CN117551165B - Preparation method of abalone viscera peptide - Google Patents
Preparation method of abalone viscera peptide Download PDFInfo
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- CN117551165B CN117551165B CN202410048724.7A CN202410048724A CN117551165B CN 117551165 B CN117551165 B CN 117551165B CN 202410048724 A CN202410048724 A CN 202410048724A CN 117551165 B CN117551165 B CN 117551165B
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- phytic acid
- shell powder
- activated carbon
- chitosan
- acid modified
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- 210000001835 viscera Anatomy 0.000 title claims abstract description 30
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 claims abstract description 62
- 239000000467 phytic acid Substances 0.000 claims abstract description 62
- 229940068041 phytic acid Drugs 0.000 claims abstract description 62
- 235000002949 phytic acid Nutrition 0.000 claims abstract description 62
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 59
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 claims abstract description 59
- 239000000843 powder Substances 0.000 claims abstract description 55
- 229920001661 Chitosan Polymers 0.000 claims abstract description 46
- 239000004005 microsphere Substances 0.000 claims abstract description 43
- 239000002131 composite material Substances 0.000 claims abstract description 41
- 229910001385 heavy metal Inorganic materials 0.000 claims abstract description 31
- 238000001179 sorption measurement Methods 0.000 claims abstract description 27
- 239000007788 liquid Substances 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000000463 material Substances 0.000 claims abstract description 19
- -1 phytic acid modified activated carbon Chemical class 0.000 claims abstract description 13
- 238000004132 cross linking Methods 0.000 claims abstract description 11
- 239000000725 suspension Substances 0.000 claims abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 33
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 31
- 238000003756 stirring Methods 0.000 claims description 24
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 14
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 7
- 239000003995 emulsifying agent Substances 0.000 claims description 6
- 229940057995 liquid paraffin Drugs 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 102000057297 Pepsin A Human genes 0.000 claims description 5
- 108090000284 Pepsin A Proteins 0.000 claims description 5
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 239000003463 adsorbent Substances 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 4
- 238000001354 calcination Methods 0.000 claims description 4
- 239000008098 formaldehyde solution Substances 0.000 claims description 4
- 229940111202 pepsin Drugs 0.000 claims description 4
- 239000012588 trypsin Substances 0.000 claims description 4
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 claims description 3
- 238000005352 clarification Methods 0.000 claims description 3
- 230000008961 swelling Effects 0.000 claims description 3
- 108091005804 Peptidases Proteins 0.000 claims description 2
- 239000004365 Protease Substances 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 2
- 239000000839 emulsion Substances 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims description 2
- 239000000413 hydrolysate Substances 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims description 2
- 238000004042 decolorization Methods 0.000 claims 1
- 230000004048 modification Effects 0.000 abstract description 6
- 238000012986 modification Methods 0.000 abstract description 6
- 230000009278 visceral effect Effects 0.000 abstract description 6
- 230000000052 comparative effect Effects 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 239000002245 particle Substances 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000003431 cross linking reagent Substances 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
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- 238000000926 separation method Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- LRWZZZWJMFNZIK-UHFFFAOYSA-N 2-chloro-3-methyloxirane Chemical compound CC1OC1Cl LRWZZZWJMFNZIK-UHFFFAOYSA-N 0.000 description 2
- 241001526627 Azumapecten farreri Species 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241001489139 Haliotis discus Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical group CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229910052793 cadmium Inorganic materials 0.000 description 2
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000007873 sieving Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000237509 Patinopecten sp. Species 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 241000282894 Sus scrofa domesticus Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- BRPQOXSCLDDYGP-UHFFFAOYSA-N calcium oxide Chemical compound [O-2].[Ca+2] BRPQOXSCLDDYGP-UHFFFAOYSA-N 0.000 description 1
- 239000000292 calcium oxide Substances 0.000 description 1
- ODINCKMPIJJUCX-UHFFFAOYSA-N calcium oxide Inorganic materials [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000004021 humic acid Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 235000020637 scallop Nutrition 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/20—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising free carbon; comprising carbon obtained by carbonising processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28004—Sorbent size or size distribution, e.g. particle size
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention belongs to the technical field of active peptide preparation, and particularly relates to a preparation method of abalone viscera peptide, which comprises the steps of carrying out enzymolysis on abalone viscera, adding an adsorption material into enzymolysis liquid obtained after the enzymolysis, and removing heavy metals; the preparation method of the adsorption material comprises the following steps: (1) obtaining shell powder; (2) preparing phytic acid modified shell powder; (3) preparing phytic acid modified activated carbon; (4) Preparing chitosan/activated carbon/shell powder composite microspheres: preparing chitosan/activated carbon/shell powder composite microspheres by adopting a reverse suspension crosslinking method; (5) preparing the phytic acid modified composite microsphere. The invention carries out phytic acid grafting modification on the solidified chitosan/activated carbon/shell powder composite microsphere, and uses the phytic acid modified composite microsphere as an adsorption material for adsorbing heavy metals in abalone visceral peptide, thereby effectively solving the problem of high heavy metal content in abalone visceral peptide.
Description
Technical Field
The invention belongs to the technical field of active peptide preparation, and particularly relates to a preparation method of abalone viscera peptide.
Background
The abalone viscera account for 1/3 of the dry weight of the abalone, and researches show that the abalone viscera contain a large amount of protein, but a large amount of abalone viscera are abandoned as scraps in the process of processing the abalone at present, so that considerable economic loss and environmental pollution are caused. The food-borne peptide has the advantages of no toxicity, low sensitization, high safety and the like, plays an increasingly important role in human nutrition and health and disease regulation, and the small molecular peptide has the characteristics of small molecule and easy absorption. The development of small molecule peptides with functional activity by abalone viscera proteins has become a hotspot for abalone viscera development.
However, abalone has higher accumulation capacity of heavy metals as marine animals, particularly in liver and kidney, and has higher heavy metal concentration, so that the problem of solving and ensuring the heavy metal content meeting the requirement is the primary problem.
At present, technologies for removing heavy metals from enzymolysis liquid of aquatic products mainly comprise an adsorption method, a complexation method, a chelating resin method, a membrane separation method and the like. In the adsorption method, common adsorbents include activated carbon, cellulose, humic acid resin, medical stone and the like, and metal ions can be firmly adsorbed on the surface by utilizing the functional groups such as-OH, -COOH, -SH and the like which can be matched or complexed with the heavy metal ions on the surface so as to be removed. However, most of the existing adsorbent materials have limited adsorption effect. In order to improve the adsorption efficiency, it is generally required to improve the specific surface area of the adsorption material, so that most of the adsorption materials have small particle sizes (such as activated carbon powder), which causes difficulty in separation and regeneration of the adsorption material, increases operation difficulty and production cost, and therefore, is difficult to realize efficient removal of heavy metals in the process.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a preparation method of abalone visceral peptide, which can effectively remove heavy metals in abalone visceral peptide.
The specific technical scheme is as follows:
the invention provides a preparation method of abalone viscera peptide, which comprises the steps of carrying out enzymolysis on abalone viscera by using protease, wherein the difference between the method and the prior art is that an adsorption material is added into enzymolysis liquid obtained after enzymolysis to remove heavy metals;
the preparation method of the adsorption material comprises the following steps:
(1) Obtaining shell powder;
(2) Preparing phytic acid modified shell powder: reacting the shell powder obtained in the step (1) with phytic acid to obtain phytic acid modified shell powder;
(3) Preparing phytic acid modified activated carbon: reacting the activated carbon powder with phytic acid to obtain phytic acid modified activated carbon;
(4) Preparing chitosan/activated carbon/shell powder composite microspheres: mixing chitosan, the phytic acid modified shell powder obtained in the step (2) and the phytic acid modified activated carbon obtained in the step (3) into an acetic acid solution, and preparing chitosan/activated carbon/shell powder composite microspheres by adopting a reverse suspension crosslinking method;
(5) Preparing phytic acid modified composite microspheres: and (3) reacting the chitosan/activated carbon/shell powder composite microsphere obtained in the step (4) with phytic acid to obtain the phytic acid modified composite microsphere.
The shell powder and the activated carbon have a large number of spatial micropores, large specific surface area and certain heavy metal adsorption capacity. After the phytic acid is modified, the original space structure has more action sites, and the adsorption capacity is greatly improved. However, both are in powder form, so that in the practical application process, solid-liquid separation and regeneration are difficult, secondary waste is easy to generate, and industrial pollution is increased. After the chitosan is crosslinked and solidified (usually, glutaraldehyde crosslinked after formaldehyde is pre-crosslinked), the chitosan is converted into porous solid microspheres from a powder state which is easy to be dissolved in an acid solution, so that the chitosan is easier to separate and regenerate, and is beneficial to industrial application. However, the amino groups in the molecules participate in the reaction, so that the heavy metal adsorption capacity is reduced. Therefore, the method carries out surface grafting modification on the cured composite microsphere, increases adsorption sites on the surface of the microsphere, and greatly increases the group capturing heavy metal on the surface of the composite microsphere after the phytic acid is introduced, and the test result shows that the adsorption effect is greatly improved.
Further, in step (1): calcining shell, converting calcium carbonate in shell into calcium oxide, and pulverizing to obtain shell powder.
Specifically, the shells are preferably calcined at 500-1000 ℃ for 2-6 hours.
Wherein the shell is preferably scallop shell, more preferably Chlamys farreri shell.
Specifically, the shell is preferably subjected to acid treatment before calcination to remove surface attachments and is cleaned.
Wherein, the particle size of the shell powder is preferably below 120 meshes after crushing and sieving.
Further, in step (2): and (3) mixing the shell powder obtained in the step (1) with a phytic acid solution, and reacting for 1-5 h at the temperature of 30-80 ℃.
Wherein, in the step (2): the concentration of the phytic acid solution is preferably 10-wt wt%.
Wherein, in the step (2): the dosage ratio of the shell powder to the phytic acid solution is preferably 1g (1-5) mL.
Wherein, in the step (2): after the reaction is finished, the product is washed by deionized water and dried for standby.
Further, in step (3): and mixing the activated carbon powder with a phytic acid solution, and reacting for 1-5 hours at the temperature of 30-80 ℃. More specifically, the activated carbon powder may be dispersed in water first, and then the phytic acid or the phytic acid solution may be added thereto.
Wherein, in the step (3): the concentration of the phytic acid in the liquid phase of the reaction system is preferably 1-3 wt%.
Wherein, in the step (3): the dosage ratio of the activated carbon powder to the dispersing water is 1g (10-60 mL).
Wherein, in the step (3): after the reaction is finished, the product is washed by deionized water and dried for standby.
Wherein, in the step (3): the particle size of the activated carbon powder is preferably below 200 mesh.
Further, in step (4): the mass ratio of the chitosan to the phytic acid modified shell powder obtained in the step (2) to the phytic acid modified activated carbon obtained in the step (3) is 6 (1-3).
Further, in the step (4), specific working conditions are as follows:
dissolving chitosan in acetic acid solution, adding the phytic acid modified shell powder obtained in the step (2) and the phytic acid modified activated carbon obtained in the step (3), and uniformly stirring; then adding liquid paraffin and an emulsifying agent, and stirring to form a uniform emulsion; adding ethyl acetate, stirring, and then adding formaldehyde solution for pre-crosslinking; and finally adding glutaraldehyde solution for crosslinking and curing.
More specifically, the working conditions of step (4) are: adding acetic acid solution into chitosan, and stirring until the chitosan is fully dissolved to obtain gel-like solution; adding the phytic acid modified shell powder and the phytic acid modified activated carbon into the shell powder, and uniformly stirring the mixture; then adding liquid paraffin into the mixture, and fully stirring the mixture for 5 to 20 minutes at the temperature of 30 to 60 ℃; then adding an emulsifier dropwise, stirring for 10-30 min at 30-60 ℃, adding ethyl acetate, stirring for 10-30 min at 30-60 ℃, heating to 40-70 ℃, adding a pre-crosslinking agent formaldehyde solution, and stirring for 20-40 min to enable partial amino groups on chitosan and formaldehyde to undergo a pre-crosslinking reaction; then heating to 50-80 ℃, adding a cross-linking agent glutaraldehyde solution, adjusting the pH value to 7.0-8.0, and reacting for 1-5 h; the aldehyde group of glutaraldehyde reacts with amino or acetamido group of chitosan to form intramolecular or intermolecular Schiff base.
Wherein the concentration of the acetic acid solution is preferably 1wt% to 3wt%.
Wherein the dosage ratio of chitosan to acetic acid solution for dissolving chitosan is preferably 1g (10-50) mL.
Wherein, the volume ratio of the acetic acid solution to the liquid paraffin is preferably 1 (1-2).
Wherein the emulsifier is preferably Span-80.
Wherein the volume of the liquid paraffin and the emulsifier is 1 (0.01-0.05).
Wherein the volume ratio of the acetic acid solution to the ethyl acetate is 1 (0.1-0.5).
Wherein the mass ratio of chitosan to formaldehyde is preferably 1 (0.1-0.5).
Wherein the mass ratio of chitosan to glutaraldehyde is preferably 1 (0.1-0.5).
Among them, naOH solution is preferably used to adjust pH.
Wherein, in the step (4): the product is preferably washed with petroleum ether, ethanol and sodium hydroxide solution and dried.
Further, in step (5): and (3) swelling the chitosan/activated carbon/shell powder composite microspheres obtained in the step (4) with water, and reacting with phytic acid at 30-80 ℃ for 1-5 h.
Wherein, in the step (5): and adding a phytic acid solution with the concentration of 10-30wt%. The dosage ratio of the chitosan/activated carbon/shell powder composite microsphere obtained in the step (4) to the phytic acid solution is preferably 1g (1-5) mL.
Still further, in step (5): before the reaction with phytic acid, sodium hydroxide solution and epichlorohydrin are added into the swelled chitosan/activated carbon/shell powder composite microsphere for activation. The addition of sodium hydroxide can drive the activation to proceed continuously.
Specifically, in the step (5), the activation method is preferably as follows: adding sodium hydroxide solution into the swelled chitosan/activated carbon/shell powder composite microspheres, controlling the temperature at 40-70 ℃ and activating for 1-3 hours; and then adding epoxy chloropropane, and performing coupling for 1-3 hours at the temperature of 30-50 ℃.
Wherein, in the step (5): the concentration of the sodium hydroxide solution is preferably 1 to 5mol/L.
Wherein, in the step (5): the dosage ratio of the chitosan/activated carbon/shell powder composite microsphere obtained in the step (4) to the sodium hydroxide solution is preferably 1g (1-2) mL.
Wherein, in the step (5): the dosage ratio of the chitosan/activated carbon/shell powder composite microsphere obtained in the step (4) to the epichlorohydrin is preferably 1g (0.1-0.6) mL.
Further, the method for removing heavy metals in the enzymolysis liquid comprises the following steps: and adding the adsorption material into the enzymolysis liquid obtained after enzymolysis, adjusting the pH value to 6-9, adjusting the temperature to 30-50 ℃, and stirring for 1.5-3 hours.
Still further, the amount of the adsorption material is 2-4 g/L calculated by the enzymolysis liquid.
Further, the adsorption material can be separated and recovered by means of acid washing, and can be used for multiple times.
Further, the abalone viscera are preferably subjected to enzymolysis by pepsin and then by trypsin to obtain enzymolysis liquid.
Specifically, the conditions for the enzymolysis of abalone viscera are preferably as follows: homogenizing viscera of abalone, and mixing with water; pepsin is added, and enzymolysis is carried out for 4-8 hours at the pH of 2-4 and the temperature of 35-45 ℃; then adding trypsin, and carrying out enzymolysis for 2-4 hours at the pH of 6-7.5 and the temperature of 35-45 ℃.
Wherein the mass ratio of the abalone viscera to the water is preferably 1 (2-5).
Further, after the abalone viscera are subjected to enzymolysis, the enzymolysis liquid is preferably subjected to clarification and decoloration treatment.
Among them, clarification treatment by centrifugation is preferable.
Among them, the enzymatic hydrolysate is preferably decolorized with activated carbon.
Further, the abalone viscera are preferably viscera of Haliotis discus makinoica.
The beneficial effects of the invention are as follows:
the invention carries out phytic acid grafting modification on the solidified chitosan/activated carbon/shell powder composite microsphere, and uses the phytic acid modified composite microsphere as an adsorption material for adsorbing heavy metals in abalone visceral peptide, thereby effectively solving the problem of high heavy metal content in abalone visceral peptide, and the average particle size of the composite microsphere obtained by the invention can reach 350 mu m, and compared with a powdery adsorbent, the composite microsphere is easier to separate and recycle. The adsorption effect of the phytic acid modified composite microsphere prepared by the invention on heavy metals in abalone viscera polypeptides is obviously better than that of the existing adsorbent.
Detailed Description
The principles and features of the present invention are described below in connection with examples, which are set forth only to illustrate the present invention and not to limit the scope of the invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
The method for preparing abalone viscera peptide comprises the following steps:
s1, raw material pretreatment: homogenizing viscera of Haliotis discus Sus Domestica, mixing with water, and pre-treating with ultrasound for 30 min; the mass ratio of the abalone viscera to the water is 1:2.
S2, enzymolysis: adding 3000U/g pepsin (EC 3.4.23.1) calculated by abalone viscera into the feed liquid obtained in the step S1, and performing enzymolysis at the pH of 2 and 42 ℃ for 6 h; then adding trypsin (EC 3.4.21.4) 100U/g based on abalone viscera, and performing enzymolysis at 37deg.C at pH7 for 2 h; enzyme deactivation; obtaining enzymolysis liquid.
S3, clarifying: centrifuging to obtain clarified enzymolysis liquid.
S4, decoloring: adding 50 g/L active carbon in the previous volume to the clarified enzymolysis liquid, and stirring for 1 h at 35 ℃ to obtain the decolorized enzymolysis liquid.
S5, preparing an adsorption material:
(1) Preparation of Shell Powder (SP): repeatedly washing the chlamys farreri shells with distilled water, cleaning, soaking in 2 moL/L hydrochloric acid for 1 h to remove impurities attached to the surfaces of the shells, cleaning with distilled water, drying, calcining the shells in a muffle furnace at 900 ℃ for 5h, crushing, and sieving with a 120-mesh sieve for later use.
(2) Preparation of phytic acid modified shell powder (PSP): 1g of shell powder is fully mixed with a phytic acid solution with the concentration of 5 mL of 20wt percent, the mixture is reacted at 35 ℃ for 3h, and after the reaction is finished, the mixture is filtered, washed by deionized water and dried for standby.
(3) Preparing phytic acid modified activated carbon: placing 5 g of 200-mesh activated carbon powder into a triangular flask, adding 200 mL deionized water, adding 25-mL of phytic acid solution with the concentration of 20wt%, and magnetically stirring until the mixture is uniformly mixed; reacting at 60 ℃ for 1 h, filtering after the reaction is finished, cleaning with deionized water, and drying for later use.
(4) Preparing chitosan/activated carbon/shell powder composite microspheres (CS-PAC-PSP): dissolving 20 g chitosan in 400-mL concentration acetic acid solution with 2wt%, and stirring until the chitosan is fully dissolved to obtain gel solution; adding 10 g phytic acid modified shell powder and 3.4 g phytic acid modified activated carbon into the shell powder, and uniformly stirring; then 400 mL liquid paraffin is added into the mixture and fully stirred for 10 min at 50 ℃; then maintaining the temperature at 50 ℃, dropwise adding 8 mL emulsifier Span-80, stirring for 30 min, then adding 80 mL ethyl acetate, maintaining the temperature at 50 ℃, continuously stirring for 15 min, raising the temperature to 60 ℃, adding 40 mL of 0.1 g/mL of pre-crosslinking agent formaldehyde solution, and stirring for 30 min to enable partial amino groups on chitosan to have pre-crosslinking reaction with formaldehyde; after the reaction, the temperature is raised to 70 ℃, 10 mL of glutaraldehyde solution of cross-linking agent with the concentration of 0.5 g/mL is added, and NaOH solution is used for adjusting the pH of the system to 8, so as to carry out cross-linking reaction 3h. After the crosslinking reaction is finished, the chitosan/activated carbon/shell powder composite microsphere is obtained after the filtration is carried out by a Buchner funnel, the washing is carried out by petroleum ether, ethanol and sodium hydroxide solution and the drying is carried out.
(5) Preparation of phytic acid modified composite microsphere (PCS-PAC-PSP): fully swelling 10 g chitosan/activated carbon/shell powder composite microspheres with deionized water, adding 10 mL sodium hydroxide solution with concentration of 3 mol/L, and activating at 60deg.C to obtain 1 h; then adding 2 mL epoxy chloropropane, and performing coupling at the temperature of 40 ℃ for 2 h; then adding 20 mL concentration of 20wt% phytic acid solution, reacting at 50 ℃ for 3h, and carrying out surface grafting modification to obtain phytic acid modified composite microspheres marked as PCS-PAC-PSP, wherein the average particle size is 350 mu m.
S6, heavy metal removal: adding 3 g/L of the phytic acid modified composite microsphere PCS-PAC-PSP obtained in the step S5 into the decolored enzymolysis liquid obtained in the step S4, regulating the pH value to 7.5, regulating the temperature to 40 ℃, and stirring for 2 hours to remove heavy metals.
S7, removing the phytic acid modified composite microsphere: and centrifuging to obtain enzymolysis liquid after heavy metal removal.
S8, molecular interception: and intercepting the enzymolysis liquid after heavy metal removal by a 1000 Da nanofiltration membrane to obtain a peptide segment with the molecular weight less than or equal to 1000 Da.
S9, drying: and freeze-drying to obtain the small molecular peptide powder.
Example 2
Referring to example 1, the difference from example 1 is that the working conditions of step S6 are: adding 2 g/L of the phytic acid modified composite microsphere PCS-PAC-PSP obtained in the step S5 into the decolored enzymolysis liquid obtained in the step S4, regulating the pH value to 6.5, regulating the temperature to 30 ℃, stirring for 1.5 h, and removing heavy metals.
The other technical features are the same as those of example 1.
Example 3
Referring to example 1, the difference from example 1 is that in step S5 (5), phytic acid was added and then reacted at 30℃with 1 h.
The remaining features are the same as in example 1.
Comparative example 1
Referring to example 1, the difference from example 1 is that steps S5, S6, S7 are not performed; namely: no adsorption material is prepared and no heavy metal is removed.
The remaining features are the same as in example 1.
Comparative example 2
Referring to example 1, the difference from example 1 is that the chitosan in step S5 (4) was replaced with the phytic acid modified chitosan in equal amount, and step S (5) was not performed, namely: and (3) not carrying out phytic acid modification on the composite microsphere prepared in the step (4).
The remaining features are the same as in example 1.
The preparation method of the phytic acid modified chitosan comprises the following steps: dissolving 10 g chitosan powder in acetic acid solution with the concentration of 2wt% of 200 mL, adding phytic acid with the concentration of 20wt% of 20 mL, stirring at 50 ℃ for reaction of 2h, vacuumizing, standing and defoaming to obtain the phytic acid modified chitosan.
Comparative example 3
Referring to example 1, the difference from example 1 is that step S5 (5) was not performed, and the phytic acid modified composite microsphere PCS-PAC-PSP used in step S6 was replaced with the chitosan/activated carbon/shell powder composite microsphere obtained in step S5 (4) in equal amount.
The other technical features are the same as those of example 1.
Test 1 heavy metal removal experiment
The heavy metal content in the small molecule peptide powders obtained in examples 1 to 3 and comparative examples 1 to 3 was tested, and the results are shown in Table 1.
TABLE 1 heavy metal content in small molecule peptide powders
| Heavy metal content | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Cadmium content (mg/kg) | 0.17 | 1.57 | 3.16 | 10.59 | 4.23 | 5.62 |
| Lead content (mg/kg) | 0.43 | 0.65 | 1.22 | 2.35 | 1.54 | 1.75 |
The data listed in the table show that the technology can remove heavy metals in abalone viscera, wherein the cadmium content removal rate can reach more than 90 percent, and the lead content removal rate can reach more than 80 percent.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (8)
1. A preparation method of abalone viscera peptide comprises enzymolysis of abalone viscera with protease, and is characterized in that adsorption material is added into enzymolysis liquid obtained after enzymolysis to remove heavy metal;
the preparation method of the adsorption material comprises the following steps:
(1) Obtaining shell powder;
(2) Preparing phytic acid modified shell powder: reacting the shell powder obtained in the step (1) with phytic acid to obtain phytic acid modified shell powder;
(3) Preparing phytic acid modified activated carbon: reacting the activated carbon powder with phytic acid to obtain phytic acid modified activated carbon;
(4) Preparing chitosan/activated carbon/shell powder composite microspheres: mixing chitosan, the phytic acid modified shell powder obtained in the step (2) and the phytic acid modified activated carbon obtained in the step (3) into an acetic acid solution, and preparing chitosan/activated carbon/shell powder composite microspheres by adopting a reverse suspension crosslinking method;
the specific working conditions are as follows:
dissolving chitosan in acetic acid solution, adding the phytic acid modified shell powder obtained in the step (2) and the phytic acid modified activated carbon obtained in the step (3), and uniformly stirring; then adding liquid paraffin and an emulsifying agent, and stirring to form a uniform emulsion; adding ethyl acetate, stirring, and then adding formaldehyde solution for pre-crosslinking; finally, glutaraldehyde solution is added for crosslinking and curing;
(5) Preparing phytic acid modified composite microspheres: and (3) reacting the chitosan/activated carbon/shell powder composite microsphere obtained in the step (4) with phytic acid to obtain the phytic acid modified composite microsphere.
2. The method according to claim 1, wherein in step (5): and (3) swelling the chitosan/activated carbon/shell powder composite microspheres obtained in the step (4) with water, and reacting with phytic acid at 30-80 ℃ for 1-5 h.
3. The method according to claim 2, wherein in step (5): before the reaction with phytic acid, sodium hydroxide solution and epichlorohydrin are added into the swelled chitosan/activated carbon/shell powder composite microsphere for activation.
4. The method according to claim 1, wherein in step (1): calcining shell, and pulverizing to obtain shell powder.
5. The preparation method according to any one of claims 1 to 4, wherein the heavy metal removal method comprises the following steps: and adding the adsorption material into the enzymolysis liquid obtained after enzymolysis, adjusting the pH value to 6-9, adjusting the temperature to 30-50 ℃, and stirring for 1.5-3 hours.
6. The method according to claim 5, wherein the amount of the adsorbent is 2-4 g/L based on the enzymatic hydrolysate.
7. The preparation method of any one of claims 1-4, wherein the abalone viscera are subjected to enzymolysis by pepsin and then by trypsin to obtain an enzymolysis liquid.
8. The method according to any one of claims 1 to 4, wherein the enzymolysis solution is subjected to clarification and decolorization after the enzymolysis of the viscera of abalone.
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| CN110102082A (en) * | 2019-05-21 | 2019-08-09 | 浙江省海洋水产研究所 | The minimizing technology of heavy metal cadmium in a kind of shellfish enzymolysis liquid |
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| CN107952416A (en) * | 2017-11-23 | 2018-04-24 | 浙江海洋大学 | A kind of oyster shell whiting chitosan compound microsphere for adsorbing heavy metal and preparation method thereof |
| CN110102082A (en) * | 2019-05-21 | 2019-08-09 | 浙江省海洋水产研究所 | The minimizing technology of heavy metal cadmium in a kind of shellfish enzymolysis liquid |
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