CN107312807A - The enzymolysis preparation of the brown alga function oligosaccharides in one main laminaria source - Google Patents
The enzymolysis preparation of the brown alga function oligosaccharides in one main laminaria source Download PDFInfo
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Abstract
The invention discloses the enzymolysis preparation of the brown alga function oligosaccharides in main laminaria source, it is, using sea-tangle as raw material, algin to be obtained using sodium carbonate alkali carries, recycles algin catenase to digest and purified by gel chromatography, the brown alga function oligosaccharides is made.The high viscosity polysaccharide utilized that can make to be difficult in sea-tangle to be absorbed by the body using the inventive method is degraded into rich in the brown alga oligose of physiologically active and some small-molecule active substances, is advantageously implemented the higher value application of brown alga resource.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to the enzymolysis system of the brown alga function oligosaccharides in main laminaria source
Preparation Method.
Background technology
Algin content in marine alga is higher, is primarily present in the cell membrane of frond and cytoplasm, from sea-tangle, horse hair
Algin is can extract in the brown algas such as algae, bulk kelp, yellow tang.The algin of commercial Application is mainly derived from sea-tangle, bulk kelp, bubble leaf
Three category of algae, the algin of American-European countries's production is largely derived from bulk kelp, and the algin of domestic product is then more to be derived from sea-tangle.Mesh
The algin of preceding commercialization is main to be existed in the form of the alginates such as sodium alginate, because alginate has unique physics and chemistry
Matter includes the characteristic such as hydrophilic, gel, sticky, can be applied to food, medicine, printing and dyeing, chemical industry etc. multi-field.
But the algal polysaccharides naturally extracted because its stickiness is big, not readily dissolve, indigestibility, big molecular weight the shortcomings of, it is difficult to quilt
Animal body is absorbed, and limits its utilization.Brown alga oligose is the functional oligose generated after algin is degraded, is generally referred to
The degree of polymerization（DP）Sugar chain less than 20, it has multiple biological activities, especially in anti-oxidant, antibacterial, antitumor, raising immunity
In terms of play an important roll.At present, the method for algin degraded is broadly divided into three classes：Physical degradation methods, chemical degradation method and
Biological degradation method.Biological degradation method is that the otherness for acting on substrate sites using algin catenase prepares brown alga oligose, its
The unsaturated oligosaccharides for acting on glycosidic bond formation by beta-elimination reaction has more more preferable than the saturation oligosaccharides that chemical degradation method is obtained
Bioactivity.Meanwhile, biological degradation method is compared to chemical degradation method with specificity is high, enzymatic hydrolysis condition is gentle, course of reaction is easy
Control, the advantage such as products collection efficiency is high, free from environmental pollution.
The present invention researches and develops the algin catenase of biodegradable algal polysaccharide using Modern microbiological engineering technology, then leads to
Crossing enzyme engineering technology replaces traditional chemical degradation method to prepare brown alga oligose so that be difficult to be absorbed by the body the height utilized in sea-tangle
Viscosity polysaccharide is degraded into the brown alga oligose rich in physiologically active, and some small-molecule active substances, realizes brown alga resource
Higher value application.
The content of the invention
It is an object of the invention to provide the enzymolysis preparation of the brown alga function oligosaccharides in main laminaria source, it can make sea
The high viscosity polysaccharide utilized that is difficult to be absorbed by the body in band is degraded into the brown alga oligose rich in physiologically active, is advantageously implemented brown alga
The higher value application of resource.
To achieve the above object, the present invention is adopted the following technical scheme that：
The enzymolysis preparation of the brown alga function oligosaccharides in one main laminaria source, it is characterised in that：Using sea-tangle as raw material, using carbonic acid
Soda carries acquisition algin, recycles algin catenase enzymolysis, and is purified by gel chromatography, the brown alga function is made few
Sugar.It specifically includes following steps：
1）Alkali carries prepare algin：Rub, added water by solid-liquid ratio 70g/L, in 90 DEG C, concentration of sodium carbonate 3% after sea-tangle is cleaned
Alkali carries 3h under conditions of w/v, obtains algin；
2）Enzymolysis prepares brown alga oligose：Phosphate buffer is added in gained algin, is dissolved by heating, it is 0.2% to be made into concentration
W/v substrate, then adds the U/mL of algin catenase 0.35, digests 3 h under the conditions of 45 DEG C, pH 7.0, then boiling water bath
Destroy the enzyme treatment 5min, removing protein is extracted with Sevage methods repeatedly, and 12000 r/min centrifuge 5 min, abandon precipitation, supernatant（50
℃）Revolving is to the 1/20 of original volume, and absolute ethyl alcohol precipitation oligosaccharides is freeze-dried rear standby；
3）Digest isolating and purifying for oligosaccharides：Gained enzymolysis product is isolated and purified using Bio-Gel P-6 gel columns, with
0.2 mol/L ammonium hydrogen carbonate is eluent, and flow velocity is 0.4 mL/min, and each component is collected respectively and is concentrated, chilled dry
Dry product must be purified after dry.
The advantage of the invention is that：The present invention is obtained after algin, using brown using sea-tangle as raw material using sodium carbonate alkali carries
Phycocolloid lyases is degraded, and uses Bio-Gel P-6 gel column separating purifications, and the brown alga oligose rich in physiologically active is made, has
Beneficial to the higher value application for realizing sea-tangle resource；Meanwhile, the algin catenase that the present invention is used voluntarily is developed for this laboratory,
Its activity is high, helps to reduce the production cost of brown alga oligose.
Brief description of the drawings
Fig. 1 is influence of the change of alkali carries time to brown alga glue yield.
Fig. 2 is influence of the change of alkali carries temperature to brown alga glue yield.
Fig. 3 is influence of the change of solid-liquid ratio to brown alga glue yield.
Fig. 4 is influence of the change of alkali concn to brown alga glue yield.
Fig. 5 is influence of the change of enzyme concentration to algin degree of hydrolysis.
Fig. 6 is influence of the change of enzymolysis time to algin degree of hydrolysis.
Fig. 7 is influence of the change of hydrolysis temperature to algin degree of hydrolysis.
Fig. 8 is influence of the pH change to algin degree of hydrolysis.
Fig. 9 is influence of the change of concentration of substrate to algin degree of hydrolysis.
Figure 10 is the response surface design figure and contour map that temperature and pH influence on algin degree of hydrolysis.
Figure 11 is the response surface design figure and contour map that temperature and enzyme concentration influence on algin degree of hydrolysis.
Figure 12 is the response surface design figure and contour map that pH and enzyme concentration influence on algin degree of hydrolysis.
Figure 13 is comparison diagram of the different brown alga products to Hydroxyl radical-scavenging ability.
Figure 14 is comparison diagram of the different brown alga products to ABTS radical scavenging activities.
Figure 15 is comparison diagram of the different brown alga products to DPPH radical scavenging activities.
Figure 16 is comparison diagram of the different brown alga products to ultra-oxygen anion free radical Scavenging activity.
Figure 17 is inhibitory action situation map of the various concentrations brown alga enzymolysis product to tyrosinase bis-phenol enzyme activity.
Figure 18 is the thin-layer chromatogram of brown alga enzymolysis product, wherein, M is brown alga oligose mark product；1 is enzymolysis product.
Figure 19 is the situation map that enzymolysis product uses gel column separating purification.
Figure 20 is the thin-layer chromatogram for respectively isolating and purifying product, wherein, M is brown alga oligose mark product；1 is component 1；2 be group
Divide 2；3 be component 3.
Embodiment
In order that content of the present invention easily facilitates understanding, with reference to embodiment to of the present invention
Technical scheme is described further, but the present invention is not limited only to this.
Algin catenase used presses document（Han Wei, Lin Juan, Xie Yong, wait the " clonal expressions of alginate lyase gene
And recombinase zymologic property ",《Microbiology is circulated a notice of》, 2017,44（5）：1074-1080）Prepared.
Sea-tangle used is provided by Fujian hundred million up to Food Co., Ltd；Bio-Gel P-6 gels used, ABTS, DPPH, junket ammonia
Sour enzyme is purchased from Sigma companies；Thin layer used is purchased from Merck companies of Germany with silica white；Brown alga oligose mark product used are purchased from green grass or young crops
Dao Bozhihui power company；Sodium carboxymethylcellulose (CMC) used is purchased from Aladdin company；Absolute ethyl alcohol used, phenol, sulfuric acid,
The conventional medications such as hydrochloric acid are domestic AR.
The alkali carries sea-tangle of embodiment 1 prepares the process optimization of algin
The computational methods of brown alga glue yield are：Brown alga glue yield（%）=algin dry weight/sea-tangle dry weight × 100%.
1. in liquid ratio 50:1, temperature 70 C under conditions of concentration of sodium carbonate 1%, investigates the change of alkali carries time to brown
The influence of phycocolloid yield, is as a result shown in Fig. 1.As seen from Figure 1, the most suitable alkali carries time is 3 h.
2. in liquid ratio 50:Under conditions of 1, the h of extraction time 3, concentration of sodium carbonate 1%, the change of temperature is investigated to brown alga
The influence of glue yield, is as a result shown in Fig. 2.From Figure 2 it can be seen that most suitable alkali carries temperature is 80 DEG C.
3. in 80 DEG C of alkali carries temperature, the h of extraction time 3 under conditions of concentration of sodium carbonate 1%, investigates the change pair of solid-liquid ratio
The influence of brown alga glue yield, is as a result shown in Fig. 3.As seen from Figure 3, most suitable liquid ratio is 60:1.
4. in 80 DEG C of alkali carries temperature, the h of extraction time 3, liquid ratio 60:Under conditions of 1, the change pair of concentration of lye is investigated
The influence of brown alga glue yield, is as a result shown in Fig. 4.From fig. 4, it can be seen that most suitable alkali concn is 2%.
5. on the basis of experiment of single factor, using orthogonal test, by L9（34）Orthogonal arrage in alkali carries algin when
Between（2 h, 3 h, 4 h）, temperature（70 DEG C, 80 DEG C, 90 DEG C）, alkali concn（1%, 2%, 3%）, liquid ratio（50:1,60:1,70:1）
Orthogonal Optimization Test is carried out, each factor level table as shown in table 1, as shown in table 2, divide for Orthogonal Experiment and Design and experimental result by variance
Analysis the results are shown in Table 3.
The factor level table of table 1
The Orthogonal Experiment and Design of table 2 and experimental result
The analysis of variance table of table 3
Knowable to variance analysis, only factor A（Time）There is conspicuousness influence, the primary and secondary order of factor effect on brown alga glue yield
For A（Time）>D（Liquid ratio）>B（Temperature）>C（Alkali concn）, by table 2 understand A, B, C, D optimal level be respectively
A2B3C3D1, i.e., the optimum process condition of alkali carries algin is from sea-tangle：Time is 3 h, 90 DEG C of temperature, alkali concn 3%, liquid
Material compares 70:1, now the highest yield of algin is 62%.
The enzymolysis optimum preparation condition of the brown alga oligose of embodiment 2
The computational methods of algin degree of hydrolysis：
,
In formula：n：Phend-sulphuric acid measures sugared content, g；
m：Phend-sulphuric acid measures total sugar content, g.
1. in the h of enzymolysis time 2, temperature 50 C, concentration of substrate 0.5% under conditions of pH value 7.0, investigates the change of enzyme concentration
Change the influence to algin degree of hydrolysis, as a result see Fig. 5.As seen from Figure 5, when enzyme concentration is 0.3 U/mL, degree of hydrolysis is maximum.
2. in 50 DEG C of hydrolysis temperature, concentration of substrate 0.5%, pH value 7.0 under conditions of the U/mL of enzyme concentration 0.3, investigates enzyme
Influence of the change of solution time to algin degree of hydrolysis, is as a result shown in Fig. 6.As seen from Figure 6, the degree of hydrolysis when enzymolysis time is 3 h
It is maximum.
3. in the h of enzymolysis time 3, concentration of substrate 0.5%, pH7.0 under conditions of the U/mL of enzyme concentration 0.3, investigates enzymolysis temperature
Influence of the change of degree to algin degree of hydrolysis, is as a result shown in Fig. 7.As seen from Figure 7, when hydrolysis temperature be 45 DEG C when degree of hydrolysis most
Greatly.
4. in the h of enzymolysis time 3, temperature 45 C, concentration of substrate 0.5% under conditions of the U/mL of enzyme concentration 0.3, investigates pH
Change the influence to algin degree of hydrolysis, as a result see Fig. 8.As seen from Figure 8, when pH is 7.5, degree of hydrolysis is maximum.
5. in the h of enzymolysis time 3, temperature 45 C, pH7.5 under conditions of the U/mL of enzyme concentration 0.3, investigates concentration of substrate
Change the influence to algin degree of hydrolysis, as a result see Fig. 9.As seen from Figure 9, when concentration of substrate is 0.2%, degree of hydrolysis is maximum.
6. on the basis of experiment of single factor, experimental design is carried out using Box-Behnken center combination designs, it is determined that
Optimal enzymolysis process.Factor level coding schedule as shown in table 4, response surface experimental program and the results are shown in Table 5.
The response surface design experimental design factor level coding schedule of table 4
The response surface experimental program of table 5 and result
The foundation and analysis of second-order model
Regression analysis is carried out to the experimental result of table 5 using Design-Expert 8.0.5b softwares, regression equation is obtained：Y=
67.86+1.42·X1-5.99·X2-15.04·X3+4.54·X1·X2-6.56·X2X3-9.98·X1 2-12.62·X2 2。
The analysis of variance table of table 6
Variance analysis is carried out to model, 6 are the results are shown in Table.From table 6, F=74.00, P of model<0.0001<0.01, show mould
Type effect is notable；Lose and intend P=0.3251>0.05, show that equation is preferable to the fitting degree of experiment.X2、X3、X1X2、X2X3、X1 2、
X2 2Influence to algin degree of hydrolysis is extremely notable, shows hydrolysis temperature（X2）、pH（X3）, enzyme concentration and hydrolysis temperature interaction work
With（X1X2）, hydrolysis temperature and pH reciprocation（X2X3）, enzyme concentration square（X1 2）, hydrolysis temperature square（X2 2）Institute
Take and algin degree of hydrolysis is had a significant impact in the range of level.Enzyme concentration（X1）Influence to regression equation is not notable.By items
F values understand, each factor is followed successively by the influence degree of algin degree of hydrolysis：X3（pH）>X2（Hydrolysis temperature）>X1（Enzyme concentration）.
According to analysis result, obtaining final optimization pass condition is：Optimal enzymolysis process condition be pH7.0,45.15 DEG C of temperature,
The U/mL of enzyme concentration 0.35, the h of enzymolysis time 3, concentration of substrate 0.2%, degree of hydrolysis predicted value are 80.35%, degree of hydrolysis measuring
It is worth for 71.22%, illustrates that the model can preferably reflect actual enzymolysis process.
The bioactivity research of the enzymolysis product of embodiment 3
The preparation of brown alga oligose acid hydrolysate：The mL of 1.5% algin substrate 100 is prepared, with salt acid for adjusting pH to 4.0,120 DEG C of bakings
4 h are reacted in case；PH to 7.0 is adjusted after reaction, precipitation, 50 DEG C of revolvings of supernatant to the 1/20 of original volume are abandoned in centrifugation；Absolute ethyl alcohol
Precipitate and acidolysis brown alga oligose is obtained after oligosaccharides, sample drying, it is standby.
1. Hydroxyl radical-scavenging capacity experimental
As seen from Figure 13, under same concentrations, Scavenging activity of the brown alga enzymolysis product to hydroxy radical（IC50For 0.182 mg/
mL）Apparently higher than acid hydrolysate（IC50For 0.448 mg/mL）With brown alga glue polysaccharide（IC50For 0.554 mg/mL）.
2. ABTS radical scavenging activities are tested
As seen from Figure 14, under same concentrations, brown alga enzymolysis product removes the ability of ABTS free radicals（IC50For 0.0798 mg/
mL）Apparently higher than acid hydrolysate（IC50For 0.125 mg/mL）, and brown alga glue polysaccharide does not remove energy substantially to ABTS free radicals
Power.
3. DPPH radical scavenging activities are tested
As seen from Figure 15, under same concentrations, brown alga enzymolysis product removes the ability of DPPH free radicals（IC50For 0.11 mg/
mL）Apparently higher than acid hydrolysate（IC50More than 0.4 mg/mL）, and brown alga glue polysaccharide does not remove energy substantially to DPPH free radicals
Power.
4. ultra-oxygen anion free radical Scavenging activity is tested
As seen from Figure 16, under same concentrations, brown alga enzymolysis product has the ability for removing ultra-oxygen anion free radical（IC50For
0.26 mg/mL）, and brown alga glue polysaccharide and acid hydrolysate be not obvious to the elimination effect of ultra-oxygen anion free radical.
5. the inhibitory action of pair tyrosinase bis-phenol enzyme activity
As seen from Figure 17, brown alga enzymolysis product is inhibited to tyrosinase bis-phenol enzymatic L-3,4 dihydroxyphenylalanine, its IC50For 1.8
Mg/mL, it is close with acid hydrolysate（IC50For 1.62 mg/mL）, and brown alga glue polysaccharide does not have obvious inhibitory action.
The constituent analysis of the enzymolysis product of embodiment 4
Obtained product progress thin-layer chromatographic analysis will be digested, solvent used is VN-butanol/VFormic acid/VWater=4：6：1.Can by Figure 18
See, brown alga oligose of the gained enzymolysis product mainly containing 3 kinds of degree of polymerization, respectively DP1, DP2, DP3, wherein DP2 and DP3 are main
Product, DP1 is presence on a small quantity.
Embodiment 5 enzymolysis product is isolated and purified
Enzymolysis product is separated with Bio-Gel P-6 gel columns, using 0.2 mol/L ammonium hydrogen carbonate as eluent, flow velocity is
0.4 mL/min, every 2.5 min collect one and managed, and light absorption value is detected under 235 nm.Shown by Figure 19 results, each component separating effect
Preferably, 3 main components, component 1 are obtained（Peak scope is 430-467.5 min）, component 2（Peak scope is 392.5-420
min）, component 3（Peak scope is 362.5-377.5 min）.Thin-layer chromatographic analysis are carried out after collecting each component concentration, as a result as schemed
20。
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
Claims (4)
1. the enzymolysis preparation of the brown alga function oligosaccharides in main laminaria source, it is characterised in that：Using sea-tangle as raw material, using carbon
Sour soda carries acquisition algin, recycles algin catenase enzymolysis, and is purified by gel chromatography, and the brown alga function is made
Oligosaccharides.
2. according to claim 1 sea-tangle source brown alga function oligosaccharides enzymolysis preparation, it is characterised in that：It is described
The condition of alkali carries is：90 DEG C of temperature, the h of time 3, solid-liquid ratio 70g/L, the w/v of concentration of sodium carbonate 3%.
3. according to claim 1 sea-tangle source brown alga function oligosaccharides enzymolysis preparation, it is characterised in that：It is described
The condition of enzymolysis is：PH7.0, temperature 45 C, the U/mL of enzyme concentration 0.35, the h of enzymolysis time 3, the w/v of concentration of substrate 0.2%.
4. according to claim 1 sea-tangle source brown alga function oligosaccharides enzymolysis preparation, it is characterised in that：It is described
Purifying uses Bio-Gel P-6 gel columns, using 0.2 mol/L ammonium hydrogen carbonate as eluent, and flow velocity is 0.4 mL/min.
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Cited By (7)
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| CN108690857A (en) * | 2018-05-22 | 2018-10-23 | 齐鲁工业大学 | A kind of preparation method of high-purity brown alga oligose |
| CN108753642A (en) * | 2018-05-10 | 2018-11-06 | 江南大学 | One plant of production algin catenase bacterial strain Yue Shi Flavobacterium |
| CN109156615A (en) * | 2018-09-18 | 2019-01-08 | 青岛农业大学 | A method of animal semen quality is improved using brown alga oligose |
| CN109221811A (en) * | 2018-09-28 | 2019-01-18 | 福州大学 | A kind of preparation method of feeding additive aquatic animal brown alga oligose |
| CN110714046A (en) * | 2019-11-22 | 2020-01-21 | 浙江丰安生物制药有限公司 | Preparation method of polypeptide for hydrolyzing pork protein of suckling pig |
| CN112501225A (en) * | 2019-09-16 | 2021-03-16 | 广东岩志生物科技有限公司 | Brown algae oligosaccharide extraction method |
| CN114532500A (en) * | 2022-02-18 | 2022-05-27 | 福建农林大学 | Preparation method of active kelp matter |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753642A (en) * | 2018-05-10 | 2018-11-06 | 江南大学 | One plant of production algin catenase bacterial strain Yue Shi Flavobacterium |
| CN108753642B (en) * | 2018-05-10 | 2020-02-11 | 江南大学 | Flavobacterium johnsonii strain for producing alginate lyase |
| CN108690857A (en) * | 2018-05-22 | 2018-10-23 | 齐鲁工业大学 | A kind of preparation method of high-purity brown alga oligose |
| CN109156615A (en) * | 2018-09-18 | 2019-01-08 | 青岛农业大学 | A method of animal semen quality is improved using brown alga oligose |
| CN109156615B (en) * | 2018-09-18 | 2021-12-28 | 青岛农业大学 | Method for improving animal semen quality by using brown algae oligosaccharide |
| CN109221811A (en) * | 2018-09-28 | 2019-01-18 | 福州大学 | A kind of preparation method of feeding additive aquatic animal brown alga oligose |
| CN112501225A (en) * | 2019-09-16 | 2021-03-16 | 广东岩志生物科技有限公司 | Brown algae oligosaccharide extraction method |
| CN110714046A (en) * | 2019-11-22 | 2020-01-21 | 浙江丰安生物制药有限公司 | Preparation method of polypeptide for hydrolyzing pork protein of suckling pig |
| CN110714046B (en) * | 2019-11-22 | 2021-06-04 | 浙江丰安生物制药有限公司 | Preparation method of polypeptide for hydrolyzing pork protein of suckling pig |
| CN114532500A (en) * | 2022-02-18 | 2022-05-27 | 福建农林大学 | Preparation method of active kelp matter |
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Application publication date: 20171103 |