CN102634463B - Saccharomycete producing xylitol and applicaton of saccharomycete - Google Patents
Saccharomycete producing xylitol and applicaton of saccharomycete Download PDFInfo
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- CN102634463B CN102634463B CN 201210079787 CN201210079787A CN102634463B CN 102634463 B CN102634463 B CN 102634463B CN 201210079787 CN201210079787 CN 201210079787 CN 201210079787 A CN201210079787 A CN 201210079787A CN 102634463 B CN102634463 B CN 102634463B
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Abstract
The invention discloses saccharomycete producing xylitol and application of the saccharomycete. The xylitol can be produced by pure xylose by Candida maltose XU316CGMCC No.5695 and can be produced by hydrolysate during xylose production. Transformation rate can be nearly equal to the theoretical value and up to 0.90 (xylitol)/gram (xylose), yield can be up to 3g (xylitol)/liter hour, concentration of the xylitol can reach more than 200g/liter, production cost can be reduced greatly, and the saccharomycete has excellent application prospect.
Description
Technical field
The present invention relates to a strain and produce yeast and the application thereof of Xylitol.
Background technology
Xylitol is a kind of five-carbon sugar alcohol, and at nature, it is present in many fruits and vegetables.The sugariness of Xylitol is 1.05 times of sucrose, and heat and sucrose are suitable, and its metabolism does not need Regular Insulin.Therefore can be used as the sweeting agent of patients with diabetes mellitus.Xylitol is by fermentation using bacteria, therefore in chewing gum as sweeting agent, anticariogenic function is not only arranged, and refrigerant sense is arranged in mouth.In addition.Xylitol reduces the liver transaminase in addition and prevents function such as respiratory tract infection.Therefore, Xylitol is not only sweeting agent, also can be used as a kind of adjuvant therapy medicaments.
Because the content of Xylitol in fruits and vegetables is all very low, directly extract difficult and uneconomical.Present industrial production Xylitol adopts chemical method, the i.e. method of wood sugar chemical catalysis hydrogenation.This method needs independent hydrogen manufacturing, reacts under High Temperature High Pressure, and complex process, poor stability, cost are higher, therefore, have become the development trend of Xylitol production with biological process substituted chemistry method.The microorganism for preparing Xylitol for fermentation method nearly all is yeast, and existing natural bacterial classification also has genetic engineering bacterium.The nature strain fermentation is substrate with the wood sugar, part wood sugar will be for regenerating coenzyme, theoretical yield is 0.887g/g, be 0.875mol Xylitol/mol wood sugar (under the anaerobic condition) and 0.917g/g, be 0.905mol Xylitol/mol wood sugar (under half anaerobic condition) (Paraj ó J C, DOMINGUEZ H, DOMINGUEZ J M.Biotechnological production of xylitol, Part 1.Interest of xylitol and fundamentals of its biosynthesis[J] .Biore Technol, 1998,65 (3): 191-201.).The substrate of genetic engineering bacterium fermentation is except wood sugar, and other has other carbon source to be used for regenerating coenzyme, is 1.013g/g to the theoretical yield of wood sugar, i.e. 1mol Xylitol/mol wood sugar.The production level of nature bacterial classification is: transformation efficiency 0.56～0.74 gram (Xylitol)/gram (wood sugar), productive rate 0.2～0.5 gram (Xylitol)/liters per hour; The production level of genetic engineering bacterium is: transformation efficiency can be near theoretical value, productive rate 0.6～1.0 gram (Xylitol)/liters per hour (Leathers:FEMS Yeast Research, 3133-140,2003).In the production of Xylitol, though genetic engineering bacterium is better than the nature bacterial classification, need adds conduct such as glucose, ethanol, acetic acid, glycerine and assist the substrates coenzyme of regenerating, thereby improved cost.No matter be genetic engineering bacterium or natural bacterial classification, its comprehensive production level still can not satisfy the requirement of industrialization.
Summary of the invention
An object of the present invention is to provide a strain and produce yeast and the application thereof of Xylitol.
The yeast of product Xylitol provided by the present invention is maltose candiyeast (Candida maltosa), and its bacterial strain number is XU316, and it is numbered CGMCC No.5695 registering on the books of China Committee for Culture Collection of Microorganisms common micro-organisms center.This bacterial classification has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on January 9th, 2012.
The existing substratum that can cultivate maltose candiyeast (Candida maltosa) all can be used for cultivating maltose candiyeast (Candida maltosa) XU316CGMCC No.5695, and its culture temperature can be 24-37 ℃, and incubation time can be 4-48h; The suitableeest culture temperature is 28-32 ℃.Carbon source in the substratum can be wood sugar and/or glucose; Nitrogenous source can be peptone and/or yeast extract and/or extractum carnis and/or corn steep liquor and/or ammonium salt.
The fixed yeast cell that obtains after maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 is fixing also belongs to protection scope of the present invention.
Maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 or its fixed yeast cell also belong to protection scope of the present invention in the application that wood sugar is converted in the Xylitol.
Another object of the present invention provides a kind of low cost and has the Xylitol production method of higher yields.
Xylitol production method provided by the present invention as reaction substrate, utilizes maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 or its fixed yeast cell that wood sugar is converted into Xylitol with the material that contains wood sugar.The described material that contains wood sugar is not limited to following 1)-3), can comprise that also other contains the liquid of wood sugar: 1) xylose solution; 2) xylose mother liquid; 3) contain the hydrolyzate of hemicellulose material, contain wood sugar in the described hydrolyzate that contains the hemicellulose material.
Xylose mother liquid is the residual sugar liquors that contains behind the xylose crystalline in the xylose production process, and its main component is wood sugar, pectinose, glucose, semi-lactosi etc.
Containing the hemicellulose material includes, but is not limited to: corn cob, bagasse, wood fragments, wood chip, various stalk, rice bran etc.Described hydrolyzate comprises acid hydrolysis thing, protease hydrolysate and sour enzyme associating hydrolyzate.
Aforesaid method can comprise the steps: that also maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 or its fixed yeast cell insert in the aqueous solution of the described material that contains wood sugar according to following proportioning: 0.3-0.5g stem cell/gram wood sugar (wherein xylose concentration is 50-250g/L).Wherein, it is 25 ℃-40 ℃ that the described temperature of reaction that wood sugar is converted into Xylitol can be, as 30 ℃-35 ℃, 30 ℃ or 35 ℃.The described reaction times that wood sugar is converted into Xylitol can be 12-500 hour, as 52-480 hour, 52-66 hour, 52 hours, 66 hours, 65 hours or 480 hours.
Maltose candiyeast of the present invention (Candida maltosa) XU316CGMCC No.5695 can not only be the raw material production Xylitol with pure wood sugar, can also utilize hydrolyzed solution in the xylose production process as the main material production Xylitol.Transformation efficiency can be near theoretical value, be up to 0.90 gram (Xylitol)/gram (wood sugar), productive rate reaches as high as 3 grams, and (Xylitol)/more than the liters per hour, Xylitol concentration reaches as high as more than 200 grams per liters, production cost is reduced significantly, have good application prospects.
Describe the present invention in detail below in conjunction with specific embodiment, these embodiment are used for understanding rather than restriction the present invention.
The preservation explanation
Strain name: maltose candiyeast
Latin name: Candida maltosa
Strain number: XU316
Preservation mechanism: China Committee for Culture Collection of Microorganisms common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on 01 09th, 2012
The preservation center numbering of registering on the books: CGMCC No.5695
Embodiment
Method among the following embodiment is ordinary method if no special instructions.
Wood sugar among the following embodiment 3-5 and Xylitol are all measured as follows: instrument be HPLC (Waters, USA), chromatographic column be Aminex HPX-87H (BioRad, CA, USA), detector be RI@8X detector (Waters, USA).Moving phase is 5mM H
2SO
4The aqueous solution, flow velocity 0.6ml/min, column temperature are 60 ℃.Measure the concentration of wood sugar and Xylitol with the external standard standard measure.The wood sugar standard substance that adopt are available from sigma company, and purity＞99%, the Xylitol standard substance of employing is available from sigma company, purity＞99%.Under this chromatographic condition, the retention time of wood sugar standard substance is 10.627 minutes, and the retention time of Xylitol standard substance is 13.293 minutes.Under this chromatographic condition, the typical curve of wood sugar is y=1452.004x, and wherein y is peak area, and x is concentration, and its unit is g/L, and sample size is 10ul; The typical curve of Xylitol is y=1538.614x, and wherein y is peak area; X is concentration, and its unit is g/L, and sample size is 10ul.
Glucose among the following embodiment 4-5 and pectinose are all measured as follows: instrument be HPLC (Waters, USA), chromatographic column be Aminex HPX-87H (BioRad, CA, USA), detector be RI@8X detector (Waters, USA).Moving phase is 5mM H
2SO
4The aqueous solution, flow velocity 0.6ml/min, column temperature are 60 ℃.Measure the concentration of glucose and pectinose with the external standard standard measure.The glucose standard substance that adopt are available from sigma company, and purity＞99%, the pectinose standard substance of employing is available from sigma company, purity＞99%.Under this chromatographic condition, the retention time of glucose standard substance is 9.898 minutes, and the retention time of pectinose standard substance is 11.689 minutes.Under this chromatographic condition, the typical curve of glucose is y=1359.440x, and wherein y is peak area, and x is concentration, and its unit is g/L, and sample size is 10ul; The typical curve of pectinose is y=1490.792x, and wherein y is peak area, and x is concentration, and its unit is g/L, and sample size is 10ul.
Total reducing sugar among the following embodiment 5 uses saccharometer to measure (refractometry).
Separation and the evaluation of embodiment 1, maltose candiyeast (Candida maltosa) XU316CGMCC No.5695
1, the separation of maltose candiyeast (Candida maltosa) XU316CGMCC No.5695
Maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 contains extraction the sample of decay of wood thing, separation, screening, purifying to obtain from Chinese Beijing area.Concrete method is as follows: soak sample with sterilized water, remove by filter insolubles, coat in the solid plate substratum that contains wood sugar, grow single bacterium colony as for being cultured in 30 ℃ of incubators, single bacterium colony is carried out the wood sugar transformation experiment one by one, choose the good bacterial strain of proterties, obtain maltose candiyeast (Candida maltosa) XU316CGMCC No.5695.
2, the evaluation of maltose candiyeast (Candida maltosa) XU316CGMCC No.5695
Maltose candiyeast (Candida maltosa) XU316CGMCC No.5695, bacterial strain is avette, oval, cylindricality, size is 2-5.5 * 3.5-12 μ m, has collarium and mould film to form; There is pseudohypha to produce; Bacterium colony cheese shape, oyster white, surface smoothing is extremely coarse, not reflective, edge tree root shape or tasselled shape.Pcr amplification 26SrDNA/D1, the D2 sequence, used PCR primer is: upstream GCATATCAATAAGCGGAGGAAAAG, downstream GGTCCGTGTTTCAAGACGG; Amplification to sequence be sequence 1 in the sequence table.
The cell cultures of embodiment 2, maltose candiyeast (Candida maltosa) XU316CGMCC No.5695
Maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 is stored in the wort agar slant medium, and substratum consists of: malt extract 100g/L, agar 20g/L, solvent are water.Cultivate maltose candiyeast XU316CGMCC No.5692 with the YPD liquid nutrient medium, substratum consists of: yeast extract 10g/L, and peptone 20g/L, glucose 50g/L, solvent are water, pH is 5.0.Cultural method is as follows: connect a ring slant strains and shake in the bottle in the 250ml triangle, interior dress 50ml liquid nutrient medium places on the rotary shaker, and is extremely saturated as first order seed in 30 ℃, 250rpm overnight incubation.Change first order seed in the 500ml liquid nutrient medium by 10% (volume ratio) inoculum size, be sub-packed in 5 500ml triangles and shake in the bottle, every bottle of 100ml substratum, in 30 ℃, 250rpm overnight incubation to saturated as secondary seed.Change secondary seed in the 3000ml liquid nutrient medium by 10% (volume ratio) inoculum size, substratum places 6L NBS BIOFLO III self-control desk type fermentor tank, and add 0.3ml polyethers defoamer, culture condition is as follows: mixing speed is that 500rpm, temperature are that 30 ℃, air flow are that 0.5vvm, dissolved oxygen are controlled more than 30%, incubation time is to go up to dissolved oxygen in 9 hours, and dry cell weight is the 100g/L fermented liquid.The dry cell weight measuring method is as follows: get the 10ml fermented liquid with 13000rpm centrifugal 5 minutes, abandon supernatant, 10ml washes once, and is centrifugal equally, abandons supernatant, and 90 ℃ of oven for drying are to constant weight, and conversion is the dry weight of every L fermented liquid behind the gravimetry.Cultivate and finish back Backman J2-HS whizzer collecting cell, centrifugal condition is: 6 pipes (every pipe 500ml), rotating speed 5000rpm, 30 minutes time.Abandon supernatant after centrifugal, cell is used for wood sugar and transforms.
Embodiment 3, maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 transform the wood sugar aqueous solution and produce Xylitol.
1, maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 cell single transforms the wood sugar aqueous solution and produces Xylitol
Take by weighing 600g wood sugar (available from Shandong Futian Medicine Industry Co., Ltd., purity＞99%) and be dissolved in the water, be dissolved in 3L surely, be made into the wood sugar aqueous solution and be used for cell transformation.This 3L wood sugar aqueous solution is placed 6L NBS BIOFLO III self-control desk type fermentor tank, the maltose candiyeast XU316CGMCCNo.5692 cell of collecting with the 1.8L fermented liquid among the embodiment 2 is resuspended in the wood sugar aqueous solution, the proportioning that makes wood sugar in maltose candiyeast XU316CGMCC No.5692 cell and the wood sugar aqueous solution is 0.3g stem cell/gram wood sugar, carries out the cell transformation reaction of wood sugar.Conversion condition is as follows: temperature is that 30 ℃, mixing speed are that 300rpm, air flow are 0.05vvm, reacts on end in 57 hours, sampling, and every pipe 1ml in desk centrifuge with 13000rpm centrifugal 5 minutes, supernatant liquor is used for measuring wood sugar and Xylitol concentration.
Above-mentioned experiment repeats 3 times, and the result shows that 57 hours Xylitol concentration of conversion reaction is 170.5g/L, and xylose concentration is 8.2g/L, and the wood sugar of consumption is 191.8g/L, and transformation efficiency 0.89 gram (Xylitol)/gram (wood sugar), productive rate are 3 gram (Xylitol)/liters per hours.Wherein, the retention time of wood sugar is 10.630 minutes in the above-mentioned supernatant liquor, and the retention time of Xylitol is 13.295 minutes.
2, maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 cell repeats to transform the wood sugar aqueous solution and produces Xylitol
Take by weighing 600g wood sugar (available from Shandong Futian Medicine Industry Co., Ltd., purity＞99%) and be dissolved in the water, be dissolved in 3L surely, be made into the wood sugar aqueous solution and be used for cell transformation.This 3L wood sugar aqueous solution is placed 6L NBS BIOFLO III self-control desk type fermentor tank, the maltose candiyeast XU316CGMCCNo.5692 cell of collecting with the 1.8L fermented liquid among the embodiment 2 is resuspended in the wood sugar aqueous solution, the proportioning that makes wood sugar in maltose candiyeast XU316CGMCC No.5692 cell and the wood sugar aqueous solution is 0.3g stem cell/gram wood sugar, carries out the cell transformation reaction of wood sugar.Conversion condition is as follows: temperature is that 30 ℃, mixing speed are that 300rpm, air flow are 0.05vvm, reacts on end in 57 hours.After conversion reaction finished, with clear liquid and the cell of Backman J2-HS whizzer centrifugation reaction solution, centrifugal condition was: 6 pipes (every pipe 500ml), rotating speed 5000rpm, 30 minutes time.Supernatant liquor is for separating of the purifying Xylitol, and cell is used for the bio-transformation of the wood sugar aqueous solution again according to the method for step 1, and except the conversion reaction asynchronism(-nization), other condition all conversion reaction conditions with step 1 is identical.Cell repeats to transform 9 times altogether, reuses 9 times, wherein transforms for the 6th, 8 time and adds the 150g wood sugar respectively, wood sugar as reaction substrate amounts to 5700 grams, and accumulative total consumes wood sugar 5409 grams, generates Xylitol 4599 grams, when sharing 480 hours, cell still had conversion capability.9 times average conversion is 0.85 (Xylitol)/gram (wood sugar), and average yield is 3.2 gram (Xylitol)/liters per hours, average tree sugar alcohol concentration 170g/L.Wherein, the 2-9 time conversion reaction time was respectively 50,48,50,53,54,55,56 and 57 hours.
3, the extraction of Xylitol in the supernatant liquor.
With the supernatant liquor that contains Xylitol (containing Xylitol 610g altogether) that obtains in the step 2, remove positively charged ion in the supernatant liquor with Hydrogen 732 strongly acidic cation-exchanges, remove negatively charged ion in the supernatant liquor with hydroxyl type D-301R weak base type anionite-exchange resin again, making supernatant liquor pH is 5.5-6.5.Carry out ultrafiltration with the supernatant liquor of hollow fiber membrane ultrafiltration device after to deionization and remove macromole, the average molecular weight cut-off of ultra-filtration membrane is 5000.Concentrate removal ion and macromolecular reaction solution with rotary vacuum evaporator, temperature is 55 ℃, and being concentrated into Xylitol concentration is 753g/L.Concentrated solution is placed beaker, slowly lower the temperature and slow agitated liquid with the programmed cooling instrument, cooling rate is 2 ℃/hour, to crystal occurring, continues slowly to be stirred to cool to 18 ℃, and restir 1 hour makes crystallization abundant.With B vacuum filtration isolation of crystalline and mother liquor, Energizer is drained, and obtains xylitol crystal 385g after 40 ℃ of dryings.Mother liquor and another time supernatant liquor merge concentrated back recrystallize.
Embodiment 4, maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 maize transformation core hydrolyzed solution are produced Xylitol.
Corn cob and 1% (quality percentage composition) dilute sulphuric acid is mixed by 1: 5 mass ratio, after 120-130 ℃ of insulation hydrolysis in 2-3 hour, by in and depickling, activated carbon decolorizing, reduction vaporization concentrate, after ion-exchange breaks away from son, obtain the corn cob hydrolyzed solution.This hydrolyzed solution contains wood sugar 160g/L, glucose 16g/L, pectinose 15g/L, does not contain Xylitol.This 3L corn cob hydrolyzed solution is placed 6L NBS BIOFLO III self-control desk type fermentor tank, the maltose candiyeast XU316CGMCC No.5692 cell of collecting with the 1.8L fermented liquid among the embodiment 2 is resuspended in the 3L corn cob hydrolyzed solution, the proportioning that makes wood sugar in maltose candiyeast XU316CGMCC No.5692 cell and the corn cob hydrolyzed solution is 0.375g stem cell/gram wood sugar, carries out the cell transformation reaction of wood sugar.Conversion condition is as follows: temperature is that 25 ℃, mixing speed are that 300rpm, air flow are 0.05vvm, react on end in 52 hours, sampling, every pipe 1ml in desk centrifuge with 13000rpm centrifugal 5 minutes, supernatant liquor is used for measuring wood sugar, Xylitol, glucose and arabinose concentrations.Experiment repeats 3 times, the result showed conversion reaction 52 hours, Xylitol concentration is 135.3g/L, xylose concentration is 9.2g/L, the wood sugar that consumes is 150.8g/L, and glucose concn is 0.15g/L, arabinose concentrations 14.5g/L, transformation efficiency 0.90 gram (Xylitol)/gram (wood sugar), productive rate are 2.6 gram (Xylitol)/liters per hours.Wherein, the retention time of wood sugar is 10.629 minutes in the above-mentioned supernatant liquor, and the retention time of Xylitol is 13.296 minutes, and the retention time of glucose is 9.901 minutes, and the retention time of pectinose is 11.690 minutes.
Embodiment 5, maltose candiyeast (Candida maltosa) XU316CGMCC No.5695 transform xylose mother liquid and produce Xylitol.
Xylose mother liquid is in the xylose production xylose crystalline liquid to be told liquid waste behind the crystal wood sugar by solid-liquid separation, and color is Vandyke brown, and total reducing sugar is 660g/L, wherein contains wood sugar 340g/L, glucose 150g/L, pectinose 170g/L, does not contain Xylitol.Water is with one times of mother liquor dilution, and the activated carbon that adds 3% (W/V) decoloured 2 hours for 60 ℃, removed by filter gac, obtained handling the back xylose mother liquid.Handle in the xylose mother liquid of back and contain wood sugar 165gL, glucose 70g/L, pectinose 82g/L.The maltose candiyeast XU316CGMCCNo.5692 cell of collecting with the 1.8L fermented liquid among the embodiment 2 is resuspended in 3L to be handled in the back xylose mother liquid (containing wood sugar 165g/L), making maltose candiyeast XU316CGMCC No.5692 cell and the proportioning of handling wood sugar in the xylose mother liquid of back is 0.364 stem cell/gram wood sugar, carries out the cell transformation reaction of wood sugar.Conversion condition is as follows: temperature is that 35 ℃, mixing speed are that 300rpm, air flow are 0.05vvm, react on end in 65 hours, sampling, every pipe 1ml in desk centrifuge with 13000rpm centrifugal 5 minutes, supernatant liquor is used for measuring wood sugar, Xylitol, glucose and arabinose concentrations.Experiment repeats 3 times, the result shows that Xylitol concentration is 136.7g/L, xylose concentration is 10.1g/L, the wood sugar that consumes is 154.9g/L, glucose concn is 0.21g/L, arabinose concentrations 81.1g/L, transformation efficiency are 0.88 gram (Xylitol)/gram (wood sugar), and productive rate is 2.1 gram (Xylitol)/liters per hours.Wherein, the retention time of wood sugar is 10.631 minutes in the above-mentioned supernatant liquor, and the retention time of Xylitol is 13.298 minutes, and the retention time of glucose is 9.897 minutes, and the retention time of pectinose is 11.691 minutes.
Sequence table
Claims (7)
1. maltose candiyeast (Candida maltosa) is characterized in that: the bacterial strain of described maltose candiyeast number is XU316, and deposit number is CGMCC No.5695.
2. the described maltose candiyeast of claim 1 is in the application that wood sugar is converted in the Xylitol.
3. method for preparing Xylitol is characterized in that: described method as reaction substrate, utilizes the described maltose candiyeast of claim 1 that wood sugar is converted into Xylitol with the material that contains wood sugar; The described material that contains wood sugar is xylose solution.
4. method according to claim 3, it is characterized in that: described xylose solution is xylose mother liquid or corn cob hydrolyzate.
5. according to claim 3 or 4 described methods, it is characterized in that: the described temperature of reaction that wood sugar is converted into Xylitol is 25 ℃-40 ℃.
6. according to claim 3 or 4 described methods, it is characterized in that: the described reaction times that wood sugar is converted into Xylitol is 12-500 hour.
7. the application of the described maltose candida cell of claim 1 in the preparation Xylitol.
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