CN115725679A - Method for preparing small molecular collagen peptide powder from fresh animal skin by using whole-process biological enzyme method - Google Patents
Method for preparing small molecular collagen peptide powder from fresh animal skin by using whole-process biological enzyme method Download PDFInfo
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Abstract
The invention relates to a method for preparing small-molecule collagen peptide powder from fresh animal skins by using a whole-process biological enzyme method. Fresh animal skins are firstly soaked in water with the aid of enzyme, then enzyme unhairing, degreasing and softening treatment are carried out, then, the skins are crushed and then subjected to enzyme hydrolysis, the obtained mixture is subjected to centrifugal separation and degreasing, deep enzymolysis is carried out continuously, and finally, filtering, concentrating and spray drying are carried out to obtain micromolecular collagen peptide powder with the molecular weight within a 0.3-1.0kDa segment. Compared with the prior art of the same type, the method has the advantages of simple process, safety, small pollution and the like, and the prepared micromolecule collagen peptide powder has controllable molecular weight and especially prominent antioxidant activity and has better application prospect in various fields.
Description
Technical Field
The invention relates to the technical field of deep processing of agricultural and sideline products, in particular to a method for preparing small-molecular collagen peptide powder from fresh animal skins by using a whole-process biological enzyme method.
Background
China is a large meat consumption country, a large number of byproducts can be produced while meat is produced, animal skins are one of main byproducts, and 8000 or more than ten thousand pigskins, 2000 or more than ten thousand cowskins and nearly hundred million sheepskins can be provided every year. In addition to tanning the animal skins into leather for utilization, a new processing and utilization method is urgently needed.
The collagen is the main component of animal skin, is the protein with the highest content in the animal body, and accounts for more than 70% of the dry weight of tissues such as skin, tendon, cartilage and the like. It is estimated that there are a total of more than 500 million tons of collagen in nature, from lower invertebrates to higher mammals. Collagen is composed of 18 kinds of alpha-amino acids, two kinds of amino acids are contained in human milk protein, the content of the amino acids is much higher, and 8 kinds of amino acids essential for human body and 2 kinds of essential amino acids for promoting growth and development of children are basically covered. Therefore, high quality collagen can be used as a high-grade nutrient for the human body, and it has been proved safe to eat collagen and products thereof. At present, the collagen is more and more widely applied to high value-added industries such as food, medicine, cosmetics, biological fertilizer, biological pesticide and the like. With the improvement of living standard of people, the collagen with low fat and high protein and the products thereof are more and more interesting to consumers.
The development of technology has led to a variety of methods for preparing collagen, but the methods can be broadly divided into two methods, extraction from animal tissues and artificial bioengineering synthesis, and the former method is mainly used at present. Since the amount and type of collagen contained in different tissues are different, the raw materials used for extracting collagen from animal tissues are different. The currently used raw materials mainly comprise: (1) Skin tissues such as cow skin, pig skin, rat skin, rabbit skin, fish skin, etc.; (2) heel and leg, such as cow heel and leg, rat tail achilles tendon, etc.; (3) Bone and cartilage tissue, such as avian cartilage, porcine cartilage, bovine nasal cartilage, and the like; and (4) connective tissues such as placenta, sclera, and pig liver. Compared with other parts, the animal skin has high protein content, and the collagen content can reach more than 80 percent, so the animal skin becomes an important raw material for extracting the collagen.
The animal skin can be divided into two parts of a hair layer (fur quilt) and a skin layer (leather plate) in appearance, and all Mao Zongchen growing on the leather plate are the fur quilt. The weight of the fur layer in the sheepskin can account for more than 70 percent of the weight of the whole sheepskin, and particularly, the fur quilt is developed in northern cold regions. The collagen extracted from animal skin is mainly used in the skin layer, and the hair is removed as useless tissue in advance (i.e., depilation). Depilation is the separation of the hair from the cuticle by physical and chemical means and can also serve to recover the hair for valuable hair. The principle of clean production is inherited, the hair removal technology is developed through a long process, and the advantages and the disadvantages of the common animal skin hair removal method are shown in the table 1.
TABLE 1 comparison table of the quality of common depilation method
In recent years, with the increasing requirement for environmental protection, enzyme-based depilation has also gained attention in China, and has made a remarkable progress. The productivity of enzymes is continuously improved due to the rapid advance of biotechnology, and the production cost and price of enzymes are continuously reduced along with the continuous expansion of the application range. In short, enzymatic depilation is the most promising hair-protecting depilation method, has the advantages that other methods cannot be replaced, and is expected to become a clean depilation technology which can be widely applied, has good effect and low cost.
The extraction principle of collagen is as follows: by changing the external environment (temperature, salt concentration, pH), collagen with different characteristics can be separated from the raw material. Common collagen extraction methods comprise a hot water method, an acid method, an alkaline method, an enzymatic method, a salt method and the like, and collagen obtained by different extraction methods has different physicochemical properties. In recent years, researchers often combine various extraction methods to improve the extraction efficiency of collagen.
(1) Hot water method
The method comprises the steps of pretreating raw materials, and then soaking and boiling the raw materials with hot water under certain conditions to obtain the water-soluble collagen. Within a certain range, the extraction rate of collagen increases with the increase of the extraction temperature. However, under the condition of hot water extraction, the supercoiled space structure of the collagen is destroyed, and the collagen does not have the original physiological function of the collagen, so that the product prepared by the method is generally called gelatin and cannot be used as a medical biomaterial.
(2) Acid process
The principle of the method is that salt bonds and Schiff base structures among collagen molecules are unstable under low-concentration acidic conditions, so dilute acid solution can be adopted to prepare the collagen. The method needs to strictly control the concentration of acid, hydrolysis temperature, hydrolysis time and other important conditions, and prevent collagen from being hydrolyzed into amino acid completely and further damaged. The acid extraction method generally uses acidic substances such as formic acid, acetic acid, hydrochloric acid, lactic acid, malic acid, tartaric acid, citric acid and the like, and the extraction rate of collagen of different acids is different. In addition, although the method has short treatment time and rapid reaction, the product yield is low and the equipment corrosion is serious.
(3) Alkaline process
The method is mainly based on the fact that alkaline solution can have saponification reaction with fat on collagen, so that non-helical terminal peptide is cut off, and collagen fiber is dissolved out. Common alkali treatment agents include calcium hydroxide, sodium carbonate, and the like. Under alkaline conditions, the peptide bonds of collagen are very susceptible to hydrolysis, destroying its supercoiled structure. In addition, there has been little research on the extraction of collagen by alkaline methods, since alkaline extraction generates toxic and even carcinogenic and teratogenic D-amino acids in the case of over-hydrolysis.
(4) Enzyme method
The enzymatic extraction mainly utilizes protease to remove non-helical terminal peptide of collagen, so that the collagen is dissolved in acid or neutral salt solution. The collagen extracted by the enzyme method has mild immunological characteristics and can be widely used for biomedical materials. Commonly used enzymes include neutral protease, papain, trypsin, pepsin and the like, and the mixed use of a plurality of enzymes is the current research hotspot.
(5) Salt method
Salt extraction is generally suitable for newly synthesized collagen with low degree of cross-linking in tissues, and salt solution can negatively affect the conformational stability of the collagen, so that the salt extraction method is not suitable for industrial application. Common neutral salt solutions include sodium chloride, potassium chloride, sodium citrate, and the like.
(6) Other methods
The existing single extraction method has certain defects and shortcomings, so that different extraction methods are combined with auxiliary technical means such as microwaves, ultrasonic waves, high-voltage pulse electric fields and the like to obtain better extraction effect.
Compared with the prior art, the preparation method adopts the whole-course biological enzyme method to prepare the micromolecular collagen peptide powder, and has the advantages of safety, controllability, small pollution, stable product quality and the like.
Disclosure of Invention
One of the purposes of the invention is to provide a method for preparing small molecular collagen peptide powder from fresh animal skin by using a whole-process biological enzyme method, which comprises the following steps: (1) washing with water; (2) enzyme soaking treatment; (3) enzyme depilation treatment; (4) enzyme degreasing and enzyme softening treatment; (5) mincing; (6) carrying out enzymatic hydrolysis treatment; (7) carrying out centrifugal degreasing treatment; (8) deep enzymatic hydrolysis treatment; (9) Separating and purifying to obtain the micromolecular collagen peptide powder.
Further, the fresh animal skin is selected from at least one of pigskin, sheepskin, yak hide and cattle hide.
Further, after the fresh leather materials in the step (1) are cleaned by clean water, enzyme-assisted soaking treatment is carried out by using soaking complex enzyme H in the step (2). The soaking compound enzyme H is formed by mixing neutral protease, cellulase and lipase according to the mass ratio of (6-10) 1:1, and the adding amount of the soaking compound enzyme H is 0.2-0.5% of the weight of the leather before being cleaned by clear water.
Further, in the enzyme unhairing treatment process in the step (3), the compound unhairing enzyme P is added for enzyme unhairing. The composite depilatory enzyme P is formed by mixing neutral protease, alkaline protease and lipase according to the mass ratio of (10-20) to 1, and the addition amount of the composite depilatory enzyme P is 0.5-2% of the weight of the leather before being cleaned by clear water.
Further, compound degreasing softening enzyme Y is added in the enzyme degreasing and enzyme softening treatment process in the step (4) for enzyme unhairing, enzyme degreasing and enzyme softening. The composite degreasing softening enzyme Y is formed by mixing neutral protease, alkaline protease and lipase according to the mass ratio of (2-5) to 1, and the addition amount of the composite degreasing softening enzyme Y is 0.2-0.6 percent of the weight of the leather before being cleaned by clear water.
Further, the hydrolase A used in the enzymatic hydrolysis treatment in the step (6) is at least one selected from trypsin, alkaline protease and neutral protease, the addition amount of the hydrolase A is 5000-8000 u/g, the enzymolysis temperature is 40-60 ℃, and the enzymolysis time is 2-4 h.
Further, the oil and the protein liquid are separated by a disk centrifuge in the centrifugal degreasing treatment in the step (7), and the separated protein liquid is continuously processed in the next step.
Further, the hydrolase B used in the deep enzymatic hydrolysis treatment in the step (8) is at least one of alkaline protease, neutral protease and papain, the addition amount of the hydrolase B is 6000 to 15000u/g, the enzymolysis temperature is 45 to 65 ℃, and the enzymolysis time is 2 to 4 hours.
Further, the hydrolase B used in the deep enzymatic hydrolysis treatment in the step (8) is specifically an alkaline protease-neutral protease mixture in an equal mass ratio, a papain-neutral protease mixture in an equal mass ratio or a papain-alkaline protease mixture in an equal mass ratio.
Further, the specific process of the separation and purification in the step (9) is as follows: carrying out pressure filtration on the liquid obtained by deep enzymatic hydrolysis by using a filter press, and then filtering by using a 1.0kDa ultrafiltration membrane; continuously concentrating the filtrate after being filtered by the 1.0kDa nanofiltration membrane by using a 0.3kDa nanofiltration membrane; finally, the membrane concentrated solution is dried by spraying to obtain the micromolecule collagen peptide powder with the molecular weight of 0.3kDa-1.0 kDa.
Compared with the prior art, the invention has the advantages that the invention has the following aspects: 1) From the perspective of green and ecological product design, a green collagen peptide product with high added value is prepared by adopting a whole-process biological enzyme method; 2) Fresh and cut pigskin, cowhide, sheepskin and the like are used as raw materials, and the raw materials can be refrigerated and stored for later use, so that the pollution of the traditional salt pickling storage method to the environment is avoided, and the salt pollution in the production process is eliminated by 100%; 3) The fur quilt on the raw leather is effectively removed by adopting a safe and efficient enzyme unhairing technology, and the leather plate after the treatment is well reserved; 4) The extraction method of multiple times of enzymatic hydrolysis and membrane separation is adopted to prepare the small-molecule protein peptide with controllable molecular weight and outstanding antioxidant activity, and the defects of damage of chemical hydrolysis to nutrient substances in the leather and large molecular weight difference of collagen obtained by chemical hydrolysis are avoided.
Drawings
FIG. 1 is a graph showing a comparison of OH radical scavenging ability of samples of different molecular weight ranges;
FIG. 2 is a graph showing a comparison of the DPPH.radical scavenging ability of samples of different molecular weight ranges;
FIG. 3 is a graph of the sample pairs O for different molecular weight ranges 2 - Comparison of radical scavenging ability.
Detailed Description
In order to make those skilled in the art fully understand the technical solutions and advantages of the present invention, the following embodiments are further described.
The enzyme preparations used in the examples of the present invention are commercially available products, each having the following activities: alkaline protease, 20 ten thousand u/g; neutral protease, 10 ten thousand u/g; 10 ten thousand u/g of cellulase and 10 ten thousand u/g of lipase; papain, 10 ten thousand u/g; trypsin, 4000u/g.
Example 1
The raw material selected in this example is fresh pigskin, and the equipment used is a rotating drum.
Cleaning with clear water: washing with clean water at a total of 200% of the weight of the leather twice for 30min, and draining.
Enzyme-assisted soaking: adding clean water which is 200% of the weight of the leather material and 0.5% of soaking complex enzyme H (formed by mixing neutral protease, cellulase and lipase according to the mass ratio of 8.
Depilation: clean water and 2% of composite depilatory enzyme P (which are mixed by neutral protease, alkaline protease and lipase according to the mass ratio of 20.
Washing and filtering wool: draining water, filtering to remove hair, washing with water 200 wt% of the leather twice for 30min each time, and draining water.
Degreasing and softening: clean water and 0.2% of composite degreasing softening enzyme Y (which are formed by mixing neutral protease, alkaline protease and lipase according to the mass ratio of 2.
Washing with water: after draining, cleaning twice with clear water which is 200 percent of the weight of the leather material, each time for 30min, and draining after cleaning; then, the leather is washed twice with pure water which is 200 percent of the weight of the leather for 30min, and the water is drained after washing.
Initial enzymatic hydrolysis: the cleaned leather is stirred and put into an enzymolysis tank, then 5000u/g trypsin (calculated by the weight of the stirred leather) is added, and the mixture is stirred and enzymolyzed for 4 hours at the temperature of 45 ℃.
Centrifugal degreasing: and carrying out centrifugal separation and degreasing on the mixture obtained by enzymolysis to obtain macromolecular collagen liquid.
Deep enzymolysis: measuring the solid content of macromolecular collagen liquid, adding 6000u/g (calculated by solid content, the same below) of hydrolase B (prepared by mixing alkaline protease and neutral protease according to the mass ratio of 1:1), and deeply stirring at 65 ℃ for enzymolysis for 2h.
And (3) post-treatment: and after the deep enzymolysis is finished, the obtained liquid is subjected to pressure filtration, then a 1.0kDa ultrafiltration membrane is used for filtering, the filtrate after 1.0kDa filtration is continuously concentrated by a 0.3kDa nanofiltration membrane, and finally the membrane concentrated solution is subjected to spray drying to obtain the pigskin micromolecule collagen peptide powder with the molecular weight of 0.3kDa-1.0 kDa. And carrying out deep enzymolysis on the concentrated solution with the molecular weight of more than 1.0kDa again, filtering and concentrating to prepare the pigskin micromolecule collagen peptide powder with the molecular weight of 0.3-1.0 kDa.
Example 2
The raw materials that this embodiment chose for use are fresh yak hide, and the equipment that uses is the rotary drum.
Cleaning with clear water: washing with clean water at a total of 200% of the weight of the leather twice for 30min, and draining.
Enzyme-assisted soaking: adding clean water which is 200% of the weight of the leather material and 0.2% of soaking complex enzyme H (formed by mixing neutral protease, cellulase and lipase according to the mass ratio of 6.
Depilation: clean water and 2% of composite depilatory enzyme P (which are mixed by neutral protease, alkaline protease and lipase according to the mass ratio of 10.
Washing and filtering wool: draining water, filtering off removed hair, and washing with clear water which is 200% of the weight of the leather twice, each time for 30min, and draining water.
Degreasing and softening: sequentially adding clear water which is 100 percent of the weight of the leather and 0.6 percent of composite degreasing softening enzyme Y (which is formed by mixing neutral protease, alkaline protease and lipase according to the mass ratio of 5.
Washing with water: after draining, cleaning twice with clear water which is 200 percent of the weight of the leather material, each time for 30min, and draining after cleaning; then, the leather is washed twice with pure water which is 200 percent of the weight of the leather for 30min, and the water is drained after washing.
Initial enzymatic hydrolysis: the cleaned leather is stirred and put into an enzymolysis tank, then 8000u/g of hydrolase A (formed by mixing alkaline protease and trypsin according to the mass ratio of 1:2, and the adding amount is calculated by the weight of the stirred leather) is added, and the stirring and enzymolysis are carried out for 2 hours at the temperature of 55 ℃.
Centrifugal degreasing: and (4) carrying out centrifugal separation and degreasing on the mixture obtained by enzymolysis to obtain macromolecular collagen liquid.
Deep enzymolysis: measuring the solid content of macromolecular collagen liquid, adding 10000u/g of hydrolase B (prepared by mixing papain and neutral protease according to the mass ratio of 1:1), and deeply performing enzymolysis at 55 deg.C for 4h under stirring.
And (3) post-treatment: and after the deep enzymolysis is finished, the obtained liquid is subjected to filter pressing, then a 1.0kDa ultrafiltration membrane is used for filtering, the filtrate after 1.0kDa filtration is continuously concentrated by a 0.3kDa nanofiltration membrane, and finally the membrane concentrated solution is subjected to spray drying to obtain the yak skin micromolecule collagen peptide powder with the molecular weight of 0.3kDa-1.0kDa segment. And carrying out deep enzymolysis on the concentrated solution with the molecular weight of more than 1.0kDa again, filtering and concentrating to obtain the yak skin micromolecule collagen peptide powder with the molecular weight of 0.3-1.0 kDa.
Example 3
The raw material selected in this example was fresh kraft, and the equipment used was a rotating drum.
Weighing: weigh and record, and subsequently base on the weight of the hide.
Washing with water: washing with clean water at a total of 200% of the weight of the leather twice for 30min, and draining.
Enzyme assisted soaking: adding clean water which is 200% of the weight of the leather material and 0.2% of soaking complex enzyme H (formed by mixing neutral protease, cellulase and lipase according to the mass ratio of 10.
Depilation: clean water and 2% of composite depilatory enzyme P (which are mixed by neutral protease, alkaline protease and lipase according to the mass ratio of 20.
Washing and filtering wool: draining water, filtering to remove hair, washing with water 200 wt% of the leather twice for 30min each time, and draining water.
Degreasing and softening: clean water and 0.3% of composite degreasing softening enzyme Y (which are formed by mixing neutral protease, alkaline protease and lipase according to the mass ratio of 3.
Washing with water: after draining, cleaning twice with clear water which is 200 percent of the weight of the leather material, each time for 30min, and draining after cleaning; then, the leather was washed with pure water in an amount of 200% by weight of the leather in two portions, each for 30 minutes, and the water was drained.
Initial enzymatic hydrolysis: the cleaned leather materials are stirred and put into an enzymolysis tank, 7000u/g of hydrolase A (prepared by mixing neutral protease, trypsin and alkaline protease according to the mass ratio of 1.
Centrifugal degreasing: and carrying out centrifugal separation and degreasing on the mixture obtained by enzymolysis to obtain macromolecular collagen liquid.
Deep enzymolysis: measuring the solid content of macromolecular collagen protein solution, adding hydrolase B (prepared by mixing papain and alkaline protease at a mass ratio of 1:1) at 15000u/g, and stirring at 45 deg.C for deep enzymolysis for 3h.
And (3) post-treatment: and after deep enzymolysis, performing pressure filtration on the obtained liquid, filtering the obtained liquid by using a 1.0kDa ultrafiltration membrane, continuously concentrating the filtrate after 1.0kDa filtration by using a 0.3kDa nanofiltration membrane, and finally performing spray drying on the membrane concentrated solution to obtain the cowhide small molecular collagen peptide powder with the molecular weight of 0.3kDa-1.0kDa segment. And carrying out deep enzymolysis on the concentrated solution with the molecular weight of more than 1.0kDa again, filtering and concentrating to obtain the cow leather micromolecule collagen peptide powder with the molecular weight of 0.3-1.0 kDa.
In order to fully understand various properties of the prepared micromolecular collagen peptide powder, samples are respectively taken for antioxidant activity test. Taking the examples 2 and 3 as examples, the pretreatment process is unchanged, the obtained liquid is subjected to pressure filtration after deep enzymolysis is finished, then 2.0kDa and 1.0kDa ultrafiltration membranes are respectively used for filtering, the filtrate after 1.0kDa filtration is continuously concentrated by a 0.3kDa nanofiltration membrane, and finally, each membrane concentrated solution is subjected to spray drying to respectively obtain sections with molecular weight of more than 2.0kDa, 1.0 kDa-2.0 kDa and 0.3kDa-1.0kDa and deep enzymolysis yak skin or cowhide collagen peptide powder. The specific test methods for each sample were as follows:
1. OH free radical scavenging Capacity test
Preparing 1.0mmol/L o-diazaphenanthrene ethanol solution and 0.75mmol/L FeSO 4 ·7H 2 O solutionLiquid and 0.01% (v/v) H 2 O 2 And (3) solution. Sequentially adding 1.0mL of 1.0mmol/L o-phenanthroline ethanol solution, 2.0mL of 0.5mol/L PBS with pH =7.4, and 1.0mL of 0.75mmol/L FeSO into a beaker 4 ·7H 2 O and 0.5mL of sample to be tested, and finally adding 1.0mL of H with the concentration of 0.01% (v/v) 2 O 2 And placing the obtained test solution in a water bath kettle at 37 ℃ for constant temperature for 60min, and taking out to measure the absorbance Aj at 536 nm. And (4) replacing the sample to be detected with deionized water, and repeating the operation, wherein the absorbance is recorded as Ai. Replacing the sample to be detected and H with deionized water 2 O 2 The above procedure was repeated and the absorbance was recorded as Ao. OH radical clearance was calculated according to equation 1-1.
2. DPPH scavenging ability test
Ethanol with the mass fraction of 95% is used for preparing DPPH solution with the concentration of 0.2 mmol/L. 4.0mL of the prepared DPPH solution, 4.0mL of PBS (with a pH of = 6.86) (the concentration is the same as above) and 0.5mL of the sample solution to be tested are mixed and shaken up. Storing the obtained mixture at room temperature in dark place for 30min, and measuring the light absorption value at 517nm as Ax; replacing a sample to be detected with 0.5mL of deionized water, and determining the absorbance value of the sample to be detected by the same method and marking as Ac; the absorbance value was measured in the same manner using 95% ethanol instead of DPPH solution and was recorded as Ay. DPPH.radical scavenging ratio was calculated according to equation 1-2.
3. O is 2 - Free radical scavenging ability test
Preparing a Tris-HCl buffer solution with the pH of 8.2 and the concentration of 50mmol/L, adding 5.6mL of the Tris-HCl buffer solution into 0.2mL of a sample to be detected, uniformly mixing, and placing the obtained mixture at 25 ℃ for heat preservation for 10-20 min to serve as a reagent A. Preparing pyrogallol solution with concentration of 3mmol/L (solvent is HCl of 10 mmol/L), and taking 0.2mLAnd placing the pyrogallol solution at 25 ℃ for 10-20 min to serve as a reagent B. The reagent A and the reagent B were mixed quickly and shaken up, and the slope Ks of the change in absorbance at 320nm was measured every 30 seconds. And (4) replacing the sample solution to be detected with deionized water in the control group, and determining the change slope Kc of the absorbance. O is 2 - The radical clearance was calculated according to equations 1-3.
The small molecular collagen peptides of the cow leather and the yak skin prepared in the embodiment 2-3 are respectively filtered by ultrafiltration membranes with the molecular weight cut-off of 2.0kDa, 1.0kDa and 0.3kDa, and three components are sequentially obtained. The antioxidant properties of the collagen peptides (i.e. samples to be tested) of the three different molecular weight segments were detected and contrastively analyzed with reference to the above method.
1. Scavenging ability of active peptide with different molecular weight segments to OH free radical
FIG. 1 shows the results of the activity test of samples with different molecular weight ranges for scavenging OH radicals. As can be seen from FIG. 1, the peptide fragments with different molecular weights after ultrafiltration all show certain OH free radical antioxidant capacity, and the peptide fragments with different molecular weights have different OH free radical scavenging capacities; the clearance rate of the ox skin collagen polypeptide and yak skin collagen polypeptide with 0.3kDa-1.0kDa segment in the three components to OH free radical is the highest. This indicates that the collagen peptide having a smaller molecular weight has better anti-OH radical activity.
2. DPPH-free radical scavenging ability of active peptides with different molecular weight segments
FIG. 2 shows the result of detecting the DPPH-free radical scavenging activity of samples with different molecular weight ranges. As can be seen from FIG. 2, the peptide fragments with different molecular weights after ultrafiltration all show certain DPPH free radical antioxidant capacity; and with the increase of molecular weight, the anti-DPPH free radical oxidation activity of the cattle/yak skin collagen polypeptide is obviously reduced.
3. Active peptide pair O with different molecular weight segments 2 - Scavenging ability of free radical
FIG. 3 is a graph of the molecular weight band for O 2 - Results of measurement of radical scavenging activity. As can be seen from FIG. 3, the three components of the collagen polypeptide of the cattle/yak skin obtained by ultrafiltration resist O with the increase of the molecular weight 2 - A significant reduction in free radical oxidation activity; the 0.3-1.0kDa segment of the collagen polypeptide pair of the cattle/yak skin of the three components 2 - The radical clearance was highest.
In conclusion, the collagen polypeptides of the cowhide and yak skin with the 0.3-1.0kDa segment have the smallest molecular weight and the strongest antioxidant activity, and have the effects of resisting DPPH, OH and O 2 - The scavenging activity of the three free radicals is: bovine hide collagen polypeptide: 39.75%, 62.1%, 68.2%, yak collagen polypeptide: 43.95%, 77.57%, 71.75%. Researches show that in the preparation process of the antioxidant active peptide of the collagen of the cow leather/yak skin, a method is needed to screen out a hydrolysate with a molecular weight range of 0.3-1.0 kDa.
Claims (10)
1. The method for preparing the small molecular collagen peptide powder from the fresh animal skin by using the whole-process biological enzyme method is characterized by comprising the following steps: (1) washing with water; (2) enzyme soaking treatment; (3) enzyme depilation treatment; (4) enzyme degreasing and enzyme softening treatment; (5) mincing; (6) carrying out enzymatic hydrolysis treatment; (7) carrying out centrifugal degreasing treatment; (8) deep enzyme hydrolysis treatment; and (9) separating and purifying to obtain the micromolecule collagen peptide powder.
2. The method of claim 1, wherein: the fresh animal skin is selected from at least one of pigskin, sheepskin, yak skin and cattle hide.
3. The method of claim 1, wherein: after the fresh leather materials in the step (1) are cleaned by clear water, adding soaking complex enzyme H in the step (2) for enzyme-assisted soaking treatment; the soaking compound enzyme H is formed by mixing neutral protease, cellulase and lipase according to the mass ratio of (6-10) 1:1, and the adding amount of the soaking compound enzyme H is 0.2-0.5% of the weight of the leather before being cleaned by clear water.
4. The method of claim 1, wherein: in the enzyme unhairing treatment process in the step (3), the compound unhairing enzyme P is added for unhairing, the compound unhairing enzyme P is formed by mixing neutral protease, alkaline protease and lipase according to the mass ratio of (10-20) to 1, and the adding amount of the compound unhairing enzyme P is 0.5-2% of the weight of the leather material before being cleaned by clear water.
5. The method of claim 1, wherein: and (3) adding a compound degreasing softening enzyme Y for degreasing and softening in the enzyme degreasing and enzyme softening treatment process in the step (4), wherein the compound degreasing softening enzyme Y is formed by mixing neutral protease, alkaline protease and lipase according to the mass ratio of (2-5) to 1, and the adding amount of the compound degreasing softening enzyme Y is 0.2-0.6% of the weight of the leather material before being cleaned by clear water.
6. The method of claim 1, wherein: the hydrolase A used in the enzymatic hydrolysis treatment in the step (6) is selected from at least one of trypsin, alkaline protease and neutral protease, the addition amount of the hydrolase A is 5000-8000 u/g, the enzymolysis temperature is 40-60 ℃, and the enzymolysis time is 2-4 h; the hydrolase B used in the deep enzymatic hydrolysis treatment in the step (8) is at least one of alkaline protease, neutral protease and papain, the addition amount of the hydrolase B is 6000 to 15000u/g, the enzymolysis temperature is 45 to 65 ℃, and the enzymolysis time is 2 to 4 hours.
7. The method of claim 1, wherein: and (5) in the step (7), oil and protein liquid in the mixture are separated by adopting a disc centrifuge in the centrifugal degreasing treatment, and the separated protein liquid is continuously subjected to the next treatment.
8. The method of claim 6, wherein: and (3) carrying out deep enzymatic hydrolysis treatment on the hydrolase B in the step (8), wherein the hydrolase B is specifically an alkaline protease-neutral protease mixture with an equal mass ratio, a papain-neutral protease mixture with an equal mass ratio, or a papain-alkaline protease mixture with an equal mass ratio.
9. The method of claim 1, wherein: the specific process of the separation and purification in the step (9) is as follows: and (3) filtering the liquid obtained by deep enzymatic hydrolysis in the step (8) by using a filter press, then filtering by using a 1.0kDa ultrafiltration membrane, continuously concentrating the filtrate by using a 0.3kDa nanofiltration membrane, and finally spray-drying the membrane concentrated solution to obtain the micromolecule collagen peptide powder with the molecular weight of 0.3-1.0kDa segment.
10. A micromolecular collagen peptide powder is characterized in that: the small molecule collagen peptide powder is prepared according to any one of the methods in claims 1 to 9.
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