WO2013125336A1

WO2013125336A1 - Method for saccharification of potato starting material and method for producing liquid fuel

Info

Publication number: WO2013125336A1
Application number: PCT/JP2013/052485
Authority: WO
Inventors: 旻天高; 矢野　伸一; 井上　宏之; 欣也坂西; 龍彦中田; 恭一千葉; 山本　淳一; 正史岡田
Original assignee: 独立行政法人産業技術総合研究所; 双日株式会社; 日立造船株式会社
Priority date: 2012-02-23
Filing date: 2013-02-04
Publication date: 2013-08-29
Also published as: JP5843264B2; CN104981546A; CN104981546B; JP2013172645A

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

Method for saccharification of potato raw material and method for producing liquid fuel

The present invention relates to a method for saccharifying potato raw material using an enzyme and a method for producing a liquid fuel based on the method.

Since liquid fuel such as ethanol produced from biomass can replace petroleum and contribute to the reduction of greenhouse gas emissions, its production and use are progressing worldwide.

Potato tubers are mainly composed of starch other than moisture, and it is relatively easy to produce a liquid fuel such as ethanol by saccharifying this starch with an amylase enzyme and fermenting it. However, potato tubers contain components such as cellulose and pectin in addition to starch, and it is difficult to decompose these components only with amylase used for starch degradation. In particular, in the residue obtained by extracting starch from potato tubers (also called potato pulp or potato starch extraction residue), the content ratio of cellulose, pectin, etc. is relatively increased, and the starch remaining in the cell wall containing these as components Utilization has become difficult, and this is a factor that limits the utilization of the residue. Non-patent document 1 describes that an enzyme secreted from Rhizopus oryzae decomposes starch in potato pulp to produce lactic acid, but the cell wall resolution of Rhizopus oryzae is low (Non-patent document 1). Table 2), and the yield of sugar is reduced by lactic acid production, so there is still room for improvement in the method for saccharification of starch inside the cell wall using Rhizopus oryzae. In addition, it has been reported that the use of a plurality of enzymes is necessary for the decomposition of potato pulp, and ideally the use of three kinds of enzymes is desirable (Non-patent Document 2). However, the use of a plurality of enzymes is a cause of cost increase, and a simpler and lower cost saccharification technique is required.

An object of the present invention is to provide a method for efficiently saccharifying a potato tuber-derived raw material and a method for producing a liquid fuel using the potato tuber-derived raw material.

As a result of intensive studies to solve the above problems, the present inventors have produced filamentous fungi that are effective in efficiently saccharifying potato-derived raw materials, particularly potato tuber-derived raw materials such as potato pulp. As a result, the present invention has been completed.

That is, the present invention includes the following.

[1] A method for producing a saccharified product, comprising treating a raw material derived from potato tuber with a secretory enzyme solution of a cell wall highly degradable filamentous fungus containing a hydrolase.

[2] The method according to [1] above, wherein the raw material derived from the potato tuber is potato pulp.

[3] The method according to [1] or [2] above, wherein the cell wall highly degradable filamentous fungus is Acremonium filamentous fungus or Penicillium sp.

[4] The method according to any one of [1] to [3] above, wherein the filamentous cell wall highly degradable fungus is Acremonium cellulolyticus.

[5] The method according to any one of [1] to [3] above, wherein the cell wall highly degradable filamentous fungus is Acremonium cellulolyticus TN strain (Accession No. FERM BP-11452) or Penicillium sp. NBRC 101300 strain .

[6] The secretory enzyme solution is prepared by culturing the filamentous fungus in a medium containing a raw material derived from potato tuber and inducing the production of hydrolase, and used for the treatment of the raw material derived from potato tuber, One of the methods from 1] to [5].

[7] A method for producing alcohol, comprising subjecting a saccharified product obtained by the methods [1] to [6] above to alcohol fermentation.

[8] The method according to [7] above, wherein alcohol fermentation is carried out using Saccharomyces yeast.

[9] The method according to [7] or [8] above, which is a method for producing ethanol, wherein the saccharified product is ethanol-fermented using Saccharomyces yeast.

[10] The method according to [7] to [9] above, wherein the fermentation microorganism is pre-cultured in a medium containing a saccharified material derived from potato tuber and alcohol fermentation is performed using the microorganism.

This specification includes the contents of Japanese Patent Application No. 2012-037404, which is the basis for the priority claim of the present application.

According to the method of the present invention, the potato tuber-derived raw material can be efficiently hydrolyzed and saccharified.

FIG. 1 is a graph showing the sugar yield obtained by saccharification of potato pulp with a commercially available cellulase enzyme. A shows glucose and B shows the yield (g / L) of galactose. FIG. 2 is a graph showing cellulase production by Acremonium cellulolyticus using powdered cellulose or potato pulp as a carbon source. FIG. 3 shows the effect of adding powdered cellulose on cellulase enzyme production. "Powdered cellulose" is a medium containing 5% powdered cellulose, "Potato pulp only" is a medium containing 5% potato pulp (powdered cellulose added 0%), "Potato pulp + 0.5%", "Potato pulp + 1.5%" “Potato pulp + 5%” indicates the results of culture using a medium containing 5% potato pulp + powdered cellulose added: 0.5%, 1.5%, 5%, respectively. FIG. 4 is a diagram showing the sugar yield by saccharification using an enzyme produced by Acremonium cellulolyticus. A: Amount of glucose (g / L), B: Amount of galactose (g / L). Each graph shows, from the left, an acremonium cellulase preparation (commercially available cellulase preparation), a powdered cellulose-derived enzyme solution (enzyme solution in which production was induced using powdered cellulose as a carbon source), and a potato pulp-derived enzyme solution (potato pulp as a carbon source) Enzyme solution whose production was induced using The white bar shows the result of saccharification for 24 hours, and the black bar shows the result of saccharification for 48 hours. FIG. 5 is a diagram showing the sugar yield by saccharification of potato pulp using an enzyme solution obtained by culturing three types of filamentous fungi using potato pulp. A: Amount of glucose (g / L), B: Amount of galactose (g / L). In the figure, diamonds indicate the results of Acremonium genus (Acremonium cellulolyticus TN strain), squares indicate Trichoderma genus (Trichoderma cho reesei RUT C-30), and triangles indicate the results of Penicillium genus (Penicillium sp. NBRC 101300). FIG. 6 is a diagram showing the growth level of yeast using a potato saccharified solution as a nutrient source for culture. The percentage described in each sample name indicates the concentration of each component in the medium. FIG. 7 is a diagram showing the concentration of ethanol obtained by saccharifying and fermenting potato pulp using an enzyme solution obtained by culturing filamentous fungi using potato pulp and cellulose as a carbon source.

Hereinafter, the present invention will be described in detail.

In the method of the present invention, a potato tuber-derived material is used as a material. The potato tuber-derived raw material refers to a potato tuber or a processed product thereof that has been processed into a state suitable for enzyme treatment. The potato tuber-derived raw material is not limited, but, for example, in addition to the form of the potato tuber as it is, any form such as slice, random cut, square cut, crushed, crushed, pulverized, or pulverized suspension The skin and buds may or may not be removed, but those containing the potato tuber-containing components (particularly starch, cellulose, etc.) as much as possible are preferred. The potato tuber-derived raw material may also be a residue generated when the potato tuber is processed. For example, potato pulp is particularly suitable. Potato pulp means a product remaining as a residue after separation of potato starch when potato starch is extracted from potato tubers. Potato pulp is also called potato extraction residue. The potato tuber-derived raw material is the above-mentioned potato tuber or processed product thereof, which is processed into a state suitable for enzyme treatment, for example, a residue generated when processing potato tubers is mixed with other biomass raw materials It may be a thing. The raw material from the potato tuber may also be in a raw state or in a heated state. The potato tuber-derived material may be in a frozen state, in a dried state, or in a lyophilized product. The potato tuber-derived material may be sterilized. As an example, the potato tuber-derived material may be autoclaved at 121 ° C. for about 10 to 30 minutes, which is usually used for sterilization treatment in microorganism culture. This is a useful treatment because the raw material is sterilized, and it can be expected to prevent the contamination of germs in the subsequent saccharification / fermentation process and increase the degradability of the raw material.

In the present invention, a potato tuber generally refers to a potato underground stem portion used for food. The potato used in the present invention may be of any varieties or variants thereof, or may be a wild species.

The filamentous fungus used in the present invention is a fungus having the ability to produce and secrete a plurality of biomass-degrading enzymes, and at least one of the secreted enzymes comprises starch, cellulose, pectin, etc., which are the main components of potato tubers It is preferable that the fungus has the ability to hydrolyze. The filamentous fungus used in the present invention is preferably a cell wall highly degradable filamentous fungus. In the present invention, the cell wall highly degradable filamentous fungus can efficiently decompose components constituting the cell wall such as cellulose and pectin, and liquefy potato tuber-derived raw materials (potato pulp, etc.) containing these cell wall components. A filamentous fungus that can be degraded to For example, filamentous fungi belonging to the genus Acremonium or Penicillium, which are anamorphs, can be mentioned as suitable filamentous fungi used in the present invention. A preferable example of the filamentous fungus used in the present invention includes Acremonium cellulolyticus. Particularly preferred examples of the filamentous fungus used in the present invention are Acremonium cellulolyticus TN strain and Penicillium sp. NBRC 101300 strain.

Various filamentous fungi can be obtained from commercial products, or from depository institutions based on the Culture Collection or the Budapest Treaty.

Acremonium® cellulolyticus® TN strain was issued on January 12, 2012 by the National Institute of Technology and Evaluation (NITE-IPOD), postal code 305-8566, 1-chome, 1-chome, East 1-chome, Tsukuba, Ibaraki, Japan. ) Under the accession number FERM BP-11452 under the Budapest Treaty.

Penicillium sp. NBRC は 101300 is a catalog of NBRC (NITE Biological 日本 Resource Center) (Japan) of the National Institute of Technology and Evaluation (NITE; 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture) [NBRC Catalog of Biological Resources, Microorganisms, Microorganism-Related DNA Resources, Human-Related DNA Resources, Second Edition (2010)] and available from NBRC under the number 101300.

In the method of the present invention, a secretory enzyme solution containing a hydrolase produced by the filamentous fungus is used for processing the raw material derived from potato tubers. In the present invention, the secretory enzyme solution of filamentous fungi refers to a solution (enzyme solution) containing a secretory enzyme group produced by culturing filamentous fungi and secreted into the medium. This secretory enzyme solution of filamentous fungi contains one or more, preferably several, enzymes having the ability to degrade polysaccharides contained in potato such as starch, cellulose, pectin, etc., and preferably contains at least cellulase. . The secretory enzyme solution of the filamentous fungus according to the present invention typically contains cellulase (cellulose hydrolase), pectinase (pectin hydrolase), and amylase (starch hydrolase). The secretory enzyme solution of the filamentous fungus according to the present invention preferably further contains a galactan degrading enzyme. In the present specification, a secretory enzyme solution containing a plurality of types of enzymes may be expressed as an enzyme system. These enzymes are produced intracellularly by filamentous fungi and secreted extracellularly after production. This secreted enzyme solution may be a filamentous fungus culture or a culture supernatant. The filamentous fungus secretory enzyme solution is obtained by culturing one or more (preferably one) filamentous fungi in the presence of a carbon source to induce hydrolase production, and secreting the enzyme into the culture solution. Can be prepared. A medium for culturing filamentous fungi can be prepared by adding a carbon source or the like to any culture medium suitable for culturing filamentous fungi. Arbitrary organic substances can be used as a carbon source in the medium necessary for culturing filamentous fungi, but potato tuber-derived raw materials (for example, potato pulp) or enzyme degradation products (for example, saccharified products) are used as the carbon source. It is more preferable to use it for inducing the production of hydrolases such as cellulase, pectinase and amylase. In the latter case, it is possible to produce an enzyme that is excellent in degradability with respect to components (starch, cellulose, pectin, etc.) that are particularly abundant in potato tubers as compared with the case of using other carbon sources. For this reason, it is preferable that the culture medium which culture | cultivates a filamentous fungus contains at least 1 (preferably all) among starch, a cellulose, and pectin as a carbon source. The medium for culturing the filamentous fungus is not only potato tuber-derived raw material (for example, potato pulp), but also its enzymatic degradation product (for example, saccharified product) or a carbon source other than the potato tuber-derived raw material (for example, purified cellulose such as powdered cellulose or purified It is also preferable to contain starch and the like. The carbon source other than the potato tuber-derived material can be added to the potato tuber-derived material at a concentration of, for example, 0.5% or more, preferably 1.5% or more, more preferably 5% or more, for example, 0.5 to 30%. By adding other carbon sources to the medium in addition to the potato tuber-derived material, the production of hydrolases such as cellulase, pectinase and amylase can be more effectively induced.

The secretory enzyme solution of the filamentous fungus may be used after being collected, separated or purified from the culture solution of such a filamentous fungus (for example, in the form of a culture supernatant) or without being collected, separated or purified. The culture solution can be used as it is for the processing of the potato tuber-derived material. An industrially produced and commercially available product (for example, Acremonium cellulase (manufactured by Meiji Seika Pharma Co., Ltd.)) may be used as the secretory enzyme solution for filamentous fungi.

In the method of the present invention, the enzyme treatment of the potato tuber-derived material can be performed by adding and reacting the secretory enzyme solution of the filamentous fungus to the solution or liquid medium containing the potato tuber-derived material or the potato tuber-derived material. it can.

Alternatively, in the method of the present invention, the filamentous fungus of the present invention is cultured in a liquid medium containing a potato tuber-derived raw material or a potato tuber-derived raw material to induce the production of a hydrolase such as cellulase and secreted into the medium. A potato tuber-derived raw material may be subjected to an enzyme treatment by adding a potato tuber-derived raw material to the secreted enzyme solution and reacting it.

The enzyme treatment is preferably performed at the optimum temperature and pH of each enzyme. For example, in the case of an enzyme system produced by Acremonium cellulolyticus, 40 to 60 ° C., about pH 4 to 6, preferably 45 to 55 ° C. It is suitable to be carried out at pH 4.5 to 5.5.

In the enzyme treatment, the potato tuber-derived material (preferably potato pulp) is 5% or more, preferably 7% or more, more preferably 10% or more, still more preferably 20% or more, still more preferably 30% or more, for example 50 It is preferable to use a solution or a medium containing a concentration of not less than 100% and not more than 100%, for example, 5 to 50%.

By the enzyme reaction as described above, polysaccharides (cellulose, starch, pectin, etc.) constituting potato tubers are hydrolyzed to produce monosaccharides or oligosaccharides (eg, glucose, galactose, maltose, cellobiose, etc.) ( Saccharification reaction). The present invention also provides a method for producing a saccharified product from such potato tuber-derived materials. The present invention also provides a method for saccharification of a potato tuber-derived raw material by such a saccharification reaction.

Thus, the saccharified product (typically, saccharified solution) obtained by saccharifying the potato tuber-derived raw material can be converted into various useful substances by fermentation. In particular, alcohol can be produced from monosaccharides or oligosaccharides in a saccharified product by alcohol fermentation. The alcohol obtained can be used, for example, as a liquid fuel or industrial material.

Alcohol fermentation can be performed using fermentation microorganisms that perform alcohol fermentation, such as yeast. Examples of the yeast include, but are not limited to, for example, Saccharomyces spp. Schizosaccharomyces, Kluyveromyces marxianus and other Kluyveromyces, Isatchenkia orientalis, etc.

For example, by using Saccharomyces yeast having ethanol fermentation ability as yeast, the saccharified product can be ethanol-fermented. In particular, Saccharomyces cerevisiae is preferable, and ethanol can be produced (manufactured) from monosaccharides such as glucose by adding it to a saccharified product and culturing. This ethanol can be used as a liquid fuel that can replace gasoline. Fermentation experiments with Saccharomyces cerevisiae show that the saccharified potato tuber-derived raw material does not show an inhibitory effect on yeast and can be used as a good fermentation raw material. In the present invention, “fermenting microorganism” refers to a microorganism having the ability to perform fermentation.

The above fermentation is not limited to alcohol fermentation in which ethanol is produced using Saccharomyces cerevisiae, and other alcohols such as butanol can be produced according to the fermentation microorganism used. Such various alcohols can also be used for liquid fuel and the like.

By fermenting a saccharified product obtained by saccharifying a raw material derived from potato tubers, useful substances that can be liquid fuel components such as substances other than alcohol, such as fatty acids, can also be produced. This fermentation is achieved by using the corresponding fermenting microorganism.

It is preferable to perform a step (generally referred to as pre-culture) in which the fermenting microorganisms are grown and prepared to a certain amount before being used for fermentation of a saccharified product. In the method of the present invention, it is preferable to pre-culture fermented microorganisms in a medium containing a potato tuber-derived raw material saccharified product, and to perform fermentation (preferably alcoholic fermentation) of the saccharified product using it. Fermentation may be performed under normal fermentation conditions of the fermenting microorganism. For example, the fermentation may be performed under anaerobic conditions or aerobic conditions. In the case of Saccharomyces cerevisiae, it is preferably carried out under anaerobic fermentation conditions.

In the pre-culture in the present invention, a medium containing a saccharified product of potato tuber-derived raw material, for example, a saccharified solution of a potato tuber-derived raw material itself may be used, or a saccharified product of a potato tuber-derived raw material may be used as a culture medium or the like It may be prepared in addition to the medium. By using the saccharified material derived from the potato tuber, better growth of the microorganisms can be obtained as compared with the case where waste molasses, which is a commonly used nutrient source, is used. Thus, alcohol fermentation can be performed very efficiently by using the microorganisms pre-cultured with the saccharified material derived from the potato tuber for the fermentation of the saccharified product as the main culture. In addition, the microorganisms pre-cultured with the saccharified material derived from the potato tuber can be widely used not only in fermentation of the saccharified material derived from the potato tuber but also in fermentation using other various materials.

Conventionally, for potato tuber-derived raw materials, particularly potato pulp, etc., there has been no technology capable of comprehensively and efficiently decomposing and saccharifying the ingredients, which has been an obstacle to their use. In the method of the present invention, by using an enzyme system derived from a filamentous fungus, the main components of cellulose, starch and pectin contained in the raw material derived from potato tubers can be efficiently decomposed, and as a result, can be converted into liquid fuel by fermentation. Monosaccharides can be obtained efficiently. The method of the present invention makes it possible to easily decompose the raw materials derived from potato tubers, which has been difficult in the past, and to produce liquid fuels including alcohol by fermenting them. Thus, it is possible to provide an effective utilization method of biomass raw materials that have been low in utilization.

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[Example 1]
(1) Analysis of the basic components of potato starch squeezed potato We obtained freeze-dried potato pulp (potato starch squeezed potato) collected at the starch factory of Kita Ara potato Group Co., Ltd. used.

Therefore, first, the component composition of the potato pulp used was analyzed by a conventional method. The results are shown in Table 1.

The lower part of Table 1 shows the monosaccharide composition after complete degradation of the polysaccharide in the potato pulp. This monosaccharide composition is an actual measurement value obtained by completely decomposing potato pulp with sulfuric acid. Since glucose is mostly produced from the degradation of starch and cellulose, the total amount of starch and cellulose is approximately consistent with the glucose content in the monosaccharide composition.

(2) Saccharification using enzyme Water was added to the freeze-dried potato pulp (sample) of (1) above to adjust the concentration to 10%, 20%, and 30% (each w / v). To this was added commercially available cellulase preparation Acremonium cellulase (manufactured by Meiji Seika Pharma Co., Ltd .; cellulase produced by Acremonium cellulolyticus) at a ratio of 10 Filter Paper Unit (FPU; filter paper degradation activity) / g substrate, and 0.05M citric acid The reaction was performed in a buffer at 50 ° C. for 24 hours or 48 hours, and the amounts of produced glucose and galactose were measured. Glucose and galactose are measured using a high-performance liquid chromatography system (detector: differential refractometer 2031Plus) manufactured by JASCO Corporation equipped with an Aminex HPX-87H (BioRad) column. did. The result is shown in FIG. As shown in FIG. 1, the production amounts of glucose and galactose increased depending on the concentration of the sample used. The potato pulp that was initially gelled by enzymatic treatment was decomposed and liquefied, producing glucose and galactose monosaccharides.

On the other hand, when Accelerase 1000 (Genencore; Trichoderma reesei) was similarly used for the enzymatic treatment of the potato pulp of (1), the residue was not liquefied even at 10% concentration. No decomposition was observed.

This result shows that cellulase produced by Acremonium spp. Is particularly suitable for saccharification of potatoes, particularly potato-derived raw materials.

[Example 2]
(1) Enzyme production using potato pulp Cellulase-producing bacteria Acremonium cellulolyticus TN strain (accession number: FERM BP-11452) was cultured using potato pulp as a carbon source to produce an enzyme. As a control, culturing was also performed using powdered cellulose, which is usually used as a carbon source for cellulase production, as a carbon source instead of potato pulp.

The composition of the medium used for the culture is as follows: 24 g / L KH ₂ PO ₄ , 1 g / L Tween 80, 5 g / L (NH ₄ ) ₂ SO ₄ , 1.2 g / L MgSO ₄ .7H ₂ O , 0.01 g / L ZnSO ₄・ 7H ₂ O, 0.01 g / L MnSO ₄・ 6H ₂ O, 0.01 g / L CuSO ₄ 7H ₂ O, 4 g / L urea, 4.7 g / L potassium tartrate monohydrate, And powdered cellulose (Solka Floc®; control) or lyophilized potato pulp (5%, 7%, 10%) as carbon source.

Cultivation was performed at a reaction temperature of 30 ° C. and a stirring speed of 200 rpm. Pre-culture was performed using powdered cellulose as a carbon source, and after 72 hours from the start of pre-culture, the pre-culture was added to the medium so as to be 5%, and main culture was performed.

The medium after 5 days, 7 days, and 14 days after the start of the main culture was collected, and the activity of enzyme cellulase secreted into the medium was measured. Cellulase activity was measured using a filter paper (Whatman No. 1) as a substrate, reacted in 50 mM mM citrate buffer (pH 5.0) for 60 minutes at 50 ° C, and the amount of produced reducing sugar was colorimetrically determined by the dinitrosalicylic acid method. The activity was expressed in FPU (FIG. 2).

As shown in FIG. 2, an enzyme having cellulase activity could be produced by culturing with potato pulp as a carbon source, although the concentration of activity was lower than when powdered cellulose was used as the carbon source.

Furthermore, culture of Acremonium cellulolyticus TN (A. cellulolyticus) TN strain as described above using lyophilized potato pulp (5%) added with 0.5%, 1.5%, or 5% powdered cellulose as the above, Was produced. When the cellulase activity (FPU activity) was measured in the same manner as described above for the cultured medium, an improvement in FPU activity according to the amount of cellulose added to the potato pulp was observed (FIG. 3).

(2) Saccharification of potato pulp by enzyme produced using potato pulp as carbon source Culture supernatant of culture obtained by culturing A. cellulolyticus TN strain using potato pulp as carbon source in (1) of this example Was collected as an enzyme solution, and this was ultrafiltered and concentrated using a tube with an ultrafiltration membrane (VIVASPIN 6, Sartorius). This was added to 30% (w / v) potato pulp at a rate of 5 FPU / g, and saccharification was carried out for 24 hours or 48 hours in the same manner as in Example 1- (2).

Similarly, the culture supernatant of the culture obtained by culturing A. cellulolyticus TN strain using powdered cellulose as a carbon source in the above (1) of this example was collected as an enzyme solution, and the reaction conditions were the same as above. A saccharification test was performed. As a control, a saccharification test using the commercially available cellulase preparation Acremonium cellulase used in Example 1 was also carried out under the same reaction conditions.

Next, the amounts of glucose and galactose were measured in the same manner as in Example 1 for the obtained saccharified solution.

The above results are shown in FIG. As shown in FIG. 4, glucose and galactose could be produced from potato pulp using an enzyme solution produced by A.ulocellulolyticus TN strain using potato pulp as a carbon source. Glucose and galactose could also be produced from potato pulp when an enzyme solution produced using powdered cellulose as a carbon source or a commercially available cellulase preparation Acremonium cellulase was used. In particular, when an enzyme solution produced using potato pulp as a carbon source was used, a sugar yield higher than that obtained using an enzyme solution produced using powdered cellulose as a carbon source or the commercially available cellulase preparation Acremonium cellulase could be obtained. For galactose, a significantly higher yield was obtained with the enzyme solution obtained using potato pulp as the carbon source than the enzyme solution produced using powdered cellulose as the carbon source. From this, when potato pulp is used as a carbon source, it is considered that the production of galactan-degrading enzyme was induced and the resulting secretory enzyme solution contained galactan-degrading enzyme.

[Example 3]
Instead of the A. cellulolyticus TN strain, the fungus Penicillium sp. NBRC 101300 strain (NBRC Catalog of Biological Resources, Microorganisms, Microorganism-Related DNA Resources, Human- Related DNA Resources, Second Edition (2010)], obtained from NBRC), and Trichoderma reesei RUT C-30 strain (the American Type Culture Collection (ATCC) no. 56765)) as in Example 2. Under these conditions, potato pulp was cultured as a carbon source, and the resulting enzyme solution was used to saccharify the potato pulp. The result of having measured the quantity of glucose and galactose about a saccharified liquid is shown in FIG.

As a result, it was shown that all three fungi produced glucose and galactose. As shown in FIG. 5, the amount of glucose in the case of using an A. 液 cellulolyticus-derived enzyme solution was the highest, indicating that it is particularly suitable for saccharification. However, for the enzyme solution derived from Penicillium sp., About 75% of the amount of glucose from the enzyme solution derived from A. cellulolyticus was obtained, and the amount of galactose was higher than that of A. cellulolyticus. This bacterium was also shown to be suitable for saccharification. These fungi are particularly useful for producing enzymes for saccharification of potato pulp.

In the enzyme solution derived from T. reesei, which is generally used industrially as a cellulase-producing bacterium, it takes 40 hours or more for the residue to liquefy and start saccharification, and the reaction is A. cellulolyticus TN strain and Penicillium sp. It was slower than NBRC 101300 shares.

[Example 4]
(1) Pre-culture of fermented microorganisms with potato saccharified liquid It was tested whether potato saccharified liquid can be used as a nutrient source for pre-culture to increase the number of microorganisms used for fermentation of saccharified liquid to a certain amount.

Deposited on April 8, 1985, in the yeast Saccharomyces cerevisiae IR-2 strain (Institute for Product Evaluation and Technology, Japan Patent Biological Deposit Center (postal code 305-8566, 1st, 1st East, 1-chome, Tsukuba City, Ibaraki Prefecture, Japan)) Incubate overnight with YPD medium (1% yeast extract, 1% polypeptone, 2% glucose) with an OD ₆₀₀ value (Optical density: an indicator of cell number by absorbance) of 0.1. The culture experiment was conducted at 30 ° C. and a stirring speed of 120 rpm.

The medium is based on YPD medium, and uses saccharified liquid (potato saccharified liquid) obtained by treating potato pulp with commercially available enzyme acremonium cellulase in Example 1- (2), and sugar beet waste molasses obtained in China. Prepared. As a control medium, YPD medium was used. The culture time was 6 hours, 18 hours, or 24 hours.

Generally, molasses is used as a suitable nutrient source for yeast culture, but by using a potato saccharified solution, a yeast growth amount exceeding the molasses was obtained (FIG. 6).

This result indicates that the saccharified material derived from the potato tuber is excellent not only as a carbon source but also as a comprehensive nutrient source including nitrogen and other inorganic components.

(2) Ethanol fermentation experiment In Example 2- (1), A. cellulolyticus TN strain was cultured using lyophilized potato pulp (5%) added with 0.5%, 1.5%, or 5% powdered cellulose as a carbon source. The yeast Saccharomyces cerevisiae IR-2 strain was added to the enzyme solution thus obtained for fermentation. In this fermentation, yeast was added to the saccharified solution so that the OD ₆₀₀ value was 0.1, and then cultured at 30 ° C. and a stirring speed of 120 rpm for 24 hours. The ethanol yield of the obtained post-culture supernatant was measured (FIG. 7). The measurement of ethanol was performed using a high-performance liquid chromatography system (detector: differential refractometer 2031Plus) manufactured by JASCO Corporation equipped with an Aminex HPX-87H (BioRad) column.

As a result, glucose in the saccharified liquid was rapidly consumed and converted to ethanol, and the yield reached 97% of the theoretical value (FIG. 7, “potato pulp only”). Therefore, it was shown that the saccharified solution obtained by this method does not contain substances that cause fermentation inhibition.

The method of the present invention can be efficiently used by enzymatic decomposition of a potato tuber-derived raw material, and can be used to treat it when it is waste. Furthermore, a novel liquid fuel can be produced by converting the sugar obtained by this saccharification by fermentation. The technology for decomposing and saccharifying potato-derived raw materials according to the present invention can be widely applied to the effective use of the raw materials and, when it is waste, the processing thereof. In addition, the saccharified product obtained by this technique is useful for efficiently producing a liquid fuel.

Claims

A method for producing a saccharified product, comprising treating a potato tuber-derived material with a secretory enzyme solution of a cell wall highly degradable filamentous fungus containing a hydrolase.
The method according to claim 1, wherein the potato tuber-derived material is potato pulp.
The method according to claim 1 or 2, wherein the cell wall highly degradable filamentous fungus is Acremonium filamentous fungus or Penicillium sp.
The method according to any one of claims 1 to 3, wherein the cell wall highly degradable filamentous fungus is Acremonium cellulolyticus.
The method according to any one of claims 1 to 3, wherein the cell wall highly degradable filamentous fungus is Acremonium cellulolyticus TN strain (Accession No. FERM BP-11452) or Penicillium sp. NBRC 101300 strain.
The secretory enzyme solution is prepared by culturing the filamentous fungus in a medium containing a potato tuber-derived material and inducing the production of hydrolase, and is used for the treatment of the potato tuber-derived material. The method of any one of these.
A method for producing alcohol, comprising subjecting the saccharified product obtained by the method according to any one of claims 1 to 6 to alcohol fermentation.
The method according to claim 7, wherein alcohol fermentation is performed using Saccharomyces yeast.
The method according to claim 7 or 8, which is a method for producing ethanol, wherein the saccharified product is subjected to ethanol fermentation using Saccharomyces yeast.
The method according to any one of claims 7 to 9, wherein the fermenting microorganism is pre-cultured in a medium containing a saccharified product derived from a potato tuber, and alcohol fermentation is performed using the microorganism.