CN110551774A - Method for preparing active beta-1, 3-xylo-oligosaccharide from sea grape by enzyme method - Google Patents
Method for preparing active beta-1, 3-xylo-oligosaccharide from sea grape by enzyme method Download PDFInfo
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Abstract
The invention discloses a method for preparing active beta-1, 3-xylo-oligosaccharide from sea grapes by an enzyme method, which comprises the following steps: (1) extracting beta-1, 3-xylan from the vitis amurensis; (2) obtaining recombinant beta-1, 3-xylanase; (3) hydrolyzing the material obtained in the step (1) by the recombinant beta-1, 3-xylanase to obtain the active beta-1, 3-xylo-oligosaccharide. In the prior art, the xylan is beta-1, 4-xylan and beta-1, 4-xylooligosaccharide (xylooligosaccharide), while the beta-1, 3-xylooligosaccharide is prepared from beta-1, 3-xylan derived from Vitis amurensis through an enzyme method, so that the domestic blank is filled, and meanwhile, through tests, the prepared beta-1, 3-xylooligosaccharide has better activity compared with the beta-1, 4-xylooligosaccharide.
Description
Technical Field
the invention belongs to the technical field of xylooligosaccharide preparation, and particularly relates to a method for preparing active beta-1, 3-xylooligosaccharide from sea grapes by an enzyme method.
Background
the plant algae in nature has a main component, hemicellulose which is the second most abundant polysaccharide, is second only to cellulose, accounts for about 20-30%, and the main component is 1, 4-xylan. The xylo-oligosaccharide or xylose can be obtained by hydrolyzing xylan by two methods, namely acid hydrolysis and enzyme hydrolysis, and the utilization rate and the economic value of xylan can be improved. The acid hydrolysis needs to be carried out at high temperature and high pressure, the product recovery rate and the instrument cost are high, and meanwhile, the method can generate byproducts in the processing process, so that the hydrolysis conversion rate of xylan is reduced. The enzymolysis method has the characteristics of high specificity, mild processing conditions and easy product recovery. In addition to being used for the production of 1, 4-xylo-oligosaccharides, the process is also widely used in the food and cosmetic industry, pharmaceutical biotechnology, agriculture, environmental protection and sewage treatment. It should be noted that the term "xylan or xylooligosaccharide" as used herein refers to 1, 4-xylan or 1, 4-xylooligosaccharide. Some seaweeds contain 1, 3-xylan with special 1, 3-bond connection, and the structure and activity of the existing 1, 4-xylan are obviously different from those of the 1, 4-xylan. The 1, 3-oligosaccharide obtained therefrom has many biological activities, and the activities thereof are influenced by Molecular Weight (MW) and chemical bond, and have certain effects in medical fields such as anticancer, antiviral and antioxidant. Algal polysaccharide is a biological component which is easy to extract, and has great difference in molecular structure. In recent years, algal polysaccharide-degraded oligosaccharides have been applied to the treatment of chronic diseases, and some reports have revealed anticancer, antiviral and anti-inflammatory activities of β -1, 3-xylan, such as the induction of apoptosis in MCF-7 human breast cancer cells.
beta-1, 3-xylan, which is composed of beta-1, 3-linkages to D-xylose, is a polysaccharide component unique to the cell wall of certain seaweeds, and is mainly present in macroalgae such as Caulerpa, Bryopans, Bangia, Porphyra, and Palmaria spp. The beta-1, 3-xylan molecules are abundantly present in the cell walls of the algal cells and mainly exist in the form of right-handed triple helical structures combined into hexagonal crystal microfibrils, and the microfibrils are randomly distributed around the algal cells and have important protection effect. Most algae, which have β -1, 3-xylan as a major component of their cell wall structure, are closely related to human activities: according to the existing research, the siphonaptera green algae contains beta-1, 3-xylan, two kinds of fern algae are common, one is the usable long neck fern sea grape which can be artificially cultured at present, and the other is the highly invasive species codium cuneatum, so the long neck fern sea grape is usually selected as the experimental raw material for extracting the beta-1, 3-xylan.
however, the prior art generally prepares the xylo-oligosaccharide (xylo-oligosaccharide) by beta-1, 4-xylan, for example, the method for preparing the xylo-oligosaccharide disclosed in CN108359696A generates 2-10 xylose molecules by bagasse fermentation liquor and is formed by connecting the (beta-1-4) glycosidic bonds; for another example, CN104762343A discloses a method for preparing xylo-oligosaccharide by an enzymatic method, which is to express endoglucanase EG I gene in Pichia Pastoris (Pichia Pastoris), and the obtained endoglucanase EG I is used for degrading xylan to prepare xylo-oligosaccharide, and the xylo-oligosaccharide is also a low-polymerization degree saccharide formed by connecting 2-10 xylose molecules by β -1, 4-glycosidic bonds, and the effective components of the xylo-oligosaccharide are β -1, 4-xylobiose, β -1, 4-xylotriose, β -1, 4-xylotetraose, β -1, 4-xylopentaose, and the like. However, the preparation and research of the prior art on beta-1, 3-xylan are not comprehensive enough: no preparation method of 1, 3-xylo-oligosaccharide exists at home, 1, 3-xylo-oligosaccharide cannot be bought from the market, and the preparation method aiming at 1, 3-xylo-oligosaccharide at foreign countries is not only complex in method and poor in effect, but also does not sell commercialized 1, 3-xylo-oligosaccharide.
disclosure of Invention
the invention aims to overcome the defects of the prior art and provides a method for preparing active beta-1, 3-xylo-oligosaccharide from Vitis heyneana by an enzyme method.
the technical scheme of the invention is as follows:
A method for preparing active beta-1, 3-xylo-oligosaccharide from sea grapes by an enzymatic method comprises the following steps:
(1) Extracting beta-1, 3-xylan from the vitis amurensis;
(2) Obtaining recombinant beta-1, 3-xylanase;
(3) hydrolyzing the material obtained in the step (1) by using the recombinant beta-1, 3-xylanase to obtain the active beta-1, 3-xylooligosaccharide, which specifically comprises the following steps:
a. adding the material obtained in the step (1) into a Tris-HCl buffer solution with the pH value of 6.9-7.2 to prepare a beta-1, 3-xylan solution with the concentration of 0.8-1.2 wt%;
b. mixing the beta-1, 3-xylan solution with a proper amount of the recombinant beta-1, 3-xylanase, reacting at 44-46 ℃ for 20-30h, and heating at 95-100 ℃ for 4-6min to inactivate the recombinant beta-1, 3-xylanase;
c. And c, centrifuging the material obtained in the step b at the speed of 11000-13000rpm for 4-6min, and obtaining supernatant which is the active beta-1, 3-xylo-oligosaccharide.
In a preferred embodiment of the present invention, the step (1) comprises:
a. Mixing the sea grape powder with a proper amount of NaOH solution, heating while stirring, and fully boiling for 25-35 min;
b. Centrifuging the material obtained in the step a at 3500-;
c. Adding a proper amount of H into the material obtained in the step b2SO4Centrifuging the solution at 3500-4500rpm for 15-25min to obtain a second precipitate;
d. Adding a proper amount of NaOH solution into the second precipitate, stirring and extracting sugar for 1.5-2h under the ice bath condition, and then centrifuging the obtained material to obtain a supernatant;
e. mixing the supernatant with anhydrous ethanol at a volume ratio of 1: 3-5, standing at 3-5 deg.C for 10-12 hr, and performing solid-liquid separation to obtain a third precipitate;
f. Adding a proper amount of high-sodium chlorate solution into the third precipitate, decoloring for 1.5-2.5h while stirring, and fully washing with distilled water to obtain a fourth precipitate;
g. centrifuging the fourth precipitate at 9500-12000rpm for 12-20min to obtain a fifth precipitate;
h. And fully washing the fifth precipitate with acetic acid solution, and fully washing with distilled water to obtain the beta-1, 3-xylan.
the sea grape powder in the step a of the step (1) is pretreated sea grape powder, and the pretreatment process comprises the following steps:
a1, mixing the sea grape powder with purified water, extracting for 5-20 min under the subcritical water state, cooling to room temperature, centrifuging, collecting supernatant, and freeze-drying.
Still further preferably, the preprocessing further comprises:
2, sequentially adding 13-14 parts by weight of distilled water, 0.1-0.2 part by weight of hydrochloric acid, 4-5 parts by weight of glucose, 9-12 parts by weight of absolute ethyl alcohol and 50-60 parts by weight of ethyl orthosilicate into a reaction vessel at room temperature, rapidly stirring for 4-6min, sequentially carrying out transparent-turbid-transparent change on liquid, cooling with room-temperature water, and fully removing ethanol to form prehydrolysis gel;
3, adding 16-17 parts by weight of the freeze-dried material into the pre-hydrolyzed gel, adjusting the pH to 5.4-5.6 by using alkali, stirring at a low speed for 25-35min, and standing at 3-5 ℃ for 45-50h to obtain wet gel;
a4, grinding and crushing the wet glue, fully washing the ground wet glue with distilled water to remove glucose in the wet glue, filtering, drying, grinding, crushing and sieving the washed wet glue to obtain the finished product.
in a preferred embodiment of the present invention, the step (1) is: extracting beta-1, 3-xylan from Vitis amurensis, preparing beta-1, 3-xylan into beta-1, 3-xylan, and mixing with the rest beta-1, 3-xylan.
Further preferably, the beta-1, 3-xylan as the hydroxyl alcohol accounts for 5-10 wt% of the material obtained in step (1).
Still more preferably, the method for preparing the beta-1, 3-xylan comprises:
I. Dissolving beta-1, 3-xylan in NaOH solution, adding a proper amount of dichloroethanol, stirring in an ice bath for 0.8-1.5h, and then stirring at room temperature for 20-25 h;
II. Adding glacial acetic acid into the material obtained in the step I, neutralizing in an ice bath, and then dialyzing;
and III, heating, stirring and concentrating the material obtained in the step III, and freeze-drying to obtain the hydroxyl alcohol beta-1, 3-xylan.
The invention has the beneficial effects that:
1. In the prior art, the xylan is beta-1, 4-xylan and beta-1, 4-xylooligosaccharide (xylooligosaccharide), while the beta-1, 3-xylooligosaccharide is prepared from beta-1, 3-xylan derived from Vitis amurensis through an enzyme method, so that the domestic blank is filled, and meanwhile, through tests, the prepared beta-1, 3-xylooligosaccharide has better activity compared with the beta-1, 4-xylooligosaccharide.
2. the invention adopts the sea grape to generate the beta-1, 3-xylan, and adopts the enzyme reaction substrate obtained by adding the dihydric alcohol group to the beta-1, 3-xylan to increase the water solubility thereof, so that the subsequent preparation of the beta-1, 3 xylo-oligosaccharide has higher yield.
3. The invention improves the original preparation process of the beta-1, 3 xylan, and the beta-1, 3 xylan is sequentially treated by the alkaline-acidic solution, so that the prepared beta-1, 3 xylan is purer and has higher yield.
4. The invention carries out the steps of subcritical water pretreatment and dispersion treatment on the sea grape powder, not only achieves the aim of simplifying the process flow, but also further improves the yield of the beta-1, 3-xylan.
5. According to the invention, the sea grape powder is selected and subjected to subcritical water pretreatment and dispersion treatment, the reaction speed of an insoluble substrate can be greatly improved by the treated sea grape powder, the purpose of simplifying the process flow can be achieved, the specific beta-1, 3-xylanase is adopted as a catalyst, and the beta-1, 3 xylan is subjected to enzymolysis to prepare the active beta-1, 3 xylooligosaccharide, so that the optimal content ratio of xylobiose and xylotriose which play main roles in the prepared active beta-1, 3 xylooligosaccharide can be realized, and the antioxidant activity is greatly improved.
drawings
Fig. 1 is a schematic diagram of results of HPLC qualitative and quantitative analysis of active β -1, 3-xylooligosaccharide, β -1, 4-xylooligosaccharide and standard xylobiose standard solution in example 1 of the present invention, wherein fig. 1a is a chromatogram of peak time of β -1, 3-xylan standard sample, and fig. 1b is a chromatogram of peak time of β -1, 4-xylan standard sample.
FIG. 2 is a graph showing the effect of active β -1, 3-xylooligosaccharide, β -1, 4 xylooligosaccharide and Vc on DPPH.RTM.in example 1 of the present invention.
FIG. 3 is a graph showing the effect of active β -1, 3-xylooligosaccharide, β -1, 4 xylooligosaccharide and Vc on DPPH.RTM.in example 1 of the present invention.
FIG. 4 is a graph showing the effect of active β -1, 3-xylooligosaccharide, β -1, 4-xylooligosaccharide and Vc on OH.in example 1 of the present invention.
FIG. 5 is a graph showing the effect of active β -1, 3-xylooligosaccharide, β -1, 4-xylooligosaccharide and Vc on OH.in example 1 of the present invention.
FIG. 6 is a graph showing the effect of active β -1, 3-xylooligosaccharide, β -1, 4-xylooligosaccharide and Vc on scavenging superoxide anions in example 1 of the present invention.
FIG. 7 is a graph comparing the reducing power of active beta-1, 3-xylo-oligosaccharides, beta-1, 4-xylan, and Vc in example 1 of the present invention.
Detailed Description
the technical solution of the present invention is further illustrated and described by the following detailed description.
example 1
A method for preparing active beta-1, 3-xylo-oligosaccharide from sea grapes by an enzymatic method comprises the following steps:
(1) extracting beta-1, 3-xylan from the sea grapes, preparing a proper amount of beta-1, 3-xylan into hydroxyl alcohol beta-1, 3-xylan, and mixing the hydroxyl alcohol beta-1, 3-xylan with the rest beta-1, 3-xylan, wherein the hydroxyl alcohol beta-1, 3-xylan accounts for 10 wt% of the material obtained in the step (1);
The extraction process of the beta-1, 3-xylan comprises the following steps:
a. mixing 5g of pretreated sea grape powder with 250mL of 0.3M NaOH solution, heating while stirring, and fully boiling for 30 min;
b. centrifuging the material obtained in the step a at 4000rpm for 20min, and fully washing the obtained first precipitate twice with distilled water;
c. adding into the material obtained in step bInto 250mL of 0.25M H2SO4Centrifuging the solution at 4000rpm for 20min to obtain a second precipitate;
d. Adding 200mL of 2.5M NaOH solution into the second precipitate, extracting sugar for 1.5h under the ice bath condition by magnetic stirring, and centrifuging the obtained material to obtain a supernatant;
e. mixing the supernatant with anhydrous ethanol at a volume ratio of 1: 4, standing at 4 deg.C for 10-12 hr, and performing solid-liquid separation to obtain a third precipitate;
f. Adding 1% sodium perchlorate solution into the third precipitate, decoloring for 2 hours while magnetically stirring, and fully washing for three times by using distilled water to obtain a fourth precipitate;
g. Centrifuging the fourth precipitate at 10000rpm for 15min to obtain a fifth precipitate;
h. fully washing the fifth precipitate with 5.7M acetic acid solution, and fully washing twice with distilled water to obtain beta-1, 3-xylan;
the pretreatment in the step a comprises the following steps:
a1, mixing the ampelopsis grossedentata powder with purified water, extracting for 5-20 min under the subcritical water state, cooling to room temperature, centrifuging, collecting supernate, and then freeze-drying;
2, sequentially adding 13.75 wt% of distilled water, 0.15 wt% of hydrochloric acid, 4.1 wt% of glucose, 10 wt% of absolute ethyl alcohol and 55 wt% of ethyl orthosilicate into a reaction vessel at room temperature, rapidly stirring for 5min, sequentially carrying out transparent-turbid-transparent state change on liquid, cooling with room-temperature water, and fully removing ethanol to form prehydrolysis gel;
3, adding 17 wt% of the freeze-dried material into the pre-hydrolyzed gel, adjusting the pH to 5.5 by using alkali, stirring at a low speed for 30min, and standing for 48h at the temperature of 3-5 ℃ to obtain wet gel;
a4, grinding and crushing the wet glue, fully washing the wet glue for 2 times by using distilled water to remove glucose in the wet glue, and filtering, drying, grinding, crushing and sieving the wet glue to obtain the product;
the preparation method of the hydroxyl alcohol beta-1, 3-xylan comprises the following steps:
I. Dissolving 1g of beta-1, 3-xylan in 50mL of 14 wt% NaOH solution, adding 6mL of dichloroethanol, stirring for 1h in ice bath, and stirring for 24h at room temperature;
II. Adding glacial acetic acid into the material obtained in the step I, neutralizing in an ice bath, and then dialyzing (placing into a basin containing distilled water for about 20h, and at least replacing the distilled water for three times);
III, heating, stirring and concentrating the material obtained in the step III, and then freeze-drying to obtain the hydroxyl alcohol beta-1, 3-xylan;
(2) Preparing recombinant beta-1, 3-xylanase with gene sequence shown as SEQ ID NO.01 from recombinant Escherichia coli E.coli BL21(DE3) containing recombinant plasmid: collecting a bacterial liquid after IPTG induced expression, centrifuging, discarding a supernatant, washing the thalli for 2 times by using a buffer solution, crushing cells by using ultrasonic waves, centrifuging, discarding a precipitate, and purifying the supernatant by using a nickel column to obtain purified recombinant beta-1, 3-xylanase; the recombinant plasmid is pET-22b containing a nucleotide sequence shown in SEQ ID NO.01, and the nucleotide sequence is connected into pET-22b through Nde I and HindIII restriction enzyme sites;
(3) Hydrolyzing the material obtained in the step (1) by using the recombinant beta-1, 3-xylanase to obtain the active beta-1, 3-xylooligosaccharide, which specifically comprises the following steps:
a. Adding 0.1g of the material obtained in the step (1) into 10mL of Tris-HCl buffer solution with the pH value of 7.0 to prepare a beta-1, 3-xylan solution with the concentration of 1 wt%;
b. Mixing 350 μ L of the beta-1, 3-xylan solution with 50 μ L of the recombinant beta-1, 3-xylanase, reacting in a water bath at 45 ℃ for 24h, and heating in a water bath at 100 ℃ for 5min to inactivate the recombinant beta-1, 3-xylanase;
c. and c, centrifuging the material obtained in the step b at 12000rpm for 5min, and obtaining supernate, namely the active beta-1, 3-xylo-oligosaccharide.
(4) Taking 3 centrifuge tubes with the volume of 2mL, adding 10 mu L of the active beta-1, 3-xylo-oligosaccharide solution into the tubes, supplementing 390 mu L of distilled water to make the total volume of the solution be 400 mu L, adding 400 mu L of DNS reagent into the tubes, placing the tubes in a boiling water bath for 5min, cooling, adding 1.6mL of distilled water into a quartz cuvette by using a liquid transfer gun, and setting the absorbance at the wavelength of 540nm by using a spectrophotometer.
In the enzymolysis process, the types of the xylan substrate and xylanase are factors which influence the structural characteristics of the xylobiose and xylotriose components in the xylo-oligosaccharide mixed liquor to be not negligible, and therefore, the beta-1, 3-xylan from the Vitis heyneana is particularly selected. Meanwhile, the invention prepares the active beta-1, 3-xylo-oligosaccharide by hydrolyzing xylan by utilizing the 1, 3-xylanase obtained by self design, thereby not only optimizing the preparation process, but also enabling the content of active substances, namely xylobiose and xylotriose to be the optimal ratio, and greatly improving the antioxidant activity of the active substances.
meanwhile, the invention also carries out the determination of the antioxidant activity of the active beta-1, 3-xylo-oligosaccharide obtained by the steps and compares the activity of the 1, 3-xylo-oligosaccharide with that of the 1, 4-xylo-oligosaccharide to visually compare the improvement effects of the invention:
FIG. 1 is an HPLC chart, which is a schematic diagram of the results of HPLC qualitative and quantitative analysis of active beta-1, 3-xylooligosaccharide, beta-1, 4 xylooligosaccharide and standard xylobiose standard solution. And comparing and analyzing the HPLC (high performance liquid chromatography) spectrum of the xylobiose with the HPLC spectrums of the active beta-1, 3-xylooligosaccharide and the beta-1, 4 xylooligosaccharide standard sample to determine the peak-off time of xylobiose and xylotriose in the active beta-1, 3-xylooligosaccharide and the beta-1, 4 xylooligosaccharide so as to judge the relative content of each component in the xylooligosaccharide. As can be seen from FIG. 1a, the peak-off time of xylobiose is about 8min, as can be seen from FIG. 1b, the peak of active beta-1, 3-xylooligosaccharide and beta-1, 4-xylooligosaccharide respectively appears at the peak-off time of xylobiose and xylotriose, the corresponding oligosaccharide concentration can be judged according to the peak area, and the analysis result shows that the concentrations of xylobiose and xylotriose in the beta-1, 3-xylooligosaccharide are higher than those in the beta-1, 4-xylooligosaccharide. Wherein the difference in the concentration of xylobiose is large; and the content of xylobiose in the active beta-1, 3-xylooligosaccharide and the beta-1, 4 xylooligosaccharide is higher.
secondly, determining the DPPH free radical scavenging capacity of the oligosaccharide by a DPPH method:
DPPH free radical is an artificially synthesized free radical with ammonia as a core, has stable properties in organic solvents, and is currently widely applied to the determination of antioxidant activity of animal and plant extracts.
The principle of determining the antioxidant scavenging capacity by the DPPH method is as follows: the lone pair electrons of DPPH & have strong absorption (deep purple color) at 517nm, and when a strong scavenging agent exists, the lone pair electrons are paired to form stable DPPH-H, so that the characteristic absorption light at 517nm is weakened or disappeared, the weakening degree of the light absorption value and the scavenging capacity of the antioxidant show a quantitative relation, and the scavenging rate and the oligosaccharide concentration also have a dose-effect relation. The scavenging ability of the radical scavenger can thus be assessed by determining the level of attenuation of the absorbance. As can be seen more clearly from FIG. 2, the scavenging ability of p-1, 4 xylo-oligosaccharide to DPPH radical is weak, so it is dark purple. Vc is a strong antioxidant, has strong capacity of scavenging DPPH free radicals, forms stable DPPH-H, and has a light yellow color change visible to naked eyes, so that the characteristic light absorption at 517nm of the stable DPPH-H is greatly reduced, and similarly, the obvious color change can also be seen in the active beta-1, 3-xylo-oligosaccharide, so that the antioxidant activity of the active beta-1, 3-xylo-oligosaccharide is obviously higher than that of the beta-1, 4 xylo-oligosaccharide and is slightly lower than Vc. Previous studies have shown that some steric structures in-OH, -COOH and carbohydrates enhance antioxidant activity.
The scavenging ability of active beta-1, 3-xylo-oligosaccharide, beta-1, 4 xylo-oligosaccharide and Vc on DPPH free radical is shown in figure 2 and figure 3. As can be seen from FIG. 3, both the active beta-1, 3-xylo-oligosaccharides and Vc have stronger scavenging capacity for DPPH free radicals, and with the increase of the concentration, the scavenging rates for DPPH free radicals are gradually increased and then become stable, the scavenging rate for Vc is kept at about 60%, the scavenging rate for the active beta-1, 3-xylo-oligosaccharides is kept above 45%, and the scavenging rate for DPPH free radicals of beta-1, 4 xylo-oligosaccharides is lower and less than 20%. The scavenging ability of the active beta-1, 3-xylo-oligosaccharide to DPPH free radicals is obviously higher than that of the beta-1, 4 xylo-oligosaccharide, the trend of the scavenging rate of the active beta-1, 3-xylo-oligosaccharide to DPPH free radicals tends to rise rapidly in the concentration range of 0.25-1.5mg/mL, the trend of the beta-1, 4 xylo-oligosaccharide does not exist, and the scavenging rate of the active beta-1, 3-xylo-oligosaccharide is always higher than that of the beta-1, 4 xylo-oligosaccharide. The scavenging ability of the active beta-1, 3-xylo-oligosaccharide to DPPH free radical is especially close to Vc when the concentration of xylo-oligosaccharide is 2 mg/mL. The research of Xue et al proves that the antioxidant activity of xylo-oligosaccharide can be influenced by different molecular weights, the research of the scholars of Wanglidong and the like proves that xylo-oligosaccharide mainly plays the role of antioxidation, xylo-oligosaccharide and xylotriose play a role in the xylo-oligosaccharide, the content of xylo-oligosaccharide directly determines the antioxidant activity of xylo-oligosaccharide, and the clearance rate of active beta-1, 3-xylo-oligosaccharide to DPPH free radical is higher than that of beta-1, 4 xylo-oligosaccharide, which is probably related to the relatively higher proportion of xylo-disaccharide and xylotriose in the active beta-1, 3-xylo-oligosaccharide.
thirdly, measuring the effect of eliminating OH free radicals
Hydroxyl radical (. OH) is a chemically very reactive radical which reacts with almost all biological macromolecules and has a relatively high reaction rate constant, and of all the radicals, it has the greatest toxicity. Inside the organism, it can directly attack the cell membrane through lipid peroxidation, and can also degrade or even completely inactivate many important enzymes inside the human body. Hydroxyl radicals can also damage DNA and can even cause cell mutation and death.
As is apparent from fig. 4, the removal effect of OH · from Vc is very good, Vc itself is colorless, and the colored substance almost completely disappears under the condition of Vc of different concentrations, and the colored substance is significantly reduced even when the Vc concentration is the lowest. It is also obvious from the figure that the OH & of the active beta-1, 3-xylo-oligosaccharide is also good in removing effect, the active beta-1, 3-xylo-oligosaccharide is yellow, the removing effect of OH & of different concentrations is good, and the colored substances can disappear. The beta-1, 4 xylo-oligosaccharide has extremely poor OH removing effect, colored substances are not faded when the color is observed by naked eyes, the color is not obviously different from the reference color, and the OH removing capability of the beta-1, 4 xylo-oligosaccharide is extremely low.
the results of FIG. 5 show that beta-1, 3-xylo-oligosaccharide has very strong scavenging effect on OH as well as Vc, and the scavenging rate is improved along with the increase of the mass concentration of the beta-1, 3-xylo-oligosaccharide and Vc, and the beta-1, 3-xylo-oligosaccharide and Vc have obvious dose-effect relationship. Research results also show that the removal effect of the active beta-1, 3-xylo-oligosaccharide on OH can be increased rapidly to 95.45 percent within the mass concentration range of 0.5-1.5 mg/mL; when the concentration is 2mg/mL, the removal effect of the active beta-1, 3-xylo-oligosaccharide on OH is kept stable, which shows that the removal efficiency of the active beta-1, 3-xylo-oligosaccharide is best at 1.5 mg/mL. However, the beta-1, 4 xylo-oligosaccharide has a 0 OH clearance in the mass concentration range of 0.25-1.5mg/mL, and has a weak clearance of 5.47% for OH only under the condition of the mass concentration of 2mg/mL, so the active beta-1, 3-xylo-oligosaccharide has better oxidation resistance to OH, and the oxidation resistance of the active beta-1, 3-xylo-oligosaccharide to OH is very poor. This is slightly different from the results obtained in the previous measurement of DPPH radical scavenging ability, which indicates that different results may be obtained for the same target even when the same index is used for the same subject, depending on the nature of the evaluation system and the evaluation principle.
determination of superoxide anion radical clearance rate by using tetrapyrogallol method and pyrogallol method
the pyrogallol can generate autooxidation under the alkaline condition to generate a colored intermediate productWhen it is releasedWhen inhibited or cleared, the accumulation of intermediates can be prevented. Therefore, the experiment can reflect the autoxidation inhibition effect of the active beta-1, 3-xylo-oligosaccharide, beta-1, 4 xylo-oligosaccharide and Vc on pyrogallol by measuring the autoxidation inhibition effectclearing away the toxic materials. As can be seen from FIG. 6, the active beta-1, 3-xylooligosaccharide and beta-1, 4-xylooligosaccharide pairs with mass concentrations ranging from 0.25 to 1.5mg/mLSlightly increased clearance rate and activity beta-1, 3-xylo-oligosaccharide pairThe clearance rate is increased slightly faster than that of beta-1, 4 xylo-oligosaccharide, and the mass concentration of the active beta-1, 3-xylo-oligosaccharide pair is in the range of 1.5-2mg/mLThe clearance tends to be stable, the clearance rate is stabilized at about 77.77 percent, and the beta-1, 4 xylo-oligosaccharide pairthe clearance rate is lower than 40 percent. Vc mass concentration of 0.25mg/mLthe clearance rate is up to 96.79 percent, and a reference product Vc pair can be seenthe scavenging effect is very strong, when the mass concentration is 2mg/mL, the scavenging rates of the active beta-1, 3-xylo-oligosaccharide and Vc on superoxide anions are 97.42% and 71.66% respectively, and compared with the scavenging rate of beta-1, 4 xylo-oligosaccharide, the scavenging capacities of the active beta-1, 3-xylo-oligosaccharide and Vc on superoxide anions are closer, which shows that the active beta-1, 3-xylo-oligosaccharide has better scavenging capacity of superoxide anions.
compared with Vc, the active beta-1, 3-xylo-oligosaccharide and beta-1, 4 xylo-oligosaccharide pairthe clearance effect is weak, but the activity beta-1, 3-xylo-oligosaccharide pairThe clearance capacity is far larger than that of beta-1, 4 xylo-oligosaccharide. The active beta-1, 3-xylo-oligosaccharide has good capability of eliminating superoxide anions.
Fifthly, determining the reduction capability by potassium ferricyanide reduction method
the reduction capacity was measured according to the potassium ferricyanide reduction method, and the greater the absorbance measured at a wavelength of 700nm, the stronger the reduction capacity of the sample was indicated. As can be seen from FIG. 7, in the mass concentration range of 0.25-2.0mg/mL, the absorbance of the active beta-1, 3-xylo-oligosaccharide at 700nm gradually increases with the increase of the concentration, which indicates that the active beta-1, 3-xylo-oligosaccharide can convert Fe into Fe3reduction of the iron cyanide complex to the ferrous form (Fe)2+) The beta-1, 3-xylo-oligosaccharide and the beta-1, 4-xylo-oligosaccharide have strong reducing capability, the reducing capability is improved along with the increase of the mass concentration of the beta-1, 3-xylo-oligosaccharide, the clearance rate of the beta-1, 3-xylo-oligosaccharide can reach 64.27% at most along with the increase of the concentration, the quantity-effect relationship is obvious, the reducing capability of the beta-1, 3-xylo-oligosaccharide is obviously higher than that of the beta-1, 4-xylo-oligosaccharide, the clearance rate of the beta-1, 4-xylo-oligosaccharide is only 12.44% at most, the clearance rate of Vc can be always higher than 83% and 86.24% at most, and therefore, the reducing capability of the beta-1, 3-xylo-oligosaccharide and the beta-1, 4.
In the experiment, the beta-1, 3 xylan hydrolysate is mainly xylobiose and xylotriose, and the beta-1, 4 xylan hydrolysate is mainly xylotriose and xylotetraose according to thin layer chromatography and HPLC. The active beta-1, 3-xylo-oligosaccharide has certain antioxidant capacity, and the antioxidant capacity presents a good dose-effect relationship in a certain sample concentration range. Particularly, the clearance rate of hydroxyl free radical can reach more than 90 percent at most, but the clearance ability to DPPH free radical is limited, and the clearance rate is not more than 50 percent at most. In the experiment, through researching the scavenging capacity of DPPH free radicals, superoxide free radicals and hydroxyl free radicals and the reducing capacity of ferric ions in a certain concentration range, the antioxidant capacity is found to be the reducing capacity > hydroxyl free radical scavenging capacity > superoxide anion scavenging capacity > DPPH free radical scavenging capacity in sequence, Vc is used as a contrast, although the antioxidant activity of the Vc is slightly lower than that of Vc, the antioxidant activity is obviously higher than that of beta-1, 4 xylo-oligosaccharide, and the active beta-1, 3-xylo-oligosaccharide has better antioxidant activity.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Sequence listing
<110> university of Chinese
<120> method for preparing active beta-1, 3-xylo-oligosaccharide from Vitis heyneana by enzyme method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 951
<212> DNA
<213> Artifical Sequence
<400> 1
catatgtatg gttgcctgcc gacaaaacct ctgccgaaca gcgagcgcaa gattgccaag 60
tttgaaccgg ccgacggcaa atgcctggtg tttattggcc aggagctgaa tgccatcggc 120
ggtctggacg actacaatga cggctatctg gaccacttcc agcagcgccc ggccggtttc 180
accgcatata ccgttctgac cccgggcagc gagagcttcg gtttcatcca taaaggcctg 240
gatggcgtga ccaccacaga cgattggggc gacaacaaaa gcaacatgag cctgcagctg 300
gccgacgagg actataaaaa catggccctg gccatcggtc tgggcatggt gcaccacgat 360
agcgcagtgg cctatggtaa acgtgaccag ctgatccgcg agctgggcaa tttcatcaag 420
gaacagagcc cgcgcccgat cttcctgcgc attggctacg agtttgacgg ccatgactgg 480
aaccattatg atcgcgataa ttacatcaaa gcctacaaac gtatcaaaaa tatttatgat 540
gagatggaga tcaccaacgt ggcctacgtg tggcagagtt gcggctttat gagcagcctg 600
gaagaactgg agaaatggta tccgggtgac gagtacgtgg attggtgtgc ctttagcttc 660
tttggcgcct ggaagaagca gaacatgatc gcctttgcca agcagaaggg caaaccggtg 720
tttatcgccg aagcaacccc ggccctggaa gagaacaaag agaccgttct gagcaatcaa 780
gatcaggcac gtgtggcctg ggataactgg ttcaccccgt tctttgccac cattcaccag 840
aatccggaaa ccgtgaaagc catcagctac attaattgta actggaaagc ccaccgcatg 900
tggtttgata atccgacctt taaatatatc gatagccgta tccagaccaa c 951
Claims (7)
1. A method for preparing active beta-1, 3-xylo-oligosaccharide from sea grapes by an enzyme method is characterized by comprising the following steps: the method comprises the following steps:
(1) extracting beta-1, 3-xylan from the vitis amurensis;
(2) obtaining recombinant beta-1, 3-xylanase;
(3) Hydrolyzing the material obtained in the step (1) by using the recombinant beta-1, 3-xylanase to obtain the active beta-1, 3-xylooligosaccharide, which specifically comprises the following steps:
a. Adding the material obtained in the step (1) into a Tris-HCl buffer solution with the pH value of 6.9-7.2 to prepare a beta-1, 3-xylan solution with the concentration of 0.8-1.2 wt%;
b. Mixing the beta-1, 3-xylan solution with a proper amount of the recombinant beta-1, 3-xylanase, reacting at 44-46 ℃ for 20-30h, and heating at 95-100 ℃ for 4-6min to inactivate the recombinant beta-1, 3-xylanase;
c. And c, centrifuging the material obtained in the step b at the speed of 11000-13000rpm for 4-6min, and obtaining supernatant which is the active beta-1, 3-xylo-oligosaccharide.
2. The method of claim 1, wherein: the step (1) comprises the following steps:
a. Mixing the sea grape powder with a proper amount of NaOH solution, heating while stirring, and fully boiling for 25-35 min;
b. centrifuging the material obtained in the step a at 3500-;
c. Adding a proper amount of H into the material obtained in the step b2SO4centrifuging the solution at 3500-4500rpm for 15-25min to obtain a second precipitate;
d. adding a proper amount of NaOH solution into the second precipitate, stirring and extracting sugar for 1.5-2h under the ice bath condition, and then centrifuging the obtained material to obtain a supernatant;
e. Mixing the supernatant with anhydrous ethanol at a volume ratio of 1: 3-5, standing at 3-5 deg.C for 10-12 hr, and performing solid-liquid separation to obtain a third precipitate;
f. Adding a proper amount of high-sodium chlorate solution into the third precipitate, decoloring for 1.5-2.5h while stirring, and fully washing with distilled water to obtain a fourth precipitate;
g. Centrifuging the fourth precipitate at 9500-12000rpm for 12-20min to obtain a fifth precipitate;
h. and fully washing the fifth precipitate with acetic acid solution, and fully washing with distilled water to obtain the beta-1, 3-xylan.
3. the method of claim 2, wherein: the sea grape powder in the step a of the step (1) is pretreated sea grape powder, and the pretreatment process comprises the following steps:
a1, mixing the sea grape powder with purified water, extracting for 5-20 min under the subcritical water state, cooling to room temperature, centrifuging, collecting supernatant, and freeze-drying.
4. the method of claim 3, wherein: the preprocessing process further comprises the following steps:
2, sequentially adding 13-14 parts by weight of distilled water, 0.1-0.2 part by weight of hydrochloric acid, 4-5 parts by weight of glucose, 9-12 parts by weight of absolute ethyl alcohol and 50-60 parts by weight of ethyl orthosilicate into a reaction vessel at room temperature, rapidly stirring for 4-6min, sequentially carrying out transparent-turbid-transparent change on liquid, cooling with room-temperature water, and fully removing ethanol to form prehydrolysis gel;
3, adding 16-17 parts by weight of the freeze-dried material into the pre-hydrolyzed gel, adjusting the pH to 5.4-5.6 by using alkali, stirring at a low speed for 25-35min, and standing at 3-5 ℃ for 45-50h to obtain wet gel;
a4, grinding and crushing the wet glue, fully washing the ground wet glue with distilled water to remove glucose in the wet glue, filtering, drying, grinding, crushing and sieving the washed wet glue to obtain the finished product.
5. the method of claim 1, wherein: the step (1) is as follows: extracting beta-1, 3-xylan from Vitis amurensis, preparing beta-1, 3-xylan into beta-1, 3-xylan, and mixing with the rest beta-1, 3-xylan.
6. The method of claim 5, wherein: the hydroxyl alcohol beta-1, 3-xylan accounts for 5-10 wt% of the material obtained in the step (1).
7. The method of claim 5 or 6, wherein: the preparation method of the hydroxyl alcohol beta-1, 3-xylan comprises the following steps:
I. Dissolving beta-1, 3-xylan in NaOH solution, adding a proper amount of dichloroethanol, stirring in an ice bath for 0.8-1.5h, and then stirring at room temperature for 20-25 h;
II. Adding glacial acetic acid into the material obtained in the step I, neutralizing in an ice bath, and then dialyzing;
and III, heating, stirring and concentrating the material obtained in the step III, and freeze-drying to obtain the hydroxyl alcohol beta-1, 3-xylan.
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|---|---|---|---|---|
| EP1469066A2 (en) * | 1996-01-11 | 2004-10-20 | Diversa Corporation | Glycosidase enzymes |
| WO2007029773A1 (en) * | 2005-09-08 | 2007-03-15 | Kabushiki Kaisha Yakult Honsha | Cholesterol absorption inhibitor |
| WO2017110975A1 (en) * | 2015-12-25 | 2017-06-29 | 東レ株式会社 | Method for producing xylooligosaccharide composition |
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2019
- 2019-09-24 CN CN201910907836.2A patent/CN110551774A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1469066A2 (en) * | 1996-01-11 | 2004-10-20 | Diversa Corporation | Glycosidase enzymes |
| WO2007029773A1 (en) * | 2005-09-08 | 2007-03-15 | Kabushiki Kaisha Yakult Honsha | Cholesterol absorption inhibitor |
| WO2017110975A1 (en) * | 2015-12-25 | 2017-06-29 | 東レ株式会社 | Method for producing xylooligosaccharide composition |
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| Title |
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| CAI ZW等: "Characterization of a novel psychrophilic and halophilic β-1, 3-xylanase from deep-sea bacterium, Flammeovirga pacifica strain WPAGA1", 《INT J BIOL MACROMOL.》 * |
| IRIKI Y等: "Xylan of siphonaceous green algae", 《NATURE》 * |
| YAMAURA I等: "Purification and some properties of endo-1,3-.BETA.-D-xylanase from Pseudomonas sp. PT-5", 《AGRICULTURAL AND BIOLOGICAL CHEMISTRY》 * |
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