CN114601171A - Preparation method of rana japonica bone meat polypeptide chelated zinc microcapsule - Google Patents
Preparation method of rana japonica bone meat polypeptide chelated zinc microcapsule Download PDFInfo
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- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 75
- 235000013372 meat Nutrition 0.000 title claims abstract description 74
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 66
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 61
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 61
- 229940038879 chelated zinc Drugs 0.000 title claims abstract description 46
- 239000003094 microcapsule Substances 0.000 title claims abstract description 30
- 241000270930 Rana japonica Species 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 241000191896 Rana sylvatica Species 0.000 claims abstract description 66
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims abstract description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 241001336827 Rana chensinensis Species 0.000 claims abstract description 13
- 238000005238 degreasing Methods 0.000 claims abstract description 8
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- 238000000034 method Methods 0.000 claims abstract description 3
- 239000000843 powder Substances 0.000 claims description 37
- 239000007788 liquid Substances 0.000 claims description 34
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000002244 precipitate Substances 0.000 claims description 14
- 238000009210 therapy by ultrasound Methods 0.000 claims description 14
- 238000001816 cooling Methods 0.000 claims description 13
- 238000004108 freeze drying Methods 0.000 claims description 13
- 239000012153 distilled water Substances 0.000 claims description 12
- 238000000108 ultra-filtration Methods 0.000 claims description 12
- 230000009920 chelation Effects 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 229920000858 Cyclodextrin Polymers 0.000 claims description 10
- 239000001116 FEMA 4028 Substances 0.000 claims description 10
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 10
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 10
- 229960004853 betadex Drugs 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 235000005074 zinc chloride Nutrition 0.000 claims description 10
- 239000011592 zinc chloride Substances 0.000 claims description 10
- 108010019160 Pancreatin Proteins 0.000 claims description 8
- 108090000526 Papain Proteins 0.000 claims description 8
- 239000004365 Protease Substances 0.000 claims description 8
- 229940055695 pancreatin Drugs 0.000 claims description 8
- 229940055729 papain Drugs 0.000 claims description 8
- 235000019834 papain Nutrition 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000004090 dissolution Methods 0.000 claims description 7
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- 238000002156 mixing Methods 0.000 claims description 7
- 238000002474 experimental method Methods 0.000 claims description 6
- 238000000227 grinding Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 239000004570 mortar (masonry) Substances 0.000 claims description 6
- 239000008055 phosphate buffer solution Substances 0.000 claims description 6
- 238000005057 refrigeration Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
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- 238000010298 pulverizing process Methods 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims 1
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- 239000011701 zinc Substances 0.000 abstract description 14
- 229910052725 zinc Inorganic materials 0.000 abstract description 14
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- 206010048259 Zinc deficiency Diseases 0.000 abstract description 5
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- 239000013589 supplement Substances 0.000 abstract 1
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- 235000019441 ethanol Nutrition 0.000 description 10
- 230000002255 enzymatic effect Effects 0.000 description 7
- 108010009736 Protein Hydrolysates Proteins 0.000 description 5
- 239000000413 hydrolysate Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000000717 retained effect Effects 0.000 description 5
- 238000007873 sieving Methods 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 241000269350 Anura Species 0.000 description 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 206010013883 Dwarfism Diseases 0.000 description 1
- 206010013911 Dysgeusia Diseases 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 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 description 1
- 241000269435 Rana <genus> Species 0.000 description 1
- 101000924393 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Vacuolar aminopeptidase 1 Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 231100000001 growth retardation Toxicity 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
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- 150000003751 zinc Chemical class 0.000 description 1
- 229940091251 zinc supplement Drugs 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
- A23L33/165—Complexes or chelates
-
- 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
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Dispersion Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the field of deep processing of wood frog applied to wood frog bone and meat, and discloses a preparation method of a wood frog bone and meat polypeptide chelated zinc microcapsule and a microcapsule prepared by an embedding technology. The method comprises the following steps of 1, wood frog residue pretreatment; 2. degreasing; 3. performing enzymolysis to obtain Rana temporaria chensinensis polypeptide solution; 4. ultrafiltering to obtain small molecular polypeptide; 5. preparing polypeptide chelated zinc; 6. alcohol precipitation of polypeptide chelated zinc; 7. and (4) preparing the microcapsule. The polypeptide chelated zinc is used as a novel trace element supplement, can avoid zinc precipitation, can be absorbed through an intestinal peptide absorption way and can regulate the proliferation and differentiation of intestinal cells through interaction of the intestinal peptide absorption way, and the utilization rate of organisms is improved. The rana japonica bone and meat polypeptide chelated zinc microcapsule prepared by the invention is expected to provide a feasible zinc supplementation scheme for zinc deficiency patients and improve the comprehensive utilization rate of rana japonica.
Description
Technical Field
The invention belongs to the field of deep processing of wood frog products, and researches a preparation method of a wood frog bone and meat polypeptide chelated zinc microcapsule.
Background
The rare frog species of the forest frogs in the northeast of China has been recorded in the compendium of materia Medica for pharmacological action, and the forest frog oil is known as one of the three treasures in the northeast. However, the deep processing of the current wood frog products mainly uses wood frog oil as a main material, and most of the wood frog bone and meat are discarded. This not only pollutes the environment, but also wastes resources. Therefore, how to improve the deep processing and added value of the wood frog products and create greater economic and ecological benefits is one of the important problems concerned in the wood frog product processing field.
Zinc is a trace element necessary for human body, and its content is second to iron. Dietary zinc is primarily absorbed from the intestinal tract and distributed to the tissues by circulating blood. Zinc functions to maintain protein structure and function by coordinating with zinc finger and ring finger domains, and about 200 enzyme activities in human body are related to zinc. Zinc deficiency is known to cause various symptoms of immune disorders, skin development and taste disturbances. In addition, growth retardation occurs when zinc is deficient in children, and severe deficiency can lead to dwarfism.
Like other trace elements, zinc deficiency is an urgent problem to be solved in China. However, the bioavailability of inorganic zinc salts in the intestinal tract may be negatively affected by plant fiber and phytate, which can form insoluble complexes with zinc ions and reduce the absorption rate of zinc. The chelating peptide and the zinc can be combined to form a soluble chelate, so that zinc precipitation is avoided; and secondly, the zinc-peptide compound can be absorbed through an intestinal peptide absorption way and can regulate the proliferation and differentiation of intestinal cells through interaction of the intestinal peptide absorption way, so that the biological utilization of the body to zinc element can be greatly improved by the polypeptide chelated zinc.
The preparation method takes the wood frog bone and meat and zinc chloride as raw materials, explores the preparation process of the wood frog bone and meat polypeptide chelated zinc microcapsule, and aims to provide a feasible zinc supplementing scheme and improve the utilization rate of the wood frog bone and meat.
Disclosure of Invention
The invention takes the wood frog bone and meat and zinc chloride as raw materials to prepare the wood frog bone and meat polypeptide chelated zinc microcapsule. Aiming at the low utilization rate of wood frog bone and meat and the current situation of zinc deficiency in China, a solution strategy is provided.
In order to solve the technical problems, the invention adopts the technical scheme that:
(1) pretreatment: cleaning appropriate amount of Rana temporaria chensinensis bone and meat residue with distilled water, and oven drying at 40-60 deg.C for 1-4 hr. Pulverizing the dried wood frog residue with a multifunctional pulverizer for 5min, wherein the operation is stopped for 3min to cool the equipment every 1min, the pulverized wood frog bone meat is sequentially sieved with a 50-200 mesh sieve, and the obtained wood frog bone meat powder is sealed and refrigerated.
(2) Degreasing: putting the wood frog bone meat powder into a beaker, and adding 0.5mol/L N according to the ratio of the material to the liquid of 1:5-1:20aHCO3The solution is soaked overnight to remove lipid from the wood frog bone and meat powder. The supernatant was then centrifuged off, the precipitate was washed twice with distilled water, and the precipitate was retained by centrifugation.
(3) Enzymolysis: adding distilled water into the degreased wood frog bone meat powder according to the ratio of the material to the liquid of 1:5-1:20, and firstly carrying out enzymolysis for 1-3h by using 0.5% of pancreatin, wherein the enzymolysis temperature is 30-50 ℃, and the pH value is 6.0-9.0. Then adding 1% papain for enzymolysis for 60-120min again, wherein the enzymolysis temperature is 40-70 deg.C, and pH is 6.0-9.0. After sufficient enzymolysis, the enzymatic hydrolysate was heated to 95 ℃ for 15min to inactivate the enzyme. And (3) rapidly cooling and centrifuging, wherein the supernatant is the wood frog bone and meat polypeptide, and the centrifugation conditions are as follows: centrifuging at 10000rpm at 4 deg.C for 10 min.
(4) And (3) ultrafiltration: the prepared rana chensinensis bone and meat polypeptide liquid sequentially passes through ultrafiltration membranes of 20kD, 10kD and 5kD, and the ultrafiltered polypeptide liquid can be immediately used for a chelation experiment or temporary refrigeration in a refrigerator and can also be stored for a long time after being freeze-dried.
(5) Chelating: adding 2-10% zinc chloride powder according to the volume of the polypeptide liquid, fully dissolving, performing ultrasonic treatment for 5min, and then placing in a 45 ℃ water bath for chelation for 30 min.
(6) Alcohol precipitation: adding 5-20 times volume of anhydrous ethanol into the chelated polypeptide liquid to carry out alcohol cooling on the chelated zinc, standing at room temperature for 6h, then centrifuging to remove supernatant, placing the precipitate in a freeze dryer, and carrying out sealing and cold storage after freeze drying.
(7) Preparing microcapsules: taking a proper amount of beta-cyclodextrin, putting the beta-cyclodextrin into a phosphate buffer solution for full dissolution, then adding a proper amount of wood frog bone and meat chelated zinc powder, uniformly mixing, and carrying out ultrasonic treatment at 30-60 ℃ for 25 min. Then freeze-drying, grinding into powder by using a mortar, and obtaining the powder, namely the rana japonica bone meat polypeptide chelated zinc microcapsule.
Preferably, the method comprises the following steps:
in the step (1), the cleaned wood frog residual bodies are dried in an oven at 60 ℃ for 1 hour, and are crushed and sieved by a 50-mesh sieve.
In the step (2), degreasing is carried out according to the ratio of material to liquid of 1: 10.
In the step (3), distilled water is added according to the ratio of material to liquid of 1:10 for enzymolysis, and pancreatin is used for enzymolysis for 2 hours, wherein the enzymolysis temperature is 40 ℃, and the pH value is 8.0. Enzymolysis with papain for 90min at 60 deg.C and pH 8.0.
In the step (5), the addition amount of the zinc chloride powder is 5%.
In the step (6), the addition amount of the absolute ethyl alcohol is 7 times the volume of the chelating solution.
In the step (7), the ratio of the beta-cyclodextrin to the wood frog bone and meat chelated zinc powder is 1:20, and the preparation temperature of the microcapsule is 50 ℃.
Compared with the prior art, the invention has the advantages that:
(1) the invention uses the wood frog residual body as the polypeptide raw material, thereby not only avoiding the pollution of the wood frog residual body to the environment, but also improving the comprehensive utilization rate of the wood frog.
(2) The polypeptide chelated zinc prepared by the invention can avoid the negative influence of dietary plant fibers on the zinc utilization rate, and meanwhile, the polypeptide chelated zinc can improve the content of soluble zinc in intestinal tracts, promote the absorption of zinc, and provide a zinc supplement product beneficial to the absorption and utilization of human bodies for improving the current situation of zinc deficiency of residents in China.
(2) The invention uses the wood frog bone and meat polypeptide chelated zinc prepared by microcapsule embedding, which is beneficial to protecting the bioactivity of the polypeptide chelated zinc and prolonging the storage time.
Detailed Description
The following are specific embodiments of the present invention:
example 1
(1) Pretreatment: and (3) taking 30g of wood frog bone and meat residues, cleaning, and drying in an oven at 60 ℃ for 1 h. Crushing the dried wood frog residues for 5min, and sieving with a 50-mesh sieve to obtain 26.3g of wood frog bone and meat powder.
(2) Degreasing: putting the wood frog bone meat powder into a beaker, adding 263mL of 0.5mol/L NaHCO3The solution was soaked overnight. The precipitate was then retained after centrifugal washing.
(3) Enzymolysis: 263mL of distilled water is added into degreased wood frog bone meat powder, 1.315g of pancreatin is firstly used for enzymolysis for 2h, the enzymolysis temperature is 40 ℃, and the pH value is 8.0. Then 2.63g papain was added for another 90min, the enzymolysis temperature was 60 deg.C, pH 8.0. After sufficient enzymolysis, the enzymatic hydrolysate was heated to 95 ℃ for 15min to inactivate the enzyme. Quickly cooling and centrifuging to obtain 255mL of wood frog bone and meat polypeptide hydrolysate, wherein the centrifugation conditions are as follows: centrifuging at 10000rpm at 4 deg.C for 10 min.
(4) And (3) ultrafiltration: the prepared rana chensinensis bone and meat polypeptide liquid sequentially passes through ultrafiltration membranes of 20kD, 10kD and 5kD, and the ultrafiltered polypeptide liquid can be immediately used for a chelation experiment or temporary refrigeration in a refrigerator and can also be stored for a long time after being freeze-dried.
(5) Chelating: adding 10g of zinc chloride powder into 200mL of the ultrafiltered enzymolysis liquid, fully dissolving, performing ultrasonic treatment for 5min, and then placing in a 45 ℃ water bath for chelation for 30 min.
(6) Alcohol reduction: and adding 1400mL of absolute ethyl alcohol into the chelated polypeptide liquid to carry out alcohol cooling on the chelated zinc, standing at room temperature for 6 hours, centrifuging to remove supernatant, placing the precipitate in a freeze dryer, and freeze-drying to obtain 23.7g of rana chensinensis polypeptide chelated zinc.
(7) Preparing microcapsules: taking 100g of beta-cyclodextrin, putting into a phosphate buffer solution for full dissolution, then adding 5g of wood frog bone and meat chelated zinc powder, uniformly mixing, and carrying out ultrasonic treatment at 50 ℃ for 25 min. Then freeze-drying, grinding into powder by using a mortar, and obtaining 104.7g of the rana japonica bone meat polypeptide chelated zinc microcapsule.
Example 2
(1) Pretreatment: 50g of wood frog bone and meat residues are cleaned and then dried in an oven for 1 hour at 60 ℃. Crushing the dried wood frog residues for 5min, and sieving with a 50-mesh sieve to obtain 43.1g of wood frog bone and meat powder.
(2) Degreasing: putting the wood frog bone meat powder into a beaker, adding 431mL of 0.5mol/L NaHCO3The solution was soaked overnight. The precipitate was then retained after centrifugal washing.
(3) Enzymolysis: adding 431mL of distilled water into the degreased wood frog bone meat powder, and firstly performing enzymolysis for 2h by using 2.155g of pancreatin, wherein the enzymolysis temperature is 40 ℃, and the pH value is 8.0. Then adding 4.31g papain for enzymolysis for 90min again, wherein the enzymolysis temperature is 60 deg.C, and pH is 8.0. After sufficient enzymolysis, the enzymatic hydrolysate was heated to 95 ℃ for 15min to inactivate the enzyme. Quickly cooling and centrifuging to obtain 417mL of wood frog bone and meat polypeptide hydrolysate, wherein the centrifugation conditions are as follows: centrifuging at 10000rpm at 4 deg.C for 10 min.
(4) And (3) ultrafiltration: the prepared wood frog bone and meat polypeptide liquid sequentially passes through ultrafiltration membranes of 20kD, 10kD and 5kD, and the ultrafiltered polypeptide liquid can be immediately used for chelation experiments or temporary refrigeration in a refrigerator, and can also be stored for a long time after freeze-drying.
(5) Chelating: adding 10g of zinc chloride powder into 200mL of the ultrafiltrated enzymatic hydrolysate, fully dissolving, performing ultrasonic treatment for 5min, and chelating in a 45 ℃ water bath for 30 min.
(6) Alcohol reduction: and adding 1400mL of absolute ethyl alcohol into the chelated polypeptide liquid to carry out alcohol cooling on the chelated zinc, standing at room temperature for 6 hours, then centrifuging to remove supernatant, placing the precipitate in a freeze dryer, and freeze-drying to obtain 23.4g of rana chensinensis polypeptide chelated zinc.
(7) Preparing microcapsules: taking 100g of beta-cyclodextrin, putting into a phosphate buffer solution for full dissolution, then adding 5g of wood frog bone and meat chelated zinc powder, uniformly mixing, and performing ultrasonic treatment at 50 ℃ for 25 min. Then freeze-drying, grinding into powder by using a mortar, and obtaining 104.5g of the rana japonica bone meat polypeptide chelated zinc microcapsule.
Example 3
(1) Pretreatment: 100g of wood frog bone and meat residues are cleaned and then dried in an oven for 1 hour at 60 ℃. Crushing the dried wood frog residues for 5min, and sieving with a 50-mesh sieve to obtain 88.8g of wood frog bone and meat powder.
(2) Degreasing: putting the wood frog bone meat powder into a beaker, adding 888mL of 0.5mol/L NaHCO3The solution was soaked overnight. The precipitate was then retained after centrifugal washing.
(3) Enzymolysis: adding 888mL distilled water into defatted bone powder of Rana temporaria chensinensis David, and performing enzymolysis with 4.44g pancreatin at 40 deg.C and pH of 8.0 for 2 hr. Then 8.88g of papain is added for enzymolysis for 90min again, the enzymolysis temperature is 60 ℃, and the pH value is 8.0. After sufficient enzymolysis, the enzymatic hydrolysate was heated to 95 ℃ for 15min to inactivate the enzyme. Quickly cooling and centrifuging to obtain 862mL of wood frog bone and meat polypeptidase hydrolysate, wherein the centrifugation conditions are as follows: centrifuging at 10000rpm at 4 deg.C for 10 min.
(4) And (3) ultrafiltration: the prepared rana chensinensis bone and meat polypeptide liquid sequentially passes through ultrafiltration membranes of 20kD, 10kD and 5kD, and the ultrafiltered polypeptide liquid can be immediately used for a chelation experiment or temporary refrigeration in a refrigerator and can also be stored for a long time after being freeze-dried.
(5) Chelating: adding 10g of zinc chloride powder into 200mL of the ultrafiltrated enzymatic hydrolysate, fully dissolving, performing ultrasonic treatment for 5min, and chelating in a 45 ℃ water bath for 30 min.
(6) Alcohol precipitation: and adding 1400mL of absolute ethyl alcohol into the chelated polypeptide liquid to carry out alcohol cooling on the chelated zinc, standing at room temperature for 6 hours, then centrifuging to remove the supernatant, placing the precipitate in a freeze dryer, and freeze-drying to obtain 23.6g of rana polypeptide chelated zinc.
(7) Preparing microcapsules: taking 100g of beta-cyclodextrin, putting into a phosphate buffer solution for full dissolution, then adding 5g of wood frog bone and meat chelated zinc powder, uniformly mixing, and performing ultrasonic treatment at 50 ℃ for 25 min. Then freeze-drying, grinding into powder by using a mortar, and obtaining 104.8g of the rana japonica bone meat polypeptide chelated zinc microcapsule.
Example 4
(1) Pretreatment: 100g of wood frog bone and meat residues are taken and cleaned, and then dried in an oven for 1 hour at 60 ℃. Crushing the dried wood frog residues for 5min, and sieving with a 50-mesh sieve to obtain 87.9g of wood frog bone and meat powder.
(2) Degreasing: putting wood frog bone meat powder into a beaker, adding 879mL of 0.5mol/L NaHCO3The solution was soaked overnight. The precipitate was then retained after centrifugal washing.
(3) Enzymolysis: 879mL of distilled water is added into the degreased wood frog bone meat powder, 4.395g of pancreatin is firstly used for enzymolysis for 2h, the enzymolysis temperature is 40 ℃, and the pH value is 8.0. Then 8.79g of papain is added for enzymolysis for 90min again, the enzymolysis temperature is 60 ℃, and the pH value is 8.0. After sufficient enzymolysis, the enzymatic hydrolysate was heated to 95 ℃ for 15min to inactivate the enzyme. Rapidly cooling and centrifuging to obtain 855mL of wood frog bone and meat polypeptide enzymolysis liquid, wherein the centrifugation conditions are as follows: centrifuging at 10000rpm at 4 deg.C for 10 min.
(4) And (3) ultrafiltration: the prepared rana chensinensis bone and meat polypeptide liquid sequentially passes through ultrafiltration membranes of 20kD, 10kD and 5kD, and the ultrafiltered polypeptide liquid can be immediately used for a chelation experiment or temporary refrigeration in a refrigerator and can also be stored for a long time after being freeze-dried.
(5) Chelating: adding 10g of zinc chloride powder into 200mL of the ultrafiltered enzymolysis liquid, fully dissolving, performing ultrasonic treatment for 5min, and then placing in a 45 ℃ water bath for chelation for 30 min.
(6) Alcohol reduction: and adding 1400mL of absolute ethyl alcohol into the chelated polypeptide liquid to carry out alcohol cooling on the chelated zinc, standing at room temperature for 6 hours, centrifuging to remove supernatant, placing the precipitate in a freeze dryer, and freeze-drying to obtain 23.7g of rana chensinensis polypeptide chelated zinc.
(7) Preparing microcapsules: taking 100g of beta-cyclodextrin, putting into a phosphate buffer solution for full dissolution, then adding 5g of wood frog bone and meat chelated zinc powder, uniformly mixing, and carrying out ultrasonic treatment at 50 ℃ for 25 min. Then freeze-drying, grinding into powder by using a mortar, and obtaining 104.6g of the rana japonica bone meat polypeptide chelated zinc microcapsule.
Claims (6)
1. A preparation method of a rana japonica bone meat polypeptide chelated zinc microcapsule is characterized by comprising the following steps:
(1) pretreatment: cleaning appropriate amount of Rana temporaria chensinensis bone and meat residue with distilled water, and oven drying at 40-60 deg.C for 1-4 hr; pulverizing the dried wood frog residue with a multifunctional pulverizer for 5min, wherein the operation is stopped for 3min to cool the equipment every 1min, the pulverized wood frog bone meat is sequentially sieved with a 50-200 mesh sieve, and the obtained wood frog bone meat powder is sealed, refrigerated and preserved;
(2) degreasing: putting the wood frog bone meat powder into a beaker, and adding 0.5mol/L N according to the ratio of the material to the liquid of 1:5-1:20aHCO3Soaking the solution overnight to remove lipid in the bone and meat powder of Rana temporaria chensinensis David; centrifuging to remove supernatant, washing the precipitate with distilled water twice, centrifuging and retaining the precipitate;
(3) enzymolysis: adding distilled water into the degreased wood frog bone meat powder according to the ratio of material to liquid of 1:5-1:20, firstly carrying out enzymolysis for 1-3h by using 0.5% of pancreatin, wherein the enzymolysis temperature is 30-50 ℃, and the pH value is 6.0-9.0; adding 1% papain for enzymolysis for 60-120min at 40-70 deg.C and pH 6.0-9.0; heating the enzymolysis solution to 95 deg.C for 15min to inactivate enzyme; and (3) rapidly cooling and centrifuging, wherein the supernatant is the wood frog bone and meat polypeptide, and the centrifugation conditions are as follows: centrifuging at 10000rpm at 4 ℃ for 10 min;
(4) and (3) ultrafiltration: the prepared rana chensinensis bone and meat polypeptide liquid sequentially passes through ultrafiltration membranes of 20kD, 10kD and 5kD, and the ultrafiltered polypeptide liquid can be immediately used for a chelation experiment or temporary refrigeration in a refrigerator and can also be stored for a long time after being freeze-dried;
(5) chelating: adding 2-10% zinc chloride powder according to the volume of the polypeptide liquid, fully dissolving, performing ultrasonic treatment for 5min, and then placing in a 45 ℃ water bath for chelation for 30 min;
(6) alcohol precipitation: adding 5-20 times volume of anhydrous ethanol into the chelated polypeptide liquid to carry out alcohol cooling on the chelated zinc, standing at room temperature for 6h, then centrifuging to remove supernatant, placing the precipitate in a freeze dryer, and carrying out sealing and cold storage after freeze drying;
(7) preparing microcapsules: taking a proper amount of beta-cyclodextrin, putting the beta-cyclodextrin into a phosphate buffer solution for full dissolution, then adding a proper amount of wood frog bone and meat chelated zinc powder, uniformly mixing, and performing ultrasonic treatment at 30-60 ℃ for 25 min; then freeze-drying, grinding into powder by using a mortar, and obtaining the powder, namely the rana japonica bone meat polypeptide chelated zinc microcapsule.
2. The preparation method of the rana japonica bone meat polypeptide chelated zinc microcapsule according to claim 1, wherein 0.5mol/L N is added into the microcapsule (2) according to the ratio of the feed liquid to the feed liquid of 1:10aHCO3The solution is degreased.
3. The preparation method of the rana japonica bone and meat polypeptide chelated zinc microcapsule as claimed in claim 1, wherein the defatted rana japonica bone and meat powder in the step (3) is added with distilled water according to a feed-liquid ratio of 1:10, and is subjected to enzymolysis for 2h by using 0.5% of pancreatin at 40 ℃ and with the pH value of 8.0; then adding 1% papain for enzymolysis for 90min again, wherein the enzymolysis temperature is 60 deg.C, and pH is 8.0.
4. The method for preparing the rana japonica bone meat polypeptide chelated zinc microcapsule as claimed in claim 1, wherein 5% of zinc chloride powder is added according to the volume of the polypeptide liquid in the step (5), ultrasonic treatment is performed for 5min after sufficient dissolution, and then chelation is performed for 30min in a 45 ℃ water bath.
5. The preparation method of the rana japonica bone and meat polypeptide chelated zinc microcapsule as claimed in claim 1, wherein 7 times volume of absolute ethanol is added into the polypeptide liquid chelated in (6) to carry out alcohol cooling on the chelated zinc, and the solution is placed at room temperature for 6 hours.
6. The preparation method of the rana japonica bone and meat polypeptide chelated zinc microcapsule according to claim 1, wherein the addition amount of the beta-cyclodextrin and the rana japonica bone and meat chelated zinc powder in the step (7) is 20:1, and the ultrasonic treatment is performed for 25min at 50 ℃ after the uniform mixing.
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| CN111254178A (en) * | 2020-03-23 | 2020-06-09 | 吉林大学 | Preparation method of wood frog skin small-molecular peptide chelated iron |
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