CN108968023A - A kind of glycosylation minced fish gel reinforcing agent and preparation method thereof - Google Patents
A kind of glycosylation minced fish gel reinforcing agent and preparation method thereof Download PDFInfo
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- CN108968023A CN108968023A CN201810725808.4A CN201810725808A CN108968023A CN 108968023 A CN108968023 A CN 108968023A CN 201810725808 A CN201810725808 A CN 201810725808A CN 108968023 A CN108968023 A CN 108968023A
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- glycosylation
- reinforcing agent
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- minced fish
- albumen
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- 238000006206 glycosylation reaction Methods 0.000 title claims abstract description 58
- 230000013595 glycosylation Effects 0.000 title claims abstract description 56
- 241000251468 Actinopterygii Species 0.000 title claims abstract description 35
- 239000012744 reinforcing agent Substances 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 38
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 38
- 150000004676 glycans Chemical class 0.000 claims abstract description 33
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 33
- 239000005017 polysaccharide Substances 0.000 claims abstract description 33
- 238000006366 phosphorylation reaction Methods 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 26
- 230000026731 phosphorylation Effects 0.000 claims abstract description 24
- 230000008569 process Effects 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000013019 agitation Methods 0.000 claims abstract description 15
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 14
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 14
- 210000000969 egg white Anatomy 0.000 claims abstract description 14
- 229920000161 Locust bean gum Polymers 0.000 claims abstract description 13
- 239000000711 locust bean gum Substances 0.000 claims abstract description 13
- 235000010420 locust bean gum Nutrition 0.000 claims abstract description 13
- 229920002752 Konjac Polymers 0.000 claims abstract description 12
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims abstract description 10
- 235000014103 egg white Nutrition 0.000 claims abstract description 10
- 235000019832 sodium triphosphate Nutrition 0.000 claims abstract description 8
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 claims abstract description 7
- 244000247812 Amorphophallus rivieri Species 0.000 claims abstract description 7
- 235000001206 Amorphophallus rivieri Nutrition 0.000 claims abstract description 7
- 229920002581 Glucomannan Polymers 0.000 claims abstract description 7
- 238000005352 clarification Methods 0.000 claims abstract description 7
- 239000008367 deionised water Substances 0.000 claims abstract description 7
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 7
- 210000002969 egg yolk Anatomy 0.000 claims abstract description 7
- 229940046240 glucomannan Drugs 0.000 claims abstract description 7
- 239000000252 konjac Substances 0.000 claims abstract description 7
- 235000010485 konjac Nutrition 0.000 claims abstract description 7
- 238000000502 dialysis Methods 0.000 claims abstract description 6
- 238000001914 filtration Methods 0.000 claims abstract description 3
- 230000001279 glycosylating effect Effects 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000000654 additive Substances 0.000 claims description 9
- 230000000996 additive effect Effects 0.000 claims description 9
- 108010093031 Galactosidases Proteins 0.000 claims description 8
- 102000002464 Galactosidases Human genes 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 7
- 230000007062 hydrolysis Effects 0.000 claims description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- 238000002242 deionisation method Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 21
- 230000002708 enhancing effect Effects 0.000 abstract description 20
- 102000004169 proteins and genes Human genes 0.000 abstract description 20
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- 239000000523 sample Substances 0.000 description 12
- 239000000047 product Substances 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 235000019465 surimi Nutrition 0.000 description 6
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical class OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 5
- 229940054441 o-phthalaldehyde Drugs 0.000 description 5
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000001879 gelation Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 150000003016 phosphoric acids Chemical class 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 3
- 102000005927 Cysteine Proteases Human genes 0.000 description 2
- 108010005843 Cysteine Proteases Proteins 0.000 description 2
- 102000003505 Myosin Human genes 0.000 description 2
- 108060008487 Myosin Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
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- 239000004365 Protease Substances 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 102000035101 Aspartic proteases Human genes 0.000 description 1
- 108091005502 Aspartic proteases Proteins 0.000 description 1
- 108010001682 Dextranase Proteins 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- VQLYBLABXAHUDN-UHFFFAOYSA-N bis(4-fluorophenyl)-methyl-(1,2,4-triazol-1-ylmethyl)silane;methyl n-(1h-benzimidazol-2-yl)carbamate Chemical compound C1=CC=C2NC(NC(=O)OC)=NC2=C1.C=1C=C(F)C=CC=1[Si](C=1C=CC(F)=CC=1)(C)CN1C=NC=N1 VQLYBLABXAHUDN-UHFFFAOYSA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000015168 fish fingers Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 108010000416 ovomacroglobulin Proteins 0.000 description 1
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- 230000000630 rising effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000004328 sodium tetraborate Substances 0.000 description 1
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- 238000005829 trimerization reaction Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
-
- 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
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/70—Comminuted, e.g. emulsified, fish products; Processed products therefrom such as pastes, reformed or compressed products
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Dispersion Chemistry (AREA)
- Marine Sciences & Fisheries (AREA)
- Zoology (AREA)
- Meat, Egg Or Seafood Products (AREA)
Abstract
The present invention relates to gel enhancing agents, disclose a kind of glycosylation minced fish gel reinforcing agent and preparation method thereof, it should, preparation method contains following steps: 1) separating fresh albumen with yolk, agitation and filtration obtains clarification egg white solution, egg white solution and deionized water are mixed, sodium tripolyphosphate solution is added, phosphorylation albumen solution is obtained after dialysis；2) locust bean gum and/or konjac glucomannan digest and enzymatic hydrolysis polysaccharide is made；3) phosphorylation albumen and enzymatic hydrolysis polysaccharide are subjected to wet process glycosylation.The present invention can significantly reduce preparation cost and the time of glycosylation albumen, it greatly enhances and utilizes the feasibility for glycosylating protein modified obtained minced fish gel reinforcing agent, the technical issues of improving frozen minced fillets quality especially gelling performance and whiteness is solved, is had a good application prospect.This prepares that material is cheap and easy to get, and processing step is simple, easily implements, to equipment without particular/special requirement, preparation cost is low, is conducive to industrialization promotion.
Description
Technical field
The present invention relates to gel enhancing agents more particularly to a kind of glycosylation minced fish gel reinforcing agent and preparation method thereof.
Background technique
Minced fillet is a kind of novel aquatic products conditioning food raw material.Minced fillet is cut after mixing, salt, auxiliary material etc. is added to be beaten
It bursts, at heating after sticky pasted fish meat reshaping, becomes flexible gelinite, this based article includes fish ball, breaded fish stick, with sweet and sour flavor
Intestines, paupiette etc..It is delicate delicious since surimi product improves simplicity, and storage endurance, quite it is suitble to urban consumption, this based article can
Large-scale industrialized manufacture, and can family manual production.The economic value of Optimization of Low Value Fish can be improved, and can be connect by the people
By, thus be a kind of rising aquatic product.But due to that can have following two side after frozen minced fillets long-term storage
Freeze denaturation can occur for face problem, one side protein, therefore will affect its gelling performance after surimi product is made；On the other hand,
Surimi product is needed in process by heat aging, and there are endogenous thermostable protease meetings in minced fillet fribrillin
Gel collapse is induced in 50-70 DEG C of temperature band, reduces its gelling performance.
Albumen plays an important role in the food industry, with good gelation, emulsibility and foaming characteristic etc.
Functional characteristic, and the consistency and quality of food can be improved by forming gel, egg white is also commonly used in surimi product
Albumen increases its gel strength as gel enhancing agent, improves combinations color and lustre and quality.It is in the prior art usually by egg white
Powder is added in surimi product, can generate one with the gelation for improving frozen minced fillets to a certain extent, but for its color
Determine the influence of degree.The glycosylation modification effect of protein can effectively improve the gelation of albumen protein, but dry method is sugared
Protein after glycosylation reaction can generate certain brown stain, and the high production cost reaction time is long, be added in minced fillet and often result in
The decline of minced fillet aesthetic quality, especially color.
Summary of the invention
In order to solve the above-mentioned technical problems, the present invention provides a kind of glycosylation minced fish gel reinforcing agent and its preparation sides
Method, by adjusting the component and ratio of reinforcing agent, modified using wet process glycosylation, production cost is low, and feasibility is high, by it
It is added in frozen minced fillets, the gel strength of frozen minced fillets can be effectively improved, and does not influence whiteness.
The specific technical proposal of the invention is: the preparation method of the glycosylation minced fish gel reinforcing agent contains following steps:
1) fresh albumen is separated with yolk, after stirring 10-20min, clarification egg white solution is obtained by filtration, by egg white solution and deionization
Water is mixed, and sodium tripolyphosphate solution is added while stirring, phosphorylation albumen solution is obtained after dialysis；
2) locust bean gum and/or konjac glucomannan digest and enzymatic hydrolysis polysaccharide is made；
3) phosphorylation albumen and enzymatic hydrolysis polysaccharide are subjected to wet process glycosylation, form glycosylation minced fish gel reinforcing agent.
Due to containing endogenous thermostable protease in minced fillet, when being in 50-70 DEG C of temperature band, gel occurs for minced fillet
Deterioration, the enzyme induction of endogenous heat-stable protein glycosylate the albumen in minced fish gel reinforcing agent to myosin of degrading
Containing cysteine proteinase, egg mother cell globulin and ovomacroglobulin can specific effect in fribrillin
Cysteine proteinase, serine protease and aspartic protease, so that myosin is inhibited to degrade, on the other hand, phosphorus
After being acidified the enzymatic hydrolysis polysaccharide generation glycosylation of albumen and locust bean gum and/or konjac glucomannan, gelation is improved, in minced fillet
In play the role of adhesive, the gap of gel protein network structure is filled with, so that minced fish gel structure is finer and close.Phosphorus
Albumen and enzymatic hydrolysis polysaccharide are acidified after glycosylation modification, the whiteness of gel enhancing agent itself can improve, and it is visually observed, from
And the color of minced fish gel is influenced, improve the whiteness of surimi product.
Preferably, egg white solution and the mass ratio of deionized water are 1:1 in the step 1).
Preferably, it is 9-11 that sodium tripolyphosphate solution is added in the step 1) to adjust pH.
Preferably, dialysis procedure is room temperature 2-5 hours in the step 1), reaction was completed is adjusted to 7 for pH.
Preferably, locust bean gum is digested using galactosidase in the step 2), konjac glucomannan uses β-sweet dew
Dextranase is digested.
Preferably, locust bean gum concentration is 0.5-1.2wt% in the step 2), galactosidase additive amount is 50-
100U/g；Konjaku gum concentration is 0.5-1.5wt%, and 'beta '-mannase additive amount is 60-120U/g.
Preferably, enzymatic hydrolysis pH is 4-7 in the step 2), hydrolysis temperature is 30-60 DEG C, enzymolysis time 2-5h.
Preferably, enzymatic hydrolysis polysaccharide additive amount is the 1-5wt% of phosphorylation albumen in the step 3).
Preferably, in the step 3) wet process glycosylation concrete operations are as follows: take phosphorylation albumen solution,
Enzymatic hydrolysis polysaccharide is added in magnetic agitation to being uniformly mixed, 30-60 DEG C after water-bath 0-24 hour, is taken out cooling rapidly.
With the extension of reaction time, the trend that gradually rises is presented in glycosylation degree, continue to heat influence to reaction compared with
Small, because the time that glycosylation occurs is longer, the substrate of consumption is more, then remaining substrate is fewer, so reaction process
Slow down, in addition, the too long time makes gel enhancing agent form protein film, and the albumen film thickness and toughness of time longer formation
Stronger, moisture is evaporated in film, affects the process of glycosylation.
It is compared with the prior art, the beneficial effects of the present invention are: the present invention is by carrying out phosphorylation reaction to albumen
Obtain phosphorylation albumen, digest that locust bean gum and konjac glucomannan, enzymatic hydrolysis polysaccharide is made, by phosphorylation albumen with
Enzymatic hydrolysis polysaccharide carries out wet process glycosylation and obtains glycosylation minced fish gel reinforcing agent, and the present invention can significantly reduce glycosylation egg
Albuminised preparation cost and time greatly enhance using the feasibility for glycosylating protein modified obtained minced fish gel reinforcing agent,
The technical issues of improving frozen minced fillets quality especially gelling performance and whiteness is solved, is had a good application prospect.The preparation
Method material is cheap and easy to get, and processing step is simple, easily implements, to equipment without particular/special requirement, preparation cost is low, is conducive to industrialize
It promotes.
Detailed description of the invention
Fig. 1 is the variation of 1 differential responses time of embodiment of the present invention gel enhancing agent grafting degree；
Fig. 2 is one of the SDS-PAGE map that the embodiment of the present invention 1 glycosylates gel enhancing agent；
Fig. 3 is the two of the SDS-PAGE map that the embodiment of the present invention 1 glycosylates gel enhancing agent.
Specific embodiment
The present invention will be further described with reference to the examples below.
Embodiment 1
One, glycosylation minced fish gel reinforcing agent preparation
1. albumen carries out phosphorylation reaction and obtains modified albumen.
Fresh albumen is separated with yolk, insoluble impurities is removed with gauze after magnetic agitation 10min and obtains clarification egg white
Egg white solution and deionized water are mixed in 1:1 ratio and carry out magnetic agitation, be slowly added to the trimerization phosphorus of 10wt% while stirring by liquid
Acid sodium solution, while adjusting pH is 10, room temperature 3 hours after stablizing, reaction was completed is adjusted to 7 for pH, and dialysis is to remove dephosphorization
Hydrochlorate obtains phosphorylation albumen solution.
2. enzymatic hydrolysis obtains enzymatic hydrolysis polysaccharide.
0.9wt% locust bean gum solution is taken, the galactosidase of 60U/g is added, adjusting pH is 5, is placed in 45 DEG C of water-baths
In digested, boil after 5 hours of time to remove enzyme activity, supernatant taken to be freeze-dried to obtain enzymatic hydrolysis polysaccharide.
3. phosphorylation albumen and enzymatic hydrolysis polysaccharide, which are carried out wet process glycosylation, obtains glycosylation minced fish gel enhancing
Agent.
Phosphorylation albumen solution 50mL is taken, side magnetic agitation is added the enzymatic hydrolysis polysaccharide of 3wt% to being uniformly mixed, places
In 55 DEG C of water-baths, respectively after wet process glycosylation 0,4,8,12,16,20,24 hour, cooling rapidly, formation sugar is taken out
Base minced fish gel reinforcing agent.
Two, the measurement of gel enhancing agent grafting degree is glycosylated
Configuration reagent one: the OPA (o-phthalaldehyde) of 40mg being dissolved in the methanol of 1mL, the pure water of 3mL is added, and is mixed
It is placed in spare in brown bottle；
The SDS of reagent two: 2.5mL 20% (w/w), the borax of 25mL0.1mol/L and the mixing of 100 μ L beta -mercaptoethanols, pure water
It is settled to 50mL.
When measurement, 0.3mL reagent one and 3.7mL reagent two is taken to mix in test tube respectively, 200 μ L of sample liquid is added, mixed
In 35 DEG C of reaction 2min after even, its light absorption value is surveyed at 340nm, replaces sample liquid as blank using pure water, and the difference between the two is freely
The net light absorption value of amino.Lysine makes standard curve, and the content C of free amino in sample is calculated according to net light absorption value.Grafting
Spend the relative percentage that DG is free amino content in each sample, formula are as follows:
In formula: DG- grafting degree
C0--- the free amino total amount of sample (μ ɡ/mL) when unreacted
C1--- the content (μ ɡ/mL) of the reaction free amino of t moment sample
The production of OPA standard curve: weighing lysine 50mg, is settled to 100mL with distilled water, and being made into concentration is 500 μ g/mL's
Solution.It is diluted on this basis, pipettes 8mL, 6mL, 4mL, 2mL, 1mL respectively and be settled to 10mL, be diluted to and correspond to
400 μ g/mL of concentration, 300 μ g/mL, 200 μ g/mL, 100 μ g/mL, 50 μ g/mL.OPA examination is added according still further to preceding sample handling procedure
Agent reacts 2min under conditions of temperature is 35 DEG C after mixing, surveys its light absorption value, at 340nm to be added in OPA solution
It is blank that 200 μ L water, which replace sample liquid,.Lysine concentration is as abscissa, A340Value is used as ordinate, draws standard curve, obtains
The regression equation arrived are as follows:
Y=0.0027x+0.0102, R2=0.9997
Three, the SDS- polyacrylamide gel electrophoresis analysis of gel enhancing agent is glycosylated
Glue, 12wt% separation gel is concentrated using 5wt%.Sample lysate is 0.01mol/L pH8.0Tris-HCl buffer, interior
Containing 2wt%SDS, 10wt% glycerol, 0.02wt% bromophenol blue, (non-reduced SDS-PAGE is free of beta -mercaptoethanol, restores SDS-
PAGE beta -mercaptoethanol containing 5wt%), sample is dissolved in sample solution and is tuned into protein concentration 2mg/m L.It is heated in boiling water bath
Ice bath is cooling after 5min, direct electrophoresis sample introduction, and applied sample amount is 15 μ L, and it is 80V that voltage in glue, which is concentrated, into separation gel later by it
120V is increased to, until instruction band, which is located at separation gel lowermost end, stops electrophoresis.After electrophoresis, with dyeing liquor (45wt% methanol,
10wt% glacial acetic acid, 0.25wt% coomassie brilliant blue R_250) in dyeing 1h on shaking table.Finally in using destainer on shaking table
(25wt% methanol, 10wt% glacial acetic acid) decoloration is clear to band, and carries out scanning imagery analysis.
Four, interpretation of result
During glycosylation, the free amine group of protein can be grafted with glucide or sugared catabolite
Reaction, so that free amine group is reduced, therefore the carry out degree of glycosylation can be obtained preferably by measuring the variation of free amine group
Display.As shown in Figure 1, with the extension of reaction time, the trend gradually risen is presented in grafting degree, grafting degree when reacting 12h
For (23.37 ± 1.62) %, it is smaller to continue the influence heated to grafting degree, it may be possible to because the time that glycosylation occurs gets over
Long, the substrate of consumption is more, then remaining substrate is fewer, so reaction process slows down after 12h hours, in addition, when too long
Between make gel enhancing agent form protein film, and the albumen film thickness of time longer formation and toughness are stronger, and moisture is evaporated in film,
Affect the process of glycosylation.So determining that the reaction time is 8-16h.
SDS-PAGE map is often used to the constituent and relative molecular weight of analysis protein, usually uses Coomassie brilliant blue
It dyes to analyze the access of macromolecular sugar chain in graft product, relative mobility of the protein in running gel is opposite with it to be divided
There is good linear relationship between protonatomic mass, has the characteristics that easy to operate, high resolution, reproducible.Fig. 2-3 is differential responses
Glycosylation gel enhancing agent SDS-PAGE map corresponding to time, swimming lane 1 are standard relative molecular mass albumen, swimming lane 2-8
Respectively glycosylation 0h, 4h, 8h, 12h, 16h, 20h, graft product for 24 hours.The electrophoresis of beta -mercaptoethanol is free of by Fig. 2
Band can be seen that a, c, d band in gel enhancing agent than unreacted position have it is slight move up, i.e. molecule relative mass
Increase slightly, it may be because the substance average molecular does not enter greatly very much separation gel that 7,8 swimming lanes do not occur a band above.With
The glycosylation time extend, the content of protein e is lower and lower, show that protein molecule migrates upwards and form aggregation,
Due to not destroying disulfide bond without beta -mercaptoethanol, explanation is that the formation of some aggregations is resulted in by other covalent bonds.By
Fig. 3 can be seen that protein f, g, i by clearly gradually thickening or disappear, it may be possible to which protein f, g, i are divided
The bigger protein aggregate of molecular weight that has been cross-linked to form between son illustrates sugar secondly, beta -mercaptoethanol can destroy disulfide bond
Formation a part of protein aggregate is also due to caused by disulfide bond during glycosylation reaction.
Embodiment 2
1. albumen carries out phosphorylation reaction and obtains modified albumen.
Fresh albumen is separated with yolk, insoluble impurities is removed with gauze after magnetic agitation 10min and obtains clarification egg white
Egg white solution and deionized water are mixed in 1:1 ratio and carry out magnetic agitation, be slowly added to 10% tripolyphosphate while stirring by liquid
Sodium solution, while adjusting pH is 10, room temperature 3 hours after stablizing, reaction was completed is adjusted to 7 for pH, dialyses to remove phosphoric acid
Salt obtains phosphorylation albumen solution.
2. enzymatic hydrolysis obtains enzymatic hydrolysis polysaccharide.
0.9wt% locust bean gum solution is taken, the galactosidase of 60U/g is added, adjusting pH is 5, is placed in 45 DEG C of water-baths
In digested, boil after 5 hours of time to remove enzyme activity, supernatant taken to be freeze-dried to obtain enzymatic hydrolysis polysaccharide.It takes dense
Degree be 1.2wt% konjaku sol solution, 'beta '-mannase additive amount be 100U/g, pH 6,50 DEG C of hydrolysis temperature, enzymolysis time
3h is boiled to remove enzyme activity, and supernatant is taken to be freeze-dried to obtain enzymatic hydrolysis polysaccharide.
3. phosphorylation albumen and enzymatic hydrolysis polysaccharide, which are carried out wet process glycosylation, obtains glycosylation minced fish gel enhancing
Agent.
Phosphorylation albumen solution 50mL is taken, side magnetic agitation is added the enzymatic hydrolysis polysaccharide of 3wt% to being uniformly mixed, places
In 55 DEG C of water-baths, respectively after wet process glycosylation 12 hours, cooling rapidly, formation glycosylation minced fish gel enhancing is taken out
Agent.
Embodiment 3
1. albumen carries out phosphorylation reaction and obtains modified albumen.
Fresh albumen is separated with yolk, insoluble impurities is removed with gauze after magnetic agitation 20min and obtains clarification egg white
Egg white solution and deionized water are mixed in 1:1 ratio and carry out magnetic agitation, be slowly added to 8% tripolyphosphate while stirring by liquid
Sodium solution, while adjusting pH is 11, room temperature 5 hours after stablizing, reaction was completed is adjusted to 7 for pH, dialyses to remove phosphoric acid
Salt obtains phosphorylation albumen solution.
2. enzymatic hydrolysis obtains enzymatic hydrolysis polysaccharide.
0.5wt% locust bean gum solution is taken, the galactosidase of 50U/g is added, adjusting pH is 7, is placed in 60 DEG C of water-baths
In digested, boil after 2 hours of time to remove enzyme activity, supernatant taken to be freeze-dried to obtain enzymatic hydrolysis polysaccharide.It takes dense
Degree be 0.5wt% konjaku sol solution, 'beta '-mannase additive amount be 60U/g, pH 4,30 DEG C of hydrolysis temperature, enzymolysis time
5h is boiled to remove enzyme activity, and supernatant is taken to be freeze-dried to obtain enzymatic hydrolysis polysaccharide.
3. phosphorylation albumen and enzymatic hydrolysis polysaccharide, which are carried out wet process glycosylation, obtains glycosylation minced fish gel enhancing
Agent.
Phosphorylation albumen solution 50mL is taken, side magnetic agitation is added the enzymatic hydrolysis polysaccharide of 5wt% to being uniformly mixed, places
In 30 DEG C of water-baths, respectively after wet process glycosylation 8 hours, cooling rapidly, formation glycosylation minced fish gel enhancing is taken out
Agent.
Embodiment 4
1. albumen carries out phosphorylation reaction and obtains modified albumen.
Fresh albumen is separated with yolk, insoluble impurities is removed with gauze after magnetic agitation 15min and obtains clarification egg white
Egg white solution and deionized water are mixed in 1:1 ratio and carry out magnetic agitation, be slowly added to 15% tripolyphosphate while stirring by liquid
Sodium solution, while adjusting pH is 9, room temperature 2 hours after stablizing, reaction was completed is adjusted to 7 for pH, dialyses to remove phosphoric acid
Salt obtains phosphorylation albumen solution.
2. enzymatic hydrolysis obtains enzymatic hydrolysis polysaccharide.
1.2wt% locust bean gum solution is taken, the galactosidase of 100U/g is added, adjusting pH is 4, is placed in 30 DEG C of water-baths
In digested, boil after 4 hours of time to remove enzyme activity, supernatant taken to be freeze-dried to obtain enzymatic hydrolysis polysaccharide.It takes dense
Degree be 1.5wt% konjaku sol solution, 'beta '-mannase additive amount be 120U/g, pH 7,60 DEG C of hydrolysis temperature, enzymolysis time
2h is boiled to remove enzyme activity, and supernatant is taken to be freeze-dried to obtain enzymatic hydrolysis polysaccharide.
3. phosphorylation albumen and enzymatic hydrolysis polysaccharide, which are carried out wet process glycosylation, obtains glycosylation minced fish gel enhancing
Agent.
Phosphorylation albumen solution 50mL is taken, side magnetic agitation is added the enzymatic hydrolysis polysaccharide of 1wt% to being uniformly mixed, places
In 60 DEG C of water-baths, respectively after wet process glycosylation 16 hours, cooling rapidly, formation glycosylation minced fish gel enhancing is taken out
Agent.
Raw materials used in the present invention, equipment is unless otherwise noted the common raw material, equipment of this field；In the present invention
Method therefor is unless otherwise noted the conventional method of this field.
The above is only presently preferred embodiments of the present invention, is not intended to limit the invention in any way, it is all according to the present invention
Technical spirit any simple modification, change and equivalent transformation to the above embodiments, still fall within the technology of the present invention side
The protection scope of case.
Claims (10)
1. a kind of preparation method for glycosylating minced fish gel reinforcing agent, which is characterized in that this method contains following steps:
1) fresh albumen is separated with yolk, after stirring 10-20min, clarification egg white solution is obtained by filtration, by egg white solution and deionization
Water is mixed, and sodium tripolyphosphate solution is added while stirring, phosphorylation albumen solution is obtained after dialysis；
2) locust bean gum and/or konjac glucomannan digest and enzymatic hydrolysis polysaccharide is made；
3) phosphorylation albumen and enzymatic hydrolysis polysaccharide are subjected to wet process glycosylation, form glycosylation minced fish gel reinforcing agent.
2. the preparation method of glycosylation minced fish gel reinforcing agent as described in claim 1, which is characterized in that in the step 1)
Egg white solution and the mass ratio of deionized water are 1:1.
3. the preparation method of glycosylation minced fish gel reinforcing agent as described in claim 1, which is characterized in that in the step 1)
It is 9-11 that sodium tripolyphosphate solution, which is added, and adjusts pH.
4. the preparation method of glycosylation minced fish gel reinforcing agent as claimed in claim 1 or 3, which is characterized in that the step
1) dialysis procedure is room temperature 2-5 hours in, and reaction was completed is adjusted to 7 for pH.
5. the preparation method of glycosylation minced fish gel reinforcing agent as described in claim 1, which is characterized in that in the step 2
Locust bean gum is digested using galactosidase, and konjac glucomannan is digested using 'beta '-mannase.
6. the preparation method of glycosylation minced fish gel reinforcing agent as claimed in claim 1 or 5, which is characterized in that the step
2) locust bean gum concentration is 0.5-1.2wt% in, and galactosidase additive amount is 50-100U/g；Konjaku gum concentration is 0.5-
1.5wt%, 'beta '-mannase additive amount are 60-120U/g.
7. the preparation method of glycosylation minced fish gel reinforcing agent as described in claim 1, which is characterized in that in the step 2
Enzymatic hydrolysis pH is 4-7, and hydrolysis temperature is 30-60 DEG C, enzymolysis time 2-5h.
8. the preparation method of glycosylation minced fish gel reinforcing agent as described in claim 1, which is characterized in that in the step 3)
Digest the 1-5wt% that polysaccharide additive amount is phosphorylation albumen.
9. the preparation method of glycosylation minced fish gel reinforcing agent as described in claim 1, which is characterized in that in the step 3)
The concrete operations of wet process glycosylation are as follows: take phosphorylation albumen solution, enzymatic hydrolysis polysaccharide is added in magnetic agitation to mixed
It closes uniformly, 30-60 DEG C after water-bath 8-16 hour, is taken out cooling rapidly.
10. a kind of glycosylation minced fish gel reinforcing agent, which is characterized in that the reinforcing agent is -9 to be prepared according to claim 1.
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| CN110074353A (en) * | 2019-06-04 | 2019-08-02 | 吉林大学 | A kind of preparation method of high grafting degree pectin egg white product |
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Application publication date: 20181211 |