阅读说明：本技术 一种从海葡萄中酶法制备活性β-1,3-木寡糖的方法 (Method for preparing active beta-1, 3-xylo-oligosaccharide from sea grape by enzyme method ) 是由 张光亚 刘婷 葛慧华 于 2019-09-24 设计创作，主要内容包括：本发明公开了一种从海葡萄中酶法制备活性β-1,3-木寡糖的方法,括如下步骤：(1)从海葡萄中提取β-1,3-木聚糖；(2)获得重组β-1,3-木聚糖酶；(3)以上述重组β-1,3-木聚糖酶水解步骤(1)所得的物料,以获得所述活性β-1,3-木寡糖。现有技术中的木聚糖都是β-1,4-木聚糖及β-1,4-木寡糖(低聚木糖),而本发明从来源于海葡萄的β-1,3-木聚糖中利用酶法制备β-1,3-木寡糖,填补了国内空白,同时经过测验,本发明制得的β-1,3-木寡糖与β-1,4-木寡糖相比,具有更好的活性。(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.)

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.

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.