CN104981546A - Method for saccharification of potato starting material and method for producing liquid fuel - Google Patents

Method for saccharification of potato starting material and method for producing liquid fuel Download PDF

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CN104981546A
CN104981546A CN201380010643.8A CN201380010643A CN104981546A CN 104981546 A CN104981546 A CN 104981546A CN 201380010643 A CN201380010643 A CN 201380010643A CN 104981546 A CN104981546 A CN 104981546A
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potato
raw material
filamentous fungus
fermentation
enzyme
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CN104981546B (en
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高旻天
矢野伸一
井上宏之
坂西欣也
中田龙彦
千叶恭一
山本淳一
冈田正史
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Hitachi Zosen Corp
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Hitachi Zosen Corp
National Institute of Advanced Industrial Science and Technology AIST
Sojitz Corp
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/14Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/02Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06Ethanol, i.e. non-beverage
    • C12P7/08Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00Technologies for the production of fuel of non-fossil origin
    • Y02E50/10Biofuels, e.g. bio-diesel

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Provided are a method for the efficient saccharification of a starting material derived from potato tubers, and a method for producing liquid fuel using a starting material derived from potato tubers. In particular, provided is a method for producing a saccharification product, characterized in that a starting material derived from potato tubers is treated with a secretase solution of a mold having a highly degradable cell wall containing a hydrolytic enzyme.

Description

The method for saccharifying of raw potatoes and the manufacture method of liquid fuel
Technical field
The present invention relates to the manufacture method of the method for saccharifying of the raw potatoes utilizing enzyme and the liquid fuel based on the method.
Background technology
The liquid fuels such as the ethanol manufactured by biomass are due to can petroleum replacing and contribute to the output of cutting down greenhouse effect gas, and thus it is produced and utilize and is developed in the world.
The stem tuber of potato is principal constituent with starch except moisture, and by the amylase saccharification of this starch, and to make it fermentation to manufacture the liquid fuels such as ethanol be comparatively be easy to.But potato tuber also containing the composition such as Mierocrystalline cellulose, pectin, decomposes these compositions and only leans on amylase used in amylolysis to be difficult except starch.In residue (liquid is called potato residues or yam starch extracts residue) particularly after extracting starch from potato tuber, relatively increasing containing proportional of Mierocrystalline cellulose, pectin etc., the utilization of the starch residual in the cell walls of composition using them becomes difficulty, and this becomes the major cause of the utilization limiting above-mentioned residue.Describe secretion in non-patent literature 1, from the enzyme of Rhizopus oryzae, the amylolysis in potato residues is generated lactic acid, but the cell wall breakdown ability of Rhizopus oryzae low (table 2 of non-patent literature 1) and lactic acid generation can cause the yield of sugar to reduce, and thus use the method for saccharifying of the starch of the cell walls inside of Rhizopus oryzae still to there is the leeway of improvement.In addition, need in the decomposition of this potato residues to use multiple enzyme, it is reported and it is desirable to expect use 3 kinds of enzymes (non-patent literature 2).But the use of multiple enzyme is the major cause that cost raises, thus need the more simple and saccharification technology of low cost.
Prior art document
Non-patent literature
Non-patent literature 1:Oda Y., et al., Curr Microbiol., (2002) 45 (1): p.1-4
Non-patent literature 2:Miyaji et al., J. Agric. Sci., Tokyo Univ. Agric., 52 (3) 147-150 (2007).
Summary of the invention
The problem that invention will solve
Problem of the present invention be to provide by the raw material deriving from potato tuber effectively saccharification method and utilize the manufacture method deriving from the liquid fuel of the raw material of potato tuber.
For the method for dealing with problems
The present inventor furthers investigate repeatedly in order to solve above-mentioned problem, found that, filamentous fungus can produce the raw material deriving from taro class, especially take potato residues as the effective enzyme group of the efficient saccharification deriving from the raw material of potato tuber of representative, thus complete the present invention.
That is, the present invention comprises following.
[1] manufacture method of saccharides, is characterized in that, is processed by the raw material deriving from potato tuber with the Secretases liquid of the cell walls high de-agglomeration filamentous fungus containing lytic enzyme.
[2] method of above-mentioned [1], wherein, the raw material deriving from potato tuber is potato residues.
[3] above-mentioned [1] or the method described in [2], wherein, cell walls high de-agglomeration filamentous fungus is top spore mould genus filamentous fungus or Penicillium filamentous fungus.
[4] method any one of above-mentioned [1] ~ [3], wherein, cell walls high de-agglomeration filamentous fungus separates fiber top spore mould (Acremonium cellulolyticus).
[5] method any one of above-mentioned [1] ~ [3], wherein, cell walls high de-agglomeration filamentous fungus separates the mould TN strain of fiber top spore (deposit number FERM BP-11452) or Penicillium notatum NBRC 101300 strain.
[6] method any one of above-mentioned [1] ~ [5], wherein, the generation of induced hydrolysis enzyme with the aforementioned filamentous fungus of the culture medium culturing containing the raw material deriving from potato tuber, prepares aforementioned Secretases liquid thus, and uses it for the process of the raw material deriving from potato tuber.
[7] manufacture method of alcohol, is characterized in that, the saccharides obtained the method by above-mentioned [1] ~ [6] carries out alcohol fermentation.
[8] method of above-mentioned [7], wherein, uses Saccharomycodes yeast to carry out alcohol fermentation.
[9] method of above-mentioned [7] or [8], it is the manufacture method using Saccharomycodes yeast aforementioned saccharides to be carried out to the ethanol of ethanol fermentation.
[10] method of above-mentioned [7] ~ [9], wherein, carries out front cultivation with the substratum of saccharides containing the raw material deriving from potato tuber to organism of fermentation, and carries out alcohol fermentation with it.
This specification sheets comprises the content of the Japanese Patent Application 2012-037404 as the application's priority request basis.
Invention effect
According to method of the present invention, the raw material deriving from potato tuber can be hydrolyzed efficiently and saccharification.
Accompanying drawing explanation
[Fig. 1] Fig. 1 is the figure of the sugar receipts amount of the saccharification gained that the potato residues utilizing commercially available cellulase is shown.The receipts amount (g/L) that A represents glucose, B represents semi-lactosi.
[Fig. 2] Fig. 2 is the figure of the cellulase generation of the solution fiber top spore mould (Acremonium cellulolyticus) illustrating that utilization is carbon source with cellulose powder or potato residues.
[Fig. 3] Fig. 3 illustrates that cellulose powder adds figure cellulase being produced to the impact caused." cellulose powder " represents the result of the cultivation of the substratum used containing 5% cellulose powder, " only potato residues " represents the result of cultivation of the substratum (cellulose powder addition 0%) used containing 5% potato residues, and " potato residues+0.5% ", " potato residues+1.5% ", " potato residues+5% " represent respectively to use and contain 5% potato residues+cellulose powder addition: 0.5%, the result of cultivation of substratum of 1.5%, 5%.
[Fig. 4] Fig. 4 illustrates the figure employed by the sugar receipts amount of the saccharification of separating the enzyme that fiber top spore mould (Acremonium cellulolyticus) produces.A: glucose amount (g/L), B: semi-lactosi amount (g/L).In each figure, from being from left to right, the top mould cellulase preparation of spore (marketed cellulose zymin), cellulose powder inducible enzyme liquid (using the enzyme liquid that cellulose powder produces as carbon source induction), potato residues inducible enzyme liquid (using the enzyme liquid that potato residues produces as carbon source induction).White bars represents the result of the saccharification of 24 hours, and black bar represents the result of the saccharification of 48 hours.
[Fig. 5] Fig. 5 illustrates to employ to cultivate 3 kinds of filamentous funguss with potato residues and the figure of the sugar receipts amount of the saccharification of the potato residues of the enzyme liquid obtained.A: glucose amount (g/L), B: semi-lactosi amount (g/L).In figure, the result that rhombus represents the result on top spore mould genus bacterium (separate fiber top spore mould TN strain), square represents Trichoderma bacterium (Trichodermareesei RUT C-30 strain), trilateral represent the result of Penicillium bacterium (Penicillium notatum NBRC 101300 strain).
[Fig. 6] Fig. 6 is the figure of the propagation level that the yeast using potato saccharified liquid as cultivation nutrition source is shown.The percentage ratio recorded in each sample name represent each composition in substratum containing concentration.
[Fig. 7] Fig. 7 illustrates to use with potato residues and Mierocrystalline cellulose as the enzyme liquid that carbon source cultivates filamentous fungus and obtain, and carries out diastatic fermentation and the figure of the concentration of ethanol that obtains to potato residues.
Embodiment
Below, the present invention is described in detail.
In method of the present invention, use the raw material deriving from potato tuber as raw material.The raw material deriving from potato tuber refers to stem tuber or its handled thing of potato, and is processed into the state being suitable for ferment treatment.The raw material deriving from potato tuber does not limit, such as, except the form of potato tuber itself, can also be section, disorderly chop thing, stripping and slicing, stamping, broken thing, crushed material or pulverize arbitrary forms such as being suspended thing, can remove or not except peeling, bud, be preferably the state containing composition (particularly starch, Mierocrystalline cellulose etc.) keeping potato tuber as far as possible.In addition, the residue produced when the raw material deriving from potato tuber can be processing potato stem tuber, such as, is particularly preferably potato residues.Potato residues means to take potato tuber as the residue after raw material is separated as yam starch when carrying out the extraction of yam starch and residual product.Potato residues is also referred to as potato and extracts residue.The raw material deriving from potato tuber is the stem tuber of above-mentioned potato or its handled thing and is processed into the state being suitable for ferment treatment, such as, can be the material residue produced during processing potato stem tuber and other biomass material are mixed together.In addition, the raw material deriving from potato tuber can be raw state, also can be through heating condition.The raw material deriving from potato tuber can be through freezing state, also can be the state of drying, can also be lyophilize thing.The raw material deriving from potato tuber can carry out germicidal treatment.As an example, for the raw material deriving from potato tuber, can the autoclave process of 121 DEG C, about 10 ~ 30 minutes that adopts of the germicidal treatment of carrying out in usual microorganism culturing.By this, the living contaminants in the diastatic fermentation step after this raw material can prevent through sterilization, also can expect the effect of the decomposability improving raw material, thus it is useful process simultaneously.
In the present invention, potato tuber refers to usually edible potato subterraneous stem part.The potato used in the present invention can be any kind or its mutation, also can be wild species.
Filamentous fungus used in the present invention is the fungi with the ability producing the multiple biomass decomposition enzyme of secretion, and at least a kind that is preferably its Secretases possesses the fungi main composition composition of potato tuber and starch, Mierocrystalline cellulose, pectin etc. being hydrolyzed to the ability forming sugar.The filamentous fungus used in the present invention is preferably cell walls high de-agglomeration filamentous fungus.In the present invention, cell walls high de-agglomeration filamentous fungus refers to and high-efficiency decomposition of cellulose, pectin etc. can form the composition of cell walls, and the raw material (potato residues etc.) such as deriving from potato tuber containing these cell wall constituents can be decomposed to the filamentous fungus of aqueousization.Such as, belong to and can be enumerated as preferred filamentous fungus used in the present invention as the mould genus of top spore of anamorph or the filamentous fungus of Penicillium.As the filamentous fungus used in the present invention, preference can be enumerated and separate fiber top spore mould (Acremonium cellulolyticus).The particularly preferred example of the filamentous fungus used in the present invention separates mould (Acremonium cellulolyticus) the TN strain of fiber top spore and mould (Penicillium) bacterium NBRC 101300 strain.
Various filamentous fungus by commercially available product or culture presevation or can obtain based on the preservation mechanism of budapest treaty.
Separating the mould TN strain of fiber top spore is on January 12nd, 2012, and based on budapest treaty, with deposit number FERM BP-11452, international accession is in independent administrative corporation's goods evaluation technique fundamental mechanism Patent Organism preservation center (NITE-IPOD; Ci Cheng Ken つ く ば Shi East 1 fourth order a kind of ground 1 central authorities the 6th of postcode 305-8566 this country).
Penicillium notatum NBRC 101300 strain is recorded in independent administrative corporation's goods evaluation technique fundamental mechanism (NITE; Thousand Leaf Ken Mu Geng Jinshi City か ず さ Sickle foot 2-5-8) NBRC(NITE Biological Resource Center) catalogue [ the NBRC Catalogue of Biological Resources of (Japan), Microorganisms, Microorganism-Related DNA Resources, Human-Related DNAResources, Second Edition (2010) ] in, can be obtained by NBRC with numbering 101300.
In method of the present invention, in the process of raw material deriving from potato tuber, the Secretases liquid containing lytic enzyme using aforementioned filamentous fungus to produce.In the present invention, the Secretases liquid of filamentous fungus refers to by cultivating filamentous fungus and produces, containing the liquid (enzyme liquid) secreted to the Secretases group in substratum.This Secretases liquid of filamentous fungus contain one or more, the preferred multiple enzyme (lytic enzyme) with the ability of the polyose contained by potato such as starch-splitting, Mierocrystalline cellulose, pectin, at least containing cellulase.The Secretases liquid of filamentous fungus of the present invention is typically containing cellulase (cellulolytic enzyme), polygalacturonase (pectin hydrolase) and amylase (amylolytic enzyme).The Secretases liquid of filamentous fungus of the present invention is also preferably further containing galactan lytic enzyme.Should illustrate, in this specification sheets, sometimes the Secretases liquid containing multiple enzyme is expressed as enzyme system.These enzymes are that filamentous fungus produces and secretes after manufacture to extracellular in cell.This Secretases liquid can be filamentous fungal cultures or culture supernatant.The Secretases liquid of aforementioned filamentous fungus can, by cultivating the filamentous fungus of more than one (preferably 1 kinds) and the production of induced hydrolysis enzyme under the existence of carbon source, make it to secrete this enzyme to prepare in its nutrient solution.The substratum cultivating filamentous fungus can add carbon source etc. and prepare in any culture medium being suitable for filamentous fungus cultivation.As the carbon source in the substratum cultivated needed for filamentous fungus, arbitrary organism can be used, use derive from the raw material (such as potato residues) of potato tuber or its enzyme resolvent (such as saccharides) as carbon source in the generation of the lytic enzymes such as cellulase induction, polygalacturonase and amylase more preferably.In the situation of the latter, compared with using the situation of other carbon source, the enzyme of the decomposability excellence to the composition (starch, Mierocrystalline cellulose, pectin etc.) existed a large amount of especially in potato tuber can be produced.Therefore, the substratum cultivating filamentous fungus preferably containing at least a kind (preferably whole) in starch, Mierocrystalline cellulose and pectin as carbon source.Cultivate in the substratum of filamentous fungus, except the raw material (such as potato residues) deriving from potato tuber, also preferably containing its enzyme resolvent (such as saccharides) or derive from potato tuber raw material beyond carbon source refined cellulose or purified starchs etc. such as (such as) cellulose powders.Carbon source beyond the raw material deriving from potato tuber can with such as more than 0.5%, preferably more than 1.5%, preferably the concentration of more than 5%, such as 0.5 ~ 30% makes an addition to and derives from the raw material of potato tuber further.Except the raw material deriving from potato tuber, also other carbon source is added into substratum, thus can the generation of the lytic enzyme such as cellulase induction, polygalacturonase and amylase effectively further.
The Secretases liquid of filamentous fungus can carry out gathering, be separated or refine using from the nutrient solution of this kind of filamentous fungus (such as with the form of culture supernatant), also can not carry out gathering, to be separated or refining and nutrient solution is directly used in the process of the raw material deriving from potato tuber.As the Secretases liquid of filamentous fungus, industrial commercial articles (such as, pushing up the mould cellulase of spore (Meiji Seika Off ァ Le マ Co., Ltd. system)) can be used.
In method of the present invention, by to derive from potato tuber raw material or containing in the solution of raw material deriving from potato tuber or liquid nutrient medium, add the Secretases liquid of filamentous fungus and make it reaction and ferment treatment is carried out to the raw material deriving from potato tuber.
Or, in method of the present invention, also can derive from potato tuber raw material or containing deriving from the liquid nutrient medium of raw material of potato tuber, cultivate the generation of filamentous fungus of the present invention and the lytic enzymes such as cellulase induction, and make it secretion to substratum, form Secretases liquid, add the raw material deriving from potato tuber further wherein and make it reaction, thus ferment treatment being carried out to the raw material deriving from potato tuber.
Ferment treatment preferably carries out under the optimum temps, pH of each enzyme, such as, in the situation of enzyme system of separating fiber top spore mould generation, suitably 40 ~ 60 DEG C, about pH4 ~ 6, preferably 45 ~ 55 DEG C, pH4.5 ~ 5.5 time carry out.
Ferment treatment preferably uses with more than 5%, preferably more than 7%, more preferably more than 10%, further preferably more than 20%, also preferably more than 30%, such as more than 50% and the concentration of the concentration of less than 100%, such as 5 ~ 50% contains the solution of the raw material (being preferably potato residues) deriving from potato tuber or substratum carries out.
By enzyme reaction as above, the polyose (Mierocrystalline cellulose, starch, pectin etc.) forming potato tuber is hydrolyzed and generates monose or oligosaccharide kind (such as, glucose, semi-lactosi, maltose, cellobiose etc.) (saccharification react).The raw material that the present invention also provides this origin to come from potato tuber manufactures the method for saccharides.In addition, the present invention also provides the method for saccharifying deriving from the raw material of potato tuber utilizing this saccharification react.
So, saccharides (the typically being saccharified liquid) fermentation obtained by making to derive from the raw saccharified of potato tuber, can change various useful matter into.Especially, fermented by alcohol, alcohol can be produced by the monose in saccharides or oligosaccharide kind.The alcohol of gained can be used as such as liquid fuel or Industrial materials etc.
Alcohol fermentation can use the organism of fermentation carrying out alcohol fermentation, such as yeast, carries out.Yeast indefinite, can enumerate such as: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomycodes (Saccharomyces) bacterium such as saccharomyces pastorianus (Saccharomyces pastorianus), schizosaccharomyces pombe (Saccharomyces pombe)) etc. Schizosccharomyces (Schizosaccharomyces) bacterium, genus kluyveromyces (Kluyveromyces) bacterium such as kluyveromyces marxianus (Kluyveromyces marxianus), Issatchenkia (Issatchenkia) bacterium etc. such as Issatchenkia orientalis (Issatchenkia orientalis).
Such as, by using the Saccharomycodes yeast with ethanol fermentation ability as yeast, ethanol fermentation can be carried out to above-mentioned saccharides.Particularly preferably yeast saccharomyces cerevisiae, is cultivated by adding in saccharides, can produce (manufacture) ethanol by monoses such as glucose.This ethanol can be used as can replacing gasoline liquid fuel use.By utilizing the fermenting experiment of yeast saccharomyces cerevisiae to show, the saccharides deriving from the raw material of potato tuber does not observe the inhibition to yeast, can be used as good fermentation raw material.In the present invention, " organism of fermentation " refers to the microorganism having and carry out the ability of fermenting.
Above-mentioned fermentation is not limited to use yeast saccharomyces cerevisiae to generate the alcohol fermentation of ethanol, also may correspond in organism of fermentation used and manufactures other alcohol such as butanols.Various alcohol so also may be used for liquid fuel etc.
By to saccharides that is that obtain ferments by deriving from the raw saccharified of potato tuber, can also produce the material beyond alcohol, such as, lipid acid etc. can be used as the useful matter of liquid fuel component.This fermentation realizes by using corresponding organism of fermentation.
Organism of fermentation, before the fermentation for saccharides, preferably carries out propagation and is prepared as a certain amount of step (cultivating before so-called).In method of the present invention, preferably with the substratum of saccharides containing the raw material deriving from potato tuber, front cultivation is carried out to organism of fermentation, and use its fermentation carrying out saccharides (preferred alcohols fermentation).Fermentation can be carried out under the usual fermentation condition of this organism of fermentation, such as, under anaerobic can carry out, also can under aerobic conditions carry out.In the situation of yeast saccharomyces cerevisiae, preferably carry out under the fermentation condition of anaerobism.
In cultivating before of the present invention, containing the substratum of saccharides of raw material deriving from potato tuber, such as, can be the saccharified liquid of the raw material deriving from potato tuber itself, also the saccharides of the raw material deriving from potato tuber can be added in other substratum such as culture medium and prepare.By using the saccharides deriving from the raw material of potato tuber, compared with using the situation of nutrition source usually used and waste molasses, the better propagation of microorganism can be obtained.So, by the microorganism having carried out front cultivation with the saccharides of the raw material deriving from potato tuber being used for, as in the fermentation of the formal saccharides cultivated, efficiency to carry out alcohol fermentation very well.Should illustrate, not only can be used for the fermentation of the saccharides of the raw material deriving from potato tuber with the microorganism that the saccharides of the raw material deriving from potato tuber has carried out front cultivation, also can be widely used in the fermentation using other various raw material.
In the past, for raw material, the particularly potato residues etc. that derive from potato tuber, do not exist and by the technology of its composition comprehensively efficient-decomposition saccharification, the obstacle that it utilizes can be become.In method of the present invention, by using the enzyme system deriving from filamentous fungus, efficiency can decompose the main component such as Mierocrystalline cellulose, starch, pectin contained in the raw material deriving from potato tuber well, consequently, efficiency the monose that can be changed into liquid fuel by fermentation can be obtained well.By method of the present invention, the comprehensive decomposition deriving from the raw material of potato tuber of difficulty in the past becomes and can easily carry out, by fermenting to it, the manufacture of the liquid fuel being representative can be carried out with alcohol, effective Application way of the biomass material that availability was low in the past can be provided thus.
The whole distribution publications quoted in this specification sheets, patent and patent application are all incorporated in this specification sheets by reference to by it.
Embodiment
Below, enumerate embodiment and the present invention is described in more detail, but the present invention is not by their restriction.
[ embodiment 1 ]
(1) yam starch squeezes the analysis of slag basal component
Obtain the lyophilize thing of the potato residues (yam starch squeezes slag) gathered in the starch plants of Heilongjiang Province of China Beidahuang Potato Industry Group Co., Ltd., for aftermentioned mashing test.
Therefore, first by well-established law the one-tenth of potato residues used is grouped into and analyzes.The results are shown in table 1.
[table 1]
Should illustrate, the bottom of table 1 illustrates that the monose after being decomposed completely by the polysaccharide in above-mentioned potato residues forms.This monose composition is the measured value being decomposed completely by potato residues sulfuric acid and obtain.Glucose is generated by starch and cellulosic decomposition substantially, thus the total of starch and cellulosic amount and monose form in glucose content basically identical.
(2) saccharification of enzyme is used
In the lyophilize potato residues (sample) of above-mentioned (1), add water, make concentration be 10%, 20%, each w/v of 30%().Wherein, with 10 Filter Paper Unit(FPU; Filter paper degrading activity) ratio of/g substrate adds the mould cellulase of marketed cellulose zymin top spore (Meiji Seika Off ァ Le マ Co., Ltd. system; Separate the cellulase of the mould generation of fiber top spore), in the citrate buffer solution of 0.05M, carry out 24 hours or reaction in 48 hours at 50 DEG C, measure the glucose of generation, the amount of semi-lactosi.The mensuration of glucose and semi-lactosi uses the Japanese light splitting society highly effective liquid phase chromatographic system (detector: Differential refractometer 2031Plus) being provided with Aminex HPX-87H (BioRad society) post, with respective standard substance for benchmark carries out quantitatively.The results are shown in Fig. 1.As shown in Figure 1, the growing amount of glucose and semi-lactosi depends on the concentration of sample used and increases.By ferment treatment, originally for gelatinous potato residues decomposes and aqueousization, generated the monose such as glucose, semi-lactosi.
On the other hand, identical marketed cellulose zymin and Accellerase 1000(Genencore society system is used; Derive from Trichodermareesei), similarly carry out the experiment potato residues of (1) being carried out to ferment treatment, even result 10% concentration, residue also not aqueousization, does not observe decomposition.
This result shows, the saccharification that the cellulase that top spore mould genus bacterium produces is very suitable for deriving from taro class, particularly derives from the raw material of potato.
[ embodiment 2 ]
(1) enzyme of potato residues is used to produce
Be that carbon source is cultivated the mould TN strain of cellulase production fungi degradation fiber top spore (deposit number: FERM BP-11452) with potato residues, make it to produce enzyme.In addition, in contrast, potato residues is also carried out the cellulose powder of the carbon source being typically used as cellulase production to substitute and the cultivation that uses as carbon source.
Substratum used in cultivation composed as follows described in: 24 g/L KH 2pO 4, 1 g/L Tween 80,5 g/L (NH 4) 2sO 4, 1.2 g/L MgSO 47H 2o, 0.01 g/L ZnSO 47H 2o, 0.01 g/L MnSO 46H 2o, 0.01 g/L CuSO 47H 2o, 4 g/L ureas, 4.7 g/L soluble tartrate monohydrates and the cellulose powder (Solka Floc(registered trademark) as carbon source; Contrast) or lyophilize potato residues (5%, 7%, 10%).
Cultivate and carry out under temperature of reaction 30 DEG C, stirring velocity 200rpm.Be that carbon source carries out front cultivation with cellulose powder, cultivate after having started 72 hours in the past, front nutrient solution is added in substratum in the mode reaching 5%, formally cultivates.
The formal cultivation of cultivating of collection has started the substratum after 5 days, after 7 days, after 14 days, measures the activity of secretion to the enzyme-cellulose enzyme in substratum.The mensuration of cellulase activity is for substrate with filter paper (Whatman No.1), 50 DEG C of reactions 60 minutes in 50 mM citrate buffer solutions (pH 5.0), by dinitrosalicylic acid system, colorimetric, is quantitatively carried out to the amount of reducing sugar generated, and activity FPU is represented (Fig. 2).
As shown in Figure 2, although low with the concentration taking cellulose powder as carbon source phase specific activity, by being that the cultivation of carbon source can produce the enzyme with cellulase activity with potato residues.
And then, to add the material of 0.5%, 1.5% or 5% cellulose powder as carbon source in lyophilize potato residues (5%), carry out the cultivation of separating fiber top spore mould (A. cellulolyticus) TN strain in the same manner as described above, make it to produce enzyme.For the substratum after cultivation, measure cellulase activity (FPU is active) in the same manner as described above, results verification is to the raising (Fig. 3) of the FPU activity corresponding with the Mierocrystalline cellulose addition in potato residues.
(2) saccharification of the potato residues being the enzyme that carbon source produces is utilized with potato residues
To be that carbon source cultivates the culture supernatant collection of the culture that the mould TN strain of solution fiber top spore obtains as enzyme liquid with potato residues in above-mentioned (1) of the present embodiment, and use the pipe (VIVASPIN 6, Sartorius society) with ultra-filtration membrane to carry out ultrafiltration with concentrated to it.It is respectively added into 30%(w/v with the ratio of 5 FPU/g) in potato residues, with embodiment 1-(2) carry out the saccharification of 24 hours or 48 hours in the same manner.
In the same manner, using being that carbon source cultivates the culture supernatant collection of the culture that the mould TN strain of solution fiber top spore obtains as enzyme liquid with cellulose powder in above-mentioned (1) of the present embodiment, under reaction conditions same as described above, mashing test is carried out.In addition, in contrast, the mashing test employing the mould cellulase of marketed cellulose zymin top spore used in embodiment 1 is also implemented under the same reaction conditions.
Then, for gained saccharified liquid, measure the amount of glucose and semi-lactosi respectively identically with embodiment 1.
Above result is shown in Fig. 4.As shown in Figure 4, using with potato residues is carbon source by the enzyme liquid separated the mould TN strain of fiber top spore and produce, and can produce glucose and semi-lactosi by potato residues.In addition, use with cellulose powder is that the enzyme liquid of carbon source generation or the situation of the mould cellulase of marketed cellulose zymin top spore also can produce glucose and semi-lactosi by potato residues.Particularly use with potato residues be carbon source produce enzyme liquid time, can obtain higher than use take cellulose powder as the enzyme liquid that produces of carbon source or marketed cellulose zymin top spore mould cellulase time sugar receipts amount.For semi-lactosi, and taking cellulose powder as the enzyme liquid phase ratio that carbon source produces, is that the enzyme liquid that carbon source obtains can obtain significantly higher receipts amount with potato residues.Think thus, when taking potato residues as carbon source, the generation of galactan lytic enzyme is induced, containing galactan lytic enzyme in gained Secretases liquid.
[ embodiment 3 ]
Substitute and separate the mould TN strain of fiber top spore, by filamentous fungus Penicillium notatum NBRC 101300 strain (catalogue [ the NBRC Catalogue of Biological Resources of the NBRC of independent administrative corporation's goods evaluation technique fundamental mechanism (NITE), Microorganisms, Microorganism-Related DNA Resources, Human-Related DNA Resources, Second Edition (2010) ] in, obtained by NBRC), Trichodermareesei RUT C-30 strain (the American Type Culture Collection (ATCC) no. 56765)) under the same conditions as in practical example 2, potato residues is used to cultivate as carbon source, and use gained enzyme liquid to make potato residues saccharification.For saccharified liquid, the result measuring the amount of glucose and semi-lactosi is shown in Fig. 5.
Its result shows, 3 kinds of filamentous funguss all produce glucose and semi-lactosi.As shown in Figure 5, use the glucose amount derived from when separating the mould enzyme liquid of fiber top spore the highest, in saccharification particularly preferably.But for deriving from the enzyme liquid of Penicillium notatum, obtain derive from separate the mould enzyme liquid of fiber top spore about 75% glucose amount, and for semi-lactosi amount, obtain higher receipts amount more mould than solution fiber top spore, therefore show that this bacterium is also preferred to saccharification.These bacterium are particularly useful for the generation of the saccharification enzyme of potato residues.
For deriving from the enzyme liquid of industrially usual the used Trichodermareesei as cellulase producing bacteria, residue aqueousization, saccharification start to need more than 40 hours, its reaction comparatively separate the spore mould TN strain of fiber top and Penicillium notatum NBRC 101300 strain slow.
[ embodiment 4 ]
(1) the front cultivation of the organism of fermentation of potato saccharified liquid is utilized
For for make microorganism used in the fermentation of saccharified liquid be increased to a certain amount of before cultivate in potato saccharified liquid whether can be used to test as nutrition source.
By yeast Saccharomyces cerevisiae IR-2 strain (on April 8th, 1985, in independent administrative corporation's goods evaluation technique fundamental mechanism Patent Organism preservation center (postcode 305-8566 day this country's thatch city Ken つ く ば city East 1 fourth order a kind of ground 1 central authorities the 6th) preservation.Deposit number FERM BP-754) cultivate a Dinner with YPD substratum (1% yeast extract, 1% polyprotein peptone, 2% glucose), and with OD 600the mode that value (optical density(OD): utilize absorbancy to represent the index of somatic cells number) reaches 0.1 makes an addition to substratum, 30 DEG C, carry out culture experiment under stirring velocity 120 rpm.
Substratum is based on YPD substratum, uses potato residues at embodiment 1-(2) in the saccharified liquid (potato saccharified liquid) that obtains by the mould cellulose treatment of commercially available enzyme top spore and the waste molasses of sugar beet that obtains in China prepare.Use YPD substratum substratum in contrast.Incubation time is set to 6 hours, 18 hours or 24 hours.
Usually, in the cultivation of yeast, waste molasses is used by as preferred nutrition source, but by using potato saccharified liquid, obtains the proliferative amount (Fig. 6) of the yeast higher than waste molasses.
This result shows, derives from the saccharides of the raw material of potato tuber not only as carbon source, and also excellent as the comprehensive nutrition source comprising nitrogen or other inorganic components.
(2) ethanol fermentation experiment
Embodiment 2-(1) in, in the enzyme liquid that the material adding 0.5%, 1.5% or 5% cellulose powder in lyophilize potato residues (5%) obtains as the carbon source cultivation solution mould TN strain of fiber top spore, add yeast Saccharomyces cerevisiae IR-2 strain and ferment.In this fermentation, with OD 600yeast is added into after in aforementioned saccharified liquid by the mode that value reaches 0.1,30 DEG C, carry out cultivating for 24 hours under stirring velocity 120 rpm.Supernatant after cultivating for gained, measures ethanol receipts amount (Fig. 7).The mensuration of ethanol uses the Japanese light splitting society highly effective liquid phase chromatographic system (detector: Differential refractometer 2031Plus) being provided with Aminex HPX-87H (BioRad society) post to carry out.
Glucose in result saccharified liquid is promptly consumed and changes ethanol into, and its receipts amount reaches 97%(Fig. 7 relative to theoretical value, " only potato residues ").Therefore, show in the saccharified liquid of the method gained not containing the material causing fermentation to hinder.
Industrial applicability
Method of the present invention realizes efficient utilization by carrying out enzyme decomposition to the raw material deriving from potato tuber, and can be used for when it is waste processing it.And then, changing by fermenting to the sugar obtained by this saccharification, the manufacture of novel liquid fuel can also be implemented.Utilize the decomposition saccharification technology deriving from the raw material of potato of the present invention can be widely used in effective utilization of its raw material, its for waste time its process in.The saccharides obtained by this technology is in addition useful for manufacturing liquid fuel efficiently.
PCT/RO/134 shows

Claims (10)

1. the manufacture method of saccharides, is characterized in that, is processed by the raw material deriving from potato tuber with the Secretases liquid of the cell walls high de-agglomeration filamentous fungus containing lytic enzyme.
2. method according to claim 1, wherein, the raw material deriving from potato tuber is potato residues.
3. the method described in claim 1 or 2, wherein, cell walls high de-agglomeration filamentous fungus is top spore mould genus filamentous fungus or Penicillium filamentous fungus.
4. the method according to any one of claims 1 to 3, wherein, cell walls high de-agglomeration filamentous fungus separates fiber top spore mould (Acremonium cellulolyticus).
5. the method according to any one of claims 1 to 3, wherein, cell walls high de-agglomeration filamentous fungus separates the mould TN strain of fiber top spore (deposit number FERM BP-11452) or Penicillium notatum NBRC 101300 strain.
6. the method according to any one of Claims 1 to 5, wherein, the generation of induced hydrolysis enzyme with the aforementioned filamentous fungus of the culture medium culturing containing the raw material deriving from potato tuber, prepares aforementioned Secretases liquid thus, and uses it for the process of the raw material deriving from potato tuber.
7. the manufacture method of alcohol, is characterized in that, carries out alcohol fermentation to the saccharides obtained by the method according to any one of claim 1 ~ 6.
8. method according to claim 7, wherein, uses Saccharomycodes yeast to carry out alcohol fermentation.
9. the method described in claim 7 or 8, it is the manufacture method using Saccharomycodes yeast aforementioned saccharides to be carried out to the ethanol of ethanol fermentation.
10. the method according to any one of claim 7 ~ 9, wherein, carries out front cultivation with the substratum of saccharides containing the raw material deriving from potato tuber to organism of fermentation, and carries out alcohol fermentation with it.
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