CN113880890B - Hawthorn seed acetylated xylo-oligosaccharide and preparation method thereof - Google Patents

Abstract

The invention discloses hawthorn seed acetylated xylo-oligosaccharide and a preparation method thereof. The acetylated xylo-oligosaccharide has a structural formula shown as the following formula I:

in the formula I, n is a natural number of 0-3, R ₁ 、R ₂ Hydrogen or acetyl, but not both. The preparation method comprises the following steps: (1) Soaking hawthorn seed powder in water, putting the soaked hawthorn seed powder into a subcritical water pretreatment container, heating and preserving heat to obtain subcritical water-treated hawthorn seed powder liquid; (2) And (3) hydrolyzing the subcritical water-treated hawthorn seed powder feed liquid by adopting endo beta-1,4-xylanase to obtain the acetylated xylo-oligosaccharide. According to the invention, the subcritical water pretreatment and enzymolysis combined technology is utilized to convert the cheap hawthorn seeds into the acetylated xylo-oligosaccharide with high added value, xylan in the biomass is effectively extracted by the subcritical water pretreatment means without breaking the side chain on the xylan, and the acetylated xylo-oligosaccharide is prepared, so that the method has great application value and potential economic value.

Description

Hawthorn seed acetylated xylo-oligosaccharide and preparation method thereof

Technical Field

The invention relates to hawthorn seed acetylated xylo-oligosaccharide and a preparation method thereof, belonging to the field of food and feed processing.

Background

The hawthorn is a plant of hawthorn of Rosaceae, and is mainly distributed in east Asia, europe, north America and the like with north temperate zone of 30-50 degrees north latitude. According to the climatic characteristics, cultivation utilization, geographical position and other conditions of China, the method can be roughly divided into Beijing jin Ji, jiliao, lusu, yunhui plateau and 5 hawthorn producing areas (Qijing, etc. the food science and technology report, 2019, 1. The hawthorn fruit has high nutritive value and unique flavor, has certain health care function, can be processed into different foods, is an ideal processing raw material, but generates a large amount of hawthorn seed byproducts in the processing process. The hawthorn seeds account for 30 percent of the dry weight of the hawthorn fruits, and the annual output of China is about 150 ten thousand tons according to statistics. The current major utilization route for hawthorn seeds is combustion and as a low nutritional value feed, which not only wastes resources but also causes environmental pollution (Liu et al, food Chemistry,2016, 212. Although there are documents and patents that flavonoids and fats can be produced from hawthorn seeds, the yields are relatively low, and 100g of hawthorn seeds can only produce 3-5g of flavonoids or 1-2g of fats, which have the problems of insignificant economic benefit, complex Technology and serious pollution (Pan et al, adhesives Science, 2012,19, salvanian et al, journal of Agricultural Science and Technology,2014,1.

Hemicellulose is a polysaccharide substance that is second only to cellulose in nature, and xylan is the main component of hemicellulose, and accounts for about 15% to 50% of lignocellulose (Biley et al, biotechnology Advances,2016, 34. The hawthorn seeds, as an agricultural byproduct, were determined to have a xylan content of about 28% (dry basis) and abundant acetyl side chains on the xylan. Xylans have a complex structure, with the main chain being xylose molecules linked by β -1,4-glycosidic bonds, and the side chains containing different substituents, such as arabinose, glucuronic acid, ferulic acid, acetyl, etc. (Kalsson et al, applied Microbiology Biotechnology,2018, 9081-9088. Xylan can be used for preparing some products with higher added value, such as xylo-oligosaccharide, by a biotechnology means. Xylo-oligosaccharides, also known as xylo-oligosaccharides, are oligosaccharides with 2-10 xylose chains linked as backbone by β -1,4-glycosidic linkages (Zhou et al, 2019, 289. Xylo-oligosaccharides can promote the proliferation of intestinal probiotics of human or animals, inhibit the production of harmful bacteria, improve the micro-ecological environment of colon, improve the immunity of organism, resist tumor, reduce blood pressure, blood sugar and serum cholesterol (Amorim et al, biotechnology Advances,2019, 107397-107404).

Generally, the methods for producing xylo-oligosaccharides using xylan in lignocellulose can be classified into three methods, acid degradation, oxidative degradation, and enzymatic degradation (Amorim et al, biotechnology Advances,2019, 37. The enzyme degradation method is the most concerned method due to the characteristics of uniform products, environmental friendliness and the like. Xylans in lignocellulose mostly form crystalline structures with cellulose and lignin, so that pretreatment is required to expose the xylans in lignocellulose before enzymatic degradation (Liu et al, food Chemistry,2018, 264. The pretreatment methods commonly used at present mainly comprise two modes of alkali pretreatment and high-temperature cooking pretreatment, the two methods release xylan, simultaneously break side chains on the xylan easily, and generate some harmful substances such as furfurals in the pretreatment process (Jacquet et al, industrial & Engineering Chemistry Research,2015, 2593-2598. Therefore, some documents and patents report that agricultural byproducts such as corncobs, straws, peanut shells and tea fruit shells are used for preparing xylooligosaccharides at home and abroad, but most of the prepared xylooligosaccharides are unsubstituted xylooligosaccharides (patent application No. 201510831144.6, 201110213461.3, amorim et al, biotechnology Advances,2019, 37. To date, only a few reports have been made of xylooligosaccharides containing glucuronic acid side chains and arabinose side chains, and no documents and patents for xylooligosaccharides containing abundant acetyl side chains have been found (Kalsson et al, applied Microbiology Biotechnology,2018,102, 9081-9088).

Disclosure of Invention

The invention aims to provide hawthorn seed acetylated xylo-oligosaccharide and a preparation method thereof, cheap hawthorn seeds are converted into acetylated xylo-oligosaccharide with high added value by using a subcritical water pretreatment and enzymolysis technology, xylan in biomass is effectively extracted by a subcritical water pretreatment means without breaking the side chain on the xylan, and the acetylated xylo-oligosaccharide is prepared and has high application value and potential economic value.

The invention provides acetylated xylo-oligosaccharide, which has a structural formula shown as the following formula I:

in the formula I, n is a natural number of 0-3, R ₁ 、R ₂ Hydrogen or acetyl, but not both.

The invention also provides a preparation method of the acetylated xylo-oligosaccharide, which comprises the following steps: (1) Soaking hawthorn seed powder in water, putting the soaked hawthorn seed powder into a subcritical water pretreatment container, heating and preserving heat to obtain subcritical water treatment hawthorn seed powder feed liquid;

(2) And (3) hydrolyzing the subcritical water-treated hawthorn seed powder feed liquid by adopting endo beta-1,4-xylanase to obtain the acetylated xylo-oligosaccharide.

In the method, the particle size of the hawthorn seed powder can be 0-20 meshes and is not zero;

the preparation method of the hawthorn seed powder comprises the following steps: selecting fructus crataegi seed without mould, washing with clear water to remove surface dirt, draining water, naturally air drying, pulverizing, and sieving.

In the above method, in the step (1), the solid-to-liquid ratio of the hawthorn seed powder to the water may be 1g: 6-20 mL, specifically 1g:10mL, 1g: 6-10 mL, 1g: 10-20 mL or 1g:8 to 15mL.

In the above method, the soaking time may be 8 to 12 hours, specifically 12 hours or 10 to 12 hours.

In the above method, the heating temperature may be 150 to 195 ℃, specifically 150 ℃, 185 ℃ and 150 to 190 ℃.

In the above method, the incubation conditions may be: the temperature can be 150-195 ℃ and the time can be 15-35 min; the heat preservation temperature can be 150 ℃, 165 ℃, 180 ℃ and 195 ℃, and the heat preservation time can be 15min, 20min, 25min, 30min and 35min; the optimal condition is heat preservation at 185 ℃ for 25min.

In the above method, the hydrolysis conditions are as follows: the pH value can be 4.5-7.5, the temperature can be 40-80 ℃, and the time can be 2-12 h; preferably, the pH value can be 5.5, the hydrolysis temperature can be 50 ℃, and the hydrolysis time can be 4-8 h, 4h and 8h.

In the method, the preparation of the endo-beta-1,4-xylanase comprises the following steps: carrying out fermentation culture on natural bacteria or recombinant bacteria capable of expressing endo-beta-1,4-xylanase, and obtaining the endo-beta-1,4-xylanase from a fermentation product.

Wherein the native bacteria can be Paecilomyces viridissus (e.g., paecilomyces viridis (Paecilomyces aerugineus) GY 701), paecilomyces thermophilus, cladosporium camphoratum, chaetomium fortunei (e.g., chaetomium CQ 31), thermotoga maritima, or Paecilomyces bajunensis Ge Ci, which can express endo-beta-1,4-xylanase.

The recombinant strain can be obtained by introducing an encoding gene of endo-beta-1,4-xylanase into a recipient strain; the recipient bacterium can be Pichia pastoris (such as Pichia pastoris GS 115), escherichia coli or Bacillus subtilis. The coding gene of the endo-beta-1,4-xylanase can be derived from Paecilomyces viridonii (such as Paecilomyces viridonii (Paecilomyces aerugineus) GY 701), paecilomyces thermophilus, cladosporium camphorata, chaetomium globosum (such as Chaetomium fortunei CQ 31), thermotoga maritima or Palonon Ge Ci Paenibacillus sp.

In the method, the enzyme adding amount of the endo-beta-1,4-xylanase is 5-100U per mL, and the subcritical water treatment hawthorn seed powder feed liquid can be 5-25U/mL (such as 10U/mL).

In the method, the step (2) further comprises the step of carrying out post-treatment on the enzymatic hydrolysate obtained after hydrolysis;

the post-treatment process comprises the following steps: and filtering the enzymolysis solution, taking filtrate for decoloring, performing ion exchange after decoloring, concentrating the solution after ion exchange, and finally drying to obtain the acetylated xylo-oligosaccharide.

In the invention, the filtrate is decolorized by using active carbon, the addition amount of the active carbon is 0.5 percent of the filtrate, and the specific condition of the decolorization is that the temperature is kept at 70 ℃ for 15min.

In the invention, the specific conditions for obtaining the acetylated xylo-oligosaccharide by adopting ion exchange in the post-treatment are as follows:

the ion exchange treatment specifically adopts column chromatography separation; the column chromatography separation is carried out according to the sequential treatment of cation, anion and cation resin, wherein the cation resin is 001 multiplied by 7, and the anion resin is D301.

The invention has the following advantages:

1. the subcritical water pretreatment method provided by the invention can effectively extract xylan in the lignocellulose material, can effectively reduce the breakage of xylan side chains, and can maintain the structural integrity of the extracted xylan;

2. the endo beta-1,4-xylanase provided by the invention has good stability and high specific enzyme activity, and has great application value in the industries of food, feed and the like;

3. the preparation method provided by the invention can be used for preparing the acetylated xylo-oligosaccharide with uniform product and polymerization degree of 2-5, and the conversion rate is up to 67%.

4. The preparation method has the advantages of simple preparation process, mild reaction conditions and convenience for industrial production.

Drawings

FIG. 1 is a graph showing the growth conditions (measured by wet weight of bacteria) and the protein produced by fermentation and the secreted endo-xylanase of recombinant Pichia pastoris GS115 (taking Paecilomyces viridissima endo-xylanase as an example);

FIG. 2 is a scanning electron microscope image of hawthorn seed powder before and after subcritical water pretreatment, wherein A is before pretreatment and B is after pretreatment;

FIG. 3 shows the yield of acetylated xylo-oligosaccharide of an enzymatic hydrolysate obtained by performing subcritical water pretreatment on hawthorn seed powder at different temperatures and times and hydrolyzing the hawthorn seed powder with endo-xylanase;

FIG. 4 is a diagram showing the enzymatic hydrolysis process of acetylated xylo-oligosaccharide of the present invention;

FIG. 5 is an ion chromatogram of acetylated xylooligosaccharide of the present invention;

FIG. 6 is a MALDI-TOF analysis chart of acetylated xylo-oligosaccharide prepared by the present invention;

FIG. 7 is an NMR one-dimensional hydrogen spectrum analysis chart of acetylated xylooligosaccharide of the present invention, (A) is the characteristic peak of hydrogen on acetyl group, (B) is the characteristic peak of hydrogen on acetylated xylooligosaccharide;

FIG. 8 is a diagram of NMR two-dimensional heteronuclear multiple carbon correlation spectroscopy (HMBC) analysis for preparing acetylated xylo-oligosaccharides;

FIG. 9 is a process flow diagram of the expanded production of acetylated xylo-oligosaccharide from hawthorn seed powder.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 cloning of the beta-1,4-xylanase Gene and Pichia expression

1. Obtaining of recombinant bacteria (taking Paecilomyces aeruginosa endoxylanase as an example)

The biological materials involved in the experimental procedure:

green blue Paecilomyces GY701 was screened in soil and stored in the laboratory (Katrolia et al, journal of Molecular Catalysis B: enzymatic,2011, 69.

The vector pPIC9K is available from Invitrogen corporation, USA.

1. A nucleotide sequence coding a mature protein is amplified by using a primer pair consisting of Paxyn10AF (5'-CCGGAATTCGCTCCAGCTGAAACTTTGGAGTCTA-3') and Paxyn10AR (5'-GATACGCGGCCGCTTACAAAGCAGCAGTGATAGCGTTG-3') by taking the GY701cDNA of Paecilomyces viridis as a template. The amplification conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and circulating for 35 times; finally, extension is carried out for 5min at 72 ℃. Recovering the product by 1% agarose gel electrophoresis, carrying out double digestion by EcoR I and Not I, and recovering the DNA fragment after digestion; carrying out double enzyme digestion on the pPIC9K vector by using restriction enzymes EcoRI and Not I, and recovering the skeleton vector after enzyme digestion; the skeleton vector is connected with the DNA fragment to obtain a recombinant vector A (namely the DNA fragment of beta-1,4-xylanase from paecilomyces viridochromosus is inserted between EcoRI and Not I enzyme cutting sites of a pPIC9K vector).

2. And (3) converting the recombinant vector A into Pichia pastoris GS115 to obtain the recombinant strain Pichia pastoris GS115 containing the recombinant vector A.

2. Fermentation of recombinant bacteria

1. Fermentation process

Fermenting recombinant bacteria Pichia pastoris GS115 containing recombinant vector A to produce beta-1,4-xylanase. The Fermentation method is described in "Pichia Fermentation Process Guidelines (Version B,053002, invitrogen)". The fermentation tank is a 5L fermentation tank. Seed medium, fermentation minimal medium, glycerol fed-batch medium and 100% methanol induction medium were prepared according to the methods described in the above documents. The whole fermentation process adopts three stages of batch culture, glycerol fed-batch culture and 100% methanol induction culture.

The enzymatic activity of β -1,4-xylanase was determined by reference to Liu et al (Liu et al, food Chemistry,2018,264: adding 900 μ L of 1% (w/v, unit is g/mL) beech xylan (pH 5.5) into a cuvette, preheating at 55 deg.C for 3min, then adding 100 μ L of an enzyme solution to be measured (pH 5.5) diluted appropriately (pH 5.5) (i.e., a solution of a protein to be measured), reacting at 55 deg.C for 10min, adding 1000 μ L of DNS reagent (containing 10g of 3, 5-dinitrosalicylic acid, 10g of NaOH and 2g of phenol per 1L of aqueous solution), boiling for 15min, adding 1000 μ L of saturated potassium sodium potassium tartrate solution, cooling, and measuring the absorbance at a wavelength of 540nm, using xylose as a standard. Definition of enzyme activity: the amount of enzyme required to hydrolyze beechwood xylan to 1. Mu. Mol xylose at 55 ℃ and pH5.5 per minute was 1U.

2. Results of fermentation

The fermentation process is shown in figure 1, (1) is the enzyme activity of beta-1,4-endo-xylanase in the fermentation supernatant (the supernatant obtained by centrifuging the fermentation liquor for 10min under the condition of 10000 Xg after the fermentation is finished); (2) the protein content in the fermentation supernatant; (3): the wet weight of the cells. The enzyme activity reaches the maximum on day 6, the highest enzyme activity reaches 20100U/mL, the protein content in the fermentation supernatant reaches 19mg/mL, and the wet weight of the thallus is 380g/L.

Example 2 optimization of conditions for preparing acetylated xylooligosaccharide from Hawthorn seed

1. Experimental method

Washing fructus crataegi seed (with xylan content of 28%) with water, draining, pulverizing, and sieving with 20 mesh sieve. 100g of sieved hawthorn seed powder is taken, distilled water is added into the hawthorn seed powder according to the solid-to-liquid ratio of 1 (g: mL) for soaking for 12 hours, the materials are transferred into a subcritical water pretreatment container after soaking, the container is heated to the set temperature (150-190 ℃), the temperature is kept for the set time (15-35 min), the materials in the container are released instantly after the temperature is kept, and the materials are stored for standby.

Scanning electron microscope images of the hawthorn seed powder before and after subcritical water pretreatment are shown in figure 2, as can be seen from figure 2, the originally flat hawthorn seed surface after pretreatment becomes rough and porous, and the endo-xylanase can contact xylan in the hawthorn seed through the holes.

Carrying out enzymolysis treatment on the hawthorn seed material liquid pretreated under different conditions, wherein the enzyme adding amount is 25U/mL, the hydrolysis pH is 5.5, the hydrolysis temperature is 50 ℃, the hydrolysis time is 8h, and the hydrolysate is stored for later analysis.

And optimizing the enzyme adding amount under the optimal pretreatment condition, wherein the enzyme adding amount has the variation range of 5-25U/mL, the hydrolysis pH value of 5.5, the hydrolysis temperature of 50 ℃, the hydrolysis time of 8h, and the hydrolysate is stored for later analysis.

The hydrolysis process is optimized under the optimal pretreatment condition and the enzyme adding amount condition, the hydrolysis pH is 5.5, the hydrolysis temperature is 50 ℃, the hydrolysis time is 8h, and samples are taken at different times for later analysis.

The above sampleThe product adopts HPAEC-PAD (ISC-5000) ⁺ Thermo, USA) in combination with CarboaPac ^TM PA1 (4X 250mm, thermo, USA) anion exchange column assay, elution conditions: eluting with 100mM NaOH solution for 8min, adding 0-150mM sodium acetate, and linearly eluting for 12min at a total elution time of 20min, flow rate: 1mL/min, the column temperature is 30 ℃, and the xylose-xylopentaose is used as a standard substance for analysis and quantification.

Conversion rate of acetylated xylooligosaccharide: (xylobiose + xylotriose + xylotetraose + xylopentaose) (g)/xylan (g) x 100% in hawthorn seed powder.

2. Results of the experiment

Optimizing a sample by quantitative pretreatment conditions (figure 3), preserving heat for 15min at 150 ℃, preparing 1.9g of xylo-oligosaccharide per 100g of hawthorn seed powder, continuously increasing the yield of the xylo-oligosaccharide along with the increase of the heat preservation time, and preparing 3.9g of xylo-oligosaccharide per 100g of hawthorn seed powder when the temperature is increased to 35min; the incubation is carried out at 165 ℃, and the yield of xylooligosaccharide is increased along with the increase of the incubation time. The temperature is kept for 15min at 180 ℃, 11.9g of xylo-oligosaccharide can be prepared from every 100g of hawthorn seed powder, the conversion rate of the xylo-oligosaccharide is 42.5%, 17.5g of xylo-oligosaccharide can be prepared from every 100g of hawthorn seed powder along with the increase of the temperature keeping time to 25min, the conversion rate reaches the highest value at the moment and is 62.5%, the temperature keeping time is continuously increased, the yield of the xylo-oligosaccharide is not increased, but is reduced, and the problem that the xylan in the hawthorn seed powder is subjected to self-hydrolysis to generate a large amount of xylose possibly exists in the hawthorn seed powder after the pretreatment time is too long. Similarly, the temperature is continuously increased to 195 ℃, and the yield of the xylo-oligosaccharide is lower than 185 ℃ for 25min. Therefore, the optimal conditions for pretreatment are 185 ℃ for 25min.

The result of optimizing the enzyme adding amount under the optimal subcritical water pretreatment condition is shown in table 1, the yield of the xylo-oligosaccharide reaches the highest when the enzyme adding amount is 10U/mL, 18.5g of xylo-oligosaccharide can be prepared by every 100g of hawthorn seed powder, the conversion rate of the xylo-oligosaccharide reaches 66.1%, and the conversion rate of the xylo-oligosaccharide is not improved by continuously increasing the enzyme adding amount.

TABLE 1 yield and conversion of xylo-oligosaccharide from hawthorn seeds pretreated by hydrolyzing subcritical water with different enzyme dosages

Under the optimized conditions, the enzymolysis process is shown in figure 4, when the enzymolysis time is 4 hours, the yield of xylo-oligosaccharide can reach 18.7g per 100g of hawthorn seed powder, the conversion rate of xylo-oligosaccharide is 66.8%, and the conversion rate of xylo-oligosaccharide is not improved by increasing the enzymolysis time.

As shown in figure 5, after the hawthorn seed powder is subjected to subcritical water pretreatment, xylan in the hawthorn seed powder is degraded, but the polymerization degree is not uniform, and after the hawthorn seed powder is subjected to enzymolysis by using beta-1,4-xylanase, the prepared xylooligosaccharide product is specific, has low polymerization degree and is respectively acetylated xylobiose, xylotriose, xylotetraose and xylopentaose.

EXAMPLE 3 MALDI-TOF and NMR analysis of Crataegus pinnatifida seed acetylated xylooligosaccharide

1. Experimental methods

Mass spectrometry analysis: the analysis of the molecular weight of the components in the enzymolysis sample is carried out by matrix assisted laser desorption/ionization-ionization mass spectrometry (MALDI-TOF), the mode is positive ions, the solvent is water, and the matrix is 2,5-dihydroxybenzoic acid.

Nuclear Magnetic Resonance (NMR) analysis: the sample is decolorized by active carbon and desalted by anion and cation resin and then is freeze-dried. 20mg of the lyophilized sample was dissolved in 750. Mu.L of dimethyl sulfoxide (DMSO) and 5mM 4, 4-dimethyl-4-silapentane (DSS) was added as an internal standard. One-dimensional 1H and 13C spectra of the samples were analyzed using an Agilent DD2 MHz NMR spectrometer, and two-dimensional 1H and 13C heteronuclear multiple carbon correlation (HMBC) spectra were obtained using standard pulse sequences and related parameters provided by Agilent.

2. Results of the experiment

The prepared hawthorn seed acetylated xylo-oligosaccharide has a polymerization degree of 2-5 by MALDI-TOF analysis, and is shown in FIG. 6 and Table 2, and the polymerization degree of the acetylated xylo-oligosaccharide is 2-5, and the acetylated xylo-oligosaccharide is xylobiose (X2), xylobiose (X2 Ac 1) containing one acetyl group, xylotriose (X3 Ac 2) containing two acetyl groups, xylotetraose (X4 Ac 2) containing two acetyl groups, xylopentaose (X4 Ac 3) containing three acetyl groups, xylopentaose (X5 Ac 2) containing two acetyl groups and xylopentaose (X5 Ac 3) containing three acetyl groups.

TABLE 2 inference of xylooligosaccharide composition from Mass Spectroscopy

To further confirm the presence of acetyl groups of the hawthorn seed acetylated xylo-oligosaccharides, NMR analysis of one-dimensional H spectra and two-dimensional HMBC was performed. As shown in FIG. 7 and FIG. 8, the xylo-oligosaccharide has distinct characteristic peaks on substituent No. 2 and substituent No. 3, which indicates that the acetyl substitution positions of the prepared acetylated xylo-oligosaccharide are 2-, 3-and 2,3-, respectively.

Example 4 Scale-Up production of Hawthorn seed acetylated xylooligosaccharide

According to the scheme shown in figure 9, 10kg of crushed hawthorn seed powder which is sieved by a 20-mesh sieve is put into a subcritical water reaction container, 100L of distilled water is added, the temperature of the reaction container is set to 185 ℃, the temperature is kept for 25min after the temperature is reached, the material is released instantly after the temperature keeping time is reached, the pH value of the material liquid is adjusted to 5.5, 10U/mL of beta-1,4-xylanase is added for enzymolysis at 50 ℃ for 4h, enzyme is inactivated at 80 ℃ after the enzymolysis is completed, and the material liquid is cooled to room temperature after the enzyme inactivation is completed. Then, carrying out plate-and-frame filtration (other filtration methods can be selected) on the feed liquid, collecting filtrate, decolorizing with active carbon, adding 0.5% of active carbon, and keeping the temperature at 70 ℃ for 15min; filtering to remove active carbon after decolorization, and performing ion exchange treatment according to the order of cation, anion and cation resin, wherein the cation resin is 001 × 7, and the anion resin is D301; ion exchange is followed by concentration by rotary evaporation or thin film. The concentration step can prepare the sugar solution into high-concentration xylo-oligosaccharide syrup; or concentrating, spray drying (or other drying method) to obtain sugar powder.

Through calculation, the conversion rates of the xylo-oligosaccharide after the steps of enzymolysis, filtration, decoloration, ion exchange and concentration are 66.8%, 62%, 60%, 57% and 55% respectively. Spray drying to obtain 1.4kg of acetylated xylo-oligosaccharide.

In addition, endoxylanases from Chaetomium globosum CQ31 (natural fermentation, 521.6U/mg), cladosporium camphoratum (natural fermentation, 350.7U/mg), valencia Ge Ci Paenibacillus (Escherichia coli, 292.5U/mg), paecilomyces aeruginosa (Pichia pastoris: 20100U/mL), paecilomyces thermophilus (Pichia pastoris: 52940U/mL) and Thermotoga maritima (Pichia pastoris: 40020U/mL) are respectively tried, and all the endoxylanases can hydrolyze pretreated hawthorn seeds to produce acetylated xylooligosaccharides.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (9)

1. Acetylated xylo-oligosaccharides having the formula I:

in the formula I, n is a natural number of 0-3, R ₁ 、R ₂ Hydrogen or acetyl, but not both.

2. A process for the preparation of acetylated xylo-oligosaccharides as defined in claim 1, comprising the steps of: (1) Soaking hawthorn seed powder in water, putting the soaked hawthorn seed powder into a subcritical water pretreatment container, heating and preserving heat to obtain subcritical water-treated hawthorn seed powder liquid;

heating to the temperature of 150-195 ℃;

the heat preservation conditions are as follows: the temperature is 150-195 ℃ and the time is 15-35 min;

(2) Hydrolyzing the subcritical water-treated hawthorn seed powder liquid by adopting endo beta-1,4-xylanase to obtain the acetylated xylo-oligosaccharide;

the hydrolysis conditions were as follows: the pH value is 4.5-7.5, the temperature is 40-80 ℃, and the time is 2-12 h.

3. The method of claim 2, wherein: the particle size of the hawthorn seed powder is 0-20 meshes and is not zero;

the preparation method of the hawthorn seed powder comprises the following steps: selecting fructus crataegi seed without mould, washing with clear water to remove surface dirt, draining water, naturally air drying, pulverizing, and sieving.

4. A method according to claim 2 or 3, characterized in that: in the step (1), the solid-to-liquid ratio of the hawthorn seed powder to the water is 1g: 6-20 mL;

the soaking time is 8-12 h.

5. A method according to claim 2 or 3, characterized in that: the endo beta-1,4-xylanase is prepared by the following steps: carrying out fermentation culture on natural bacteria or recombinant bacteria capable of expressing endo-beta-1,4-xylanase, and obtaining the endo-beta-1,4-xylanase from a fermentation product.

6. The method of claim 5, wherein: the natural bacteria are paecilomyces viridochromous, paecilomyces thermophilus, cladosporium camphoratus, chaetomium cupreum, thermotoga maritima or Paenibacillus barnacanthus Ge Ci capable of expressing endo-beta-1,4-xylanase;

the recombinant strain is obtained by introducing an encoding gene of endo-beta-1,4-xylanase into a recipient strain; the recipient bacterium is Pichia pastoris, escherichia coli or Bacillus subtilis.

7. The method of claim 6, wherein: the coding gene of the endo-beta-1,4-xylanase is derived from paecilomyces viridissus, paecilomyces thermophilus, cladosporium camphorata, chaetomium globosum, thermotoga maritima or Paenibacillus Ge Ci.

8. A method according to claim 2 or 3, characterized in that: the enzyme dosage of the endo-beta-1,4-xylanase is 5-100U/mL, and the subcritical water treatment is performed on the hawthorn seed powder feed liquid.

9. A method according to claim 2 or 3, characterized in that: the step (2) also comprises a step of carrying out post-treatment on the enzymolysis liquid obtained after hydrolysis;

the post-treatment process comprises the following steps: and filtering the enzymolysis liquid, decoloring the filtrate, performing ion exchange after decoloring, concentrating the solution after ion exchange, and finally drying to obtain the acetylated xylo-oligosaccharide.

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