CN110628846B - Method for preparing xylooligosaccharide by high-temperature high-pressure treatment - Google Patents
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Abstract
The invention relates to a method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment, which comprises the following steps: pulverizing corn cob, sieving, adding water, and concocting to obtain premix; carrying out high-temperature high-pressure treatment on the premix to obtain a crude xylan extract, wherein the treatment temperature is 95-140 ℃, and the treatment pressure is 0.05-0.25 MPa; adjusting the mass concentration of the obtained crude xylan extracting solution to 4-6% and the pH value to 4.2-4.8, and performing microwave treatment to obtain xylan liquid; wherein the microwave frequency is 2450MHz, the treatment temperature is 40-55 deg.C, and the microwave time is 10-25 min; adding xylanase into the xylan liquid for enzymolysis to obtain crude xylo-oligosaccharide liquid; and (3) performing enzyme deactivation treatment on the crude xylo-oligosaccharide solution, and performing decoloration treatment, ion exchange treatment and concentration treatment to obtain the xylo-oligosaccharide solution. The xylo-oligosaccharide is prepared by using a high-temperature high-pressure process, the defects of the traditional production process are overcome, the traditional chemical method treatment is replaced, acid and alkali are not used in the preparation process, the large amount of sewage discharge is avoided, and the environmental protection pressure is reduced.
Description
Technical Field
The invention relates to a preparation method of xylo-oligosaccharide, belonging to the field of food raw material preparation.
Background
The health of the intestinal tract of a human body is closely related to the distribution of bacterial flora in the intestinal tract, wherein after the probiotics in the intestinal tract are propagated in a large quantity, pseudomembranous enteritis caused by using a large quantity of antibiotics can be treated; treating constipation and chronic diarrhea; protecting the liver; preventing hypertension and arteriosclerosis, resisting aging, reducing serum cholesterol, preventing cancer, and inhibiting tumor growth. The probiotics in the intestinal tract of human body mainly include lactic acid bacteria, bifidobacteria and the like.
Xylo-oligosaccharide is one of the varieties with the strongest function for proliferating bifidobacteria in polymeric saccharides, has the efficacy which is nearly 20 times that of other polymeric saccharides, and has no enzyme for hydrolyzing xylo-oligosaccharide in the gastrointestinal tract of a human body, so that xylo-oligosaccharide can be directly introduced into the large intestine to be preferentially utilized by bifidobacteria to promote the proliferation of the bifidobacteria and simultaneously generate a plurality of organic acids. The pH value of the intestinal tract is reduced, the growth of harmful bacteria is inhibited, and the probiotics are greatly proliferated in the intestinal tract to achieve the health-care effect, so that the health care mysterious place of the xylo-oligosaccharide is formed.
At present, the preparation of xylo-oligosaccharide mostly takes corncobs as raw materials, xylan is obtained by chemical methods such as alkali or acid treatment, and the crude xylo-oligosaccharide solution is prepared by secondary enzymolysis of xylanase and complex enzyme. There are also many patent documents on the preparation of xylo-oligosaccharides, such as: chinese patent document CN109354628A A preparation method of xylo-oligosaccharide, comprising the following steps: alkaline crushing corncobs, freezing to break cell walls, then placing the corncobs in an acidic environment for ultrasonic treatment, and obtaining a xylan solution for later use after the corncobs are treated by AMBERJET 1200H and Amberlite XAD-16 in sequence; and (3) carrying out enzymolysis on the xylan solution by using xylanase to obtain a xylo-oligosaccharide solution, and crystallizing the xylo-oligosaccharide solution.
For another example: chinese patent document CN108949860A discloses an efficient enzymatic hydrolysis preparation method of functional xylo-oligosaccharide. Which comprises the following steps: preparing xylan, hydrolyzing the xylan after preparing a crude enzyme solution, preparing xylanase by taking trapped fluid obtained by ultrafiltration as a carbon source, loading the xylanase on a mesoporous silicon carrier, and performing enzymolysis on the xylan to obtain xylo-oligosaccharide.
Chinese patent document CN108359696A discloses a preparation method of xylo-oligosaccharide, which comprises the following steps: crushing bagasse into powder, and mixing the bagasse powder, water and cane molasses to obtain a first mixture; after the pH value of the first mixture is adjusted and the temperature is raised, adding a complex enzyme preparation, and carrying out constant temperature treatment to obtain a second mixture; performing moist-heat sterilization on the second mixture, cooling, adding an aspergillus niger strain, and performing stable culture and fermentation to obtain a fermentation liquid; filter pressing the fermentation liquor by a plate frame, and adding a filtering and color removing auxiliary agent to obtain filter residue and clear liquid; drying and crushing filter residues to obtain feeding xylo-oligosaccharide; and carrying out vacuum spray drying on the clear liquid to obtain the high-purity xylo-oligosaccharide.
Chinese patent document CN108004284A discloses a preparation method of xylo-oligosaccharide with polymerization degree of 2-6, which comprises the following steps: (1) adding water into the raw material rich in xylan, carrying out high-temperature hydrolysis, and centrifuging to obtain hydrolysate; (2) performing nanofiltration treatment on the hydrolysate to obtain xylo-oligosaccharide trapped fluid; (3) and (3) carrying out enzymolysis on the xylo-oligosaccharide trapped fluid by using endo-xylanase to obtain the xylo-oligosaccharide fluid with the polymerization degree of 2-6.
Chinese patent document CN106755615A discloses a method for degrading lignocellulosic biomass by combining a hydrothermal method and a dilute acetic acid hydrolysis method, comprising the following steps: (1) drying and crushing lignocellulose biomass, performing hydrothermal pretreatment by taking water as a reaction solvent, quickly cooling after the reaction is finished until solid-liquid separation is realized, and collecting solids; (2) taking an acetic acid aqueous solution with the concentration of less than 100mmol/L as a reaction solvent, carrying out hydrothermal hydrolysis reaction on the solid obtained in the step (1), rapidly cooling after the reaction is finished, carrying out solid-liquid separation, and collecting hydrolysate; (3) and refining the collected hydrolysate to obtain a xylo-oligosaccharide product.
Chinese patent document CN106636480A discloses a method for preparing xylo-oligosaccharide from corncobs, A, selecting qualified corncobs, and crushing the corncobs; B. b, placing the corncobs obtained in the step A into water for soaking, and adding acetic acid; C. b, performing high-pressure cooking on the mixture obtained in the step B to obtain hydrolysate; D. cooling the hydrolysate obtained in the step C; E. d, performing heat preservation saccharification on the hydrolysate treated in the step D; F. e, heating and sterilizing the saccharified liquid obtained in the step E; G. cooling the saccharified liquid obtained in the step F; H. g, squeezing and dehydrating the saccharified liquid obtained in the step G; J. carrying out primary decoloring treatment on the liquid obtained in the step H; K. sequentially carrying out membrane filtration and membrane concentration treatment on the liquid obtained in the step J; l, purifying the liquid obtained in the step K; m, evaporating and concentrating the liquid obtained in the step L; and N, performing chromatographic separation on the liquid obtained in the step M to obtain xylo-oligosaccharide.
However, the above prior art has many disadvantages, such as: the preparation process is complex, a large amount of acid and alkali are used in the process, a large amount of sewage is discharged, serious environmental pollution is caused, side reactions are more, byproducts are more, the product purification is difficult, and the extraction rate of xylo-oligosaccharide is low.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for preparing xylooligosaccharide by high-temperature high-pressure treatment. According to the invention, the corncob is treated by a high-temperature and high-pressure process to obtain xylan, xylanase is added for enzymolysis to prepare the crude xylo-oligosaccharide solution, acid and alkali are not used in the process, a large amount of sewage is avoided being discharged, the environmental protection pressure is reduced, meanwhile, only one time of enzymatic hydrolysis is added in the process, the production cost is reduced, the xylanase produced by newly developed trichoderma reesei is used for enzymolysis, the enzyme activity is as high as 508U/ml, and the extraction efficiency of xylo-oligosaccharide is further improved.
The technical scheme of the invention is as follows:
a method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment comprises the following steps:
pulverizing corn cob, sieving, adding water, and concocting to obtain premix;
carrying out high-temperature high-pressure treatment on the premix to obtain a crude xylan extract, wherein the treatment temperature is 95-140 ℃, and the treatment pressure is 0.05-0.25 MPa;
adjusting the mass concentration of the obtained crude xylan extracting solution to 4-6% and the pH value to 4.2-4.8, and performing microwave treatment to obtain xylan liquid; wherein the microwave frequency is 2450MHz, the treatment temperature is 40-55 deg.C, and the microwave time is 10-25 min;
adding xylanase into the xylan liquid for enzymolysis to obtain crude xylo-oligosaccharide liquid;
and (3) carrying out enzyme deactivation treatment on the crude xylo-oligosaccharide solution, and then carrying out decoloration treatment, ion exchange treatment and concentration treatment to obtain the xylo-oligosaccharide solution.
According to the invention, the corn cob is preferably ground into particles with a size of 80-120 meshes, and the mass concentration of the premix is 8-12%.
According to the invention, the high-temperature high-pressure treatment temperature is 115-128 ℃, the treatment pressure is 0.09-0.18MPa, and the treatment time is 4-8 hours.
According to the invention, preferably, the mass concentration of the xylan liquid is adjusted to 4-6% before enzymolysis; preferably, the xylanase is added in an amount of 4-6g/kg dry matter; preferably, the enzymolysis reaction temperature is 50-60 ℃, the enzymolysis reaction time is 20-40h, and the enzymolysis reaction is a standing reaction.
According to the invention, preferably, in the process of adding xylanase into the xylan liquid for enzymolysis, the xylanase is produced by the following strains, and the enzyme activity reaches 508U/ml;
the strain is Trichoderma reesei (Trichoderma reesei) BLCY-007 which is preserved in China general microbiological culture Collection center (CGMCC) in 6 months and 14 days in 2019, the preservation number is CGMCC No.17970, and the address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
The screening process of the trichoderma reesei is as follows:
(1) screening of original strains:
enrichment culture
Selecting soil near a xylo-oligosaccharide production workshop of Bailong Chuang of Dezhou, Shandong, removing surface soil by using a small shovel, taking about 10g of soil 10-20 cm away from the ground, diluting by 10 times by using sterile water, adding a PDA culture medium for enrichment culture, and culturing for 24-48h at the temperature of 24-28 ℃.
The PDA culture medium comprises the following raw materials:
1.0L of potato extract, 20.0g of glucose and 15.0g of agar.
The potato extract is prepared by the following method: removing peel of potato 200g, cutting into small pieces, adding water 1.0L, boiling for 30min, filtering to remove potato pieces, and adding filtrate to 1.0L.
Separation of pure seeds
Adopting a scribing separation method, taking a large test tube containing 5ml of sterile water, taking 2ml of the bacterial liquid after enrichment culture in the step (1), diluting the test tube, fully oscillating and dispersing, selecting an inoculating loop by aseptic operation, firstly carrying out primary parallel scribing on one side of a plate culture medium by using the diluting liquid, carrying out 3-4 parallel scribing on one side of the plate culture medium by using the inoculating loop, rotating the culture dish by an angle of about 60 degrees, burning off the remainder on the inoculating loop, carrying out secondary scribing by using the same scribing method after cooling, and sequentially carrying out third scribing and fourth scribing by using the same method. And after the lineation is finished, covering a dish cover, inverting the culture dish, culturing for 24 hours at the temperature of 28-38 ℃, picking a single colony, and inoculating the single colony on 10 slant culture media to obtain slant seeds which are respectively numbered 01-10.
Inoculating the 01-10 slant seeds into a shake flask culture medium, culturing for 36h at 24-28 ℃, and performing xylanase enzyme activity determination on the 01-10 shake flask fermentation liquor, wherein the 03 shake flask enzyme activity is the highest and reaches 105U/ml.
The plate culture medium comprises the following raw materials:
1.0L of potato extract, 20.0g of glucose and 15.0g of agar.
The slant culture medium comprises the following components in percentage by weight:
1.0L of potato extract, 20.0g of glucose and 15.0g of agar.
The shake flask culture medium comprises the following components:
peeled potato 200g, glucose 20g, KH2PO43g，MgSO4·7H2O1.5 g. Mixing the above materials, adding 1.0L water, boiling for 30min, filtering to remove potato pieces, and adding filtrate to 1.0L.
Inoculating the 01-10 shake flask fermentation liquor into a seed culture medium, and carrying out propagation culture for 10-20 h at the temperature of 24-28 ℃ to prepare a seed solution;
the seed culture medium comprises the following raw materials:
peeled potato 200g, glucose 20g, KH2PO43g，MgSO4·7H2O1.5 g. Mixing the above materials, adding 1.0L water, boiling for 30min, filtering to remove potato pieces, and adding filtrate to 1.0L.
Inoculating the seed liquid into a fermentation culture medium according to the volume ratio of 1-10%, and carrying out amplification culture at 24-28 ℃ for 24-36 h to obtain a thallus fermentation liquid.
The fermentation medium comprises the following raw materials in percentage by weight:
25% of corncob, 4% of glucose, 6% of beef extract, 1% of dried egg white, 0.01% of anhydrous magnesium sulfate, 0.02% of dipotassium hydrogen phosphate, 0.02% of ammonium sulfate and the balance of water, wherein the pH value is 5.0-6.0.
(2) Mutagen or ultraviolet irradiation induced mutation process:
mutagenesis screening
And (2) carrying out ultraviolet mutagenesis on the No. 03 strain, wherein the ultraviolet mutagenesis is carried out by irradiating the strain by adopting a 15W ultraviolet lamp for 20cm for 180s, carrying out ethyl methanesulfonate mutagenesis treatment on the obtained high-yield strain, and finally obtaining the strain with high xylanase yield, namely BLCY-007, wherein the xylanase production activity of the strain reaches 508U/ml under the optimal condition.
And (3) enzyme activity determination:
(i) definition of xylanase Activity Unit
The enzyme amount required for releasing 1 mu mol of reducing sugar from 5mg/ml xylan solution per minute at 37 ℃ and pH 5.5 is an enzyme activity unit U.
(ii) Enzyme activity measuring method
Taking 2ml of xylan substrate with the concentration of 1% (prepared by acetic acid-sodium acetate buffer solution with the pH value of 5.5), adding the xylan substrate into a colorimetric tube, balancing for 10min at 37 ℃, adding 2ml of acidic xylanase enzyme solution which is properly diluted by the acetic acid-sodium acetate buffer solution with the pH value of 5.5 and well balanced at 37 ℃, uniformly mixing, and accurately preserving the temperature at 37 ℃ for reaction for 30 min. After the reaction was completed, 5ml of DNS reagent was added and mixed well to terminate the reaction. Then boiling in boiling water bathCooling to room temperature with tap water for 5min, adding distilled water to constant volume of 25ml, mixing, measuring absorbance A at 540nm with standard blank as blank controlE。
The enzyme activity calculation formula is as follows:
XD＝[(AE－AB)×K+C0]×N×1000/(M×t)
in the formula: xDFor the activity of xylanase in the diluted enzyme solution, U/ml; a. theEThe absorbance of the enzyme reaction solution; a. theBThe absorbance of the enzyme blank liquid; k is the slope of the standard curve; c0Is the intercept of the standard curve; m is the molar mass of xylose, 150.2 g/mol; t is enzymolysis reaction time, min; n is the dilution multiple of enzyme solution; 1000 is conversion factor, 1mmol ═ 1000 μmol.
According to the invention, preferably, the enzyme deactivation treatment mode is high-temperature enzyme deactivation, the enzyme deactivation temperature is 85-98 ℃, and the enzyme deactivation time is 10-15 min.
According to the invention, preferably, the decoloring treatment process is to use activated carbon for decoloring, the adding amount of the activated carbon is 0.8-5% of the dry basis weight of the crude xylo-oligosaccharide solution, the decoloring temperature is 78-85 ℃, the decoloring heat preservation time is 15-30min, and the liquid flow rate during decoloring is 20-30 mL/min;
preferably, the ion exchange column used in the ion exchange treatment process is a combined column of a cation exchange column, an anion exchange column and a cation exchange column, the temperature of the ion exchange treatment is 25-35 ℃, and the flow rate of the ion exchange treatment is 15-25 mL/min; further preferably, the cation exchange column is a strong acid positive resin, and the anion exchange column is a weak base negative resin;
preferably, the concentration treatment mode is vacuum rotary concentration, the working pressure is-0.1 MPa, the working temperature is 60-80 ℃, and the concentration treatment is carried out until the concentration of dry matters is 60-78%.
According to the present invention, a preferred embodiment comprises the steps of:
(1) size mixing: weighing a certain amount of corncobs, crushing, sieving with a 80-120-mesh sieve, and mixing to obtain a premix with a mass concentration of 8-12%, wherein water for size mixing is purified water;
(2) high-temperature high-pressure treatment: carrying out high-temperature high-pressure treatment on the premix in the step (1) to obtain xylan liquid; wherein the treatment temperature is 115-128 ℃, the treatment time is 4-8 hours, and the treatment pressure is 0.09-0.18 MPa;
(3) secondary size mixing: adjusting the xylan liquid obtained in the step (2) into a reaction front liquid with the mass concentration of 4-6%, and adjusting the pH to 4.2-4.8;
(4) enzymolysis: adding xylanase into the pre-reaction solution obtained in the step (3) for enzymolysis reaction, wherein the addition amount of the xylanase is 4-6g/kg of dry matter, so as to obtain crude xylo-oligosaccharide solution; wherein the enzymolysis reaction temperature is 50-60 ℃, the enzymolysis reaction time is 20-40h, the enzymolysis reaction is a standing reaction, and the xylanase is produced by Trichoderma reesei (Trichoderma reesei) BLCY-007;
(5) enzyme deactivation: carrying out enzyme deactivation treatment on the crude xylo-oligosaccharide solution in the step (4), wherein the enzyme deactivation mode is high-temperature enzyme deactivation, the enzyme deactivation temperature is 85-98 ℃, and the enzyme deactivation time is 10-15 min;
(6) refining treatment: carrying out decoloring treatment, ion exchange treatment and concentration treatment on the liquid subjected to enzyme deactivation in the step (5) to obtain xylo-oligosaccharide liquid; wherein the decolorization treatment uses activated carbon for decolorization, the addition amount of the activated carbon is 0.8-5% of the dry basis weight of the liquid after enzyme deactivation, the decolorization temperature is 78-85 ℃, the decolorization heat preservation time is 15-30min, and the liquid flow rate during decolorization is 20-30 mL/min; the ion exchange column used in the ion exchange treatment is a combined column of a cation exchange column, an anion exchange column and a cation exchange column, the temperature of the ion exchange treatment is 25-35 ℃, and the flow rate of the ion exchange treatment is 15-25 mL/min;
the concentration mode is vacuum rotary concentration, the working pressure is-0.1 MPa, the working temperature is 60-80 ℃, and the concentration is carried out until the mass concentration of dry matters is 60-78%.
The invention has the advantages of
1. The traditional production process usually needs acid and alkali resistant equipment, and one-time investment is large; the preparation process is complex, the control of the reaction is not facilitated, the side reactions are more, the byproducts are more, the product purification is difficult, a large amount of acid and alkali is used in the process, a large amount of sewage is discharged, and serious environmental pollution is caused; the xylo-oligosaccharide is prepared by using a high-temperature high-pressure process, the defects of the traditional production process are overcome, the traditional chemical method treatment is replaced, acid and alkali are not used in the preparation process, the large amount of sewage discharge is avoided, and the environmental protection pressure is reduced.
2. The Trichoderma reesei BLCY-007 with high xylanase yield is used, the xylanase activity in the fermentation liquor can reach 508U/ml, is improved by more than 60% compared with the activity of the traditional xylanase, the production cost is obviously reduced, and the yield of xylooligosaccharide is further improved.
3. Compared with the traditional production mode, the yield of xylan and the extraction rate of xylo-oligosaccharide are both greatly improved, the yield of xylan reaches more than 64 percent and reaches as high as 83 percent; the extraction rate of xylo-oligosaccharide reaches over 72 percent and reaches up to 87 percent; the enzyme is added for enzymolysis only once in the process, thereby effectively reducing the production cost.
Detailed Description
The present invention is further illustrated by, but is not limited to, the following specific examples.
The starting materials used in the examples are all conventional commercial products, in which: the xylanase used in examples 1-3 was xylanase SP-min produced by Ulva Neurosa Vahl. The xylanase used in example 4 was a xylanase produced by Trichoderma reesei (Trichoderma reesei) BLCY-007.
The cation exchange column is strong acid positive resin, and the anion exchange column is weak base negative resin;
in the examples, "%" is used in mass% unless otherwise specified.
Xylan yield: the mass of xylan actually obtained/the mass of xylan theoretically obtained × 100%
The extraction rate of the xylo-oligosaccharide is × 100 percent of the mass of the xylo-oligosaccharide/the mass of the xylan.
Example 1
A method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment comprises the following steps:
(1) size mixing: 5.5g of corncob is weighed and prepared into 10 percent premix, wherein the water for size mixing is purified water.
(2) High-temperature high-pressure treatment: and (3) carrying out high-temperature high-pressure treatment on the liquid, wherein the treatment temperature is 121 ℃, the treatment time is 5 hours, and the treatment pressure is 0.10 MPa.
(3) Secondary size mixing: the liquid after the high-temperature high-pressure treatment was adjusted to a pre-reaction liquid having a concentration of 5%, and the pH was adjusted to 4.5.
(4) Enzymolysis: adding xylanase into the solution before reaction, and carrying out enzymolysis reaction to obtain crude xylo-oligosaccharide solution, wherein the addition amount of the xylanase is 4g/kg dry matter; the enzymolysis reaction temperature is 55 ℃, the enzymolysis reaction time is 23 hours, and the enzymolysis reaction is a standing reaction.
(5) Enzyme deactivation: and (3) carrying out enzyme deactivation treatment on the crude xylo-oligosaccharide solution. The enzyme deactivation temperature is 85 deg.C, and the enzyme deactivation time is 10 min.
(6) Refining treatment: and (4) refining the liquid. The method comprises the following steps: decolorizing at 85 deg.C with the addition of active carbon of 1% of dry basis for 20min, and keeping the flow rate of liquid at 25 mL/min;
performing ion exchange treatment, wherein the used ion exchange column is a combined column of a cation exchange column, an anion exchange column and a cation exchange column, the temperature of the ion exchange treatment is 25 ℃, and the flow rate of the ion exchange treatment is 20 mL/min;
vacuum rotary concentration is carried out, the working pressure is-0.1 MPa, and the working temperature is 75 ℃. Concentrating until the concentration of dry matter is 75% to obtain xylo-oligosaccharide solution.
The detection proves that the yield of the xylan is 64 percent, and the extraction rate of the xylo-oligosaccharide is 72 percent.
Example 2
A method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment comprises the following steps:
(1) size mixing: 5g of corncob is weighed and prepared into 9 percent premix, wherein the water for size mixing is purified water.
(2) High-temperature high-pressure treatment: and (3) carrying out high-temperature high-pressure treatment on the liquid, wherein the treatment temperature is 121 ℃, the treatment time is 6 hours, and the treatment pressure is 0.10 MPa.
(3) Secondary size mixing: the liquid after the high-temperature high-pressure treatment was adjusted to a pre-reaction liquid having a concentration of 4%, and the pH was adjusted to 4.7.
(4) Enzymolysis: adding xylanase into the solution before reaction, and carrying out enzymolysis reaction to obtain crude xylo-oligosaccharide solution, wherein the addition amount of the xylanase is 5g/kg dry matter; the enzymolysis reaction temperature is 52 ℃, the enzymolysis reaction time is 24 hours, and the enzymolysis reaction is a standing reaction.
(5) Enzyme deactivation: and (3) carrying out enzyme deactivation treatment on the crude xylo-oligosaccharide solution. The enzyme deactivation temperature is 85 deg.C, and the enzyme deactivation time is 10 min.
(6) Refining treatment: and (4) refining the liquid. The method comprises the following steps: decolorizing at 85 deg.C with the addition of active carbon of 1% of dry basis for 20min, and keeping the flow rate of liquid at 25 mL/min;
performing ion exchange treatment, wherein the used ion exchange column is a combined column of a cation exchange column, an anion exchange column and a cation exchange column, the temperature of the ion exchange treatment is 25 ℃, and the flow rate of the ion exchange treatment is 20 mL/min;
vacuum rotary concentration is carried out, the working pressure is-0.1 MPa, and the working temperature is 75 ℃. Concentrating to obtain xylooligosaccharide solution with dry matter concentration of 75%.
The detection proves that the yield of the xylan is 66 percent, and the extraction rate of the xylo-oligosaccharide is 78 percent.
Example 3
A method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment comprises the following steps:
(1) size mixing: 5g of corncob is weighed and prepared into a premix with the concentration of 10 percent, wherein the water for size mixing is purified water.
(2) High-temperature high-pressure treatment: and (3) carrying out high-temperature high-pressure treatment on the liquid, wherein the treatment temperature is 121 ℃, the treatment time is 1 hour, and the treatment pressure is 0.10 MPa.
(3) Secondary size mixing: the liquid after the high-temperature high-pressure treatment was adjusted to a pre-reaction liquid having a concentration of 4%, and the pH was adjusted to 4.7.
(4) Enzymolysis: adding xylanase into the solution before reaction, and carrying out enzymolysis reaction to obtain crude xylo-oligosaccharide solution, wherein the addition amount of the xylanase is 6g/kg dry matter; the enzymolysis reaction temperature is 52 ℃, the enzymolysis reaction time is 12 hours, and the enzymolysis reaction is a standing reaction.
(5) Enzyme deactivation: and (3) carrying out enzyme deactivation treatment on the crude xylo-oligosaccharide solution. The enzyme deactivation temperature is 85 deg.C, and the enzyme deactivation time is 10 min.
(6) Refining treatment: and (4) refining the liquid. The method comprises the following steps: decolorizing at 85 deg.C with the addition of active carbon of 1% of dry basis for 20min, and keeping the flow rate of liquid at 25 mL/min;
performing ion exchange treatment, wherein the used ion exchange column is a combined column of a cation exchange column, an anion exchange column and a cation exchange column, the temperature of the ion exchange treatment is 25 ℃, and the flow rate of the ion exchange treatment is 20 mL/min;
vacuum rotary concentration is carried out, the working pressure is-0.1 MPa, and the working temperature is 75 ℃. Concentrating to obtain xylooligosaccharide solution with dry matter concentration of 75%.
Detection shows that the yield of xylan is 65% and the extraction rate of xylo-oligosaccharide is 77%.
Example 4
A method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment comprises the following steps:
(1) size mixing: 5g of corncob is weighed and prepared into 9 percent premix, wherein the water for size mixing is purified water.
(2) High-temperature high-pressure treatment: and (3) carrying out high-temperature high-pressure treatment on the liquid, wherein the treatment temperature is 121 ℃, the treatment time is 6 hours, and the treatment pressure is 0.10 MPa.
(3) Secondary size mixing: the liquid after the high-temperature high-pressure treatment was adjusted to a pre-reaction liquid having a concentration of 4%, and the pH was adjusted to 4.7.
(4) Enzymolysis: adding xylanase into the solution before reaction, and carrying out enzymolysis reaction to obtain crude xylo-oligosaccharide solution, wherein the addition amount of the xylanase is 5g/kg dry matter; the enzymolysis reaction temperature is 52 ℃, the enzymolysis reaction time is 24 hours, and the enzymolysis reaction is a standing reaction.
The xylanase is produced by Trichoderma reesei (Trichoderma reesei) BLCY-007 which is preserved in China general microbiological culture collection center (CGMCC) in 2019, 6 months and 14 days, the preservation number is CGMCC No.17970, and the address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
The screening process of the trichoderma reesei is as follows:
A. screening of original strains:
enrichment culture
Selecting soil near a xylo-oligosaccharide production workshop of Bailong Chuang of Dezhou, Shandong, removing surface soil by using a small shovel, taking about 10g of soil 10-20 cm away from the ground, diluting by 10 times by using sterile water, adding a PDA culture medium for enrichment culture, and culturing for 24-48h at the temperature of 24-28 ℃.
The PDA culture medium comprises the following raw materials:
1.0L of potato extract, 20.0g of glucose and 15.0g of agar.
The potato extract is prepared by the following method: removing peel of potato 200g, cutting into small pieces, adding water 1.0L, boiling for 30min, filtering to remove potato pieces, and adding filtrate to 1.0L.
Separation of pure seeds
Adopting a scribing separation method, taking a large test tube containing 5ml of sterile water, taking 2ml of the bacterial liquid after enrichment culture in the step (1), diluting the test tube, fully oscillating and dispersing, selecting an inoculating loop by aseptic operation, firstly carrying out primary parallel scribing on one side of a plate culture medium by using the diluting liquid, carrying out 3-4 parallel scribing on one side of the plate culture medium by using the inoculating loop, rotating the culture dish by an angle of about 60 degrees, burning off the remainder on the inoculating loop, carrying out secondary scribing by using the same scribing method after cooling, and sequentially carrying out third scribing and fourth scribing by using the same method. And after the lineation is finished, covering a dish cover, inverting the culture dish, culturing for 24 hours at the temperature of 28-38 ℃, picking a single colony, and inoculating the single colony on 10 slant culture media to obtain slant seeds which are respectively numbered 01-10.
Inoculating the 01-10 slant seeds into a shake flask culture medium, culturing for 36h at 24-28 ℃, and performing xylanase enzyme activity determination on the 01-10 shake flask fermentation liquor, wherein the 03 shake flask enzyme activity is the highest and reaches 105U/ml.
The plate culture medium comprises the following raw materials:
1.0L of potato extract, 20.0g of glucose and 15.0g of agar.
The slant culture medium comprises the following components in percentage by weight:
1.0L of potato extract, 20.0g of glucose and 15.0g of agar.
The shake flask culture medium comprises the following components:
peeled potato 200g, glucose 20g, KH2PO43g，MgSO4·7H2O1.5 g. Mixing the above materials, adding 1.0L water, boiling for 30min, filtering to remove potato pieces, and adding filtrate to 1.0L.
Inoculating the 01-10 shake flask fermentation liquor into a seed culture medium, and carrying out propagation culture for 10-20 h at the temperature of 24-28 ℃ to prepare a seed solution;
the seed culture medium comprises the following raw materials:
peeled potato 200g, glucose 20g, KH2PO43g，MgSO4·7H2O1.5 g. Mixing the above materials, adding 1.0L water, boiling for 30min, filtering to remove potato pieces, and adding filtrate to 1.0L.
Inoculating the seed liquid into a fermentation culture medium according to the volume ratio of 1-10%, and carrying out amplification culture at 24-28 ℃ for 24-36 h to obtain a thallus fermentation liquid.
The fermentation medium comprises the following raw materials in percentage by weight:
25% of corncob, 4% of glucose, 6% of beef extract, 1% of dried egg white, 0.01% of anhydrous magnesium sulfate, 0.02% of dipotassium hydrogen phosphate, 0.02% of ammonium sulfate and the balance of water, wherein the pH value is 5.0-6.0.
B. Mutagen or ultraviolet irradiation induced mutation process:
mutagenesis screening
And (2) carrying out ultraviolet mutagenesis on the No. 03 strain, wherein the ultraviolet mutagenesis is carried out by irradiating the strain by adopting a 15W ultraviolet lamp for 20cm for 180s, carrying out ethyl methanesulfonate mutagenesis treatment on the obtained high-yield strain, and finally obtaining the strain with high xylanase yield, namely BLCY-007, wherein the xylanase production activity of the strain reaches 508U/ml under the optimal condition.
And (3) enzyme activity determination:
(i) definition of xylanase Activity Unit
The enzyme amount required for releasing 1 mu mol of reducing sugar from 5mg/ml xylan solution per minute at 37 ℃ and pH 5.5 is an enzyme activity unit U.
(ii) Enzyme activity measuring method
Taking 2ml of xylan substrate with the concentration of 1% (prepared by acetic acid-sodium acetate buffer solution with the pH value of 5.5), adding the xylan substrate into a colorimetric tube, balancing for 10min at 37 ℃, adding 2ml of acidic xylanase enzyme solution which is properly diluted by the acetic acid-sodium acetate buffer solution with the pH value of 5.5 and well balanced at 37 ℃, uniformly mixing, and accurately preserving the temperature at 37 ℃ for reaction for 30 min. After the reaction was completed, 5ml of DNS reagent was added and mixed well to terminate the reaction. Boiling in boiling water bath for 5min, cooling to room temperature with tap water, adding distilled water to constant volume to 25ml, mixing, measuring absorbance A at 540nm with standard blank as blank controlE。
The enzyme activity calculation formula is as follows:
XD＝[(AE－AB)×K+C0]×N×1000/(M×t)
in the formula: xDFor the activity of xylanase in the diluted enzyme solution, U/ml; a. theEThe absorbance of the enzyme reaction solution; a. theBThe absorbance of the enzyme blank liquid; k is the slope of the standard curve; c0Is the intercept of the standard curve; m is the molar mass of xylose, 150.2 g/mol; t is enzymolysis reaction time, min; n is the dilution multiple of enzyme solution; 1000 is conversion factor, 1mmol ═ 1000 μmol.
(5) Enzyme deactivation: and (3) carrying out enzyme deactivation treatment on the crude xylo-oligosaccharide solution. The enzyme deactivation temperature is 85 deg.C, and the enzyme deactivation time is 10 min.
(6) Refining treatment: and (4) refining the liquid. The method comprises the following steps: decolorizing at 85 deg.C with the addition of active carbon of 1% of dry basis for 20min, and keeping the flow rate of liquid at 25 mL/min;
performing ion exchange treatment, wherein the used ion exchange column is a combined column of a cation exchange column, an anion exchange column and a cation exchange column, the temperature of the ion exchange treatment is 25 ℃, and the flow rate of the ion exchange treatment is 20 mL/min;
vacuum rotary concentration is carried out, the working pressure is-0.1 MPa, and the working temperature is 75 ℃. Concentrating to obtain xylooligosaccharide solution with dry matter concentration of 75%.
The detection proves that the yield of the xylan is 83 percent, and the extraction rate of the xylo-oligosaccharide is 87 percent.
Comparative example 1
As described in example 1, except that:
high-temperature treatment is not performed, and only high-pressure treatment is performed.
The results of xylan yield and xylo-oligosaccharide extraction in this comparative example are as follows:
the yield of xylan is 31 percent, and the extraction rate of xylo-oligosaccharide is 38 percent.
The xylan in the corncob is a biological macromolecule, and exists in a complex way with other components such as cellulose and wood rope in a natural state. The three components are in discontinuous layered structure and have the function of inhibiting the hydrolysis of acid or alkali. The discontinuous lamellar structure cannot be destroyed by simply carrying out high-pressure treatment, so that the yield of xylan and the extraction rate of xylo-oligosaccharide are not high.
Comparative example 2
As described in example 1, except that:
high-pressure treatment is not performed, and only high-temperature treatment is performed.
The results of xylan yield and xylo-oligosaccharide extraction in this comparative example are as follows:
the yield of xylan is 40%, and the extraction rate of xylo-oligosaccharide is 41%.
For the reasons as in comparative example 1, the discontinuous layered structure could not be destroyed by the high temperature treatment alone, and thus neither the xylan yield nor the xylo-oligosaccharide extraction rate was high.
Comparative example 3
As described in example 1, except that:
the high-temperature treatment temperature is 80 ℃.
The results of xylan yield and xylo-oligosaccharide extraction in this comparative example are as follows:
the yield of xylan is 44%, and the extraction rate of xylo-oligosaccharide is 42%.
As in comparative example 1, the high temperature treatment temperature was too low to destroy the discontinuous layered structure, and thus both the xylan yield and the xylo-oligosaccharide extraction rate were not high.
Comparative example 4
As described in example 1, except that:
the high-temperature treatment temperature is 150 ℃.
The results of xylan yield and xylo-oligosaccharide extraction in this comparative example are as follows:
the yield of xylan is 36%, and the extraction rate of xylo-oligosaccharide is 35%.
Due to the high temperature, the xylan is hydrolyzed excessively to generate xylose. Thereby reducing the yield of xylan and the extraction rate of xylo-oligosaccharide.
Comparative example 5
As described in example 1, except that:
the high-pressure treatment pressure was 0.04 MPa.
The results of xylan yield and xylo-oligosaccharide extraction in this comparative example are as follows:
the yield of xylan is 42%, and the extraction rate of xylo-oligosaccharide is 41%.
As in comparative example 1, the high pressure treatment was too low to destroy the discontinuous layered structure, and thus both the xylan yield and the xylo-oligosaccharide extraction rate were not high.
Comparative example 6
As described in example 1, except that:
the high-pressure treatment pressure was 0.26 MPa.
The results of xylan yield and xylo-oligosaccharide extraction in this comparative example are as follows:
the yield of xylan is 38%, and the extraction rate of xylo-oligosaccharide is 36%.
Excessive hydrolysis of xylan to xylose occurs due to too high pressure treatment. Thereby reducing the yield of xylan and the extraction rate of xylo-oligosaccharide.
Claims (8)
1. A method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment comprises the following steps:
pulverizing corn cob, sieving, adding water, and concocting to obtain premix;
carrying out high-temperature high-pressure treatment on the premix to obtain a crude xylan extract, wherein the treatment temperature is 115-128 ℃, the treatment pressure is 0.09-0.18MPa, and the treatment time is 4-8 hours;
adjusting the mass concentration of the obtained crude xylan extracting solution to 4-6% and the pH value to 4.2-4.8, and performing microwave treatment to obtain xylan liquid; wherein the microwave frequency is 2450MHz, the treatment temperature is 40-55 deg.C, and the microwave time is 10-25 min;
adding xylanase into the xylan liquid for enzymolysis to obtain crude xylo-oligosaccharide liquid; the xylanase used was a xylanase produced by the following strains: the strain is Trichoderma reesei (Trichoderma reesei) BLCY-007 which is preserved in China general microbiological culture Collection center (CGMCC) in 6 months and 14 days in 2019, the preservation number is CGMCC 17970, and the address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing;
and (3) carrying out enzyme deactivation treatment on the crude xylo-oligosaccharide solution, and then carrying out decoloration treatment, ion exchange treatment and concentration treatment to obtain the xylo-oligosaccharide solution.
2. The method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment according to claim 1, wherein the crushed particle size of the corncobs is 80-120 meshes, and the mass concentration of the premix is 8-12%.
3. The method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment according to claim 1, wherein the mass concentration of the xylan liquid is adjusted to 4% -6% before enzymolysis, and the addition amount of xylanase is 4-6g/kg dry matter.
4. The method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment according to claim 1, wherein the enzymolysis reaction temperature is 50-60 ℃, and the enzymolysis reaction time is 20-40 h.
5. The method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment according to claim 1, characterized in that the enzyme deactivation treatment mode is high-temperature enzyme deactivation, the enzyme deactivation temperature is 85-98 ℃, and the enzyme deactivation time is 10-15 min.
6. The method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment according to claim 1, wherein the decolorization treatment process is decolorization by using active carbon, the addition amount of the active carbon is 0.8-5% of the dry mass of the crude xylo-oligosaccharide solution, the decolorization temperature is 78-85 ℃, the decolorization heat preservation time is 15-30min, and the flow rate of the liquid during decolorization is 20-30 mL/min.
7. The method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment according to claim 1, wherein the ion exchange column used in the ion exchange treatment process is a combination column of cation exchange column-anion exchange column-cation exchange column, the ion exchange treatment temperature is 25-35 ℃, and the flow rate of the ion exchange treatment is 15-25 mL/min.
8. The method for preparing xylo-oligosaccharide by high-temperature high-pressure treatment according to claim 1, wherein the concentration treatment mode is vacuum rotary concentration, the working pressure is-0.1 MPa, the working temperature is 60-80 ℃, and the concentration of dry substances after concentration treatment is 60-78%.
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