CN106497901B - Compound carrageenase and the method for preparing selenide of carragheen oligosaccharides using the enzyme - Google Patents
Compound carrageenase and the method for preparing selenide of carragheen oligosaccharides using the enzyme Download PDFInfo
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- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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Abstract
The present invention relates to compound carrageenase and the methods for preparing selenide of carragheen oligosaccharides using the enzyme, wherein it is 2.1 × 10 that every gram of compound carrageenase, which contains enzyme activity,5~1.7 × 106κ type carrageenase, the enzyme activity of U is 6.0 × 104~4.8 × 105The ι type carrageenase and enzyme activity of U is 3.0 × 104~2.4 × 105The λ type carrageenase of U.Compound carrageenase of the present invention has the function of Synergistic degradation selenide of carragheen, so that degradation is more full and uniform, have many advantages, such as that reaction condition is mild, easily controllable, environmentally protective and single-minded efficient, prepared oligosaccharide molecular amount is more uniform, and its yield is much higher than selenide of carragheen oligosaccharides prepared by chemical method and single enzyme method.
Description
Technical field
The present invention relates to compound carrageenase and the methods for preparing selenide of carragheen oligosaccharides using the enzyme, belong to marine organisms
Technical field.
Background technique
Currently, the understanding to selenium as essential trace element in life is just increasingly being deepened.Selenium is micro member needed by human
Element, referred to as " king of anticancer ", selenium-supply helps to improve immunity of organisms, the effect of building up health.Selenium is a pole again simultaneously
Malicious element, animal are 0.1~0.3ppm to the nutritional need range of selenium, and 2~10ppm will cause slow poisoning, and more than 10ppm
When can cause acute poisoning so that die by visitation of God.Se content is being reduced in mankind nowadays society food chain, animal and human body selenium deficiency
Disease increases, and selenium deficiency trend is being aggravated.Obviously, seek low toxicity and the good selenides of bioavilability for further disclosing selenium
Physiological function and its in human health care's cause application be very significant.
Selenium in nature can be divided into inorganic selenium and organic selenium.Inorganic selenium toxicity is big, is not easy to absorb, bioavailability
Low, application is restricted；In contrast organic selenium toxicity is low, Small side effects, is easier to be absorbed by the body, can preferably play
The physiological activity of selenium.CN1121414C combines inorganic selenium and marine polysaccharide by artificial synthesized method, and inorganic selenium is converted
For organic selenium compounds, new selenium compound of polysaccharide is had been successfully prepared.The case provides the production of seaweed selenium poly saccharide marine drug
Product and preparation method thereof, the new drug have significant curative effect to renal failure, angiosis and tumour, are low toxicity or even nontoxic drug.Its
In with antiviral, AntiHIV1 RT activity, antitumor and provide the selenizing algal polysaccharide of the wide variety of functions such as immunity of organism, selenium chitosan and
Selenizing red seaweed polysaccharide, wherein selenizing red seaweed polysaccharide carragheen also has significant facilitation to the division of lymphocyte, has suppression
Sufferings virus replication effect processed.
The selenium compound of polysaccharide of CN1121414C preparation is as the unique ocean selenium fortification agent in China, in food
Industrialized production is realized with field of health care products, and major product is selenizing red seaweed polysaccharide, i.e. selenide of carragheen.Although selenide of carragheen
Immunity of organisms and antitumor action are improved with good, and is had no toxic side effect, but since its molecular weight is big, bioavailability
It is relatively low, limit the application of selenide of carragheen.Carragheen be extracted from the cell wall of certain red algaes it is a kind of by 1,
The linear sulfated polysaccharide that 3- β-D- galactopyranose and Isosorbide-5-Nitrae-α-D- galactopyranose are alternately formed by connecting, and carragheen degradation shape
At oligosaccharides have various new physiological activity, such as anti-oxidant, antiviral and antitumor, immunological regulation and promotion plant growth
Function.Compared with carragheen, the bioavailability of carrageenan oligosaccharide is higher, and has more physiological activity.Therefore, it needs to grind
Send out the high selenide of carragheen oligosaccharides of bioavailability.
Foreign countries have no the report of selenide of carragheen oligosaccharides at present, and there is the research report about selenide of carragheen oligosaccharides in the country
Road, " preparation of selenide of carragheen oligosaccharides and its inhibition angiogenesis function that the 9th phase in 2014 " chemistry and bioengineering " delivers
Research ", this article prepares selenide of carragheen oligosaccharides by the following method: in acid condition, by sodium selenite, sulfuric acid and carragheen
It is reacted；After reaction, reaction solution is concentrated under reduced pressure and passes through the bag filter dialysis 48h that molecular cut off is 500, then through second
Alcohol precipitating, freeze-drying, obtain selenide of carragheen oligosaccharides.The reaction condition that the chemical method prepares selenide of carragheen oligosaccharides is not easy
Environment is polluted in control, and low using bag filter retention yield, the selenide of carragheen oligosaccharides Se content of preparation is low, in industrialized production
In be difficult to apply.Therefore, those skilled in the art urgently find the new mode for preparing selenide of carragheen oligosaccharides.
Summary of the invention
The main purpose of the present invention is to provide a kind of compound carrageenase, which can effectively hydrolyze selenium
Change carragheen, provides a kind of new mode for the preparation of selenide of carragheen oligosaccharides.
Another object of the present invention is to provide the methods for preparing selenide of carragheen oligosaccharides using the compound carrageenase.
A further object of the present invention is to provide the selenide of carragheen oligosaccharides being prepared by the method and its applications.
To achieve the above object, a kind of compound carrageenase of the present invention, wherein every gram of compound carrageenase contains enzyme
Vigor is 2.1 × 105~1.7 × 106κ (Kappa) type carrageenase, the enzyme activity of U is 6.0 × 104~4.8 × 105The ι of U
(Iota) type carrageenase and enzyme activity are 3.0 × 104~2.4 × 105λ (Lambda) type carrageenase of U.
Experimental study of the present invention shows that compound carrageenase has the function of Synergistic degradation selenide of carragheen, so that degradation is more
Add full and uniform, has many advantages, such as that reaction condition is mild, easily controllable, environmentally protective and single-minded efficient, prepared oligosaccharides point
Son amount is more uniform, and its yield is much higher than selenide of carragheen oligosaccharides prepared by chemical method and single enzyme method.
Preferably, according to the optimization ratio and its cost of different type carrageenase, every gram of the present invention compound carrageenase
It is 2.1 × 10 containing enzyme activity5~8.5 × 105κ type carrageenase, the enzyme activity of U is 6.0 × 104~2.4 × 105The ι type of U
Carrageenase and enzyme activity are 3.0 × 104~1.2 × 105The λ type carrageenase of U.
Specific embodiment according to the present invention, compound carrageenase of the present invention are 2.7 × 10 by enzyme activity5~
2.7×106κ type carrageenase raw material, the enzyme activity of U/g is 2.3 × 105~2.3 × 106The ι type carrageenase raw material of U/g and
Enzyme activity is 2.0 × 105~2.0 × 106The λ type carrageenase raw material of U/g compounds to obtain；
Preferably, active highest of the κ type carrageenase raw material in 20 DEG C~45 DEG C, pH6.0~9.0；
Preferably, active highest of the ι type carrageenase raw material in 15 DEG C~40 DEG C, pH6.0~9.0；
Preferably, active highest of the λ type carrageenase raw material in 25 DEG C~45 DEG C, pH6.0~10.0.
κ type carrageenase raw material of the present invention, ι type carrageenase raw material and λ type carrageenase raw material are commercially available or adopt
It is obtained with existing bacterial strain by conventional technical means in the art.The enzyme classes of above-mentioned amount are made according to a certain percentage in an aseptic environment
Compound carrageenase of the present invention can be obtained with the mixing of mechanical blending machine.Compound carrageenase quality standard symbol of the invention
Close hygienic requirements of the National Standard of the People's Republic of China GB 25594-2010 to food industry enzyme preparation.Its physicochemical property
Appearance: white powder；Total number of bacteria < 50000/g；Escherichia coli < 30CFU/g；Pathogenic escherichia coli is not detected；Salmonella
It is not detected.
Specific embodiment according to the present invention, κ type carrageenase raw material of the present invention, the ι type carragheen proenzyme
Material, the λ type carrageenase raw material are by producing κ type carrageenase bacterial strain, producing ι type carrageenase bacterial strain or producing λ type OK a karaoke club respectively
Glue enzyme bacterial strain generates and purified, drying obtains.
Production κ type carrageenase bacterial strain, production ι type carrageenase bacterial strain and production λ type carrageenase bacterial strain of the present invention are existing
There is bacterial strain, generate the condition of enzyme, purifying and drying process are this field routine techniques, are not repeated separately herein.
Preferably, producing the κ type carrageenase bacterial strain is Pseudoalteromonas sp NJ62.
Preferably, producing the ι type carrageenase bacterial strain is Pseudoalteromonas sp NJ64.
Preferably, producing the λ type carrageenase bacterial strain is Pseudoalteromonas sp NJ276.
Bacterial strain Pseudoalteromonas sp NJ62 of the present invention, bacterial strain Pseudoalteromonas sp NJ64
Belong to Alteromonas Zoopagales (Alteromonadales), Alteromonad with bacterial strain Pseudoalteromonas sp NJ276
Section (Alteromonadaceae), Pseudoalteromonas (Pseudoalteromonas sp.).These bacterial strains are existing bacterium
Strain, bacterial strain Pseudoalteromonas sp NJ62, Pseudoalteromonas sp NJ64 and Pseudoalteromonas
For sp NJ276 available from key lab, bioactive substance National Bureau of Oceanography, the prior art of these bacterial strains includes Zheng Zhou, Miao
Brocade etc., influence of the mercury to Antarctic bacteria Pseudoalteromonas sp NJ62 activities of antioxidant enzymes, Marine Sciences progress,
The supplementary issue of volume 23, in December, 2005；Gao Cong, Miao Jinlai etc., a plant height produce low-temperature cellulase Antarctic bacteria screening, identification and
Zymologic property Primary Study, chemistry and bioengineering, the 2nd phase of volume 29,2 months 2012；Zheng Zhou, Liu Fangming etc., South Pole petroleum
The screening of alkane degradation psychrophile and its research of degradation characteristic, Marine Sciences progress, the 3rd phase of volume 25, in July, 2007.As before
The active highest in 20 DEG C~45 DEG C, pH6.0~9.0 can be produced by Pseudoalteromonas sp NJ62 bacterial strain
It is raw；The highest ι type carrageenase raw material of activity in 15 DEG C~40 DEG C, pH6.0~9.0 can be by Pseudoalteromonas
Sp NJ64 is generated；The highest λ type carrageenase raw material of activity in 25 DEG C~45 DEG C, pH6.0~10.0 can be by
Pseudoalteromonas sp NJ276 is generated.Enzyme digestion reaction can be made to carry out under mild conditions using preferred strain.
On the other hand, the present invention provides a kind of preparation method of selenide of carragheen oligosaccharides, wherein the method includes using
The compound carrageenase of the present invention of effective catalytic amount hydrolyzes selenide of carragheen.
Specific embodiment according to the present invention, the method for the invention include by the compound carrageenase according to 80~
The additional amount of 800U/ml be added to mass fraction be 5~20% selenide of carragheen aqueous solution in, in 25~45 DEG C of reaction 6h~
12h hydrolyzes selenide of carragheen, and post-processing obtains the selenide of carragheen oligosaccharides.Preferably, the post-processing includes after hydrolyzing
Reaction solution heating boil, cool down, evaporate and obtain selenide of carragheen oligosaccharides concentrate, the concentrate is then passed through into 3~9 weight
Times ethanol precipitation, be collected by centrifugation sediment, dry the selenide of carragheen oligosaccharides.
Specifically, such as with distilled water prepare the selenide of carragheen solution that mass fraction is 5~20%, then according to 80~
Compound carragheen enzyme solutions are added in the additional amount of 800U/ml, hydrolyze selenide of carragheen in 25~45 DEG C of reaction 6h~12h, then
Heating boils 10min and terminates reaction, is cooled to room temperature, is placed in 60 DEG C of rotary evaporations and is concentrated, and obtains selenide of carragheen oligosaccharides
Sediment is collected by centrifugation, then through 60 in logical 3~9 times of the ethanol precipitation of the selenide of carragheen oligosaccharides concentrate of acquisition by concentrate
DEG C drying after obtain selenide of carragheen oligosaccharides.
The enzyme process that the method that the present invention prepares selenide of carragheen oligosaccharides is has essence with prior art chemical preparation process
It is different.Make degradation more full and uniform using compound carragheen enzyme hydrolysis selenide of carragheen of the present invention, there is reaction item
The advantages that part is mild, easily controllable, environmentally protective and single-minded efficient, prepared oligosaccharide molecular amount is more uniform, and its yield
The selenide of carragheen oligosaccharides prepared much higher than chemical method and single enzyme method.
In another aspect, the present invention provides a kind of selenide of carragheen oligosaccharides, it is to be prepared by the method for the invention.
Preferably, the molecular weight of the selenide of carragheen oligosaccharides is 900~2700Da, preferably 900~1500Da.
In another aspect, the present invention provides the selenide of carragheen oligosaccharides as Selenium supplement agent in production rice containing selenium, tealeaves
Or the application in egg.
In summary, the present invention compounds 3 kinds of different types of carrageenases, and multiple using the carrageenase of compounding
Synthase degradation prepares selenide of carragheen oligosaccharides, not only have reaction condition it is mild, it is easily controllable, environmentally protective and it is single-minded efficiently etc.
Advantage, and its yield is much higher than selenide of carragheen oligosaccharides prepared by chemical method and single enzyme method.Selenizing card prepared by the present invention
Draw glue oligosaccharides that not only there are whole physiological activity of selenide of carragheen polysaccharide, but also its bioavailability is much higher than selenide of carragheen
Polysaccharide can be used as novel safe and efficient Selenium supplement agent applied to industries such as Se-enriched egg, tealeaves and rice, it is few to have opened up seaweed selenium
The frontier of sugar application.
Specific embodiment
In order to which technical characteristic of the invention, purpose and beneficial effect are more clearly understood, now in conjunction with specific implementation
Example carries out technical solution of the present invention described further below, it should be understood that these examples are merely to illustrate the present invention rather than limit
The scope of the present invention processed.In embodiment, each Starting reagents material is commercially available, and test method without specific conditions is
Conventional method and normal condition known to fields, or according to condition proposed by apparatus manufacturer.
Kappa-carrageenan proenzyme material, ι-carrageenase raw material, lambda-carrageenan proenzyme material point in following embodiment or comparative example
Not by bacterial strain Pseudoalteromonas sp NJ62, bacterial strain Pseudoalteromonas sp NJ64 and bacterial strain
Pseudoalteromonas sp NJ276 (Pseudoalteromonas sp NJ62 bacterial strain, Pseudoalteromonas sp
NJ64 and Pseudoalteromonas sp NJ276 bacterial strain is all obtained from key lab, bioactive substance National Bureau of Oceanography) training
Support obtained three kinds of carrageenases, through ammonium sulfate precipitation, desalination, ion-exchange chromatography and etc. purifying, specific steps are as follows:
The Pseudoalteromonas sp NJ62 of kappa-carrageenan enzyme, ι-carrageenase and lambda-carrageenan enzyme will be produced respectively
Bacterial strain, the inoculation of Pseudoalteromonas sp NJ64 and Pseudoalteromonas sp NJ276 bacterial strain South Pole pyschrophile
Into fermentation medium, fermentation medium is 2216E fluid nutrient medium, 15 DEG C of shake culture 60h.9800 × g of fermentation liquid centrifugation
10min, supernatant are carrageenase crude enzyme liquid；2216E fluid nutrient medium (g/L): peptone 5, yeast powder 1, Chen Haishui are prepared；
Solid medium is that 1.5% agar powder is added in enriched medium.
Carrageenase isolates and purifies: acquisition being contained kappa-carrageenan enzyme, ι-carrageenase and lambda-carrageenan enzyme respectively
Supernatant, ammonium sulfate is slowly gradually added in ice bath to 30% saturation degree, it is stirring while adding, be further continued for stirring after adding
1h, 4 DEG C of standing 5h；Then, 12000 × g is centrifuged 20min at 4 DEG C, takes supernatant, continuously adds ammonium sulfate to 90% saturation degree,
1h stirring while adding is placed in 4 DEG C of refrigerators and stands overnight；12000 × g is centrifuged 20min at 4 DEG C, precipitating is taken, with pH8.0's
Tris-HCl (0.05mol/L)-buffer solution, dialysis.Solution after dialysis is splined on DEAE Sepharose Fast
Flow carries out ion-exchange chromatography, disodium hydrogen phosphate-phosphoric acid that the buffer solution system A liquid of chromatography is 0.05mol/L pH 7.0
Sodium dihydrogen (PBS) buffer, B liquid are the PBS of the 0.05mol/L pH7.0 containing 117g/L NaCl, carry out gradient elution, stream
For speed to carry out gradient elution, fraction collection eluent measures a component enzyme activity.Obtained active component is concentrated by ultrafiltration laggard
Row gel permeation chromatography, filler is Superdex G75, with the PBS (0.05mol/L) of the pH7.0 containing 0.15mol/L NaCl
Buffer balance, is eluted with equilibrium liquid, and the above operation is carried out at 4 DEG C, and the active part vacuum freeze drying of collection obtains
Kappa-carrageenan enzyme, ι-carrageenase and lambda-carrageenan enzyme.Carrageenase vigor is measured with DNS method, pure specific activity of enzyme is respectively
2742,2336 and 2075U/mg.Enzyme activity is defined as: enzyme amount needed for catalysis generates 1 μm of ol reduced sugar per minute is an enzyme
Unit of activity (U).
Embodiment 1
The preparation method of selenide of carragheen oligosaccharides, comprising the following steps:
(1) composition of compound carrageenase: 7 parts by weight of kappa-carrageenan proenzyme material, ι -2 parts by weight of carrageenase raw material, λ -
1 parts by weight of carrageenase raw material.It mixes according to a certain percentage in an aseptic environment.Appearance is white powder；Total number of bacteria <
50000/g；Escherichia coli < 30CFU/g；Pathogenic escherichia coli is not detected；Salmonella is not detected.Through DNS (dinitrosalicylic
Acid) method test, it is 5.7 × 10 that every gram of compound carrageenase, which contains enzyme activity,5κ type carrageenase, the enzyme activity 1.4 of U
×105The ι type carrageenase and enzyme activity of U is 6.0 × 104The λ type carrageenase of U.
(2) it utilizes compound carragheen enzyme hydrolysis selenide of carragheen: preparing the selenizing card that mass fraction is 15% with distilled water
It draws sol solution that compound carragheen enzyme solutions are added then according to the additional amount of 300U/ml, hydrolyzes selenizing card in 35 DEG C of reaction 9h
Glue is drawn, then heating is boiled 10min and terminated and reacts, and is cooled to room temperature, is placed in 60 DEG C of rotary evaporations and is concentrated, and obtains selenizing card
Draw glue oligosaccharides concentrate.
(3) the selenide of carragheen oligosaccharides of different molecular weight is prepared using ethanol precipitation: by the selenide of carragheen of acquisition
Oligosaccharides concentrate passes through the ethanol precipitation of 5 times of weight, and sediment is collected by centrifugation, and then obtains selenide of carragheen after 60 DEG C of drying
Oligosaccharides；The selenide of carragheen oligosaccharides yield of preparation is 81%, and molecular weight is 900~1500Da.
Embodiment 2
The preparation method of selenide of carragheen oligosaccharides, comprising the following steps:
(1) composition of compound carrageenase: 8 parts by weight of kappa-carrageenan proenzyme material, ι -1 parts by weight of carrageenase raw material, λ -
1 parts by weight of carrageenase raw material.It mixes according to a certain percentage in an aseptic environment.Appearance is white powder；Total number of bacteria <
50000/g；Escherichia coli < 30CFU/g；Pathogenic escherichia coli is not detected；Salmonella is not detected.It is tested through DNS method, every gram
It is 6.5 × 10 that the compound carrageenase, which contains enzyme activity,5κ type carrageenase, the enzyme activity of U is 6.9 × 104The ι type OK a karaoke club of U
Glue enzyme and enzyme activity are 6.0 × 104The λ type carrageenase of U.
(2) it utilizes compound carragheen enzyme hydrolysis selenide of carragheen: preparing the selenizing OK a karaoke club that mass fraction is 5% with distilled water
Suitable compound carragheen enzyme solutions are added then according to the additional amount of 300U/ml in sol solution, hydrolyze selenium in 30 DEG C of reaction 9h
Change carragheen, then heating is boiled 10min and terminated and reacts, and is cooled to room temperature, is placed in 60 DEG C of rotary evaporations and is concentrated, and obtains selenium
Change carrageenan oligosaccharide concentrate.
(3) the selenide of carragheen oligosaccharides of different molecular weight is prepared using ethanol precipitation: by the selenide of carragheen of acquisition
Oligosaccharides concentrate passes through the ethanol precipitation of 5 times of weight, and sediment is collected by centrifugation, and then obtains selenide of carragheen after 60 DEG C of drying
Oligosaccharides；The selenide of carragheen oligosaccharides yield of preparation is 76%, and molecular weight is 900~1800Da.
Embodiment 3
The preparation method of selenide of carragheen oligosaccharides, comprising the following steps:
(1) composition of compound carrageenase: 6 parts by weight of kappa-carrageenan proenzyme material, ι -2 parts by weight of carrageenase raw material, λ -
2 parts by weight of carrageenase raw material.It mixes according to a certain percentage in an aseptic environment.Appearance is white powder；Total number of bacteria <
50000/g；Escherichia coli < 30CFU/g；Pathogenic escherichia coli is not detected；Salmonella is not detected.It is tested through DNS method, every gram
It is 4.9 × 10 that the compound carrageenase, which contains enzyme activity,5κ type carrageenase, the enzyme activity of U is 1.4 × 105The ι type OK a karaoke club of U
Glue enzyme and enzyme activity are 1.2 × 105The λ type carrageenase of U.
(2) it utilizes compound carragheen enzyme hydrolysis selenide of carragheen: preparing the selenizing OK a karaoke club that mass fraction is 5% with distilled water
Suitable compound carragheen enzyme solutions are added then according to the additional amount of 300U/ml in sol solution, hydrolyze selenium in 30 DEG C of reaction 9h
Change carragheen, then heating is boiled 10min and terminated and reacts, and is cooled to room temperature, is placed in 60 DEG C of rotary evaporations and is concentrated, and obtains selenium
Change carrageenan oligosaccharide concentrate.
(3) the selenide of carragheen oligosaccharides of different molecular weight is prepared using ethanol precipitation: by the selenide of carragheen of acquisition
Oligosaccharides concentrate passes through the ethanol precipitation of 5 times of weight, and sediment is collected by centrifugation, and then obtains selenide of carragheen after 60 DEG C of drying
Oligosaccharides；The selenide of carragheen oligosaccharides yield of preparation is 73%, and molecular weight is 900~2100Da.
Embodiment 4
The preparation method of selenide of carragheen oligosaccharides, comprising the following steps:
(1) composition of compound carrageenase: 3 parts by weight of κ type carrageenase raw material, 4 parts by weight of ι type carrageenase raw material, λ
3 parts by weight of type carrageenase raw material.It mixes according to a certain percentage in an aseptic environment.Appearance is white powder；Total number of bacteria <
50000/g；Escherichia coli < 30CFU/g；Pathogenic escherichia coli is not detected；Salmonella is not detected.It is tested through DNS method, every gram
It is 2.4 × 10 that the compound carrageenase, which contains enzyme activity,5κ type carrageenase, the enzyme activity of U is 2.8 × 105The ι type OK a karaoke club of U
Glue enzyme and enzyme activity are 1.8 × 105The λ type carrageenase of U.
(2) it utilizes compound carragheen enzyme hydrolysis selenide of carragheen: preparing the selenizing card that mass fraction is 15% with distilled water
Sol solution is drawn, then according to the additional amount of 300U/ml, suitable compound carragheen enzyme solutions are added, are hydrolyzed in 35 DEG C of reaction 9h
Selenide of carragheen, then heating is boiled 10min and is terminated and reacts, and is cooled to room temperature, is placed in 60 DEG C of rotary evaporations and is concentrated, and obtains
Selenide of carragheen oligosaccharides concentrate.
(3) the selenide of carragheen oligosaccharides of different molecular weight is prepared using ethanol precipitation: by the selenide of carragheen of acquisition
Oligosaccharides concentrate passes through the ethanol precipitation of 5 times of weight, and sediment is collected by centrifugation, and then obtains selenide of carragheen after 60 DEG C of drying
Oligosaccharides；The selenide of carragheen oligosaccharides yield of preparation is 71%, and molecular weight is 900~2700Da.
Comparative example 1
This comparative example is with compound carrageenase of the invention and single its effect of carrageenase comparative study, specifically:
This comparative example using compound carrageenase prepare selenide of carragheen oligosaccharides method the following steps are included:
(1) composition of compound carrageenase: 7 parts by weight of kappa-carrageenan proenzyme material, ι -2 parts by weight of carrageenase raw material, λ -
1 parts by weight of carrageenase raw material.It mixes according to a certain percentage in an aseptic environment.Appearance is white powder；Total number of bacteria <
50000/g；Escherichia coli < 30CFU/g；Pathogenic escherichia coli is not detected；Salmonella is not detected.It is tested through DNS method, every gram
It is 5.7 × 10 that the compound carrageenase, which contains enzyme activity,5κ type carrageenase, the enzyme activity of U is 1.4 × 105The ι type OK a karaoke club of U
Glue enzyme and enzyme activity are 6.0 × 104The λ type carrageenase of U.
(2) it utilizes compound carragheen enzyme hydrolysis selenide of carragheen: preparing the selenizing card that mass fraction is 15% with distilled water
Sol solution is drawn, then according to the additional amount of 300U/ml, suitable compound carragheen enzyme solutions are added, are hydrolyzed in 35 DEG C of reaction 9h
Selenide of carragheen, then heating is boiled 10min and is terminated and reacts, and is cooled to room temperature, is placed in 60 DEG C of rotary evaporations and is concentrated, and obtains
Selenide of carragheen oligosaccharides concentrate.
(3) the selenide of carragheen oligosaccharides of different molecular weight is prepared using ethanol precipitation: by the selenide of carragheen of acquisition
Oligosaccharides concentrate passes through the ethanol precipitation of 5 times of weight, and sediment is collected by centrifugation, and then obtains selenide of carragheen after 60 DEG C of drying
Oligosaccharides；The selenide of carragheen oligosaccharides yield of preparation is 81%, and molecular weight is 900~1500Da.
The method for preparing selenide of carragheen oligosaccharides of single carrageenase and aforementioned using compound is respectively adopted in this comparative example
The method that carrageenase prepares selenide of carragheen oligosaccharides is identical, only difference is that respectively with kappa-carrageenan enzyme, ι-OK a karaoke club
Glue enzyme and the compound carrageenase of lambda-carrageenan enzymes extraction, enzyme additive amount are also identical；Reaction condition and result are as shown in table 1 below:
Table 1
Embodiment 5
The application of selenide of carragheen oligosaccharides, including the following contents:
(1) selenium-rich rice is prepared, is added to water for the selenide of carragheen oligosaccharides of preparation as a kind of safe and efficient Selenium supplement agent
In rice, selenium-rich rice is produced.The selenide of carragheen oligosaccharides of preparation is made into 0.5% aqueous solution, foliage-spray in rice, thus
Produce selenium-rich rice；Or imposed in rice field using selenide of carragheen oligosaccharides as fertilizer, produce selenium-rich rice.
(2) selenium-enriched tea leaf is prepared, is added to tea for the selenide of carragheen oligosaccharides of preparation as a kind of safe and efficient Selenium supplement agent
In tree, selenium-enriched tea leaf is produced.The selenide of carragheen oligosaccharides of preparation is made into 0.1% aqueous solution, foliage-spray on tea tree, from
And produce selenium-enriched tea leaf；Or imposed in tea place using selenide of carragheen oligosaccharides as fertilizer, produce selenium-enriched tea leaf.
(3) Se-enriched egg is prepared, is added to egg for the selenide of carragheen oligosaccharides of preparation as a kind of safe and efficient Selenium supplement agent
In chicken feed, Se-enriched egg is produced.The selenide of carragheen oligosaccharides of preparation is added in egg feedstuff according to 0.5wt%, thus
Laying hen is set to produce Se-enriched egg.
Finally, it is stated that: above embodiments are merely to illustrate implementation process and feature of the invention, rather than limit this hair
Bright technical solution, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art answer
Work as understanding: it is still possible to modify or equivalently replace the present invention, without departing from the spirit and scope of the present invention any
Modification or part replacement, should all cover in protection scope of the present invention.
Claims (6)
1. a kind of compound carrageenase, wherein it is 2.1 × 10 that every gram of compound carrageenase, which contains enzyme activity,5~1.7 ×
106κ type carrageenase, the enzyme activity of U is 6.0 × 104~4.8 × 105The ι type carrageenase and enzyme activity of U is 3.0 × 104~
2.4×105The λ type carrageenase of U；
The compound carrageenase is 2.7 × 10 by enzyme activity5~2.7 × 106κ type carrageenase raw material, the enzyme activity of U/g be
2.3×105~2.3 × 106The ι type carrageenase raw material and enzyme activity of U/g is 2.0 × 105~2.0 × 106The λ type OK a karaoke club of U/g
Glue proenzyme material compounds to obtain；
The κ type carrageenase raw material, the ι type carrageenase raw material, the λ type carrageenase raw material are by producing κ type respectively
Carrageenase bacterial strain, production ι type carrageenase bacterial strain or production λ type carrageenase bacterial strain generate and purified, drying obtains；
Producing the κ type carrageenase bacterial strain is Pseudoalteromonas sp NJ62；
Producing the ι type carrageenase bacterial strain is Pseudoalteromonas sp NJ64；
Producing the λ type carrageenase bacterial strain is Pseudoalteromonas sp NJ276.
2. compound carrageenase according to claim 1, wherein every gram of compound carrageenase contain enzyme activity be 2.1 ×
105~8.5 × 105κ type carrageenase, the enzyme activity of U is 6.0 × 104~2.4 × 105The ι type carrageenase and enzyme activity of U be
3.0×104~1.2 × 105The λ type carrageenase of U.
3. compound carrageenase according to claim 1 or 2, wherein the κ type carrageenase raw material is 20 DEG C~45
DEG C, pH6.0~9.0 when active highest；
Active highest of the ι type carrageenase raw material in 15 DEG C~40 DEG C, pH6.0~9.0；
Active highest of the λ type carrageenase raw material in 25 DEG C~45 DEG C, pH6.0~10.0.
4. a kind of preparation method of selenide of carragheen oligosaccharides, wherein the method includes using the claim 1 of effective catalytic amount
Compound carrageenase described in any one of~3 hydrolyzes selenide of carragheen.
5. the preparation method according to claim 4, wherein the method includes by the compound carrageenase according to 80~
The additional amount of 800U/ml be added to mass fraction be 5~20% selenide of carragheen aqueous solution in, in 25~45 DEG C of reaction 6h~
12h hydrolyzes selenide of carragheen, and post-processing obtains the selenide of carragheen oligosaccharides.
6. preparation method according to claim 5, wherein the post-processing includes boiling the reaction solution heating after hydrolysis
Boiling, cooling, evaporation obtain selenide of carragheen oligosaccharides concentrate, then by the concentrate by the ethanol precipitations of 3~9 times of weight,
It is collected by centrifugation sediment, dry the selenide of carragheen oligosaccharides.
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| CN103627752A (en) * | 2013-10-29 | 2014-03-12 | 中国海洋大学 | Method for preparing carrageenin oligosaccharides by compositely degrading eucheuma with carrageenanase and cellulase |
| CN104894100A (en) * | 2015-06-17 | 2015-09-09 | 集美大学 | Immobilized kappa-carrageenan enzyme and method for preparing kappa-carrageenan oligosaccharide by adopting immobilized kappa-carrageenan enzyme |
| CN105950590A (en) * | 2016-07-26 | 2016-09-21 | 集美大学 | Method for preparing t-carrageenase |
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| CN103627752A (en) * | 2013-10-29 | 2014-03-12 | 中国海洋大学 | Method for preparing carrageenin oligosaccharides by compositely degrading eucheuma with carrageenanase and cellulase |
| CN104894100A (en) * | 2015-06-17 | 2015-09-09 | 集美大学 | Immobilized kappa-carrageenan enzyme and method for preparing kappa-carrageenan oligosaccharide by adopting immobilized kappa-carrageenan enzyme |
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