protein composed of fish protein peptides and soybean peptides Peptide Production Method" discloses a method for preparing aquatic protein active peptides by combining solid-state fermentation and liquid-state enzymatic hydrolysis.

The following deficiencies exist in the existing aquatic protein active peptide preparation technology. First of all, the existing techniques for the preparation of active peptides from aquatic proteins all have a process of high temperature treatment. All of the above preparation technologies have the process of high temperature inactivation of enzymes; the raw materials of the microbial fermentation method need to be sterilized. The above high-temperature treatments will weaken the biological activity of the prepared functional peptides or the functional components contained in the aquatic protein itself to a certain extent; in addition, the high-temperature treatment will significantly enhance the Maillard reaction, making the color of the enzymatic hydrolyzate darker, which is suitable for the finished product processing process adds to the burden. Secondly, the method of enzymatic hydrolysis with external enzymes requires the addition of proteases, which increases the cost; autolytic enzymatic hydrolysis is incomplete due to the lack of endogenous enzymes. In addition, although the microbial fermentation method does not require external proteases, the raw materials need to be sterilized to inactivate the endogenous enzymes, and the endogenous enzymes cannot be fully and effectively utilized.

Contents of the invention

The invention aims at the problems existing in the existing preparation technology of aquatic protein active peptides. The main purpose of the present invention is to provide a low-temperature method for preparing active peptides from aquatic proteins, so as to better retain the biological activity of the prepared functional peptides and the functional components contained in the aquatic proteins themselves. Another object of the present invention is to provide a method for preparing active peptides by fermentation, so that microbial fermentation and endogenous enzyme enzymatic hydrolysis can be organically combined. Another purpose of the present invention is to simplify the preparation process of the active peptide and reduce its production cost.

In order to solve the above problems, the technical scheme adopted in the present invention is as follows: a method for preparing active peptides by high-temperature fermentation of aquatic proteins, comprising the following steps:

(1) Insert Bacillus licheniformis HL-3, which is capable of producing thermostable protease, preserved on the slant into the LB liquid medium cooled after sterilization, and culture it at 55°C and 150r/min for 12h to prepare licheniformis spores Bacillus licheniformis HL-3 seed liquid for use;

(2) Grind the fresh aquatic protein for later use;

(3) Put the minced aquatic protein prepared in step (2) into a fermenter, add water and nutrients into the fermenter, and mix well to obtain a fermentation medium;

(4) adding the Bacillus licheniformis HL-3 seed solution prepared in step (1) to the fermentation culture obtained in step (3);

(5) Control of the liquid fermentation process: the fermentation temperature is 55°C-60°C, the sterile air intake is 0.5vvm-2.5vvm, the rotation speed of the fermentation tank is 150rpm-350rpm, and the fermentation time is 42h-48h;

(6) Finished product treatment: filter the fermented liquid obtained in step (5) to remove sediment to obtain a supernatant; The permeated liquid is then filtered through a nanofiltration membrane with a molecular weight cut-off of 200 Daltons to remove amino acids to obtain a concentrated liquid; the concentrated liquid is freeze-dried to prepare a finished product of active peptide.

The Bacillus licheniformis HL-3 capable of producing thermostable protease used in the step (1) has been preserved in the Guangdong Provincial Microbial Culture Collection Center on January 18, 2016, and the preservation number is GDMCCNo: 60003. The address is Guangdong Institute of Microbiology, 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City.

The aquatic protein in the step (2) is tilapia leftovers, Antarctic krill, shrimp head, scallop skirt, cod leftovers, squid leftovers, herring leftovers, herring leftovers, silver carp leftovers , grass carp scraps and skipjack scraps.

The amount of aquatic protein added in the step (3) is 10% to 30% based on the mass percentage of the added aquatic protein in the total mass of the fermentation medium. The nutrient in the step (3) is a mixture composed of glucose, food-grade calcium chloride, food-grade ferrous chloride and food-grade dipotassium hydrogen phosphate. The amount of nutrients added in the step (3) is based on the weight of the added nutrients as a percentage of the total mass of the fermentation medium, which are: 0.01% to 5% of glucose, 0.01% to 5% of food grade calcium chloride, Food grade ferrous chloride 0.01% ~ 5%, food grade dipotassium hydrogen phosphate 0.01% ~ 5%.

The inoculum amount of Bacillus licheniformis HL-3 seed liquid in the step (4) is 1%-15% based on the weight of the inserted seed liquid in the total mass percentage of the fermentation medium.

Compared with the prior art, the present invention has the following advantages.

(1) There is no high-temperature treatment in the whole preparation process, which not only can better retain the biological activity of the functional components contained in the aquatic protein itself, such as astaxanthin in shrimp heads and Antarctic krill; The biological activity of the prepared functional peptide is retained. The application of the present invention uses high-temperature-resistant Bacillus licheniformis to ferment, which can inhibit the growth of pathogenic bacteria, spoilage bacteria and other miscellaneous bacteria under high-temperature conditions, so the culture medium in this technology does not need to be sterilized, which is also the existing normal-temperature bacteria. It is impossible to prepare active peptides by fermentation. In addition, this technology uses ultrafiltration membranes to remove proteases in the enzymatic hydrolysis solution, without the need to use high temperature methods to inactivate enzymes.

(2) The process is simplified and the cost is reduced. Because the application of the present invention does not have the process of high-temperature enzyme elimination, this not only reduces energy consumption, but also greatly reduces the Maillard reaction of the enzymolysis solution, so the color of the fermentation solution is relatively light. The subsequent membrane separation has a certain decolorization effect, so the decolorization procedure can be eliminated in this technology, which simplifies the process and reduces the cost.

(3) The perfect combination of endogenous enzyme hydrolysis and microbial fermentation. In the existing fermentation technology, all raw materials need to be sterilized, which not only increases energy consumption, but also inactivates endogenous enzymes of aquatic proteins. However, in this technology, the raw materials do not need to be sterilized, and the activity of endogenous enzymes in the aquatic protein is retained. Moreover, the temperature of microbial fermentation is consistent with the optimum enzymolysis temperature of endogenous enzymes, so that endogenous enzyme enzymolysis and microbial fermentation can be perfectly combined. It is precisely because the enzymatic hydrolysis of endogenous enzymes is fully utilized in the present invention that no additional protease is needed in the present invention.

(4) The preparation cost is not only lower than the existing fermentation technology, but also the fermentation effect is better than the existing fermentation technology. Compared with the existing technology of preparing active peptides by fermentation, the raw materials for the application of the present invention do not need to be extinct, which can reduce energy consumption and cost. Since the technical scheme provided by the application of the present invention does not require high-temperature extinction, the enzyme activity of the endogenous protease of the aquatic protein is retained, which is impossible to prepare active peptides by using normal temperature fermentation strains. In addition, the activity of the enzyme is higher at high temperature Strong, therefore, this technology uses high-temperature-resistant strains for fermentation, and the degree of hydrolysis of aquatic proteins is higher than that of the existing normal-temperature strain fermentation technology.

The invention provides a method for preparing active peptides through high-temperature fermentation of aquatic proteins. The technology of the invention has mild action conditions, retains more of the biological activity of the prepared functional peptides and the functional components contained in the aquatic protein itself, simplifies the preparation process of the active peptides and reduces the production cost of the active peptides, and has good advantages Application prospects.

detailed description

In order to better understand the present invention, the following will further describe the present invention with regard to the selection of aquatic protein types, fermentation parameters and fermentation methods in conjunction with examples, but the protection scope of the present invention is not limited thereto.

Example 1: Inoculate Bacillus licheniformis HL-3, which is capable of producing thermostable protease, preserved on a slant into LB liquid medium cooled after sterilization, and cultivated at 55°C and 150r/min for 12h to prepare a lichen The seed liquid of Bacillus licheniformis HL-3 was reserved. Mince fresh Antarctic krill for later use. Add 20kg of minced Antarctic krill and 100kg of water to the fermenter and add 300g of glucose, 300g of food grade calcium chloride, 300g of food grade ferrous chloride and 500g of food grade Dipotassium hydrogen phosphate to obtain the fermentation medium; add 8 kg of Bacillus licheniformis HL-3 seed solution to the fermentation medium in the fermenter; the fermentation temperature is 55°C, and the sterile air intake is 1.5vvm～ 2.5vvm, the rotation speed of the fermenter is 250rpm-350rpm, and the fermentation time is 42h; after the fermentation, the fermentation liquid is filtered to remove the precipitate to obtain the supernatant; the supernatant is removed by ultrafiltration with an ultrafiltration membrane with a molecular weight cut-off of 5000 Daltons Macromolecular protein to obtain the permeate; the permeate is then filtered through a nanofiltration membrane with a molecular weight cut-off of 200 Daltons to remove amino acids to obtain a concentrate; the concentrate is freeze-dried to obtain the finished active peptide.

Example 2: Insert Bacillus licheniformis HL-3, which is capable of producing thermostable protease, preserved on a slant into LB liquid medium cooled after sterilization, and cultivated at 55°C and 150r/min for 12h to prepare a lichen The seed liquid of Bacillus licheniformis HL-3 was reserved. Mince fresh shrimp heads and set aside. Add 22 kg of minced shrimp heads and 100 kg of water to the fermenter, and add 200 g of glucose, 400 g of food grade calcium chloride, 400 g of food grade ferrous chloride and 600 g of food grade phosphoric acid into the fermenter dipotassium hydrogen to obtain the fermentation medium; add 9 kg of Bacillus licheniformis (Bacillus licheniformis) HL-3 seed solution to the fermentation medium in the fermenter; vvm, the rotation speed of the fermenter is 250rpm-350rpm, and the fermentation time is 48h; after the fermentation, the fermentation liquid is filtered to remove the precipitate to obtain the supernatant; Molecular protein to obtain the permeate; the permeate is then filtered through a nanofiltration membrane with a molecular weight cut-off of 200 Daltons to remove amino acids to obtain a concentrated solution; the concentrated solution is freeze-dried to obtain the finished active peptide.

Example 3: Insert the Bacillus licheniformis HL-3 capable of producing thermostable protease preserved on the slant into the LB liquid medium cooled after sterilization, and cultivate it at 55°C and 150r/min for 12h to prepare a lichen The seed liquid of Bacillus licheniformis HL-3 was reserved. The fresh tilapia leftovers are minced and set aside. 25 kg of minced tilapia scraps and 100 kg of water were added to the fermenter, and 450 g of glucose, 450 g of food grade calcium chloride, 350 g of food grade ferrous chloride and 580 g of Food grade dipotassium hydrogen phosphate to obtain fermentation medium; add 10 kg of Bacillus licheniformis HL-3 seed solution to the fermentation medium in the fermenter; the fermentation temperature is 57°C, and the sterile air intake is 1.5 vvm～2.5vvm, the rotation speed of the fermenter is 250rpm～350rpm, and the fermentation time is 45h; after the fermentation, the fermentation liquid is filtered to remove the precipitate to obtain the supernatant; The permeate is obtained by filtering out macromolecular proteins; the permeate is then filtered through a nanofiltration membrane with a molecular weight cut-off of 200 Daltons to remove amino acids to obtain a concentrate; the concentrate is freeze-dried to obtain a finished active peptide.

Claims (7)

1. A method for preparing active peptide by high-temperature fermentation of aquatic protein, characterized in that: the method comprises the steps: (1) Insert Bacillus licheniformis HL-3, which is capable of producing thermostable protease, preserved on the slant into the LB liquid medium cooled after sterilization, and culture it at 55°C and 150r/min for 12h to prepare licheniformis spores Bacillus licheniformis HL-3 seed liquid for use; (2) Grind the fresh aquatic protein for later use; (3) Put the minced aquatic protein prepared in step (2) into a fermenter, add water and nutrients into the fermenter, and mix well to obtain a fermentation medium; (4) adding the Bacillus licheniformis HL-3 seed solution prepared in step (1) to the fermentation culture obtained in step (3); (5) Control of the liquid fermentation process: the fermentation temperature is 55°C-60°C, the sterile air intake is 0.5vvm-2.5vvm, the rotation speed of the fermentation tank is 150rpm-350rpm, and the fermentation time is 42h-48h; (6) Finished product treatment: filter the fermented liquid obtained in step (5) to remove sediment to obtain a supernatant; The permeated liquid is then filtered through a nanofiltration membrane with a molecular weight cut-off of 200 Daltons to remove amino acids to obtain a concentrated liquid; the concentrated liquid is freeze-dried to prepare a finished product of active peptide. 2. A method for preparing active peptides by high-temperature fermentation of aquatic proteins according to claim 1, characterized in that: the Bacillus licheniformis HL-3 capable of producing high-temperature resistant protease used in the step (1) The preservation number in the Guangdong Microbial Culture Collection Center is GDMCCNo: 60003. 3. A method for preparing active peptides by high-temperature fermentation of aquatic proteins according to claim 1, characterized in that: the aquatic proteins in the step (2) are tilapia leftovers, Antarctic krill, shrimp heads, One of scallop skirt, cod scraps, squid scraps, herring scraps, herring scraps, silver carp scraps, grass carp scraps, and skipjack scraps. 4. A method for preparing active peptides by high-temperature fermentation of aquatic proteins according to claim 1, characterized in that: the amount of aquatic proteins added in the step (3) is based on the mass of the added aquatic proteins in the total amount of the fermentation medium In terms of mass percentage, it is 10% to 30%. 5. A method for preparing active peptides by high-temperature fermentation of aquatic proteins according to claim 1, characterized in that the nutrients in the step (3) are glucose, food-grade calcium chloride, and food-grade ferrous chloride and food grade dipotassium hydrogen phosphate. 6. A method for preparing active peptides by high-temperature fermentation of aquatic proteins according to claim 1, characterized in that: the amount of nutrients added in the step (3) is based on the quality of the added nutrients in the fermentation medium In terms of total mass percentage, they are: glucose 0.01%-5%, food-grade calcium chloride 0.01%-5%, food-grade ferrous chloride 0.01%-5%, food-grade dipotassium hydrogen phosphate 0.01%-5%. 7. A method for preparing active peptides by high-temperature fermentation of aquaproteins according to claim 1, characterized in that: in the step (4), the inoculum of Bacillus licheniformis HL-3 seed solution is inoculated with The quality of the seed liquid accounts for 1% to 15% of the total mass percentage of the fermentation medium.