CN107176992A - A kind of preparation method and application for the antifreeze glycopeptide modified based on FOS - Google Patents

A kind of preparation method and application for the antifreeze glycopeptide modified based on FOS Download PDF

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CN107176992A
CN107176992A CN201710483488.1A CN201710483488A CN107176992A CN 107176992 A CN107176992 A CN 107176992A CN 201710483488 A CN201710483488 A CN 201710483488A CN 107176992 A CN107176992 A CN 107176992A
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antifreeze
fos
glycopeptide
preparation
antifreeze glycopeptide
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汪少芸
陈旭
张凌拓
王文龙
蔡茜茜
方卫东
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Fuzhou University
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Abstract

The invention discloses a kind of preparation method and application for the antifreeze glycopeptide modified based on FOS, belong to biological, food processing technology field.FOS sugar chain is incorporated into the antifreeze peptide of separate sources by glycosylation grafting means, antifreeze glycopeptide is synthesized.Antifreeze glycopeptide of the present invention has good antifreezing function, and the cryogenic freezing available for cell, tissue, organ, microorganism and frozen food is preserved.Cell, tissue, organ, the freeze survival rate of microorganism of addition antifreeze glycopeptide of the present invention are significantly increased;Apply the invention in freezing food, the stability of the original quality of frozen food can be kept well, food quality deterioration caused by freezing injury is significantly reduced.

Description

A kind of preparation method and application for the antifreeze glycopeptide modified based on FOS
Technical field
Present invention relates particularly to a kind of preparation method and application for the antifreeze glycopeptide modified based on FOS, belong to it is biological, Food processing technology field.
Background technology
Antifreeze protein, is commonly called as ice structural protein, is called and makees thermal hysteresis protein, first in 1969 by Devries in the South Pole A kind of polar region fish in straits(Trematomus borchgrevinki)Blood in find.Antifreeze protein has low-temperature protection work Property, the necks such as medical science, biological and food processing can be applied to as cryoprotector.As antifreeze protein is applied to tissue, cell And the Cord blood of microorganism, its freeze survival rate can be significantly improved;Antifreeze protein can reduce frozen food in low temperature cold storage The quality deterioration that the ice crystal and its recrystallization of process formation are caused to food, improves the stability of frozen food.
The Activity of Antifreeze of antifreeze protein is mainly derived from some of antifreeze protein active fragment, is not that whole albumen exists Work.Few rue of Wang et al. is digested from the collagen of the separate sources such as ox-hide, pigskin and fish-skin by zymolysis technique, separated Go out the polypeptide fragment with high anti-freezing activity.
In recent years studies have found that recrystallization inhibition activity is served to pass for antifreeze protein performance cryoprotection effect Important effect, but the heat stagnation of antifreeze protein activity and ice binding characteristic when storage temperature for being less than heat stagnation active temperature area Between biomaterial(Such as cell, blood, tissue)Destruction even lethal effect is played on the contrary, is primarily due to when storage temperature is less than During heat stagnation active temperature range, ice-crystal growth is uncontrolled and is quickly converted to acicular texture, and then causes cellular damage to be conciliate The decline of cell survival rate after jelly.This is also to hinder natural antifreeze protein(Or peptide)Practical application is carried out as cryoprotector Principal element.Therefore, international research in recent years starts to be directed to exploitation there is recrystallization inhibition activity to be lived without heat stagnation for some The novel antifreeze proteins of property, are exactly one kind of this kind of active material based on glycosylation modified antifreeze protein.Such as one by 4 The peptide chain that Ser-Gly-Gly tripeptides is repeated for skeleton and connects what a galactoside base was formed by the carbon teminal of Ser residues Antifreeze peptide is found that with very strong recrystallization inhibition activity without heat stagnation activity.In addition, research it has also been found that glycosyl species and Glycosylation position and amino acid residue type have significant impact effect, such as gala to recrystallization inhibitory activity in peptide backbone Glycosyl modified antifreeze peptide recrystallization inhibition activity is stronger than the antifreeze peptide that mannose group is modified, and proline is conditional due to having The anglec of rotation can make albumen have obvious secondary structure in the solution, therefore some have stronger recrystallization inhibition containing proline Activity.
FOS is a kind of cheap mixture of price, is made up of the oligosaccharide of fructose and low polymerization degree, and its quilt It is classified as the food with specific function nutrition and specific function.In terms of nutrition, they are the sources of dietary fiber, low energy, It is adapted to diabetic, no carcinogenesis, and is the benefit rhzomorph of stimulation of bifidobacteria growth.FOS also can be steady by improving Qualitative, quality, mouthfeel, taste etc. increase the feature of food, while can also replace sugar and fat.It is a kind of successfully freeze proof many Peptide-FOS copolymer may possess better functional character.
Therefore, the present invention is by the antifreeze peptide and FOS of separate sources by glycosylation modified means, and chemical synthesis resists Freeze glycopeptide.The Activity of Antifreeze of synthesized antifreeze glycopeptide is significantly higher than original antifreeze peptide, can be widely applied to cell, group Knit, organ, the cryogenic freezing of microorganism and frozen food are preserved;, can be with large-scale industry and this method is safe, easily controllable Metaplasia is produced.
The content of the invention
Present invention aims in view of the shortcomings of the prior art, providing a kind of preparation for the antifreeze glycopeptide modified based on FOS Method and application.Cell, tissue, organ, the freeze survival rate of microorganism of addition antifreeze glycopeptide of the present invention are significantly increased;Will The present invention is applied in freezing food, and the stability of the original quality of frozen food can be kept well, is significantly reduced cold Freeze food quality deterioration caused by damage.
To achieve the above object, the present invention is adopted the following technical scheme that:
A kind of preparation method for the antifreeze glycopeptide modified based on FOS, is prepared as follows:By antifreeze peptide powder and fruit Between oligosaccharides according to mass ratio be 1:1 ~ 1:5 ratio is dissolved in phosphate buffer, and solution ultimate density is 5 ~ 15% (w/v), stirring makes it fully dissolve;Then 1 ~ 2 h is reacted under the conditions of 60 ~ 80 DEG C, pH is 8.0 ~ 9.0;Reaction knot Cooled down in Shu Hou, the water for being immediately placed at 0 DEG C with terminating reaction, reacted solution freeze-drying obtains described FOS and repaiied The antifreeze glycopeptide freeze-dried powder of decorations.
The raw material sources of described antifreeze peptide powder are in pigskin, ox-hide, fish-skin or fish scale.
The pH of described phosphate buffer is 7.6 ~ 8.0, wherein KH2PO4For 2.6 mM, K2PO4For 32.5 mM.
Described mixing time is 10 ~ 20 min;The cool time is 15 ~ 30 min.
The purposes of antifreeze glycopeptide made from preparation method as described above, described antifreeze glycopeptide is used for cell, tissue, device The cryogenic freezing of official, microorganism and frozen food are preserved.
Technical scheme provided by the present invention has following advantage:
(1)Antifreeze peptide raw material sources used in the present invention are in food source leftover bits and pieces, with safety non-toxic, wide material sources, price The advantage such as cheap;
(2)Antifreeze glycopeptide synthetic method that the present invention is provided efficiently, safety, cost it is low, it is easy to control;
(3)The antifreeze glycopeptide that the present invention is synthesized is widely used, and can be applied to cell, tissue, organ, microorganism and frozen food Cryogenic freezing is preserved;And compared with antifreeze peptide, its Activity of Antifreeze is significantly improved;
(4)FOS in the present invention is a kind of food with specific function nutrition and specific function, can be stable by improving Property, quality, mouthfeel, taste etc., increase the feature of food, while sugar and fat can also be replaced.It is adapted to diabetic, and And be the benefit rhzomorph of stimulation of bifidobacteria growth.
Specific implementation measure
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not limited only to these embodiments.
Embodiment 1
A kind of preparation for the antifreeze glycopeptide modified based on FOS, is carried out as follows:
(1)According to antifreeze peptide powder(From pigskin)Mass ratio is 1 between FOS:2.7 ratio is by antifreeze peptide powder With FOS mixing, pH 7.6 phosphate buffer is then dissolved in(2.6 mM KH2PO4, 32.5 mM K2PO4)In, it is molten Liquid ultimate density is 10%(w/v), stirring 15 min makes it fully dissolve;
(2)Antifreeze peptide after dissolving reacts 67 min with FOS mixed liquor under the conditions of 77 DEG C, pH is 8.74;Reaction knot Shu Hou, is immediately placed at frozen water(0 ℃)20 min of middle cooling, terminating reaction, reacted solution freeze-drying obtains freeze proof sugar Peptide freeze-dried powder.
Embodiment 2
A kind of preparation for the antifreeze glycopeptide modified based on FOS, is carried out as follows:
(1)According to antifreeze peptide powder(From ox-hide)Mass ratio is 1 between FOS:1 ratio by antifreeze peptide powder and FOS is mixed, and is then dissolved in pH 7.8 phosphate buffer(2.6 mM KH2PO4, 32.5 mM K2PO4)In, solution Ultimate density is 5%(w/v), stirring 10 min makes it fully dissolve;
(2)Antifreeze peptide after dissolving reacts 60 min with FOS mixed liquor under the conditions of 60 DEG C, pH is 8.0;Reaction terminates Afterwards, it is immediately placed at frozen water(0 ℃)15 min of middle cooling, terminating reaction, reacted solution freeze-drying obtains antifreeze glycopeptide Freeze-dried powder.
Embodiment 3
A kind of preparation for the antifreeze glycopeptide modified based on FOS, is carried out as follows:
(1)According to antifreeze peptide powder(From fish-skin)Mass ratio is 1 between FOS:5 ratio by antifreeze peptide powder and FOS is mixed, and is then dissolved in pH 8.0 phosphate buffer(2.6 mM KH2PO4, 32.5 mM K2PO4)In, solution Ultimate density is 15%(w/v), stirring 20 min makes it fully dissolve;
(2)Antifreeze peptide after dissolving reacts 120 min with FOS mixed liquor under the conditions of 80 DEG C, pH is 9.0;Reaction knot Shu Hou, is immediately placed at frozen water(0 ℃)30 min of middle cooling, terminating reaction, reacted solution freeze-drying obtains freeze proof sugar Peptide freeze-dried powder.
Application of the antifreeze glycopeptide of the present invention of application examples 1 in microorganism freezen protective
Carry out as follows:
Test process:A. blank control(Deionized water), B. FOSs, C. do not carry out glycosylation modified antifreeze peptide, and D. is real Apply the antifreeze glycopeptide of the preparation of example 1.
Test method:
(1)Prepare seed liquor:The streptococcus thermophilus deposited of going bail for is recovered in M17 fluid nutrient mediums, 37 DEG C, 180 r/min cultures One night, OD values are about 1.3.
(2)Preparing experiment bacterium solution:50 μ L seed liquor is taken to cultivate 4 in the test tube for filling 4 mL M17 fluid nutrient mediums H, now the OD values of bacterium solution are about 1.2.
(3)Experiment bacterium solution is diluted 100 times with sterile deionized water, takes 100 μ L dilution bacterium solution to be transferred to 2 mL modeling Expect in cryovial, mixed with 900 μ L freezeproof protectant, turbine mixer is mixed.
(4)The μ L of sample mixed liquor 50 are taken to cultivate the OD values at 7 h, detection 600nm in 4 mL M17 fluid nutrient mediums. Sample after taking is put into after -24 DEG C of h of freezing processing 24,37 DEG C of water-baths 10 min of defrosting, and 50 μ L inoculated and cultureds are taken again Determine 7 h OD values.
OD values and the relative amount of lactic acid bacteria under the conditions of this meet following relational expression:
y = 0.2152Ln(x)+0.0594, R2 = 0.9972 (2 - 1)
Y is the relative amount of viable bacteria in sample in formula, and x is the OD values that 7 h cultivate bacterium solution.Calculate bacterium in the front and rear sample of freezing Relative amount C0After C, the survival rate formula of bacterium(2-2)Calculate:
(2 - 2)
C0, freeze the relative amount of bacterium in preceding sample(%);After C, freezing 24 hours in sample bacterium relative amount(%).Calculate cold Freeze the bacterium survival rate of processing sample, to detect the Activity of Antifreeze of additive.
It the results are shown in Table 1, FOS(Handle B)And antifreeze peptide(Handle C)There is certain freeze proof guarantor to streptococcus thermophilus Protect effect, the antifreeze glycopeptide trim that the present invention is modified based on FOS(Handle D)Freeze survival rate be up to 95.7%, significantly Higher than single FOS(51.2%)And antifreeze peptide(66.5%).Illustrate the antifreeze glycopeptide modified based on FOS of the present invention With good antifreezing function, there is good low-temperature protection to act on the freezing of microorganism.
The antifreeze glycopeptide of the present invention of table 1 is to microorganism cryoprotection comparative test result
Application of the antifreeze glycopeptide of the present invention of application examples 2 in porcine oocytes freezen protective
Porcine oocytes cryoprotector basic recipe, contains in every 100 parts:3 parts of glucose, 1 part of sodium citrate, penicillin 80,000 Unit.
Test process:On porcine oocytes cryoprotector basic recipe in embodiment 4, deionization is added respectively Antifreeze glycopeptide prepared by water, FOS, antifreeze peptide, embodiment 1, is accordingly named as protective agent A, B, C, D, with protective agent by it A is control group.
Test method:
(1)Tubulature:Egg mother cell is obtained from discarded pig ovary, is cleaned, moves into the cryopreservation tube equipped with cryoprotector and puts down Weigh 15 min.
(2)Freeze:Cryopreservation tube equipped with egg mother cell after balance is put into frozen cooling box(2 DEG C of rate of temperature fall/ min), it is cooled to after -80 DEG C and is put into preservation in liquid nitrogen container.
(3)As a result identify:Cultivated after defrosting, calculate survival rate, the results are shown in Table 2.
The antifreeze glycopeptide of the present invention of table 2 is to egg mother cell cryoprotection comparative test result
From table 2 it can be seen that the antifreeze glycopeptide modified based on FOS of the present invention has good antifreezing function, it is female to ovum thin The ultralow temperature preservation of born of the same parents has good protective effect.
Application of the antifreeze glycopeptide of the present invention of application examples 3 in freezing potato dough products
Raw material and formula used in a kind of freezing potato dough of low temperature resistant refrigeration is as follows:100 parts of Plain flour, fresh potato 120 parts, 2 parts of high activity dried yeast, embodiment 1 prepare 4 parts of antifreeze glycopeptide, 80 parts of distilled water.
Test method:
(1) prepared by mashed potatoes:Fresh potato is cleaned and drained, the potato drained is weighed in proportion, is put into high-temperature steam cooker and steams 30 min are boiled, is removed the peel, is put into tissue mashing machine and is pounded mashed potatoes, be cooled to room temperature standby;
(2) activated yeast:Dried yeast powder needed for weighing activates 20min in 37 DEG C of distilled water;
(3) dough preparing:Antifreeze glycopeptide prepared by embodiment 1 is added to molten in the yeast juice after activation process in required ratio Solution, then mashed potatoes and flour are together poured into dough mixing machine are sufficiently stirred for, knead dough smooth tack-free to dough surface;
(4) split:It is divided into dosage to be 20g or so dough semifinished product in the dough mixed;
(5) shaping:Dough semifinished product is rubbed with the hands circle to be wrapped up with preservative film;
(6) it is quick-frozen:The dough semifinished product wrapped is placed in the quick-frozen 60min of -80 DEG C of refrigerators;
(7) cold storage:Dough base after will be quick-frozen is placed in cold storage in -20 DEG C of refrigerators;
(8) thaw:It is 35 DEG C that freezing flour-dough is placed in into temperature, and relative humidity is the 30min that thawed in 85% insulating box;
(9) one time fermentation:The dough thawed is placed in temperature for 37 DEG C, and relative humidity is ferments 100 in 85% insulating box min;
(10) knead dough:The good dough of one time fermentation is pressed 10 times with oodle maker;
(11) shaping:The face pressed is made into required steamed bun shape;
(12) secondary fermentation:Dough after whole good type is placed in temperature for 37 DEG C, and relative humidity is proofs 30 in 85% insulating box min;
(13) steam:The steamed bun semifinished product fermented is placed in big fire boiling 30min in food steamer;Wait taking-up of being boiled after 5min.
Proofing period needed for the freezing flour-dough making steamed bun of addition antifreeze glycopeptide of the present invention is short, steam obtained by steamed bun specific volume Greatly, steamed bun center portion is in spongy, and delicate mouthfeel is flexible, close with the steamed bread quality made of fresh dough.
Application of the antifreeze glycopeptide of the present invention of application examples 4 in frozen minced fish product
Raw material and formula used in a kind of frozen minced fish product of low temperature resistant refrigeration is as follows:60 parts of fish slurry, soybean protein isolate, reality Apply 5 parts of antifreeze glycopeptide, 30 parts of gelatin, 20 parts of pig ridge fat, 8 parts of starch, 0.4 part of garlic, 1.5 parts of taste beautiful jade, the white sugar 3 of the preparation of example 1 Part, 3 parts of edible salt, 0.3 part of monosodium glutamate, surplus is frozen water.
Test method:
(1)Thaw:By fish slurry thaw to can chopped, but still there is the semi thawing state that part trash ice is advisable in centre;
(2)Cut and mix:By the fish slurry after defrosting and pig ridge fat, garlic, taste beautiful jade, white sugar, edible salt, monosodium glutamate and frozen water in cutmixer Cut and mix, during which treat that material temperature reaches that more than 15 DEG C are added starch, are continued to cut and are mixed, pulp temperature is 20 DEG C;
(3)Soybean protein isolate is broken into after emulsion form, the antifreeze glycopeptide prepared with embodiment 1 is together added in cutmixer, in Speed, which is cut, mixes uniform;
(4)Shaping:The mould that the shaping is used, including support, the support are provided with minced fillet feed screw structure, material The top and the bottom thermal bonding roller of pusher plate, pressure roller and the thermal bonding device of rotation is equipped with above conduit, the guide portion is set In the side of framework, minced fillet will be pressed into by sheet by roller machine, then minced fillet piece is rolled into oriental cherry shape by trussing machine.
(5)Boiling:First water at low temperature is boiled:Boiled water at low temperature is carried out in the raw fish ball product feeding low temperature water-boiling machine after shaping, It is 40 DEG C that water, which boils temperature, and water boils the time for 15 min;Then high-temperature water is boiled:Fish ball product feeding high temperature after water at low temperature is boiled Carry out high-temperature water in water-boiling machine to boil, it is 95 DEG C that water, which boils temperature, water boils the time for 4 min;Fish ball after finally high-temperature water is boiled Thermophilic digestion is carried out in product feeding thermophilic digestion machine, boiling temperature is 121 DEG C, and digestion time is 15 min.
(6)Cooling:The technique of the cooling is as follows:By on the fish ball product feeding cooler after thermophilic digestion, make fish ball The temperature of product is reduced to room temperature.
(7)It is quick-frozen:The quick-frozen technique is as follows:Fish ball product after cooling is sent into instant freezer, quick-frozen hangar temperature Less than -30 DEG C, fish ball article center temperature is less than -18 DEG C after the completion of freezing.
(8)Packaging:To examining qualified product to pack, packaged product passes through the detection product of metal detector In whether be mixed with metallics;
(9)Storage:Qualified product of casing is placed on to less than -18 DEG C of freezer refrigeration.
Antifreeze glycopeptide of the present invention is added in frozen minced fish product can effectively suppress slightly to become due to the quality of cold storage initiation, coagulate The problems such as colloidality can be reduced, had a poor flavour;Antifreeze glycopeptide can improve meat products quality, while make surimi product nutrition more rich, It is more popular with consumers.
It the foregoing is only the preferable embodiment of the present invention, all equivalent changes done according to scope of the present invention patent With modification, it should all belong to the covering scope of the present invention.

Claims (5)

1. a kind of preparation method for the antifreeze glycopeptide modified based on FOS, it is characterised in that:It is prepared as follows:Will be anti- It is 1 to freeze between polypeptide powder and FOS according to mass ratio:1 ~ 1:5 ratio is dissolved in phosphate buffer, and solution is final Concentration is 5 ~ 15%(w/v), stirring makes it fully dissolve;Then 1 is reacted under the conditions of 60 ~ 80 DEG C, pH is 8.0 ~ 9.0 ~ 2 h;After reaction terminates, cooled down in the water for being immediately placed at 0 DEG C with terminating reaction, reacted solution freeze-drying obtains institute The antifreeze glycopeptide freeze-dried powder for the FOS modification stated.
2. the preparation method of antifreeze glycopeptide according to claim 1, it is characterised in that:The raw material of described antifreeze peptide powder From pigskin, ox-hide, fish-skin or fish scale.
3. the preparation method of antifreeze glycopeptide according to claim 1, it is characterised in that:Described phosphate buffer PH is 7.6 ~ 8.0, wherein KH2PO4For 2.6 mM, K2PO4For 32.5 mM.
4. the preparation method of antifreeze glycopeptide according to claim 1, it is characterised in that:Described mixing time be 10 ~ 20 min;The cool time is 15 ~ 30 min.
5. the purposes of antifreeze glycopeptide made from any described preparation method of claim 1 ~ 4, it is characterised in that:Described The cryogenic freezing that antifreeze glycopeptide is used for cell, tissue, organ, microorganism and frozen food is preserved.
CN201710483488.1A 2017-06-23 2017-06-23 A kind of preparation method and application for the antifreeze glycopeptide modified based on FOS Pending CN107176992A (en)

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CN115505037A (en) * 2022-09-29 2022-12-23 广东海洋大学 Fish skin collagen antifreeze peptide improved by glycosylation and preparation method and application thereof

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