CN113736837A - Method for preparing new agaro-oligosaccharide from agar - Google Patents
Method for preparing new agaro-oligosaccharide from agar Download PDFInfo
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- CN113736837A CN113736837A CN202111183330.5A CN202111183330A CN113736837A CN 113736837 A CN113736837 A CN 113736837A CN 202111183330 A CN202111183330 A CN 202111183330A CN 113736837 A CN113736837 A CN 113736837A
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- oligosaccharide
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- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 47
- 229920001817 Agar Polymers 0.000 title claims abstract description 36
- 239000008272 agar Substances 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 27
- 241000206671 Gelidium amansii Species 0.000 claims abstract description 18
- 238000006243 chemical reaction Methods 0.000 claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 238000000265 homogenisation Methods 0.000 claims abstract description 5
- 108010045649 agarase Proteins 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
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- 238000011144 upstream manufacturing Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims 1
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- 239000011344 liquid material Substances 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract 1
- 150000002482 oligosaccharides Chemical class 0.000 description 14
- 239000000047 product Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 238000010586 diagram Methods 0.000 description 6
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- 239000000126 substance Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 241001428166 Eucheuma Species 0.000 description 2
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- 238000004458 analytical method Methods 0.000 description 2
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- 238000005119 centrifugation Methods 0.000 description 2
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- 238000002156 mixing Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 241000223600 Alternaria Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000914318 Gilvimarinus chinensis Species 0.000 description 1
- 241000206581 Gracilaria Species 0.000 description 1
- 241000132152 Polymyxa Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000206613 Pyropia yezoensis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003579 anti-obesity Effects 0.000 description 1
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- 229930027917 kanamycin Natural products 0.000 description 1
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- 239000005017 polysaccharide Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 150000004044 tetrasaccharides Chemical class 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01081—Beta-agarase (3.2.1.81)
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Abstract
The invention discloses a method for preparing new agaro-oligosaccharide from gelidium amansii, which comprises the following steps of homogenizing gelidium amansii with the following liquid-material ratio: 9: 100-13: 100, and the treatment time is 10-20 min. And (3) carrying out enzymolysis on the product subjected to homogenization treatment at 37 ℃ by using a mixed enzyme solution (GH16-1: GH16-2: GH50 in a ratio of 2:1:1), and finishing enzymolysis after 2-3 h to successfully obtain a product (10.0mg/g wet weight) containing the new agaro-oligosaccharide. The invention directly takes fresh agar as raw material, the reaction condition of the production process is simple, the conversion period is short, no pollution is caused, the environment is protected, and the invention has important industrial application prospect.
Description
Technical Field
The invention relates to the field of biological fermentation, in particular to a method for preparing new agaro-oligosaccharide from agar.
Background
The agar oligosaccharide is derived from agar polysaccharide (mainly separated from some red algae such as Gelidium amansii, Porphyra yezoensis and Gracilaria) and has various biological activities. The agar oligosaccharide is formed by alternating (1 → 3) -O-beta-D-galactose residues and (1 → 4) -O-3, 6-diether-alpha-L-galactose residues. According to the difference of the non-reducing end of the agar oligosaccharide, the agar oligosaccharide can be divided into new agar oligosaccharide with the non-reducing end of 3, 6-lacton-L-galactose and agar oligosaccharide with the non-reducing end of D-galactose. Agar oligosaccharides have various physiological activities such as anticancer, antioxidant, anti-obesity and whitening.
The existing methods for preparing agar oligosaccharides mainly comprise a chemical method and an enzymatic method, the agar oligosaccharides prepared by the chemical method have the advantages of rapid reaction, high concentration of treated substrates and the like, but the products are relatively complex after acidolysis, and the separation is relatively difficult. Therefore, the development of a specific and efficient preparation technology of the new agaro-oligosaccharides is of great significance, and a method for preparing the new agaro-oligosaccharides by using agar needs to be designed urgently to solve the problems.
Disclosure of Invention
The invention aims to provide a method for preparing new agaro-oligosaccharide from agar.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
according to the method, the agar is subjected to homogenization treatment for 10-20 min, the homogenized agar is directly used as a reaction substrate for enzymolysis, the added mixed enzyme solution is (GH16-1: GH16-2: GH50 ═ 2:1:1), and after an enzymolysis product is purified, new agaro-oligosaccharide is prepared, wherein the sequence of GH16-1 agarase is SEQ ID NO: 1. The sequence of GH16-2 is SEQ ID NO. 2, and GH16-1 and GH16-2 are novel agarases, have the similarity of less than 60% with the known agarases, and are the first cloning and expression of the invention.
Further, performing enzymolysis by using an agarase mixed solution with heterologous expression, wherein the ratio of the used enzyme solution is 2:1:1.
A method for preparing new agaro-oligosaccharide from agar comprises the steps of homogenizing agar for 20min, adding a homogenate product serving as a reaction substrate into a mixed enzyme solution for enzymolysis, and purifying an enzymolysis product to obtain the new agaro-oligosaccharide, wherein the sequence of GH16-2 agarase is SEQ ID NO. 2.
Further, the reaction primer sequence of the GH16-2 agarase is
GH16-2 TCGCGGATCCGAATTCAAGCAAGGGCACTCGC upstream primer
GH16-2 GTGCGGCCGCAAGCTTGTCACACTTTTTGCAGCCCG downstream primer
The reaction primer sequence of the GH16-1 agarase is
GH16-1 TCGCGGATCCGAATTCGCCGATTGGGATGG
GH16-1 GTGCGGCCGCAAGCTTGTTATTGCGGTGTAAATATAAAACGG
The GH16-1 agarase is applied to the conversion of agar.
Compared with the prior art, the invention has the following beneficial effects:
(1) the conversion speed of the agar oligosaccharide is high, only about 6 hours are needed, and the consumed time is short. The reported agar oligosaccharide preparation method needs the steps of drying and extracting agar, and often needs 3-5 days. Compared with the prior method, the method has the advantage of greatly shortening the period.
(2) The invention has mild reaction condition and simple required equipment. The invention can carry out enzymolysis only at 37 ℃, does not need high temperature and high pressure and strong acid and strong alkali treatment, and simultaneously needs a refiner as the main equipment with low price.
(3) The invention is green, environment-friendly and pollution-free. In the prior agar oligosaccharide preparation, the agar is extracted by a chemical method, so that the environmental pollution is caused, and the generated residues cannot be reused. The invention does not need to extract agar, reduces pollution and can recycle residual products.
Drawings
FIG. 1 is a schematic diagram showing the effect of different enzyme dosages of a method for preparing new agaro-oligosaccharides from gelidium amansii on the yield of new agaro-oligosaccharides;
FIG. 2 is a schematic diagram showing comparison of TLC analysis of product changes before and after enzymolysis in the method for preparing new agaro-oligosaccharide from gelidium amansii;
FIG. 3 is a schematic flow diagram of a process for preparing novel agaro-oligosaccharides from gelidium amansii;
FIG. 4 is a schematic diagram showing the effect of different liquid-material ratios on the yield of new agaro-oligosaccharides in the method for preparing new agaro-oligosaccharides from gelidium amansii;
FIG. 5 is a schematic diagram showing the effect of different treatment times on the yield of new agaro-oligosaccharides in the method for preparing new agaro-oligosaccharides from gelidium amansii;
Detailed Description
The present invention will be further described with reference to the following description and examples, which include but are not limited to the following examples.
As shown in fig. 1 and 2, in this example, gelidium amansii is subjected to homogenization treatment for 20min, the homogenized gelidium amansii is directly used as a reaction substrate for enzymolysis, the added mixed enzyme solution is (GH16-1: GH16-2: GH50 ═ 2:1:1), and after the enzymolysis product is purified, new agaro-oligosaccharide is prepared, wherein the sequence of GH16-1 agarase is SEQ ID NO: 1. GH16-2 sequence is SEQ ID NO 2.
The primer of the GH16-2 agarase is
GH16-1 TCGCGGATCCGAATTCGCCGATTGGGATGG
GH16-1 GTGCGGCCGCAAGCTTGTTATTGCGGTGTAAATATAAAACGG
The primer of the GH16-1 agarase is
GH16-2 TCGCGGATCCGAATTCAAGCAAGGGCACTCGC
GH16-2 GTGCGGCCGCAAGCTTGTCACACTTTTTGCAGCCCG
The invention adopts Escherichia coli BL21(DE3) as a host bacterium for protein expression, and the Escherichia coli is cultured in Luria-Bertani (LB) culture medium containing 100 mu g/mL kanamycin, and the process schematic diagram of the method for preparing the new agaro-oligosaccharide by using agar is shown in figure 3.
Homogenizing Eucheuma Gelatinosum with different liquid-material ratios, adding 20mM Tris-HCl (pH7.0) into Eucheuma Gelatinosum with wet weights of 5g, 11g, 13g and 17g respectively until the total volume is 150mL, and homogenizing for 15-20min to obtain reaction substrate. Adding 7.7U of mixed enzyme liquid, uniformly mixing, carrying out table shaking reaction at 37 ℃, carrying out post-centrifugation treatment, and inactivating the supernatant in boiling water bath for 3-10 min, as shown in figure 4, for the influence of different liquid materials on the yield of the new agaro-oligosaccharide, adding 13g of agar into 150mL of aqueous solution as reference data, and the rest is analogized.
Optimized time for homogenate treatment 5 parts of 9g agar are weighed, 20mM Tris-HCl (pH7.0) is added into the agar until the total volume is 150mL, and the crushing time is respectively 5min, 15min, 20min and 25min, and the mixture is used as a reaction substrate. Adding 7.7U of GH16-1 and GH16-2 enzyme solutions, immediately mixing uniformly, timing, reacting in a shaking table at 37 ℃, sampling and centrifuging at each time period, inactivating the supernatant in a boiling water bath for 3-10 min, and sampling to determine the content of reducing sugar. As shown in FIG. 1, the effect of different enzyme addition on the yield of new agaro-oligosaccharides, and as shown in FIG. 5, the effect of different treatment times on the yield of new agaro-oligosaccharides during homogenization.
In the experiment of degrading red algae breaking solution by enzyme combination, the enzyme activities of GH16-1, GH16-2 and GH50 enzyme solutions are combined and degraded by three agarases according to a proportion (U/U/U is 2:1:1) to 30mL of homogenated agar, the reaction is completed at 37 ℃ for 4h, the supernatant is collected by centrifugation, reducing sugar is detected, and the degree of polymerization of agar oligosaccharide in a sample is detected by TLC.
TABLE 1 Effect of different enzyme combinations on the yield of New agar oligosaccharides
As depicted in fig. 2, for TLC analysis of product changes before and after enzymatic hydrolysis, M: the new agaro-oligosaccharide standard substance comprises NA2 which is new agaro disaccharide, NA4 which is new agaro tetrasaccharide, NA6 which is new agaro hexaose, C which is a sample before enzymolysis, and 1 which is a sample after enzymolysis.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the embodiments of the invention without departing from the spirit and scope of the invention, which is to be covered by the claims.
Sequence listing
<110> university of Chengdu
<120> method for preparing new agaro-oligosaccharide from agar
<141> 2021-10-09
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<213> Huanghai fungus (Gilvimarinus chinensis)
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atttccgatg actttaacta cacatcatcg ctcaacaatt accataacga atttaccagt 180
cgttggcatg agggctttat taacccatgg actggccctg ggcttaccga gtggaccgac 240
ggtcatgcct acgtcaccgg tggcaactta ggtattgccg ccacccgcaa accaggcacc 300
gataaggtgc gagcgggctc tatcacttcg cacgatactt ttacctaccc actgtacgta 360
gaaacccgag caaagatcag caagcttgtg ctcgcttcgg acgtttggtt attaagcgcc 420
gactccaccc aagagattga tattcttgaa gcctacggca gtgatcgtcc tagtgagtca 480
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tatcaaccaa cagatgcagg aagctggtat accgatggac aaggtacggt gtggagcgat 600
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ggccaattag ttcgaagtgt atccggcccc aacattatcg accccaacgg cttcaccaat 720
ggcactggtt taagcaaacc tatgcatctg atcataaaca ctgaagatca agattggcgc 780
tcagataacg gcatctctcc aaccgacacc gagttagccg acaccaataa aagtatctat 840
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gatcatgatt ggcgctcaga agcgggtaat attgcaactg atgcagacct agccgatgac 840
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acaagcactg ttactagtgg cgcgcaaatt aaagcaaaac acagtcaaaa gtgtatcgat 960
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aacgagaacc aagcatttga gctcgttgaa ctcgcaaata acgaatatgc tatcagttca 1080
caattaactg gcttgtgtat gcaaattgca aattcaagta cctcaaatgg cgctggggtt 1140
gaacagtggg tgtgtgatca cacaaaagcc aatcaacgtt ttacactgaa caatacagga 1200
gatggctact ttgagcttag atcaagctta agtaataaat gtattgatat agcaggtaaa 1260
ttacaaacca atggcgcgtc agttgtgcag tggcagtgtt ataacggcga taatcaacgt 1320
tttcagctga ttgagtaa 1338
Claims (6)
1. A method for preparing new agaro-oligosaccharide from gelidium amansii is characterized in that: the agar is homogenized for 10-20 min, the obtained homogenate product can be directly used for enzymolysis, mixed enzyme liquid is added for enzymolysis (GH16-1: GH16-2: GH 50: 2:1:1), and new agaro-oligosaccharide is obtained after purification, wherein the sequence of GH16-1 agarase is SEQ ID NO:1, and the sequence of GH16-2 agarase is SEQ ID NO:2.
2. The method for preparing new agaro-oligosaccharides from gelidium amansii as claimed in claim 1, wherein the homogenization treatment is carried out in a mass ratio of gelidium amansii to water of 1: and 16, homogenizing for 10-20 min.
3. The method for preparing new agaro-oligosaccharides from gelidium amansii as claimed in claim 1, wherein the enzymolysis is performed by using mixed enzyme solution of agarase expressed heterologously.
4. The method for preparing new agaro-oligosaccharides from gelidium amansii as claimed in claim 1, wherein the primer sequence of the reaction of GH16-1 agarase is
GH16-1 TCGCGGATCCGAATTCGCCGATTGGGATGG
GH16-1 GTGCGGCCGCAAGCTTGTTATTGCGGTGTAAATATAAAACGG。
5. The method for preparing new agaro-oligosaccharides from gelidium amansii as claimed in claim 1, wherein the primer sequence of the reaction of GH16-2 agarase is
GH16-2 TCGCGGATCCGAATTCAAGCAAGGGCACTCGC upstream primer
GH16-2 GTGCGGCCGCAAGCTTGTCACACTTTTTGCAGCCCG downstream primer.
6. The method for preparing new agaro-oligosaccharides from gelidium amansii as claimed in claim 1, wherein the mixed enzyme solution is used for transforming gelidium amansii.
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| CN202111183330.5A CN113736837A (en) | 2021-10-11 | 2021-10-11 | Method for preparing new agaro-oligosaccharide from agar |
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| CN202111183330.5A CN113736837A (en) | 2021-10-11 | 2021-10-11 | Method for preparing new agaro-oligosaccharide from agar |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834379A (en) * | 2016-09-26 | 2017-06-13 | 中国海洋大学 | A kind of preparation method and applications of new fine jade tetrose |
| CN107794158A (en) * | 2017-11-01 | 2018-03-13 | 中国海洋大学 | A kind of preparation method of health marine oligosaccharide yellow rice wine |
| CN109182414A (en) * | 2018-08-16 | 2019-01-11 | 国家海洋局第三海洋研究所 | A method of producing new fine jade disaccharides |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834379A (en) * | 2016-09-26 | 2017-06-13 | 中国海洋大学 | A kind of preparation method and applications of new fine jade tetrose |
| CN107794158A (en) * | 2017-11-01 | 2018-03-13 | 中国海洋大学 | A kind of preparation method of health marine oligosaccharide yellow rice wine |
| CN109182414A (en) * | 2018-08-16 | 2019-01-11 | 国家海洋局第三海洋研究所 | A method of producing new fine jade disaccharides |
Non-Patent Citations (2)
| Title |
|---|
| "MULTISPECIES: RICIN domain-containing protein [Pseudoalteromonas]", NCBI, pages 016709218 * |
| "RICIN domain-containing protein [Gilvimarinus chinensis]", NCBI, pages 020208752 * |
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Application publication date: 20211203 |