CN110628854A - Enzyme method green process for producing chitosan oligosaccharide, astaxanthin, protein and calcium powder by using shrimp shells - Google Patents
Enzyme method green process for producing chitosan oligosaccharide, astaxanthin, protein and calcium powder by using shrimp shells Download PDFInfo
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Abstract
The invention provides an enzyme method green process for producing chitosan oligosaccharide, astaxanthin, protein and calcium powder by using shrimp shells. After the shrimp shells are efficiently hydrolyzed by protease and chitinase, the protein-chitin-mineral compound structure in the shrimp shells is opened, so that the protein and the chitin in the shrimp shells can be effectively recycled, the chitin tightly surrounded by the protein is exposed, and a chitin hydrolysate which is low in deacetylation degree, rich in chitosan oligosaccharide distribution and contains chitosan oligosaccharides with high polymerization degree is obtained; the astaxanthin is fully exposed and is easy to extract, the extraction rate of the astaxanthin reaches 101.3 mug/g of shrimp shell powder, the extraction time is obviously shortened, the contact time of the astaxanthin and an organic solvent is reduced, the quality of the extracted astaxanthin is improved, and the antioxidant activity is strong. Meanwhile, the enzymolysis scheme of the invention is scientific and ingenious, the enzyme dosage is low, the protein recovery effect is good, the environment is protected, the efficiency is high, and the method has good application prospect in the recycling of shrimp shell wastes.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a novel green recovery process of shrimp shell waste.
Background
Guangdong province is the major aquatic product province in China, and according to statistics, the total production amount of the Guangdong aquatic products in 2016 years reaches 882 million tons, wherein various shrimps and crabs are included, and only the yield of the shrimps is nearly 100 million tons. The edible part of the shrimps is less than 50 percent, a large amount of waste such as shrimp heads, shrimp shells and the like can be generated in the processing process of the shrimp meat, the shrimp heads account for more than one third of the weight of the whole shrimps, and the leftovers in the processing of the shrimps in Guangdong province can reach dozens of million tons every year. With the development of the marine industry and the artificial shrimp and crab industry in China, the amount of the wastes is getting larger and larger, and serious environmental pollution is caused (Ferraro et al, 2010, Valrisation of natural sources from marine source on marine by-products: A review). On the other hand, they are valuable biological resources, and the waste shrimp shells contain rich biological resources, such as chitin, protein, astaxanthin and the like, and have high development and utilization values (Shahidi and Synoweecki, 1991). Therefore, how to realize the comprehensive utilization of the waste shrimp shells and recover the active ingredients in the waste shrimp shells has become a hot point of research and development.
As a high-quality protein resource, the shrimp shells can be directly used as feed after being crushed, but the digestion and absorption rate of the shrimp shells is low, so that the application of the shrimp shells is influenced. Generally, shrimp shell protein can be recovered by deproteinizing to obtain a protein hydrolysate, which is then further processed into powder, paste, fluid, and the like for use as an additive for shrimp flavor in feed. Unlike the strong acid and strong base (such as 1M HCl and 4% NaOH) treatment of the traditional process, biocatalysis and microbial fermentation are also used in shrimp shell protein recovery as novel treatment modes. The shrimp shell protein is recovered by microbial fermentation, can be used as a carbon-nitrogen source of the microbes, is easy to excessively hydrolyze and consume the protein, and is easy to bring other substances during fermentation, so the enzymatic hydrolysis is more popular. Some of the processes and effects of enzymatic treatment of shrimp shells in recent years are shown in table 1, and it can be seen from the table that the current processes for enzymatic hydrolysis of shrimp shells have extremely large enzyme dosage (e.g. 2 ten thousand U/g for commercial alcalase enzyme substrate ratio and 5 ten thousand U/g for pepsin) due to low hydrolysis efficiency of the current commercial enzymes, and the cost is too high and the recovery effect is not ideal. In addition, when alkaline protease is used, the alkaline condition has a certain influence on the structures of proteins and astaxanthin in the shrimp shells, and the biological activity of the proteins and the astaxanthin is influenced. In addition, chitin in the shrimp shell is tightly surrounded by protein, and the chitin in the shrimp shell is difficult to be hydrolyzed and utilized.
TABLE 1 comparison of enzymatic recovery of shrimp Shell protein
The current technology focuses on producing chitin (chitin) by using shrimp and crab shells and the like, or producing chitin oligosaccharide with low polymerization degree by using chitin, and rarely directly producing chitin oligosaccharide with various polymerization degrees by using shrimp and crab shells. The preparation of the chitosan oligosaccharide is a difficult problem in the industry, and no large-scale industrialized enterprise for producing the chitosan oligosaccharide exists in China at present. In the existing research technology, the preparation method of the chitosan oligosaccharide mainly comprises a chemical degradation method, a chitosan oligosaccharide acetylation method and an enzyme degradation method. The chemical degradation method generally hydrolyzes chitin by using concentrated acid, usually uses concentrated hydrochloric acid, although the process is simple and is early developed, the method has the defects of harsh reaction conditions, difficult control, low yield, high equipment requirement, environmental pollution and the like, and the polymerization degree of the chitosan oligosaccharide product is below 4; the acetylation method of chitosan oligosaccharide (patent CN201110409312.4) has complex steps, time and labor waste, high cost and low possibility of industrialization; the enzyme degradation method is the most potential method at present, however, the specific enzymes used in the existing enzyme degradation method, such as chitinase (patent CN2011102588936) and chitosanase (patent CN200610080091.x), generally have the problems of insufficient enzyme activity and low enzyme yield; and about thirty enzymes such as cellulase, protease, lipase and the like are mixed to realize partial or complete hydrolysis of chitin and chitosan, but the mutual synergistic action of the non-specific enzymes is needed, so that the method is often a composite application of a plurality of enzymes in an experiment, the uniformity and stability of hydrolysate in different batches are low, the product quality cannot be controlled, even if the enzymes are the same, the enzyme effects of different batches or manufacturers are greatly different, and the fixed experiment process is not favorable to be established. Therefore, the chitinase with high yield and high enzyme activity is a key technology for preparing the chitosan oligosaccharide and the industrialization thereof. The chitinase with high expression and the application thereof in the recovery of shrimp shell chitin have great research value and application potential. On the other hand, the chitooligosaccharide with the polymerization degree of 6-8 is considered to have the highest biological activity, and particularly, the chitooligosaccharide with the polymerization degree of less than 5 basically has no binding activity in the aspect of binding with related receptors. However, it has been reported that the degree of polymerization of hydrolyzed chitin oligosaccharides is substantially below 5 (nutritional et al, 2014, An acid, thermal expression oligosaccharide with beta-N-acetyl glucose activity from Paenibacillus genus) to N-acetyl glucose, Nguide et al, 2015, Microwave-induced enzymatic degradation of expression Loop housing, Sinha et al, Microbial expression of Protein genes, calcium phosphate, calcium. Therefore, how to obtain the chitooligosaccharide with higher polymerization degree also becomes a difficult point for research.
Astaxanthin in shrimp shells, protein and chitin are tightly combined with each other and are not easy to extract. The extraction of astaxanthin, which is treated by strong acid and strong base used in the traditional process, can destroy the structure of astaxanthin. It is currently preferred to extract astaxanthin after removal of protein using enzymatic or microbial fermentation. The extraction rate of astaxanthin reaches 2.4mg/g after the shells of the penaeus vannamei boone are fermented by using lactobacillus plantarum; the extraction rate of astaxanthin of the shrimp shell of the penaeus vannamei boone hydrolyzed by endogenous enzyme reaches 0.8 mg/g; the extraction rate of astaxanthin reaches 6.6mg/g by using the combination of Pediococcus pentosaceus fermentation, Segwei protease and Libo lipase. Chitin is the major supporting structure of crustacean exoskeletons and it has been reported that Chitin washed out of Crab shells with Ionic liquids (excluding proteins and minerals) still has a structure similar to that of natural Crab shells (Pei Xu et al, 2019, Double-Chitin Hydrolysis of Crab Shell Chitin Pretreated by Ionic Liquid production Chito-Oligosaccharide). Therefore, chitin in the shrimp shells after protein removal may still affect the extraction of astaxanthin. For example, after removing the protein of the shrimp shell in CN1715255A, the astaxanthin is extracted by using an organic solvent for 6 to 12 hours; after removing proteins and minerals in shrimp shells in CN103172763A, extracting astaxanthin by using ethanol requires ultrasonic treatment, and then extracting for 2-5 hours by using an organic solvent. The structure of astaxanthin is destroyed by intense physicochemical treatment, and the activity of astaxanthin is reduced by organic solvents.
Until now, no one applies combined acid protease and chitinase to the hydrolysis and recovery of shrimp shell wastes, and no one simultaneously recovers shrimp shell protein hydrolysate, chitosan oligosaccharide and astaxanthin from the shrimp shell wastes.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provide a novel green recovery process for shrimp shell waste.
The invention also aims to provide a shrimp shell powder protein hydrolysate, chitin hydrolysate, astaxanthin or calcium powder prepared by the novel green recovery process of the shrimp shell waste.
The purpose of the invention is realized by the following technical scheme:
a novel green recovery process of shrimp shell waste comprises the following steps:
(1) adding acid protease to hydrolyze the shrimp shell waste powder for the first time, and performing solid-liquid separation to obtain a first supernatant and a first precipitate, wherein the first supernatant is a shrimp shell powder protein hydrolysate;
(2) adding chitinase to carry out secondary hydrolysis on the first precipitate, and carrying out solid-liquid separation to obtain a second supernatant and a second precipitate, wherein the second supernatant is a chitin hydrolysate;
(3) and adding an organic solvent into the second precipitate for extraction, carrying out solid-liquid separation, collecting an extracting solution, and drying to obtain the astaxanthin.
The acidic protease used in step (1) is preferably at least one of the acidic protease P6281 described in Chinese patent application CN201710088628.5 and the acidic protease Saccharopepsin (GeneBank accession No.: EPB 83353).
The acid protease P6281 can be obtained by heterologous expression and purification method described in Chinese patent application CN 201710088628.5.
The shrimp shell waste source is preferably one or at least two of crustaceans such as shrimp, crab, shellfish and the like.
Preferably, the shrimp shell waste powder in the step (1) is prepared into a solution with the mass volume concentration of 10% and then subjected to the first hydrolysis; the solvent used is preferably 50mM sodium lactate buffer or sodium phosphate buffer.
The particle size of the shrimp shell waste powder in the step (1) is preferably 50 meshes.
The amount of the acidic protease added in step (1) is preferably 1000U/g substrate.
The pH value of the first hydrolysis in the step (1) is preferably 3-5; when the acid protease is acid protease P6281, the pH is preferably 3; when the acidic protease is Saccharomyces pastin, pH5 is preferred.
The temperature of the first hydrolysis in step (1) is preferably 40 ℃.
The time for the first hydrolysis in the step (1) is preferably 4-10 h.
The first hydrolysis in the step (1) is preferably performed by first stage hydrolysis with acid protease P6281, and the precipitate obtained by solid-liquid separation is continuously subjected to second stage hydrolysis with acid protease Saccharopepsin.
The chitinase in the step (2) is preferably chitinase Chit46 described in Chinese patent application CN 201910064040.5; can be obtained by the expression and purification method described in Chinese patent application CN 201910064040.5.
The chitinase to be added in the step (2) is preferably added in an amount of 50U/g substrate.
In the second hydrolysis in the step (2), the first precipitate is preferably prepared into a solution with the mass volume concentration of 10% and then subjected to the hydrolysis; the solvent used is preferably 50mM sodium phosphate buffer.
The pH of the second hydrolysis described in step (2) is preferably pH6.
The temperature of the second hydrolysis in step (2) is preferably 45 ℃.
The time for the second hydrolysis in step (2) is preferably 6 hours.
The chitin hydrolysate in the step (2) is preferably chitosan oligosaccharide with the polymerization degree of at least 2; further preferably, the chitosan oligosaccharide has a degree of polymerization of 4 or more, and particularly preferably has a degree of polymerization of 6 or more.
The organic solvent in the step (3) is preferably at least one of ethyl acetate, dichloromethane, acetone and petroleum ether.
The extraction time in step (3) is preferably 2 h.
The extraction temperature in the step (3) is preferably 16-30 ℃; further preferably 25 ℃.
The drying in the step (3) is preferably carried out under the condition of keeping out of the sun; the temperature for drying is preferably room temperature.
And (4) performing decoloration, drying and other steps on the precipitate obtained by solid-liquid separation in the step (3) to obtain calcium powder.
The solid-liquid separation in the steps (1), (2) and (3) is preferably realized by centrifugation; the centrifugation conditions are preferably 12000rpm for 2 minutes.
A shrimp shell powder protein hydrolysate, chitin hydrolysate, astaxanthin or calcium powder is prepared by the novel green recovery process of the shrimp shell waste.
The shrimp shell meal protein hydrolysate can be further processed into powder, paste and fluid, for example, for use as a shrimp flavor additive for food, feed and the like.
The determination method of the protein hydrolysate is to determine the protein concentration by a BCA method and determine the amino acid concentration by an ninhydrin method.
The method for measuring the chitin hydrolysate is to measure the concentration of reducing sugar and the deacetylation degree by a DNS method.
The detection method of the chitin hydrolysate is an HPLC method, a chromatographic column is Asahipak NH2P-504E, and a chromatographic system is waters E2695.
The detection conditions of the chitin hydrolysate are preferably as follows: mobile phase 70% acetonitrile, sample loading 20 uL, flow rate 0.7mL/min, temperature 30 ℃, elution time 40min, difference detector.
The method for measuring the oxidation resistance of the astaxanthin is a DPPH method.
The method for detecting the purity and the yield of the astaxanthin is an HPLC method, a chromatographic column is Waters SunFire C18, and a chromatographic system is Shimadzu-LC-20A.
The detection conditions of astaxanthin are preferably as follows: mobile phase methanol: dichloromethane: acetonitrile: water 85: 5: 5: 5, the sample loading amount is 20 mu L, the flow rate is 1.5mL/min, the temperature is 37 ℃, the elution time is 20min, and the detection wavelength is 475 nm.
The influence of the mineral substances of the shrimp shells on the hydrolysis of the protease is firstly researched, and the chitin tightly surrounded by the protein in the shrimp shells is exposed, so that the utilization rate of the chitin is obviously improved; compared with the prior art that the astaxanthin is extracted only after the protein is removed, the astaxanthin is extracted after the protein and the chitin are removed for the first time. The research of the invention finds that after the shrimp shells are hydrolyzed by protease and chitinase, the compound structure of protein-chitin-mineral substance in the shrimp shells can be opened, so that the astaxanthin is fully exposed and is easy to extract, and the efficiency of the method is obviously higher than that of the existing method. Compared with the existing enzyme method and fermentation method, the method is simpler in operation, is a more ideal astaxanthin recovery mode, improves the astaxanthin extraction efficiency, reduces the contact time of the astaxanthin with an organic solvent, and can improve the quality of the astaxanthin obtained by extraction.
Compared with the prior art, the invention has the following advantages and effects:
1. at present, no combination of human acid protease and chitinase is used for protein hydrolysis of shrimp shell waste, preparation of chitosan oligosaccharide and extraction of astaxanthin. The invention successfully applies the acid protease P6281, the Saccharomyces cerevisiae and the chitinase Chit46 to the recovery of the shrimp shell waste for the first time.
2. The hydrolysis effect of the acid protease P6281 and the Saccharomyces pastsin used in the invention is better than that of commercial acid protease, alkaline protease, papain and neutral protease rNpI under the condition of the same enzyme adding amount in the hydrolysis of shrimp shell wastes. The enzyme addition amount is 1000U/g, which is lower than that reported in most reports, and the protein recovery effect is better than that reported in most reports.
3. After the shrimp shell protein is hydrolyzed, the chitin in the shrimp shell protein is easily hydrolyzed by chitinase Chit46, and the use of strong alkali reagents in the traditional process is avoided. Furthermore, P6281 and Saccharomyces pastin have poor activity at pH6.0 and poor thermostability, and a small amount of protease remaining when chitinase treatment is performed does not have a significant effect on Chit 46.
4. The chitinase Chit46 has good hydrolytic activity, stable hydrolysate, low deacetylation degree and rich distribution of chitooligosaccharide, and can obtain chitooligosaccharide with polymerization degree of 6-8 (the activity is the best, and the affinity with related receptors is the strongest).
5. After the shrimp shell protein and the chitin are hydrolyzed, the astaxanthin in the shrimp shell protein and the chitin is easily extracted, the extraction rate of the astaxanthin reaches 101.3 mug/g of the shrimp shell powder, the damage of chemical reagents to the astaxanthin in the traditional process is avoided, the extraction time is effectively shortened, and the influence of organic solvents on the astaxanthin is reduced.
Drawings
FIG. 1 is a diagram showing the results of amino acid analysis of a product of shrimp shell waste enzymolysis by acidic protease P6281.
FIG. 2 is a graph showing the results of amino acid analysis of the shrimp shell waste product by the enzymatic hydrolysis of the shrimp shell with the acid protease Saccharomyces pastsin.
FIG. 3 is a HPLC analysis chart of the product of chitin enzyme Chit46 hydrolyzed shrimp shell waste, wherein the upper chart is chitin hydrolysate sample and the lower chart is standard product.
FIG. 4 is an HPLC analysis chart of astaxanthin extracted from shrimp shell waste, wherein A is an extraction product, B is a saponification product, and C is a standard.
FIG. 5 is a photograph showing the recovered products of shrimp shell wastes, wherein A is the recovered product of shrimp shell protein hydrolysis (P6281 hydrolysis) in the first step, B is the recovered product of shrimp shell protein hydrolysis (Saccharomyces cerevisiae hydrolysis) in the second step, C is the recovered product of shrimp shell chitin hydrolysis (chitooligosaccharide), and D is the astaxanthin extract.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1 measurement of Components of shrimp Shell waste
The shrimp shell waste is purchased from Zhanjiang Union aquatic product development Co., Ltd, and is crushed through a 50-mesh sieve to obtain shrimp shell waste powder, which is stored at 4 ℃ for later use. The method for measuring protein, ash, chitin and moisture in the shrimp shell waste comprises the following steps:
(1) protein: because chitin contains nitrogen elements, in order to eliminate interference, the shrimp shell powder is firstly treated by 1M NaOH solution at 40 ℃ for 3h, and the protein concentration of the supernatant is determined by using a Kjeldahl method (GB 50095-2010), so that the protein content in the shrimp shell is determined to be 48.84%;
(2) ash content: the ash content in the shrimp shells is measured to be 20.75 percent by using the 'determination of total ash in the first method food' in the national food safety standard (GB 5009.4-2016);
(3) chitin: treating shrimp shell powder with 1M NaOH solution at 40 deg.C for 3 hr to remove protein, treating the precipitate with 1M HCl solution at room temperature for 3 hr to obtain white chitin precipitate, and measuring the chitin content in shrimp shell to be 18.9%;
(4) moisture content: the water content in the shrimp shells was determined to be 4.9% using direct drying (GB 5009.3-2016).
Example 2 acid protease hydrolysis of shrimp shell meal and analysis of hydrolysate
The acidic protease P6281 is prepared by referring to the heterologous expression and purification method of Chinese patent application CN 201710088628.5.
The neutral protease rNpI is disclosed in the literature Ke, Y et al, 2012, enzymic characteristics of a recombinant neutral protease I (rNpI) from Aspergillus oryzae expressed in Pichia pastoris, and is prepared by the method described in the literature.
GeneBank accession number of Saccharopepsin: EPB83353, which can be prepared by expression purification technical means conventional in the art; for example, the expression vector can be obtained by cloning a coding gene of Saccharopepsin, constructing an expression strain, expressing and purifying the gene by referring to the heterologous expression and purification method of chinese patent application CN 201710088628.5.
The substrate used in the example of hydrolyzing shrimp shell meal with acidic protease P6281 was the shrimp shell waste powder prepared in example 1. Adding 50mM sodium lactate buffer solution with pH3.0 into shrimp shell waste powder to ensure that the concentration (mass volume fraction) of the shrimp shell powder is 10%, adding 1000U/g substrate, oscillating at 200rpm under the conditions of pH3.0 and 40 ℃ for reaction for 4h, centrifuging at 12000rpm for 2 min after reaction, and taking the obtained supernatant as a shrimp shell powder protein hydrolysate and precipitate as a next reaction substrate.
The effect of hydrolyzing shrimp shell powder by the acid protease P6281 under the respective optimum reaction conditions is shown in Table 2 in comparison with the hydrolysis effect of other proteases; wherein the protein recovery rate is determined by a weight loss method, and the protein recovery rate (%) is (weight of shrimp shell powder-weight of precipitate)/weight of shrimp shell powder.
TABLE 2 comparison of the hydrolysis effects of different proteases on shrimp shell protein
The shrimp shell powder is hydrolyzed by using acid protease P6281, the protein recovery rate is 71.2 percent measured by a weight reduction method, and the total nitrogen recovery rate of the solution is 71.5 percent measured by a Kjeldahl method. The protein concentration of the hydrolysate was determined by using BCA protein quantitative detection kit (C503021) purchased from Biotechnology engineering (Shanghai) GmbH, and the protein concentration was 39.05mg/mL and accounted for 64% of the total protein of the shrimp shell; after the hydrolysate was precipitated with 0.4M trichloroacetic acid, the total amount of amino acids in the supernatant was measured by GB/T8314-1987 (i.e., the ninhydrin method), and the concentration of amino acids was found to be 3.07mg/mL, which is 6.3% of the total protein in shrimp shells. The hydrolysate is detected by amino acid analysis of MembraPure A300 advanced in Germany, the essential amino acid in the hydrolysate accounts for 35.33 percent of the amino acid, and the hydrolysate has higher nutritional value; the fresh amino acid accounts for 40.42%, and has good flavor enhancing effect.
TABLE 3 amino acid composition analysis of shrimp shell protein hydrolysate
Hydrolyzing the precipitate with acid protease Saccharopepsin, wherein the concentration of the shrimp shell powder is 5% (the solvent is 50mM pH5.0 sodium phosphate buffer solution), the enzyme amount is 1000U/g substrate, the reaction is carried out under the conditions of pH5.0 and 40 ℃ and 200rpm oscillation for 4h, after the reaction, the shrimp shell powder is centrifuged at 12000rpm for 2 min, and the obtained supernatant is the shrimp shell powder protein hydrolysate, and the precipitate is used as the next reaction substrate. The protein recovery rate measured by a weight loss method is 20.2 percent, and the total nitrogen recovery rate of the solution measured by a Kjeldahl nitrogen determination method is 19.6 percent. The protein concentration of the hydrolysate was determined by using BCA protein quantitative detection kit (C503021) purchased from Biotechnology engineering (Shanghai) GmbH, and the protein concentration was 4.16mg/mL and accounted for 15.9% of the total protein of the shrimp shell; after the hydrolysate was precipitated with 0.4M trichloroacetic acid, the supernatant was assayed for the total amount of amino acids by GB/T8314-. The hydrolysate is detected by amino acid analysis of MembraPure A300 advanced in Germany, the essential amino acid in the hydrolysate accounts for 38.13% of the amino acid, has high nutritional value, the delicious amino acid accounts for 37.23%, and has good freshness and taste increasing effects.
TABLE 4 amino acid composition analysis of shrimp shell protein hydrolysate
In the course of the study on the hydrolysis of shrimp shell protein, the inventor prepares an enzymolysis substrate with the concentration (mass volume fraction) of the shrimp shell powder of 10% by respectively using a sodium lactate buffer solution with the pH value of 3.0, a sodium citrate buffer solution with the pH value of 4.0 and a sodium citrate buffer solution with the pH value of 5.0; in the 3 enzymolysis substrates, acid protease P6281 (with the enzyme adding amount of 1000U/g substrate) and acid protease Saccharopepsin (with the enzyme adding amount of 1000U/g substrate) are respectively and simultaneously added for mixed enzymolysis for 6 hours at the temperature of 40 ℃, and the protein recovery rates measured by a weight loss method are respectively 78.5%, 76.3% and 68.2%. It can be seen that hydrolysis with P6281 followed by hydrolysis with Saccharomyces pastin is more effective (total protein recovery of 91.4%).
The inventor also tried to hydrolyze the protein by using the acidic protease Saccharopepsin firstly, and then hydrolyze the obtained precipitate by using the acidic protease P6281 (except for different enzymolysis sequences and enzymolysis time, other conditions are the same as the operation of hydrolyzing by using P6281 and then hydrolyzing by using Saccharopepsin), so that the time for achieving the same protein recovery rate is longer, the first step of the hydrolysis of the Saccharopepsin needs 6 hours, and the recovery rate is 54.8%; the second step of hydrolysis of P6281 took 12 hours with a recovery of 34.1% and an overall recovery of 88.9%. This phenomenon indicates that the enzymatic sequence of the present invention has an effect on the enzymatic effect, probably because the mutual hydrolysis between P6281 and Saccharomyces cerevisiae affects their interaction. P6281 has a lower activity at pH5.0 and has a smaller effect on Saccharomyces pastsin. Thus, the enzymatic hydrolysis sequence is more preferably performed first with P6281 and then with Saccharomyces pastsin.
EXAMPLE 3 chitinase Chit46 hydrolysis of shrimp shell meal and analysis of the hydrolysate
Chitinase Chit46 was prepared by the method described in example 1 of Chinese patent application CN 201910064040.5.
Chitinase Chit46 hydrolyzes shrimp shell powder the substrate used in the example is the precipitate obtained in example 2 after hydrolysis by acid protease P6281 and then by acid protease Saccharopepsin. Adding 50U/g substrate into original shrimp shell powder (which refers to the shrimp shell powder which is not subjected to any hydrolysis step and is convenient to calculate) with the concentration of 10% (50 mM of solvent, pH6.0 sodium phosphate buffer solution), oscillating at 150rpm under the conditions of pH6.0 and 45 ℃ for 6h, centrifuging at 12000rpm for 2 min after reaction, and obtaining supernatant which is a chitin hydrolysate of the shrimp shell powder, wherein the precipitate is used as a reaction substrate in the next step. The weight loss method determines that the precipitation mass is reduced by 25.86 percent, and the recovery rate of chitin reaches 88.92 percent.
And (3) determining the content of reducing sugar in the chitin hydrolysate of the shrimp shell powder by adopting a Ghose DNS method. 1mL of the hydrolysate diluted with distilled water as appropriate was taken, 1mL of distilled water and 3mL of DNS reagent were added, a boiling water bath was carried out for 10 minutes, the supernatant was centrifuged at 12000rpm for 2 minutes, and then the absorbance was measured at a wavelength of 540nm by taking the supernatant, and the reducing sugar content was calculated from a standard N-acetylglucosamine gradient concentration solution-absorbance curve prepared before the experiment, using distilled water as a blank. The hydrolysis product was found to have a reducing sugar content of 5.08mg/mL, 16.25mg/mL after complete digestion of the hydrolysis product with 1% snailase (purchased from Biotechnology engineering, Shanghai, Ltd., product No. A600870), and a chitin recovery of 85.98%.
The deacetylation degree of the hydrolysate is measured by using food additive chitosan (chitosan) in national standard GB 29941-2013 for food safety, and the deacetylation degree is 8.6%, which indicates that most of the hydrolysate is indeed chitosan oligosaccharide.
The hydrolysate was added with 2 volumes of ethanol to remove the enzymes from the product, centrifuged at 12000rpm for 2 minutes, the supernatant was placed in an oven at 65 ℃ for 1 hour to remove ethanol, and the resulting product was assayed by HPLC using Asahipak NH2P-504E as a column and waters E2695 as a system. The detection conditions are 70% acetonitrile of mobile phase, 20 mul of sample loading, 0.7mL/min of flow rate, 30 ℃ of temperature, 40min of elution time and a differential detector. The detection result shows that the product consists of chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose and three chitooligosacchrides with higher polymerization degrees, wherein the chitobiose accounts for most of the chitobiose, and the chitotetraose, chitotriose, chitopentaose, chitohexaose and the like are used as the second chitosan.
In the hydrolysis process of the shrimp shell chitin, 50mM sodium lactate buffer solution with the pH value of 3.0 is added into the shrimp shell waste powder to ensure that the concentration (mass volume fraction) of the shrimp shell powder is 10 percent, acid protease P6281 is added, the amount of the added enzyme is 1000U/g substrate, the pH value is 3.0, the vibration reaction is carried out at 200rpm and 40 ℃ for 4 hours, the reaction is centrifuged at 12000rpm for 2 minutes after the reaction, the solid-liquid separation is carried out to obtain a precipitate, and chitinase Chit46 is added into the precipitate for enzymolysis according to the method of the embodiment. The study found that samples treated with P6281 alone without saccharomyces cerevisiae were also hydrolyzed by Chit46 with similar results (chitin recovery 85.6%), indicating that similar chitosan oligosaccharide recovery could be achieved without complete removal of the proteins from the shrimp shells as long as the shrimp shell proteins were hydrolyzed to a degree sufficient to break down the protein-chitin complex and expose the chitin to binding with chitinase.
During the research process, the inventor uses the precipitates obtained by solid-liquid separation of the enzymolysis products of acidic protease (SDG-242110000U/g, M type acidic protease for Xiaheng feed), alkaline protease, papain and neutral protease rNpI in Table 2 as the reaction substrate for the hydrolysis of Chit46, and finds that the recovery rate of chitin is below 20%.
Example 4 astaxanthin extraction and product analysis
The substrate used for astaxanthin extraction was the precipitate prepared in example 3 (i.e. the precipitate after hydrolysis with the acid protease P6281, the acid protease Saccharomyces pastin, chitinase Chit 46). Soaking the original shrimp shell powder (which is not subjected to any hydrolysis step) with the concentration of 10% (the solvent is ethyl acetate) in ethyl acetate at room temperature (25 ℃) for 2 hours, centrifuging at 12000rpm for 2 minutes after reaction, and drying the obtained supernatant at room temperature in a dark place to obtain an extraction product, wherein the extraction product can obtain a good astaxanthin extraction effect within the temperature range of 16-30 ℃; and (4) decoloring the precipitate obtained after centrifugation by using ethanol and drying the decolored precipitate to obtain a product, namely calcium powder.
Antioxidant assay reference cosmetic-free radical (DPPH) scavenging test method (T/SHRH006-2018) in which solvent is changed from ethanol to methanol, EC of vitamin C on DPPH scavenging is determined50EC of the resulting extract on DPPH clearance at 15.47. mu.g/mL500.21 μ g/mL, and is resistant to oxidationThe sex is 72 times of that of vitamin C.
Adding equal volume of 10% KOH methanol solution into the obtained product, reacting for 15min at room temperature to saponify astaxanthin, and detecting the saponified product by using HPLC, wherein a chromatographic column is waters sunfire C18, and a chromatographic system is Shimadzu-LC-20A. The detection conditions were mobile phase methanol: dichloromethane: acetonitrile: water 85: 5: 5: 5, the sample loading amount is 20 mu L, the flow rate is 1.5mL/min, the temperature is 37 ℃, the elution time is 20min, and the detection wavelength is 475 nm. The detection result shows that the purity of the astaxanthin in the product is high, and the yield of the astaxanthin is 101.3 mug/g of shrimp shell powder.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A novel green recovery process of shrimp shell waste is characterized by comprising the following steps:
(1) adding acid protease to hydrolyze the shrimp shell waste powder for the first time, and performing solid-liquid separation to obtain a first supernatant and a first precipitate, wherein the first supernatant is a shrimp shell powder protein hydrolysate;
(2) adding chitinase to carry out secondary hydrolysis on the first precipitate, and carrying out solid-liquid separation to obtain a second supernatant and a second precipitate, wherein the second supernatant is a chitin hydrolysate;
(3) and adding an organic solvent into the second precipitate for extraction, carrying out solid-liquid separation, collecting an extracting solution, and drying to obtain the astaxanthin.
2. The novel green recycling process of shrimp shell waste as claimed in claim 1, wherein:
the acid protease in the step (1) is at least one of acid protease P6281 described in Chinese patent application CN201710088628.5 and acid protease Saccharopepsin with GeneBank accession number of EPB 83353;
the chitinase in the step (2) is chitinase Chit46 described in Chinese patent application CN 201910064040.5.
3. A novel green recovery process of shrimp shell waste as claimed in any one of claims 1 or 2, characterized in that:
the enzyme adding amount of the acid protease in the step (1) is 1000U/g substrate;
the enzyme adding amount of the chitinase in the step (2) is 50U/g substrate.
4. The novel green recycling process of shrimp shell waste as claimed in claim 1, wherein:
the waste source of the shrimp shell is one or at least two of shrimp, crab and shellfish;
the time for the first hydrolysis in the step (1) is 4-10 h;
the time of the second hydrolysis in the step (2) is 6 hours;
and (4) decoloring and drying the precipitate obtained by solid-liquid separation in the step (3) to obtain calcium powder.
5. The novel green recycling process of shrimp shell waste as claimed in claim 1, wherein:
the pH value of the first hydrolysis in the step (1) is 3-5;
the pH of the second hydrolysis in step (2) is pH 6;
the temperature of the second hydrolysis in the step (2) is 45 ℃;
the chitin hydrolysate in the step (2) is chitosan oligosaccharide with the polymerization degree of at least 2.
6. The novel green recycling process of shrimp shell waste as claimed in claim 1, wherein:
the temperature of the first hydrolysis in the step (1) is 40 ℃;
the chitin hydrolysate in the step (2) is chitin oligosaccharide with the polymerization degree of more than 4;
the organic solvent in the step (3) is at least one of ethyl acetate, dichloromethane, acetone and petroleum ether.
7. The novel green recycling process of shrimp shell waste as claimed in claim 1, wherein:
the first hydrolysis in the step (1) is first stage hydrolysis by using acid protease P6281, and the precipitate obtained by solid-liquid separation is continuously hydrolyzed by using acid protease Saccharopepsin in a second stage;
the shrimp shell waste powder in the step (1) is prepared into a solution with the mass volume concentration of 10 percent and then is subjected to the first hydrolysis;
the second hydrolysis in the step (2) is to prepare the first precipitate into a solution with the mass volume concentration of 10% and then perform the hydrolysis;
the chitin hydrolysate in the step (2) is chitin oligosaccharide with the polymerization degree of more than 6.
8. The novel green recycling process of shrimp shell waste as claimed in claim 7, wherein:
when the acid protease is acid protease P6281, the pH of the first hydrolysis is pH 3;
when the acid protease is Saccharomyces pastin, the pH of the first hydrolysis is 5;
the shrimp shell waste powder in the step (1) is prepared into a solution with the mass volume concentration of 10% by using a sodium lactate buffer solution or a sodium phosphate buffer solution with the solvent of 50 mM;
the second hydrolysis in step (2) is to prepare the first precipitate into a solution with a mass volume concentration of 10% by first using a 50mM sodium phosphate buffer as a solvent.
9. The novel green recycling process of shrimp shell waste as claimed in claim 1, wherein:
the granularity of the shrimp shell waste powder in the step (1) is 50 meshes;
the extraction time in the step (3) is 2 h;
the extraction temperature in the step (3) is 16-30 ℃;
and (4) drying in the step (3) under the condition of keeping out of the sun.
10. A shrimp shell powder protein hydrolysate, chitin hydrolysate, astaxanthin or calcium powder, characterized in that it is obtained by the novel green recovery process of shrimp shell waste as claimed in any one of claims 1 to 9.
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