CN113951516A - Preparation method of marine fish skin collagen oligopeptide and weight-losing coffee based on same - Google Patents
Preparation method of marine fish skin collagen oligopeptide and weight-losing coffee based on same Download PDFInfo
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- CN113951516A CN113951516A CN202111249745.8A CN202111249745A CN113951516A CN 113951516 A CN113951516 A CN 113951516A CN 202111249745 A CN202111249745 A CN 202111249745A CN 113951516 A CN113951516 A CN 113951516A
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F5/00—Coffee; Coffee substitutes; Preparations thereof
- A23F5/10—Treating roasted coffee; Preparations produced thereby
- A23F5/14—Treating roasted coffee; Preparations produced thereby using additives, e.g. milk, sugar; Coating, e.g. for preserving
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention discloses a preparation method of marine fish skin collagen oligopeptide and weight-losing coffee based on the same, and belongs to the technical field of oligopeptide. The marine fish skin collagen oligopeptide is a marine fish skin collagen peptide containing an amino acid sequence shown as SEQ ID NO.1, and the preparation method comprises the following steps: dispersing marine fish skin powder in water, adjusting pH, adding an enzyme mixture, reacting at constant temperature, heating to inactivate enzyme after reaction, filtering, and drying to obtain collagen oligopeptide; wherein the enzyme mixture comprises neutral protease, papain, and trypsin. The marine fish skin collagen oligopeptide prepared by the method has low molecular weight, has the functions of beautifying and improving immunity of most collagen oligopeptides, and also has the functions of reducing body fat percentage, reducing triglyceride level in blood, protecting gastric mucosa and promoting recovery of gastric mucosa.
Description
Technical Field
The invention relates to the technical field of oligopeptide, and particularly relates to a preparation method of marine fish skin collagen oligopeptide and weight-losing coffee based on the marine fish skin collagen oligopeptide.
Background
Collagen is a biological macromolecule, a main component in the connective tissue of animals, and is also a functional protein with the largest content and the widest distribution in mammals, and accounts for 25 to 30 percent of the total amount of protein, and certain organisms even reach more than 80 percent. The collagen polypeptide has high nutritive value, and can protect gastric mucosa and prevent ulcer.
The preparation method of the marine biological active peptide mainly comprises two ways: firstly, the protein in the organism is degraded by utilizing the bioactive enzyme to obtain some biological polypeptides with physiological functions, and secondly, the biological polypeptides are separated from some inherent marine organisms with some natural peptide substances. The direct extraction method for preparing the bioactive peptide is difficult to realize large-scale industrial production. Moreover, the active peptide extracted by the method necessarily needs to use a large amount of organic solvent, and the organic solvent not only causes environmental pollution, but also causes problems such as subsequent treatment of organic solvent residue in the obtained bioactive polypeptide substance. The enzymolysis method can perform localized hydrolysis under simple conditions to produce specific peptide fragments, and has high safety, no side effect and easy control of reaction process. Therefore, the invention adopts an enzymolysis method to prepare the marine fish skin collagen oligopeptide.
Nowadays, obesity seriously harms human health. At present, six main weight-losing methods are available all over the world, wherein exercise weight-losing is the first choice and effective weight-losing method, however, modern life with high efficiency and rapidness makes people difficult to extract time to implement; the traditional Chinese medicine has long treatment course and is difficult to insist; the surgery weight loss has certain hidden troubles such as surgery risk and complication; the adverse side effects of drug weight reduction can damage the physical health of the organism; diet and weight loss can cause malnutrition of the body; the weight-losing method of the nutrition catering is complicated and the steps are complex and difficult to implement. Most of the current weight-reducing medicines are appetite suppressants, diuretics, laxatives and the like, the illusion of weight reduction is achieved by discharging water and protein in a human body, the root of obesity cannot be reached, and the long-term taking of the weight-reducing medicines can cause damage to the functions of the human body. Therefore, there is an urgent need for a safe and efficient food that can regulate dietary habits without side effects.
Disclosure of Invention
An object of the present invention is to provide a marine fish skin collagen peptide having effects of reducing fat absorption, enhancing immunity, and protecting gastric mucosa; the invention also aims to provide a preparation method of the marine fish skin collagen oligopeptide with high extraction efficiency, and the obtained marine fish skin collagen oligopeptide has the effects of reducing fat absorption, enhancing immunity and protecting gastric mucosa; the invention also aims to provide the weight-losing coffee containing the marine fish skin collagen oligopeptide, which can improve the immunity of the organism, reduce the body fat rate and protect the gastric mucosa.
In order to achieve the purpose of the invention, the following technical scheme is adopted.
A marine fish skin collagen peptide has an amino acid sequence shown in SEQ ID NO. 1.
The marine fish skin collagen peptide sequence is Leu-Val-Arg-Glu-Arg, and has the effects of reducing fat absorption, enhancing immunity and protecting gastric mucosa.
Preferably, the preparation method of the marine fish skin collagen peptide comprises the following steps:
dispersing marine fish skin powder in water, adjusting pH, adding an enzyme mixture, reacting at constant temperature, heating to inactivate enzyme after reaction, filtering, and drying to obtain marine fish skin collagen oligopeptide; detecting and separating the active peptide segment from the marine fish skin collagen oligopeptide to obtain the marine fish skin collagen peptide.
Preferably, the method for obtaining the marine fish skin collagen peptide by detecting and separating the active peptide segment from the marine fish skin collagen oligopeptide comprises the following steps:
(1) separating peptide fragments:
separating the peptide segment of the marine fish skin collagen oligopeptide amino acid by using a reversed-phase high performance liquid chromatography, wherein the chromatographic conditions are as follows: c18A reverse phase chromatography column, mobile phase a being water containing 1% trifluoroacetic acid by volume, mobile phase B being water; collecting obvious component peaks, naming, and drying for later use;
(2) detecting the peptide fragment:
determining the fat absorption reducing capacity of the peptide fragments corresponding to the component peaks collected in the step (1), and selecting a group with the fat absorption reducing capacity for sequence identification;
(3) and (3) identifying the peptide fragment:
and (3) carrying out structural identification on the peptide fragments corresponding to the component peaks selected in the step (2) by using an orthogonal acceleration electrospray tandem mass spectrometer.
The invention also discloses a marine fish skin collagen oligopeptide which contains the marine fish skin collagen peptide with the amino acid sequence shown as SEQ ID NO. 1.
The marine fish skin collagen peptide has multiple beneficial effects, wherein the effects of improving immunity, protecting gastric mucosa and reducing weight are most obvious, and the marine fish skin collagen peptide can be used as a raw material of a medicine, a health-care product and a food for preparing the medicine, the health-care product or the food with the effects of reducing fat absorption, improving immunity and nourishing stomach.
The invention also discloses a preparation method of the marine fish skin collagen oligopeptide, which comprises the following steps:
dispersing marine fish skin powder in water, adjusting pH, adding an enzyme mixture, reacting at constant temperature, heating to inactivate enzyme after reaction, filtering, and drying to obtain collagen oligopeptide; wherein the enzyme mixture comprises neutral protease, papain, and trypsin.
Preferably, the weight ratio of the fish skin powder, the water, the neutral protease, the papain and the trypsin is as follows: 100, 500, 300, 1200, 0.1-0.5; the constant temperature reaction time is 3-5 h.
Preferably, the preparation method further comprises adding corilagin and baicalein. The oligopeptide prepared by the method does not influence the original biological activity and function, and the enzyme mixture consisting of the protease, the corilagin and the baicalein has better extraction efficiency and can improve the degree of proteolysis.
More preferably, the weight ratio of the fish skin powder, the water, the neutral protease, the papain, the trypsin, the corilagin and the baicalein is as follows: 100, 500, 300, 1200, 0.1-0.5, 5-25; the constant temperature reaction time is 3-5 h.
Preferably, the reaction temperature is 20-30 ℃.
Preferably, the reaction pH is 6 to 7.
Preferably, the marine fish skin is deep sea salmon skin.
Preferably, the preparation method of the marine fish skin collagen oligopeptide comprises the following steps:
dispersing 500 portions of marine fish skin powder of 100 weight portions in 1200 portions of water of 300 weight portions, adjusting the pH value to 6-7, adding 0.1-0.5 portion of neutral protease, 0.1-0.5 portion of papain and 0.1-0.5 portion of trypsin, and reacting for 3-5h at a constant temperature of 20-30 ℃; heating to 90 ℃ after the reaction is finished to inactivate the enzyme, filtering, and drying the filtrate to obtain the collagen oligopeptide.
More preferably, the enzymatic activity used is 2400-2600U/g.
More preferably, the filtration is performed by a 60-80 mesh filter screen for the first filtration and a 1500-2000Da filter membrane for the second filtration.
The invention also discloses application of the marine fish skin collagen peptide in preparation of medicines and/or health-care products and/or foods for improving immunity and/or reducing subcutaneous fat.
The invention also discloses application of the marine fish skin collagen oligopeptide in preparing a medicament and/or a health-care product and/or a food for improving immunity and/or reducing subcutaneous fat.
The invention also discloses weight-losing coffee which is characterized by comprising any one of the following compositions (1), (2) or (3):
(1) marine fish skin collagen oligopeptide, medium chain triglyceride microcapsule powder and coffee powder;
(2) marine fish skin collagen peptide, medium chain triglyceride microcapsule powder and coffee powder;
(3) marine fish skin collagen peptide, marine fish skin collagen oligopeptide, medium chain triglyceride microcapsule powder and coffee powder.
The coffee is added with marine fish skin collagen oligopeptide and/or protein peptide, so that the coffee is rich in nutrition and has the effects of improving immunity and nourishing stomach; the product can be used together with medium chain triglyceride microcapsule powder and coffee powder, and has effects of reducing weight, reducing weight and fat, reducing triglyceride in blood, regulating blood pressure, and reducing blood pressure fluctuation.
Preferably, the medium chain triglyceride microencapsulation powder comprises coconut oil, isomaltooligosaccharide, casein, resistant dextrin, potassium citrate, mono-diglycerol fatty acid ester, silicon dioxide.
The medium chain triglyceride microcapsule powder is beneficial to absorption by human body, has good health care effect, and can reduce or eliminate inflammation in vivo; when taken together with other ingredients, the health food can also increase satiety and relieve hunger sensation, and is beneficial to losing weight and reducing fat.
More preferably, the weight ratio of each component in the medium chain triglyceride microcapsule powder is 40-60 parts of coconut oil, 8-10 parts of oligomeric isomaltose, 6-9 parts of casein, 5-7 parts of resistant dextrin, 1-2.5 parts of potassium citrate, 2-5 parts of monoglyceride and diglyceride fatty acid ester and 0.5-2 parts of silicon dioxide.
More preferably, the coconut oil is extracted by a cold pressing process.
Preferably, the preparation method of the medium chain triglyceride microcapsule powder comprises the following steps:
preparing a raw material mixture → dissolving the raw material → emulsifying → sterilizing → homogenizing → spray drying → cooling → sieving.
More preferably, the preparation method of the medium chain triglyceride microcapsule powder comprises the following steps:
mixing 40-60 parts by weight of coconut oil with 2-5 parts by weight of monoglyceride and diglyceride fatty acid ester to obtain a solution A, mixing 8-10 parts by weight of isomaltooligosaccharide, 6-9 parts by weight of casein, 5-7 parts by weight of resistant dextrin, 1-2.5 parts by weight of potassium citrate and 0.5-2 parts by weight of silicon dioxide to obtain a mixture, adding 50-100 parts by weight of water, and stirring uniformly to obtain a solution B; slowly adding the solution B into the solution A while stirring; then carrying out water bath at 60-70 ℃ for 10-20 min; carrying out ultraviolet sterilization for 1-3h after water bath and homogenizing at the speed of 500-; spray drying is carried out after treatment, and the air inlet temperature is 150-; drying, cooling, and sieving with 1000 mesh sieve to obtain medium chain triglyceride microcapsule powder.
The medium chain triglyceride microcapsule powder prepared by the method microencapsulates the coconut oil which is oil difficult to eat directly, has good embedding effect and is very stable, can prevent damage to the coconut oil caused by light, heat, oxidation and other substances, and has good quality and flavor preservation after long-term storage; and the coconut oil can fully exert the health-care effect of the coconut oil, is easy to absorb and has a certain anti-inflammatory effect.
Preferably, the weight-reducing coffee comprises the following components in parts by weight: 50-80 parts of medium chain triglyceride microcapsule powder, 20-35 parts of coffee powder and 5-10 parts of marine fish skin collagen oligopeptide.
Preferably, the weight-reducing coffee comprises the following components in parts by weight: 50-80 parts of medium-chain triglyceride microcapsule powder, 20-35 parts of coffee powder and 5-10 parts of marine fish skin collagen peptide.
Preferably, the weight-reducing coffee comprises the following components in parts by weight: 50-80 parts of medium-chain triglyceride microcapsule powder, 20-35 parts of coffee powder, 1-5 parts of marine fish skin collagen oligopeptide and 1-5 parts of marine fish skin collagen peptide.
Preferably, the preparation method of the coffee powder comprises the following steps:
the coffee beans are dried in a low-temperature roasting manner and then ground into powder to form coffee powder.
Compared with the prior art, the invention has the beneficial effects that:
the invention uses the enzyme mixture containing corilagin and baicalein in the process of preparing the marine fish skin collagen oligopeptide, can increase the hydrolysis degree of protein, obtains the oligopeptide with smaller molecular weight, and prepares the medicine, the health care product or the food by using the oligopeptide, thereby being more beneficial to the absorption of human bodies. In addition, the marine fish skin collagen oligopeptide prepared by the invention has the functions of beautifying the skin and improving the immunity of most collagen oligopeptides, and also has the functions of reducing the body fat rate, reducing the level of triglyceride in blood, protecting gastric mucosa and promoting the recovery of gastric mucosa; the medium chain triglyceride microcapsule powder prepared by the invention has small particle size, so that the microcapsule powder is used for preparing medicines, health products or foods, and is more beneficial to absorption by human bodies. In addition, the medium chain triglyceride microcapsule powder also has the function of diminishing inflammation and can effectively inhibit the secretion of inflammatory factors TFN-alpha and IL-6. In addition, the weight-losing coffee prepared by the invention has good effects of reducing weight and body fat rate.
Drawings
FIG. 1 shows the results of reversed-phase HPLC separation of marine fish skin collagen oligopeptide;
FIG. 2 shows the measurement results of the particle size distribution of medium chain triglyceride microcapsules.
Detailed Description
The exemplary embodiments will be described herein in detail, and the embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.
The experimental procedures in the following examples are conventional or according to the manufacturer's recommendations unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. In the following test examples, the experimental animals were purchased from Jiangsu Ailing phenanthrene Biotech Co.
Example 1
Preparation of weight-reducing coffee
1. Preparation of marine fish skin collagen oligopeptide
Dispersing 200g of salmon skin powder in 500g of water, adjusting the pH to 6.5, adding 0.2g of neutral protease, 0.1g of papain, 0.5g of trypsin, 9g of corilagin and 10.7g of baicalein, wherein the enzyme activity is 2500U/g, and reacting for 3 hours at a constant temperature of 24 ℃; heating to 90 ℃ to inactivate the enzyme after the reaction is finished; filtering with 80 mesh filter screen for the first time, filtering with 2000Da filter membrane for the second time, and freeze drying the filtrate to obtain collagen oligopeptide.
2. Preparing coconut oil
Taking out coconut meat from mature coconut, cleaning, cutting into pieces, freeze-drying, and squeezing the dried coconut meat with a squeezer to obtain coconut oil.
3. Preparation of medium chain triglyceride microcapsule powder
Adding 5g of monoglyceride and diglyceride fatty acid ester into 60g of coconut oil, mixing uniformly to obtain solution A, adding 100g of water into 10g of isomaltooligosaccharide, 9g of casein, 7g of resistant dextrin, 2g of potassium citrate and 2g of silicon dioxide, mixing uniformly, and stirring uniformly to obtain solution B; slowly adding the solution B into the solution A while stirring; then carrying out water bath at 70 ℃ for 15 min; performing ultraviolet sterilization for 3h after water bath, and homogenizing at 1000 r/min; after treatment, spray drying is carried out, and the air inlet temperature is 170 ℃; drying, cooling and sieving with 1000 mesh sieve to obtain medium chain triglyceride microcapsule powder.
4. Preparation of coffee powder
Baking fresh coffee beans at 120 deg.C for 30min, and grinding with bean grinder to obtain coffee powder.
5. Preparation of weight-reducing coffee
And (3) uniformly mixing 60g of medium-chain triglyceride microcapsule powder, 30g of coffee powder and 10g of marine fish skin collagen oligopeptide, sterilizing for 1 hour by using ultraviolet rays, and bagging to obtain the weight-reducing coffee.
Example 2
Preparation of weight-reducing coffee
This example differs from example 1 in that corilagin and baicalein were not added to the enzyme mixture in step 1.
Example 3
Preparation of weight-reducing coffee
This example differs from example 1 in that step 3 is as follows:
preparation of medium chain triglyceride microcapsule powder
Adding 2g of monoglyceride and diglyceride fatty acid ester into 40g of coconut oil, mixing uniformly to obtain a solution A, adding 80g of water into 8g of isomaltooligosaccharide, 6g of casein, 5g of resistant dextrin, 1g of potassium citrate and 0.5g of silicon dioxide after mixing uniformly, and stirring uniformly to obtain a solution B; slowly adding the solution B into the solution A while stirring; then carrying out water bath at 65 ℃ for 20 min; performing ultraviolet sterilization for 2h after water bath, and homogenizing at 1000 r/min; after treatment, spray drying is carried out, and the air inlet temperature is 170 ℃; drying, cooling and sieving with 1000 mesh sieve to obtain medium chain triglyceride microcapsule powder.
Example 4
Preparation of weight-reducing coffee
1. Preparation of marine fish skin collagen peptide
(1) Separating peptide fragments:
separating the peptide fragment of the marine fish skin collagen oligopeptide amino acid prepared in the example 1 by using a reversed phase high performance liquid chromatography, wherein the chromatographic conditions are as follows: c18Reverse phase chromatography column, flowPhase a was water containing 1% by volume trifluoroacetic acid, mobile phase B was water: acetonitrile: trifluoroacetic acid 80: 20: 0.1 (V/V/V); the gradient elution conditions were:
0-10min, mobile phase B: 0% -5%;
10-20min, mobile phase B: 5 percent;
20-35min, mobile phase B: 5% -10%;
35-45min, mobile phase B: 10% -15%;
45-60min, mobile phase B: 15 percent;
60-70min, mobile phase B: 15% -75%;
mass concentration of the sample: 10 mg/mL; detection wavelength: UV 220 nm; flow rate: 0.5 mL/min; sample introduction volume: 100 μ L. Collecting main component peak, volatilizing organic solvent, and freeze drying.
The measurement results are shown in FIG. 1. As can be seen from the figure, the marine fish skin collagen oligopeptide prepared by the invention has 9 obvious component peaks, is respectively collected and named as oligopeptide 1-9, and is frozen and dried for subsequent determination.
(2) Detecting the peptide fragment:
and (2) measuring the fat absorption regulating capacity of the peptide fragments corresponding to the component peaks collected in the step (1), and selecting a group with the obviously reduced fat absorption capacity for sequence identification. The influence of each peptide segment on the apparent digestibility of the fat of the mice is measured to judge the strength of the fat absorption adjusting capability of the peptide segment.
Taking 55 SPF mice with the weight of 25 +/-2 g, adaptively feeding for 1 week, and randomly dividing the mice into 11 groups of 5 mice each; 1-10 groups of the feed are fed with high-fat feed, and the feed formula is as follows: 79.8% of basal feed, 0.2% of ox gall salt, 10% of lard and 10% of egg yolk powder, wherein 11 groups of the basal feed are fed with the feed for free feeding, and the daily feed intake of the mice is recorded. After continuously feeding for 30 days, 1-9 groups are filled with 30mg/kg of 1-9 peptide fragments every day, and the peptide fragments are dispersed in 3mL of distilled water and filled; 10. 3mL of distilled water was poured into 11 groups; gavage was continued for 4 weeks, during which time the diet of each group of mice was unchanged; continuously collecting feces of each group of mice for 72 hours after four weeks, and taking 1g of feces of each mouse for detection; drying the excrement to constant weight, recording the weight, extracting fat after excrement by using a Soxhlet extraction method, recording the weight of the excrement after extraction, calculating the fat apparent digestibility by calculating the fat apparent digestibility and taking an average value to record, wherein the difference value of the two weighing is the fat content in the excrement.
Apparent digestibility of fat (lard content in feed-fat content in feces)/lard content in feed x 100%
The measurement results are shown in table 1.
TABLE 1 apparent digestibility of mouse fat
| Group of | Apparent digestibility of fat (%) |
| 1 | 79.2 |
| 2 | 77.6 |
| 3 | 77.1 |
| 4 | 78.5 |
| 5 | 77.9 |
| 6 | 78.3 |
| 7 | 79.1 |
| 8 | 71.3 |
| 9 | 78.8 |
| 10 | 78.4 |
| 11 | 76.2 |
As can be seen from Table 1, except group 8, the effect of each group of peptide fragments on the apparent digestibility of the fat of the mice is not obvious, and the apparent digestibility of the fat of the mice taking 8 groups of peptide fragments is obviously reduced, which indicates that the group 8 peptide fragments reduce the digestive absorption of the fat intake of the mice and are beneficial to losing weight and reducing fat. Therefore 8 was selected for identification.
(3) And (3) identifying the peptide fragment:
the oligopeptide 8 was structurally characterized using an orthogonal acceleration electrospray tandem mass spectrometer. The mass spectrum conditions are as follows:
an ionization mode: nanoliter electrospray positive ions; atomizing gas: n is a radical of2(ii) a Collision gas: ar; source temperature: 80 ℃; taper hole voltage: 50V; TOF acceleration voltage: 9.1 kV; MCP detector voltage: 2150V; capillary voltage: 800V; mass accuracy of MS and MS/MS: 0.1 u.
The sequence of the oligopeptide 8 is shown in SEQ ID NO.1 through determination.
2. Preparing coconut oil
The same as in example 1.
3. Preparation of medium chain triglyceride microcapsule powder
The same as in example 1.
4. Preparation of coffee powder
The same as in example 1.
5. Preparation of weight-reducing coffee
And (3) uniformly mixing 60g of medium-chain triglyceride microcapsule powder, 30g of coffee powder and 10g of marine fish skin collagen peptide, sterilizing for 1 hour by using ultraviolet rays, and bagging to obtain the weight-reducing coffee.
Example 5
Preparation of weight-reducing coffee
This example differs from example 1 in that 5g of the marine fish skin collagen oligopeptide prepared in this example and 5g of the marine fish skin collagen oligopeptide prepared in example 1 were used in place of 10g of the marine fish skin collagen oligopeptide in step 5.
(1) Preparation of marine fish skin collagen peptide
Dispersing 200g of salmon skin powder in 500g of water, adjusting the pH to 6.5, adding 0.2g of neutral protease, 0.1g of papain and 0.5g of trypsin, wherein the enzyme activity is 2500U/g, and reacting for 3 hours at a constant temperature of 24 ℃; heating to 90 ℃ to inactivate the enzyme after the reaction is finished; filtering with 80 mesh filter screen for the first time, filtering with 2000Da filter membrane for the second time, and freeze drying the filtrate to obtain collagen oligopeptide.
(2) Separating peptide fragments:
separating the peptide segments of the marine fish skin collagen oligopeptide amino acid prepared in the step (1) by using a reversed-phase high performance liquid chromatography, wherein the chromatographic conditions are as follows: c18Reverse phase chromatography column, mobile phase a is water containing 1% by volume trifluoroacetic acid, mobile phase B is water: acetonitrile: trifluoroacetic acid 80: 20: 0.1 (V/V/V); the gradient elution conditions were:
0-10min, mobile phase B: 0% -5%;
10-20min, mobile phase B: 5 percent;
20-35min, mobile phase B: 5% -10%;
35-45min, mobile phase B: 10% -15%;
45-60min, mobile phase B: 15 percent;
60-70min, mobile phase B: 15% -75%;
mass concentration of the sample: 10 mg/mL; detection wavelength: UV 220 nm; flow rate: 0.5 mL/min; sample introduction volume: 100 μ L. Collecting main component peak, volatilizing organic solvent, and freeze drying.
Through determination, 9 obvious component peaks are totally collected and named as oligopeptide 1-9 respectively, the influence of the oligopeptide 1-9 on the apparent fat digestibility of the mice is detected according to the peptide segment detection in the step (2) in the embodiment 4, wherein the apparent fat digestibility of the mice taking the oligopeptide 8 is the lowest, and 8 is selected for peptide segment identification, and the sequence of the peptide segment is shown as SEQ ID NO. 1. Thus, group 8 was taken after freeze-drying for the preparation of diet coffee.
Comparative example 1
Preparation of weight-reducing coffee
This comparative example is different from example 1 in that it contains only marine fish skin collagen oligopeptide prepared in example 1.
Comparative example 2
Preparation of weight-reducing coffee
This comparative example differs from example 1 in that only the medium chain triglyceride microcapsule powder prepared in example 1 was contained.
Comparative example 3
Preparation of weight-reducing coffee
This comparative example differs from example 1 in that it contained only the coffee powder prepared in example 1.
Comparative example 4
Preparation of weight-reducing coffee
This comparative example differs from example 1 in that no coffee powder was present.
Comparative example 5
Preparation of weight-reducing coffee
This comparative example differs from example 1 in that no medium chain triglyceride microcapsule powder was included.
Comparative example 6
Preparation of weight-reducing coffee
This comparative example differs from example 1 in that marine fish skin collagen oligopeptide was not contained.
Test example 1
Performance determination of marine fish skin collagen oligopeptide
Relative molecular mass distribution of marine fish skin collagen oligopeptide
The molecular mass distribution of the marine fish skin collagen oligopeptide prepared in example 1 was measured using high performance gel filtration chromatography. The measurement conditions were:
chromatographic column 7.8 × 300mm, 5 μm; mobile phase acetonitrile: water: trifluoroacetic acid ═ 45: 55: 0.1(V/V/V), flow rate 1 mL/min. Wherein the standard substances are cytochrome C (molecular weight of 12400Da), aprotinin (molecular weight of 6512Da), bacitracin (molecular weight of 1450Da), glycine-tryptophan-arginine (molecular weight of 451Da) and glycine-glycine (molecular weight of 189Da), respectively.
The measurement results are shown in Table 2. (2000Da filter membrane for the second filtration, will be composed of 2 ~ 10 amino acids called oligopeptide, the maximum amino acid molecular weight 201.213)
TABLE 2 Marine Fish skin collagen oligopeptide relative molecular mass distribution
As can be seen from Table 2, the marine fish skin collagen oligopeptide prepared by the preparation method of the embodiment 1-2 of the invention is mostly 180-500Da, and has smaller molecular weight, which indicates that the obtained oligopeptide has good quality. In addition, the ratio of the marine fish skin collagen oligopeptide prepared by the preparation method in example 1 in 180-500Da is much higher than that in example 2, which is probably because the addition of corilagin and baicalein can increase the hydrolysis degree of the marine fish skin collagen, and a peptide segment with smaller molecular weight is obtained.
II, marine fish skin collagen oligopeptide amino acid composition
The amino acid composition of the marine fish skin collagen oligopeptide obtained in example 1 was measured according to the method described in GB/T5009.124-2016 (determination of amino acids in food), and the measurement results are shown in Table 3.
TABLE 3 Marine Fish skin collagen oligopeptide amino acid composition
| Name of amino acid | Amino acid content (%) | Name of amino acid | Amino acid content (%) |
| Aspartic acid (Asp) | 5.26 | Lysine (Lys) | 3.17 |
| Serine (Ser) | 2.97 | Histidine (His) | 0.27 |
| Glycine (Gly) | 24.11 | Glutamic acid (Glu) | 10.31 |
| Tyrosine (Tyr) | 1.03 | Alanine (Ala) | 11.27 |
| Arginine (Arg) | 5.58 | Proline (Pro) | 0.79 |
| Leucine (Leu) | 3.70 | Cysteine (Cys) | 0.12 |
| Valine (Val) | 1.94 | Threonine (Thr) | 2.04 |
| Methionine (Met) | 2.03 | Phenylalanine (Phe) | 0.15 |
| Tryptophan (Trp) | 0.11 | Isoleucine (Ile) | 1.14 |
As can be seen from Table 3, the marine fish skin collagen oligopeptide is rich and reasonable in amino acid composition, contains essential amino acids for human body, and therefore has good health care function.
Third, determination of basic composition of marine fish skin collagen oligopeptide
The contents of protein, fat, water and ash in the marine fish skin collagen oligopeptide sample prepared in example 1 were measured according to GB/T5009.5-2016 (determination of protein in food), GB/T5009.6-2016 (determination of fat in food), GB/T5009.3-2016 (determination of water in food), and GB/T5009.4-2016 (determination of ash in food). The results were: 89.16% of protein, 0.22% of fat, 4.12% of water and 3.53% of ash.
Fourthly, determination of extraction rate of marine fish skin collagen oligopeptide
The degree of hydrolysis of the marine fish skin oligopeptide prepared in step 1 of examples 1 and 2 was determined by ninhydrin colorimetry, and the specific operating steps were as follows:
preparing a salmon skin complete hydrolysate: taking salmon skin powder 50mg, hydrolyzing at 120 ℃ for 24h under vacuum condition by using 10mL of hydrochloric acid with concentration of 6mol/L, and evaporating to remove hydrochloric acid after hydrolysis to obtain salmon skin complete hydrolysate;
drawing a standard curve: diluting the salmon skin complete hydrolysate with water to different concentrations to obtain standard solution, adding ninhydrin developer into the standard solution, measuring absorbance of the standard solution at 575nm, and drawing a standard curve using the measurement result to obtain y-0.7336 x +0.1876, R2＝0.997。
And (3) measuring the hydrolysis degree: respectively taking the solution obtained after the enzyme inactivation in the step 1 in the embodiment 1 and the embodiment 2 as a solution to be detected, adding 49mL of water into 1mL of the solution to be detected, uniformly mixing, adding 0.5mL of water into 1.5mL of water and 1mL of ninhydrin color developing agent, measuring the absorbance at 575nm, and calculating the hydrolysis degree, wherein the calculation formula of the hydrolysis degree is as follows:
degree of hydrolysis DH (%) (CW/1000) V1(50/V2) × 100
Wherein C is the mass concentration (g/L) of the liquid to be detected;
w is the mass (g) of the salmon skin powder;
v1 is the volume (mL) of salmon skin hydrolysate;
v2 is the volume of the solution to be measured (mL).
The measurement results are as follows: the degree of protein hydrolysis in example 1 was 30.52%, and the degree of hydrolysis in example 2 was 23.77%; the degree of proteolysis in example 1 is significantly higher than that in example 2, indicating that the enzyme mixture of example 1 with corilagin and baicalein added has the effect of increasing the degree of proteolysis.
Fifth, determination of capability of marine fish skin collagen oligopeptide to protect gastric mucosa
Taking 50 SPF SD rats, adaptively feeding for 3d, dividing into 5 groups, performing intragastric administration once with 2mL of 20mmol/L sodium deoxycholate for 1-4 groups every day, and performing intragastric administration once with 2mL of 50% alcohol for 3d for 8 weeks; feeding in 5 groups, and performing intragastric gavage with distilled water; after 8 weeks, 1-3 groups administered 100mg/kg marine fish skin collagen oligopeptide powder prepared in step 1 of example 1-2, dispersed in 3mL distilled water for intragastric administration, and 3, 4 groups administered distilled water for intragastric administration for 4 weeks; after 4 weeks, the mice were sacrificed and the gastric mucosa lesion area was measured after fasting for 12 hours after the last administration, and the percentage of the lesion area was calculated using a grid and the average value was recorded, and the measurement results are shown in table 4.
TABLE 4 area of gastric mucosal injury in rats
As can be seen from Table 4, the damaged area of the gastric mucosa of the mice fed with the marine fish skin collagen oligopeptide prepared in examples 1-2 is much smaller than that of the mice not fed with the marine fish skin collagen oligopeptide, and the damaged area is close to that of the mice fed with the marine fish skin collagen oligopeptide normally, which indicates that the marine fish skin collagen oligopeptide prepared by the invention has the effects of protecting the gastric mucosa and promoting the recovery of the gastric mucosa. In addition, it can be seen that the recovery effect of the marine fish skin collagen oligopeptide prepared in example 1 on the gastric mucosa is better than that of example 2, which is probably because the addition of corilagin and baicalein can increase the hydrolysis degree of the marine fish skin collagen, obtain a peptide segment with smaller molecular weight, and is more beneficial to the absorption of the stomach, thereby playing a better role in protecting the gastric mucosa.
Test example 2
Measurement of particle size distribution of medium-chain triglyceride microcapsules
The particle size distribution of the medium chain triglyceride micro-capsule powder in the mixed solution before spray drying in example 1 was measured using a laser particle scatterometer. The measurement results are shown in FIG. 2.
As can be seen from FIG. 2, the medium chain triglyceride microcapsule powder is mostly distributed around 10 μm, wherein about 84.61% of the medium chain triglyceride microcapsule powder distributed at 5-15 μm, which indicates that the size distribution of the medium chain triglyceride microcapsule powder particles is relatively uniform and concentrated.
Test example 3
First, the effect of diet coffee on body weight and triglycerides in blood of obese mice
Taking 65 SPF mice with the weight of 25 +/-2 g, adaptively feeding for 1 week, and randomly dividing the mice into 13 groups of 5 mice each; 1-12 groups of the feed are fed with high-fat feed, and the feed formula is as follows: 79.8% of basal feed, 0.2% of ox gall salt, 10% of lard oil, 10% of egg yolk powder and 13 groups of feeding basal feed. Continuously feeding for 30 days, performing intragastric administration, and injecting the weight-reducing coffee prepared in the example 1-3 into 1-3 groups with the injection amount of 100mg/kg, dispersing in 3mL distilled water, and injecting; 4. the weight-reducing coffee prepared in examples 4 and 5 was poured into 5 groups, the amount of the poured coffee was 100mg/kg, and the coffee was dispersed in 3mL of distilled water and poured; the weight-reducing coffee prepared in the comparative examples 1 to 6 was poured into 6 to 11 groups, the poured amount was 100mg/kg, and the coffee was dispersed in 3mL of distilled water and poured; 12. the 13 groups were filled with 3mL of distilled water. After continuous gavage for 30 days, fasting is carried out for 12 hours without water prohibition, the weight of each group of mice and triglyceride in blood are measured, the triglyceride in the blood is detected by using a mouse Triglyceride (TG) ELISA kit (purchased from Shanghai research Biotechnology Co., Ltd.) according to the operation steps of the instruction, and the average value is taken and recorded. The measurement results are shown in Table 5.
TABLE 5 measurement of body weight and triglyceride in blood of mice
As can be seen from Table 5, the results of the tests of the comparative groups 1-3 and 12 and 13 show that the diet coffee prepared in examples 1-3 has good weight loss and triglyceride reduction effects, and the weight and triglyceride content in blood of obese mice reach the level of normal mice; comparing the detection results of the groups 4, 5 and 1-3, it can be known that the weight-reducing coffee only containing the marine fish skin collagen peptide has poor weight-reducing effect, and the weight-reducing effect of the marine fish skin collagen oligopeptide can be deduced from other peptide segments contained in the marine fish skin collagen oligopeptide; comparing the test results of the groups 6-11 and the groups 12 and 13, it can be seen that the marine fish skin collagen oligopeptide, the medium chain triglyceride microcapsule powder and the coffee have certain effects of reducing weight and reducing the content of triglyceride in blood when used alone or in combination of two of the marine fish skin collagen oligopeptide, the medium chain triglyceride microcapsule powder and the coffee, but the effects are not as good as that of the three groups.
Second, the influence of weight-reducing coffee on the fat rate of human body
Selecting 9 adult subjects without cardiovascular diseases and lacking physical exercise experience, and randomly dividing the adult subjects into 3 groups, wherein the first group takes the weight-reducing coffee prepared in example 1, the second group takes the weight-reducing coffee prepared in example 4, and the third group takes the weight-reducing coffee prepared in example 5; taking weight-reducing coffee twice a day under the condition of not changing the original dietary habit, taking 8g each time, keeping for 4 months, not doing exercise, measuring and recording the changes of the weight, BMI and body fat rate of the subject. The measurement results are reported in table 6.
TABLE 6 changes in subject body weight, BMI, body fat Rate
As can be seen from table 6, the weight, BMI index and body fat percentage of 9 subjects were all decreased, indicating that the diet caffeine prepared by the present invention has the effects of reducing weight and body fat percentage, wherein the weight reduction of the first group of subjects, regardless of men and women, was much greater than that of the other two groups, while the weight reduction of the second group was less than that of the other two groups, indicating that the effect of adding marine fish skin collagen oligopeptide in the diet coffee is better than that of adding marine fish skin collagen peptide alone.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Jiangsu elastic animal life science and technology limited
<120> preparation method of marine fish skin collagen oligopeptide and weight-losing coffee based on marine fish skin collagen oligopeptide
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Leu Val Ala Gly Ala
1 5
Claims (10)
1. A marine fish skin collagen peptide has an amino acid sequence shown in SEQ ID NO. 1.
2. A marine fish skin collagen oligopeptide contains marine fish skin collagen peptide with amino acid sequence shown as SEQ ID NO. 1.
3. A method for preparing the marine fish skin collagen oligopeptide according to claim 2, which comprises the following steps:
dispersing marine fish skin powder in water, adjusting pH, adding an enzyme mixture, reacting at constant temperature, heating to inactivate enzyme after reaction, filtering, and drying to obtain collagen oligopeptide; wherein the enzyme mixture comprises neutral protease, papain, and trypsin.
4. The method for preparing marine fish skin collagen oligopeptide according to claim 3, wherein the method further comprises adding corilagin and baicalein.
5. The method for preparing marine fish skin collagen oligopeptide according to claim 4, wherein the weight ratio of the fish skin powder, water, neutral protease, papain, trypsin, corilagin and baicalein is as follows: 100, 500, 300, 1200, 0.1-0.5, 5-25; the constant temperature reaction time is 3-5 h.
6. Use of the marine fish skin collagen peptide according to claim 1 for the preparation of a medicament and/or health product and/or food for enhancing immunity and/or reducing subcutaneous fat.
7. Use of the marine fish skin collagen oligopeptide according to claim 3 in the preparation of medicaments and/or health products and/or foods for improving immunity and/or reducing subcutaneous fat.
8. A weight-reducing coffee is characterized by comprising any one of the following compositions (1), (2) and (3):
(1) marine fish skin collagen oligopeptide, medium chain triglyceride micro-capsule powder, coffee powder according to claim 2;
(2) marine fish skin collagen peptide, medium chain triglyceride micro-capsule powder, coffee powder according to claim 1;
(3) the marine fish skin collagen peptide according to claim 1, the marine fish skin collagen oligopeptide according to claim 2, the medium chain triglyceride micro-capsule powder, and the coffee powder.
9. The diet coffee of claim 8, wherein the medium chain triglyceride microencapsule powder comprises coconut oil, isomalto-oligosaccharide, casein, resistant dextrin, potassium citrate, mono-diglycerol fatty acid ester, silicon dioxide.
10. The weight-reducing coffee as claimed in claim 8, wherein the weight ratio of the weight-reducing coffee is as follows: 50-80 parts of medium chain triglyceride microcapsule powder, 20-35 parts of coffee powder and 5-10 parts of marine fish skin collagen oligopeptide.
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Country or region after: China Address after: C403-404, Xidong Chuangrong Building, No. 88 Danshan Road, Anzhen Street, Xishan District, Wuxi City, Jiangsu Province, 214000 Applicant after: Bullet beast life technology Wuxi Co.,Ltd. Address before: Room 401-404, block C, Xidong chuangrong building, No. 88, Danshan Road, anzhen street, Xishan District, Wuxi City, Jiangsu Province, 214000 Applicant before: Jiangsu bullet animal life Technology Co.,Ltd. Country or region before: China |