CN107556364B - Method for extracting abalone protein peptide by subcritical water assisted enzymolysis and product - Google Patents
Method for extracting abalone protein peptide by subcritical water assisted enzymolysis and product Download PDFInfo
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Abstract
The invention discloses a method for extracting abalone protein peptide by subcritical water assisted enzymolysis and a product thereof. The technology of the invention improves the traditional technology for preparing protein peptide by enzymolysis, adopts subcritical water to assist enzymolysis to extract abalone protein peptide, not only can reduce the usage amount of enzyme, but also can greatly shorten the production time and reduce the production cost. In addition, the prepared abalone protein peptide has good antioxidant and antitumor functions, and provides conditions for application and popularization of the abalone protein peptide.
Description
Technical Field
The invention relates to a technology for extracting abalone protein peptide by subcritical water assisted enzymolysis and product development thereof, in particular to a method for extracting abalone protein peptide by subcritical water assisted enzymolysis and a product thereof.
Background
The subcritical water is also called pressure hot water, the temperature of the pressure hot water is 100-374 ℃, and the water is kept in a liquid state by controlling the pressure of a system. The subcritical water has low dielectric constant and is rich in hydrogen ions and hydroxyl ions, so that the subcritical water has an acid-base catalysis function and can rapidly decompose organic matters. Compared with the traditional extraction method, the method for extracting by using subcritical water has the advantages of low cost, low energy consumption, high extraction rate of active ingredients, high extraction speed, economy, environmental protection and the like. On the other hand, in the abalone processing and production process, a large amount of abalone viscera are inevitably generated, and account for about 25% of the abalone weight. The abalone viscera is rich in protein and polysaccharide, but is mainly used for producing products with low added value, such as seasonings, feeds and the like at present. Although the utilization of an enzymolysis mode to extract abalone viscera active substances is reported at home and abroad, the applicant also discloses a preparation method for coproducing abalone polysaccharide, lipid and protein peptide by adopting an enzymolysis mode at the earlier stage (Chinese patent publication No. CN 103255186A), but the problems of high production cost and the like exist in practical application due to long enzymolysis time, low extraction rate and the like.
Therefore, the abalone viscera are extracted by subcritical water, the extracted protein is subjected to enzymolysis, then the abalone protein peptide is refined by activated carbon decoloration and column chromatography, the abalone protein peptide prepared by spray drying has good antioxidation and anticancer functions, and the prepared abalone protein peptide is used for developing functional health-care products.
Disclosure of Invention
The invention aims to provide a method for extracting abalone protein peptides by subcritical water assisted enzymolysis, which is a technology for preparing the abalone protein peptides with bioactivity by using abalone viscera as raw materials and mainly adopting high-temperature and high-pressure subcritical water assisted treatment and enzymolysis to extract proteins and then developing functional health-care products by using the extracted abalone protein peptides.
The invention also aims to provide a product prepared by subcritical water-assisted enzymolysis extraction of abalone protein peptide, which has good functional activity and mouthfeel.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention relates to a subcritical water assisted enzymolysis extraction method of abalone protein peptides, which comprises the following steps of (1) subcritical water treatment, adding purified water into abalone viscera according to a material-liquid ratio of 1 ׃ 3-1 ׃ 5 (w/v), extracting for 15 min-60 min under a subcritical water state, cooling to room temperature, removing filter residues by using a stainless steel filter screen to obtain protein extract, drying the filter residues in the sun to obtain seaweed fertilizer, (2) enzymolysis, adding complex enzyme into the protein extract obtained in the step (1), wherein the addition of the complex enzyme is 0.1-0.5% (w/v) of the protein extract, performing enzymolysis reaction for 1 h-3 h, separating, concentrating and spray drying, refining the protease obtained in the step (2) by using activated carbon hydrolysate and resin, removing fishy smell, removing heavy metals, removing arsenic and the like, concentrating, performing vacuum concentration and spray drying to prepare abalone protein peptide powder, and (4) preparing a product, namely adding β -cyclodextrin, collagen peptide, soybean lecithin, olive green pulp extract and honey into the extracted protein peptide powder, uniformly mixing and preparing a chewable tablet by adding magnesium stearate, uniformly mixing and pressing into a chewable tablet product with a mass percent of 1-5%.
In the step (1), the subcritical water is water with the temperature of 120-160 ℃ and the pressure of 1.0-7.0 MPa.
In the step (2), the compound enzyme refers to the compound of any protease of papain, flavourzyme, neutral protease, bromelain and the like and pectinase, and the compound ratio of the protease to the pectinase is 3: 1-5: 1; .
In the step (2), the enzymolysis condition of the complex enzyme is pH 5.0-7.0 and the temperature is 40-50 ℃.
In the step (3), the activated carbon decoloration and deodorization conditions are as follows: adjusting the pH value of the protein enzymolysis liquid to 5.0-6.0, adding active carbon in an amount of 0.5-2.0% (w/v) of the volume of the protease enzymolysis liquid, preserving the heat at 40-50 ℃ for 15-60 min, and removing the active carbon by using a disc centrifuge; the conditions for removing fishy smell, heavy metal and arsenic by the resin are as follows: the mixed filler consists of macroporous adsorption resin, chelate resin for adsorbing heavy metal and chelate fiber for adsorbing arsenic, and the mixing ratio of the macroporous adsorption resin to the chelate resin for adsorbing heavy metal to the chelate fiber for adsorbing arsenic is 7:2: 1; the pH value of the protein enzymolysis liquid is 5.0-6.0, and the residence time of the protein enzymolysis liquid in the mixed filler is 15-30 min.
In the step (4), the product particles comprise, by weight, 20% -30% of abalone protein peptide powder, 55% -70% of β -cyclodextrin, 2% -10% of collagen peptide, 3% -5% of soybean lecithin, 1.0% -2.5% of green olive pulp extract and 0.5% -2.0% of honey.
The abalone viscera is one of fresh abalone viscera, frozen abalone viscera or heat-treated abalone viscera.
The invention relates to a product prepared by subcritical water assisted enzymolysis extraction of abalone protein peptide, which comprises, by weight, 20% -30% of abalone protein peptide powder, 55% -70% of β -cyclodextrin, 2% -10% of collagen peptide, 3% -5% of soybean lecithin, 1.0% -2.5% of green olive pulp extract and 0.5% -2.0% of honey.
By adopting the scheme, because subcritical water is adopted to extract protein in abalone viscera, and then protein peptide is prepared by an enzymolysis method, the using amount of enzyme and enzymolysis time can be reduced, and the temperature and pressure of the subcritical water adopted by the invention can not enable the indigestible and complete seaweed in the abalone viscera to be dissolved, the subsequent separation of enzymolysis liquid does not need to be carried out through a ceramic membrane and an ultrafiltration membrane, the production time and the production cost of abalone protein peptide can be greatly shortened, moreover, the seaweed in the abalone viscera is digested by the abalone to a certain degree, the filter residue after protein extraction can be directly used as seaweed fertilizer by drying in the sun, and the emission of waste is reduced, meanwhile, because protease hydrolysate is decolorized and deodorized by activated carbon, and is further decolorized, deodorized by heavy metal and dearsenized by mixed resin in a deep level, the quality of the product can be improved, the safety of the product can be ensured, on the other hand, the concern of people to health gradually extends from diseases to a sub-health state along with the deterioration of environment, the acceleration of life rhythm and the improvement of the health care of the taste of the collagen peptide, the collagen peptide and the collagen peptide added into a small collagen peptide tablet, so that people can easily absorb the collagen peptide from the small collagen peptide, the collagen peptide-vitamin E β, and the collagen peptide, the collagen peptide has the collagen peptide, the collagen peptide has the advantages of the collagen peptide, the:
1. the method utilizes abalone viscera to extract abalone protein peptides, and residues can be used for producing seaweed fertilizer, so that high-efficiency comprehensive utilization can be achieved, and environmental pollution is reduced.
2. The method adopts subcritical water to assist enzymolysis to extract the abalone protein peptide, so that the use amount of enzyme can be reduced, the production time can be greatly shortened, and the production cost can be further reduced.
3. The abalone protein peptide prepared by the invention has good oxidation resistance and anti-tumor functions.
4. The product prepared from the abalone protein peptide has enhanced functional activity, is not easy to absorb moisture, has good taste and is easy to popularize and apply in practice.
The invention is further described with reference to the following figures and specific examples.
Description of the figures
Fig. 1 is a diagram of the in vitro antioxidant activity of the abalone protein peptide produced by the present invention.
FIG. 2 is a graph showing the effect of abalone protein peptide produced by the present invention on the survival rate of breast cancer MDA-MB-231 cells.
Table 1 shows the basic components of the abalone protein peptides produced by the present invention.
Table 2 shows the molecular weight distribution of the basic components of the abalone protein peptides produced by the present invention.
Detailed Description
The method comprises the following steps:
example 1
The invention relates to a method for extracting abalone protein peptide by subcritical water assisted enzymolysis, which comprises the following steps:
(1) subcritical water treatment, namely adding 0.4 kg of abalone viscera and 1.2L kg of purified water into a high-temperature reaction kettle, rapidly increasing the temperature and the pressure of the reaction kettle to 120 ℃ and 7.0MPa, keeping the temperature and the pressure for 60min, cooling to room temperature, releasing pressure, and filtering with gauze to obtain protein extract and filter residue;
(2) and (2) enzymolysis, namely adding 1.0g of papain and 0.2g of pectinase into the protein extract liquid of about 1.2L obtained in the step (1), and carrying out enzymolysis reaction for 1 hour under the condition that the pH value is 5.0 and the temperature is 50 ℃.
(3) Separation, concentration and spray drying: adding 6 g of activated carbon into the protease hydrolysate obtained in the step (2), preserving the heat at 50 ℃ for 60min, and then removing the activated carbon by using a disk centrifuge; cooling to normal temperature, confirming pH and adjusting to 5.0, performing decolorization, deodorization, heavy metal removal and arsenic removal through a column chromatography column with mixed filler (the mixing ratio is 7:2:1) consisting of macroporous adsorption resin, heavy metal adsorption chelating resin and arsenic adsorption chelating fiber, enabling the liquid to stay in the column chromatography column for 15min, performing desalination and concentration by using a nanofiltration membrane with the molecular weight cutoff of 150 Da until the solid concentration is 10%, performing vacuum concentration until the solid concentration is 30%, and finally performing spray drying to prepare the abalone protein peptide powder;
(4) the production method comprises the steps of adding and uniformly mixing 20% of abalone protein peptide powder, β -cyclodextrin 70%, collagen peptide 5%, soybean lecithin 3%, green olive pulp extract 1.0% and honey 1.0% into the extracted abalone protein peptide powder, preparing into granules, adding magnesium stearate accounting for 1% of the mass of the granules, uniformly mixing, and pressing into chewable tablets.
Example 2
The invention relates to a method for extracting abalone protein peptide by subcritical water assisted enzymolysis, which comprises the following steps:
(1) subcritical water treatment, namely adding 0.3 kg of abalone viscera and 1.2L kg of purified water into a high-temperature reaction kettle, rapidly increasing the temperature and the pressure of the reaction kettle to 140 ℃ and 4.0 MPa, keeping the temperature and the pressure for 40 min, cooling to room temperature, releasing pressure, and filtering with gauze to obtain protein extract and filter residue;
(2) and (2) enzymolysis, namely adding 4.5g of flavourzyme and 1.5g of pectinase into the protein extract liquid of about 1.2L obtained in the step (1), and carrying out enzymolysis reaction for 3 hours at the pH of 7.0 and the temperature of 45 ℃.
(3) Separation, concentration and spray drying: adding 12 g of activated carbon into the protease hydrolysate obtained in the step (2), preserving the heat at 45 ℃ for 15min, and then removing the activated carbon by using a disc centrifuge; cooling to normal temperature, confirming pH and adjusting to 7.0, performing decolorization, deodorization, heavy metal removal and arsenic removal through a column chromatography column with mixed filler (the mixing ratio is 7:2:1) consisting of macroporous adsorption resin, heavy metal adsorption chelating resin and arsenic adsorption chelating fiber, enabling the liquid to stay in the column chromatography column for 35min, performing desalination and concentration by using a nanofiltration membrane with the molecular weight cutoff of 150 Da until the solid concentration is 10%, performing vacuum concentration until the solid concentration is 30%, and finally performing spray drying to prepare the abalone protein peptide powder;
(4) the production method comprises the steps of adding 30% of abalone protein peptide powder, 60% of β -cyclodextrin, 2% of collagen peptide, 5% of soybean lecithin, 2.5% of olive pulp extract and 0.5% of honey into the extracted abalone protein peptide powder, uniformly mixing, preparing into granules, adding magnesium stearate accounting for 3% of the mass of the granules, uniformly mixing, and pressing into chewable tablets.
Example 3
The invention relates to a method for extracting abalone protein peptide by subcritical water assisted enzymolysis, which comprises the following steps:
(1) subcritical water treatment, namely adding 0.3 kg of abalone viscera and 1.5L kg of purified water into a high-temperature reaction kettle, rapidly increasing the temperature and the pressure of the reaction kettle to 160 ℃ and 1.0 MPa, keeping the temperature and the pressure for 15min, cooling to room temperature, releasing pressure, and filtering with gauze to obtain protein extract and filter residue;
(2) and (2) enzymolysis, namely adding 3.2g of bromelain and 0.8g of pectinase into the protein extract liquid obtained in the step (1), and carrying out enzymolysis reaction for 2 hours at the pH of 6.0 and the temperature of 40 ℃.
(3) Separation, concentration and spray drying: adding 28 g of activated carbon into the protease hydrolysate obtained in the step (2), preserving the heat at 40 ℃ for 30min, and then removing the activated carbon by using a disk centrifuge; cooling to normal temperature, confirming pH and adjusting to 6.0, performing decolorization, deodorization, heavy metal removal and arsenic removal through a column chromatography column with mixed filler (the mixing ratio is 7:2:1) consisting of macroporous adsorption resin, heavy metal adsorption chelating resin and arsenic adsorption chelating fiber, enabling the liquid to stay in the column chromatography column for 60min, performing desalination and concentration by using a nanofiltration membrane with the molecular weight cutoff of 150 Da until the solid concentration is 10%, performing vacuum concentration until the solid concentration is 30%, and finally performing spray drying to prepare the abalone protein peptide powder;
(4) the production method comprises the steps of adding 27% of abalone protein peptide powder, β -cyclodextrin 55%, collagen peptide 10%, soybean lecithin 4%, green olive pulp extract 2% and honey 2% into the extracted abalone protein peptide powder, uniformly mixing, preparing into granules, adding magnesium stearate accounting for 5% of the mass of the granules, uniformly mixing, and pressing into chewable tablets.
II, product
The invention relates to a product prepared by subcritical water assisted enzymolysis extraction of abalone protein peptide, which comprises, by weight, 20% -30% of abalone protein peptide powder, 55% -70% of β -cyclodextrin, 2% -10% of collagen peptide, 3% -5% of soybean lecithin, 1.0% -2.5% of green olive pulp extract and 0.5% -2.0% of honey.
The filter residue produced by extracting the protein from the abalone viscera with subcritical water can be directly used as the seaweed fertilizer after being dried in the sun.
The physical and chemical properties of the abalone protein peptide produced by the invention are analyzed as follows:
subcritical water has higher concentration of H+And OH-Has strong decomposition capability on organic matters such as protein, polysaccharide and the like in the abalone viscera. This application makes the albumen of abalone viscera can dissolve out through control pressure and temperature, but the complete marine alga of undigested can not take place to dissolve and can get rid of through the filter screen filtration, and this filter residue can directly regard as the fertile use of marine alga through the sun-drying in addition, can not produce the discarded object. The protein content of the protein peptide prepared by carrying out compound enzyme enzymolysis on the dissolved protein by protease and pectinase is as high as about 65%, but the protein peptide also contains a small amount of polysaccharide (table 1), which is mainly that abalone live on ingestion of seaweed and plankton, so that the viscera also contain polysaccharide and metabolites thereof besides abundant protein. The molecular weight of the prepared abalone protein peptide is detected, and the result shows that the molecular weight of the protein in the abalone peptide is mainly distributed in 180 Da-1000 Da, the protein peptide with high abundance is distributed in 180 Da-500 Da (table 2), and the obtained abalone protein peptide is mainly small peptide consisting of 2-4 amino acids. Oligopeptides, particularly dipeptides or tripeptides, are reported to be readily absorbed and utilized by the human body through the small intestinal mucosa. Therefore, the abalone protein peptide prepared by the method is easy to be absorbed by the human body, so that the abalone protein peptide can exert the biological activity function.
The hydroxyl radical scavenging ability and DPPH radical scavenging ability of the abalone protein peptide are detected, and the result shows thatIC50The value is very low (figure 1), and can approach typical small peptide glutathione (hydroxyl radical scavenging capability IC) with antioxidant activity50Value = 2.46; DPPH free radical scavenging ability IC50The value =0.72) indicates that the abalone protein peptide prepared by the method has good antioxidant activity, and according to the influence of the abalone protein peptide on the proliferation inhibition of the breast cancer MDA-MB-231 cells (figure 2), the survival rate of the breast cancer MDA-MB-231 cells is seen to be in a descending trend along with the increase of the content of the abalone protein peptide in the culture solution, when the concentration of the abalone protein peptide in the culture solution is 4 mg/m L, the survival rate of the breast cancer MDA-MB-231 cells is only 40% -50%, and the results indicate that the proliferation inhibition effect of the abalone protein peptide on the breast cancer cells is remarkable (the concentration of the abalone protein peptide in the culture solution is 4 mg/P<0.05) And is dose dependent.
The chewable tablets prepared from the abalone protein peptide are subjected to sensory evaluation, do not feel bitter taste and astringent taste, only have delicate flavor and sweet flavor, show good mouthfeel and are easily accepted by consumers.
In conclusion, subcritical water is used for treating abalone viscera to extract protein, pectinase and protease are used for carrying out enzymolysis on the extracted protein, the enzymolysis liquid is subjected to column chromatography separation, nanofiltration membrane concentration, vacuum concentration and spray drying to prepare abalone protein peptide powder, and the abalone protein peptide powder is used for developing functional health-care products, so that reference is provided for realizing high-value utilization of the abalone viscera.
The above description is only an embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by using the design concept should fall within the scope of infringing the present invention.
TABLE 1 basic Components of abalone protein peptides
| Example 1 | Example 2 | Example 3 | |
| Protein content (%) | 65.45 | 63.11 | 67.85 |
| Polysaccharide content (%) | 14.90 | 15.31 | 12.09 |
| Moisture content (%) | 7.12 | 8.36 | 7.02 |
Table 2 molecular weight distribution (%) of the basic components of the abalone protein peptide
| Molecular weight distribution (Da) | Example 1 | Example 2 | Example 3 |
| ≥1000 | 6.95 | 6.08 | 6.45 |
| 500-1000 | 27.27 | 26.14 | 25.87 |
| 180-500 | 60.71 | 62.92 | 63.09 |
| ≤180 | 5.07 | 4.86 | 4.59 |
Claims (6)
1. A method for extracting abalone protein peptide through subcritical water assisted enzymolysis is characterized by comprising the following steps of (1) carrying out subcritical water treatment, namely adding purified water into abalone viscera according to a material-liquid ratio of 1 ׃ -1 ׃ (w/v), extracting for 15-60 min under a subcritical water state, cooling to room temperature, removing filter residues through a stainless steel filter screen to obtain protein extract, drying the filter residues in the sun to obtain seaweed fertilizer, carrying out enzymolysis reaction for 1 h-3 h, carrying out separation, concentration and spray drying, namely adding complex enzyme into the protein extract obtained in the step (1), adding the complex enzyme into the protein extract with an addition amount of 0.1% -0.5% (w/v) of the protein extract, carrying out refining on decolorization, fishy smell removal, dearsenization and the like by using activated carbon and resin, carrying out concentration, vacuum concentration and spray drying to prepare a product, namely carrying out nanofiltration membrane, vacuum concentration and spray drying on the protein peptide powder, adding a heavy metal protease extracted from the protease obtained in the step (2), adding the complex enzyme into a mixture of abalone protein extract, pectin protease, pectin, and the like, wherein the mixture of the abalone protein extract is prepared in the abalone protein extract, the steps of 0.1-5% of the pectin, 634, the pectin, the oyster shell protein extract, the pectin, the mixture of the pectin.
2. The subcritical water-assisted enzymatic hydrolysis extraction method of abalone protein peptides according to claim 1, characterized in that: in the step (2), the enzymolysis condition of the complex enzyme is pH 5.0-7.0 and the temperature is 40-50 ℃.
3. The subcritical water-assisted enzymatic hydrolysis extraction method of abalone protein peptides according to claim 1, characterized in that: in the step (3), the activated carbon decoloration and deodorization conditions are as follows: adjusting the pH value of the protein enzymolysis liquid to 5.0-6.0, adding active carbon in an amount of 0.5-2.0% (w/v) of the volume of the protease enzymolysis liquid, preserving the heat at 40-50 ℃ for 15-60 min, and removing the active carbon by using a disc centrifuge; the conditions for removing fishy smell, heavy metal and arsenic by the resin are as follows: the mixed filler consists of macroporous adsorption resin, chelate resin for adsorbing heavy metal and chelate fiber for adsorbing arsenic, and the mixing ratio of the macroporous adsorption resin to the chelate resin for adsorbing heavy metal to the chelate fiber for adsorbing arsenic is 7:2: 1; the pH value of the protein enzymolysis liquid is 5.0-6.0, and the residence time of the protein enzymolysis liquid in the mixed filler is 15-30 min.
4. The subcritical water assisted enzymolysis method for extracting abalone protein peptide according to claim 1, wherein in step (4), the product particles comprise, by weight, 20% -30% of abalone protein peptide powder, 55% -70% of β -cyclodextrin, 2% -10% of collagen peptide, 3% -5% of soybean lecithin, 1.0% -2.5% of green olive pulp extract, and 0.5% -2.0% of honey.
5. The subcritical water-assisted enzymatic hydrolysis extraction method of abalone protein peptides according to claim 1, characterized in that: the abalone viscera is one of fresh abalone viscera, frozen abalone viscera or heat-treated abalone viscera.
6. The product prepared by subcritical water assisted enzymolysis extraction of abalone protein peptide according to claim 1 is characterized by comprising, by weight, 20% -30% of abalone protein peptide powder, 55% -70% of β -cyclodextrin, 2% -10% of collagen peptide, 3% -5% of soybean lecithin, 1.0% -2.5% of green olive pulp extract and 0.5% -2.0% of honey.
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| CN115094110A (en) * | 2022-07-19 | 2022-09-23 | 韩耀辉 | Method for assisting directional enzymolysis of abalone protein peptide by using subcritical water |
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| CN103255186A (en) * | 2013-04-23 | 2013-08-21 | 集美大学 | Combined production method for abalone polysaccharide, lipid and protein peptide |
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| CN103255186A (en) * | 2013-04-23 | 2013-08-21 | 集美大学 | Combined production method for abalone polysaccharide, lipid and protein peptide |
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| CN106046196A (en) * | 2016-04-15 | 2016-10-26 | 海南大学 | Preparation method of small-molecule anti-angiosclerosis mango pectin |
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