CN101914591A - Application of candida tropicalis to preparation of xylitol and application of high-purity xylitol product to pharmacy and health care - Google Patents

Abstract

The invention relates to application of candida tropicalis, a microbial fermentation and separation method for preparing xylitol by using the candida tropicalis, and application of a high-purity xylitol product prepared by using the method to pharmacy and health care. The xylitol has high output quality and high yield, and the product can be used as a sweetener, a nutritional supplement and an auxiliary treatment agent for diabetes patients. Moreover, the xylitol product can serve as a medicinal intermediate for preparing health-care toothpaste, health-care chewing gum, health-care sweet food or sweet beverage and the like.

Description

A kind of candida tropicalis prepares the purposes of Xylitol and pharmacy, the health caring use of high-purity xylitol product

Technical field

The invention belongs to biological technical field, relate to candida tropicalis and prepare the purposes of Xylitol and pharmacy, the health caring use of high-purity xylitol product.

Background technology

Hemicellulose is the glycan class except that Mierocrystalline cellulose in the plant cell wall, and content is difference to some extent with different plants, roughly accounts for the 20-35% of plant materials weight, be on the earth except that Mierocrystalline cellulose the abundantest polysaccharide.

Mierocrystalline cellulose is the homogeneity glycan that is aggregated into by glucose unit fully, hemicellulose then mainly be by wood sugar so that β-(1-4) glycosidic link constitutes main chain, the heterogencity glycan of Ah 's sugar, seminose and semi-lactosi formation branched structure.The xylan backbone of hemicellulose generally contains a 50-150 wood sugar unit, and no crystalline texture is combined in the surface of cellulose micro-fibers.

Hemicellulose than the easy hydrolysis of Mierocrystalline cellulose many,, under 100-130 ℃ of condition, just be easy to diluted acid hemicellulose, it is the hemicellulose hydrolysate of main component that hydrolysis generates with the five-carbon sugar.All kinds of Chemicals such as the Xylitol that fermentation hemicellulose hydrolysate production market capacity is big, ethanol are to utilize hemicellulose resources effective approach on a large scale.

Though with diluted acid hydrolysis of hemicellulose being generated with monose is that the process of hydrolyzate of main component is not difficult, but, the dilute acid hydrolysis process can be supervened a series of microbial metabolism inhibitions, improve the leavening property of hemicellulose hydrolysate, must remove microbial metabolism inhibition wherein, i.e. detoxification process is absolutely necessary.

The poisonous substance composition of having identified in hemicellulose hydrolysate has at present had tens kinds, and wherein representative poisonous substance comprises three major types, that is: furfural compounds, and as furfural, 5 hydroxymethyl furfural; Fatty acid compound, as formic acid, acetate, propionic acid; A series of compounds that contain phenyl ring that lignin degradation generates, as methyl catechol, phenyl aldehyde, forulic acid etc.

Reported vacuum-evaporation, solvent extraction, charcoal absorption, macroporous resin adsorption, ion exchange resin treatment can be sloughed some poisonous substance, improves the leavening property of hydrolyzate.Yet a kind of physics or chemical process can only be removed a certain class toxic substance, need unite usually and adopt multiple physics and chemistry measure to handle, and hemicellulose hydrolysate just can reach reasonable detoxification efficiency.But detoxification is a process that expends cost, uses multiple physics and chemistry measure that hemicellulose hydrolysate is carried out detoxification treatment simultaneously, must increase substantially the cost of product fermentation.

In fact, more natural microorganisms have degrading activity to some poisonous substance in the hemicellulose hydrolysate.For example, for example whiterot fungi (vast stretch of wooded country Lu Gang Zhang Qingna, the Screening and Identification and the application of degraded paper waste xylogen bacterial classification, University of Science ﹠ Technology, Beijing's journal of lignin degrading and a series of phenolic compounds of generating by lignin degradation.2007,29 (6): 569-573; Tao Yang Liao Jun and Luo Xuegang, bio-pulping technology more recent application progress.The Mierocrystalline cellulose science and technology.2007,15 (1): 70-74, degraded furfural, 5 hydroxymethyl furfural bacterium, yeast (tinkling of pieces of jade of writing legal document for others, Zhang Lujia, furfural and derivative microbiological deterioration (conversion) progress thereof.Amino acid and Biological resources 2007,29 (4): 41-45; Liu Aiping is permitted to learn book Fan Hangxue, utilizes yeast bio to transform vegetable fibre and is the alcoholic acid progress.The biotechnology communication, 2004,15 (2): 193--196), (what firm stifled state becomes Liu Liming to the ascomycetes of degraded short chain fatty acid, and thermophilic ascomycete utilizes short chain organic acid production at.The biotechnology journal, 2008,24 (5): 821-828)

In recent years the someone begins to attempt to remove poisonous substance in the hydrolyzate with biological enzyme.Human laccase and peroxidase combination treatment timber acid hydrolysis things such as Jonsson can have been removed most monocycle phenol thing (monoaromatic phenolic copounds).Hydrolyzate after the processing is used for ethanol fermentation, glucose consumption speed and ethanol generating rate have improved nearly 5 times (Jonsson J L as a result, Palmqvist E, Nilvebrant N O., Detoxification of wood hydrolysates with laccase andperoxidase from the white-rot fungus Trametes versicolor.Appl.Microbiol Biotechnol.1998, (49): 691-697)).But complicated poisonous substance composition needs the combined degradation of complicated enzyme system, and this has increased the cost of detoxification undoubtedly greatly.

People such as Lopez are with microorganism Conichaeta ligniaria NRRL 30616 fermentative processing wood fibre dilute acid hydrolysis things, and the effect of not only removing furfural, 5 hydroxymethyl furfural is remarkable, also obviously reduced the aldehydes matter in the hydrolyzate simultaneously.Use the producing and ethanol yeast fermentation again through the hydrolyzate that this bacterial strain is handled, 80h produces 1.66% ⁰Ethanol, and untreatedly do not have ethanol to produce (Lopez J M, Nichols N N, Dien B Set al.Isolation of microorganisms for biological detoxification oflignocellulosec hydrolysates.Appl Microbiol Biotechnol.2004,64 (1): 125-131).

Above-mentioned studies show that utilizes that complicated toxic substance is in the cards in the microbiological deterioration wood fibre hydrolysis thing.Especially, if can obtain a kind of metabolic system that possesses three big representative poisonous substances in the degraded hemicellulose hydrolysate simultaneously, promptly can degrade simultaneously furfural, lipid acid and contain the microorganism of benzene ring structure compound, and this microorganism can not utilize wood sugar again, so directly utilize this microbial fermentation hemicellulose hydrolysate to carry out the detoxification mass treatment, just can effectively overcome the existing shortcoming that physics and chemistry poison-removing method technology is loaded down with trivial details, cost is high, and have eco-friendly advantage.

In addition, the easy hydrolysis of hemicellulose, 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 can be used as the intermediate of the synthesis of nucleoside medicine, and range of application is arranged 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 ofxylose from solutions, 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) 6,872,316 (United StatesPatent (2005) 6 for the recovery of wood sugar, Recovery of xylose, 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 1-arabinose from sugar beet 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, introduces 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.

(patent name: the weakly acidic cationic exchanger resin chromatography reclaims monose to Chinese patent 01816511.7 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.) introduces utilization 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 on the Zeo-karb as sugar the chromatographic separation filler, 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 pentose/hexose mixture; Chang; Chin-Hsiung, Process for separating arabinose from a pentose/hexosemixture ().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 pentose/hexose mixture, 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, Process for separating arabinose from a mixture of otheraldoses, 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 separatingarabinose from a mixture of aldoses, 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, and 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 the preparation of crystalline L-arabinose fromarabinoxylan by enzymatic hydrolysis and selective fermentation withyeast.Biotechnology Letters 2001,23:411-416] be rich in araboxylan (Arabinoxylan with the crude enzyme liquid hydrolysis that derives from penicillium funiculosum (Penicillium funiculosum) 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.Hydrolyzate is 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 wood sugar only residual quantity only be equivalent to 0.002% of original araboxylan weight.This fermented liquid is through decolorizing with activated carbon, and ion is imitated and changed the 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 Hollingsworth with preparation pectinose and wood sugar from mixture of monosaccharides; Rawle I, Process for the preparation and separation ofarabinose and xylose from a mixture of saccharides; United States PatentApplication, 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 introduce [Feng Yaqing, Liu Yantong, Zhang Xiaodongs etc. extract L-arabinose from the L-Sudan Gum-arabic, fine chemistry industry, 2003,20 (5): 288-290], with Microcrystalline Cellulose, or silica gel is stationary phase, mixture with propyl carbinol, ethyl acetate, Virahol and water is a moving phase, carries out two dimensional chromatography continuously and separates, and can 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 zymotechnique Xylitol prepares the problem that combines with pectinose.

The inventor [Cai Aihua, Zhang Hourui, what Cheng Xin 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 separation problem between Xylitol and the 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 be that main component is a hemicellulose hydrolysate with wood sugar-pectinose-glucose, 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), Xylitol is relative with the purity of pectinose to be 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, and the level of comprehensive utilization of raising raw material, saved production cost.

Summary of the invention

The objective of the invention is in order to solve the existing process of existing hemicellulose hydrolysate physics and chemistry detoxification process loaded down with trivial details, the problem that cost is high.

Content of the present invention comprises: the contriver is from the paper mill, wood sugar factory, and furfural mill surrounding soil mud sample is rich in cultivation, separation, screening, and acquisition can effectively improve two isolate: the S-7 and the Lj-3 of wood fibre hydrolysis thing leavening property.The contriver identifies that isolate S-7 belongs to Issatchenkia orientalis (Issatchenkiaorientalis), it is saved to the Chinese typical culture collection center (CCTCC) of Chinese Wuhan Wuhan University on September 18th, 2006, preserving number is CCTCC NO:M206098, can not utilize wood sugar; Isolate Lj-3 belongs to her Sa yeast (Issa tchenkia occidentalis) of west, it is saved to the Chinese typical culture collection center (CCTCC) of Chinese Wuhan Wuhan University on September 18th, 2006, preserving number is CCTCCNO:M206097, utilize the ability of wood sugar atomic a little less than.The contriver has set up the biological detoxication method of hemicellulose hydrolysate with the active new bacterial strain of this detoxification; Detoxification active bacterial strain and xylose-fermenting are generated the combined inoculation of Xylitol microorganism, realize the synchronous detoxification xylitol zymolysis production of hemicellulose hydrolysate; Detoxification active bacterial strain and xylose-fermenting are generated the combined inoculation of alcoholic acid microorganism, realize the synchronous detoxification fermentative production of ethanol of hemicellulose hydrolysate.The preservation information of above-mentioned two primary yeasts is accepted letter of information referring to two parts of appended preservations of present patent application.

Belong to two biological detoxication active bacterial strain S-7 of the present invention (CCTCC NO:M206098) and Lj-3 (CCTCC NO:M206097), its separation screening, and the process identified of classification as follows:

With the bagasse hemicellulose hydrolyzate is the basal component of isolation medium, suitably regulates pH5-6 with alkali after the vacuum.If make flat board, add agar 20g/L as solid agent.

Soil, the mud of the environment collection of polluting from pulp mill wastewater are made sample separation.250ml triangular flask loading amount 25ml inserts waste water or the about 1g of mud sample separation, and 30 ℃, 200rpm shakes a bottle enrichment culture 72h.The separation of ruling on same culture medium flat plate of the nutrient solution of getting thalli growth, the type that selection can be grown fast forwards the inclined-plane to and preserves.

Slant culture is transferred in the half fiber dilute acid hydrolysis thing and was cultivated 24 hours shaking under bottle condition, the centrifugal thalline of removing, insert the yeast strain that can assimilate wood sugar then and ferment, selecting and remain, those can effectively improve the detoxification active bacterial strain of hemicellulose dilute acid hydrolysis thing leavening property.So through surplus ten batches sample separation, two the strongest isolates of the hemicellulose hydrolysate leavening property activity that is improved, numbering is respectively S-7 and Lj-3.

The detected result of high performance liquid chromatography shows, S-7 and Lj-3 are to the representative poisonous substance in the hemicellulose dilute acid hydrolysis thing: acetic acid, and furfural and phenol thing all have good degrading activity, and wherein S-7 does not utilize wood sugar, and Lj-3 can only faintly utilize wood sugar.

Aforesaid method separates two bacterial strains that obtain and refrigerates or the lyophilization preservation in 4 ℃.

According to the method that " yeast identification handbook " (press of Qingdao Marine University) book provides, identified cell, bacterium colony and the physiological and biochemical property (table 1, table 2) of S-7 and Lj-3.Issatchenkia orientalis (Issatchenkiaorientalis) described in the physiology of isolate S-7, biochemical character and " the yeast identification handbook " is identical; Her Sa yeast (Issatchenkia occidentalis) of west described in the physiology of isolate Lj-3, biochemical character and the same book is identical.

With primer 5 '-GCATATCAAAAGCGGAGGAAAAG-3 ' and 5 '-GGTCCGTGTTTCAAGACGG-3 ', (method is with reference to Kurtzman C P for the 26S rDNA D1/D2 zone nucleotide sequence of polymerase chain reaction (PCR) amplification S-7 and Lj-3, Four new Candida species from geographically diverselocations.Antonie van Leeuwenhoek, 2001,79:353-361).Measure the nucleotide sequence (table 3, table 4) of amplified production, and in GenBank nucleic acid sequence data storehouse, carry out homologous sequence search (Nucleotide-nucleotide BLAST) with this sequence.Search Results shows, the nucleotide sequence in the 26S rDNA D1/D2 zone of S-7 all reaches 100% with the existing Issatchenkia orientalis the same area nucleic acid sequence homology of GenBank; Lj-3 with wherein reach 100% with the homology of Issatchenkia occidentalis the same area nucleotide sequence.

According to can determining of above-mentioned cell, colonial morphology, Physiology and biochemistry and molecular biological characteristics, the classification position of S-7 belongs to Issatchenkia orientalis (Issatchenkia orientalis); Her Sa yeast (Issatchenkia occidentalis) of the west that the classification position of Lj-3 belongs to.

The preserving number of Issatchenkia orientalis (Issatchenkia orientalis) S-7 at China typical culture collection center (CCTCC) is CCTCC NO:M206098, and the registration number that the nucleotides sequence in its 26S rDNA D1/D2 zone is listed in GenBank is EF030708

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi？db＝nucleotide?&?val＝116834296；

She Sa yeast (Issatchenkia occidentalis) Lj-3 of west is CCTCC NO:M206097 at the preserving number of CCTCC, and the GenBank registration number of the nucleotide sequence in its 26S rDNA D1/D2 zone is EF030710

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi？db＝nucleotide?&?val＝116834298。

Cell and the colony morphology characteristic of table 1 isolate S-7, Lj-3

	Colonial morphology	Cell characteristic
			S-7	Surface drying, the fringe radiation shape.	Multiterminal sprout, and the cell mean length is: between 10～20um.
Lj-3	Glossy, surperficial thickness, neat in edge is level and smooth.	Multiterminal sprout, and the cell mean length is: 5～15um.

The physiological and biochemical property of table 2 isolate S-7, Lj-3 (+, utilize;-, do not utilize)

The nucleotide sequence (GenBank registration number EF030708) in Issatchenkia orientalis (Issatchenkia orientalis) S-7 (CCTCC NO:M206098) 26SrDNA D1/D2 zone:

1 taagcggagg?aaaagaaacc?aacagggatt?gcctcagtag?cggcgagtga?agcggcaaga

61 gctcagattt?gaaatcgtgc?tttgcggcac?gagttgtaga?ttgcaggttg?gagtctgtgt

121?ggaaggcggt?gtccaagtcc?cttggaacag?ggcgcccagg?agggtgagag?ccccgtggga

181?tgccggcgga?agcagtgagg?cccttctgac?gagtcgagtt?gtttgggaat?gcagctccaa

241?gcgggtggta?aattccatct?aaggctaaat?actggcgaga?gaccgatagc?gaacaagtac

301?tgtgaaggaa?agatgaaaag?cactttgaaa?agagagtgaa?acagcacgtg?aaattgttga

361?aagggaaggg?tattgcgccc?gacatgggga?ttgcgcaccg?ctgcctctcg?tgggcggcgc

421?tctgggcttt?ccctgggcca?gcatcggttc?ttgctgcagg?agaaggggtt?ctggaacgtg

481?gctcttcgga?gtgttatagc?cagggccaga?tgctgcgtgc?ggggaccgag?gactgcggcc

541?gtgtaggtca?cggatgctgg?cagaacggcg?caacaccgcc?cgtcttgaaa?cacgga

The 26S rDNA D1/D2 zone nucleotide sequence (GenBank registration number EF030710) of she Sa yeast (Issatchenkia occidentalis) Lj-3 (CCTCC NO:M206097) of west:

1 tatcaataag?cggaggaaaa?gaaaccaaca?gggattgcct?cagtagcggc?gagtgaagcg

61 gcaaaagctc?agatttgaaa?tcgtgtttcg?gcacgagttg?tagattgcag?gttggagtct

121?ttgtggaagc?gtgtgtctaa?gtcccttgga?acagggtgcc?attgagggtg?agagccccgt

181?gagacgcgtg?cggaagctgt?aaggcccttc?tgacgagtcg?agttgtttgg?gaatgcagct

241?ctaagtgggt?ggtaaattcc?atctaaggct?aaatattggc?gagagaccga?tagcgaacaa

301?gtactgtgaa?ggaaagatga?aaagcacttt?gaaaagagag?tgaaacagca?cgtgaaattg

361?ttgaaaggga?agggtattgg?gctcgacatg?ggatttgcgc?accgctgctc?cttgtgggcg

421?gcgctctgtg?cttttcctgg?gccagcatcg?gtttttgccg?caggagaagg?cgtgctggaa

481?tgtggctctt?cggagtgtta?tagccagtgc?gagatgctgc?gtgcggggac?cgaggactgc

541?gacatctgtc?tcggatgctg?gcacaacggc?gcaataccgc?ccgtcttgta?a

The present invention prepares needed hemicellulose hydrolysate as follows:

Bagasse, corn cob, utilizing rice straw or other crops ' stalk are crushed to suitable granularity, the dilute sulphuric acid or the dilute hydrochloric acid solution that add 0.3～3% (w/w) by solid-to-liquid ratio 1: 6～7, stir,, kept 0.5～3 hour acid proof pressurized vessel internal heating to 100～130 ℃.Hydrolysis finishes back filtering residue, and filtrate is hemicellulose hydrolysate.With solid carbonic acid calcium or calcium hydroxide hemicellulose dilute acid hydrolysis liquid is transferred to pH value 3～8, can directly or after concentrating carry out biological detoxication again.Usually, can use lower hydrolysis temperature than the condition of high acid concentration, or corresponding shortening hydrolysis time, lower acid concentration then must improve hydrolysis temperature or prolong hydrolysis time.

The present invention cultivates as follows and is used for the hemicellulose hydrolysate biological detoxication, xylitol fermentation, and the microbial strains of ethanol fermentation:

Detoxification bacterial strain culture of seed liquid: the slant culture of CCTCC NO:M206098 or CCTCC NO:M206097 is inserted liquid seed culture medium, liquid amount is to shake 20% of bottle capacity, at 27--30 ℃, shaking table was cultivated 12～18 hours under the rotating speed 200rmp condition, can obtain can be used for the kind liquid of detoxification.Seed culture medium consists of: glucose 50g/L, MgSO ₄.7H ₂O 2g/L, K ₂HPO ₄4g/L, KH ₂PO ₄6g/L, yeast extract paste 5g/L.

Xylose-fermenting generates the bacterial strain seed liquor of Xylitol and cultivates: used bacterial strain is candida tropicalis (Candida tropicalis) CCTCC NO:M205067, it is saved to the Chinese typical culture collection center (CCTCC) of Chinese Wuhan Wuhan University on June 15th, 2005, preserving number is CCTCC NO:M205067, classification called after candida tropicalis 1-18 Candida tropicalis 1-18, (see patent of invention " separation of candida tropicalis bacterial strain and be used for the method that Xylitol is produced ", application for a patent for invention numbers 200510037580.2), contain glucose 20g/L, wood sugar 20g/L, all the other compositions and culture condition are with detoxification bacterial strain seed culture.

Xylose-fermenting generates alcoholic acid bacterial strain seed liquor and cultivates: used bacterial strain is had a liking for tannin pipe capsule yeast (Pachysolen tannophilus) CICC 1770 for what be preserved in Chinese industrial microbial strains preservation administrative center (CICC), and medium component and culture condition generate the spawn culture of Xylitol with xylose-fermenting.

The present invention carries out the hemicellulose hydrolysate biological detoxication as follows:

The hemicellulose hydrolysate total sugar content is 4～20% (w/w), and wherein reductive monosaccharide accounts for total sugar content＞more than 90%, be principal constituent with the wood sugar.With alkali lye (calcium hydroxide, ammoniacal liquor etc.) be adjusted to pH3-7, insert cultured CCTCC NO:M206098 or CCTCC NO:M206097 seed liquor by 10% (v/v) inoculum size, at 25～35 ℃, aeration condition bottom fermentation detoxification 5～20 hours, most of microorganism toxic ingredient wherein can be degraded and remove.The thalline of collecting centrifugation is recycled and reused for the fresh hemicellulose hydrolysate detoxification of next batch, and centrifuged supernatant can directly or after concentrating be used for xylitol fermentation, or ethanol fermentation.

The hemicellulose hydrolysate that the present invention is fermented after the detoxification is as follows produced Xylitol:

Passed through CCTCC NO:M206098, or the hemicellulose hydrolysate of CCTCC NO:M206097 fermentation detoxification, vacuum concentration is to the about 150g/L of wood sugar, and ammoniacal liquor is adjusted to pH=6, other adds yeast extract paste 5g/L, obtains being used for the hemicellulose hydrolysate substratum of xylitol fermentation.Xylitol fermentation is carried out with shaking bottle, shakes bottled amount 10%, inserts candida tropicalis (Candida tropicalis) CCTCCNO:M205067 seed liquor by 5% (v/v) inoculum size, 200rmp, and 33 ℃ ferment to wood sugar and run out of.Collect the thalline of centrifugation, put into the xylitol fermentation that continues a new round in the fresh hemicellulose hydrolysate substratum, centrifuged supernatant is used to prepare Xylitol.

The present invention utilizes hemicellulose hydrolysate to carry out synchronous detoxification xylitol zymolysis production as follows:

The vacuum concentration hemicellulose hydrolysate is to the about 100--150g/L of wood sugar content, transfers to pH3--pH7 with alkali (calcium hydroxide, or ammoniacal liquor),, centrifugal or remove by filter precipitation, add yeast extract paste 5g/L, the hemicellulose hydrolysate substratum, be sub-packed in triangular flask; Get cultured strain CCTCC NO:M206098 seed liquor, the perhaps seed liquor of CCTCC NO:M206097, and mix with isopyknic CCTCC NO:M205067 seed liquor, by in 5% (v/v) and the sowing quantity access hemicellulose hydrolysate, shake-flask culture to wood sugar runs out of then.The yeast cell of centrifugal collecting precipitation also is used for fresh substratum, continues synchronous detoxification fermentation.So constantly circulation can not utilize until yeast cell.

The follow-up xylitol fermentation of carrying out of the hemicellulose hydrolysate that carries out in advance obtaining after the detoxification treatment is comprised the steps: with the method for preparing pectinose

S1. to through the hemicellulose hydrolysate vacuum concentration of CCTCC NO:M206098 fermentation detoxification to the about 150g/L of wood sugar, ammoniacal liquor is adjusted to pH=6, other adds yeast extract paste 5g/L, obtains being used for the hemicellulose hydrolysate substratum of xylitol fermentation;

S2. xylitol fermentation is carried out with shaking bottle, shakes bottled amount 10%, inserts the CCTCCNO:M205067 seed liquor by 5% (v/v) inoculum size, 200rmp, and 33 ℃ ferment to wood sugar and run out of;

S3. collect the thalline of centrifugation, put into the xylitol fermentation that continues a new round in the fresh hemicellulose hydrolysate substratum, centrifuged supernatant prepares Xylitol;

S4. after xylitol fermentation liquor being removed cell, purify through ion-exchange, carry out chromatographic separation with calcium type resin cation (R.C.) again, obtain purified Xylitol shunting, the xylitol fermentation liquor of described shunting obtains crystalline xyhose alcohol through concentrated, crystallization;

S5. after shunting xylitol fermentation liquor, acquisition pectinose shunting fermented liquid carries out chromatographic separation to pectinose shunting fermented liquid with ammonium type resin cation (R.C.), improves pectinose purity, again by concentrated, crystallization, obtains crystallized arabinose.

The method of utilizing hemicellulose hydrolysate to carry out synchronous detoxification xylitol zymolysis production and pectinose comprises the steps:

S1. the vacuum concentration hemicellulose hydrolysate is to the about 100--150g/L of wood sugar content, transfers to pH3--pH7 with alkali (calcium hydroxide, or ammoniacal liquor),, centrifugal or remove by filter precipitation, add yeast extract paste 5g/L, the hemicellulose hydrolysate substratum;

S2. get cultured CCTCC NO:M206098 seed liquor, and mix with isopyknic CCTCC NO:M205067 seed liquor, insert in the hemicellulose hydrolysate by 5% (v/v) and sowing quantity then, shake-flask culture to wood sugar runs out of;

S3. the yeast cell of centrifugal collecting precipitation and be used for fresh substratum continues synchronous detoxification fermentation; So constantly circulation can not utilize until yeast cell;

S4. after xylitol fermentation liquor being removed cell, purify through ion-exchange, carry out chromatographic separation with calcium type resin cation (R.C.) again, obtain purified Xylitol shunting, the xylitol fermentation liquor of described shunting obtains crystalline xyhose alcohol through concentrated, crystallization;

S5. after shunting xylitol fermentation liquor, acquisition pectinose shunting fermented liquid carries out chromatographic separation to pectinose shunting fermented liquid with ammonium type resin cation (R.C.), improves pectinose purity, again by concentrated, crystallization, obtains crystallized arabinose.

The present invention utilizes hemicellulose hydrolysate to carry out synchronous detoxification fermentative production of ethanol as follows:

The substratum of the hemicellulose hydrolysate that synchronous detoxification fermentative production of ethanol is used is identical with the substratum of synchronous detoxification xylitol zymolysis production.Get cultured strain CCTCC NO:M206098 seed liquor, the perhaps seed liquor of CCTCCNO:M206097, mix with isopyknic CICC 1770 seed liquor, insert in the hemicellulose hydrolysate culture media shaking vase by 5% (v/v) and sowing quantity then, be cultured to wood sugar and run out of.The yeast cell circulation of centrifugal collecting precipitation is used to the fresh substratum that ferments, and continues synchronous detoxification fermentation, and so constantly circulation can not utilize until yeast cell.The ethanol in the centrifuged supernatant is reclaimed in distillation.

The present invention to the representative poisonous substance matter in the hemicellulose hydrolysate, reaches main aldehydes matter degrading activity with high performance liquid chromatography detection of biological detoxification bacterial strain.

Select the representative of acetate, furfural and methyl catechol as dissimilar inhibitions in the hemicellulose hydrolysate, these three kinds of compounds are joined in the common yeast culture base together, and access detoxification active bacterial strain (CCTCCNO:M206097, or CCTCC NO:M206098) ferments.Utilize these three kinds of compounds that the characteristics of stronger uv-absorbing are all arranged at the 198nm place, at following chromatographic condition: the Waters486 high performance liquid chromatograph, ZORBAX XDB-C18 chromatographic column, mobile phase methanol: phosphoric acid (0.2%, w/w)=75: 25 (v/v), the content of these three kinds of compounds of comparison and detection before and after fermentation judges that these two bacterial strains all have degrading activity (Fig. 1) to these three kinds of representative toxic ingredients.

Because phenolic compound all has strong uv-absorbing near 270nm, so with 270nm is to detect wavelength, with the bagasse hemicellulose hydrolyzate of high performance liquid phase comparison and detection operation 1, operation 3 through Issatchenkia orientalis CCTCC NO:M206098 biological detoxication, the bagasse hemicellulose hydrolyzate (Fig. 2) of her Sa yeast CCTCC NO:M206097 biological detoxication through the west.And analyze the information (table 4) of some characteristic peaks under this detection wavelength condition, and hemicellulose hydrolysate is after biological detoxication is handled as can be known, and furfural wherein and many deleterious aldehydes matters are degraded.

The present invention's detoxification efficiency of the method detection of biological detoxification bacterial strain of contrast biological fermentation to hemicellulose hydrolysate:

Ferment through CCTCC NO:M206097 with CCTCC NO:M205067 contrast, or, detect the tunning Xylitol through CCTCCNO:M206098 detoxification treatment front and back hemicellulose hydrolysate; Or with CICC1770 contrast fermentation through CCTCC NO:M206097, or before and after CCTCC NO:M206098 detoxification treatment hemicellulose hydrolysate, detect tunning ethanol.Can find no matter the hemicellulose hydrolysate through after biological bacterial strain provided by the present invention and the poison-removing method processing is used for xylitol fermentation or ethanol fermentation, output, the generating rate of fermented product all are improved largely.Can affirm that thus biological detoxication bacterial strain provided by the invention and biological detoxification method can improve the leavening property of hemicellulose hydrolysate effectively.

Substantive distinguishing features and marked improvement that the present invention gives prominence to are:

1. the present invention Issatchenkia orientalis CCTCC NO:M206097 of finding and providing, her these two bacterial strains of Sa yeast CCTCC NO:M206098 of west, to three big representative microorganism metabolic antagonists in the hemicellulose hydrolysate: with acetic acid is the organic acid of representative, with the furfural is the carbohydrate degraded product of representative, reaching with the methyl catechol is the phenolic compound of representative, all have degrading activity simultaneously, they all have outstanding biological detoxication activity to hemicellulose hydrolysate.

2. the present invention utilizes Issatchenkia orientalis CCTCC NO:M206097, or the multiple toxic ingredient in her the Sa yeast CCTCCNO:M206098 biological degradation hemicellulose hydrolysate of west, and the fact has reduced the impurity in the matrix.Compare with traditional physics and chemistry detoxification process, hemicellulose hydrolysate is carried out detoxification, obviously have simple and direct, low-cost, and eco-friendly outstanding advantage with the present invention.

3. two detoxification active bacterial strains provided by the invention, wherein CCTCC NO:M206097 can not assimilate wood sugar fully, CCTCC NO:M206098 is also very faint to the metabolic capacity of wood sugar, utilize them to the hemicellulose hydrolysate detoxification, and can't cause wood sugar loss wherein.Because these characteristics, they and xylose-fermenting are generated Xylitol, or xylose-fermenting generation alcoholic acid microorganism places same fermentation system, can realize hemicellulose hydrolysate detoxification process and xylitol fermentation, or with ethanol fermentation coupling mutually, greatly simplify fermentation hemicellulose hydrolysate production xylitol fermentation, or produce alcoholic acid technology.

Particularly, the present invention also provides a cover to 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 the raw material that is rich in xylan with acid or enzymic hydrolysis, obtains with wood sugar, and pectinose is a principal constituent, contains glucose simultaneously, seminose, the hydrolyzate 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.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 liquid of purity more than 99% respectively, and with pectinose--assorted sugar soln.High-purity xylitol liquid concentrating under reduced pressure, decrease temperature crystalline obtains the crystal Xylitol.It is sorbent material that pectinose--assorted sugar soln is used ammonium type Zeo-karb instead, still is that eluent carries out chromatographic separation with the pure water, obtains the higher Arabic liquid glucose of purity 1.With the pectinose condensing crystal, obtain the crystal pectinose at last.

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.

Being used for hemicellulose hydrolysate of the present invention prepares as follows.The material of hemicellulose such as maize peel (corn fiber), corn cob (corn 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 Candida tropicalis CCTCC M205067[number of patent application at Chinese typical culture collection center: 200510037580.2, publication number: CN1982460].

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 sugar component 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.

Aspect the preparation pectinose, 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.

After testing, Xylitol output object heat of the present invention only is 38% of a white sugar, need not insulin action after eating and just can directly be utilized by cell, can do diabetics's sweeting agent, nutritious supplementary and auxiliary therapeutical agent.In addition, this Xylitol output object can suppress streptococcus mutans, anti-streptococcus pneumoniae, suppress campylobacter pyloridis, the prevention of dental caries effect surpasses 90%, also can improve liver function, the prevention pulmonary bacterial, this Xylitol output object can serve as medicine intermediate, makes health-care toothpaste, health-care chewing gum, health care sweet food or sweet drink etc.

But, experimentation on animals shows that too fast, excessive absorption Xylitol can cause metabolic acidosis, causes injury of the kidney, even causes the brain function damage.Through measuring and calculating, Xylitol metabolite of the present invention is taken in speed, intake should meet following standard: per kilogram of body weight per hour intake should be no more than 0.3g, and every day, intake should be no more than 100g.

Therefore, if Xylitol output object of the present invention is taken in by the disposable apace heavy dose of people, taking in carrier so should have slow-release capability.For example, for the medicine that contains Xylitol, the present invention selects Xylitol effective constituent suspendible (dispersion) in a kind of hydrophobic oil, wherein some effective constituent is in dissolved state in oil, the oil that will contain medicine again is filled in a kind of polyethylene, in the cavity in the polyvinyl chloride framework (cavity communicates with the external world), oil phase communicates with the external world simultaneously, and this molectron that includes oil body and effective constituent thereof promptly is to can be used for pharmaceutical preparation oral or that implant.After medicine was taken, oil phase can not overflow from framework yet and can not digested, and it is constant that the contact area of oil phase and people's body fluid keeps, and pharmaceutical cpd spreads to the body fluid constant speed from oil phase.This can guarantee that human body is being no more than on the basis of limiting the quantity of, and evenly absorbs, utilizes Xylitol.

Description of drawings

Fig. 1 is that the high performance liquid chromatography of inhibition after the degraded of detoxification active bacterial strain changes.Wherein parameter is: (A) inhibition, and (B) substratum (C) has added the substratum of inhibition, and the substratum that (D) contains inhibition is through CCTCC NO:M206097 fermentation detoxification, and E contains inhibition and cultivates it through CCTCC NO:M206098 fermentation detoxification.Peak number and compound: 1 acetate, 2 furfurals, 1,3 methyl catechol, 4,5 medium components, 6 furfural degraded products, 7 methyl catechol degraded products.

Fig. 2 is the HPLC collection of illustrative plates (detecting wavelength 270nm) before and after the bagasse hemicellulose hydrolysate biological detoxication.Wherein parameter is: A, furfural standard specimen; B, the bagasse hemicellulose hydrolysate; C is through the bagasse hemicellulose hydrolysate of CCTCC NO:M206098 detoxification; D is through the bagasse hemicellulose hydrolysate of CCTCC NO:M206097 detoxification.

Embodiment

Embodiment 1

Operation 1

Claim to have washed the new dry bagasse 3kg that crosses that lays equal stress on, by solid-to-liquid ratio (w/v) is 1: 7 adding H2SO4 solution (2.5%, w/w), 120 ℃ of hydrolysis 2h, centrifugal collection filtrate, filter residue is washed once, merging filtrate, with lime carbonate solid adjust pH to 3, suction filtration is removed precipitation, gets the bagasse hemicellulose hydrolyzate.This hydrolyzate reducing sugar content 3%, wood sugar monose accounts for more than 80% of total reducing sugar.

Operation 2

Get already pulverised dried corn cob 2kg, by the H2SO4 solution of solid-to-liquid ratio adding in 1: 7 2% (w/w), 120 ℃ of hydrolysis 2h collect liquid portion, and lime carbonate is neutralized to pH value 3, and the suction filtration disgorging is able to corn cob hemicellulose dilute acid hydrolysis liquid.This hydrolyzed solution total sugar content 5%, wherein wood sugar accounts for 60% of total reducing sugar.

Operation 3

Issatchenkia orientalis CCTCC NO:M206098 inclined-plane is inoculated on the liquid seed culture medium, 200rmp, 30 ℃ of shaking tables are cultivated 12h, get activated seed liquid.By the inoculum size access operation 1 of 15% (v/v), in the bagasse hemicellulose dilute acid hydrolysis liquid of gained, 200rmp, 33 ℃ of shaker fermentation detoxification 45h, centrifuged supernatant is bagasse half hydrolyzate through Issatchenkia orientalis CCTCC NO:M206098 biological detoxication.

(,, obtain the bagasse hemicellulose hydrolyzate of her Sa yeast CCTCC NO:M206097 biological detoxication through the west with the bagasse hemicellulose hydrolyzate of her Sa yeast CCTCC NO:M206097 processing operation 1 of west perhaps by same condition.)

Operation 4

Operate the bagasse hemicellulose hydrolyzate through the biological detoxication processing of 3 gained, continue vacuum concentration to wood sugar 150g/L, ammoniacal liquor is adjusted to pH=6, other adds yeast extract paste 5g/L, insert candida tropicalis (Candidatropicalis) CCTCC NO:M205067 seed liquor, the access amount is 5% (v/v), 200rmp, and 33 ℃ ferment.Contrast HPLC detected result (table 3) is used for xylitol fermentation through the hemicellulose hydrolysate after the biological detoxication processing, and not only the product generating rate is more than doubled, and product yield is also near doubling.

Table 3. biological detoxication is handled influence (initial xylose concentration, the 150g/L to xylitol fermentation; Fermentation time, 61.5h)

Operation 5

Hemicellulose hydrolysate with operation 1 gained, be evaporated to the about 120g/L of soluble solid wood sugar content, ammoniacal liquor is adjusted to pH5, one group of equivalent inserts the Issatchenkia orientalis CCTCCNO:M206098 and the candida tropicalis CCTCC NO:M205067 cell of centrifugal collection, and another is organized then equivalent and inserts her Sa yeast CCTCC NO:M206097 cell and candida tropicalis CCTCC NO:M205067 cell of west.Their total inoculum size all is about stem cell 50g/L.33 ℃, shake bottle synchronous detoxification fermentation 30 hours under the 200rpm condition.Result's (table 4) shows that the detoxification fermentation energy obviously improves tunning concentration, product generating rate and product yield synchronously.

Comparison (initial wood sugar, the 120g/L of Xylitol performance produced in synchronous detoxification fermentation of table 4. hemicellulose hydrolysate and direct fermentation; Fermentation time, 30 hours)

Operation 6.

Hemicellulose hydrolysate with operation 2 gained, be evaporated to the about 60g/L of soluble solid wood sugar content, ammoniacal liquor is adjusted to pH5, press Issatchenkia orientalis CCTCC NO:M206098 and candida tropicalis CCTCC NO:M205067 combination respectively, the mode of her Sa yeast CCTCC NO:M206097 of west and candida tropicalis CCTCC NO:M205067 combination, carry out the balanced mix cell inoculation, total inoculum size is about cultivates 5% of its volume, 33 ℃, shake bottle synchronous detoxification fermentation 80 hours under the 200rpm condition.Result's (table 5) shows that the detoxification fermentation energy obviously improves tunning alcoholic acid generating rate and alcohol concn synchronously.

Table 5 biological detoxication is handled the influence to lignocellulose hydrolytic residue simultaneous saccharification and fermentation alcohol performance

Referring to Fig. 1. the high performance liquid chromatography of inhibition after the degraded of detoxification active bacterial strain changes.Wherein parameter is: (A) inhibition, and (B) substratum (C) has added the substratum of inhibition, and the substratum that (D) contains inhibition is through CCTCC NO:M206097 fermentation detoxification, and E contains inhibition and cultivates it through CCTCC NO:M206098 fermentation detoxification.Peak number and compound: 1 acetate, 2 furfurals, 1,3 methyl catechol, 4,5 medium components, 6 furfural degraded products, 7 methyl catechol degraded products.Fig. 2 is the HPLC collection of illustrative plates (detecting wavelength 270nm) before and after the bagasse hemicellulose hydrolysate biological detoxication.Wherein parameter is: A, furfural standard specimen; B, the bagasse hemicellulose hydrolysate; C is through the bagasse hemicellulose hydrolysate of CCTCC NO:M206098 detoxification; D is through the bagasse hemicellulose hydrolysate of CCTCCNO:M206097 detoxification.

The content of some uv-absorbing thing (detecting wavelength 270nm) in the bagasse hemicellulose hydrolysate after table 6 biological detoxication is handled

Embodiment 2

Concrete steps 1

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.

Concrete steps 2

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 concrete steps 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.

Concrete steps 3

Get the maize peel hemicellulose hydrolysate of concrete steps 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 BaoxinBioengineering.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.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 seven so continuously.

Table seven selective conversion maize peel hydrolyzate generates Xylitol

Concrete steps 4

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 concrete steps 3.Carry out 5 round-robin fermentation result such as table eight continuously.

The selective conversion of table Yagi spark gap sugar crystalline mother solution generates Xylitol

Concrete steps 5

Get the fermented liquid 10L of concrete steps 3 gained, the ultra-filtration membrane ultrafiltration deproteinated through molecular weight cut-off 5Kdal by order is: positively charged ion--negatively charged ion-positively charged ion---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 part, Xylitol content 〉=99%, concentration 12-13%; 2. pectinose-assorted sugar moieties, total solubility substrate concentration 5-7%, pectinose content 55-60% wherein, other impurity 30-40%.Crystallization after Xylitol liquid directly concentrates obtains the crystal Xylitol, and pectinose-assorted sugar moieties then need be further purified.

The rapid pectinose that obtains of previous step-assorted liquid glucose 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 part after the system balancing, the purity of its pectinose is brought up to 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 (9)

1. the application of microorganism strains candida tropicalis Candida tropicalis, it is characterized in that, the culture of described microorganism strains candida tropicalis is seeded to hemicellulose hydrolysate, transforms from described hemicellulose hydrolysate and generate high-purity xylitol product.

2. application according to claim 1 is characterized in that, described microorganism strains candida tropicalis Candida tropicalis is CCTCC M 205067 and naturalized strain thereof.

3. application according to claim 1 is characterized in that, the seed preparation technology of described microorganism strains candida tropicalis Candida tropicalis is:

S1. prepare seed culture medium, it consists of wood sugar 20g/L, glucose 20-30g/L, yeast extract paste 10g/L, KH2PO4,5g/L, NH4H2PO4,3g/L, MgSO4.7H2O, 0.1g/L, pH 5-6, the liquid loading amount generally is controlled at triangular flask volumetrical 10-20%, sterilizes 15 minutes for 115 ℃;

S2. get the slant culture of described candida tropicalis Candida tropicalis and inoculate cooled substratum, 28-35 ℃ of shaking table activation culture 10--12 hour;

S3. the 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.

4. application according to claim 1 is characterized in that, described hydrolyzate is inserted after Issatchenkia orientalis or her the Sa yeast-inoculated detoxification of west, perhaps inserts the culture of described microorganism strains candida tropicalis synchronously.

5. described application transforms the high-purity xylitol product that generates and is used for the treatment of application in the medicine of blood sugar disorders in preparation according to each of claim 1 to 4, it is characterized in that, described medicine comprises Xylitol, and the Xylitol intake standard of described medicine is: per kilogram of body weight per hour intake is no more than 0.3g, and every day, intake was no more than 100g.

6. described application transforms the high-purity xylitol product that generates and is used for the treatment of application in the medicine of carious tooth disease in preparation according to each of claim 1 to 4, it is characterized in that, described medicine comprises described high-purity xylitol product, and the Xylitol intake standard of described medicine is: per kilogram of body weight per hour intake is no more than 0.3g, and every day, intake was no more than 100g.

7. described application transforms the high-purity xylitol product that generates and is used for the treatment of application in the fat medicine in preparation according to each of claim 1 to 4, it is characterized in that, described medicine comprises described high-purity xylitol product, and the Xylitol intake standard of described medicine is: per kilogram of body weight per hour intake is no more than 0.3g, and every day, intake was no more than 100g.

8. each the described application according to claim 1 to 4 transforms the high-purity xylitol product of generation in preparation prevention blood sugar disorders, prevention of obesity, and the healthcare products or the Application in Food of preventing and treating carious tooth, it is characterized in that, described healthcare products or food comprise described high-purity xylitol product, and the intake standard of described xylitol product is: per kilogram of body weight per hour intake is no more than 0.3g, and every day, intake was no more than 100g.

9. use according to each described pharmacy of claim 5 to 7, it is characterized in that, described medicine is by the slow administration of injection, perhaps by the disposable apace oral administration of controlled release means.

Images