CN113135992A - Preparation method of fish scale collagen peptide - Google Patents
Preparation method of fish scale collagen peptide Download PDFInfo
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- CN113135992A CN113135992A CN202110508700.1A CN202110508700A CN113135992A CN 113135992 A CN113135992 A CN 113135992A CN 202110508700 A CN202110508700 A CN 202110508700A CN 113135992 A CN113135992 A CN 113135992A
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- 241000251468 Actinopterygii Species 0.000 title claims abstract description 78
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- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 3
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- 241000283690 Bos taurus Species 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 108010028690 Fish Proteins Proteins 0.000 description 1
- 102000006587 Glutathione peroxidase Human genes 0.000 description 1
- 108700016172 Glutathione peroxidases Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
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- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
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- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of preparation of collagen peptides, and discloses a fish scale collagen peptide preparation method, which comprises the following steps of firstly, preprocessing fish scales; step two, preparing a fermentation medium; step three, preparing bacterial liquid; step four, fermentation is combined with an enzyme method; step five, inactivating enzyme; and step six, purifying. According to the preparation method of the fish scale collagen peptide, the fish scale collagen peptide which is free of bitter taste, small in molecular weight, high in purity and strong in free radical scavenging capacity is prepared by combining a microbial fermentation method and an enzymolysis method, so that the purity and the quality of the peptide are improved, the cost is reduced, and continuous production can be realized.
Description
Technical Field
The invention relates to the technical field of preparation of collagen peptides, in particular to a preparation method of fish scale collagen peptides.
Background
The collagen peptide has higher absorption and utilization rate than collagen. The peptide contains a large amount of bioactive components, can resist oxidation, repair skin, enhance immunity of human body, enhance bone density and the like. Peptides are widely used in cosmetics, foods and health foods. The main sources of the present peptides are: soybean, bone and skin (pig, cattle and fish), etc. Wherein the fish protein is high quality protein, and the amino acid species are abundant. The marine fish resources in China are very rich, and a large amount of fish leftovers are produced while the aquaculture industry is developed at a high speed. Due to the low proportion of further processing, these fish offal are mostly discarded. This not only wastes resources but also causes environmental pollution. The fish scale is a novel protein resource, and the economic value of the fish scale can be deeply explored.
The prior art has the following defects and shortcomings:
at present, the main preparation methods of collagen peptide are an enzymolysis method and a microbial fermentation method, the enzymolysis method is applied more, but the prepared product has bitter and fishy smell, is difficult to be imported, and the enzyme preparation is expensive, so that the price of the protein peptide product on the market is high, and the daily requirement of people cannot be met.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of fish scale collagen peptide, which can solve the problems that the main preparation methods of the collagen peptide at present are an enzymolysis method and a microbial fermentation method, the enzymolysis method is applied more, but the prepared product has bitter and fishy smell, is difficult to enter the mouth, and the enzyme preparation is expensive, so that the price of the protein peptide product on the market is high and cannot meet the daily requirements of people; the method prepares the fish scale collagen peptide by combining a microbial fermentation method with an enzymolysis method, not only improves the purity and the quality of the peptide, but also reduces the cost, and can realize continuous production.
In order to achieve the purpose of the preparation method of the fish scale collagen peptide, the invention provides the following technical scheme: a method for preparing fish scale collagen peptide comprises the following steps,
step one, fish scale pretreatment, namely cleaning fish scales, soaking the fish scales in a 10% sodium chloride solution to remove foreign proteins, selecting 6% citric acid, and performing ultrasonic-assisted deliming; selecting 0.4mol/L hydrochloric acid, stirring for 1 time at intervals, decalcifying fish scales, filtering to obtain wet fish scales, and drying to obtain purified fish scales;
step two, preparing a fermentation culture medium, namely adding water into the fish scale superfine powder, adding 1-3% of maltose, glucose or sucrose, cooking for a period of time at high temperature, and cooling to obtain the fermentation culture medium;
step three, preparing a bacterium liquid, namely activating the bacillus natto strain for 2 times; inoculating the strain on a sterilized culture medium, and performing shake culture at a proper temperature for a period of time to obtain seed bacteria suspension;
step four, a fermentation combined enzyme method is adopted, the seed bacterium liquid is inoculated into a fermentation medium according to the amount of 2-5% of the volume of the fermentation medium, the pH is controlled between 7-8, the fermentation liquid is obtained after the fermentation is finished and the aeration culture is carried out for a period of time, water is added, the pH is adjusted, the flavourzyme is added, and the secondary enzymolysis is carried out on the fermentation liquid for a period of time at the appropriate temperature;
step five, inactivating enzyme, heating the fermentation liquor to 90 ℃, inactivating enzyme, then centrifuging, and taking supernatant fluid as enzymolysis liquid;
and step six, purifying, namely performing ceramic membrane microfiltration on the enzymolysis liquid, removing thalli, performing ultrafiltration by using an activated carbon fiber membrane, performing nanofiltration by using a spiral membrane to obtain a fish scale collagen peptide concentrated solution, and finally performing spray drying to obtain fish scale collagen peptide powder.
Preferably, the ratio of the 6% citric acid to the liquid is 1: 20, the power of the assisted ultrasonic wave is 120w, the auxiliary deashing time is 3h, the material-liquid ratio of 0.4mol/L hydrochloric acid is 1:15, the interval time of stirring for 1 time is 10min, and the decalcification time of fish scales is 90 min.
Preferably, the weight of the added water is 10 times of that of the water, the cooking temperature is 115 ℃, and the cooking time is 30 min.
Preferably, the strain is inoculated on a sterilized culture medium, the culture temperature is 37 ℃, the rotation speed of a shaking table is 180rpm, and the culture time is 24 h.
Preferably, the ventilating culture temperature of inoculating the seed bacterial liquid into the fermentation culture medium is 37 ℃, the culture time is 36-72 hours, the weight of the added flavourzyme is 0.07% of the weight of the fish scales, the secondary enzymolysis temperature is 45 ℃, and the secondary enzymolysis time is 2 hours.
Preferably, the enzyme deactivation time is 10min, the centrifugation time is 20min, and the centrifugation revolution is set to 4000 r/min.
Preferably, the fish scale collagen peptide concentrated solution is obtained, and the solid content is about 20%.
Compared with the prior art, the invention provides a preparation method of fish scale collagen peptide, which has the following beneficial effects:
the preparation method of the fish scale collagen peptide uses a microbial fermentation method and an enzymolysis method, and prepares the fish scale collagen peptide which has no bitter taste, small molecular weight, high purity and stronger free radical scavenging capacity, not only improves the purity and the quality of the peptide, but also can reduce the cost, and can realize continuous production.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A method for preparing fish scale collagen peptide comprises the following steps,
step one, fish scale pretreatment, namely cleaning fish scales, soaking the fish scales in a 10% sodium chloride solution to remove foreign proteins, selecting 6% citric acid, and performing ultrasonic-assisted deliming; selecting 0.4mol/L hydrochloric acid, stirring for 1 time at intervals, decalcifying fish scales, filtering to obtain wet fish scales, and drying to obtain purified fish scales;
step two, preparing a fermentation culture medium, namely adding water into the fish scale superfine powder, adding 1-3% of maltose, glucose or sucrose, cooking for a period of time at high temperature, and cooling to obtain the fermentation culture medium;
step three, preparing a bacterium liquid, namely activating the bacillus natto strain for 2 times; inoculating the strain on a sterilized culture medium, and performing shake culture at a proper temperature for a period of time to obtain seed bacteria suspension;
step four, a fermentation combined enzyme method is adopted, the seed bacterium liquid is inoculated into a fermentation medium according to the amount of 2-5% of the volume of the fermentation medium, the pH is controlled between 7-8, the fermentation liquid is obtained after the fermentation is finished and the aeration culture is carried out for a period of time, water is added, the pH is adjusted, the flavourzyme is added, and the secondary enzymolysis is carried out on the fermentation liquid for a period of time at the appropriate temperature;
step five, inactivating enzyme, heating the fermentation liquor to 90 ℃, inactivating enzyme, then centrifuging, and taking supernatant fluid as enzymolysis liquid;
and step six, purifying, namely performing ceramic membrane microfiltration on the enzymolysis liquid, removing thalli, performing ultrafiltration by using an activated carbon fiber membrane, performing nanofiltration by using a spiral membrane to obtain a fish scale collagen peptide concentrated solution, and finally performing spray drying to obtain fish scale collagen peptide powder.
The invention is further illustrated by the following examples:
example 1:
taking 10kg fish scale ultra-fine powder, adding 100L water, adding 0.2kg maltose, cooking at 115 deg.C for 30min, cooling to obtain fermentation medium, performing activation culture of natto strain to obtain seed bacteria suspension, inoculating 2L seed bacteria liquid into fermentation medium, controlling pH between 7-8, performing ventilation culture at 37 deg.C for 36h, adding water after fermentation, and adjusting pH. Adding flavourzyme (7g), and carrying out secondary enzymolysis on the fermentation liquor at 45 ℃ for 2 h. Inactivating enzyme at 90 deg.C for 10min, centrifuging at 4000r/min for 20min, and collecting supernatant as enzymolysis solution. Then, carrying out ceramic membrane microfiltration (thallus removal), activated carbon fiber membrane ultrafiltration (decolorization and the like) and spiral membrane nanofiltration on the enzymatic hydrolysate to obtain a fish scale collagen peptide concentrated solution (about 20 percent of solid matters), and finally carrying out spray drying to obtain powder, namely fish scale collagen peptide powder, so as to obtain 2.3kg of peptide powder.
Example 2:
taking 10kg fish scale ultra-fine powder, adding 100L water, adding 0.2kg glucose, cooking at 115 deg.C for 30min, cooling to obtain fermentation medium, performing activation culture on natto strain to obtain seed bacteria suspension, inoculating 3L seed bacteria liquid into fermentation medium, controlling pH between 7-8, performing ventilation culture at 37 deg.C for 54h, adding water after fermentation, and adjusting pH. Adding flavourzyme (7g), and carrying out secondary enzymolysis on the fermentation liquor at 45 ℃ for 2 h. Inactivating enzyme at 90 deg.C for 10min, centrifuging at 4000r/min for 20min to obtain supernatant as enzymolysis solution, performing ceramic membrane microfiltration (to remove thallus), activated carbon fiber membrane ultrafiltration (to decolorize), and spiral membrane nanofiltration to obtain fish scale collagen peptide concentrated solution (solid content about 20%), and spray drying to obtain fish scale collagen peptide powder 2.4 kg.
Example 3:
taking 10kg of fish scale ultra-fine powder, adding 100L of water, adding 0.2kg of cane sugar, cooking for 30min at 115 ℃, cooling to obtain a fermentation medium, carrying out activated culture on natto strains to obtain seed strain suspension, taking 4L of seed strain liquid, inoculating the seed strain liquid into the fermentation medium, controlling the pH between 7 and 8, carrying out ventilation culture for 72h at 37 ℃, adding water after fermentation is finished, adjusting the pH, adding flavourzyme (7g), carrying out secondary enzymolysis on fermentation liquor at 45 ℃, carrying out secondary enzymolysis for 2h, and carrying out enzyme deactivation for 10min at 90 ℃. Centrifuging at 4000r/min for 20min to obtain supernatant as enzymolysis solution, performing ceramic membrane microfiltration (for removing thallus), activated carbon fiber membrane ultrafiltration (for decolorizing, etc.), and spiral membrane nanofiltration to obtain fish scale collagen peptide concentrated solution (solid content is about 20%), and spray drying to obtain fish scale collagen peptide powder (2.6 kg).
For the fish scale collagen peptide prepared in example 3, the molecular weight distribution of the peptide and the heavy metal content of GB 5009.268 were determined according to GB/T22492, and the results are shown in tables 1 and 2.
TABLE 1 peptide molecule distribution
TABLE 2 heavy metal content
| Heavy metals | Content (mg/kg) |
| Arsenic (in As) | 0.059 |
| Lead (in Pb) | 0.031 |
| Chromium (in terms of Cr) | Not detected (< 0.02) |
| Cadmium (in Cd) | 0.0061 |
| Nickel (in terms of Ni) | 0.09 |
| Mercury (in Hg) | Not detected (< 0.0003) |
The enzyme activity of the fish scale collagen peptide prepared in example 3 was measured, and the results are shown in table 3.
TABLE 3 enzyme Activity
| Detecting items | Index (I) |
| SOD superoxide dismutase (U/mL) | 5.69±0.23 |
| Glutathione peroxidase (GSH-PX) Activity (U/mL) | 40.49±0.86 |
| Catalase (CAT) Activity (U/mL) | 0.19±0.023 |
Example 4, animal experiments for relieving physical fatigue were performed on the peptide powder,
materials and methods
Sample preparation: fish scale peptide powder of example 3.
Experimental animals: the male mice are 150 mice, and the body weight is 18-22 g.
Experiment design: animal experiments are designed according to the function evaluation program and the inspection method of the health food for relieving physical fatigue.
Results of the experiment
2.1. Effect of fish scale peptide on mouse swimming time under load (table 4, n-10,
)
TABLE 4 swimming time under load
| Group of | Dosage mg/(kg. d) | Swimming time (min) |
| Blank space | Physiological saline | 7.23±0.41 |
| Positive for | Taurine 50 | 9.42±1.32 |
| Low dose | 50 | 11.58±1.35 |
| In | 100 | 13.19±0.71 |
| Height of | 150 | 12.32±0.56 |
The swimming time of the mice is improved compared with that of the control group in the experimental group;
2.2. effect of fish scale peptides on mouse liver glycogen (table 5, n-10,
)
TABLE 5 hepatic glycogen
| Group of | Hepatic glycogen (mg/g) |
| Blank space | 3.19±0.58 |
| Positive for | 4.30±0.77 |
| Low dose | 5.06±0.67 |
| In | 5.21±0.82 |
| Height of | 5.34±0.65 |
Each experimental group is higher than the control group, which shows that the peptide powder can promote the synthesis of hepatic glycogen; 2.3. effect on serum urea nitrogen content in mice (table 6, n-10,
)
TABLE 6 serum Urea Nitrogen
Each experimental group is lower than the blank group, which shows that the peptide powder can reduce the serum urea nitrogen level;
and (4) conclusion:
according to the function evaluation program and the test method for relieving physical fatigue, more than 1 (including) exercise test and more than 2 (including) biochemical indexes are positive, the tested object can be judged to have the function of relieving the physical fatigue, and therefore, the fish scale peptide powder can be obtained to have the function of relieving the physical fatigue.
When the preparation method of the fish scale collagen peptide is used, a microbial fermentation method is combined with an enzymolysis method to prepare the fish scale collagen peptide which has no bitter taste, small molecular weight, high purity and stronger free radical removal capacity, so that not only is the purity and the quality of the peptide improved, but also the cost can be reduced, and continuous production can be realized.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A preparation method of fish scale collagen peptide is characterized by comprising the following steps: comprises the following steps of (a) carrying out,
step one, fish scale pretreatment, namely cleaning fish scales, soaking the fish scales in a 10% sodium chloride solution to remove foreign proteins, selecting 6% citric acid, and performing ultrasonic-assisted deliming; selecting 0.4mol/L hydrochloric acid, stirring for 1 time at intervals, decalcifying fish scales, filtering to obtain wet fish scales, and drying to obtain purified fish scales;
step two, preparing a fermentation culture medium, namely adding water into the fish scale superfine powder, adding 1-3% of maltose, glucose or sucrose, cooking for a period of time at high temperature, and cooling to obtain the fermentation culture medium;
step three, preparing a bacterium liquid, namely activating the bacillus natto strain for 2 times; inoculating the strain on a sterilized culture medium, and performing shake culture at a proper temperature for a period of time to obtain seed bacteria suspension;
step four, a fermentation combined enzyme method is adopted, the seed bacterium liquid is inoculated into a fermentation medium according to the amount of 2-5% of the volume of the fermentation medium, the pH is controlled between 7-8, the fermentation liquid is obtained after the fermentation is finished and the aeration culture is carried out for a period of time, water is added, the pH is adjusted, the flavourzyme is added, and the secondary enzymolysis is carried out on the fermentation liquid for a period of time at the appropriate temperature;
step five, inactivating enzyme, heating the fermentation liquor to 90 ℃, inactivating enzyme, then centrifuging, and taking supernatant fluid as enzymolysis liquid;
and step six, purifying, namely performing ceramic membrane microfiltration on the enzymolysis liquid, removing thalli, performing ultrafiltration by using an activated carbon fiber membrane, performing nanofiltration by using a spiral membrane to obtain a fish scale collagen peptide concentrated solution, and finally performing spray drying to obtain fish scale collagen peptide powder.
2. The method for preparing fish scale collagen peptide according to the step one of claim 1, wherein the method comprises the following steps: the material-liquid ratio of the selected 6% citric acid is 1: 20, the power of the assisted ultrasonic wave is 120w, the auxiliary deashing time is 3h, the material-liquid ratio of 0.4mol/L hydrochloric acid is 1:15, the interval time of stirring for 1 time is 10min, and the decalcification time of fish scales is 90 min.
3. The method for preparing fish scale collagen peptide according to the step two of claim 1, wherein the method comprises the following steps: the weight of the added water is 10 times of that of the water, the cooking temperature is 115 ℃, and the cooking time is 30 min.
4. The method for preparing fish scale collagen peptide according to the third step of claim 1, wherein the method comprises the following steps: the strain is inoculated on a sterilized culture medium, the culture temperature is 37 ℃, the rotating speed of a shaking table is 180rpm, and the culture time is 24 h.
5. The method for preparing fish scale collagen peptide according to the fourth step of claim 1, wherein the method comprises the following steps: the seed bacterial liquid is inoculated into a fermentation medium, the ventilation culture temperature is 37 ℃, the culture time is 36-72 hours, the weight of the added flavourzyme is 0.07% of the weight of the fish scales, the secondary enzymolysis temperature is 45 ℃, and the secondary enzymolysis time is 2 hours.
6. The method for preparing fish scale collagen peptide according to the fifth step of claim 1, wherein the method comprises the following steps: the enzyme deactivation time is 10min, the centrifugation time is 20min, and the centrifugation revolution is set to 4000 r/min.
7. The method for preparing fish scale collagen peptide according to the sixth step of claim 1, wherein the method comprises the following steps: the fish scale collagen peptide concentrated solution is obtained, and the solid content is about 20 percent.
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