AU2021101626A4 - Multi-stage enzymolysis method and use of animal proteins - Google Patents
Multi-stage enzymolysis method and use of animal proteins Download PDFInfo
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Abstract
The present disclosure relates to a multi-stage enzymolysis method and use of animal proteins,
and belongs to the technical field of proteolysis. The multi-stage enzymolysis method of animal
proteins according to the present disclosure includes the following steps: pretreating an animal
source raw material; mixing a resulting slurry with water and subjecting a resulting mixture to
hydrolysis; and subjecting a primary hydrolyzed slurry successively to a first-stage biochemical
degradation, a second-stage biochemical degradation, a third-stage biochemical degradation, a
fourth-stage biochemical degradation, and a fifth-stage biochemical degradation; and filtering a
resulting slurry to obtain a protein product from multi-stage enzymolysis. Compared with existing
enzymolysis methods of animal proteins, the present disclosure has the advantages of stable
enzymolysis effect, high molecular weight uniformity, high target molecule yield, low relative cost,
and so on.
Description
The present disclosure relates to the technical field of proteolysis, and in particular to a multi
stage enzymolysis method and use of animal proteins.
With the development and extensive use of biotechnologies, it is very common to acquire natural nutritional raw material by extracting specific natural substances from natural animal and plant bodies. Plant extracts are mostly obtained by subcritical, supercritical, alcohol, or ether extraction solutions, which can efficiently isolate specific characteristic molecules. Animal extracts are mainly obtained by the enzymolysis technology and fermentation technology. A common enzymolysis technology focuses on activity and average analysis amount without considering molecular weight distribution (MWD).
Taking collagen production as an example, it is difficult to achieve a uniform and stable MWD in conventional large-scale production, which limits the purification and extraction of small molecule products and thus limits the development of the small molecule protein market. In many studies, it is impossible to realize industrialized transformation and technology development due to the failure to obtain a large number of products with an accurate molecular weight.
The present disclosure is intended to provide a multi-stage enzymolysis method and use of animal proteins. Compared with existing enzymolysis methods of animal proteins, the present disclosure has the advantages of stable enzymolysis effect, high molecular weight uniformity, high target molecule yield, low relative cost, and so on.
The present disclosure provides a multi-stage enzymolysis method of animal proteins, including the following steps:
1) subjecting an animal source raw material successively to physical shredding, crushing, and wall-breaking with a colloid mill to obtain a slurry;
2) mixing the slurry obtained in step 1) with water, stirring a resulting mixture and heating to °C, and conducting hydrolysis at a constant temperature for 3 h to obtain a primary hydrolyzed slurry;
3) adjusting a temperature of the primary hydrolyzed slurry in step 2) to 50°C to 54°C and adjusting a pH to 8.0 to 9.0; adding an alkaline protease and stirring a resulting mixture; and conducting reaction at a constant temperature for 20 min to 50 min to obtain a first-stage biochemically-degraded slurry; where, the alkaline protease is added at a mass 0.02% to 0.04% of a mass of the animal source raw material;
4) after a pH of thefirst-stage biochemically-degraded slurry in step 3) becomes 7.5, adding the alkaline protease at 50°C to 53°C and stirring a resulting mixture; and conducting reaction at a constant temperature for 20 min to 40 min to obtain a second-stage biochemically-degraded slurry; where, the alkaline protease is added at a mass 0.02% to 0.05% of the mass of the animal source raw material;
5) after a pH of the second-stage biochemically-degraded slurry in step 4) becomes 6.8 to 7.2, adding papain at 50°C to 53°C and stirring a resulting mixture; and conducting reaction at a constant temperature for 30 min to 40 min to obtain a third-stage biochemically-degraded slurry; where, the papain is added at a mass 0.03% to 0.06% of the mass of the animal source raw material;
6) after a pH of the third-stage biochemically-degraded slurry in step 5) becomes 6.4 to 6.8, adding the papain at 50°C and stirring a resulting mixture; and conducting reaction at a constant temperature for 20 min to 30 min to obtain a fourth-stage biochemically-degraded slurry; where, the papain is added at a mass 0.03% to 0.04% of the mass of the animal source raw material;
7) after a pH of the fourth-stage biochemically-degraded slurry in step 6) becomes 5.5 to 6.0, adding an acidic protease at 45°C to 50°C and stirring a resulting mixture; and conducting reaction at a constant temperature for 30 min to 50 min to obtain a fifth-stage biochemically-degraded slurry; where, the acidic protease is added at a mass 0.05% to 0.08% of the mass of the animal source raw material; and
8) after a pH of the fifth-stage biochemically-degraded slurry in step 7) becomes 4.0 to 7.0, filtering a resulting slurry to obtain a protein product from multi-stage enzymolysis.
Preferably, the animal source raw material may include animal residues left over from agricultural and fishery processing.
Preferably, the animal residues left over from agricultural and fishery processing may include offal residues from slaughter plants and small fish and shrimps and trash fish caught at fishery quays; and the offal residues from slaughter plants may include chicken intestines left over from chicken-killing and/or fish intestines and scraps left over from fish-killing in aquatic product plants.
Preferably, in step 2), the slurry and water may be mixed at a mass ratio of 1:1.
Preferably, in step 3), the temperature may be adjusted by mixing the primary hydrolyzed slurry with cold water.
Preferably, in step 8), the filtration may include fabric filtration or plate and frame pressure filtration, and a residue may be removed by filtering through a filter screen or a filter cloth.
Preferably, in step 8), the filtration may be conducted 2 times, with 100 mesh for the first time and 200 mesh for the second time.
Preferably, the alkaline protease may have enzyme activity of 200,000 U/g.
Preferably, the papain may have enzyme activity of 200,000 U/g.
Preferably, the acidic protease may have enzyme activity of 100,000 U/g.
The present disclosure provides a multi-stage enzymolysis method of animal proteins. The present disclosure adopts step-by-step enzymolysis and a distributed control system (DCS) to improve the control for enzymolysis, which changes the understanding of enzymolysis processes and brings a fundamental change to traditional enzymolysis process ideas. The present disclosure is a brand-new attempt and exploration. The present disclosure pays more attention to MWD, and improves the molecular weight uniformity of enzymolysis and increases a yield of target molecular fragments through more accurate control to the progress and intensity of enzymolysis. Moreover, in the present disclosure, a stepped or staged enzymolysis process is accurately used to better realize industrial production. Specifically, on the basis of traditional techniques, the present disclosure adopts 3 enzymes, multi-stage control, and simple equipment to achieve accurate control of biological enzymolysis, which improves enzyme activity and reduces a side reaction rate of enzymolysis to improve the stability of an enzymolysis effect. With multi-stage enzymolysis, enzymatic cleavage sites for protein molecules show a high synchronization rate, and a yield of target molecules is increased by about 20%. Moreover, due to the stepped or staged enzymolysis, an enzyme amount used is reduced, resulting in a reduced production cost.
Therefore, the multi-stage enzymolysis system of the present disclosure can effectively produce protein peptides with specified characteristics (a specific molecular weight range), and thus can be used in a wide range of fields, such as agricultural feed additives, organic fertilizers, and nutritional extracts.
General
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. None of the cited material or the information contained in that material should, however be understood to be common general knowledge.
The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products and methods are clearly within the scope of the invention as described herein.
Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.
FIG. 1 shows the optimal pH control curve provided in the present disclosure;
FIG. 2 shows the optimal temperature control curve provided in the present disclosure; and
FIG. 3 shows the optimal enzyme dosage control curve provided in the present disclosure.
The present disclosure provides a multi-stage enzymolysis method of animal proteins, including the following steps:
1) subjecting an animal source raw material successively to physical shredding, crushing, and wall-breaking with a colloid mill to obtain a slurry;
2) mixing the slurry obtained in step 1) with water, stirring a resulting mixture and heating to
°C, and conducting hydrolysis at a constant temperature for 3 h to obtain a primary hydrolyzed slurry;
3) adjusting a temperature of the primary hydrolyzed slurry in step 2) to 50°C to 54°C and adjusting a pH to 8.0 to 9.0; adding an alkaline protease and stirring a resulting mixture; and conducting reaction at a constant temperature for 20 min to 50 min to obtain a first-stage biochemically-degraded slurry; where, the alkaline protease is added at a mass 0.02% to 0.04% of a mass of the animal source raw material;
4) after a pH of the first-stage biochemically-degraded slurry in step 3) becomes 7.5, adding the alkaline protease at 50°C to 53°C and stirring a resulting mixture; and conducting reaction at a constant temperature for 20 min to 40 min to obtain a second-stage biochemically-degraded slurry; where, the alkaline protease is added at a mass 0.02% to 0.05% of the mass of the animal source raw material;
5) after a pH of the second-stage biochemically-degraded slurry in step 4) becomes 6.8 to 7.2, adding papain at 50°C to 53°C and stirring a resulting mixture; and conducting reaction at a constant temperature for 30 min to 40 min to obtain a third-stage biochemically-degraded slurry; where, the papain is added at a mass 0.03% to 0.06% of the mass of the animal source raw material;
6) after a pH of the third-stage biochemically-degraded slurry in step 5) becomes 6.4 to 6.8, adding the papain at 50°C and stirring a resulting mixture; and conducting reaction at a constant
temperature for 20 min to 30 min to obtain a fourth-stage biochemically-degraded slurry; where, the papain is added at a mass 0.03% to 0.04% of the mass of the animal source raw material;
7) after a pH of the fourth-stage biochemically-degraded slurry in step 6) becomes 5.5 to 6.0, adding an acidic protease at 45°C to 50°C and stirring a resulting mixture; and conducting reaction at a constant temperature for 30 min to 50 min to obtain a fifth-stage biochemically-degraded slurry; where, the acidic protease is added at a mass 0.05% to 0.08% of the mass of the animal source raw material; and
8) after a pH of the fifth-stage biochemically-degraded slurry in step 7) becomes 4.0 to 7.0, filtering a resulting slurry to obtain a protein product from multi-stage enzymolysis.
In the present disclosure, an animal source raw material is subjected successively to physical shredding, crushing, and wall-breaking with a colloid mill to obtain a slurry. In the present disclosure, the animal source raw material may preferably include animal residues left over from agricultural and fishery processing. In the present disclosure, the animal residues left over from agricultural and fishery processing may preferably include offal residues from slaughter plants and small fish and shrimps and trash fish caught at fishery quays; and the offal residues from slaughter plants may preferably include chicken intestines left over from chicken-killing and/or fish intestines and scraps left over from fish-killing in aquatic product plants. The present disclosure has no specific limitation on a method of the physical shredding, crushing, and wall-breaking with a colloid mill, and a conventional method of physical shredding, crushing, and wall-breaking with a colloid mill well known by a person skilled in the art may be adopted. The crushing allows the raw material to be more fully hydrolyzed, which can shorten hydrolysis time and adjust errors.
In the present disclosure, after a slurry is obtained, the slurry is mixed with water, a resulting mixture is stirred and heated to 75°C, and then hydrolysis is conducted at a constant temperature for 3 h to obtain a primary hydrolyzed slurry. In the present disclosure, the slurry and water may be mixed at a mass ratio preferably of 1:1. In the present disclosure, hydrolysis conditions are set to reduce the nutrient loss caused by excessive hydrolysis of proteins during the hydrolysis process.
In the present disclosure, after a primary hydrolyzed slurry is obtained, a temperature of the primary hydrolyzed slurry is adjusted to 50°C to 54°C and a pH is adjusted to 8.0 to 9.0; an alkaline protease is added and a resulting mixture is stirred; and reaction is conducted at a constant temperature for 20 min to 50 min to obtain a first-stage biochemically-degraded slurry; where, the alkaline protease is added at a mass 0.02% to 0.04% and preferably 0.026% of a mass of the animal source raw material. In the present disclosure, the temperature may be adjusted preferably by mixing the primary hydrolyzed slurry with cold water. In the present disclosure, after the mixing, a resulting mixture may preferably be thoroughly stirred. The present disclosure has no specific limitation on a method for adjusting the pH, and a conventional pH adjustment method may be adopted, such as adjusting with a hydrochloric acid or sodium hydroxide solution. In the present disclosure, during the whole multi-stage enzymolysis method of animal proteins, the manual pH adjustment is conducted only once. In the present disclosure, the temperature may more preferably be 53.8°C, the pH may more preferably be 8.8, and the reaction at a constant temperature may be conducted more preferably for 40 min. A preferred solution of the present disclosure can avoid excessive dissociation of protein molecules during an biochemical enzymolysis process through actual measurement, and plays a decisive role in the final control of increasing a content of target protein peptides within a specific molecular weight range (the specific molecular weight and the specific molecular weight range in the present disclosure refers to the molecular weight or molecular weight range of protein peptides with specified biological functions and activities, such as 470 Daltons, 490 to 520 Daltons, and 690 to 740 Daltons, and these protein peptides can inhibit the growth of microorganisms, quickly enter cell macromolecule pathways, and so on) in a product. In the present disclosure, the alkaline protease may have enzyme activity preferably of 200,000
U/g.
In the present disclosure, after a pH of the first-stage biochemically-degraded slurry is 7.5, the alkaline protease is added at 50°C to 53°C and a resulting mixture is stirred; and reaction is conducted at a constant temperature for 20 min to 40 min to obtain a second-stage biochemically degraded slurry; where, the alkaline protease is added at a mass 0.02% to 0.05% of the mass of the animal source raw material. When the pH is 7.5, it reflects a first-stage protein dissociation of 60% to 65%, achieving a target progress, and a second-stage enzymolysis is introduced at this point to control the stability of specific molecular weight protein peptide output in a final product and avoid excessive degradation, thus avoiding the runaway of subsequent multi-stage enzymolysis caused by excessive degradation. In the present disclosure, the alkaline protease may have enzyme activity preferably of 200,000 U/g. In the present disclosure, after two alkaline protease treatments, enough hydrogen ions can be released from cleavage sites of proteolysis, so that the pH of the solution returns to a neutral zone beyond the optimal zone for alkaline protease reaction. Moreover, in the present disclosure, the two alkaline protease enzymolysis treatments are conducted to effectively avoid excessive addition of the alkaline protease, thus avoiding the instantaneous local contact of a substrate with a large amount of high-concentration enzymes added (it takes time to thoroughly stir). A local high enzyme content will cause excessive enzymolysis of partial proteins and finally result in too different enzymolysis progresses of all proteins. Moreover, excess alkaline proteases can only show an unsatisfactory reaction rate and effect in the neutral zone and will not play a due role, resulting in a waste of biological enzymes. The alkaline protease enzymolysis of the present disclosure requires a reduced biological enzyme dosage and shows an improved enzymolysis effect.
In the present disclosure, after a pH of the second-stage biochemically-degraded slurry becomes 6.8 to 7.2, papain is added at 50°C to 53°C and a resulting mixture is stirred; and reaction is conducted at a constant temperature for 30 min to 40 min to obtain a third-stage biochemically degraded slurry; where, the papain is added at a mass 0.03% to 0.06% of the mass of the animal source raw material. In the present disclosure, according to the design purpose of increasing target proteins with a specific molecular weight, a third-stage biochemical degradation is started when a measured pH value becomes 6.8 to 7.2. In the present disclosure, an enzyme amount added may preferably be determined according to the following method and a pH value: the lower the pH, the larger the reaction progress and the greater the number of small molecule proteins, in which case, a lower enzyme amount and a shorter reaction time are required for the third-stage biological enzymolysis to avoid excessive enzymolysis. In the present disclosure, the papain may have enzyme activity preferably of 200,000 U/g.
In the present disclosure, after a pH of the third-stage biochemically-degraded slurry becomes 6.4 to 6.8, papain is added at 50°C and a resulting mixture is stirred; and reaction is conducted at a constant temperature for 20 min to 30 min to obtain a fourth-stage biochemically-degraded slurry; where, the papain is added at a mass 0.03% to 0.04% of the mass of the animal source raw material. In the present disclosure, the papain may have enzyme activity preferably of 200,000 U/g. In the present disclosure, the papain is added in batches to avoid excessive enzymolysis caused by too much papain added at a time, which not only reduces a required amount of biological enzymes, but also improves the control of an enzymolysis process, thereby avoiding the protein peptide fragmentation in a final product. In the present disclosure, papain enzymolysis is conducted twice so that a reaction process can be monitored to avoid local excessive enzymolysis. Moreover, an enzymolysis process is controlled by adding the biological enzyme in batches to improve the control of enzyme consumption, which avoids the situation where an enzymolysis reaction needs to be terminated through enzyme inactivation due to excessive addition of enzymes in a traditional enzymolysis process. Therefore, the present disclosure can improve the overall reaction accuracy and reduce the usage of papain, which requires a biological enzyme amount that is 10% to 15% less than that of traditional composite enzymolysis.
In the present disclosure, after a pH of the fourth-stage biochemically-degraded slurry becomes 5.5 to 6.0, an acidic protease is added at 45°C to 50°C and a resulting mixture is stirred; and reaction is conducted at a constant temperature for 30 min to 50 min to obtain a fifth-stage biochemically-degraded slurry; where, the acidic protease is added at a mass 0.05% to 0.08% of the mass of the animal source raw material. In the present disclosure, the acidic protease may have enzyme activity preferably of 100,000 U/g. After the first four stages of biochemical degradation are completed, main proteins in the slurry have been degraded into small and medium molecular weight (1,500 to 3,500 Daltons) polypeptides and a pH of the reaction system has entered a slightly-acidic range, at which point, the alkaline protease and papain do not have the optimal biological activity and reaction conditions, so the acidic protease is needed to complete the final part of the enzymolysis process. Given that the acidic protease shows high activity under acidic conditions and protein polypeptides show high stability under acidic conditions, excessive enzymolysis can be avoided and the present disclosure adopts one-step acidic protease degradation to complete the final stage of enzymolysis. The key for the control lies in the control of temperature and time. In the present disclosure, the setting of enzymolysis temperature and time can ensure that a final product has a relatively-concentrated MWD, and can further reduce the usage of biological enzymes.
In the present disclosure, after a pH of the fifth-stage biochemically-degraded slurry becomes
4.0 to 7.0, a resulting slurry is filtered to obtain a protein product from multi-stage enzymolysis. In the present disclosure, the filtration may preferably include fabric filtration or plate and frame pressure filtration, and a residue may be removed by filtering through a filter screen or a filter cloth. In the present disclosure, the filtration may be conducted 2 times, with preferably 100 mesh for the first time and preferably 200 mesh for the second time. The setting offiltration conditions in the present disclosure allows to efficiently filter out impurities. The protein product obtained from multi-stage enzymolysis in the present disclosure is rich in protein nutrition and is very easy to breed bacteria, causing corruption of a protein liquid. Subsequently, the product of the present disclosure can preferably be processed into a corresponding product in combination with anti corrosion technology; and the product, if provided to a downstream plant as a semi-finished product, can be stored and transported by freezing and other means to avoid deterioration of a finished product.
The present disclosure adopts a first-stage to a fifth-stage of enzymolysis with alkaline protease, papain, and acidic protease to realize full enzymolysis of animal proteins and better cleave proteins. Moreover, the specific setting of enzymolysis conditions allows the present disclosure to avoid excessive enzymolysis, and the method of adding in batches can achieve the optimal enzymolysis effect for each enzyme. The process of using alkaline protease, papain, and acidic protease successively can avoid the destruction of a reaction system due to adjusting the pH of the reaction solution twice or multiple times (in the present disclosure, after enzymolysis is started, a reaction process is tracked according to the change of pH and step-by-step enzymolysis is conducted by changing the enzyme amount, temperature, and reaction time, where, the substrate changes with the control of the reaction process and the pH changes according to the reaction process). In the present disclosure, the setting of the pH range is an important data index for monitoring a reaction process. In the multi-stage enzymolysis method of the present disclosure, the pH is continuously reduced through the continuous release of amino acid termini during the enzymolysis process to form hydrogen ions in the solution. In the present disclosure, after a pH of the primary hydrolyzed slurry is adjusted manually, there is no need to adjust the pH subsequently, and the pH is used as an index for indicating when to change the temperature and enzymolysis conditions. The present disclosure also avoids problems caused by multiple pH adjustments such as a too cumbersome process, a reduced fault tolerance rate, and inconvenience to industrial production control. The present disclosure retains a fault tolerant workspace for workers to a certain extent and is more convenient for the initial training of workers in industrial production.
The protein product obtained from multi-stage enzymolysis in the present disclosure is rich in natural polypeptides and small peptides (preferably small molecule peptides composed of 2 to 10 amino acids with a molecular weight of 350 Daltons to 1,880 Daltons, protein peptide molecules with the molecular weight can be directly absorbed by cells through special channels on the cell membrane); includes a variety of natural free amino acids required by plants and animals (left handed amino acids: glycine, lysine, alanine, leucine, isoleucine, phenylalanine, glutamate, valine, aspartic acid, proline, hydroxyproline, etc.); and also includes some natural minerals (mainly mineral elements among the 17 essential elements necessary for plants other than nitrogen, phosphorus, sulfur, carbon, hydrogen, oxygen, and chlorine: calcium, magnesium, zinc, iron, boron, copper, potassium, molybdenum, manganese, and nickel; and a small amount of silicon and sodium). As determined, a proteolysis solution has a protein polypeptide content (calculated based organic nitrogen) < 210 mg/L, a free amino acid content < 120 mg/L, and a trace element content (magnesium, zinc, calcium, boron, and the like) > 20 mg/L, which can be used as a feed additive, a raw material for organic fertilizers, a raw material for nutritional protein products, etc.
Compared with the traditional proteolysis technology that adopts a fuzzy average molecular weight description, the present disclosure has the following technical advantage: a concentrated distribution of small molecule protein peptides can be better controlled so that an overall content of small molecule proteins with superior biological functions in a product is substantially increased. The control method in the present disclosure includes: controlling a pH at the start of the reaction, starting the next stage of enzymolysis at a key time point determined according to the pH change during the multi-stage enzymolysis process, and accurately controlling the temperature, time, and enzyme amount for each stage of enzymolysis, which involves the accumulation and actual measurement of a large amount of production data and experimental data. For example, an initial pH of 9.0 in the example enables a reduced minimum molecular weight of protein molecules in a final product, that is, smaller peptide molecules can be obtained; the second-stage and third-stage enzymolysis allows the process to be stable so that excessive enzymolysis can be avoided to leave enough room for the fourth-stage and fifth-stage enzymolysis; and the enzyme amount and enzymolysis time at the final stage determine the size of final macromolecular protein peptides, that is, the content and molecular weight of macromolecular proteins in a final product are controlled.
The multi-stage enzymolysis method and use of animal proteins according to the present disclosure will be further described in detail below with reference to specific examples. The technical solutions of the present disclosure include, but are not limited to, the following examples.
Example 1
Raw material: 100 kg of fish intestines.
Pretreatment: the animal source raw material was subjected to physical shredding, crushing, and wall-breaking with a colloid mill to obtain a slurry.
Primary hydrolysis: water was added to the slurry at a mass 1 time that of the slurry, a resulting mixture was thoroughly stirred and heated to 75°C, and hydrolysis was conducted at a constant temperature for 3 h.
First-stage biochemical degradation: an appropriate amount of cold water was added, a resulting slurry was thoroughly stirred to make the slurry at 50°C, and a pH was adjusted to 9.0; an alkaline protease was added at a mass 0.04% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 40 min.
Second-stage biochemical degradation: a pH of the slurry was adjusted to 7.5 and a temperature was adjusted to 53°C; the alkaline protease was added at a mass 0.05% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 30 min.
Third-stage biochemical degradation: a pH of the slurry was adjusted to 6.8 and a temperature was adjusted to 50°C; papain was added at a mass 0.05% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for min.
Fourth-stage biochemical degradation: a pH of the slurry was adjusted to 6.4 and a temperature was adjusted to 50°C; the papain was added at a mass 0.03% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 30 min.
Fifth-stage biochemical degradation: a pH of the slurry was adjusted to 6.0 and a temperature was adjusted to 48°C; an acidic protease was added at a mass 0.08% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 40 min.
Filtration: fabric filtration or plate and frame pressure filtration was used to filter out a residue through a filter screen or a filter cloth.
Blending: a pH was adjusted to 5.0, and an appropriate preservative was added or a pasteurizer was used for preservative treatment.
Bottle filling and warehousing: a resulting product was filled in bottles, packaged and sealed according to specifications, and then warehoused.
As detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS), the product had an MWD of 365 to 1,740 Daltons and a relative average molecular weight of 1,204 Daltons.
Example 2
Raw material: 100 kg of minced chicken (including skin and cartilage).
Pretreatment: the animal source raw material was subjected to physical shredding, crushing, and wall-breaking with a colloid mill to obtain a slurry.
Primary hydrolysis: water was added to the slurry at a mass 1 time that of the slurry, a resulting mixture was thoroughly stirred and heated to 75°C, and hydrolysis was conducted at a constant temperature for 3 h.
First-stage biochemical degradation: an appropriate amount of cold water was added, a resulting slurry was thoroughly stirred to make the slurry at 53°C, and a pH was adjusted to 9.0; an alkaline protease was added at a mass 0.04% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 50 min.
Second-stage biochemical degradation: a pH of the slurry was adjusted to 7.5 and a temperature was adjusted to 53°C; the alkaline protease was added at a mass 0.05% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 40 min.
Third-stage biochemical degradation: a pH of the slurry was adjusted to 7.0 and a temperature was adjusted to 50°C; papain was added at a mass 0.06% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for min.
Fourth-stage biochemical degradation: a pH of the slurry was adjusted to 6.4 and a temperature was adjusted to 50°C; the papain was added at a mass 0.04% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 30 min.
Fifth-stage biochemical degradation: a pH of the slurry was adjusted to 5.7 and a temperature was adjusted to 48°C; an acidic protease was added at a mass 0.08% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 40 min.
Filtration: fabric filtration or plate and frame pressure filtration was used to filter out a residue through a filter screen or a filter cloth.
Blending: a pH was adjusted to 4.5, and an appropriate preservative was added or a pasteurizer was used for preservative treatment.
Bottle filling and warehousing: a resulting product was filled in bottles, packaged and sealed according to specifications, and then warehoused.
As detected by HPLC-MS, the product had an MWD of 560 to 1,520 Daltons and a relative average molecular weight of 1,132 Daltons.
Example 3
Raw material: 100 kg of small trash fish and minced fish.
Pretreatment: the animal source raw material was subjected to physical shredding, crushing, and wall-breaking with a colloid mill to obtain a slurry.
Primary hydrolysis: water was added to the slurry at a mass 1 time that of the slurry, a resulting mixture was thoroughly stirred and heated to 75°C, and hydrolysis was conducted at a constant temperature for 3 h.
First-stage biochemical degradation: an appropriate amount of cold water was added, a resulting slurry was thoroughly stirred to make the slurry at 54°C, and a pH was adjusted to 8.5; an alkaline protease was added at a mass 0.03% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 30min.
Second-stage biochemical degradation: a pH of the slurry was adjusted to 7.5 and a temperature was adjusted to 52°C; the alkaline protease was added at a mass 0.04% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 25 min.
Third-stage biochemical degradation: a pH of the slurry was adjusted to 7.0 and a temperature was adjusted to 50°C; papain was added at a mass 0.05% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for min.
Fourth-stage biochemical degradation: a pH of the slurry was adjusted to 6.8 and a temperature was adjusted to 50°C; the papain was added at a mass 0.03% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 25 min.
Fifth-stage biochemical degradation: a pH of the slurry was adjusted to 5.8 and a temperature was adjusted to 46°C; an acidic protease was added at a mass 0.06% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 40 min.
Filtration: fabric filtration or plate and frame pressure filtration was used to filter out a residue through a filter screen or a filter cloth.
Blending: a pH was adjusted to 4.2, and an appropriate preservative was added or a pasteurizer was used for preservative treatment.
Bottle filling and warehousing: a resulting product was filled in bottles, packaged and sealed according to specifications, and then warehoused.
As detected by HPLC-MS, the product had an MWD of 455 to 1180 Daltons and a relative average molecular weight of 670 Daltons.
Example 4
Raw material: 100 kg of fish skin and scales.
Pretreatment: the animal source raw material was subjected to physical shredding, crushing, and wall-breaking with a colloid mill to obtain a slurry.
Primary hydrolysis: water was added to the slurry at a mass 4 times that of the slurry, a resulting mixture was thoroughly stirred and heated to 95°C, and hydrolysis was conducted at a constant temperature for 2 h.
Biochemical degradation: a temperature was adjusted to 56°C and a pH was adjusted to 8.5; then a collagenase was added at a mass 0.4% of the mass of the raw material, and a resulting mixture was thoroughly stirred; and reaction was conducted at a constant temperature for 2 h.
Enzyme inactivation: a reaction solution was heated to a temperature above 80°C and then stirred at a constant temperature for 15 min.
Decolorization: active carbon was added at a mass ratio of 1.5% and a resulting mixture was stirred at a constant temperature for 35 min to fully mix materials.
Filtration: fabric filtration or plate and frame pressure filtration was used to filter out a residue through a filter screen or a filter cloth.
Blending: a pH was adjusted to 6.5, and an appropriate preservative was added or a pasteurizer was used for preservative treatment.
Bottle filling and warehousing: a resulting product was filled in bottles, packaged and sealed according to specifications, and then warehoused.
As detected by HPLC-MS, the product had an MWD of 260 to 6830 Daltons and a relative average molecular weight of 1560 Daltons.
Contrastive analysis of the examples shows that the method of the present disclosure well solves the problem of selectivity for different proteins in a protein engineering process, well controls an MWD range of proteolysis, improves a yield of target small molecule protein peptides, and reduce the consumption of biological enzymes, which is more applicable to industrial treatment. That is, Examples 1 to 3 were products prepared using the method of the present disclosure, and molecular weight results obtained by mass spectrometry (MS) were shown in Table 1. Example 4 was an existing common collagen extraction test. Contrastive analysis shows that the present disclosure can achieve a more accurate enzymolysis result and a higher target protein peptide content through multi-stage enzymolysis. Moreover, a molecular weight can be controlled and adjusted by controlling the enzymolysis conditions at different stages, so as to achieve the directional production of a product. For example, in Example 3, as small molecule protein extraction, the product has the lowest average molecular weight, and the process requires the smallest total enzyme consumption and the optimal cost.
Table 1 Contrastive analysis of MS results for Examples 1 to 4
Example 1 Example 2 Example 3 Example 4
Method method in the method in the method in the general collagen
present disclosure present disclosure present disclosure method
Protein type fish intestine protein muscle protein compound fish collagen
protein
MWD 365 D to 1,740 D 560 D to 1,520 D 455 D to 1,180 D 260 D to 6,830 D
Average 1,204 D 1,132 D 670 D 1,560 D
molecular weight
Biological 250 g 270 g 200 g 400 g
enzyme dosage
Comparative Example 1
The same operating conditions as in Example 3 were adopted except that different pH values, temperatures, enzyme dosages, or animal source materials were set, and different change curves were obtained through determination. A product with a more desired molecular weight can be obtained under conditions for the optimal cost. The pH was used as a monitorable index to determine a reaction process, thereby controlling the reaction process to ensure accurate progress of an enzymolysis process, and the optimal pH control curve was shown in FIG. 1. Similarly, the temperature was used as a monitorable index, and the optimal temperature control curve was
shown in FIG. 2. The enzyme dosage was used as a monitorable index, and the optimal enzyme dosage control curve was shown in FIG. 3. The animal source raw material was used as a monitorable index, and it was finally concluded that the optimal treating material was fish offal.
The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.
Claims (8)
1. A multi-stage enzymolysis method of animal proteins, comprising the following steps:
1) subjecting an animal source raw material successively to physical shredding, crushing, and wall-breaking with a colloid mill to obtain a slurry;
2) mixing the slurry obtained in step 1) with water, stirring a resulting mixture and heating to °C, and conducting hydrolysis at a constant temperature for 3 h to obtain a primary hydrolyzed slurry;
3) adjusting a temperature of the primary hydrolyzed slurry in step 2) to 50°C to 54°C and adjusting a pH to 8.0 to 9.0; adding an alkaline protease and stirring a resulting mixture; and conducting reaction at a constant temperature for 20 min to 50 min to obtain a first-stage biochemically-degraded slurry; wherein, the alkaline protease is added at a mass 0.02% to 0.04% of a mass of the animal source raw material;
4) after a pH of the first-stage biochemically-degraded slurry in step 3) becomes 7.5, adding the alkaline protease at 50°C to 53°C and stirring a resulting mixture; and conducting reaction at a constant temperature for 20 min to 40 min to obtain a second-stage biochemically-degraded slurry; wherein, the alkaline protease is added at a mass 0.02% to 0.05% of the mass of the animal source raw material;
5) after a pH of the second-stage biochemically-degraded slurry in step 4) becomes 6.8 to 7.2, adding papain at 50°C to 53°C and stirring a resulting mixture; and conducting reaction at a
constant temperature for 30 min to 40 min to obtain a third-stage biochemically-degraded slurry; wherein, the papain is added at a mass 0.03% to 0.06% of the mass of the animal source raw material;
6) after a pH of the third-stage biochemically-degraded slurry in step 5) becomes 6.4 to 6.8, adding the papain at 50°C and stirring a resulting mixture; and conducting reaction at a constant
temperature for 20 min to 30 min to obtain a fourth-stage biochemically-degraded slurry; wherein, the papain is added at a mass 0.03% to 0.04% of the mass of the animal source raw material;
7) after a pH of the fourth-stage biochemically-degraded slurry in step 6) becomes 5.5 to 6.0, adding an acidic protease at 45°C to 50°C and stirring a resulting mixture; and conducting reaction at a constant temperature for 30 min to 50 min to obtain a fifth-stage biochemically-degraded slurry; wherein, the acidic protease is added at a mass 0.05% to 0.08% of the mass of the animal source raw material; and
8) after a pH of the fifth-stage biochemically-degraded slurry in step 7) becomes 4.0 to 7.0, filtering a resulting slurry to obtain a protein product from multi-stage enzymolysis.
2. The multi-stage enzymolysis method according to claim 1, wherein, the animal source raw material comprises animal residues left over from agricultural and fishery processing; the animal residues left over from agricultural and fishery processing comprise offal residues from slaughter plants and small fish and shrimps and trash fish caught at fishery quays; and the offal residues from slaughter plants comprise chicken intestines left over from chicken-killing and/or fish intestines and scraps left over from fish-killing in aquatic product plants.
3. The multi-stage enzymolysis method according to claim 1, wherein, in step 2), the slurry and water are mixed at a mass ratio of 1:1;
wherein, in step 3), the temperature is adjusted by mixing the primary hydrolyzed slurry with cold water.
4. The multi-stage enzymolysis method according to claim 1, wherein, in step 8), the filtration comprises fabric filtration or plate and frame pressure filtration, and a residue is removed by filtering through a filter screen or a filter cloth;
wherein, in step 8), the filtration is conducted 2 times, with 100 mesh for the first time and 200 mesh for the second time.
5. The multi-stage enzymolysis method according to claim 1, wherein, the alkaline protease has enzyme activity of 200,000 U/g; the papain has enzyme activity of 200,000 U/g; the acidic protease has enzyme activity of 100,000 U/g.
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