STRAIN OF TRICHODERMA REESEI AND CULTURE METHOD AND USE THEREOF

Abstract

The invention relates to a strain of Trichoderma reesei BLCY-007 and its application in the production of xylooligosaccharides.

Claims

1. A strain of Trichoderma reesei BLCY-007, which has an accession number: CGMCC No. 17970, wherein said accession number was obtained when the strain was deposited on Jun. 14, 2019 in China General Microbiological Culture Collection Center (CGMCC).

2. A method for culturing the Trichoderma reesei BLCY-007 according to claim 1, comprising the following steps: (i) inoculating the Trichoderma reesei BLCY-007 into a PDA medium, performing an activating cultivation under a temperature from 24° C. to 28° C. for 12 to 24 hours to obtain an activated strain; (ii) inoculating the activated strain obtained in step (i) into a seed culture medium, and performing a proliferating cultivation under a temperature from 24° C. to 28° C. for 24 to 36 hours to obtain a seed broth; (iii) inoculating the seed broth obtained in step (ii) into a fermentation medium at a volume percentage of 1% to 10%, and performing an expanding cultivation at a temperature from 24° C. to 28° C. for 24 to 36 hours to obtain a bacterial fermentation broth.

3. The method according to claim 2, characterized in that, said seed culture medium in step (ii) has raw material components as follows: 200 g of peeled potato, 20 g of glucose, 3 g of KH.sub.2PO.sub.4, 1.5 g of MgSO.sub.4.7H.sub.2O; the above components are mixed, added with 1.0 L of water and boiled for 30 min, filtered to remove potato pieces, and the filtrate is supplemented to 1.0 L.

4. The method according to claim 2, characterized in that, said fermentation medium in step (iii) has raw material components as follows, in terms of weight percentages: corncobs 25%, glucose 4%, beef extract 6%, peptone 1%, anhydrous magnesium sulfate 0.01%, dipotassium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance of water, pH=5.0 to 6.0.

5. The method according to claim 2, characterized in that the PDA medium in step (i) has raw material components as follows: 1.0 L of potato extract liquid, 20.0 g of glucose, 15.0 g of agar; the potato extract liquid is prepared by the following method: 200 g of peeled potato is taken, cut into small pieces, added with 1.0 L of water and boiled for 30min, filtered to remove potato pieces, and the filtrate is supplemented to 1.0 L.

6. Use of the Trichoderma reesei BLCY-007 according to claim 1 in the production of xylanase.

7. The use according to claim 6, characterized in that the use comprises steps as follows: providing the bacterial fermentation broth obtained by the method according to claim 2, subjecting it to centrifugation separation, washing the obtained bacterial cells, performing a second centrifugation, and retaining the precipitate as the crude xylanase enzyme preparation.

8. The use according to claim 6, characterized in that the use comprises steps are as follows: (1) providing the bacterial fermentation broth obtained by the method according to claim 2; (2) subjecting the bacterial fermentation broth to centrifugation separation; (3) collecting a supernatant from the product of the previous step, wherein the supernatant is the crude xylanase enzyme preparation.

9. The use according to claim 8, wherein the centrifugation separation is performed at 4° C. and 10000 r/min for 10 min.

10. Use of the Trichoderma reesei BLCY-007 according to claim 1 in the production of xylooligosaccharides.

11. A method for preparing a crude enzyme preparation, comprising: (1) providing the bacterial fermentation broth obtained by the method according to claim 2; (2) subjecting the bacterial fermentation broth to centrifugation separation; (3) collecting a supernatant from the product of the previous step, wherein the supernatant is the crude xylanase enzyme preparation.

12. A method for preparing xylooligosaccharides, comprising: (1) preparing a crude xylanase enzyme preparation by the method according to claim 11; (2) subjecting a xylan to enzymolysis treatment by using said crude xylanase enzyme preparation to obtain xylanoligosaccharides; preferably, the enzymolysis treatment is performed at a temperature from 50° C. to 60° C.; preferably, the enzymolysis treatment is performed at a pH of 5.5 to 6.5.

13. A method for preparing xylooligosaccharides, comprising the following steps: preparing a premix by crushing and sieving corncobs and adding water thereto; subjecting the premix to high-temperature and high-pressure treatment to obtain a crude extract liquid of xylan, wherein the treatment is performed at a treatment temperature from 95° C. to 140° C. and a treatment pressure of 0.05 to 0.25 MPa; adjusting the crude extract liquid of xylan to a mass concentration of 4% to 6% and to a pH between 4.2 and 4.8, and performing a microwave treatment to obtain a xylan solution, wherein the microwave treatment is performed at a microwave frequency of 2450 MHz and a treatment temperature from 40° C. to 55° C. for a microwaving time of 10 to 25 minutes; adding a xylanase into the xylan solution to perform enzymolysis to obtain a crude xylooligosaccharide solution; wherein the xylanase used is a xylanase produced by the following strain: said strain is Trichoderma reesei BLCY-007, which has an Accession number: CGMCC No. 17970, which was obtained when the strain was deposited on Jun. 14, 2019 in China General Microbiological Culture Collection Center (CGMCC); subjecting the crude xylooligosaccharide solution to enzyme inactivation treatment, decolorization treatment, ion exchange treatment, and concentration treatment to obtain an xylooligosaccharide solution.

14. (canceled)

15. The method for preparing xylooligosaccharides according to claim 13, characterized in that the high-temperature and high-pressure treatment is performed at a treatment temperature from 115° C. to 128° C. and a treatment pressure of 0.09 to 0.18 MPa for a treatment time of 4 to 8 hours.

16-20. (canceled)

21. The method for preparing xylooligosaccharides according to claim 13, characterized in that the concentration treatment is performed by vacuum rotary concentration at a working pressure of −0.1 MPa, a working temperature from 60° C. to 80° C., and the concentration of dry matter in the crude xylooligosaccharide solution is 60% to 78% after the concentration treatment.

22. The method for preparing xylooligosaccharides according to claim 13, wherein the xylanase used for the enzymolysis is a crude enzyme preparation isolated from the fermentation product of Trichoderma reesei BLCY-007.

23. The method for preparing xylooligosaccharides according to claim 22, wherein the crude enzyme preparation is prepared by a method comprising: (1) providing the bacterial fermentation broth obtained; (2) subjecting the bacterial fermentation broth to centrifugation separation; (3) collecting a supernatant from the product of the previous step, wherein the supernatant is the crude xylanase enzyme preparation.

24. The method for preparing xylooligosaccharides according to claim 23, wherein the method for preparing the fermentation broth of Trichoderma reesei BLCY-007 in step (1) comprises: (i) inoculating the Trichoderma reesei BLCY-007 into a PDA medium, performing an activating cultivation under a temperature from 24° C. to 28° C. for 12 to 24 hours to obtain an activated strain; (ii) inoculating the activated strain obtained in step (i) into a seed culture medium, and performing a proliferating cultivation under a temperature from 24° C. to 28° C. for 24 to 36 hours to obtain a seed broth; (iii) inoculating the seed broth obtained in step (ii) into a fermentation medium at a volume percentage of 1% to 10%, and performing an expanding cultivation at a temperature from 24° C. to 28° C. for 24 to 36 hours to obtain a bacterial fermentation broth.

25-27. (canceled)

28. The method for preparing xylooligosaccharides according to claim 13, characterized in one or more of the following: the corncobs are crushed to a particle size that is capable of passing through 80 to 120 mesh sieve, and the premix has a mass concentration of 8% to 12%; the xylan solution is adjusted to a mass concentration of 4% to 6% before enzymolysis, wherein the xylanase is added in an amount of 4 to 6 g/kg dry matter; the enzymolysis is performed at an enzymolysis temperature from 50° C. to 60° C. for an enzymolysis time from 20 to 40 hours; the enzyme inactivation treatment is performed at an enzyme inactivation temperature from 85° C. to 98° C. for an enzyme inactivation time from 10 to 15 minutes; the decolorization treatment is performed by using activated carbon, the activated carbon is added in an amount of 0.8% to 5% of the dry mass of the crude xylooligosaccharide solution, the decolorization is performed at a temperature from 78° C. to 85° C., at a liquid flow rate of 20 to 30 mL/min for a time from 15 to 30 minutes; the ion exchange treatment is performed on an ion exchange column that is a combined column of cation exchange column-anion exchange column-cation exchange column, at a temperature from 25° C. to 35° C. and at a flow rate of 15 to 25 mL/min.

29. The method for preparing xylooligosaccharides according to claim 24, characterized in one or more of the following: wherein the PDA medium in step (i) has raw material components as follows: 1.0 L of potato extract liquid, 20.0 g of glucose, 15.0 g of agar; wherein the seed culture medium in step (ii) has raw material components as follows: 200 g of peeled potato, 20 g of glucose, 3 g of KH.sub.2PO.sub.4, 1.5 g of MgSO.sub.4.7.sub.2O; the above components are mixed, added with 1.0 L of water and boiled for 30 min, filtered to remove potato pieces, and the filtrate is supplemented to 1.0 L; wherein the fermentation medium in step (iii) has raw material components as follows, in terms of weight percentages: corncobs 25%, glucose 4%, beef extract 6%, peptone 1%, anhydrous magnesium sulfate 0.01%, dipotassium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance of water, pH=5.0 to 6.0.

Description

EXAMPLE 1

[0134] A strain of Trichoderma reesei BLCY-007, which has an Accession number: CGMCC No. 17970, is provided. The number was obtained when the strain was deposited on Jun. 14, 2019 in China General Microbiological Culture Collection Center, the Institute of Microbiology, Chinese Academy of Sciences, at the address: No.1, West Beichen Road, Chaoyang District, Beijing.

[0135] The mutagenesis and screening process of the aforementioned Trichoderma reesei BLCY-007 was as follows:

[0136] (1) Screening of Original Strain:

[0137] Enrichment Cultivation

[0138] The soil, which is near the xylooligosaccharides production workshop of Bailong Chuangyuan, Dezhou City, Shandong Province, was selected, and the top layer of the soil was removed with a small shovel; about 10 g of the soil was taken from the ground at a depth of 10˜20 cm, diluted 10 times with sterile water, and added to a PDA medium (Potato Dextrose Agar) to for enrichment cultivation, and the cultivation was performed at a temperature from 24° C. to 28° C. for 36 h.

[0139] The raw material components of the PDA culture medium were as follows:

[0140] 1.0 L of potato extract liquid, 20.0 g of glucose, 15.0 g of agar.

[0141] The potato extract liquid was prepared by a method as follows: 200 g of peeled potato was taken, cut into small pieces, added with 1.0 L of water and boiled for 30 min, filtered to remove potato pieces, and the filtrate was supplemented to 1.0 L.

[0142] Separation of Pure Strain

[0143] A streaking method was used in this step. A large test tube containing 5 ml of sterile water was taken, 2 ml of the bacterial solution obtained from the enrichment cultivation in step (1) was taken and added to the test tube and diluted, shaken thoroughly for dispersion, a loop of the diluted solution was aseptically picked up by using an inoculation loop and subjected to the first parallel streaking of 3 to 4 streaks on one side of a plate medium of a petri dish; then the petri dish was turned about 60 degrees, and the remainder on the inoculation loop was burned off.

[0144] After cooling, the second streaking was performed by the same method as that of the first streaking; and the third and fourth streakings were performed in sequence by the same method. After the end of streaking, the petri dish was covered with a lid, turned upside down, and incubated at 28° C. to 38° C. for 24 hours, then a single colony was picked up and inoculated on 10 slant culture medias to obtain slant seeds, numbered as 01 to 10, respectively.

[0145] The 01 to 10 slant seeds of were separately inoculated in shake flask culture medium and cultured at a temperature from 24° C. to 28° C. for 36 hours to obtain 01 to 10 shake flask fermentation broths. The xylanase enzyme activities of 01 to 10 shake flask fermentation broths were measured, and the 03 shake flask fermentation broth showed the highest enzyme activity, which is 105 U/ml.

[0146] The raw material components of the plate medium were as follows: 1.0 L of potato extract liquid, 20.0 g of glucose, and 15.0 g of agar.

[0147] Potato extract liquid: 200 g of peeled potato was taken, cut into small pieces, added with 1.0 L of water and boiled for 30 min, filtered to remove potato pieces, and the filtrate was supplemented to 1.0 L.

[0148] The components of the slant medium were as follows: 1.0 L of potato extract liquid, 20.0 g of glucose, and 15.0 g of agar.

[0149] The components of the shake flask culture medium were as follows: 200 g of peeled potato, 20 g of glucose, 3 g of KH.sub.2PO.sub.4, 1.5 g of MgSO.sub.4.7H.sub.2O. After the above components were mixed, 1.0 L of water was added and boiled for 30 min, filtered to remove potato pieces, and the filtrate was supplemented to 1.0 L.

[0150] (2) Mutation Induced by Mutagenic Agent or Ultraviolet Radiation:

[0151] Mutagenesis Screening

[0152] The strain in the fermentation broth of 03 shake flask was subjected to ultraviolet mutagenesis. The ultraviolet mutagenesis was performed by irradiating with a 15 W ultraviolet lamp at a distance of 20 cm for a radiation time of 180 seconds, the obtained high-yield strain was then mutated with ethyl methyl sulfonate, and the finally obtained high-yield xylanase-producing strain was named as BLCY-007.

EXAMPLE 2

[0153] The method for culturing the Trichoderma reesei BLCY-007 described in Example 1 comprised the following steps:

[0154] (1) the Trichoderma reesei BLCY-007 was taken and inoculated in a PDA medium, an activating cultivation was performed for 12 hours at a temperature of 24° C. to obtain an activated strain;

[0155] the PDA medium had raw material components as follows:

[0156] 1.0 L of potato extract liquid, 20.0 g of glucose, 15.0 g of agar;

[0157] the potato extract liquid was prepared by a method as follows: 200 g of peeled potato was taken, cut into small pieces, added with 1.0 L of water and boiled for 30 min, filtered to remove potato pieces, and the filtrate was supplemented to 1.0 L;

[0158] (2) the activated strain obtained in step (1) was taken and inoculated into a seed culture medium, a proliferating cultivation was performed for 24 hours at a temperature of 24° C. to obtain a seed broth;

[0159] the seed culture medium had raw material components as follows:

[0160] 200 g of peeled potato, 20 g of glucose, 3 g of KH.sub.2PO.sub.4, 1.5 g of MgSO.sub.4.7H.sub.2O; the above components were mixed, then added with 1.0 L of water and boiled for 30 min, filtered to remove potato pieces, and the filtrate was supplemented to 1.0 L;

[0161] (3) the seed broth prepared in step (2) was taken and inoculated in a fermentation medium at a volume percentage of 2%, and subjected to an expanding cultivation at a temperature of 24° C. for 24 hours to obtain a bacterial fermentation broth;

[0162] the fermentation medium had raw material components as follows, in terms of percentages by weight:

[0163] corncobs 25%, glucose 4%, beef extract 6%, peptone 1%, anhydrous magnesium sulfate 0.01%, dipotassium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance of water, pH =5.0 to 6.0.

EXAMPLE 3

[0164] The method for culturing the Trichoderma reesei BLCY-007 described in Example 1 comprised the following steps:

[0165] (1) the Trichoderma reesei BLCY-007 was taken and inoculated in a PDA medium, an activating cultivation was performed for 24 hours at 28° C. to obtain an activated strain;

[0166] the PDA medium had raw material components as follows:

[0167] 1.0 L of potato extract liquid, 20.0 g of glucose, 15.0 g of agar;

[0168] the potato extract liquid was prepared by a method as follows: 200 g of peeled potato was taken, cut into small pieces, added with 1.0 L of water and boiled for 30 min, filtered to remove potato pieces, and the filtrate was supplemented to 1.0 L;

[0169] (2) the activated strain obtained in step (1) was taken and inoculated into a seed culture medium, a proliferating cultivation was performed for 36 hours at 28° C. to obtain a seed broth;

[0170] the seed culture medium had raw material components as follows:

[0171] 200 g of peeled potato, 20 g of glucose, 3 g of KH.sub.2PO.sub.4, 1.5 g of MgSO.sub.4.7H.sub.2O; the above components were mixed, then added with 1.0 L of water and boiled for 30 min, filtered to remove potato pieces, and the filtrate was supplemented to 1.0 L;

[0172] (3) the seed broth prepared in step (2) was taken and inoculated in a fermentation medium at a volume percentage of 8%, and subjected to an expanding cultivation at 28° C. for 36 hours to obtain a bacterial fermentation broth;

[0173] the fermentation medium had raw material components as follows, in terms of percentages by weight:

[0174] corncobs 25%, glucose 4%, beef extract 6%, peptone 1%, anhydrous magnesium sulfate 0.01%, dipotassium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, balance of water, pH =5.0 to 6.0.

COMPARATIVE EXAMPLE 1

[0175] The strain in 03 shake flask fermentation broth of Example 1 was used. Cultivation was performed by reference to the method of Example 2 to obtain a bacterial fermentation broth.

COMPARATIVE EXAMPLE 2

[0176] The Trichoderma reesei purchased from Beijing Beina Chuanglian Biotechnology Research Institute was used as the culture strain. Cultivation was performed by reference to the method of Example 2 to obtain a bacterial fermentation broth.

EXAMPLE 4

[0177] Preparation of crude enzyme preparation: the bacterial fermentation broths prepared in Example 2, Comparative Example 1 and Comparative Example 2 were subjected to centrifugation separation, and the centrifugation separation was performed under conditions of: crude enzyme preparation temperature: 4° C., centrifugal rotation speed: 10000 r/min, centrifugal time: 10 min. After centrifugation, supernatants were collected to obtain crude enzyme preparations.

EXPERIMENTAL EXAMPLE 1

[0178] The crude enzyme preparations obtained in Example 4 were tested for their xylanase activities.

[0179] In the present disclosure, “enzyme activity” and “enzymatic activity” have the same meaning, and both refer to xylanase enzyme activity.

[0180] The determination of xylanase activity was performed according to “GBT 23874-2009, determination of xylanase activity in feed additives—spectrophotometric method”. The determination method was briefly described as follows:

[0181] (i) Definition of Xylanase Activity Unit

[0182] Under condition of 37° C. and pH 5.5, an amount of enzyme required to release 1 μmol of reducing sugar per minute from a xylan solution with a concentration of 5 mg/ml was an enzyme activity unit, U.

[0183] (ii) Preparation of Reaction Enzyme Solution of Liquid Sample

[0184] The above crude enzyme preparations were diluted with acetic acid-sodium acetate buffer solution (pH=5.5) to a certain volume, and the xylanase enzyme activity in the liquid sample after dilution was controlled between 0.04 U/mL and 0.10 U/mL.

[0185] (iii) Method for Determination of Enzyme Activity

[0186] 2 ml of 100 mg/ml xylan substrate (pH=5.5, acetic acid-sodium acetate buffer solution) was taken and added to a colorimetric tube, equilibrated at 37° C. for 10 min, then added with 2 ml of the reaction enzyme solution of the liquid sample that had been equilibrated at 37° C. They were mixed well and incubated accurately at 37° C. for 30 min. After the reaction was completed, 5 ml of DNS reagent was added and mixed well to stop the reaction. Then the reaction mixture were boiled in a boiling water bath for 5 minutes, cooled to room temperature with tap water, added with distilled water to a certain volume of 25 ml, mixed well, and tested for the absorbance A.sub.E at 540 nm by using a standard blank as blank control.

[0187] Enzyme activity calculation formula:

X.sub.D=[(A.sub.E−A.sub.B)×K+C.sub.0]×1000/(M×t)

X=X.sub.D×N

[0188] wherein: X represents the xylanase activity of the liquid sample, U/ml; X.sub.D represents the xylanase activity of the reaction enzyme solution of the liquid sample, U/ml; A.sub.E represents the absorbance of the enzyme reaction solution; A.sub.B represents the absorbance of the enzyme blank solution; K represents the slope of the standard curve; C.sub.0 represents the intercept of the standard curve; M represents the molar mass of xylose, 150.2 g/mol; t represents the enzymolysis time, min; N represents the enzyme dilution multiple; 1000 represents the conversion factor, 1 mmol=1000 μmol.

[0189] The results were shown in Table 1:

TABLE-US-00001 TABLE 1 Comparative Example 2 Comparative Beina Chuanglian Example 2 Example 1 Biotechnology No. BLCY-007 Original strain Research Institute Enzyme activity, 506 101 163 U/mL

[0190] It could be seen from Table 1 that compared with the original strain and other commercially available strain, the xylanase activity of the crude enzyme preparation obtained based on the Trichoderma reesei BLCY-007 had been greatly improved.

EXPERIMENTAL EXAMPLE 2

[0191] A comparison experiment between the Trichoderma reesei strain disclosed in Chinese Patent CN1185336C and the BLCY-007 strain of present invention was performed.

[0192] The BLCY-007 strain of Example 1 was used as raw material. A liquid fermentation and a solid fermentation were performed respectively according to the methods described in step (3) and (4) of Examples, section 5 on pages 4 to 5 of the description of CN1185336C, and a liquid fermentation product-B and a solid fermentation product-B were obtained.

[0193] According to the xylanase enzyme activity determination method described in section 6 and the glucanase enzyme activity determination method described in section 7 on pages 5 to 8 of the description of CN1185336C, the liquid fermentation product-B and the solid fermentation product-B were respectively tested for their xylanase enzyme activities and glucanase enzyme activities, and the results were as follows:

TABLE-US-00002 TABLE 2 Enzyme activity Enzyme activity of xylanase of glucanase BLCY-007 Liquid 587 U/ml Without glucanase strain fermentation enzyme activity product-B Solid 37605 U/g Without glucanase fermentation enzyme activity product-B CN1185336C Liquid 480 U/ml  610 U/ml strain fermentation product Solid 30000 U/g 35000 U/g fermentation product

[0194] The methods for preparing xylooligosaccharides using corncobs as raw material are described below through specific examples.

[0195] The xylanase used in Example B1 was a crude enzyme preparation obtained based on Trichoderma reesei BLCY-007. The xylanase used in Comparative Examples B1 to B7 was xylanase SP-min, which was produced by Qingdao Vland Biological Co., Ltd.

[0196] In the cation exchange column used in the following examples, a strong acid cation resin, which was D001 macroporous cation exchange resin produced by Zhejiang Zhengguang Industrial Co., Ltd, was adopted.

[0197] In the anion exchange column used in the following examples, a weak base anion resin, which was D354FD macroporous weak base anion resin produced by Zhejiang Zhengguang Industrial Co., Ltd, was adopted.

[0198] The device used for microwave treatment in the following examples was MDS-6 microwave digestion/extraction instrument produced by Shanghai Sineo Microwave Chemistry Technology Co., Ltd., and its parameters were as follows: output power, 0 to 1000 W; temperature control range: 0° C. to 250° C., temperature accuracy: ±1° C.; pressure control range: 0.1 to 5 MPa, pressure accuracy: 0.1 MPa.

[0199] Unless otherwise specified, “%” used in the following examples was a mass percentage.

[0200] In the following examples, the mass concentration referred to a mass concentration based on dry matter of corncobs.

[0201] In the following examples, the treatment pressure value should be understood as an incremental value based on 1 standard atmospheric pressure. For example, a treatment pressure of 0.1 Mpa referred to an increase of 0.1 Mpa on the basis of 1 standard atmosphere.

EXAMPLE B1

[0202] The method for preparing xylooligosaccharides by high-temperature and high-pressure treatment comprised the following steps:

[0203] (1) Pulping: 5 g of corncobs (15% water content) was provided, crushed and the crushed product was sieved by a 100-mesh sieve, and the corncob powder that passed through the sieve was collected. The corncob powder was mixed with purified water to prepare a premix with a dry matter concentration of 9 wt %, wherein the water used for the pulping was purified water.

[0204] (2) High-temperature and high-pressure treatment: the premix was placed in a pressure container and subjected to high-temperature and high-pressure treatment with a treatment temperature of 121° C. and a treatment pressure of 0.10 MPa for a treatment time of 6 hours to obtain a crude extract liquid of xylan.

[0205] (3) Microwave treatment: the dry matter concentration of the crude extract liquid of xylan was adjusted to 4%, and the pH was adjusted to 4.7 to obtain 106 ml of pre-reaction solution; the pre-reaction solution was placed in a microwave treatment device for microwave treatment to obtain a xylan solution; the microwave power of the microwave treatment device was 800 W, the microwave frequency was 2450 MHz, the treatment temperature was 50° C., and the microwaving time was 15 min.

[0206] (4) Enzymolysis: a xylanase was added to the xylan solution and an enzymolysis was performed to obtain a crude xylooligosaccharide solution, in which the xylanase was added in an amount of 5 g/kg dry matter; the enzymolysis temperature was 52° C., the enzymolysis time was 24 h, the pH value of the xylan solution was controlled at 5.5 to 6.5, and the enzymolysis was a static reaction.

[0207] The xylanase was a crude enzyme preparation obtained from the fermentation broth of Trichoderma reesei BLCY-007. The Trichoderma reesei BLCY-007 has an Accession number: CGMCC No. 17970, which was obtained when the strain was deposited on Jun. 14, 2019 in China General Microbiological Culture Collection Center, the Institute of Microbiology, Chinese Academy of Sciences, at the address: No.1, West Beichen Road, Chaoyang District, Beijing.

[0208] (5) Enzyme inactivation: the above crude xylooligosaccharide solution was subjected to enzyme inactivation treatment. The enzyme inactivation temperature was 85° C., and the enzyme inactivation time was 10 minutes.

[0209] (6) Refining treatment: the product after enzyme inactivation was subjected to refining treatment, comprising:

[0210] decolorization, the product after enzyme inactivation passed through an activated carbon filter element, the activated carbon was added in an amount of 1% of dry basis, the temperature of the decolorization was 85° C., the time of the decolorization was 20 min, and the liquid flow rate was 25 mL/min during decolorization;

[0211] ion exchange treatment, the ion exchange column used herein was a combined column of cation exchange column-anion exchange column-cation exchange column, the temperature of the ion exchange treatment was 25° C., and the flow rate for the ion exchange treatment was 20 mL/min;

[0212] rotary concentration under vacuum, a rotary thin film vacuum concentration device was used for concentration treatment, in which the device had a working pressure of −0.1 MPa, and a working temperature of 75° C. The concentration was performed until the product had a dry matter concentration of 75%, thereby obtaining an xylooligosaccharide solution.

[0213] Theoretically, the mass of xylan obtained from 5 g of corncobs was 1.8 g. After testing, the mass of xylan in the xylan solution actually obtained in step (3) was 1.494 g, and the mass of xylooligosaccharides obtained in step (6) was 1.299 g. It can be calculated that the yield of xylan was 83%, and the extraction rate of xylooligosaccharides was 87%.

COMPARATIVE EXAMPLE B1

[0214] Comparative Example B1 was similar to Example B1, except that: in step (2), the treatment temperature was 60° C., and the treatment pressure was 0.10 MPa. In step (4), the xylanase used was xylanase SP-min produced by Qingdao Vland Biotech INC, and the amount of xylanase added was 5 g/kg dry matter.

[0215] The test results showed that the yield of xylan and the extraction rate of xylooligosaccharides in this example were as follows: the yield of xylan was 31%, and the extraction rate of xylooligosaccharides was 38%.

[0216] The xylan in corncobs are a biological macromolecule, which in its natural state exists as a complex with other components such as cellulose and lignin. The three have a discontinuous laminate structure, which hinders the acid or alkali hydrolysis. Merely performing a high-pressure treatment could not destroy the discontinuous laminate structure, therefore, the yield of xylan and the extraction rate of xylooligosaccharides were not high.

COMPARATIVE EXAMPLE B2

[0217] Comparative Example B2 was similar to Example B1, except that: in step (2), the treatment temperature was 121° C., and the treatment pressure was 0.01 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the amount of xylanase added was 5 g/kg dry matter.

[0218] The test results showed that the yield of xylan and the extraction rate of xylooligosaccharides in this example were as follows: the yield of xylan was 40%, and the extraction rate of xylooligosaccharides was 41%.

[0219] Similar to the reason for Comparative Example B 1, merely performing a high-temperature treatment could not destroy the discontinuous laminate structure, therefore, the yield of xylan and the extraction rate of xylooligosaccharides were not high.

COMPARATIVE EXAMPLE B3

[0220] Comparative Example B3 was similar to Example B1, except that: in step (2), the treatment temperature was 80° C., and the treatment pressure was 0.10 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the amount of xylanase added was 5 g/kg dry matter.

[0221] The test results showed that the yield of xylan and the extraction rate of xylooligosaccharides in this example were as follows: the yield of xylan was 44%, and the extraction rate of xylooligosaccharide was 42%.

[0222] Because the treatment temperature was too low to destroy the discontinuous laminate structure, the yield of xylan and the extraction rate of xylooligosaccharides were not high.

COMPARATIVE EXAMPLE B4

[0223] Comparative Example B4 was similar to Example B1, except that: in step (2), the treatment temperature was 150° C., and the treatment pressure was 0.10 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the amount of xylanase added was 5 g/kg dry matter.

[0224] The test results showed that the yield of xylan and the extraction rate of xylooligosaccharides in this example were as follows: the yield of xylan was 36%, and the extraction rate of xylooligosaccharides was 35%.

[0225] Because the high-temperature treatment adopted an excessively high temperature, the xylan was excessively hydrolyzed to generate xylose, which reduced the yield of xylan and the extraction rate of xylooligosaccharides.

COMPARATIVE EXAMPLE B5

[0226] Comparative Example B5 was similar to Example B1, except that: in step (2), the treatment temperature was 121° C., and the treatment pressure was 0.04 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the amount of xylanase added was 5 g/kg dry matter.

[0227] The test results showed that the yield of xylan and the extraction rate of xylooligosaccharides in this example were as follows: the yield of xylan was 42%, and the extraction rate of xylooligosaccharides was 41%.

[0228] Because the treatment pressure was too low to destroy the discontinuous laminate structure, the yield of xylan and the extraction rate of xylooligosaccharides were not high.

COMPARATIVE EXAMPLE B6

[0229] Comparative Example B6 was similar to Example B1, except that: in step (2), the treatment temperature was 121° C., and the treatment pressure was 0.26 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the amount of xylanase added was 5 g/kg dry matter.

[0230] The test results showed that the yield of xylan and the extraction rate of xylooligosaccharide in this example were as follows: the yield of xylan was 38%, and the extraction rate of xylooligosaccharides was 36%.

[0231] Because the treatment pressure was too high, the xylan was excessively hydrolyzed to produce xylose, so that the yield of xylan and the extraction rate of xylooligosaccharides were reduced.

COMPARATIVE EXAMPLE B7

[0232] Comparative Example B7 was similar to Example B1, except that:

[0233] in step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the amount of xylanase added was 5 g/kg dry matter.

[0234] Analysis and Test

[0235] In the above examples, the yield of xylan and the extraction rate of xylooligosaccharides were obtained by the following calculation formulas:

yield of xylan=detected mass of xylan/theoretically obtainable mass of xylan×100%   Formula 1

extraction rate of xylooligosaccharides=mass of xylooligosaccharides/mass of xylan×100%.   Formula 2

[0236] In the Formula 1, the mass of theoretically obtainable xylan=mass of corncobs×xylan content of corncob. Corncobs usually has a xylan content of 35% to 40%. The xylan content of the corncobs used in the above examples and comparative examples was 36%.

[0237] In the Formula 1, the method for detecting xylan content of the xylan solution was as follows: the xylan solution was adjusted to a pH value of 5, added with 95% (by volume) ethanol in a volume four times of the that of the xylan solution, subjected to alcohol precipitation overnight and then centrifuged at 3000 r/min for 10 minutes, and the precipitate was collected, added with a certain amount of 7% H.sub.2SO.sub.4 for hydrolysis at 100° C. for 2 hours, then neutralized, supplemented to a certain volume, filtered, tested for the mass of reducing sugar in the filtrate, then the mass was multiplied by the xylan polymerization factor 0.9 to give the mass of xylan. DNS method was used for determination of reducing sugar.

[0238] In the Formula 1, the detected mass of xylan=0.9× mass of reducing sugar determined by DNS method.

[0239] In the Formula 2, the method for detection of xylooligosaccharides was in accordance with GB/T 35545-2017 (Appendix A: High performance liquid chromatography) for detection of xylooligosaccharides.

[0240] The parameters of some steps and the product yields of Example B1 and Comparative Examples B1 to B7 were shown in Table 3 below:

TABLE-US-00003 TABLE 3 Yield Step (2) Yield of Treatment Treatment Yield of xylo- Temperature Pressure Step (4) xylan oligosaccharides (° C.) (MPa) Enzyme (%) (%) Example B1 121 0.10 Crude enzyme 83 87 preparation of BLCY-007 Comparative 60 0.10 SP-min 31 38 Example B1 Qingdao Vland Comparative 121 0.01 Biotech INC 40 41 Example B2 Comparative 80 0.10 44 42 Example B3 Comparative 150 0.1 36 35 Example B4 Comparative 121 0.04 42 41 Example B5 Comparative 121 0.26 38 36 Example B6 Comparative 121 0.10 61 64 Example B7

[0241] It can be seen from the experimental results in Table 3 that the preparation of xylooligosaccharides using corncobs as raw material in Example B1 had a significantly improved yield of xylooligosaccharides, which indicated that unexpected technical effects were obtained.

[0242] Although the specific embodiments of the present disclosure have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been disclosed, and these changes are within the protection scope of the present disclosure. The full scope of the present disclosure is given by the appended claims and any equivalents thereof.