WO2021000963A1 - 一株里氏木霉及其培养方法与应用 - Google Patents

一株里氏木霉及其培养方法与应用 Download PDF

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WO2021000963A1
WO2021000963A1 PCT/CN2020/100482 CN2020100482W WO2021000963A1 WO 2021000963 A1 WO2021000963 A1 WO 2021000963A1 CN 2020100482 W CN2020100482 W CN 2020100482W WO 2021000963 A1 WO2021000963 A1 WO 2021000963A1
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treatment
xylo
xylanase
crude
oligosaccharides
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PCT/CN2020/100482
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English (en)
French (fr)
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窦宝德
窦光朋
干昭波
邵先豹
李方华
杜倩
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山东百龙创园生物科技股份有限公司
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Priority claimed from CN201910996538.5A external-priority patent/CN110564629A/zh
Priority claimed from CN201910997521.1A external-priority patent/CN110628846B/zh
Application filed by 山东百龙创园生物科技股份有限公司 filed Critical 山东百龙创园生物科技股份有限公司
Priority to CA3145113A priority Critical patent/CA3145113A1/en
Priority to CN202080003222.2A priority patent/CN112930391A/zh
Priority to EP20835360.7A priority patent/EP3995567A4/en
Priority to US17/253,082 priority patent/US20220186272A1/en
Publication of WO2021000963A1 publication Critical patent/WO2021000963A1/zh

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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12P21/00—Preparation of peptides or proteins
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma

Definitions

  • the invention relates to a strain of Trichoderma reesei and its cultivation method and application, and belongs to the technical field of microorganisms.
  • Oligosaccharides also known as oligosaccharides, are the general term for linear or branched low-degree polymerized sugars formed by 2-10 monosaccharides connected by glycosidic bonds, with a molecular weight of about 300-2000. Oligosaccharides have special biological functions, in particular, they can promote the proliferation of bifidobacteria in the intestine, which is beneficial to human intestinal health. Among them, xylo-oligosaccharides have the best effect.
  • Xylooligosaccharides are oligosaccharides made from natural dietary fibers such as corn cobs, cottonseed hulls, bagasse, etc., using xylanase to saccharify and decompose hemicellulose.
  • Chinese patent document CN105154412A discloses a method for extracting xylanase from the waste fungus bag of Tremella, which belongs to the field of biological fermentation engineering. Specifically, the waste fungus bag is added with water to extract, and the extract is salted out with ammonium sulfate. After dialysis to remove salt, DEAE-cellulose column chromatography is used for purification to obtain xylanase.
  • the patented xylanase extraction, separation and purification process is complicated, which is not conducive to large-scale industrial production.
  • Penicillium occitanis Pol6 Pulenicillium occitanis Pol6
  • Aspergillus niger BCC14405 Aspergillus niger BCC14405
  • Trichoderma can also produce xylanase.
  • the patent document IN201741043810A discloses a new Trichoderma strain GAMSII M501, the deposit number is MTCC25104.
  • the strain can be used to produce an enzyme mixture containing higher activity cellulase and xylanase.
  • the production method includes the following steps: a) Culturing cells of the new natural strain T.gamsii M501 with the deposit number MTCC25104 in a modified Vogel's medium, which is supplemented with 1% microcrystalline cellulose and adjusted to pH 5.5 , B) culturing the cells at a temperature of about 28°C for 3 days to obtain a culture, c) obtaining a culture supernatant from a culture containing cellulase and xylanase.
  • the maximum levels of filter paper enzyme activity FPase, carboxymethyl cellulase activity CMCase and xylanase activity produced by the new strain of T.gamsii were 2.0 U/ml, 45.3 U/ml and 600 U/ml, respectively.
  • the resulting enzyme mixture can be used for the hydrolysis of alkali-pretreated lignocellulosic biomass.
  • the enzyme obtained from this strain is a mixture. It cannot be directly applied to the production of xylo-oligosaccharides.
  • the present invention provides a Trichoderma reesei strain and its culture method and application.
  • the crude enzyme preparation obtained based on the Trichoderma reesei has high xylanase activity and does not contain other miscellaneous enzymes, and can be directly used to produce xylo-oligosaccharides.
  • CGMCC General Microbiology Center
  • Trichoderma reesei is a fungus of the genus Trichoderma.
  • Trichoderma fungi belong to the class of Myxosporae (Gloiosporae) of the class Deuteromycotina, Hyphomy-cetes, and Hyphomycetales.
  • the original strain of the novel Trichoderma reesei BLCY-007 of the present invention was isolated from the soil near the R&D pilot plant of Bailong Chuangyuan, Shandong Dezhou. After the original strain was mutagenized by ultraviolet radiation and mutagen treatment, the novel strain was obtained Trichoderma reesei BLCY-007.
  • the bacterial colony of this strain is like a spread cotton wool, at first it is a white dense flat hyphae, then a light green sporulation cluster appears on the back edge, and the reverse side is colorless.
  • the crude enzyme preparation can be obtained by simple centrifugation/filtration of the bacterial fermentation broth of this strain.
  • the crude enzyme preparation can hydrolyze xylan at an optimal pH value range of 5.5 to 6.5. It can reach 508U/ml.
  • the cultivation method (or fermentation method) of Trichoderma reesei BLCY-007 includes the following steps:
  • step (ii) Take the activated strain obtained in step (i), inoculate it into a seed culture medium, and then multiply and cultivate it for 24 to 36 hours at a temperature of 24°C to 28°C to prepare a seed liquid;
  • step (iii) Take the seed liquid prepared in step (ii), inoculate the fermentation medium at a volume ratio of 1 to 10%, and expand the culture at 24° C. to 28° C. for 24 to 36 hours to obtain the bacterial fermentation broth.
  • the method for preparing the bacterial fermentation broth of Trichoderma reesei BLCY-007 includes:
  • step (ii) Take the activated strain obtained in step (i), inoculate it into a seed culture medium, and then multiply and cultivate it for 24 to 36 hours at a temperature of 24°C to 28°C to prepare a seed liquid;
  • step (iii) Take the seed liquid prepared in step (ii), inoculate the fermentation medium at a volume ratio of 1 to 10%, and expand the culture at 24° C. to 28° C. for 24 to 36 hours to obtain the bacterial fermentation broth.
  • the seed medium raw material components in the step (ii) are as follows:
  • the seed medium raw material components in the step (ii) are as follows:
  • the raw material components of the fermentation medium in the step (iii) are as follows, all of which are percentages by weight:
  • Corncob 25%, glucose 4%, beef extract 6%, protein aging 1%, anhydrous magnesium sulfate 0.01%, dipotassium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, remaining water, pH 5.0-6.0.
  • the raw material components of the fermentation medium in the step (iii) are as follows, all of which are percentages by weight:
  • Corncob 20-30%, glucose 3-5%, beef extract 5-7%, protein aging 0.5-2%, anhydrous magnesium sulfate 0.01-0.02%, dipotassium hydrogen phosphate 0.01-0.03%, ammonium sulfate 0.01-0.03 %, balance water, pH 5.0 ⁇ 6.0.
  • the raw material components of the PDA medium in the step (i) are as follows:
  • the potato extract is prepared as follows: take 200 g of peeled potatoes, cut into small pieces, add 1.0L of water and boil for 30min, filter out the potato pieces, and make up the filtrate to 1.0L.
  • the raw material components of the PDA medium in the step (i) are as follows:
  • Each 1000 parts of potato extract is prepared as follows: Take 200 parts of peeled potatoes, cut into small pieces, add 1000 parts of water and boil for 20-40 minutes, filter out the potato pieces, and make up the filtrate to 1000 parts.
  • the present disclosure provides the application of the above-mentioned Trichoderma reesei BLCY-007 in the preparation of xylanase.
  • xylanase refers to an enzyme preparation with xylanase enzyme activity.
  • the above application steps are as follows: take the bacterial fermentation broth prepared above and centrifuge, and collect the supernatant to obtain a crude enzyme preparation.
  • the aforementioned centrifugal separation is performed at 4° C. and 10000 r/min for 10 min.
  • the application steps are as follows: take the bacterial fermentation broth prepared above and centrifuge, wash the bacterial cells, centrifuge for a second time, and retain the precipitate, which is the crude xylanase enzyme preparation.
  • the centrifugal separation is performed at 4° C. and 10000 r/min for 10 min.
  • the application of the xylanase (such as a crude xylanase enzyme preparation) prepared by the present disclosure in the preparation of xylo-oligosaccharides is provided.
  • Trichoderma reesei BLCY-007 prepared by the present disclosure in the preparation of xylo-oligosaccharides is provided.
  • the present disclosure provides a method for preparing a crude enzyme preparation, including:
  • the present disclosure provides a method for preparing xylo-oligosaccharides, including:
  • the temperature of the enzymatic hydrolysis treatment is 50-60°C.
  • the pH of the enzymatic hydrolysis treatment is 5.5-6.5.
  • the invention also provides a method for preparing xylo-oligosaccharides by high-temperature and high-pressure treatment.
  • the method uses a high temperature and high pressure process to process corn cobs to obtain xylan, and then adds xylanase enzyme hydrolysis to prepare a crude xylan oligosaccharide solution. Acid and alkali are not used in the process, thereby avoiding a large amount of sewage discharge and reducing environmental protection pressure. At the same time, there is only one enzymatic hydrolysis in the process, which reduces the production cost.
  • the present invention uses the newly developed xylanase produced by Trichoderma reesei for enzymatic hydrolysis.
  • the enzyme activity is as high as 508U/ml, which further improves the xylanase The extraction efficiency.
  • a method for preparing xylo-oligosaccharides by high temperature and high pressure treatment includes the following steps:
  • the premix is subjected to high temperature and high pressure treatment to obtain a crude xylan extract, the treatment temperature is 95°C-140°C, and the treatment pressure is 0.05-0.25MPa;
  • xylo-oligosaccharide crude liquid is subjected to enzyme inactivation treatment, decolorization treatment, ion exchange treatment, and concentration treatment are performed to obtain a xylo-oligosaccharide solution.
  • a method for preparing xylo-oligosaccharides by high temperature and high pressure treatment includes the following steps:
  • the premix is subjected to high temperature and high pressure treatment to obtain a crude xylan extract, the treatment temperature is 95°C-140°C, and the treatment pressure is 0.05-0.25MPa;
  • xylo-oligosaccharide crude liquid is subjected to enzyme inactivation treatment, decolorization treatment, ion exchange treatment, and concentration treatment are performed to obtain a xylo-oligosaccharide solution.
  • a method for preparing xylo-oligosaccharides by high temperature and high pressure treatment includes the following steps:
  • xylo-oligosaccharide crude liquid After the xylo-oligosaccharide crude liquid is subjected to enzyme inactivation treatment, it is subjected to decolorization treatment, ion exchange treatment, and concentration treatment to obtain a xylo-oligosaccharide solution.
  • the crushed particle size of the corncob is 80-120 mesh sieve, and the mass concentration of the premix is 8%-12%.
  • the high temperature and high pressure treatment temperature is 115°C-128°C
  • the treatment pressure is 0.09-0.18 MPa
  • the treatment time is 4-8 hours.
  • the xylan solution is adjusted to a mass concentration of 4%-6% before enzymolysis; preferably, the amount of xylanase added is 4-6g/kg dry matter; preferably, the enzymatic hydrolysis reaction temperature It is 50°C-60°C, the enzymolysis reaction time is 20-40h, and the enzymolysis reaction is a static reaction.
  • the xylanase used is the xylanase produced by the following strains, with an enzyme activity of 508U/ml;
  • the strain is Trichoderma reesei BLCY-007, which was deposited in the General Microbiology Center (CGMCC) of the China Microbial Culture Collection and Management Committee on June 14, 2019, with the preservation number CGMCC No.17970, address: Chaoyang, Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 1, Yard 3, Beichen West Road, District.
  • CGMCC General Microbiology Center
  • the method of preparing xylo-oligosaccharides by high-temperature and high-pressure treatment refers to a method of preparing xylo-oligosaccharides by high-temperature and high-pressure treatment of corn cobs.
  • the premix of step (1) does not contain acid or alkali.
  • step (1) the moisture content of the corncob is 14-25%.
  • the pH of the premix in step (1) is 7-8.
  • the dry matter concentration of the premix in step (1) is 8%-12%.
  • the formula for calculating the mass concentration of dry matter in the premix is corncob dry matter mass/total mass of the premix liquid ⁇ 100%.
  • dry matter refers to corncob dry matter
  • dry matter weight is the dry weight of corncob
  • the dry matter mass concentration refers to the mass concentration based on the dry weight of the corncob.
  • step (2) is performed in a closed pressure vessel.
  • high temperature refers to a processing temperature of 95°C to 140°C.
  • high pressure refers to a processing pressure of 0.05-0.25 MPa.
  • the treatment temperature is 121°C and the treatment pressure is 0.1Mpa.
  • the mass concentration in step (3) refers to the dry matter mass concentration of the crude xylan extract.
  • microwave treatment refers to placing the crude xylan extract in a closed container, and then placing the container in a microwave processing equipment for microwave processing.
  • step (3) the dry matter concentration of the crude xylan extract is adjusted to 4%-6%, and the pH is adjusted to between 4.2-4.8 to obtain the pre-reaction liquid, which is used during microwave treatment
  • the ratio of the microwave power to the pre-reaction liquid is 500-1000: 80-120 (W/ml), for example 700-900: 100-110 (W/ml).
  • step (3) the temperature of the crude xylan extract during the microwave treatment is 40-55°C.
  • the temperature of the crude xylan extract is maintained at 40-55°C.
  • step (4) the dry matter concentration of the xylan solution is adjusted to 4%-6% before enzymolysis.
  • the dry matter quality is calculated based on the dry weight of corncob.
  • step (4) in step (4), the enzymatic hydrolysis is performed at a temperature of 50°C to 60°C, the enzymatic hydrolysis time is 20-40h, and the xylan solution is kept standing during the enzymatic hydrolysis process.
  • step (4) only uses the crude enzyme preparation obtained from Trichoderma reesei BLCY-007 bacterial fermentation broth as the xylanase.
  • the processing temperature range of 95°C-140°C and the pressure range of 0.05-0.25 MPa in step (1) are critical for increasing the yield of xylo-oligosaccharides.
  • the xylan in the corncob is a biological macromolecule, which exists in a natural state in a compound with other components such as cellulose and lignin.
  • the three have a discontinuous layered structure, which hinders hydrolysis. If the treatment is outside the above-mentioned temperature and pressure range, the discontinuous layered structure cannot be destroyed, and the yield of xylan and the extraction rate of xylo-oligosaccharide are not high.
  • step (1) when the processing pressure of step (1) is lower than 0.05 MPa, the discontinuous layered structure will not be destroyed, and the yield of xylan and the extraction rate of xylo-oligosaccharides are not high.
  • the treatment pressure in step (1) is higher than 0.25 MPa, xylan will be excessively hydrolyzed to produce xylose. Thereby reducing the yield of xylan and the extraction rate of xylo-oligosaccharides.
  • the crude enzyme preparation obtained from the fermentation broth of Trichoderma reesei BLCY-007 bacteria used in step (4) is also critical for improving the yield of xylo-oligosaccharides.
  • the xylo-oligosaccharide meets the Chinese standard GB/T 35545-2017.
  • xylan refers to a heterogeneous polysaccharide present in the cell wall of plants, accounting for about 15% to 35% of the dry weight of plant cells, and is the main component of plant hemicellulose.
  • the enzyme inactivation treatment method is high temperature inactivation, the enzyme inactivation temperature is 85°C-98°C, and the enzyme inactivation time is 10-15 minutes.
  • the decolorization process is the use of activated carbon for decolorization
  • the added amount of activated carbon is 0.8-5% of the dry matter mass in the crude xylo-oligosaccharide liquid
  • the decolorization temperature is 78-85°C
  • the decolorization holding time is 15-30 min
  • the liquid flow rate during decolorization is 20-30 mL/min.
  • the dry matter quality is calculated based on the dry weight of corncob.
  • the decolorization process is decolorization using activated carbon
  • the addition amount of activated carbon is 0.8-5% of the dry matter mass in the crude xylo-oligosaccharide liquid
  • the decolorization temperature is 78-85° C.
  • the decolorization holding time is 15-30 min.
  • the decolorization treatment refers to passing the crude xylo-oligosaccharide liquid through an activated carbon filter element.
  • the flow rate of the crude xylo-oligosaccharide liquid through the filter element is 20-30 mL/min.
  • the ion exchange column used in the ion exchange treatment process is a combined column of cation exchange column-anion exchange column-cation exchange column, the ion exchange treatment temperature is 25-35°C, and the ion exchange treatment flow rate is 15-25 mL/min; further
  • the cation exchange column is a strong acid positive resin, and the anion exchange column is a weak base negative resin.
  • the strong acid-positive resin is D001 macroporous cation exchange resin produced by Zhejiang Zhengguang Industrial Co., Ltd.
  • the cation exchange resin is a styrene-divinylbenzene copolymer with a sulfonic acid group (-SO 3 H).
  • the weak base negative resin is D354FD macroporous weak base negative resin produced by Zhejiang Zhengguang Industrial Co., Ltd.
  • the weakly basic negative resin is a weakly basic anion exchange resin with a polystyrene macroporous structure.
  • the concentration treatment method is vacuum rotary concentration
  • the working pressure is -0.1MPa
  • the working temperature is 60-80°C
  • the concentration of dry matter in the crude xylo-oligosaccharide liquid is 60%-78% after the concentration treatment.
  • the method for preparing xylo-oligosaccharides by high-temperature and high-pressure treatment includes the following steps:
  • step (1) High temperature and high pressure treatment: The premix in step (1) is subjected to high temperature and high pressure treatment to obtain a crude xylan extract; wherein the treatment temperature is 115°C-128°C, and the treatment time is 4-8 hours , The processing pressure is 0.09-0.18MPa;
  • Microwave treatment adjust the mass concentration of the crude xylan extract in step (2) to 4%-6%, and adjust the pH to between 4.2-4.8 to obtain a pre-reaction liquid; Carry out microwave treatment to obtain xylan liquid; wherein the microwave frequency is 2450MHz, the treatment temperature is 40-55°C, and the microwave time is 10-25min;
  • Enzymatic hydrolysis add xylanase to the xylan solution described in step (3) for enzymatic hydrolysis, and the amount of xylanase added is 4-6g/kg dry matter to obtain xylan oligosaccharides Crude liquid; wherein the enzymolysis reaction temperature is 50°C-60°C, the enzymolysis reaction time is 20-40h, the enzymolysis reaction is a standing reaction, and the xylanase used is Trichoderma reesei BLCY- Xylanase produced by 007;
  • Enzyme inactivation the crude xylo-oligosaccharide liquid described in step (4) is subjected to enzymatic inactivation, wherein the enzymatic inactivation method is high-temperature enzymatic inactivation, the enzymatic inactivation temperature is 85°C-98°C, and the enzymatic inactivation time is 10- 15min;
  • Refining treatment decolorize, ion exchange, and concentrate the liquid after the enzyme inactivation described in step (5) to obtain a xylo-oligosaccharide liquid; wherein the decolorization treatment uses activated carbon for decolorization, and the added amount of activated carbon is after enzyme inactivation 0.8-5% of the mass of the liquid dry basis, the decolorization temperature is 78-85°C, the decolorization holding time is 15-30min, and the liquid flow rate during decolorization is 20-30mL/min; the ion exchange column used for ion exchange treatment is cation exchange column- Anion exchange column-cation exchange column combination column, ion exchange treatment temperature is 25-35°C, ion exchange treatment flow rate is 15-25mL/min;
  • the concentration method is vacuum rotary concentration, the working pressure is -0.1MPa, the working temperature is 60-80°C, and the concentration of dry matter is 60%-78%.
  • the xylanase-producing Trichoderma reesei BLCY-007 is obtained for the first time in the present invention, and the xylanase activity of the crude enzyme preparation extracted from its bacterial fermentation broth is 60% higher than that of the traditional Trichoderma reesei % Above, it can reach 508U/ml, significantly reducing production costs.
  • the optimum pH value of the crude enzyme preparation is 5.5-6.5, and this pH value range is conducive to the control of pollution during production.
  • the xylanase produced by Trichoderma reesei BLCY-007 of the present invention is an extracellular enzyme.
  • the separation process is simple. After simple centrifugation and washing, the enzyme preparation can be obtained, which saves production costs and reduces Power loss.
  • the crude enzyme preparation obtained based on Trichoderma reesei BLCY-007 does not contain cellulases and other miscellaneous enzymes.
  • the crude enzyme preparation obtained from Trichoderma reesei BLCY-007 has no filter paper enzyme activity FPase and no carboxymethyl cellulase activity CMCase.
  • the crude enzyme preparation obtained based on Trichoderma reesei BLCY-007 does not contain toxins and can be safely used in the production of food raw material xylo-oligosaccharides.
  • the traditional production process requires acid treatment or alkali treatment of the corncob, and requires acid and alkali resistant equipment, which requires a large one-time investment; the preparation process is complicated, which is not conducive to controlling the progress of the reaction, and many side reactions cause many by-products and product purification Difficulty, the use of a large amount of acid and alkali in the process causes a large amount of sewage discharge and brings serious environmental pollution; the present invention uses a high temperature and high pressure process to prepare xylo-oligosaccharides, overcomes the shortcomings of the traditional production process, and replaces the traditional chemical method Treatment, no acid or alkali is used in the preparation process, avoiding a large amount of sewage discharge and reducing environmental protection pressure.
  • the high-yield xylanase-producing Trichoderma reesei BLCY-007 is used in the present invention, and the xylanase activity in the bacterial fermentation broth can reach 508U/ml, which is 60% higher than the traditional xylanase activity. % Above, significantly reducing production costs, while further improving the yield of xylo-oligosaccharides.
  • the yield of xylan and the extraction rate of xylan oligosaccharides of the present invention have been greatly improved compared with traditional production methods.
  • the yield of xylan reaches more than 64%, and the highest is 83%;
  • the extraction rate is more than 72%, and the highest is 87%; only one enzymatic hydrolysis is added in the process, which effectively reduces the production cost.
  • CGMCC General Microbiology Center
  • Trichoderma reesei BLCY-007 The mutagenesis and screening process of the above Trichoderma reesei BLCY-007 is as follows:
  • the raw material components of the PDA medium are as follows:
  • the potato extract is prepared as follows: take 200 g of peeled potatoes, cut into small pieces, add 1.0L of water and boil for 30min, filter out the potato pieces, and make up the filtrate to 1.0L.
  • step (1) take a large test tube containing 5ml of sterile water, take 2ml of the enriched and cultured bacterial solution in step (1), and dilute it, shake it thoroughly, and use an inoculating loop to pick it up aseptically.
  • the slant seeds of No. 01-10 were inoculated in a shake flask culture medium and cultured at 24-28°C for 36 hours to obtain the fermentation broth of No. 01-10 shake flask.
  • the xylanase enzyme activity of the 01-10 shake flask fermentation broth was measured, and the 03 shake flask fermentation broth showed the highest enzyme activity, reaching 105 U/ml.
  • the raw material components of the plate medium are as follows: potato extract 1.0L, glucose 20.0g, and agar 15.0g.
  • Potato extract Take 200g of peeled potatoes, cut into small pieces, add 1.0L of water and boil for 30min, filter out the potato pieces, and make up the filtrate to 1.0L.
  • the components of the slant medium are as follows, all in weight percentage: 1.0L potato extract, 20.0g glucose, and 15.0g agar.
  • the components of the shake flask culture medium are as follows: 200 g peeled potatoes, 20 g glucose, 3 g KH 2 PO 4 and 1.5 g MgSO 4 ⁇ 7H 2 O. After mixing the above components, add 1.0L of water and boil for 30min, filter out the potato pieces, and make up the filtrate to 1.0L.
  • the strains in the fermentation broth of No. 03 shake flask were subjected to ultraviolet mutagenesis.
  • the ultraviolet mutagenesis was irradiated with 15W ultraviolet lamp 20cm and the irradiation time was 180s.
  • the obtained high-yielding strains were subjected to ethyl methanesulfonate mutagenesis treatment, and finally high yield
  • the xylanase strain was named BLCY-007.
  • the culturing method of Trichoderma reesei BLCY-007 described in Example 1, includes the following steps:
  • Trichoderma reesei BLCY-007 was inoculated into PDA medium, and activated and cultured for 12 hours at 24°C to obtain activated strains;
  • the raw material components of PDA medium are as follows:
  • the potato extract is prepared as follows: take 200g of peeled potatoes, cut into small pieces, add 1.0L of water and boil for 30min, filter out the potato pieces, and make up the filtrate to 1.0L;
  • step (2) Take the activated strain obtained in step (1), inoculate it into a seed culture medium, multiply and culture it for 24 hours at 24°C to prepare a seed liquid;
  • the basic components of seed culture materials are as follows:
  • step (3) Take the seed liquid prepared in step (2), inoculate it in the fermentation medium at a volume ratio of 2%, and expand the culture at 24°C for 24 hours to obtain the bacterial fermentation liquid;
  • the raw material components of the fermentation medium are as follows, all in weight percentage:
  • Corncob 25%, glucose 4%, beef extract 6%, protein aging 1%, anhydrous magnesium sulfate 0.01%, dipotassium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, remaining water, pH 5.0-6.0.
  • the culturing method of Trichoderma reesei BLCY-007 described in Example 1, includes the following steps:
  • Trichoderma reesei BLCY-007 was inoculated into PDA medium, and activated and cultured at 28°C for 24 hours to obtain activated strains;
  • the raw material components of PDA medium are as follows:
  • the potato extract is prepared as follows: take 200g of peeled potatoes, cut into small pieces, add 1.0L of water and boil for 30min, filter out the potato pieces, and make up the filtrate to 1.0L;
  • step (2) Take the activated strain obtained in step (1), inoculate it in a seed culture medium, and proliferate and cultivate 36 hours at 28°C to prepare a seed liquid;
  • the raw material components of the seed culture medium are as follows:
  • step (3) Take the seed liquid prepared in step (2), inoculate it in the fermentation medium at a volume ratio of 8%, and expand the culture at 28°C for 36 hours to obtain the bacterial fermentation liquid;
  • the raw material components of the fermentation medium are as follows, all in weight percentage:
  • Corncob 25%, glucose 4%, beef extract 6%, protein aging 1%, anhydrous magnesium sulfate 0.01%, dipotassium hydrogen phosphate 0.02%, ammonium sulfate 0.02%, remaining water, pH 5.0-6.0.
  • Example 1 The strain in the fermentation broth of Example 1 in the No. 03 shake flask was used.
  • the culture was carried out with reference to the method of Example 2 to obtain the bacterial fermentation broth.
  • Trichoderma reesei purchased from Beijing Beina Chuanglian Biotechnology Research Institute was used as the culture strain. The culture was carried out with reference to the method of Example 2 to obtain the bacterial fermentation broth.
  • the xylanase activity of the crude enzyme preparation obtained in Example 4 was measured.
  • enzyme activity and “enzyme activity” have the same meaning, and both refer to xylanase enzyme activity.
  • the amount of enzyme required to release 1 ⁇ mol reducing sugar from a xylan solution with a concentration of 5 mg/ml per minute is an enzyme activity unit U.
  • X is the activity of xylanase in the liquid sample, U/ml
  • X D is the activity of xylanase in the reaction enzyme solution of the liquid sample, U/ml
  • a E is the absorbance of the enzyme reaction solution
  • AB is the absorbance of the enzyme blank solution
  • K is the slope of the standard curve
  • C 0 is the intercept of the standard curve
  • M is the molar mass of xylose, 150.2g/mol
  • t is the enzymatic hydrolysis reaction time, min
  • N is the enzyme Solution dilution factor
  • liquid fermentation and solid fermentation were carried out according to the methods described in (3) and (4) in the 5th part of the example on page 4-5 of the CN1185336C manual to obtain liquid fermentation products. -B and solid fermentation product-B.
  • part 6 xylanase enzyme activity determination method and part 7 glucanase activity test method the xylanase of liquid fermentation product-B and solid fermentation product-B were detected respectively.
  • the enzyme activity of the enzyme and the enzyme activity of glucanase the results are as follows:
  • 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 produced by Qingdao Weilan Biological Co., Ltd.
  • the cation exchange column used in the following examples is a strong acid-positive resin D001 macroporous cation exchange resin produced by Zhejiang Zhengguang Industrial Co., Ltd.
  • the anion exchange column used in the following examples is a weak base negative resin, which is D354FD macroporous weak base anion resin produced by Zhejiang Zhengguang Industrial Co., Ltd.
  • the microwave processing equipment used in the following examples is the MDS-6 microwave digestion/extraction instrument produced by Shanghai Xinyi Microwave Chemical Technology Co., Ltd.
  • the parameters are: output power, 0-1000W, temperature, control range: 0-250°C, accuracy ⁇ 1°C, pressure, control range: 0.1-5MPa, accuracy 0.1MPa;
  • % used in the following examples is a mass percentage.
  • the mass concentration refers to the mass concentration of corncob dry matter.
  • the processing pressure value should be understood as an incremental value based on 1 standard atmospheric pressure.
  • a processing pressure of 0.1Mpa means an increase of 0.1Mpa on the basis of 1 standard atmosphere.
  • a method for preparing xylo-oligosaccharides by high-temperature and high-pressure treatment includes the following steps:
  • (1) Sizing Provide 5g corn cob (15% moisture content), crush the corn cob and pass the crushed product through a 100-mesh sieve, and collect the corn cob powder that passes through the sieve.
  • the corncob powder is mixed with purified water to prepare a premix with a dry matter concentration of 9% by weight, wherein the water used for the pulping is purified water.
  • Microwave treatment Adjust the dry matter concentration of the crude xylan extract to 4%, and adjust the pH to 4.7 to obtain 106ml of the pre-reaction liquid; place the pre-reaction liquid in a microwave treatment equipment for microwave treatment to obtain Xylan liquid; the microwave power of the microwave processing equipment is 800W, the microwave frequency is 2450MHz, the processing temperature is 50°C, and the microwave time is 15min.
  • Enzymatic hydrolysis add xylanase to the xylan solution and perform enzymatic hydrolysis to obtain a crude xylan-oligosaccharide solution.
  • the amount of xylanase added is 5 g/kg dry matter;
  • the enzymatic hydrolysis reaction time is 24h, the pH value of the xylan solution is controlled at 5.5-6.5, and the enzymatic hydrolysis reaction is a static reaction.
  • the xylanase is a crude enzyme preparation obtained from the fermentation broth of Trichoderma reesei BLCY-007 bacteria.
  • the Trichoderma reesei BLCY-007 was deposited in the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on June 14, 2019, with the preservation number CGMCC No.17970, address: Beichen, Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 1, Yard 3, West Road.
  • CGMCC General Microbiology Center
  • Enzyme inactivation the above crude xylo-oligosaccharide liquid is subjected to enzyme inactivation treatment.
  • the enzyme inactivation temperature is 85°C, and the enzyme inactivation time is 10 minutes.
  • Refining treatment Refining the product after enzyme inactivation. include:
  • the product after enzyme inactivation is passed through an activated carbon filter element.
  • the amount of activated carbon added is 1% of the dry basis, the decolorization temperature is 85°C, the decolorization holding time is 20min, and the liquid flow rate during decolorization is 25mL/min;
  • the ion exchange column used is a combination of cation exchange column-anion exchange column-cation exchange column, the ion exchange treatment temperature is 25°C, and the flow rate of the ion exchange treatment is 20 mL/min;
  • Vacuum rotary concentration the use of rotary thin film vacuum concentration equipment for concentration treatment, equipment working pressure -0.1MPa, working temperature is 75 °C. Concentrate to a dry matter concentration of 75% in the product to obtain a xylo-oligosaccharide liquid.
  • the xylan mass that can be obtained theoretically from 5g corncob is 1.8g.
  • the mass of xylan in the xylan solution actually obtained in step (3) is 1.494g
  • the oligomeric wood obtained in step (6) The mass of the sugar is 1.299g, based on this calculation, the yield of xylan is 83%, and the extraction rate of xylo-oligosaccharide is 87%.
  • Comparative Example B1 is similar to Example B1, except that: in step (2), the treatment temperature is 60° C. and the treatment pressure is 0.10 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the added amount of xylanase was 5 g/kg dry matter.
  • the detection results of the xylan yield and xylo-oligosaccharide extraction rate in this example are as follows: the xylan yield is 31%, and the xylo-oligosaccharide extraction rate is 38%.
  • the xylan in the corncob is a biological macromolecule, which exists in a complex with other components such as cellulose and lignin in its natural state.
  • the three have a discontinuous layered structure, which hinders the hydrolysis of acid or alkali.
  • the high-pressure treatment alone cannot destroy the discontinuous layered structure, so the xylan yield and the extraction rate of xylo-oligosaccharides are not high.
  • Comparative Example B2 is similar to Example B1, except that: in step (2), the treatment temperature is 121°C and the treatment pressure is 0.01 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the added amount of xylanase was 5 g/kg dry matter.
  • the detection results of the xylan yield and xylo-oligosaccharide extraction rate in this example are as follows: the xylan yield is 40%, and the xylo-oligosaccharide extraction rate is 41%.
  • Comparative Example B3 is similar to Example B1, except that: in step (2), the treatment temperature is 80° C. and the treatment pressure is 0.10 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the added amount of xylanase was 5 g/kg dry matter.
  • the detection results of the xylan yield and xylo-oligosaccharide extraction rate in this example are as follows: the xylan yield is 44%, and the xylo-oligosaccharide extraction rate is 42%.
  • Comparative Example B4 is similar to Example B1, except that: in step (2), the treatment temperature is 150° C. and the treatment pressure is 0.10 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the added amount of xylanase was 5 g/kg dry matter.
  • the detection results of the xylan yield and the xylo-oligosaccharide extraction rate in this example are as follows: the xylan yield is 36%, and the xylo-oligosaccharide extraction rate is 35%.
  • the xylan Due to the high temperature of the high-temperature treatment, the xylan is excessively hydrolyzed to produce xylose, which reduces the yield of xylan and the extraction rate of xylo-oligosaccharides.
  • Comparative Example B5 is similar to Example B1, except that: in step (2), the treatment temperature is 121° C. and the treatment pressure is 0.04 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the added amount of xylanase was 5 g/kg dry matter.
  • the detection results of the xylan yield and xylo-oligosaccharide extraction rate in this example are as follows: the xylan yield is 42%, and the xylo-oligosaccharide extraction rate is 41%.
  • Comparative Example B6 is similar to Example B1, except that: in step (2), the treatment temperature is 121°C and the treatment pressure is 0.26 MPa. In step (4), the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the added amount of xylanase was 5 g/kg dry matter.
  • the detection results of the xylan yield and xylo-oligosaccharide extraction rate in this example are as follows: the xylan yield is 38%, and the xylo-oligosaccharide extraction rate is 36%.
  • the treatment pressure is too high, the xylan is hydrolyzed to produce xylose, so the yield of xylan and the extraction rate of xylo-oligosaccharides are reduced.
  • Comparative Example B7 is similar to Example B1, except that:
  • step (4) the xylanase SP-min produced by Qingdao Vland Biotech INC was used, and the added amount of xylanase was 5 g/kg dry matter.
  • the theoretically obtainable xylan mass corn cob mass x corn cob half fiber content.
  • the semi-fiber content in the corncob is usually 35%-40%, and the semi-fiber content in the corncob used in the above examples and comparative examples is 36%.
  • the detection method of the xylan content in the xylan solution is as follows: the xylan solution, adjust the pH value to 5, add four times the volume of 95% (volume fraction) ethanol, alcohol precipitation overnight and then 3000r/ Centrifuge for 10 minutes in 1 min, take the precipitate, add a certain amount of 7% H 2 SO 4 and hydrolyze at 100 °C for 2 hours to neutralize, constant volume, and filter. Sugar quality.
  • the DNS method is used for the determination of reducing sugar.
  • the detection method of xylo-oligosaccharides is in accordance with GB/T 35545-2017 (Appendix A high performance liquid chromatography) for detection.
  • Example B1 Part of the step parameters and product yields of Example B1 and Comparative Examples B1 to B7 are shown in Table 3 below:

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Abstract

一株里氏木霉BLCY-007及其在制备低聚木糖中的应用。

Description

一株里氏木霉及其培养方法与应用
本申请是以CN申请号为201910597371.5，申请日为2019年7月4日；CN申请号为201910596633.6，申请日为2019年7月4日；CN申请号为201910996538.5，申请日为2019年10月19日；和CN申请号为201910997521.1，申请日为2019年10月19日的申请为基础，并主张其优先权，上述CN申请的公开内容在此作为整体引入本申请中。
技术领域
本发明涉及一株里氏木霉菌种及其培养方法与应用，属于微生物技术领域。
背景技术
随着经济社会的高速发展，人们对食品的营养和功能的认识逐步提高，更加重视通过改善膳食条件和发挥食品本身的生理调节功能来达到提高自身健康的目的。低聚糖(oligosaccharides)，又称寡糖，是由2～10个单糖经糖苷键连接而成的直链或支链低度聚合糖的总称，分子量约为300～2000。低聚糖具有特殊的生物学功能，特别是能促进肠内双歧杆菌的增殖，有益于人体肠道健康，其中功效最好的是低聚木糖。它的功效性是其他聚合糖类的近20倍。人体肠胃道内没有能够水解低聚木糖的酶，所以其可直接进入大肠内优先为双歧杆菌所利用，促进双歧杆菌增殖，同时产生多种有机酸，降低肠道PH值，抑制有害菌生长，使益生菌在肠道大量增殖，达到保健功效。
低聚木糖(Xylooligosaccharide)是由2-9个木糖单元以β-1,4糖苷键连接而成的低聚糖的混合物。其结构式如下，n＝2～9，
Figure PCTCN2020100482-appb-000001
低聚木糖是从玉米芯、棉籽壳、甘蔗渣等天然食物纤维中采用木聚糖酶糖化分解半纤维素制取的一种低聚糖。
中国专利文件CN105154412A公开了一种从银耳废菌包中提取木聚糖酶的方法，其属于生物发酵工程领域，具体是将银耳废菌包加水提取后，利用硫酸铵对提取液进行盐 析，再经透析除盐后利用DEAE-纤维素柱层析进行纯化，得木聚糖酶。但是，该专利木聚糖酶提取、分离与纯化工艺复杂，不利于大规模工业化生产。
此外，通过微生物生产木聚糖酶也是一种高效的方式。例如：真菌里的青霉菌Pol6(Penicillium occitanis Pol6)和黑曲霉BCC14405(Aspergillus niger BCC14405)在生产木聚糖酶方面具有明显的产量优势，但是其在产木聚糖过程中常常会伴随着毒素的产生，使木聚糖酶在应用上具有一定的隐患。
一些木霉也能产生木聚糖酶，例如：专利文件IN201741043810A公开了一株新的木霉菌株GAMSII M501，保藏号为MTCC25104。该菌株可用于生产含有较高活性纤维素酶和木聚糖酶的酶混合物。生产方法包括以下步骤：a)在改良Vogel’s培养基中培养具有保藏号MTCC25104的新型天然菌株T.gamsii M501的细胞，该改良的培养基中添加了1％的微晶纤维素并调节pH至5.5，b)在约28℃的温度下培养细胞3天以获得培养物，c)从含有纤维素酶和木聚糖酶的培养物中获得培养上清液。T.gamsii新菌株产生的滤纸酶活FPase，羧甲基纤维素酶活CMCase和木聚糖酶酶活的最大水平分别为2.0U/ml，45.3U/ml和600U/ml。得到的酶混合物可用于碱预处理的木质纤维素生物质的水解。但是，该菌株得到的酶为混合物。无法直接应用到低聚木糖的生产当中。
发明内容
本发明针对现有技术的不足，提供一株里氏木霉及其培养方法与应用。基于该里氏木霉获得的粗酶制剂具有较高的木聚糖酶酶活，且不含其它杂酶，可直接用于生产低聚木糖。
本发明的技术方案如下：
一株里氏木霉(Trichoderma reesei)BLCY-007，2019年6月14日保存于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号CGMCC No.17970，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
里氏木霉(Trichoderma reesei)是木霉属(Trichoderma)真菌。木霉属(Trichoderma)真菌属于半知菌亚门(Deuteromycotina)、丝孢纲(Hyphomy-cetes)、丝孢目(Hyphomycetales)的粘孢菌类(Gloiosporae)。
本发明的新型里氏木霉(Trichoderma reesei)BLCY-007的原始菌株分离于山东德州百龙创园研发中试车间附近的土壤，该原始菌株经过紫外线照射和突变剂处理诱变后，获得新型里氏木霉(Trichoderma reesei)BLCY-007。
该菌株的菌落呈广铺的棉絮状，起初为白色致密的平坦菌丝，而后边缘出现浅绿的产孢子丛束区，反面无色。分生孢子梗菌丝的短侧枝，透明，多分枝；小梗瓶形，中部弯曲；分生孢子椭圆形或长形，单细胞，透明，无色，壁光滑，成堆时绿色。
该菌株的菌体发酵液经简单的离心/过滤即可得到粗酶制剂，该粗酶制剂能够水解木聚糖，在最适pH值范围5.5～6.5，该粗酶制剂的木聚糖酶酶活能达到508U/ml。
上述里氏木霉(Trichoderma reesei)BLCY-007的培养方法(或发酵方法)，包括步骤如下：
(i)取里氏木霉(Trichoderma reesei)BLCY-007接种于PDA培养基中，在24℃～28℃的条件下，活化培养12～24h，制得活化菌株；
(ii)取步骤(i)制得的活化菌株，接种于种子培养基中，在24℃～28℃的条件下，增殖培养24～36h，制得种子液；
(iii)取步骤(ii)制得的种子液，按体积比1～10％的比例接种于发酵培养基中，在24℃～28℃，扩大培养24～36h，即得菌体发酵液。
在一些实施方案中，制备氏木霉(Trichoderma reesei)BLCY-007的菌体发酵液的方法包括：
(i)取里氏木霉(Trichoderma reesei)BLCY-007接种于PDA培养基中，在24℃～28℃的条件下，活化培养12～24h，制得活化菌株；
(ii)取步骤(i)制得的活化菌株，接种于种子培养基中，在24℃～28℃的条件下，增殖培养24～36h，制得种子液；
(iii)取步骤(ii)制得的种子液，按体积比1～10％的比例接种于发酵培养基中，在24℃～28℃，扩大培养24～36h，即得菌体发酵液。
在一些实施方案中，所述步骤(ii)中的种子培养基原料组分如下：
去皮马铃薯200g，葡萄糖20g，KH 2PO 4 3g，MgSO 4·7H 2O 1.5g。将上述组分混合后加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L。
在一些实施方案中，所述步骤(ii)中的种子培养基原料组分如下：
按重量份计，去皮马铃薯150～250份，葡萄糖15～25份，KH 2PO 4 2～4份，MgSO 4·7H 2O 1～2份，将上述组分混合后加水1000份煮沸30min，滤去马铃薯块，将滤液补足至1000份。
在一些实施方案中，所述步骤(iii)中的发酵培养基原料组分如下，均为重量百分比：
玉米芯25％，葡萄糖4％，牛肉膏6％，蛋白陈1％，无水硫酸镁0.01％，磷酸氢二钾0.02％，硫酸铵0.02％，余量水，pH＝5.0～6.0。
在一些实施方案中，所述步骤(iii)中的发酵培养基原料组分如下，均为重量百分比：
玉米芯20～30％，葡萄糖3～5％，牛肉膏5～7％，蛋白陈0.5～2％，无水硫酸镁0.01～0.02％，磷酸氢二钾0.01～0.03％，硫酸铵0.01～0.03％，余量水，pH＝5.0～6.0。
在一些实施方案中，所述步骤(i)中的PDA培养基原料组分如下：
马铃薯提取液1.0L，葡萄糖20.0g，琼脂15.0g。
马铃薯提取液按如下方法制备：取去皮马铃薯200g，切成小块，加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L。
在一些实施方案中，所述步骤(i)中的PDA培养基原料组分如下：
按重量份计，马铃薯提取液800～120份，葡萄糖25～25份，琼脂10～20份。
每1000份马铃薯提取液按如下方法制备：取去皮马铃薯200份，切成小块，加水1000份煮沸20～40min，滤去马铃薯块，将滤液补足至1000份。
在一些方面，本公开提供上述里氏木霉(Trichoderma reesei)BLCY-007在制备木聚糖酶中的应用。
在一些实施方案中，木聚糖酶是指具有木聚糖酶酶活力的酶制剂。
在一些实施方案中，上述应用步骤如下：取上述制备的菌体发酵液离心分离，收集上清液后得到粗酶制剂。
在一些实施方案中，上述离心分离均为在4℃、10000r/min条件下，离心10min。
在一些实施方案中，应用步骤如下：取上述制备的菌体发酵液经离心分离，洗涤菌体，二次离心，保留沉淀物，即为木聚糖酶粗酶制剂。在一些实施方案中，洗涤菌体采用pH＝8.0、浓度50mmol/L的Tris-HCl缓冲液，然后经离心分离，保留沉淀，制得粗酶制剂。
在一些实施方案中，离心分离均为在4℃、10000r/min条件下，离心10min。
在一些实施方案中，提供本公开制备的木聚糖酶(例如木聚糖酶粗酶制剂)在制备低聚木糖中的应用。
在一些实施方案中，提供本公开制备的里氏木霉(Trichoderma reesei)BLCY-007在制备低聚木糖中的应用。
在一些方面，本公开提供一种粗酶制剂的制备方法，包括：
(1)提供上述培养方法获得菌体发酵液；
(2)对所述菌体发酵液进行离心处理；
(3)收集上一步产物的上清液，该上清液即为木聚糖酶粗酶制剂。
在一些方面，本公开提供一种制备低聚木糖的方法，包括：
(1)根据本公开任一项的方法制备木聚糖酶粗酶制剂；
(2)使用所述木聚糖酶粗酶制剂对木聚糖进行酶解处理，获得低聚木糖。
在一些实施方案中，酶解处理的温度为50～60℃。
在一些实施方案中，酶解处理的pH为5.5～6.5。
本发明还提供一种高温高压处理制备低聚木糖的方法。该方法使用高温高压工艺处理玉米芯得到木聚糖，再加入木聚糖酶酶解制备得到低聚木糖粗液，过程中不使用酸碱，避免了污水的大量排放，减小环保压力，同时过程中仅有一次加酶酶解，减少了生产成本，本发明使用新研发的里氏木霉产生的木聚糖酶进行酶解，酶活高达508U/ml，进一步提高了低聚木糖的提取效率。
在一些方面，提供一种高温高压处理制备低聚木糖的方法，包括步骤如下：
将玉米芯粉碎后过筛加入水调成预混料；
将预混料进行高温高压处理，得木聚糖粗提液，处理温度95℃-140℃，处理压力0.05-0.25MPa；
将得到的木聚糖粗提液调整质量浓度至4％-6％，pH为4.2-4.8之间，进行微波处理，得到木聚糖液；其中微波频率为2450MHz，处理温度为40-55℃，微波时间10-25min；
将木聚糖液加入木聚糖酶进行酶解，得低聚木糖粗液；
将低聚木糖粗液进行灭酶处理后，进行脱色处理、离子交换处理、浓缩处理，即得到低聚木糖溶液。
在一些方面，提供一种高温高压处理制备低聚木糖的方法，包括步骤如下：
将玉米芯粉碎后过筛加入水调成预混料；
将预混料进行高温高压处理，得木聚糖粗提液，处理温度95℃-140℃，处理压力0.05-0.25MPa；
将得到的木聚糖粗提液调整质量浓度至4％-6％，pH为4.2-4.8之间，进行微波处理，得到木聚糖液；处理温度为40-55℃，微波时间10-25min；
将木聚糖液加入木聚糖酶进行酶解，得低聚木糖粗液；
将低聚木糖粗液进行灭酶处理后，进行脱色处理、离子交换处理、浓缩处理，即得到低聚木糖溶液。
在一些方面，提供一种高温高压处理制备低聚木糖的方法，包括步骤如下：
(1)将玉米芯粉碎后过筛加入水调成预混料；
(2)对预混料进行高温高压处理，得木聚糖粗提液，处理温度95℃-140℃，处理压力0.05-0.25MPa；
(3)将得到的木聚糖粗提液的干物质质量浓度调整至4％-6％，pH调整至4.2-4.8之间，进行微波处理，得到木聚糖液；其中微波的频率为2450MHz，微波处理的温度为40-55℃，微波处理时间10-25min；
(4)将木聚糖液加入木聚糖酶进行酶解，得低聚木糖粗液；
(5)将低聚木糖粗液进行灭酶处理后，进行脱色处理、离子交换处理、浓缩处理，即得到低聚木糖溶液。
在一些实施方案中，玉米芯的粉碎粒径为过80-120目筛，预混料的质量浓度为8％-12％。
在一些实施方案中，高温高压处理温度为115℃-128℃，处理压力0.09-0.18MPa，处理时间为4-8小时。
在一些实施方案中，酶解前将木聚糖液调节质量浓度为4％-6％；优选的，木聚糖酶的加入量为4-6g/kg干物质；优选的，酶解反应温度为50℃-60℃，酶解反应时间为20-40h，酶解反应为静置反应。
在一些实施方案中，木聚糖液加入木聚糖酶进行酶解的过程中，所用的木聚糖酶为如下菌株产生的木聚糖酶，酶活达508U/ml；
所述菌株为里氏木霉(Trichoderma reesei)BLCY-007，2019年6月14日保存于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号CGMCC No.17970，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
在一些实施方案中，高温高压处理制备低聚木糖的方法是指通过高温高压处理玉米芯制备低聚木糖的方法。
在一些实施方案中，步骤(1)的预混料中不含有酸或碱。
在一些实施方案中，步骤(1)中，玉米芯的含水率为14-25％。
在一些实施方案中，步骤(1)的预混料的pH值为7-8。
在一些实施方案中，步骤(1)的预混料的干物质质量浓度为8％-12％。
在一些实施方案中，预混料中干物质质量浓度计算公式为玉米芯干物质质量/预混料液总质量×100％。
在一些实施方案中，干物质是指玉米芯干物质，干物质重量为玉米芯的干重。
在一些实施方案中，干物质质量浓度是指基于玉米芯干重的质量浓度。
在一些实施方案中，步骤(2)在封闭压力容器中进行。
在一些实施方案中，高温是指处理温度为95℃-140℃。
在一些实施方案中，高压是指处理压力为0.05-0.25MPa。
在一些实施方案中，步骤(2)中，处理温度为121℃，处理压力为0.1Mpa。
在一些实施方案中，步骤(3)中的质量浓度是指木聚糖粗提液的干物质质量浓度。
在一些实施方案中，步骤(3)中，微波处理是指将木聚糖粗提液置于密闭容器中，然后将容器置于微波处理设备中进行微波处理。
在一些实施方案中，步骤(3)中，将木聚糖粗提液的干物质质量浓度调整至4％-6％，pH调整至4.2-4.8之间，获得反应前液，微波处理时使用的微波功率与反应前液的比例为500～1000:80～120(W/ml)，例如700～900:100～110(W/ml)。
在一些实施方案中，步骤(3)中，微波处理过程中木聚糖粗提液的温度为40-55℃。
在一些实施方案中，微波处理过程中，木聚糖粗提液的温度维持在40-55℃。
在一些实施方案中，步骤(4)中，酶解前将木聚糖液的干物质质量浓度调整至4％-6％。干物质质量按玉米芯干重计算。
在一些实施方案中，步骤(4)中，步骤(4)中，酶解在50℃-60℃温度进行，酶解的时间为20-40h，酶解过程中保持木聚糖液静置。
在一些实施方案中，步骤(4)仅使用从里氏木霉(Trichoderma reesei)BLCY-007菌体发酵液中获得的粗酶制剂作为所述木聚糖酶。
步骤(1)中的处理温度范围95℃-140℃和压力范围0.05-0.25MPa对于提高低聚木糖产率是关键的。玉米芯中的木聚糖为生物大分子，在自然状态与其它成分如纤维素、木质索复合存在，三者呈不连续的层状结构，对水解有阻碍作用。如在上述温度和压力范围之外处理，无法破坏不连续的层状结构，获得木聚糖收率和低聚木糖提取率均不高。
实验发现，步骤(1)的处理压力低于0.05MPa时，将无法破坏不连续的层状结构，木聚糖收率和低聚木糖提取率均不高。步骤(1)的处理压力高于0.25MPa时，木聚糖会过度水解生成木糖。从而降低木聚糖收率和低聚木糖提取率。
步骤(4)中使用的从里氏木霉(Trichoderma reesei)BLCY-007菌体发酵液中获得的粗酶制剂对于提高低聚木糖产率也是关键的。
在一些实施方案中，低聚木糖是由2-9个木糖单元以β-1,4糖苷键连接而成的低聚糖的混合物。其结构式如下，n＝2～9，
Figure PCTCN2020100482-appb-000002
在一些实施方案中，低聚木糖符合中国标准GB/T 35545-2017低聚木糖。
在一些实施方案中，木聚糖是指是一种存在于植物细胞壁中的异质多糖，约占植物细胞干重的15％～35％，是植物半纤维素的主要成分。
在一些实施方案中，灭酶处理方式为高温灭酶，灭酶温度为85℃-98℃，灭酶时间为10-15min。
在一些实施方案中，脱色处理过程为使用活性炭脱色，活性炭添加量为低聚木糖粗液中干物质质量的0.8-5％，脱色温度为78-85℃，脱色保温时间为15-30min，脱色时液体流速为20-30mL/min。干物质质量按玉米芯干重计算。
在一些实施方案中，脱色处理过程为使用活性炭脱色，活性炭添加量为低聚木糖粗液中干物质质量的0.8-5％，脱色温度为78-85℃，脱色保温时间为15-30min。
在一些实施方案中，脱色处理是指使低聚木糖粗液通过活性炭滤芯。
在一些实施方案中，低聚木糖粗液通过滤芯的流速为20-30mL/min。
优选的，离子交换处理过程使用的离子交换柱是阳离子交换柱-阴离子交换柱-阳离子交换柱的组合柱，离子交换处理温度25-35℃，离子交换处理的流速为15-25mL/min；进一步优选的，所述的阳离子交换柱为强酸阳性树脂，所述的阴离子交换柱为弱碱阴性树脂。
在一些实施方案中，强酸阳性树脂为浙江争光实业股份有限公司生产的D001大孔阳离子交换树脂。
在一些实施方案中，阳离子交换树脂为带有磺酸基(-SO 3H)的苯乙烯-二乙烯苯共聚体。
在一些实施方案中，弱碱阴性树脂为浙江争光实业股份有限公司生产的D354FD大孔弱碱阴树脂。
在一些实施方案中，弱碱阴性树脂为聚苯乙烯大孔结构的弱碱性阴离子交换树脂。
优选的，浓缩处理方式为真空旋转浓缩，工作压力-0.1MPa，工作温度为60-80℃，浓缩处理后至低聚木糖粗液中干物质浓度为60％-78％。
根据本发明一种优选的实施方案，高温高压处理制备低聚木糖的方法包括步骤如下：
(1)调浆：称取一定量玉米芯粉碎后过80-120目筛，调成质量浓度为8％-12％的预混料，其中调浆所用水为纯净水；
(2)高温高压处理：将步骤所述的(1)中的预混料进行高温高压处理，得到木聚糖粗提液；其中处理温度为115℃-128℃，处理时间为4-8小时，处理压力为0.09-0.18MPa；
(3)微波处理：将步骤(2)所述的木聚糖粗提液的质量浓度调至4％-6％，pH调至为4.2-4.8之间，获得反应前液；对反应前液进行微波处理，得到木聚糖液；其中微波频率为2450MHz，处理温度为40-55℃，微波时间10-25min；
(4)酶解：向步骤(3)所述的木聚糖液中加入木聚糖酶进行酶解反应，木聚糖酶的加入量为4-6g/kg干物质，得到低聚木糖粗液；其中，酶解反应温度为50℃-60℃，酶解反应时间为20-40h，酶解反应为静置反应，所用木聚糖酶为使用里氏木霉(Trichoderma reesei)BLCY-007产生的木聚糖酶；
(5)灭酶：将步骤(4)所述的低聚木糖粗液进行灭酶处理，其中灭酶方式为高温灭酶，灭酶温度为85℃-98℃，灭酶时间为10-15min；
(6)精制处理：将步骤(5)所述的灭酶后液体进行脱色处理、离子交换处理、浓缩处理，得到低聚木糖液体；其中脱色处理使用活性炭脱色，活性炭添加量为灭酶后液体干基质量的0.8-5％，脱色温度为78-85℃，脱色保温时间为15-30min，脱色时液体流 速为20-30mL/min；离子交换处理使用的离子交换柱是阳离子交换柱-阴离子交换柱-阳离子交换柱的组合柱，离子交换处理温度25-35℃，离子交换处理的流速为15-25mL/min；
浓缩方式为真空旋转浓缩，工作压力-0.1MPa，工作温度为60-80℃，浓缩至干物质质量浓度为60％-78％。
有益效果
1、本发明首次获得的高产木聚糖酶的里氏木霉BLCY-007，从其菌体发酵液中提取的粗酶制剂的木聚糖酶酶活与传统里氏木霉相比提高60％以上，可达到508U/ml，显著降低生产成本。
2.上述粗酶制剂的最适pH值为5.5～6.5，该pH值范围有利于生产中对污染的控制。
3、本发明所述的里氏木霉BLCY-007，产生的木聚糖酶为胞外酶，分离工艺简单，经简单的离心、洗涤、即可得到酶制剂，节约了生产成本，降低了动力损耗。
4、基于本发明里氏木霉BLCY-007获得的粗酶制剂不含有纤维素酶(cellulases)等杂酶。里氏木霉BLCY-007获得的粗酶制剂无滤纸酶活FPase，无羧甲基纤维素酶活CMCase。
5、基于本发明里氏木霉BLCY-007获得的粗酶制剂不含有毒素，能安全用于食品原料低聚木糖的生产。
6、传统的生产工艺需要对玉米芯进行酸处理或碱处理，需要耐酸碱的设备，一次性投资大；制备工艺复杂，不利于控制反应的进行，副反应多造成副产物多，产品提纯困难，过程中使用大量的酸碱，造成大量污水排放，带来严重的环境污染；本发明使用高温高压工艺处理工艺制得低聚木糖，克服传统生产工艺的缺点，取代了传统的化学法处理，制备过程中不使用酸碱，避免了污水的大量排放，减小环保压力。
7、本发明使用获得的高产木聚糖酶的里氏木霉BLCY-007，其菌体发酵液中的木聚糖酶酶活可达到508U/ml，较传统木聚糖酶酶活提高60％以上，显著降低生产成本，同时进一步提高了低聚木糖的收率。
8、本发明的木聚糖收率和低聚木糖提取率均较传统生产方式有了大幅度的提升，木聚糖的收率达到64％以上，最高达83％；低聚木糖的提取率达72％以上，最高达87％；过程中仅有一次加酶酶解，有效地降低了生产成本。
具体实施方式
下面结合和实施例对本公开的实施方案进行详细描述，但是，本领域技术人员将理解，下列实施例仅用于说明本公开，而不是对本公开的范围的限定。根据和优选实施方案的下列详细描述，本公开的各种目的和有利方面对于本领域技术人员来说将变得显然。
实施例1
一株里氏木霉(Trichoderma reesei)BLCY-007，2019年6月14日保存于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号CGMCC No.17970，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
上述里氏木霉(Trichoderma reesei)BLCY-007的诱变和筛选过程如下：
(1)原始菌种的筛选：
富集培养
选取山东德州百龙创园低聚木糖生产车间附近的土壤，用小铲子除去表土，取离地面10～20cm处的土壤约10g，用无菌水稀释10倍，加入PDA培养基(Potato Dextrose Agar)进行富集培养，24℃～28℃的条件下培养36h。
所述PDA培养基原料组分如下：
马铃薯提取液1.0L，葡萄糖20.0g，琼脂15.0g。
马铃薯提取液按如下方法制备：取去皮马铃薯200g，切成小块，加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L。
纯种分离
采用划线分离法，取一支盛有5ml无菌水的大试管，取步骤(1)中富集培养后的菌液2ml放入其中稀释，充分振荡分散，用接种环以无菌操作挑取稀释液一环先在平板培养基的一边做第一次平行划线3～4条，再转动培养皿约60度角，将接种环上剩余物烧掉，待冷却后同一次划线方法做第二次划线，同法依次做第三次和第四次划线。划线完毕，盖上皿盖，将培养皿倒置，28～38℃培养24h后，挑取单个菌落接种于10个斜面培养基上，得斜面种子，分别编号01～10。
将01～10号斜面种子接种于摇瓶培养基中培养24～28℃培养36h，获得01～10号摇瓶发酵液。对01～10摇瓶发酵液进行木聚糖酶酶活测定，03号摇瓶发酵液表现出最高的酶活，达到105U/ml。
所述平板培养基原料组分如下：马铃薯提取液1.0L，葡萄糖20.0g，琼脂15.0g。
马铃薯提取液:取去皮马铃薯200g，切成小块，加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L。
所述斜面培养基组分如下，均为重量百分比：马铃薯提取液1.0L，葡萄糖20.0g，琼脂15.0g。
所述摇瓶培养基组分如下：去皮马铃薯200g，葡萄糖20g，KH 2PO 4 3g，MgSO 4·7H 2O 1.5g。将上述组分混合后加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L。
(2)突变剂或紫外线照射诱导突变：
诱变筛选
对03号摇瓶发酵液中菌种进行紫外线诱变，紫外线诱变采用15W紫外线灯20cm照射，照射时间为180s，得到的高产菌种再进行甲基磺酸乙酯诱变处理，最终得到高产木聚糖酶的菌株命名为BLCY-007。
实施例2
实施例1中所述的里氏木霉(Trichoderma reesei)BLCY-007的培养方法，包括步骤如下：
(1)取里氏木霉(Trichoderma reesei)BLCY-007接种于PDA培养基中，在24℃的条件下，活化培养12h，制得活化菌株；
PDA培养基原料组分如下：
马铃薯提取液1.0L，葡萄糖20.0g，琼脂15.0g。
马铃薯提取液按如下方法制备：取去皮马铃薯200g，切成小块，加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L；
(2)取步骤(1)制得的活化菌株，接种于种子培养基中，在24℃的条件下，增殖培养24h，制得种子液；
种子培养原料基组分如下：
去皮马铃薯200g，葡萄糖20g，KH2 PO4 3g，MgSO4·7H2 O 1.5g；将上述组分混合后加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L；
(3)取步骤(2)制得的种子液，按体积比2％的比例接种于发酵培养基中，在24℃扩大培养24h，即得菌体发酵液；
发酵培养基原料组分如下，均为重量百分比：
玉米芯25％，葡萄糖4％，牛肉膏6％，蛋白陈1％，无水硫酸镁0.01％，磷酸氢二钾0.02％，硫酸铵0.02％，余量水，pH＝5.0～6.0。
实施例3
实施例1中所述的里氏木霉(Trichoderma reesei)BLCY-007的培养方法，包括步骤如下：
(1)取里氏木霉(Trichoderma reesei)BLCY-007接种于PDA培养基中，在28℃的条件下，活化培养24h，制得活化菌株；
PDA培养基原料组分如下：
马铃薯提取液1.0L，葡萄糖20.0g，琼脂15.0g。
马铃薯提取液按如下方法制备：取去皮马铃薯200g，切成小块，加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L；
(2)取步骤(1)制得的活化菌株，接种于种子培养基中，在28℃的条件下，增殖培养36h，制得种子液；
种子培养基原料组分如下：
去皮马铃薯200g，葡萄糖20g，KH 2PO 4 3g，MgSO 4·7H 2O 1.5g；将上述组分混合后加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L；
(3)取步骤(2)制得的种子液，按体积比8％的比例接种于发酵培养基中，在28℃，扩大培养36h，即得菌体发酵液；
发酵培养基原料组分如下，均为重量百分比：
玉米芯25％，葡萄糖4％，牛肉膏6％，蛋白陈1％，无水硫酸镁0.01％，磷酸氢二钾0.02％，硫酸铵0.02％，余量水，pH＝5.0～6.0。
对比例1
采用实施例1的03号摇瓶发酵液中的菌种。参考实施例2的方法进行培养，得到菌体发酵液。
对比例2
以从北京北纳创联生物技术研究院购得到的里氏木霉作为培养菌株。参考实施例2的方法进行培养，得到菌体发酵液。
实施例4
粗酶制剂的制备：取实施例2、对比例1、对比例2制备的菌体发酵液经离心分离，离心分离条件为：粗酶制剂温度4℃、离心转速10000r/min，离心时间10min。离心后采集上清液，即为粗酶制制剂。
试验例1
测定实施例4中得到的粗酶制剂的木聚糖酶酶活。
本公开中“酶活”和“酶活力”具有相同的含义，均指木聚糖酶酶活力。
按照GBT 23874-2009饲料添加剂木聚糖酶活力的测定分光光度法进行木聚糖酶活力的测定。该测定方法简述如下：
(i)木聚糖酶活单位的定义
在37℃、pH值为5.5的条件下，每分钟从浓度为5mg/ml的木聚糖溶液中释放1μmol还原糖所需要的酶量即为一个酶活力单位U。
(ii)液体样品的反应用酶液制备
将上述粗酶制剂用乙酸-乙酸钠缓冲溶液(pH＝5.5)稀释定容，稀释定容后的液体样品中木聚糖酶酶活控制在0.04U/mL～0.10U/mL之间。
(iii)酶活测定方法
取2ml浓度为100mg/ml的木聚糖底物(pH＝5.5乙酸-乙酸钠缓冲液配制)，加入到比色管中，37℃平衡10min，再加入2ml 37℃平衡好的液体样品的反应用酶液，混匀于37℃精确保温反应30min。反应结束后，加入5ml DNS试剂，混匀以终止反应。然后沸水浴煮沸5min，用自来水冷却至室温，加蒸馏水定容至25ml，混匀后，以标准空白样为空白对照，在540nm处测定吸光值A E。
酶活计算公式：
X D＝[(A E－A B)×K+C 0]×1000/(M×t)
X＝X D×N
式中：X为液体样品中木聚糖酶的活力，U/ml；X D为液体样品的反应用酶液中木聚糖酶的活力，U/ml；A E为酶反应液的吸光度；A B为酶空白液的吸光度；K为标准曲线的斜率；C 0为标准曲线的截距；M为木糖的摩尔质量，150.2g/mol；t为酶解反应时间，min；N为酶液稀释倍数；1000为转化因子，1mmol＝1000μmol。
结果如表1所示：
表1
Figure PCTCN2020100482-appb-000003
由上述表1可知，与原址菌株和市售其它菌株相比，基于里氏木霉BLCY-007获得的粗酶制剂的木聚糖酶酶活有大幅提升。
试验例2
与中国专利CN1185336C中记载里氏木霉菌株进行对比。
以实施例1的BLCY-007菌株为原料，按CN1185336C说明书第4-5页第5部分实施例中(3)和(4)记载的方法，分别进行液体发酵和固体发酵，获得了液体发酵产物-B和固体发酵产物-B。
按照CN1185336C说明书第5-8页第6部分木聚糖酶酶活性测定方法和第7部分葡聚糖酶活性测试方法，分别检测了液体发酵产物-B和固体发酵产物-B的木聚糖酶的酶活力和葡聚糖酶的酶活力，结果如下：
表2
Figure PCTCN2020100482-appb-000004
下面通过具体实施例描述以玉米芯为原料制备低聚木糖的方法。
实施例B1所用的木聚糖酶为基于里氏木霉(Trichoderma reesei)BLCY-007获得的粗酶制剂。对比例B1～B7所用木聚糖酶为青岛蔚蓝生物股份有限公司生产的木聚糖酶SP-min。
以下实施例使用的阳离子交换柱为强酸阳性树脂为浙江争光实业股份有限公司生产的D001大孔阳离子交换树脂。
以下实施例使用的阴离子交换柱为弱碱阴性树脂，为浙江争光实业股份有限公司生产的D354FD大孔弱碱阴树脂。
以下实施例使用的微波处理设备为上海新仪微波化学科技有限公司生产的MDS-6微波消解/萃取仪，参数为：输出功率，0-1000W，温度，控制范围:0-250℃，精度±1℃，压力，控制范围:0.1-5MPa，精度0.1MPa；
以下实施例中所用“％”如无特殊说明，均为质量百分比。
以下实施例中，质量浓度是指玉米芯干物质的质量浓度。
以下实施例中，处理压力值应理解为是基于1个标准大气压的增量值。比如处理压力为0.1Mpa是指在1个标准大气压的基础上增加0.1Mpa。
实施例B1
一种高温高压处理制备低聚木糖的方法，包括步骤如下：
(1)调浆：提供5g玉米芯(含水率15％)，粉碎玉米芯并将粉碎产物过100目筛，收集通过筛网的玉米芯粉料。将玉米芯粉料与纯净水混合，调成干物质浓度为9wt％的预混料，其中调浆所用水为纯净水。
(2)高温高压处理：将预混料置于压力容器中进行高温高压处理，处理温度为121℃，处理时间为6小时，处理压力为0.10MPa，得到木聚糖粗提液。
(3)微波处理：将木聚糖粗提液的干物质浓度调至4％，并将pH调至4.7，得到106ml反应前液；将反应前液置于微波处理设备中进行微波处理，得到木聚糖液；其中微波处理设备的微波功率为800W，微波频率为2450MHz，处理温度为50℃，微波时间15min。
(4)酶解：向木聚糖液中加入木聚糖酶，进行酶解反应，得到低聚木糖粗液，木聚糖酶的加入量为5g/kg干物质；酶解反应温度为52℃，酶解反应时间为24h，木聚糖液的pH值控制在5.5～6.5，酶解反应为静置反应。
所述的木聚糖酶为从里氏木霉(Trichoderma reesei)BLCY-007菌体发酵液中分离获得的粗酶制剂。所述里氏木霉(Trichoderma reesei)BLCY-007，2019年6月14日保存于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号CGMCC No.17970，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
(5)灭酶：将上述的低聚木糖粗液进行灭酶处理。灭酶温度为85℃，灭酶时间为10min。
(6)精制处理：对灭酶后的产物进行精制处理。包括：
脱色，使灭酶后的产物通过活性炭滤芯，活性炭添加量为干基的1％，脱色温度为85℃，脱色保温时间为20min，脱色时液体流速为25mL/min；
离子交换处理，使用的离子交换柱是阳离子交换柱-阴离子交换柱-阳离子交换柱的组合柱，离子交换处理温度25℃，离子交换处理的流速为20mL/min；
真空旋转浓缩，使用旋转式薄膜真空浓缩设备进行浓缩处理，设备工作压力-0.1MPa，工作温度为75℃。浓缩至产物中干物质浓度为75％，得到低聚木糖液。
5g玉米芯理论上可以得到的木聚糖质量为1.8g，经检测，步骤(3)实际得到的木聚糖液中的木聚糖质量为1.494g，步骤(6)实际得到的低聚木糖的质量为1.299g，据此计算得到，木聚糖收率为83％，低聚木糖提取率87％。
对比例B1
对比例B1的与实施例B1相似，区别仅在于：步骤(2)中，处理温度为60℃，处理压力为0.10MPa。步骤(4)使用的木聚糖酶青岛蔚蓝生物股份有限公司(Qingdao Vland Biotech INC)生产的木聚糖酶SP-min，木聚糖酶的加入量为5g/kg干物质。
本例中木聚糖收率和低聚木糖提取率检测结果如下：木聚糖收率为31％，低聚木糖提取率38％。
玉米芯中的木聚糖为生物大分子，在自然状态与其它成分如纤维素、木质索复合存在。三者呈不连续的层状结构，对酸或碱的水解有阻碍作用。单纯进行高压处理，无法破坏不连续的层状结构，因此木聚糖收率和低聚木糖提取率均不高。
对比例B2
对比例B2的与实施例B1相似，区别仅在于：步骤(2)中，处理温度为121℃，处理压力为0.01MPa。步骤(4)使用的木聚糖酶青岛蔚蓝生物股份有限公司(Qingdao Vland Biotech INC)生产的木聚糖酶SP-min，木聚糖酶的加入量为5g/kg干物质。
本例中木聚糖收率和低聚木糖提取率检测结果如下：木聚糖收率为40％，低聚木糖提取率41％。
如对比例B1中的原因，单纯进行高温处理，无法破坏不连续的层状结构，因此木聚糖收率和低聚木糖提取率均不高。
对比例B3
对比例B3的与实施例B1相似，区别仅在于：步骤(2)中，处理温度为80℃，处理压力为0.10MPa。步骤(4)使用的木聚糖酶青岛蔚蓝生物股份有限公司(Qingdao Vland Biotech INC)生产的木聚糖酶SP-min，木聚糖酶的加入量为5g/kg干物质。
本例中木聚糖收率和低聚木糖提取率检测结果如下：木聚糖收率为44％，低聚木糖提取率42％。
由于处理温度过低，无法破坏不连续的层状结构，因此木聚糖收率和低聚木糖提取率均不高。
对比例B4
对比例B4的与实施例B1相似，区别仅在于：步骤(2)中，处理温度为150℃，处理压力为0.10MPa。步骤(4)使用的木聚糖酶青岛蔚蓝生物股份有限公司(Qingdao Vland Biotech INC)生产的木聚糖酶SP-min，木聚糖酶的加入量为5g/kg干物质。
本例中木聚糖收率和低聚木糖提取率检测结果如下：木聚糖收率为36％，低聚木糖提取率35％。
由于高温处理温度过高，造成木聚糖过分水解生成木糖，从而木聚糖收率和低聚木糖提取率降低了。
对比例B5
对比例B5的与实施例B1相似，区别仅在于：步骤(2)中，处理温度为121℃，处理压力为0.04MPa。步骤(4)使用的木聚糖酶青岛蔚蓝生物股份有限公司(Qingdao Vland Biotech INC)生产的木聚糖酶SP-min，木聚糖酶的加入量为5g/kg干物质。
本例中木聚糖收率和低聚木糖提取率检测结果如下：木聚糖收率为42％，低聚木糖提取率41％。
由于处理压力过低，无法破坏不连续的层状结构，因此木聚糖收率和低聚木糖提取率均不高。
对比例B6
对比例B6的与实施例B1相似，区别仅在于：步骤(2)中，处理温度为121℃，处理压力为0.26MPa。步骤(4)使用的木聚糖酶青岛蔚蓝生物股份有限公司(Qingdao Vland Biotech INC)生产的木聚糖酶SP-min，木聚糖酶的加入量为5g/kg干物质。
本例中木聚糖收率和低聚木糖提取率检测结果如下：木聚糖收率为38％，低聚木糖提取率36％。
由于处理的压力过高，造成木聚糖过分水解生成木糖，因此木聚糖收率和低聚木糖提取率降低了。
对比例B7
对比例B7的与实施例B1相似，区别仅在于：
步骤(4)使用的木聚糖酶青岛蔚蓝生物股份有限公司(Qingdao Vland Biotech INC)生产的木聚糖酶SP-min，木聚糖酶的加入量为5g/kg干物质。
分析检测
上述实施例中，木聚糖收率和低聚木糖提取率通过以下计算公式获得：
式1：木聚糖收率＝检测获得的木聚糖质量/理论上可以得到的木聚糖质量×100％
式2：低聚木糖提取率＝低聚木糖质量/木聚糖质量×100％。
式1中，理论上可以得到的木聚糖质量＝玉米芯质量×玉米芯半纤维含量。玉米芯中半纤维含量通常为35％-40％，上述实施例和对比例中使用的玉米芯中半纤维含量为36％。
式1中，木聚糖液中木聚糖含量的检测方法按照以下进行检测：木聚糖液，调节pH值至5，加入四倍体积95％(体积分数)乙醇,醇沉过夜然后3000r/min离心10min，取沉淀，加入一定量的7％H 2SO 4于100℃水解2h中和、定容、过滤，测定滤液的还原糖质量，将其乘以木聚糖聚合因数0.9作为木聚糖质量。还原糖的测定采用DNS法。
式1中，检测获得的木聚糖质量＝0.9×DNS法测得还原糖质量
式2中，低聚木糖的检测方法按照GB/T 35545-2017低聚木糖中(附录A高效液相色谱法)进行检测。
实施例B1和对比例B1～B7部分步骤参数和产物收率如下表3所示：
表3
Figure PCTCN2020100482-appb-000005
由表3的实验结果可知，实施例B1以玉米芯为原料制备低聚木糖具有显著提高的低聚木糖收率，取得了预料不到的技术效果。
尽管本公开的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本公开的保护范围之内。本公开的全部范围由所附权利要求及其任何等同物给出。

Claims (27)

  1. 一株里氏木霉BLCY-007，2019年6月14日保存于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号CGMCC No.17970。
  2. 一种培养权利要求1所述的里氏木霉BLCY-007的方法，包括步骤如下：
    (i)取里氏木霉BLCY-007接种于PDA培养基中，在24℃～28℃的条件下，活化培养12～24h，制得活化菌株；
    (ii)取步骤(i)制得的活化菌株，接种于种子培养基中，在24℃～28℃的条件下，增殖培养24～36h，制得种子液；
    (iii)取步骤(ii)制得的种子液，按体积比1～10％的比例接种于发酵培养基中，在24℃～28℃，扩大培养24～36h，即得菌体发酵液。
  3. 根据权利要求2所述的培养方法，其特征在于，所述步骤(ii)中的种子培养基原料组分如下：
    去皮马铃薯200g，葡萄糖20g，KH 2PO 4 3g，MgSO 4·7H 2O 1.5g；将上述组分混合后加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L。
  4. 根据权利要求2所述的培养方法，其特征在于，所述步骤(iii)中的发酵培养基原料组分如下，均为重量百分比：
    玉米芯25％，葡萄糖4％，牛肉膏6％，蛋白陈1％，无水硫酸镁0.01％，磷酸氢二钾0.02％，硫酸铵0.02％，余量水，pH＝5.0～6.0。
  5. 根据权利要求2所述的培养方法，其特征在于，所述步骤(i)中的PDA培养基原料组分如下：
    马铃薯提取液1.0L，葡萄糖20.0g，琼脂15.0g；
    马铃薯提取液按如下方法制备：取去皮马铃薯200g，切成小块，加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L。
  6. 权利要求1所述的里氏木霉BLCY-007在制备木聚糖酶中的应用。
  7. 根据权利要求6所述的里氏木霉BLCY-007的应用，其特征在于，应用步骤如下：
    取权利要求2所述的培养方法获得的菌体发酵液，经离心分离，洗涤菌体，二次离心，保留沉淀物，即为木聚糖酶粗酶制剂。
  8. 根据权利要求6所述的里氏木霉BLCY-007的应用，其特征在于，应用步骤如下：
    (1)提供根据权利要求2所述的培养方法获得的菌体发酵液；
    (2)对所述菌体发酵液进行离心处理；
    (3)收集上一步产物的上清液，该上清液即为木聚糖酶粗酶制剂。
  9. 根据权利要求7或8所述的里氏木霉BLCY-007的应用，所述离心处理是指在4℃、10000r/min条件下，离心10min。
  10. 权利要求1所述的里氏木霉BLCY-007在制备低聚木糖中的应用。
  11. 一种制备粗酶制剂的方法，包括：
    (1)提供根据权利要求2所述的培养方法获得的菌体发酵液；
    (2)对所述菌体发酵液进行离心处理；
    (3)收集上一步产物的上清液，该上清液即为木聚糖酶粗酶制剂。
  12. 一种制备低聚木糖的方法，包括：
    (1)根据权利要求11所述的方法制备木聚糖酶粗酶制剂；
    (2)使用所述木聚糖酶粗酶制剂对木聚糖进行酶解处理，获得低聚木糖；
    优选地，酶解处理的温度为50～60℃；
    优选地，酶解处理的pH为5.5～6.5。
  13. 一种制备低聚木糖的方法，包括步骤如下：
    将玉米芯粉碎后过筛，加入水，调成预混料；
    对预混料进行高温高压处理，得木聚糖粗提液，其中，处理温度为95℃-140℃，处理压力为0.05-0.25MPa；
    将得到的木聚糖粗提液的质量浓度调整至4％-6％，pH调整至4.2-4.8之间，进行微波处理，得到木聚糖液；其中，微波处理的微波频率为2450MHz，处理温度为40-55℃，微波时间为10-25min；
    向木聚糖液加入木聚糖酶进行酶解，得低聚木糖粗液；其中，所用的木聚糖酶为如下菌株产生的木聚糖酶：所述菌株为里氏木霉BLCY-007，2019年6月14日保存于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号CGMCC No.17970；
    对低聚木糖粗液进行灭酶处理后，进行脱色处理、离子交换处理、浓缩处理，即得到低聚木糖溶液。
  14. 根据权利要求13所述的制备低聚木糖的方法，其特征在于，玉米芯的粉碎粒径能通过80-120目筛，预混料的质量浓度为8％-12％。
  15. 根据权利要求13所述的制备低聚木糖的方法，其特征在于，高温高压处理的处理温度为115℃-128℃，处理压力为0.09-0.18MPa，处理时间为4-8小时。
  16. 根据权利要求13所述的制备低聚木糖的方法，其特征在于，酶解前将木聚糖液的质量浓度调至4％-6％，其中，木聚糖酶的加入量为4-6g/kg干物质。
  17. 根据权利要求13所述的制备低聚木糖的方法，其特征在于，酶解反应温度为50℃-60℃，酶解反应时间为20-40h。
  18. 根据权利要求13所述的制备低聚木糖的方法，其特征在于，灭酶处理方式为高温灭酶，灭酶温度为85℃-98℃，灭酶时间为10-15min。
  19. 根据权利要求13所述的制备低聚木糖的方法，其特征在于，脱色处理过程为使用活性炭脱色，活性炭添加量为低聚木糖粗液干基质量的0.8-5％，脱色温度为78-85℃，脱色保温时间为15-30min，脱色时液体流速为20-30mL/min。
  20. 根据权利要求13所述的制备低聚木糖的方法，其特征在于，离子交换处理过程使用的离子交换柱是阳离子交换柱-阴离子交换柱-阳离子交换柱的组合柱，离子交换处理温度25-35℃，离子交换处理的流速为15-25mL/min。
  21. 根据权利要求13所述的制备低聚木糖的方法，其特征在于，浓缩处理方式为真空旋转浓缩，工作压力-0.1MPa，工作温度为60-80℃，浓缩处理后至干物质浓度为60％-78％。
  22. 根据权利要求13所述的制备低聚木糖的方法，其中，酶解所用的木聚糖酶是从里氏木霉BLCY-007的发酵产物中分离获得的粗酶制剂。
  23. 根据权利要求22所述的制备低聚木糖的方法，所述粗酶制剂的制备方法包括：
    (1)提供里氏木霉BLCY-007的菌体发酵液；
    (2)对所述菌体发酵液进行离心处理；
    (3)收集上一步产物的上清液，该上清液即为所述粗酶制剂。
  24. 根据权利要求23所述的制备低聚木糖的方法，步骤(1)中的里氏木霉BLCY-007的菌体发酵液的制备方法包括：
    (i)取里氏木霉BLCY-007接种于PDA培养基中，在24℃～28℃的条件下，活化培养12～24h，制得活化菌株；
    (ii)取步骤(i)制得的活化菌株，接种于种子培养基中，在24℃～28℃的条件下，增殖培养24～36h，制得种子液；
    (iii)取步骤(ii)制得的种子液，按体积比1～10％的比例接种于发酵培养基中，在24℃～28℃，扩大培养24～36h，即得菌体发酵液。
  25. 根据权利要求24所述的制备低聚木糖的方法，步骤(i)中的PDA培养基原料组分如下：
    马铃薯提取液1.0L，葡萄糖20.0g，琼脂15.0g。
  26. 根据权利要求24所述的制备低聚木糖的方法，步骤(ii)中的种子培养基原料组分如下：
    去皮马铃薯200g，葡萄糖20g，KH 2PO 4 3g，MgSO 4·7H 2O 1.5g，将上述组分混合后加水1.0L煮沸30min，滤去马铃薯块，将滤液补足至1.0L。
  27. 根据权利要求24所述的制备低聚木糖的方法，步骤(iii)中的发酵培养基原料组分如下，均为重量百分比：
    玉米芯25％，葡萄糖4％，牛肉膏6％，蛋白陈1％，无水硫酸镁0.01％，磷酸氢二钾0.02％，硫酸铵0.02％，余量水，pH＝5.0～6.0。
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