CN101497903B - Method for selectively converting and shunting biological products - Google Patents
Method for selectively converting and shunting biological products Download PDFInfo
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- CN101497903B CN101497903B CN2008100067858A CN200810006785A CN101497903B CN 101497903 B CN101497903 B CN 101497903B CN 2008100067858 A CN2008100067858 A CN 2008100067858A CN 200810006785 A CN200810006785 A CN 200810006785A CN 101497903 B CN101497903 B CN 101497903B
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- xylitol
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Abstract
The invention relates to method for selectively transforming, shunting and preparing various biological products from the same bio-matrixes, in particular relates to a method for directly producing xylitol and arabinose by hemicellulose hydrolysate. The method comprises the following steps: raw materials rich in arabinoxylans are hydrolyzed by acid or enzyme to obtain hydrolysate rich in the arabinose and xylose; and the hydrolysate is inoculated into yeast strains which can ferment the xylose and produce the xylitol and can not utilize the arabinose after detoxification treatment. When the xylose concentration in fermentation liquid is lowered to a certain extend, the fermentation is stopped. Ion-exchange desalinization, discoloration, proper concentration and chromatographic resolution are carried out on the fermentation liquid so as to respectively obtain xylose solution and multi-sugar solution taking the arabinose as main ingredient, from which crystal xylitol and crystal arabinose are respectively obtained by treatment. The method combines the manufacturing processes of the xylitol and the arabinose, thereby reducing the production cost and increasing the use ratio of resources.
Description
Technical field
The invention belongs to technical field of biological fermentation, it relates to the method for selective conversion, shunting biological products, relate in particular to Xylitol, pectinose component from identical bio-matrix transformed successively, generated, shunted, concentrated, crystallization, obtain the method for different biological products.
Background technology
How same bio-matrix is carried out controlled selective conversion, shunting, obtain different biological products successively or simultaneously, this is important topic of biological technical field.The present invention has carried out initiative technology with hemicellulose and has attempted.
Hemicellulose mainly refers to the glycan class outside the Mierocrystalline cellulose in the plant cell wall, is that the plant materials intensive amount is only second to cellulosic natural polysaccharide, accounts for the 20-30% of plant trunk weight.Mierocrystalline cellulose is the homogeneous glycan that is become by single glucose polymerisation, and hemicellulose then is to constitute main chain, be made of the low-molecular-weight polysaccharide of apparatus derivatorius several sugar units of difference amount with β-(1-4)-D-wood sugar.Topmost substituting group is Arabic glycosyl in the middle of the hemicellulose apparatus derivatorius, and other a spot of substituting group also has semi-lactosi, seminose, rhamnosyl etc.
The hemicellulose ratio is easier to hydrolysis, and with diluted acid or enzyme-treated plant fibres material, being easy to generate with wood sugar, pectinose is main carbohydrate, also comprises the hydrolyzate of other assorted sugar.
Have only the wood sugar in the hemicellulose hydrolysate, pectinose after purifying comes out respectively, they could realize commercial application value separately.
Purified wood sugar main application is the preparation Xylitol.Xylitol is generated by wood sugar reduction, has sugariness with sucrose is suitable, no cariogenicity, and metabolism does not need the Regular Insulin participation in vivo, can not cause the key properties such as rapid variation of blood sugar yet, and at prevention of dental caries food, diabetic food has important use value.
Purified pectinose not only local flavor is very close with sucrose, and its many special function has also caused people's extensive concern.For example pectinose can suppress the activity of sucrase, thereby suppresses the absorption of human body to sucrose, can be used for controlling the blood sugar increasing that causes because of edible sucrose; Pectinose has the activity that suppresses lipese, can be used for prevention of obesity.In addition, pectinose also is widely used as the intermediate of the synthesis of nucleoside medicine, and scope is widely used in medicine industry.
Yet no matter wood sugar or pectinose just may embody higher commercial value after only reaching very high purity.The separation and purification of wood sugar, pectinose just becomes link very important in its manufacturing processed.
United States Patent (USP) 6,086,681, [from solution, reclaim the method for wood sugar, Method for recovery of xylose fromsolutions, United States Patent (2000) 6,086,681] provide a kind of method of separating wood sugar with crystallization processes from the dilute sulphuric acid hydrolyzate of vegetable fibre, this method is that the xylose solution of 30-60% (to total reducing sugar) is concentrated to the wood sugar supersaturation with purity, and the crystal wood sugar is separated out in cooling then.But this invention does not relate to how separating the wood sugar that remains in the mother liquor, does not relate to yet and how to separate the known pectinose that is present in the hydrolyzate.
United States Patent (USP) 6,872, the recovery of 316[wood sugar, Recovery of xylose, United States Patent (2005) 6,872,316] provide a kind of nanofiltration (nanofiltration) technology to improve the method for the wood sugar purity in the vegetable fibre hydrolyzed solution.Because wood sugar can more easily see through nanofiltration membrane, other impurity such as oligose, divalent salts, hexose, pigment etc. are trapped the filter membrane inboard then morely.Thereby the nanofiltration of vegetable fibre hydrolyzed solution sees through the wood sugar purity of liquid, and comparable original hydrolyzed solution improves more than 1 times, the ion-exchange that it is follow-up, and technologies such as decolouring burden also alleviates thereupon.In this invention, the same easy nanofiltration membrane that sees through of pectinose with wood sugar, so it can not realize separating between wood sugar and the pectinose.In addition, nanofiltration also is very limited for the separating effect of monose.
Positively charged ion has adsorption to monose (or sugar alcohol), and utilization carries cationic materials and makes different monose or the sugar alcohols of chromatogram filler separation, is one of means of people's separate targets monose (or sugar alcohol) from the mixing solutions of multiple monose.
United States Patent (USP) 6,506,897[garden beet root prepares pectinose, Method of preparing l-arabinose from sugarbeet pulp.United States Patent (2003), 6,506,897] a kind of method for preparing pectinose that provides has comprised time several steps: a) extract the beet tails that had pressed sugar with strong base solution.B) the thick arabinan that is obtained with strong acid hydrolysis previous step.C) acid hydrolysis liquid neutralization is filtered.D) as separation resin, chromatographic separation goes out the pectinose component with unit price sun subtree fat (select sodium for use, or potassium type).E) be further purified the pectinose solution that is obtained through cationic, anionic exchange resin and polymeric adsorbent.F) the crystal pectinose is reclaimed in crystallization, and purity is more than 98%.This patent is used earlier the alkaline extraction material, and is obviously more loaded down with trivial details with acid-hydrolyzed technology again, in addition, do not introduce whether there is wood sugar in the hydrolyzed solution in the patent, also do not have to introduce the problem of how to separate wood sugar.
Chinese patent 01816511.7[patent name: the weakly acidic cationic exchanger resin chromatography reclaims monose from solution; International application: PCT/FI2001/000848 2001.9.28; Patentee: Finland, Danisk Sweetener OY; Contriver: H sea base Lay, J cloud Pa Yining etc.] introduced to utilize and combined Na
+, Mg
2+, H
+Or Ca
2+The weakly acidic cation-exchange resin of type is used for chromatographic separation, use comprises the rapid technology of multistep of the chromatographic separation of at least one step, reclaims the method for the monose that is selected from rhamnosyl, pectinose (arabinose), wood sugar and its mixture from the solution of the monose that contains rhamnosyl, arabinose, wood sugar and its mixture.The text of this patent and accompanying drawing are described all and are shown that in the chromatography separating method that it provided, its wood sugar component is not separated fully with the component of pectinose.In addition, it is not addressed selective conversion sugar yet and is sugar alcohol, and how to carry out the separation problem between sugar alcohol and the sugar.
Except with the chromatographic separation filler of Zeo-karb as sugar, some combine the separation and purification that cationic inorganic materials also is used to carbohydrate.
United States Patent (USP) 4,664, the method for 718[separating arabinose from the sugar mixture of pentose/; Chang; Chin-Hsiung, Process for separating arabinose from a pentose/hexose mixture.United States Patent (1987), 4,664,718] introduced with calcium Y-type zeolite, or calcium X-type zeolite makes sorbent material, the method for separating arabinose from the sugar mixture of pentose/, but this patent does not relate to the Separation and Recovery wood sugar, or Xylitol.
United States Patent (USP) 4,857, the technology of 642[separating arabinose from other aldose mixture; Kulprathipanja, Processfor separating arabinose from a mixture of other aldoses, United States Patent (1989), 4,857,642] introduction is made sorbent material with ammonium X-type zeolite, the method for separating arabinose from other aldose mixture.United States Patent (USP) 4,880, the technology of 919[separating arabinose from the aldose mixture; Kulprathipanja, Process for separating arabinose froma mixture ofaldoses, United States Patent (1989), 4,880,919] introduction is made the sorbent material separating arabinose with calcium-ammonium type blended Zeo-karb.These two patents all do not relate to the problem that how to reclaim wood sugar or Xylitol yet.
With water is eluent, and monose, the chromatographic separation effect of sugar alcohol on resin cation (R.C.) (or zeolite) depend on the parent/hydrophobic performance of their molecular structures.Same class sugar with same carbon atoms number (for example, the wood sugar and the pectinose that belong to five-carbon ring aldehydo sugar), or have between the sugar alcohol (as the Xylitol and the arabitol of five carbon) of same carbon atoms number, isomers each other each other, its chemical property is very close, and is also just very little to the difference of adsorptive power between them with a kind of resin cation (R.C.).So, no matter use resin cation (R.C.) (to comprise Na
+, Mg
2+, H
+Or Ca
2+) directly separate the five-carbon sugar (wood sugar and pectinose) in the hemicellulose hydrolysate, or separate the pure liquid (sugar generates corresponding alcohol) that hydrolyzate is generated behind chemical hydrogenation, all exist device separates efficient not high, the difficulty that the yield of product and purity are on the low side etc. is difficult to solve.
In order to improve separation efficiency, many scholars have adopted the means of biological fermentation aided purification, promptly utilize the microbial selective that to assimilate specific monose to remove unwanted carbohydrate, the purity of target monose in the hydrolyzed solution is improved relatively, to improve the efficient of later separation operation.
People such as Nyun [Nyun Ho Park, Shigeki Yoshida, Akira Takakashi, et al.A new method for thepreparation of crystalline L-arabinose from arabinoxylan by enzymatic hydrolysis and selectivefermentation with yeast.Biotechnology Letters 2001,23:411-416] be rich in araboxylan (Arabinoxylan with the crude enzyme liquid hydrolysis that derives from penicillium funiculosum (Penicilliumfuniculosum) culture, contain 28.1% pectinose, 32.8% wood sugar) maize peel, the result has 21.3% (w/w) pectinose, and 18.7% (w/w) wood sugar is hydrolyzed out.In addition, also contain some other monose and oligose in the hydrolyzate.With can the metabolism wood sugar, but Saturn of energy metabolism pectinose was not intended the inferior yeast of Weir (Williopsis saturnus) aerated culture 96 hours, under 95% pectinose is preserved in fermented liquid as a result, and the wood sugar residual quantity only is equivalent to 0.002% of original araboxylan weight.This fermented liquid is through decolorizing with activated carbon, and the ion exchange resin desalination concentrates post crystallization, does not detect wood sugar in the crystal that is obtained.The method of optionally removing wood sugar with biological fermentation though can solve pectinose and wood sugar is difficult to isolating difficulty, has been wasted valuable wood sugar resource.
People such as Li Daoyi [Li Daoyi, Yan Qiaojuan, Jiang Zhengqiang etc. (China Agricultural University), saccharomycetes to make fermentation maize peel acid hydrolysis liquid prepares the crystallization L-arabinose.Food science, 2007,28 (4): 125-127 ,] with the dilute acid hydrolysis thing of the own yeast strain WYS15-3 fermented maize skin that screens 7 days, the result optionally removed glucose and the wood sugar in the hydrolyzed solution.Fermented liquid breaks away from steps such as son, condensing crystal through decolouring, and acquisition content is 97% pectinose, and yield is 9.6% of a maize peel butt.Bacterial strain and the method purifying corn skin hydrolyzed solution used by this experiment prepare pectinose, not only do not utilize the wood sugar resource, and production process oversize (7 days).In addition,, be present in the semi-lactosi in the pectinose supersaturated solution, directly influenced the crystallization yield of pectinose because the used yeast bacterial strain can not assimilate the semi-lactosi in the hydrolyzed solution.
Chinese patent 200510040433.0[patent name: a kind of method from xylose mother liquid or xylose hydrolysis fluid extraction wood sugar and Xylitol; Contriver: Peng Qijun] a kind of technology of separating preparation high purity wood sugar or Xylitol from xylose hydrolysis fluid or xylose mother liquid is provided.Concrete grammar is: with xylose hydrolysis fluid or xylose mother liquid is raw material, remove wherein glucose with fermentation by saccharomyces cerevisiae, with the special-purpose calcium type of synthetic resin cation (R.C.) as the chromatographic separation resin, with water is eluent, separate with impurity such as pectinoses by the simulation moving-bed wood sugar that makes, obtain being rich in the component of wood sugar.Perhaps will be rich in wood sugar, the liquid glucose direct hydrogenation of pectinose is generated as after the corresponding alcohol them, separates by simulated moving bed chromatography again, obtains different sugar alcohol components.But, in of the present invention giving an example, separating the process of hydrolyzed solution (liquid glucose) fails wood sugar is thoroughly separated with other assorted sugar (mainly being pectinose), with the sugared hydrogenating reduction in the hydrolyzed solution is the chromatographic separation that sugar alcohol carries out afterwards again, also fails different sugar alcohol (mainly being Xylitol and arabitol) is thoroughly separated.In addition,, also introduced the separation of Xylitol after the liquid glucose hydrogenation, do not related to how optionally wood sugar being converted into Xylitol though this patent has used microorganism to remove the technology of glucose, and then with Xylitol and other sugared isolating problem.
Because with water is the inherent defect of ion chromatography aspect the separation carbohydrate of eluent, people also attempt addressing this problem with other method.
United States Patent (USP) 20060100423[separates the technology with preparation pectinose and wood sugar from mixture of monosaccharides; Hollingsworth; Rawle I, Process for the preparation and separation of arabinose and xylose from amixture of saccharides; United States Patent Application, 20060100423] utilize the reaction of monose and ketone or aldehyde reagent, generate sugared acetal (acetals of the saccharides), according to the difference of different sugared acetals to polarity, non-polar organic solvent solubility property, can make different sugared acetals separated from one another, respectively it is hydrolyzed to corresponding monose at last again.But, a large amount of shortcomings in industrial production with an organic solvent are conspicuous.
People such as Feng Yaqing [Feng Yaqing, Liu Yantong, Zhang Xiaodongs etc. extract L-arabinose, fine chemistry industry from the L-Sudan Gum-arabic, 2003,20 (5): 288-290] with Microcrystalline Cellulose, or silica gel is stationary phase, is moving phase with the mixture of propyl carbinol, ethyl acetate, Virahol and water, carry out two dimensional chromatography continuously and separate, obtain the pectinose crystal of content 99% at last.But this paper does not make the hemicellulose that derives from plant cell wall make raw material, and it is introduced and utilizes the chromatography separating method of organic solvent as moving phase, also has many potential safety hazards in large-scale industry production.
In sum as seen, the chemical technology of current industrial production Xylitol or direct separating arabinose from hemicellulose hydrolysate all can't solve and be difficult to isolating problem between wood sugar and the pectinose.In addition, because the chemical reduction process is non-selective, also can't directly utilize hemicellulose hydrolysate to prepare Xylitol and pectinose simultaneously with existing chemical technology.
Microorganism also can generate Xylitol by the catalysis wood sugar.Utilize microorganism direct fermentation hemicellulose hydrolysate to produce Xylitol, energy-conservation owing to having, need not advantages such as the requisite wood sugar purge process of chemical technology institute, caused people's very big concern.But the efficient of fermentative Production Xylitol is also very low at present, although relevant research report is a lot of, great majority still are confined to the preparation method of hemicellulose hydrolysate, xylitol fermentation process optimization aspect.The research for preparing the crystalline xyhose alcohol aspect from wood-sugar fermentation liquid is very few, and also nobody's research xylitol zymolysis production prepares the problem that combines with pectinose.
The inventor [Cai Aihua, Zhang Hourui etc.: separation and purification Xylitol from bagasse hemicellulose hydrolyzate fermented liquid.Food science.2006,27 (7): 136-139] once introduced, bagasse hemicellulose hydrolyzate fermented liquid is through uf processing, ultrafiltration sees through liquid after the ion-exchange chromatography desalination, again through activated carbon decolorizing, it is 80% that this Xylitol liquid is concentrated into soluble solid content, the promptly crystallizable xylitol products that obtains content 〉=98.5%.The inventor is also noted that in this report the arabinose concentrations of crystalline mother solution surpasses 45.0% of Xylitol concentration, and xylose concentration surpasses 12.6% o'clock of Xylitol concentration, and they are all separated out with the Xylitol crystallization easily.If wood sugar in the fermented liquid or pectinose surpass this concentration ratio scope, will be difficult to effectively be purified into Xylitol by the crystalline method.This by the characteristics that fermented liquid prepares crystalline xyhose alcohol technology is, with direct condensing crystal after the fermented liquid purifying treatment, draws the xylitol crystal product, remainingly is difficult to further crystallization purifying, contains Xylitol, pectinose, the crystalline mother solution of compositions such as wood sugar.The contriver did not recognize obviously at that time that xylitol fermentation and pectinose extracted the bonded problem.
People such as Ding Xinghong [fourth emerging red dawn in summer: influence the research of separation and purification Xylitol key factor in the hemicellulose fermented liquid.The Chinese food journal.2006,6 (6): 87-90] be research object with xylitol solution and hemicellulose fermented liquid, investigated the influence of Xylitol starting point concentration, residual sugar (pectinose), crystal seed and Tc to the Xylitol kinetics of crystallization, and determined that Xylitol initial mass concentration is 750g/L, Tc is-4 ℃.In xylitol solution, add 1 ‰ xylitol seed crystals, can shorten the perdurability of induction period of crystallization, improve crystallization velocity.A spot of pectinose can improve the Xylitol crystallization velocity in the xylitol solution, but when the pectinose mass concentration was higher than 120g/L, the purity of xylitol crystal can reduce.The described operational path of this piece of writing report also is that the fermented liquid after purifying is directly concentrated, and the concentrated solution direct crystallization is made xylitol products.Though it has mentioned the purity that the existence of pectinose in the fermented liquid will influence xylitol crystal, does not discuss the problem that how not to reclaim pectinose.
People such as Ying Guoqing [Ying Guoqing, Wang Pu, Zhang Feng, YuBing Jun: the separation purifying technique of fermentative Production Xylitol.Chinese Journal of Pharmaceuticals.2002,33 (3): 117--123] then in containing the fermented liquid of remaining wood sugar (conversion fluid), add certain density NaOH, the about 2h of boiling water backflow, with remain in wood sugar in the fermented liquid be degraded to corresponding acid and and generate corresponding salt, remove with anion and cation exchange resin and desalt, last condensing crystal obtains xylitol crystal.This technology does not obviously consider to reclaim other carbohydrate in the fermented liquid.
We carry out to wood sugar, pectinose and Xylitol with calcium type resin cation (R.C.) finding that the chromatographic peak major part of wood sugar and pectinose is overlapped in the process of chromatographic separation that still, the chromatographic peak of these two kinds of sugar and Xylitol is not overlapping substantially (Fig. 1) but.Obviously, the chromatographic separation efficient between Xylitol-pectinose (sugar-alcohol), more much higher than the separation efficiency between wood sugar-pectinose (sugar-sugar).If we can optionally be converted into Xylitol with the wood sugar in the hemicellulose hydrolysate, and make pectinose not participate in reaction, so just can realize will be to the separation of raw material wood sugar-pectinose, be transformed into the separation to product Xylitol-pectinose, wood sugar also just has been readily solved with the difficult problem of separating between the pectinose.
Some yeast has metabolic exhaustion glucose, transforms wood sugar and generates Xylitol, but can not utilize the characteristic of pectinose.Originally with wood sugar-pectinose--glucose is that main component is a hemicellulose hydrolysate, and will change into after this microbial fermentation with Xylitol-pectinose is the fermented liquid of main component.So, carry out one time fermentation with double cellulose hydrolysis thing of this primary yeast, (glucose is removed in cellular metabolism consumption with biologically pure can not only to reach bio-transformation (wood sugar is converted into Xylitol), the purity of Xylitol and pectinose is improved) double effects, and the effect of follow-up product chromatographic separation also will effectively be improved.In addition, to a chromatographic separation process of fermented liquid, in fact simultaneously purifying Xylitol and two products of pectinose, thereby the strictness to material choice limits when having avoided simple production Xylitol or pectinose, improve the level of comprehensive utilization of raw material, saved production cost.
Summary of the invention
The present invention has started a cover same bio-matrix has been carried out controlled selective conversion, shunting, obtains the method for different biological products successively or simultaneously.It carries out bio-transformation by special process and bacterial classification to the part component in the bio-matrix, but keep or strengthen the natural biochemical characteristic of other components, after treating that last converted product is shunted, again other components are implemented new conversion, shunting, separation, purifying, crystallization etc., obtain new biological products.The present invention in fact also discloses first, has verified by technology controlling and process and bacterial classification and selected, the different components in the same bio-matrix implemented selective conversion, shunting, and then obtain the technology of different biological products.
Be to transform in the initiative test of matrix with the hemicellulose, the invention produce Xylitol by the fermentation hemicellulose hydrolysate, with the novel method that combines from hemicellulose hydrolysate extraction pectinose, efficiently solving chemical technology produces in the process of Xylitol, and extract by hemicellulose hydrolysate in the process of pectinose, because the close separation efficiency of bringing of chemical property is low between wood sugar and the pectinose, product yield is low, or the potential safety hazard of with an organic solvent bringing.Compare with the existing technology for preparing Xylitol from fermented liquid, the present invention overcharges and has obtained pectinose; Compare with the production technique of existing pectinose, the present invention overcharges and has obtained Xylitol.The present invention is for improving resource utilization, and the advantage that reduces Xylitol and pectinose production cost aspect is fairly obvious.
Whole process of the present invention comprises that with the raw material of acid or the rich hemicellulose of enzymic hydrolysis obtain with wood sugar, pectinose is a principal constituent, contains glucose simultaneously, seminose, the hemicellulose hydrolysate of assorted sugar such as semi-lactosi.After the detoxification treatment of this hydrolyzate through removing the microorganism growth inhibition, access can be assimilated and utilized glucose, and it is Xylitol that specificity ground transforms wood sugar, but can not utilize the yeast strain of pectinose.Stop fermentation after the fermented liquid xylose concentration is low to moderate certain limit, obtaining with Xylitol and pectinose is the fermented liquid of principal constituent.Wherein, the Xylitol amount generally is not less than 1%, and the pectinose amount generally is not less than 2%.Fermented liquid is through cellular segregation, and the ion-exchange desalination suitably concentrates after the decolouring, be sorbent material directly with calcium type resin cation (R.C.), with the pure water is that eluent carries out chromatographic separation, obtains the high-purity xylitol flow point of purity more than 99% respectively, and with pectinose--assorted sugared flow point.High-purity xylitol flow point concentrating under reduced pressure, decrease temperature crystalline obtains the crystal Xylitol.Pectinose--it is sorbent material that assorted sugared flow point is used ammonium type Zeo-karb instead, still is that eluent carries out chromatographic separation with the pure water, further improves the purity of pectinose flow point.At last that purity is higher pectinose flow point condensing crystal obtains the crystal pectinose.
Though above-mentioned chromatographic separation process can be finished having filled on the stationary chromatographic post of resin cation (R.C.), industrialized simulated movable bed chromatography device can effectively improve separation efficiency.
According to content of the present invention and testing data, Fig. 1 is a wood sugar, the calcium type resin cation (R.C.) chromatographic separation elution curve figure of pectinose and Xylitol.Fig. 2 is the process flow sheet that the bio-transformation hemicellulose hydrolysate is produced Xylitol and pectinose simultaneously.Fig. 3 is the HPLC color atlas (1. glucose, 2. wood sugar, 3. pectinose) of maize peel hemicellulose hydrolysate.Fig. 4 is the xylitol fermentation liquor HPLC color atlas (1. wood sugar, 2. pectinose, 3. Xylitol) of maize peel hemicellulose hydrolysate.Fig. 5 is the isolating Xylitol shunting of calcium type resin cation (R.C.) simulated moving bed chromatography, is shown as single chromatographic peak on HPLC.Fig. 6 is the isolating pectinose of calcium type resin cation (R.C.) simulated moving bed chromatography-assorted sugar stream HPLC color atlas (1. pectinose, 2 assorted sugar, 3. assorted sugar).Fig. 7 is the pectinose shunting HPLC color atlas of chromatographic separation (ammonium type resin cation (R.C.)) for the second time, and main peak is a pectinose.
Being used for hemicellulose hydrolysate of the present invention prepares as follows:
The material of hemicellulose such as maize peel (corn fiber), corn cob (com cob) will be rich in, bagasse (sugar cane bagasse) etc., with the massfraction is the dilute sulphuric acid of 0.5-2.5% (w/w), or hydrochloric acid, can soak material with acid solution and be advisable, hydrolysis 0.5-2.5h under 110-140 ℃ of condition.Collect liquid portion after the hydrolysis, with Ca (OH)
2Or CaCO
3Be adjusted to pH3-pH4, remove precipitation, supernatant liquor is handled with the activated carbon adsorption that is equivalent to raw material weight 1～3% again.Successively by positively charged ion, anionite-exchange resin purifies the solution of elimination activated carbon again, promptly obtains the hemicellulose hydrolysate that can be used for fermenting after concentrating.
The present invention is by following method, in fermentor tank, utilize yeast cell optionally catalysis the wood sugar in the hemicellulose hydrolysate is converted into Xylitol through biocatalysis, and pectinose does not participate in biocatalytic reaction.
If the microbial host candida tropicalis of indication of the present invention.This primary yeast can utilize the glucose in the hemicellulose hydrolysate, and semi-lactosi, hexoses such as seminose be as carbon source for growth, and can not generate corresponding sugar alcohol; For five-carbon ring aldehydo sugar, it is Xylitol that candida tropicalis can transform wood sugar, but can not utilize the pectinose that belongs to five-carbon ring aldehydo sugar together.The bacterial strain of the actual use of the present invention is screened by the inventor, and be stored in the candida tropicalis bacterial strain Candidatropicalis CCTCC M 205067 at Chinese typical culture collection center, reach the Candida tropicalis AS2.1776[ZHANGHou-Rui that the inventor once reported, ZENG Jian-Zhi HE Cheng-Xin, et.al.Utilization of Sugar Cane Bagasse Hydrolysatesfor Xylitol Production by Yeast, Chinese Journal of Biotechnology.2002,18 (6): 724-728].
Seed culture medium is formed: wood sugar 20g/L, glucose 30g/L, yeast extract paste 10g/L, KH
2PO
4, 5g/L, NH
4H
2PO
4, 3g/L, MgSO
4.7H
2O, 0.1g/L, pH5-6, the liquid loading amount generally is controlled at triangular flask volumetrical 10-20%, sterilizes 15 minutes for 115 ℃.Get the zymic slant culture and inoculate cooled substratum, 28-35 ℃ of shaking table activation culture 10--12 hour.Inoculum size by 5-10% (v/v) changes activatory yeast starter nutrient solution over to fresh culture, continues shake-flask culture to obtain the liquid seeds of q.s.
The hemicellulose hydrolysate for preparing is concentrated into total sugar concentration to about 200-250g/L, injects fermentor tank, and every liter of nutrient solution adds the hot water extract from 50-150g rice bran or wheat bran in addition, to satisfy the nutritional need of yeast cell growth.The inoculum size of pressing 5-10% (v/v) inserts yeast starter, and 33 ℃ of aerobic fermentations are cultivated, and is exhausted to wood sugar, finishes fermentation.
Each batch fermentation finishes, and with centrifugal or filtering method fermented liquid is separated with yeast cell.The fermented liquid of removing cell is used to prepare crystalline xyhose alcohol and pectinose, and collected cell then changes in the fresh hydrolyzate substratum, the fermentation of beginning next batch.Xylose concentration in the substratum, liquid amount and preceding a collection of being consistent in the prescription of substratum, fermentor tank.Circulation can not utilize until cell so repeatedly.
The present invention is by Xylitol and pectinose in the following method separate fermentation liquid.
The fermented liquid of having removed cell at first passes through ultrafiltration, removes deproteinize, polysaccharide and partial pigment, and with the activated carbon adsorption decolouring, the spent ion exchange resin desalting treatment draws water white xylitol fermentation scavenging solution then again.Concentrating under reduced pressure xylitol fermentation scavenging solution is to 40-60% (w/w) concentration, for the used in chromatograph first time.
In that the chromatographic column upper end application of sample of calcium type resin cation (R.C.) is housed, open the outlet of chromatographic column lower end, after sample enters the post bed fully, with 30-90 ℃ of pure water wash-out.First chromatographic peak at the lower end effluent liquid is to be the carbohydrate admixture of principal constituent with the pectinose, and second chromatographic peak is to contain any other impurity, the Xylitol of purity＞99% hardly.On the chromatogram elution curve, two chromatographic peaks are not overlapping substantially.
Chromatographic separation is obtained the Xylitol component be evaporated to about concentration 90% (w/w), decrease temperature crystalline obtains purified crystal Xylitol.
To total reducing sugar 40-60% (w/w), usefulness ammonium type resin cation (R.C.) still is that eluent carries out the chromatographic separation second time with the pure water as sorbent material with sugared flow point concentrating under reduced pressure that the first time, chromatographic separation obtained.In this separated, its effusive first peak was assorted sugar, and second peak is principal constituent with the pectinose.Collect second chromatographic peak component, be evaporated to total reducing sugar 60-80% then, decrease temperature crystalline, and isolate purified pectinose crystal.
, can separate more efficiently than single-column Xylitol is separated with pectinose as simulation moving-bed filler with the resin cation (R.C.) of same type, increase substantially the production efficiency of unit weight resin in single time, and reduce water consumption.
The present invention compared with prior art, outstanding substantive distinguishing features and the obvious improvement that are had are:
The first, the present invention is by specific yeast strain fermentation hemicellulose hydrolysate, and wherein the assorted sugar consumption of major part is in cellular metabolism, and wood sugar is converted into Xylitol expeditiously, and pectinose does not participate in any biocatalysis process.So, can obtain two products simultaneously by same technological process, this is that the technology institute that in the past reported is irrealizable.
Second, the present invention is by biological selectivity katalysis, with the sugar-sugar between wood sugar-pectinose in the past from, sugar-the alcohol that is converted between Xylitol-pectinose separates, fermented liquid after the purification utilizes chromatographic separation can obtain the Xylitol liquid of purity＞99%, and the product rate of recovery approaches 100%.These two index levels are that any report did not reach in the past.
The 3rd, the present invention the first time chromatographic separation process just directly obtain the pure liquid of purity of xylose, follow-up Xylitol crystallisation process is the physics moulding process of product purely, rather than the means of purification of product.So there is not the crystal mother solution of xylitol that can not utilize in the present invention, this is that in the past technology institute is irrealizable.
The 4th and since the present invention the first time chromatographic separation reclaimed Xylitol fully, so for the second time chromatographic separation process mainly is separation between pectinose-assorted sugar, it is more relatively easy than the separation between pectinose-wood sugar.The present invention by the second time chromatographic separation purity of Arabic liquid glucose is brought up to more than 85%, this also is that in the past technology institute is irrealizable.
The 5th, the present invention has widened the raw material sources of Xylitol or pectinose production.No matter prepare wood sugar or pectinose with hemicellulose hydrolysate, raw materials used target contents of monosaccharides must be higher, otherwise target monose can't be separated out in crystallization.For example, produced pectinose in the past and just can only select the very abundant raw material of arabinan content for use, and controlled sour consumption to alleviate the xylan backbone degraded, reduced the wood sugar relative content in the hydrolyzate, and just can prepare crystallized arabinose in strictness.Produce wood sugar equally, in the past and can not use the higher raw material of arabinan content.And the present invention can transform wood sugar under the very high condition of pectinose content is Xylitol, and by chromatographic separation process it is separated fully with pectinose.So,, utilize technology provided by the invention can both produce purity of xylose alcohol and pectinose easily simultaneously regardless of the ratio of selecting pectinose and wood sugar in the raw material for use.
The 6th, compare with existing pectinose production technique, the present invention has more effectively utilized the wood sugar in the fiber hydrolyzate.The present invention require can be simultaneously fully hydrolysis go out wood sugar in the raw material, be translated into the higher Xylitol of added value then, and recycle.And in the existing pectinose production technique, then reducing the hydrolysis degree of xylan in the lignocellulose raw material as far as possible, the wood sugar to hydrolysis goes out can only yeast cell consume it, or lets alone to be present in crystalline mother solution, and can't be utilized effectively.
The 7th, compare with existing Xylitol production technique, the present invention has more effectively utilized wood sugar and the pectinose in the hemicellulose hydrolysate.In existing Xylitol chemical production processes, must from hemicellulose hydrolysate, go out wood sugar by crystallization purifying, just can be further used for hydrogenation and produce Xylitol, therefore there are a large amount of wood sugars, pectinose to be mixed in and exist in the crystalline mother solution and can't utilize.Existing fermented liquid separates Xylitol technology, also exists a large amount of pectinoses not utilize in the crystalline mother solution.By the technology of the present invention, not only reclaimed Xylitol fully, effective recycling pectinose wherein.
Description of drawings
Fig. 1 wood sugar, the calcium type resin cation (R.C.) chromatographic separation elution curve of pectinose and Xylitol.
The technical process that Fig. 2 bio-transformation hemicellulose hydrolysate is produced Xylitol and pectinose simultaneously.
The HPLC chromatogram of Fig. 3 maize peel hemicellulose hydrolysate (1. glucose, 2. wood sugar, 3. pectinose).
The xylitol fermentation liquor HPLC chromatogram of Fig. 4 maize peel hemicellulose hydrolysate (1. wood sugar, 2. pectinose, 3. Xylitol).
The isolating Xylitol shunting of Fig. 5 calcium type resin cation (R.C.) simulated moving bed chromatography is shown as single chromatographic peak on HPLC.
The isolating pectinose of Fig. 6 calcium type resin cation (R.C.) simulated moving bed chromatography-assorted sugar stream HPLC chromatogram (1. pectinose, 2 assorted sugar, 3. assorted sugar).
Fig. 7 is the pectinose shunting HPLC chromatogram of chromatographic separation (ammonium type resin cation (R.C.)) for the second time, and main peak is a pectinose.
Embodiment
Following example is to be the explanation specific embodiments of the invention.
The 40kg maize peel adds clear water 160L, boils.The elimination liquid portion is washed residue one time with clear water, adds 2% (w/w) sulphuric acid soln 80L in the residue after filter is done, and 125 ℃ of hydrolysis are 2 hours in hydrolysis reactor.Remove by filter residue, filtrate is adjusted to pH3 with lime carbonate, remove by filter throw out, add the 1kg activated carbon in the filtrate, adsorption treatment was removed activated carbon after 30 minutes, successively by positively charged ion, anion-exchange chromatography post, obtain water white syrup, and under reduced pressure, be concentrated into the concentration of requirement again.This syrupy glucose, wood sugar, the pectinose content ratio is approximately 1: 2: 1 (referring to Fig. 3).
Exsiccant bagasse 40Kg adds 2.4% (w/w) sulphuric acid soln 280L, 125 ℃ of hydrolysis 2 hours.All the other follow-up treatment steps are with embodiment 1.This syrup that comes from the preparation of bagasse hemicellulose hydrolyzate, its glucose, wood sugar, the content ratio of pectinose is about 1: 10: 1.
Get the maize peel hemicellulose hydrolysate of embodiment 1 gained, and every liter of fermented liquid adds from the hot water extract of 100g rice bran in addition, be adjusted to total reducing sugar 250g/L, wherein contain the about 120g/L of wood sugar, pectinose 80g/L sterilized 10 minutes for 110 ℃.5L fermentor tank (Biotech-5BG; Shanghai Baoxin Bioengineering.Equipment, Shanghai, China) loading amount 3L, inoculate fresh candida tropicalis CCTCC M 205067 (Candida tropicalis CCTCC M 205067), inoculum size 5% (v/v), air flow 1vvm, mixing speed 300rpm, cultivate about 30h for 33 ℃, wood sugar is converted into Xylitol, and pectinose does not participate in reaction (Fig. 4).The back centrifugal collecting cell that stops to ferment precipitates, and with sedimentary cell furnishing pulpous state, annotates back fermentor tank with fresh substratum.Substratum is filled it up with the loading amount volume that ferments to for the first time, keeps and first jar of identical ventilation and stir speed (S.S.) level.Carry out 5 round-robin fermentation result such as table one so continuously.
Table one, selective conversion maize peel hydrolyzate generate Xylitol
| Cell cycle | Fermentation | Initial feed is formed | Tunning is formed |
| Wood sugar (g/L) | Pectinose (g/L) | Xylitol (g/L) | Pectinose (g/L) | ||
| 1 2 3 4 5 | 30 26 18 19 18 | 118.0 123.5 120.2 122.8 125.0 | 84.3 86.3 85.4 86.3 88.7 | 100.7 103.5 109.8 110.5 110.6 | 82.6 86.3 82.5 85.3 88.5 |
Xylose crystallization mother liquor with wood sugar factory is a substrate, and in the cultured in advance thalline fermentation of 3T fermentor tank, the centrifugal fermented liquid of industrial centrifugal machine is collected thalline, and other control condition is identical with embodiment 3.Carry out 5 round-robin fermentation result such as table two continuously.
Table two, xylose crystallization mother liquor selective conversion generate Xylitol
| The cell cycle order | Fermentation time | Initial feed is formed | Tunning is formed | ||
| Wood sugar (g/L) | Pectinose (g/L) | Xylitol (g/L) | Pectinose (g/L) | ||
| 1 2 3 4 5 | 25 20 19 17 17 | 120.5 119.5 123.6 120.4 124.6 | 45.5 44.3 46.5 45.3 46.8 | 108.4 112.5 119.4 114.9 11615 | 45.3 44.2 46.3 45.3 46.2 |
Get the fermented liquid 10L of embodiment 3 gained, the ultra-filtration membrane ultrafiltration deproteinated through molecular weight cut-off 5Kdal by order is: positively charged ion--anionic-cationic---anionic column chromatography desalination (resin cation (R.C.), 001 * 7 then; Resin anion(R.A), D301.China, resin processing plant of Nankai University), remove pigment simultaneously, obtain water white Xylitol-pectinose scavenging solution.Under the reduced pressure scavenging solution reconcentration is arrived solubility thing content 60%.
Spissated scavenging solution has been filled calcium type resin cation (R.C.) (AMBERLITE CR1320Ca) and has been the simulated moving bed chromatography system of sorbent material at first with having 20 2.5 * 50cm pillars, carries out the chromatographic separation first time.60 ℃ of separation temperatures, feeding rate 5ml/min, pure water elution speed 25ml/min.The consisting of of effluent liquid after the system balancing: 1. Xylitol flow point, Xylitol content 〉=99% (Fig. 5), concentration 12-13%; 2. pectinose-assorted sugared flow point, total solubility substrate concentration 5-7%, pectinose content 55-60% (Fig. 6) wherein, other impurity 30-45%.Crystallization after Xylitol liquid directly concentrates obtains the crystal Xylitol, and pectinose-assorted sugared flow point then need be further purified.
The rapid pectinose that obtains of previous step-assorted sugared flow point is evaporated to solubility thing content 60%, with ammonium salt AMBERLITE CR1320Ca resin is converted to the ammonium type, still uses 20 above-mentioned 2.5 * 50cm pillars to form simulated moving bed chromatography system and carry out the chromatographic separation second time.30 ℃ of separation temperatures, feeding rate 3ml/min,, pure water elution speed 23ml/min.Collect the pectinose flow point after the system balancing, the purity of its pectinose is brought up to (Fig. 7) more than 85% by the 55-60% at the beginning of the charging, and crystallization draws the pectinose product of purity 〉=99% after concentrating.
Claims (3)
1. method for preparing a plurality of biological products from same bio-matrix, it is characterized in that: with the hemicellulose hydrolysate that is rich in wood sugar and pectinose is matrix, at first above-mentioned hemicellulose hydrolysate is removed the detoxification treatment of microorganism growth inhibition, access can metabolic exhaustion glucose, the conversion wood sugar is an Xylitol, but can not utilize the candida tropicalis of pectinose, after this specific barms fermentation, principal constituent wood sugar in the hydrolyzate optionally is converted into Xylitol, pectinose does not participate in reaction, the fermented liquid of Xylitol and pectinose is rich in acquisition, described fermented liquid is through cellular segregation, the ion-exchange desalination, suitably concentrate after the decolouring, be sorbent material directly with calcium type resin cation (R.C.), with the pure water is that eluent carries out chromatographic separation, obtains the Xylitol liquid flow point of purity more than 99% respectively, and pectinose, assorted sugared flow point; Pectinose, assorted sugared mixed solution still are that eluent carries out chromatographic separation with the pure water through ammonium type resin cation (R.C.) chromatographic separation, further improve pectinose flow point purity to 50%-99%.
2. a kind of method for preparing a plurality of biological products from same bio-matrix as claimed in claim 1, it is characterized in that: described candida tropicalis is to be preserved in Wuhan University China typical culture collection center, is numbered the candida tropicalis bacterial strain of CCTCC No:M 205067.
3. as claimed in claim 1ly a kind ofly prepare the method for a plurality of biological products from same bio-matrix, it is characterized in that: described chromatographic separation is used single column chromatography device, or the higher simulation moving-bed device of efficient.
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| CN101659681B (en) * | 2009-09-30 | 2012-10-03 | 济南圣泉唐和唐生物科技有限公司 | Method for producing wood sugar product |
| CN102994574A (en) * | 2012-12-10 | 2013-03-27 | 山东大学 | Method for producing xylitol by employing corn stalks |
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| CN105439814A (en) * | 2015-12-09 | 2016-03-30 | 广西壮族自治区中国科学院广西植物研究所 | Method for preparing D-mannoheptulose and persitol by extraction from avocado |
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