CN109182413A - A kind of method of the small molecule potassium alginate of beam system for high guluronic acid content - Google Patents
A kind of method of the small molecule potassium alginate of beam system for high guluronic acid content Download PDFInfo
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- CN109182413A CN109182413A CN201811011605.5A CN201811011605A CN109182413A CN 109182413 A CN109182413 A CN 109182413A CN 201811011605 A CN201811011605 A CN 201811011605A CN 109182413 A CN109182413 A CN 109182413A
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Abstract
The invention discloses a kind of beam systems for the method for the small molecule potassium alginate of high guluronic acid content, includes the following steps: that potassium alginate powder is made into the potassium alginate aqueous solution that mass concentration is 3-4.8% by (1), stirs evenly；(2) potassium alginate aqueous solution is placed in 35-39 DEG C of water-bath, algin catenase, stirring enzymatic hydrolysis is added；(3) alcohol precipitation is carried out after digesting, obtains small molecule potassium alginate.The method of the present invention step is simple, mild condition, and in smaller range, and guluronic acid content is high, can not only be used for food additives, and can be used as drug and functional food for the small molecule potassium alginate molecular weight stabilizers being prepared.
Description
Technical field
The present invention relates to small molecule potassium alginate preparation technical field, more particularly to a kind of beam system is standby high ancient
The method of the small molecule potassium alginate of sieve glucuronic acid content.
Background technique
Potassium alginate (Potassium Alginate) is also known as potassium alginate, and molecular formula is (C6H7O6K) n is sweet by β-D-
Dew uronic acid and α-L- guluronic acid are formed by Isosorbide-5-Nitrae-glucosides key connection, mainly from marine browns such as kelp, thallus laminariaes
Plant is widely used in medicine and food service industry currently as food additives and acceptable assistant.
In clinical studies it has been observed that the antihypertensive effect of potassium alginate powder, although the potassium alginate of macromolecular has one
Determine antihypertensive effect, but since molecular weight is big, gastrointestinal reaction is more apparent and body absorption is bad, seriously affect individual medication according to
From property and clinical application；And the small molecule algin of high guluronic acid content can reduce the inflammatory effector of LPS induction, and have
Higher anti-microbial pathogen, antifungal activity.
Solidification enzyme is generallyd use in the prior art and two step enzymatic isolation method of complex enzyme prepares low molecule potassium alginate, and step is numerous
It is trivial, and the potassium alginate molecular weight ranges obtained are larger, directionality is poor.In addition, researcher attempts to pass through sour water solution or alkali
Hydrolyze method prepares small molecule potassium alginate, but is easy to damage the structure of potassium alginate.
Therefore, how to provide a kind of simple step, mild condition and can beam system for the small of high guluronic acid content
The problem of method of molecular potassium alginate is those skilled in the art's urgent need to resolve.
Summary of the invention
In view of this, the present invention provides a kind of beam systems for the side of the small molecule potassium alginate of high guluronic acid content
Method, step is simple, mild condition, the small molecule potassium alginate molecular weight stabilizers being prepared in smaller range, and ancient sieve
Glucuronic acid content is high.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of method of the small molecule potassium alginate of beam system for high guluronic acid content, includes the following steps:
(1) potassium alginate powder is made into the potassium alginate aqueous solution that mass concentration is 3-4.8%, stirred evenly；
(2) potassium alginate aqueous solution is placed in 35-39 DEG C of water-bath, algin catenase, stirring enzymatic hydrolysis is added；
(3) alcohol precipitation is carried out after digesting, obtains small molecule potassium alginate.
Preferably, the molecular weight of potassium alginate is 600-1000kDa in step (1), and guluronic acid content is 10%-
30%.
Enzymatic hydrolysis substrate molecule amount is larger, and guluronic acid content is lower, is split under specific enzymatic hydrolysis condition using algin
It solves enzyme and carries out a step enzymatic hydrolysis, the small molecule potassium alginate of high guluronic acid content, molecular weight stabilizers, biology is prepared in orientation
It is active high.
It is further preferred that the molecular weight of potassium alginate is 800kDa in step (1).
Preferably, the volume fraction that algin catenase is initially added in step (2) is 0.1-0.25%；
Algin catenase is supplemented every 0.5-0.8h in enzymolysis process, the algin catenase concentration of supplement gradually drops
It is low；Enzymolysis time is 6-8h, pH 7-9.
It is further preferred that supplementing algin catenase every 0.8h in enzymolysis process, the algin catenase of supplement is dense
Degree gradually decreases；Enzymolysis time is 6h, pH 8.
Algin catenase concentration, enzymolysis time, alternate frequency ensure that the stability of enzymolysis product.
Preferably, the nucleotide sequence such as SEQ ID NO:1 of algin catenase is encoded.
Preferably, algin catenase the preparation method is as follows:
1) using SEQ ID NO:2 and SEQ ID NO:3 as primer, algin catenase complete genome sequence is expanded；
2) EcoR I and Hind III digestion coli expression carrier pProEXHTa and extension increasing sequence, T4 ligase connect
It connects, is transferred in e. coli bl21；
3) Escherichia coli recombinant strain is chosen, is inoculated into LB liquid medium with ampicillin, 37 DEG C of culture 12-
16h；It is inoculated in LB liquid medium with ampicillin and is cultivated to OD with 2% inoculum concentration600When for 0.5-0.8, cream is added
Sugar goes to 28 DEG C of culture 25-34h, and centrifugation obtains supernatant i.e. algin catenase.
The present invention is for the fermentation step of potassium alginate setting algin catenase, and obtained algin catenase is for sea
The enzyme activity of potassium alginate is high, and hydrolysis result is good；Enzymolysis product small molecule potassium alginate molecular weight stabilizers, guluronic acid content are high.
Preferably, lactose mass concentration is 0.4%.
The high efficient expression of the addition induction algin catenase of certain concentration lactose.
Preferably, the molecular weight of small molecule potassium alginate is 1-3kDa, and guluronic acid content is 50-80%.
It is further preferred that the molecular weight of small molecule potassium alginate is 0.7-1.5kDa, guluronic acid content is
55.87-73.61%.
Preferably, alcohol precipitating method is as follows in step (3):
1) enzymolysis liquid is centrifuged, and takes supernatant, and 1 times of volume ethanol is added and stands；
2) supernatant is taken, 3 times of volume ethanols are added and stand；
3) 3 times of volume ethanols take precipitating after standing, and are dissolved in water, rotary evaporation, and freeze-drying obtains small molecule alginic acid
Potassium, i.e. product 1；
4) 3 times of volume ethanols take supernatant after standing, and 5 times of volume ethanols are added and stand, takes supernatant, obtains after freeze-drying small
Molecular potassium alginate, i.e. product 2.
It is greater than 91% by the small molecule potassium alginate purity that two step alcohol precipitations obtain.
It can be seen via above technical scheme that compared with prior art, it is standby high that the present disclosure provides a kind of beam systems
The method of the small molecule potassium alginate of guluronic acid content after macromolecular seaweed potassium powder is dissolved in water, is added algin and splits
Enzyme enzymatic hydrolysis is solved, obtained small molecule potassium alginate purity is high, guluronic acid content is high, can not only be used for food additives, and can
As drug and functional food.
Detailed description of the invention
The drawings to be used in the embodiments are briefly described below, it should be apparent that, it is described below
Attached drawing is only the embodiment of the present invention, for those of ordinary skill in the art, in the premise not made the creative labor
Under, other attached drawings can also be obtained according to the attached drawing of offer.
Fig. 1 attached drawing is that embodiment 1 digests substrate potassium alginate GPC figure；
Fig. 2 attached drawing is 1 small molecule potassium alginate GPC of embodiment figure；
Fig. 3 attached drawing is that embodiment 1 digests substrate potassium alginate guluronic acid containing spirogram；
Fig. 4 attached drawing is that 1 small molecule potassium alginate guluronic acid of embodiment contains spirogram；
Fig. 5 attached drawing is 2 small molecule potassium alginate GPC of embodiment figure；
Fig. 6 attached drawing is that 2 small molecule potassium alginate guluronic acid of embodiment contains spirogram；
Specific embodiment
Below in conjunction with attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that is retouched
The embodiment stated is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, originally
Field those of ordinary skill every other embodiment obtained without making creative work, belongs to the present invention
The range of protection.
Embodiment 1
By algin catenase beam system for the small molecule potassium alginate of high guluronic acid content:
1. the preparation of algin catenase
Upstream primer F1 and downstream primer F2 is designed according to alginate lyase gene sequence SEQ ID NO:1:
F1:5 '-ATGAAAGTAAGTTGCGCTGTC-3 ' (SEQ ID NO:2)；
R1:5 '-TTAATCGTGCGACTGCTCC-3 ' (SEQ ID NO:3)；
PCR amplification obtains alginate lyase gene complete sequence；EcoR I and Hind III digestion extension increasing sequence and large intestine
The connection of bacillus expression vector pProEXHTa, T4 ligase, is transferred in e. coli bl21.
Escherichia coli recombinant strain is chosen, is inoculated into the LB liquid medium containing 90 μ g/mL ampicillins, 37 DEG C of shaking tables
160r/min cultivates 12h；It is inoculated in LB liquid medium with ampicillin with 2% inoculum concentration, shaking table 160r/ in 37 DEG C
It cultivates in min to OD600When for 0.5-0.8, final concentration of 0.4% lactose is added and goes to 160r/min in 28 DEG C of shaking tables and cultivates
34h, 4800r/min centrifugation 20min obtain supernatant i.e. algin catenase.
2. enzymatic hydrolysis
It (1) is about 800kDa by molecular weight, the potassium alginate powder that guluronic acid content is 21.57% is made into 1L mass
The potassium alginate aqueous solution that concentration is 3% dissolves overnight；Until completely dissolved, it stirs evenly, surveying pH is 9.0, and the sea 0.8g is added
Alginic acid, adjusting pH is 7.6.
(2) the pH potassium alginate aqueous solution for being 7.6 is placed in 35 DEG C of water-baths, 2mL algin catenase is added and stirs enzyme
0.8h is solved, every 0.8h adds 0.15% algin catenase, digests 6h altogether.
(3) enzymolysis liquid 4800r/min is centrifuged 25min, removal precipitating, and obtained supernatant is added 1 times of volume ethanol and stands
12h；Supernatant is taken, 3 times of volume ethanols are added and stand 12h；Precipitating is taken, 80mL water is dissolved in, 50 DEG C of rotary evaporation 1h are freeze-dried,
Obtain small molecule potassium alginate, i.e. product 1.
Using 50 liquid phase column of high performance liquid chromatography combination PLAquagel-OH respectively to enzymatic hydrolysis substrate potassium alginate and preparation
Obtained small molecule potassium alginate molecular weight is measured, and as shown in Figs. 1-2, enzymatic hydrolysis substrate potassium alginate weight average molecular weight is
800kDa, the small molecule potassium alginate weight average molecular weight digested are 2.4kDa.
Substrate potassium alginate is digested to gained using high performance liquid chromatography combination XDB-C18 liquid phase column and is prepared small
Molecular potassium alginate guluronic acid content is measured, and as shown in Figure 3-4, digests substrate potassium alginate guluronic acid content
It is 21.57%, the small molecule potassium alginate guluronic acid content digested is 55.87%.
Potassium in flame atom spectrum measuring enzymatic hydrolysis substrate potassium alginate and the small molecule potassium alginate being prepared
Content, enzymatic hydrolysis substrate potassium alginate potassium content are 16%, the small molecule potassium alginate potassium content 16.07% digested.
Further, the small molecule potassium alginate purity that the present embodiment is prepared is 91.18%.
Embodiment 2
By algin catenase beam system for the small molecule potassium alginate of high guluronic acid content:
1. the preparation of algin catenase
Alginate lyase gene conversion process is the same as embodiment 1.
Escherichia coli recombinant strain is inoculated into the LB liquid medium containing 90 μ g/mL ampicillins, 37 DEG C of 160r/
Min cultivates 16h；It is inoculated in the LB liquid medium containing 90 μ g/mL ampicillins with 2% inoculum concentration, 37 DEG C of 160r/min
It cultivates to OD600When for 0.5-0.8, final concentration of 0.4% lactose is added and goes to 28 DEG C of culture 25h, 4800r/min centrifugation 20min
Obtain supernatant i.e. algin catenase.
2. enzymatic hydrolysis
It (2) is 800kDa by molecular weight, it is dense that the potassium alginate powder that guluronic acid content is 21.57% is made into 1L mass
The potassium alginate aqueous solution that degree is 4.8% dissolves overnight；Until completely dissolved, it stirs evenly, surveying pH is 9.2, and the sea 0.7g is added
Alginic acid, adjusting pH is 8.
(2) the pH potassium alginate aqueous solution for being 8 is placed in 38 DEG C of water-baths, 2.5mL algin catenase is added and stirs enzyme
0.5h is solved, every 0.5h adds 0.2% algin catenase, digests 8h altogether.
(3) enzymolysis liquid 4800r/min is centrifuged 20min, removal precipitating, and obtained supernatant is added 1 times of volume ethanol and stands
12h；Supernatant is taken, 3 times of volume ethanols are added and stand 12h；Supernatant is taken, 5 times of volume ethanols are added and stand 12h, take 45 DEG C of supernatant
Rotary evaporation 1h, freeze-drying, obtains small molecule potassium alginate, i.e. product 2.
Using 30 liquid phase column of high performance liquid chromatography combination PLAquagel-OH to the small molecule potassium alginate being prepared point
Son amount is measured, as shown in figure 5, the small molecule potassium alginate weight average molecular weight range that enzymatic hydrolysis obtains is 0.7-1.5kDa.
Substrate potassium alginate is digested to gained using high performance liquid chromatography combination XDB-C18 liquid phase column and is prepared small
Molecular potassium alginate guluronic acid content is measured, as shown in fig. 6, the small molecule potassium alginate gulose aldehyde that enzymatic hydrolysis obtains
Acid content is 73.61%.
Potassium contains in flame atom spectrum measuring enzymatic hydrolysis substrate potassium alginate and the small molecule potassium alginate being prepared
Amount, enzymatic hydrolysis substrate potassium alginate potassium content are 16%, the small molecule potassium alginate potassium content 16.17% digested.
Further, the small molecule potassium alginate purity that the present embodiment is prepared is 93%.
Each embodiment in this specification is described in a progressive manner, the highlights of each of the examples are with other
The difference of embodiment, the same or similar parts in each embodiment may refer to each other.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Sequence table
<110>Chinese Marine University
<120>a kind of beam system for the small molecule potassium alginate of high guluronic acid content method
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 822
<212> DNA
<213> Artificial
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atgaaagtaa gttgcgctgt cgtactgtct gcttgtattg ccagtgccaa cgcagacaac 60
aatggcgatg gcaaggccga ctccatcaag gaaaatgacc tgaatgcagg ctatgcagat 120
ggcacctact tctatactgc tgccgatggc ggcatggtgt tccgctgccc gatcgatggc 180
tataaaacat cgaccaacac gtcctatacc cgcaccgagc tgcgcgagat gctacgtcgt 240
ggcgacacca gcattgccac ccagggggtc aatggaaaca actgggtatt cggctccgca 300
cccgcttcgg cacgtgaagc agccggcggt gtcgacggtg ttttacgcgc aaccctcgcg 360
gtaaaccatg tcaccactac cggagatagc ggccaggttg gacgggtgat tgttggacag 420
attcacgcca acaacgacga accgctgcgt ctttactacc gcaagttacc gggccacagc 480
aaaggttctg tgtatatcgc ccatgagcca aacggcggca gcgacagctg gtacgacatg 540
attggcagcc gttccagcag cgcctcggac ccgtccgacg gtatcgcact ggatgaagtc 600
tggagctacg aggtcaaggt tgtcggtaac accctcaccg tgaccatctt ccgtgctggt 660
aaagacgatg tggtacaggt tgtggatatg ggcaacagcg gttacgacgt cgccgaccag 720
taccagtact tcaaggccgg ggtgtacaac cagaacaaca ccggcaatgc cagtgactat 780
gtccaggtga ccttctacgc cctggagcag tcgcacgatt aa 822
<210> 2
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ttaatcgtgc gactgctcc 19
Claims (9)
1. a kind of beam system is for the method for the small molecule potassium alginate of high guluronic acid content, which is characterized in that including as follows
Step:
(1) potassium alginate powder is made into the potassium alginate aqueous solution that mass concentration is 3-4.8%, stirred evenly；
(2) potassium alginate aqueous solution is placed in 35-39 DEG C of water-bath, algin catenase, stirring enzymatic hydrolysis is added；
(3) alcohol precipitation is carried out after digesting, obtains small molecule potassium alginate.
2. a kind of beam system according to claim 1 is for the method for the small molecule potassium alginate of high guluronic acid content,
It is characterized in that, the molecular weight of potassium alginate is 600-1000kDa in the step (1), guluronic acid content is 10%-
30%.
3. a kind of beam system according to claim 1 is for the method for the small molecule potassium alginate of high guluronic acid content,
It is characterized in that, the volume fraction for initially adding algin catenase in the step (2) is 0.1-0.25%；
Algin catenase is supplemented every 0.5-0.8h in enzymolysis process, the algin catenase concentration of supplement gradually decreases；Enzyme
The solution time is 6-8h.
4. a kind of beam system according to claim 1 is for the method for the small molecule potassium alginate of high guluronic acid content,
It is characterized in that, encoding the nucleotide sequence such as SEQ ID NO:1 of the algin catenase.
5. a kind of beam system according to claim 4 is for the method for the small molecule potassium alginate of high guluronic acid content,
It is characterized in that, algin catenase the preparation method is as follows:
1) using SEQ ID NO:2 and SEQ ID NO:3 as primer, algin catenase complete genome sequence is expanded；
2) EcoR I and Hind III digestion coli expression carrier pProEXHTa and extension increasing sequence, the connection of T4 ligase, turn
Enter in e. coli bl21；
3) Escherichia coli recombinant strain is chosen, is inoculated into LB liquid medium with ampicillin, 37 DEG C of culture 12-16h；With
2% inoculum concentration is inoculated in LB liquid medium with ampicillin and cultivates to OD600When for 0.5-0.8, lactose is added and goes to
28 DEG C of culture 25-34h, centrifugation obtain supernatant i.e. algin catenase.
6. a kind of beam system according to claim 5 is for the method for the small molecule potassium alginate of high guluronic acid content,
It is characterized in that, lactose final concentration of 0.4%.
7. a kind of beam system according to claim 1 is for the method for the small molecule potassium alginate of high guluronic acid content,
It is characterized in that, the molecular weight of small molecule potassium alginate is 1-3kDa, guluronic acid content is 50-80%.
8. a kind of beam system according to claim 7 is for the method for the small molecule potassium alginate of high guluronic acid content,
It is characterized in that, the molecular weight of small molecule potassium alginate is 0.7-1.5kDa, guluronic acid content is 55.87-73.61%.
9. a kind of beam system according to claim 1 is for the method for the small molecule potassium alginate of high guluronic acid content,
It is characterized in that, alcohol precipitating method is as follows in the step (3):
1) enzymolysis liquid is centrifuged, and takes supernatant, and 1 times of volume ethanol is added and stands；
2) supernatant is taken, 3 times of volume ethanols are added and stand；
3) 3 times of volume ethanols take precipitating after standing, and are dissolved in water, rotary evaporation, and freeze-drying obtains small molecule potassium alginate, i.e.,
Product 1；
4) 3 times of volume ethanols take supernatant after standing, and 5 times of volume ethanols are added and stand, takes supernatant, obtains small molecule after freeze-drying
Potassium alginate, i.e. product 2.
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| CN201811011605.5A CN109182413A (en) | 2018-08-31 | 2018-08-31 | A kind of method of the small molecule potassium alginate of beam system for high guluronic acid content |
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| CN201811011605.5A CN109182413A (en) | 2018-08-31 | 2018-08-31 | A kind of method of the small molecule potassium alginate of beam system for high guluronic acid content |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5460957A (en) * | 1992-04-28 | 1995-10-24 | Maruha Corporation | Calcium alginate oligosaccharide and method for producing the same from potassium or sodium alginate |
| CN106884025A (en) * | 2017-05-02 | 2017-06-23 | 中国海洋大学 | A kind of enzymatic hydrolysis beam system for algin oligosaccharide method |
-
2018
- 2018-08-31 CN CN201811011605.5A patent/CN109182413A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5460957A (en) * | 1992-04-28 | 1995-10-24 | Maruha Corporation | Calcium alginate oligosaccharide and method for producing the same from potassium or sodium alginate |
| CN106884025A (en) * | 2017-05-02 | 2017-06-23 | 中国海洋大学 | A kind of enzymatic hydrolysis beam system for algin oligosaccharide method |
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