JP7460108B2 - Methods for producing fermented products, methods for producing ethanol, methods for producing lactic acid fermented products, and fermentation promoters - Google Patents

Description

(1) Wood Science Information, Vol. 25, Supplementary Issue 1 (Publication date: November 2, 2018) (2) The 25th Japan Wood Research Society Kyushu Branch Conference (Fukuoka) (Held: November 16, 2018) (3) Fukuoka University Engineering Bulletin, No. 101 and 102 (Publication date: March 20, 2019) (4) Fukuoka University New Technology Briefing (Held: May 21, 2019) (5) https://www. jst. go. jp/tt/fair/ (Website publication date: June 18, 2019) (6) https://www. ij2019. jp/exhibitor/jss20190406. html (Website publication date: July 18, 2019) (7) Innovation Japan 2019 (Held: August 29-30, 2019) (8) AgriBio September 2019 issue (Published: September 20, 2019) (9) Kyushu-Yamaguchi Venture Market 2019 University Joint New Technology Briefing (Held: October 7, 2019)

The present invention relates to a method for producing a fermented product. In particular, the present invention relates to a method for producing ethanol by alcoholic fermentation using yeast. The present invention also relates to a method for producing a lactic acid fermented product by lactic acid fermentation using lactic acid bacteria. The present invention also relates to a fermentation promoter that promotes alcoholic fermentation, lactic acid fermentation, etc.

Microbial fermentation is used to produce food, fuel, chemicals, polymers, and other materials.

Conventionally, alcoholic fermentation using yeast has been used to produce sake, beer, etc. As a method for promoting alcohol fermentation, for example, Patent Document 1 discloses an alcohol fermentation promoter comprising an adsorbent in which volatile components of the peel or fruit of Japanese pepper are adsorbed onto an ion exchange resin or gel filtration material. is disclosed.

On the other hand, in recent years, ethanol has attracted attention as an alternative fuel to petroleum, and methods of producing ethanol from biomass raw materials such as cellulosic biomass using yeast are being considered. When producing ethanol from cellulosic biomass, it can be broadly divided into two steps: a saccharification step in which cellulosic biomass is hydrolyzed into glucose, and a fermentation step in which ethanol is obtained from glucose. Until now, various studies have been conducted on pretreatment to remove lignin, a fermentation inhibitor contained in cellulosic biomass during the saccharification step, and hydrolysis techniques that do not produce furfural, a fermentation inhibitor. .

From the viewpoint of production efficiency, simultaneous saccharification and fermentation (SSF), in which the saccharification and fermentation steps are carried out at the same time, is being considered. This method makes it possible to carry out saccharification and fermentation in a single reactor. Furthermore, because glucose, the saccharification product, is also immediately consumed in ethanol fermentation, product inhibition of enzymes such as cellulase can be suppressed. For this reason, it is thought that this method has better ethanol production efficiency than the separate method in which the saccharification and fermentation steps are carried out separately.

In the case of simultaneous saccharification and fermentation, the optimum conditions for the enzymes used in saccharification are usually acidic at about 50°C, while the optimum conditions for the yeast used in fermentation are usually neutral at about 30°C, which is the optimum condition for both steps. Suitable conditions vary widely. In simultaneous saccharification and fermentation, it is necessary to match the conditions for either saccharification or fermentation, and there is a problem that the activity of the other decreases. Therefore, research is being conducted to improve the high temperature tolerance of yeasts and fungi through genetic modification and the like. Furthermore, in order to improve the production of ethanol by simultaneous saccharification and fermentation, studies are being conducted to control conditions such as temperature and pH.

For example, in Patent Document 2, in a method for producing ethanol by simultaneous saccharification and fermentation using cellulosic raw materials, the pH value is adjusted to the target pH of the simultaneous saccharification and fermentation reaction at the stage of adjusting the raw materials for the simultaneous saccharification and fermentation reaction. A method for producing ethanol is disclosed, which is characterized in that the ethanol production method is preset to a value higher than the above value.
Furthermore, Patent Document 3 describes a simultaneous saccharification and fermentation method in which a saccharification process in which a cellulose raw material is hydrolyzed by enzymes and an ethanol fermentation process by fermentation bacteria are simultaneously carried out in one reaction tank. A simultaneous saccharification and fermentation method is disclosed, which is characterized in that the reaction temperature is lowered stepwise or continuously.

As with the production of ethanol from biomass raw materials, bioplastics are attracting attention from the perspective of environmental impact. Lactic acid can be obtained by lactic acid fermentation of glucose obtained by hydrolysis of cellulosic biomass. This lactic acid can also be used as a raw material for bioplastics.

For example, Patent Document 4 describes a method for producing D-lactic acid by fermentation production of D-lactic acid from woody biomass using D-lactic acid-producing bacteria, in which the fermentation by the D-lactic acid-producing bacteria is carried out in a medium containing cyanocobalamin. Disclosed is a method for producing D-lactic acid, which is characterized in that it is carried out in a.

Japanese Patent Application Publication No. 2013-123411 JP 2011-055715 A JP2010-246422A Japanese Patent Application Publication No. 2016-168030

As mentioned above, the production of ethanol from cellulosic biomass can be divided into a saccharification step and a fermentation step. In the fermentation step, it is possible that fermentation by yeast may be inhibited by components derived from unfractionated raw materials or by-products in the saccharification step. However, in the past, it was assumed that the saccharification step was more rate-limiting than the fermentation step, and that once glucose was produced, fermentation by yeast would naturally proceed. For this reason, additives and other methods have been considered to promote alcoholic fermentation of glucose, etc., but little research has been done on the fermentation step in the production of ethanol from cellulosic biomass.

The effects of additives such as fermentation reaction promoters may be reduced or may not be fully effective due to the effects of components that inhibit the fermentation reaction of yeast, and there is a demand for additives that are not affected by the presence of components that inhibit the fermentation reaction. However, the effects of components that inhibit fermentation by yeast have not been considered for the alcohol fermentation promoter in Patent Document 1.

Furthermore, improvements in high-temperature-resistant yeasts and fungi through genetic recombination during simultaneous saccharification and fermentation have been problematic in terms of environmental risks and costs. Also in the ethanol production methods described in Patent Documents 2 and 3, it was necessary to precisely control the temperature and pH.

Under such circumstances, an object of the present invention is to provide a method for producing ethanol using a new additive that can promote alcohol fermentation.
Another object of the present invention is to provide a method for producing ethanol that can efficiently produce ethanol using a simpler method. In particular, it is an object of the present invention to provide a method for producing ethanol that can efficiently produce ethanol from cellulosic biomass in a simpler manner.
Another object of the present invention is to provide a new alcohol fermentation promoter.

With regard to lactic acid fermentation, there was a demand for a method for efficiently producing lactic acid fermentation products, with a view to expanding its use.

Under such circumstances, an object of the present invention is to provide a method for producing a lactic acid fermentation product using a new additive capable of promoting lactic acid fermentation. Another object of the present invention is to provide a new lactic acid fermentation promoter.

The present invention aims to provide a method for producing a fermented product using a new additive. The present invention also aims to provide a new fermentation promoter.

As a result of extensive research in order to solve the above problems, the present inventors found that the following invention met the above objects, leading to the present invention.

That is, the present invention relates to the following inventions.
<1> A method for producing a fermented product, comprising fermenting sugars in the presence of a composition derived from Ephedra to obtain a fermented product.
<2> The method for producing a fermented product according to <1>, wherein the composition derived from Ephedra is an Ephedra extract.
<3> The method for producing a fermented product according to <1>, wherein the composition derived from Ephedra is an Ephedra extraction residue.
<4> An ethanol production method that utilizes the method for producing a fermented product according to any one of <1> to <3>, comprising an ethanol production step of producing ethanol by alcoholic fermentation of sugars with the yeast in the presence of the Ephedra-derived composition.
<5> The method for producing ethanol according to <4>, wherein the sugars are obtained by saccharifying cellulosic biomass.
<6> The method for producing ethanol according to <5>, wherein the ethanol production process is a saccharification and fermentation process that includes a saccharification treatment of saccharifying the cellulosic biomass with a saccharification enzyme and a fermentation treatment of alcoholically fermenting the sugars obtained by the saccharification treatment with the yeast to produce ethanol.
<7> The method for producing ethanol according to <6>, wherein in the saccharification and fermentation step, the saccharification treatment and the fermentation treatment are carried out at a temperature higher than an optimal temperature for the yeast.
<8> The method for producing ethanol according to <5>, further comprising a saccharification step of saccharifying the cellulosic biomass into sugars using a saccharification enzyme prior to the ethanol production step.
<9> The method for producing ethanol according to any one of <5> to <8>, wherein the cellulosic biomass has been pretreated to remove lignin.
<10> A method for producing a lactic acid fermented product using the method for producing a fermented product described in any one of <1> to <3>, the method including a lactic acid fermented product production step of lactic acid fermenting the sugars with lactic acid bacteria in the presence of the ephedra-derived composition to produce a lactic acid fermented product.
<11> A fermentation promoter comprising ephedra extract residue.

The present invention provides a method for producing a fermented product using a new additive that can promote fermentation. The present invention also provides a new fermentation promoter.

According to the present invention, a method for producing ethanol using a new additive capable of promoting alcohol fermentation is provided.
Furthermore, a method for producing ethanol is provided that can efficiently produce ethanol using a simpler method. According to the method for producing ethanol, ethanol can be produced efficiently from cellulosic biomass in a simpler manner.
Further, according to the present invention, a new alcohol fermentation promoter is provided.

Further, according to the present invention, a method for producing a lactic acid fermented product using a new additive capable of promoting lactic acid fermentation is provided. Further, the present invention provides a new lactic acid fermentation promoter.

FIG. 1 is a flow diagram showing an example of the method for producing ethanol of the present invention. FIG. 1 is a flow chart showing an example of a method for producing ethanol according to the present invention. FIG. 1 is a flow diagram showing an example of the method for producing ethanol of the present invention. FIG. 2 is a graph in which the glucose concentration [g/L] and the ethanol concentration [g/L] are plotted against the treatment time [h] in ethanol fermentation by simultaneous saccharification and fermentation in Experimental Example 1. FIG. 1 is a diagram showing a typical example of the relationship between the amount of carbon dioxide generated (y) and the fermentation time (t) in ethanol fermentation. FIG. 3 is a graph plotting the fermentation delay time t _s [h] of ethanol fermentation in Experimental Examples 2-1 to 2-4 against the solid component concentration [g/L]. This is a graph plotting the fermentation rate r [(g/L)/h] of the ethanol fermentation in Experimental Examples 2-1 to 2-4 against the solid component concentration [g/L]. This is a diagram in which the ethanol yield [%] after completion of ethanol fermentation in Experimental Examples 2-1 to 2-4 is plotted against the solid component concentration [g/L]. FIG. 3 is a graph plotting the fermentation rate r [(g/L)/h] of ethanol fermentation in Experimental Examples 3-1 and 3-2 against the furfural concentration [g/L]. FIG. 3 is a diagram plotting the ethanol concentration [g/L] about 6 days after fermentation in Experimental Examples 3-1 and 3-2 against the furfural concentration [g/L]. FIG. 3 is a diagram plotting the fermentation delay time t _s [h] of ethanol fermentation in Experimental Example 4 against the number of decoctions of Maoto residue. This is a graph plotting the fermentation rate r [(g/L)/h] of ethanol fermentation in Experimental Example 4 against the number of times the Maoto residue was boiled. It is a diagram showing the influence of each crude drug on the fermentation delay time, with α _s value, which is the effect of adding extract, on the horizontal axis, and β _s value, which is the effect of adding residue, on the vertical axis. This graph shows the effect of adding extract ( _αr value) on the horizontal axis and residue ( _βr value) on the vertical axis, showing the effect of adding each herb on the fermentation rate. It is a diagram showing the influence of each crude drug on the fermentation completion time, with α _e value, which is the effect of adding extract, on the horizontal axis, and β _e value, which is the effect of adding residue, on the vertical axis. The graph shows the effect of adding extract (α _EtOH value) on the horizontal axis and the effect of adding residue (β _EtOH value) on the vertical axis, showing the effect of each herb on the amount of ethanol produced. FIG. 1 is a plot of the fermentation start time (fermentation lag time) t _s [h], the fermentation rate r [(g/L)/h], and the fermentation end time t _e [h] in ethanol fermentation using blackstrap molasses. FIG. 2 is a diagram plotting the amount of organic acid produced [mol/kg] against the fermentation time [h] in lactic acid fermentation.

The embodiments of the present invention will be described in detail below, but the explanation of the constituent elements described below is an example (representative example) of the embodiments of the present invention, and the present invention will be described below unless the gist thereof is changed. is not limited to the content. Note that when the expression "~" is used in this specification, it is used as an expression including the numerical values before and after it.

1. Method for Producing a Fermented Product The present invention relates to a method for producing a fermented product, which comprises fermenting sugars in the presence of a composition derived from Ephedra to obtain a fermented product (hereinafter, may be referred to as the "method for producing the fermented product of the present invention").

One of the features of the method for producing a fermented product of the present invention is that fermentation is carried out in the presence of an Ephedra-derived composition. By carrying out fermentation in the presence of an Ephedra-derived composition, the activity of yeast and lactic acid bacteria can be increased, and the fermented product can be produced in a shorter time.

Specifically, the method for producing a fermented product of the present invention can be used for producing ethanol from sugars and for producing lactic acid fermentation products from sugars.

Hereinafter, a method for producing ethanol using the method for producing a fermented product of the present invention and a method for producing a lactic acid fermented product using the method for producing a fermented product according to the present invention will be exemplified. We will explain the ephedra-derived composition, sugars, fermentation conditions, etc. that can be used.

2. Bioethanol Production Method The present invention relates to an ethanol production method having an ethanol production step of producing ethanol by alcoholic fermentation of sugars with yeast in the presence of a composition derived from Ephedra (hereinafter, sometimes referred to as the "ethanol production method of the present invention").

One of the features of the ethanol production method of the present invention is that the ethanol production step is carried out in the presence of a composition derived from Ephedra.
By carrying out alcoholic fermentation in the presence of a composition derived from Ephedra, yeast activity can be increased, enabling ethanol to be produced in a shorter period of time.

Furthermore, even if the raw material used for alcoholic fermentation contains impurities such as furfural, which is a fermentation inhibitor, the ephedra-derived composition can exhibit the effect of enhancing the function of yeast.
When producing ethanol from biomass raw materials, ethanol can be produced by saccharifying the biomass raw materials to produce saccharides, and then subjecting the saccharides to alcoholic fermentation. In the production of ethanol from such biomass raw materials, furfural and the like are likely to be mixed into the biomass raw materials as impurities. Since the activation of yeast by the ephedra-derived composition is less affected by furfural, etc., the ethanol production method of the present invention can also be used as a method for producing ethanol from biomass raw materials to efficiently produce ethanol. .

Furthermore, we have found that by using a composition derived from ephedra, it is possible to expand the applicable range of reaction temperatures in simultaneous saccharification. As a result, simultaneous saccharification and fermentation can proceed while maintaining yeast activity even under conditions that deviate from the optimum temperature for fermentation and are closer to the optimum temperature for the saccharifying enzyme. By performing the reaction under conditions closer to the optimum temperature of the saccharifying enzyme, rate-limiting saccharification can be promoted, so ethanol can be produced in a shorter time.

[Ephedra-derived composition]
Examples of the ephedra-derived composition used in the present invention include materials containing ephedra crude drugs, ephedra extracts, ephedra extraction residues, and mixtures thereof.

Ephedra (Ephedra) is an evergreen shrub that belongs to the Ephedra family. As the ephedra-derived composition, compositions derived from the stems, leaves, flowers, fruits, roots, etc. of this ephedra can be used, and it is preferable to use compositions derived from the above-ground stems of ephedra.
Among them, dried ground stems of Ephedra sinca Stapf., Ephedra intermedia Schrenk et CA meyer, or Ephedra equisetina Bunge are used as ephedra herbal medicines, and they contain active ingredients such as pseudoephedrine and ephedrine. It is. It is preferable to use an ephedra herbal drug-containing substance containing this herbal ephedra drug, an ephedra extract derived therefrom, and an ephedra extract residue, and the ephedra extract or an ephedra extract residue is more preferable as the ephedra extract.

The substance containing Ephedra herbal medicine may be anything that contains Ephedra herbal medicine, and may be either Ephedra herbal medicine alone or a mixture of Ephedra herbal medicine and other herbal medicines. Examples of Ephedra herbal medicines include Kakkonto, Mao-to, Sho-seiryu-to, and Ma-kyo-kanseki-to.

The form of the ephedra herb or ephedra-containing herbal medicine is not particularly limited, and chopped (minced) or powdered form can be used.

The ephedra extract is derived from a liquid component obtained by solvent extraction of the active ingredients contained in ephedra from a herbal medicine-containing substance and separating it from the solid content. Ephedra extraction residue is the solid content separated during the preparation of ephedra extract. The ephedra extract and the ephedra extract residue will be described in detail later.

[yeast]
The yeast is not particularly limited as long as it can perform ethanol fermentation. For example, yeast of the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, etc. can be used. In addition, genetically recombinant products thereof may also be used.
Specific examples of these include Saccharomyces cerevisae, Saccharomyces pasteurianus, Saccharomyces bayanus, Schizosaccharomyces pombe, and Kluyveromyces lactis. ) etc.

[Sugars]
The sugar may be any sugar that can be fermented by yeast to produce ethanol and carbon dioxide, such as glucose, fructose, sucrose, etc. The sugar may also be a sugar contained in molasses or blackstrap molasses from sugar cane or sugar beet.

Further, animal milk such as cow's milk and goat's milk, skim milk thereof, skim milk powder solution, whole milk powder solution, whey, etc. may be used as raw materials containing sugars.

The sugars may also be those obtained by saccharifying biomass raw materials. There are no particular limitations on the biomass raw materials, and they may be any organic resource derived from living organisms, and biomass containing cellulose or starch can be used.

The sugars are preferably those obtained by saccharifying cellulosic biomass, since they do not compete with food. The sugars obtained by saccharifying this cellulosic biomass are the sugars produced during the reaction in the fermentation saccharification process described below, or the sugars contained in the saccharification liquid in the saccharification process. In other words, the ethanol production method of the present invention is preferably a method for producing ethanol from cellulosic biomass.

Note that saccharification is the decomposition of cellulose, starch, etc. contained in biomass raw materials into monosaccharides such as glucose and fructose, and disaccharides such as amylose and cellobiose, which can be fermented with alcohol. Saccharification can be carried out by a method using a saccharifying enzyme or a method using an acid or an alkali. Saccharification using a saccharifying enzyme is more preferable, since the load on the environment is small and over-decomposition is less likely to occur.

(cellulose biomass)
Cellulosic biomass is primarily composed of cellulose, hemicellulose, and lignin. Specifically, wood, rice straw, wheat straw, corn stover, bagasse, rice husk, palm residue, cassava residue, bamboo, pulp, etc. can be used. These may be used alone or in combination of two or more.

Cellulosic biomass is preferably pretreated to remove lignin, since it becomes an inhibitor of saccharification and fermentation. Lignin can be removed, for example, by hydrothermal decomposition. By using cellulosic biomass from which lignin has been removed, saccharification can proceed more efficiently. Note that the cellulosic biomass that has been pretreated to remove lignin does not need to have lignin completely removed, and lignin may remain as long as saccharification can be promoted.

Furthermore, as described above, in the ethanol production method of the present invention, even if furfural or the like is mixed into cellulosic biomass as an impurity due to pretreatment, the effect of improving the activity of the fermentation reaction by the ephedra-derived composition can be obtained. . Therefore, in the ethanol production method of the present invention, ethanol can be stably produced even from cellulosic biomass containing by-products resulting from pretreatment.
In the ethanol production method of the present invention, cellulosic biomass containing furfural, which is easily produced as a by-product, can be used in the pretreatment for removing lignin. Such cellulosic biomass can simplify purification after a pretreatment step to remove lignin.

(Saccharification enzyme)
The saccharification enzyme is not particularly limited as long as it is an enzyme that can hydrolyze (glycolysis) the biomass raw material, and may be appropriately determined depending on the type of biomass raw material. When cellulosic biomass is used as the biomass raw material, cellulase or hemicellulase, which is a cellulose decomposition enzyme, can be used as the saccharification enzyme. Specifically, "Cellulosin AC40" (HBI Corporation), "Cellulosin AL8" (HBI Corporation), "Sucrase" (Mitsubishi Chemical Foods Corporation), "Cellulase "Onozuka"R-10" (Yakult Pharmaceutical Co., Ltd.), "Cellulase "Onozuka"RS" (Yakult Pharmaceutical Co., Ltd.), "Cellulase A "Amano"3" (Amano Enzyme Co., Ltd.), "Cellulase T "Amano"4" (Amano Enzyme Co., Ltd.), etc. can be mentioned.

In addition, when the biomass raw material is mainly starchy, such as sugar cane, enzymes such as amylase can be used.

When the ethanol production method of the present invention is a method for producing ethanol from biomass raw materials such as cellulosic biomass, it is necessary to perform saccharification and alcohol fermentation. This saccharification and alcohol fermentation may be performed sequentially or in parallel.

After the ethanol production process is completed, the ethanol produced is obtained as an ethanol-containing liquid, which is a mixture with unreacted raw materials, saccharification enzymes, yeast, etc. Ethanol can be recovered by distilling or concentrating the ethanol-containing liquid containing the ethanol produced after the ethanol production process. Conventionally known methods can be used to recover ethanol by distillation or concentration.

The following describes in more detail the aspects of the ethanol production method of the present invention.

[Method for producing ethanol of the present invention (1)]
The ethanol production method of the present invention includes an ethanol production step, which includes a saccharification treatment in which cellulosic biomass is saccharified with a saccharifying enzyme, and alcoholic fermentation of the sugars obtained by the saccharification treatment with yeast to produce ethanol. The production method may be a saccharification and fermentation step (hereinafter referred to as "method for producing ethanol (1) of the present invention") in which a fermentation process is performed to produce ethanol.

That is, in the ethanol production method (1) of the present invention, in the ethanol production step, cellulosic biomass, saccharification enzymes, yeast, and an Ephedra-derived composition are mixed in the same reaction vessel to prepare a raw material saccharification and fermentation liquid containing cellulosic biomass, saccharification enzymes, yeast, and an Ephedra-derived composition, and then this raw material saccharification and fermentation liquid is reacted, so that while the cellulosic biomass is saccharified to produce sugars, a reaction is also carried out in parallel to produce ethanol by alcoholic fermentation of the obtained sugars. The saccharification and fermentation step in the ethanol production method (1) of the present invention is a so-called simultaneous saccharification and fermentation step in which a saccharification step (saccharification treatment) and a fermentation step (fermentation treatment) are carried out simultaneously.

The sugars produced by saccharification using the saccharifying enzyme are immediately consumed in alcoholic fermentation by yeast, reducing product inhibition during saccharification. Furthermore, in the present invention, by mixing the ephedra-derived composition, yeast activity can be increased at higher temperatures, allowing the reaction to take place under conditions closer to the optimal temperature for the saccharifying enzyme.

If there is too much saccharification enzyme relative to the cellulosic biomass, the cost becomes high, and if there is too little, the glucose production rate is slow. Therefore, it is preferable to use 100 g or more or 500 g or more per 1 kg of cellulosic biomass. Also, the upper limit is preferably 1 kg or less or 800 g or less per 1 kg of cellulosic biomass. For example, in the case of cellulosin AC40, it is possible to use about 600 g per 1 kg of cellulosic biomass.

The amount of yeast used is such that the initial yeast concentration in the raw material saccharification and fermentation liquid prepared by mixing cellulosic biomass, saccharification enzymes, yeast, and the ephedra-derived composition is approximately 2 x 10 ⁸ cells/L, but is not particularly limited as the initial yeast concentration does not have a significant effect on fermentation.

The amount (g) of the ephedra-derived composition relative to the volume (L) of the raw material saccharification and fermentation liquid (amount of the ephedra-derived composition (g)/volume of the raw material saccharification and fermentation liquid (L)) is determined based on the type of cellulosic biomass and saccharification enzyme. Although it may be adjusted as appropriate, it is preferably 0.5 g/L or more, and may be 1 g/L or more or 3 g/L or more. Further, the upper limit thereof is preferably 10 g/L or less, and may be 8 g/L or less or 7 g/L or less. If the amount of the ephedra-derived composition is too small, the effect of increasing the fermentation rate will not be sufficiently manifested, and if it is too large, there is a limit to increasing the fermentation rate, resulting in unnecessary addition.

The temperature in the saccharification and fermentation step of the ethanol production method (1) of the present invention is appropriately determined depending on the type of saccharification enzyme and yeast used. As mentioned above, when saccharification and fermentation are carried out simultaneously, saccharification of cellulosic biomass is promoted by setting the temperature conditions closer to the optimum temperature of the saccharifying enzyme, but if the temperature conditions deviate from the optimum temperature of the yeast, There was a problem in that the activity of yeast decreased when the reaction was carried out. On the other hand, in the ethanol production method (1) of the present invention, the saccharification treatment and fermentation treatment are performed in the presence of the ephedra-derived composition, and the activity of the yeast can be maintained even at a temperature higher than the optimum temperature of the yeast used. . Therefore, in the ethanol production method (1) of the present invention, in the saccharification and fermentation step, it is preferable to perform the saccharification treatment and the fermentation treatment at a temperature higher than the optimum temperature of yeast. Thereby, saccharification can proceed more efficiently.

In order to carry out saccharification and fermentation at a predetermined temperature higher than the optimum temperature of yeast, the raw material saccharification and fermentation liquid is heated to the predetermined temperature in the saccharification and fermentation process, and then the reaction is carried out while managing the raw material saccharification and fermentation liquid so that the average liquid temperature until the end of the reaction is maintained at the predetermined temperature.
The temperature for saccharification and fermentation may be in a range above the optimum temperature of yeast and below the optimum temperature of the saccharification enzyme, so long as it does not significantly reduce yeast activity. For example, it is preferable to carry out the reaction (saccharification and fermentation) so that the temperature of the raw material saccharification and fermentation liquid is at least +1°C or +2°C higher than the optimum temperature of yeast. The upper limit of the temperature range is about +10°C or lower, +7°C or lower, or +5°C or lower than the optimum temperature of yeast.
In the present invention, the "optimum temperature for yeast" refers to the temperature at which the yeast growth rate is the fastest, and the "optimum temperature for saccharifying enzymes" refers to the temperature at which the activity of saccharifying enzymes is the greatest.

Specifically, although it depends on the type of yeast used, it is preferable to carry out the saccharification and fermentation processes at a liquid temperature of the raw material saccharification and fermentation liquid of 50°C or less, and more preferably 40°C or less. It is also preferable to carry out the saccharification and fermentation processes at a liquid temperature of 30°C or more, and more preferably 35°C or more. It is also possible to carry out the saccharification and fermentation processes at a liquid temperature of 36°C or more or 37°C or more.

The pH of the raw material saccharification fermentation liquid is appropriately determined depending on the type of saccharification enzyme and yeast used. If the pH is too low or too high, the reactivity of the saccharification enzyme decreases and the yeast cannot grow. Therefore, the pH is preferably 3.0 or higher, more preferably 4.0 or higher, and even more preferably 4.5 or higher. The upper limit of the pH is preferably 7.0 or lower, more preferably 6.0 or lower, and even more preferably 5.0 or lower.

The time for the saccharification and fermentation process varies depending on the production scale, but is usually 12 to 120 hours, preferably 12 to 72 hours or 12 to 24 hours.

As the ethanol production method (1) of the present invention, for example, as shown in FIG. 1, the ethanol production method (1-1) includes a pretreatment step, a saccharification fermentation step, and an ethanol recovery step. Can be done.
In the ethanol production method (1-1) shown in FIG. 1, lignin is removed from cellulosic biomass in the pretreatment step. In the saccharification and fermentation step, first, a raw material saccharification and fermentation liquid is prepared by mixing cellulosic biomass from which lignin has been removed, a saccharification enzyme, yeast, and an ephedra-derived composition. Next, the raw material saccharification and fermentation liquid is saccharified and fermented to generate ethanol and obtain an ethanol-containing liquid containing ethanol. Furthermore, in the ethanol recovery step, ethanol can be obtained by distilling and concentrating the ethanol-containing liquid obtained in the saccharification and fermentation step.

[Method for producing ethanol of the present invention (2)]
Furthermore, the method for producing ethanol of the present invention includes an ethanol production step, and before the ethanol production step, a production method having a saccharification step of saccharifying cellulosic biomass into sugars using a saccharifying enzyme (hereinafter referred to as "the ethanol production method of the present invention"). (2). In the ethanol production process, saccharides obtained in the saccharification process are alcohol-fermented by yeast to produce ethanol, so the ethanol production process in the ethanol production method (2) of the present invention can be said to be a fermentation process.

(saccharification process)
The saccharification step of the ethanol production method (2) of the present invention is a step performed before the ethanol production step (fermentation step), and is a step in which cellulosic biomass is saccharified using a saccharifying enzyme. Specifically, a biomass raw material and a saccharifying enzyme are mixed to prepare a raw material saccharification solution, and the biomass raw material and the saccharification enzyme are reacted in this raw material saccharification solution, whereby cellulosic biomass is hydrolyzed and sugars are produced. generate. Moreover, since the ephedra-derived composition does not have an adverse effect on the saccharification rate, it may be added at the stage of preparing the raw material saccharified liquid in the saccharification process, or may be added at the fermentation process.

The temperature of the saccharification reaction in the saccharification step is appropriately adjusted depending on the type of saccharification enzyme used. If the temperature of the saccharification reaction is too high, the saccharifying enzyme is likely to be deactivated due to thermal denaturation, and if the temperature of the saccharification reaction is too low, the reaction rate tends to decrease. In the ethanol production method (2) of the present invention, it is usually preferable to carry out the reaction at the optimum temperature of the saccharifying enzyme used. Specifically, it is preferable to carry out the saccharification reaction so that the average temperature of the raw material saccharified liquid is 35°C or higher until the end of the reaction, and more preferably 40°C or higher. Moreover, the upper limit thereof is preferably 70°C or less, more preferably 60°C or less.

If there is too much saccharification enzyme relative to the cellulosic biomass, the cost becomes high, and if there is too little, the glucose production rate is slow. Therefore, it is preferable to use 100 g or more or 500 g or more per 1 kg of cellulosic biomass. Also, the upper limit is preferably 1 kg or less or 800 g or less per 1 kg of cellulosic biomass. For example, in the case of cellulosin AC40, it is possible to use about 600 g per 1 kg of cellulosic biomass.

The pH of the raw material saccharified liquid is adjusted as appropriate depending on the type of saccharified yeast used. If the pH of the raw material saccharified solution is too high or too low, the reaction rate tends to decrease. Therefore, the pH of the raw material saccharified liquid is preferably 2.5 or higher, more preferably 3 or higher. Moreover, the upper limit of the pH of the raw material saccharified liquid is preferably 7 or less, more preferably 6 or less.
The time for the saccharification step is usually 12 to 120 hours. Further, the period may be 12 to 72 hours or 12 to 24 hours.

(Fermentation process)
The fermentation step (ethanol production step) of the ethanol production method (2) of the present invention is a step of producing ethanol by alcoholic fermentation of the sugars obtained in the saccharification step with yeast in the presence of the Ephedra-derived composition. Specifically, a saccharification liquid containing the sugars obtained in the saccharification step is mixed with yeast to prepare a raw material fermentation liquid, and ethanol is produced by alcoholic fermentation of the sugars in this raw material fermentation liquid with yeast. In addition, when the Ephedra-derived composition is not mixed in the saccharification step so that alcoholic fermentation proceeds in the presence of the Ephedra-derived composition, the Ephedra-derived composition is also mixed in the stage of preparing the raw material fermentation liquid.

The amount of yeast used in the fermentation process is suitably an initial yeast concentration of about 2×10 ⁸ cells/L in the raw fermentation liquid, but is not particularly limited because the initial yeast concentration does not have a large effect on fermentation.

When the ephedra-derived composition is mixed in the saccharification step, the ephedra-derived composition is adjusted to an arbitrary amount with respect to the raw material saccharification liquid. Furthermore, when the ephedra-derived composition is mixed in the fermentation process, the ephedra-derived composition is adjusted to an arbitrary amount with respect to the raw fermentation liquid.
The amount (g) of the ephedra-derived composition relative to the volume (L) of the raw material saccharified liquid or the raw material fermented liquid ((amount of ephedra-derived composition (g)/volume of raw material saccharified liquid (L)) or (of the ephedra-derived composition) The amount (g)/volume (L) of raw material fermentation liquid is preferably 0.5 g/L or more, and may be 1 g/L or more or 3 g/L or more.The upper limit is 10 g/L. It is preferably below, and may be 8 g/L or below, or 7 g/L or below. If the ephedra-derived composition is too small, the effect of increasing the fermentation rate will not be sufficiently manifested, and if it is too large, there is a limit to increasing the fermentation rate. Therefore, it is an unnecessary addition.

The alcohol fermentation temperature in the fermentation step is adjusted as appropriate depending on the type of yeast used. If the fermentation temperature is too high or too low, yeast will not grow easily and it will be difficult to obtain ethanol. In the ethanol production method (2) of the present invention, it is usually preferable to carry out the reaction at the optimal temperature of the yeast used. Therefore, unless the yeast is a special yeast such as a high-temperature-resistant bacteria, it is preferable to perform alcohol fermentation so that the average temperature of the raw fermented liquid until the end of alcohol fermentation is 40°C or less, more preferably 35°C or less. Further, the lower limit thereof is preferably 25°C or higher, more preferably 30°C or higher.

The pH of the raw fermentation liquid is adjusted as appropriate depending on the type of yeast used. If the pH of the raw fermentation liquid is too high or too low, yeast will not grow easily and it will be difficult to obtain ethanol. Therefore, the pH of the raw fermented liquid is preferably 5 or more, more preferably 6 or more. Moreover, the upper limit of the pH of the raw material fermentation liquid is preferably 8 or less, more preferably 7 or less.

The fermentation step time is usually 12 to 120 hours, preferably 12 to 72 hours or 12 to 24 hours.

The ethanol production method (2) of the present invention can be, for example, an ethanol production method (2-1) having a pretreatment step, a saccharification step (a), a fermentation step (a), and an ethanol recovery step, as shown in FIG. 2.

In the ethanol production method (2-1) shown in FIG. 2, lignin is removed from cellulosic biomass in the pretreatment step. In the saccharification step (a), first, the cellulosic biomass from which lignin has been removed is mixed with a saccharification enzyme to prepare a raw saccharified liquid. The raw saccharified liquid is then saccharified to obtain a saccharified liquid containing sugars. In the fermentation step (a), the saccharified liquid obtained in the saccharification step (a) is mixed with yeast and an ephedra-derived composition to prepare a raw fermentation liquid, which is then fermented to produce ethanol, thereby obtaining an ethanol-containing liquid containing ethanol. Furthermore, in the ethanol recovery step, ethanol can be obtained by distilling and concentrating the ethanol-containing liquid obtained in the fermentation step (a).

Further, as an example of the ethanol production method (2) of the present invention, as shown in FIG. Production method (2-2) is mentioned.
The ethanol production method (2-2) shown in FIG. 3 is an example that differs from the ethanol production method (2-1) shown in FIG. 2 in the saccharification step and the fermentation step.
In the ethanol production method (2-2) in Figure 3, in the saccharification step (b), the cellulosic biomass from which lignin has been removed, a saccharifying enzyme, and an ephedra-derived composition are mixed to prepare a raw material saccharified liquid. , performs saccharification. Further, in the fermentation step (b), the saccharified liquid obtained in the saccharification step (b) and yeast are mixed to prepare a raw fermented liquid, and then fermentation is performed to obtain an ethanol-containing liquid.

When ethanol is produced from sugars such as glucose, which does not require a saccharification process, the ethanol production process can be a process of preparing a raw material fermentation liquid containing sugars such as glucose, a composition derived from Ephedra, and yeast, and then performing alcoholic fermentation in the same manner as the fermentation process in the ethanol production method (2) of the present invention.

3. Method for Producing a Lactic Acid Fermentation Product The present invention relates to a method for producing a lactic acid fermentation product, which includes a lactic acid fermentation product production step of producing a lactic acid fermentation product by lactic acid bacteria in the presence of an ephedra-derived composition (hereinafter, sometimes referred to as the "method for producing the lactic acid fermentation product of the present invention").

One of the features of the method for producing a lactic acid fermentation product of the present invention is that the lactic acid fermentation product production process is carried out in the presence of an ephedra-derived composition. By carrying out lactic acid fermentation in the presence of an ephedra-derived composition, a lactic acid fermentation product can be produced in a shorter time.

[Ephedra-derived composition]
In the method for producing a lactic acid fermentation product of the present invention, a composition containing an ephedra-derived composition is used as a lactic acid fermentation promoter. The ephedra-derived composition used can be the same as that used in the ethanol production method of the present invention. Among these, ephedra extract or ephedra extract residue is preferred.

[Lactic acid bacteria]
The lactic acid bacteria are not particularly limited as long as they can carry out lactic acid fermentation. For example, the lactic acid bacteria that can be used include Lactobacillus, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, Streptococcus, and Bifidobacterium. In addition, genetically recombinant products thereof may also be used.
Specific examples of these include Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus plantarum, Streptococcus thermophilus, etc. .

[Sugars]
The saccharide may be anything as long as it can be fermented by lactic acid bacteria, and examples thereof include glucose, fructose, and sucrose. Moreover, the saccharide may be a saccharide contained in molasses or blackstrap molasses such as sugar cane or sugar beet. Animal milk such as cow's milk and goat's milk, skim milk thereof, skim milk powder solution, whole milk powder solution, whey, etc. may be used as raw materials containing sugars.

Furthermore, the saccharide may be one obtained by saccharifying a biomass raw material as in the ethanol production method of the present invention, and the same biomass raw material as in the ethanol production method of the present invention can be used. Regarding the saccharifying enzyme for saccharifying the biomass raw material, the same saccharifying enzyme as in the ethanol production method of the present invention can be used depending on the type of biomass raw material.

In the lactic acid fermentation product production process, it is sufficient that a reaction occurs to produce lactic acid from sugars, and other reactions may occur in parallel depending on the raw materials used.
When sugars such as glucose are used as raw materials, the process for producing a lactic acid fermentation product can be considered a fermentation process.
In addition, when saccharification of a biomass raw material such as cellulosic biomass is performed and lactic acid fermentation is performed, saccharification and lactic acid fermentation may be performed sequentially or in parallel, as in the ethanol production method of the present invention. When saccharification and lactic acid fermentation are performed sequentially, the lactic acid fermentation product production process can be said to be a fermentation process in which sugars obtained in the saccharification process are lactic acid fermented. When saccharification and lactic acid fermentation are performed in parallel, the lactic acid fermentation product production process can be said to be a saccharification and fermentation process. Note that, when saccharification and lactic acid fermentation are performed sequentially, the reaction conditions such as temperature in the saccharification process and the timing of mixing the ephedra-derived composition can be the same as in the ethanol production method of the present invention.

The temperature of lactic acid fermentation in the lactic acid fermentation product production process is appropriately adjusted depending on the type of lactic acid bacteria used, the reaction system, etc. If the fermentation temperature is too high or too low, it will be difficult for lactic acid bacteria to grow, making it difficult to obtain lactic acid.
When saccharides are used as raw materials or when saccharification and lactic acid fermentation are performed sequentially using biomass raw materials, it is usually preferable to carry out the reaction at the optimum temperature of the lactic acid bacteria used. Therefore, unless the bacteria are special bacteria such as high-temperature-resistant bacteria, it is preferable to carry out lactic acid fermentation so that the average temperature of the raw fermented liquid until the end of lactic acid fermentation is 50°C or lower, more preferably 45°C or lower. Further, the lower limit thereof is preferably 25°C or higher, more preferably 30°C or higher.

When saccharification and lactic acid fermentation are carried out in parallel using biomass raw materials, it is preferable to carry out the reactions at a temperature above the optimum temperature of the lactic acid bacteria used and below the optimum temperature of the saccharification enzyme.

The fermentation time is usually 2 to 120 hours, preferably 5 to 72 hours or 5 to 24 hours.

The amount of the ephedra-derived composition relative to the liquid to be mixed (raw material saccharification fermentation liquid, raw material saccharification liquid, or raw material fermentation liquid), the amount of lactic acid bacteria used, the pH during lactic acid fermentation (pH of the raw material saccharification fermentation liquid or pH of the raw material fermentation liquid), etc. can be the same as in the ethanol production method of the present invention.

Further, after the lactic acid fermented product production process is completed, the produced lactic acid fermented product can be appropriately post-treated depending on the use of the lactic acid fermented product. Depending on the use, it can be used as is after sterilization treatment or the like. Furthermore, by separating the lactic acid fermented product which is a mixture with unreacted raw materials, lactic acid bacteria, etc., the lactic acid contained in the lactic acid fermented product can be recovered. Conventionally known methods can be used to recover lactic acid by solid-liquid separation by neutralization, solvent extraction, or the like.

4. Fermentation Promoter The present invention relates to a fermentation promoter containing an ephedra extract residue (hereinafter, may be referred to as "the fermentation promoter of the present invention").

The fermentation accelerator of the present invention can be used as an ephedra-derived composition in the ethanol production method of the present invention.

Furthermore, the fermentation promoter of the present invention can be used as an ephedra-derived composition in the method for producing a lactic acid fermented product of the present invention.

The present inventors have surprisingly found that the ephedra extract residue has a fermentation promoting effect, even though the content of the active ingredient contained in the ephedra herbal medicine is reduced. Furthermore, it has been found that the ephedra extraction residue obtained after performing the extraction operation of the active ingredient from the ephedra crude drug-containing substance multiple times also has a fermentation promoting effect. The fermentation promoter of the present invention is based on these findings.
Conventionally, extraction residues from the manufacturing process of Chinese herbal medicines have been discharged as waste and have been reduced in volume by incineration, landfilling, or composting, and there has been a demand for their effective use. The use of the present invention as a fermentation accelerator is a new use in which the ephedra extraction residue, which was conventionally discarded, can be effectively utilized, and is environmentally and economically preferable.

The ephedra extract residue contained in the fermentation promoter of the present invention is a solid content separated during preparation of the ephedra extract. The ephedra extract is obtained by extracting the active ingredients contained in the ephedra herbal medicine with a solvent and removing the solid content.
The extraction can be carried out by a known extraction method. For example, the Ephedra herbal medicine-containing material can be immersed in a solvent for a certain period of time while stirring as necessary, thereby extracting the active ingredient in the Ephedra herbal medicine-containing material into the solvent. The extraction temperature can be, for example, room temperature to 100°C. The extraction time can be, for example, 30 minutes to 1 hour.

Moreover, the solvent used for extraction is generally water. The ratio of the Ephedra herbal drug-containing substance to the solvent can be adjusted as appropriate, and for example, the solvent can be 20 to 40 g per 1 g of the Ephedra herbal drug-containing substance.

As described above, the product containing Ephedra herbal medicine may contain other ingredients besides Ephedra herbal medicine, so long as it contains Ephedra herbal medicine. Although it depends on the types of other ingredients contained in the product containing Ephedra herbal medicine, if the product containing Ephedra herbal medicine contains too little Ephedra herbal medicine, the effect of promoting fermentation may not be fully achieved. Therefore, the amount of Ephedra herbal medicine in the product containing Ephedra herbal medicine (mass of Ephedra herbal medicine/mass of product containing Ephedra herbal medicine) is preferably 0.1 to 1, and may also be 0.2 to 1 or 0.3 to 1.

After the active ingredient is extracted, the liquid component and the solid component are separated, whereby the liquid component can be used as the Ephedra extract and the solid component can be used as the Ephedra extract residue.
The liquid component and the solid content can be separated by filtration, centrifugation, etc. The solid content obtained by separating the liquid component may be further washed with water, dried, etc., before use.
The obtained solid matter may be used as it is as the ephedra extraction residue, or may be crushed or otherwise processed to obtain a desired shape.

In addition, the liquid component may be used as it is, or may be used after solvent extraction by appropriately concentrating or diluting to adjust the concentration of the active ingredient, or may be used as a dried product obtained by drying the liquid component. The ephedra extract derived from the liquid component may be used as an ephedra-derived composition in the method for producing ethanol of the present invention, or may be used for other purposes. The liquid components extracted with water and their dried products can be used as they are as medicines. The solid content that is discarded during the production of such pharmaceuticals can be used as the ephedra extraction residue.

The liquid component may also be used as the ephedra-derived composition in the method for producing a lactic acid fermented product of the present invention.

The ephedra extract residue may be the extract residue after one extraction operation with a solvent from the ephedra herbal medicine-containing material, or the extract residue after multiple extraction operations with a solvent. From a practical standpoint, it may be the extract residue after one to three extraction operations with a solvent.

As mentioned above, the fermentation promoter of the present invention can be used in the ethanol production method of the present invention. Furthermore, the fermentation promoter of the present invention may be used for purposes other than the ethanol production method of the present invention, and may be used for fermentation in the production process of foods such as alcoholic beverages and bread, and for fermentation in the production process of biogas such as methane gas. It's okay.

The fermentation promoter of the present invention can also be used in the method for producing a lactic acid fermented product of the present invention.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples unless the gist thereof is changed.

[Experimental Example 1]
Ethanol fermentation by simultaneous saccharification and fermentation was evaluated using ephedra extract (Ma Huang extract) extracted from the herbal medicine Ephedra.

(1-1): Preparation of Ephedra extract 15 g of the herbal medicine Ephedra (manufactured by Tochimoto Tenkaido, chopped) was boiled in 300 mL of ion-exchanged water for 30 minutes. The solution was separated from the residue, centrifuged at 15,000 rpm for 5 minutes, and the supernatant was filtered through a 0.45 μm membrane filter to obtain Ephedra extract.

(1-2): Ethanol fermentation by simultaneous saccharification fermentation The yeast is Saccharomyces cervisiae (Japan Brewing Association No. 6) (optimum temperature: 35°C), and the substrate (cellulose biomass) is powder filter paper manufactured by Advantech Toyo Co., Ltd. (cellulose powder C). ), and cellulase (Cellulosin AC40 manufactured by HBI Corporation) was used as the saccharifying enzyme.
For simultaneous saccharification and fermentation, 50 mL of YP medium (10 g of yeast extract, 20 g of peptone, 1 L of ion exchange water) adjusted to pH 4.5 with citric acid buffer, enzyme concentration 25 g/L, initial yeast concentration 2 x 10 ⁸ cells/L, substrate The cellulose concentration was 50 g/L and the ephedra extract prepared in (1-1) above was placed in a test tube with a lid, and the mixture was reacted for 72 hours at a temperature of 37°C and a rotational speed of 110 rpm using an As One Mix Rotor VMRC-5. (Experiment Example 1-1).
The initial yeast concentration was determined from the intracellular ATP value measured using an AF-100 ATP analyzer manufactured by Toa DKK.
In addition, for a control in which ephedra extract was not mixed, the reaction was similarly carried out for 72 hours (Experiment Example 1-2).

(1-3): Changes in glucose concentration and ethanol concentration over time During the experiment (1-2) above, the glucose and ethanol concentrations during the reaction were measured at regular processing times (reaction times) using a JASCO LC-NetII/ADC HPLC, and the changes in glucose and ethanol concentrations over time were determined. Figure 4 shows the results of plotting the glucose concentration [g/L] and ethanol concentration [g/L] against processing time [h] in Experimental Example 1-1 and Experimental Example 1-2.

Enzymatic saccharification produces glucose from cellulose, which is immediately consumed by yeast to produce ethanol. Therefore, as shown in FIG. 4, glucose is kept at a low concentration.
Additionally, as shown in Figure 4, Experimental Example 1-1 (with Ephedra extract added) reached an ethanol concentration of 8.3 g/L in 24 hours, which was higher than Experimental Example 1-2 (without Ephedra extract). The concentration of ethanol produced in 72 hours was 7.6 g/L or more. By adding ephedra extract, the fermentation time could be reduced to less than 1/3. In this way, even if the reaction was carried out at a temperature higher than the optimum temperature for yeast, in Experimental Example 1-1, the presence of ephedra extract suppressed the decline in yeast activity at the optimum temperature and maintained the activity. In this state, ethanol fermentation can proceed. On the other hand, in Experimental Example 1-2, the activity of the yeast was reduced due to the reaction being carried out at a temperature higher than the optimum temperature for the yeast, resulting in a decrease in the production rate of ethanol and a small amount of production. There is.

[Experimental Example 2]
As the fermentation promoter of the present invention, Kampo medicines containing Ephedra, such as Kakkonto, Mao-to, and Sho-seiryu-to, were used to prepare an Ephedra extract residue, and ethanol fermentation using the obtained Ephedra extract residue was evaluated.

(2-1): Preparation of Kakkonto residue Kakkonto, which is a Chinese herbal medicine containing ephedra, consists of 4g of ephedra, 4g of kakkon, 4g of Japanese radish, 3g of Keihi, 3g of peony, 2g of licorice, and 1g of kanshokyo (all manufactured by Tochimoto Tenkaido). ) A total of 21 g was prepared by decoction in 600 mL of ion-exchanged water for 30 minutes. It was separated into a solution and a residue (solid content), and the residue was washed with ion-exchanged water and dried at 105°C. The dried residue was ground in a planetary ball mill for 30 minutes to obtain Kakkonto residue. The obtained Kakkonto residue was designated as ephedra extraction residue (a).

(2-2): Preparation of Mao-to Residue Instead of Kakkonto, Mao-to was prepared by the same procedure using 5 g of Ephedra, 5 g of Kyonin, 4 g of Cinnamon, and 1.5 g of Licorice (all manufactured by Tochimoto Tenkaido), totaling 15.5 g, to obtain Mao-to residue. The obtained Mao-to residue was designated as Ephedra extract residue (b).

(2-3): Preparation of Shoseiryuto residue Instead of Kakkonto, use Shoseiryuto: 6 g of Hange, 3 g of Ephedra, 3 g of Peony, 3 g of Licorice, 3 g of Keihi, 3 g of Saishin, 1.5 g of Kansho Kyo, 1 Goshi A similar operation was carried out using a total of 24 g of .5 g (all manufactured by Tochimoto Tenkaido) to obtain a Shoseiryuto residue. The obtained shoseiryuto residue was designated as ephedra extraction residue (c).

(2-4): Ethanol fermentation using yeast A YPD medium (glucose 15 g, peptone 2 g, yeast extract 1 g, water 100 mL) was prepared and sterilized in an autoclave. In a flask, add sake yeast solution in an amount to give yeast (optimal temperature 35°C) 2.0 x 10 ⁸ cells/L and an amount to give the solid component concentration shown in Table 1 to YPD medium after sterilization. A sample for the ethanol fermentation test was prepared by adding a solid component (separately autoclaved sterilized ephedra extract residue (a), ephedra extract residue (b), or ephedra extract residue (c)). Each sample was reacted at 35° C. for 48 hours.
In addition, in order to consider the influence of solid components, a control was also prepared in which powdered filter paper (cellulose powder C) manufactured by Advantech Toyo Co., Ltd. was added so as to have the solid component concentration shown in Table 1, and the reaction was performed in the same manner.

(2-5) Evaluation of fermentation delay time (t _s ) and fermentation rate (r) using a logistic function (2-5-1) Evaluation method using a logistic function Figure 5 shows how carbon dioxide This is a typical example showing the relationship between generation amount (y) and fermentation time (t). Moreover, the logistic function is a function expressed by the following formula (1). The values of y _max , t _1/2 , and k can be calculated by fitting the amount of carbon dioxide generated (y) to the fermentation time (t) to a logistic curve (a) expressed by a logistic function.

k is a parameter of the logistic function expressed by the following formula (2): t _s is expressed by the following formula (3), and can be calculated from the values of t _1/2 and k.

Note that, as shown in FIG. 5, y _max represents the maximum amount of carbon dioxide generated. t _1/2 represents the fermentation time at which y _max /2 is reached.
r is the slope of the tangent line (b) when t=t _1/2 in the logistic curve, and represents the fermentation rate.
t _s is defined as the intersection of the horizontal axis when the amount of carbon dioxide generated is 0 (the vertical axis is 0) and the extension of the tangent line (b) of t= _1/2 , and represents the fermentation delay time. The smaller the value of this fermentation delay time (t _s ), the faster the time until fermentation starts.

(2-5-2) Evaluation of fermentation lag time ( _ts ) and fermentation rate (r) During the ethanol fermentation experiment in (2-4) above, the weight of the entire flask was measured over time using an electronic balance placed in an incubator at 35°C, and the decrease in weight was considered to be the amount of carbon dioxide generated during fermentation.
The amount of carbon dioxide generated (y) versus treatment time (fermentation time) (t) was corrected for the amount of water evaporation and then fitted to a logistic function to calculate the fermentation lag time ( _ts ) and fermentation rate (r), and the effect of the ephedra-derived composition was evaluated.

6 shows the results of plotting the fermentation delay time _ts [h] of the ethanol fermentation in Experimental Examples 2-1 to 2-4 against the solid component concentration [g/L]. The addition of the ephedra extract residue reduced the fermentation delay time by 0.7 to 0.9 times, shortening the time it took for fermentation to start.
In particular, the Ephedra extract residue (a) (Kakkonto residue) of Experimental Example 2-1 showed a shortened fermentation delay time regardless of the concentration of the solid components.

Figure 7 shows the fermentation rate r [(g/L)/h] of the ethanol fermentation in Experimental Examples 2-1 to 2-4 plotted against the solid component concentration [g/L]. The fermentation rate of Ephedra extract residue (b) (Maoto residue) in Experimental Example 2-2 increased by increasing the amount added. This is thought to be due to the action of the solid components as well as the medicinal components remaining in the residue and the components that cannot be extracted with water, which promoted fermentation.

The fermentation rate in Experimental Example 2-2 was approximately seven times faster than when no solid ingredients were used.

In addition, the fermentation completion time was defined and evaluated by the following formula (4) using the values of t _1/2 and k calculated by fitting the logistic function. As a result, the fermentation completion time (t _e ) in each of Experimental Examples 2-1 to 2-3 was shortened to about 1/2 compared to Experimental Example 2-4 (control), and to about 1/3 compared to the case without solid components.

(2-6): Change in the amount of ethanol produced over time In the experiment (2-4) above, the amount of ethanol produced was measured using LC-NetII/ADC type HPLC manufactured by JASCO Corporation.
Figure 8 shows the ethanol yield [%] after the completion of ethanol fermentation (about 2 days) in Experimental Examples 2-1 to 2-4 (the amount of ethanol actually produced (g) relative to the theoretical amount of ethanol produced (g)). The graph shows the results of plotting the solid component concentration (g/L) against the solid component concentration [g/L]. The ethanol produced was 70 to 80% of the theoretical yield, and the difference between the control and the one containing the ephedra extraction residue was small, and no fermentation inhibition was observed.

[Experiment example 3]
The influence of furfural on ethanol fermentation was evaluated using the ephedra extraction residue (b) (maoto residue) prepared in Experimental Example 2 as the fermentation accelerator of the present invention.

(3-2): Ethanol fermentation by yeast in the presence of furfural A YPD medium (15 g of glucose, 2 g of peptone, 1 g of yeast extract, 100 mL of water) was prepared, and after adding a predetermined amount of furfural, it was sterilized in an autoclave. . In a flask, add sake yeast solution in an amount that will give 2.0 x 10 ⁸ cells/L of yeast (optimal temperature 35°C) to YPD medium after sterilization, and ephedra extract residue (b) that has been sterilized separately in an autoclave or As a control, 7 g/L of powder filter paper (cellulose powder C) manufactured by Advantech Toyo Co., Ltd. was added, and a breathable silicone stopper was attached to prepare a sample for the ethanol fermentation test.
Note that a plurality of samples were prepared in which the amount of furfural added was changed so that the furfural concentration shown in Table 2 was obtained. Each sample was used to react at 35°C for 72 hours.

(3-3): Evaluation of fermentation delay time ( _ts ) and fermentation rate (r) As in (2-5) of Experimental Example 2, the change over time in the amount of carbon dioxide generated during the ethanol fermentation in (3-2) above was measured and fitted to a logistic function to calculate and evaluate the fermentation delay time ( _ts ) and fermentation rate (r).

FIG. 9 shows the results of plotting the fermentation rate r [(g/L)/h] of the ethanol fermentation in Experimental Examples 3-1 and 3-2 against the furfural concentration [g/L].
When 7 g/L of the ephedra extraction residue (b) of Experimental Example (3-1) was added, the fermentation rate decreased as the furfural concentration increased, but even with 2 to 3 g/L of furfural, the fermentation rate decreased to 2 to 3 (g/L). It was possible to maintain a fermentation rate of L)/h.
On the other hand, in Experimental Example (3-2) (system in which 7 g/L of powdered filter paper was added), the fermentation rate decreased as the furfural concentration increased, and when furfural was 2 to 3 g/L, r≈0.15 g/h, which was approximately At half speed and above 4.5 g/L, fermentation stopped.

Figure 10 shows the results of plotting the ethanol concentration [g/L] about 6 days after ethanol fermentation in Experimental Example (3-1) and Experimental Example (3-2) against the furfural concentration [g/L]. It shows. There was a small difference in ethanol concentration between the samples with the ephedra extract residue and the powder filter paper, indicating that the ephedra extraction residue (b) did not inhibit the production amount. It was also found that the additive was highly effective and the ethanol concentration was maintained up to a furfural concentration of about 3 g/L.

[Experiment example 4]
Instead of the total of 15.5 g of ephedra 5 g, kyonin 5 g, cinnamon bark 4 g, and licorice 1.5 g in (2-2) of Experimental Example 2, ephedra extraction residue (b) (maoto residue after one decoction) was used. Then, the same operation as in Experimental Example (2-2) was performed to prepare maoto residue that had been decooked twice. Furthermore, the same decoction operation was repeated to prepare maoto residue after decoction 3 to 8 times.
Next, ethanol fermentation was performed in the same manner as in Experimental Example 2 (2-4) using the Maoto residue that had been decooked 1 to 8 times. The fermentation delay time and fermentation rate were calculated from the amount of carbon dioxide generated relative to the fermentation time in the same manner as in Experimental Example 2 (2-5), and the effect of ethanol fermentation on the number of decoctions of the maoto residue was evaluated.
In addition, a system using powdered filter paper (cellulose powder C) manufactured by Advantech Toyo Co., Ltd. instead of maoto residue and a system using only yeast (no solid component added) were also evaluated as controls.
FIG. 11 shows the results of plotting the fermentation delay time t _s [h] of ethanol fermentation against the number of decoctions of the maoto residue. FIG. 12 shows the results of plotting the fermentation rate r [(g/L)/h] of ethanol fermentation against the number of decoctions of the maoto residue. As shown in FIGS. 11 and 12, it was confirmed that the effect of maoto residue was maintained even if the number of decoctions was increased.

[Experimental Example 5]
Ethanol fermentation was evaluated using the extract and extraction residue of Ephedra sp. Ethanol fermentation was also evaluated for 10 kinds of herbs other than Ephedra contained in the herbal medicines (Kakkonto, Maoto, and Shoseiryuto) used in Experimental Example 2 (Pueraria lobate, Zingiber officinale, Glycyrrhize uralensis, Prumus sp., Cinnamomum cassia, Schisandra Chinensis, Asiasarum sp., Paeonia lactiflora, Zizyphus jujuba, and Pinellia ternata).

(5-1): Preparation of each herbal extract and extraction residue Using a Chinese herbal medicine decoction device (Bunka Rakuraku EK-SA10, manufactured by Tochimoto Tenkaido), 30 g of each herbal medicine was decoctioned in 600 mL of ion-exchanged water for 30 minutes. After that, the solution and residue were separated using a 30-mesh wire net, the solution was centrifuged at 15,000 rpm for 5 minutes, and the supernatant was filtered through a 0.45 μm membrane filter to obtain the extract of each herbal medicine. The extraction residue was washed with ion-exchanged water, dried at 105 ° C for 24 hours, and then ground for 30 minutes with a planetary ball mill (P-6, manufactured by Fritsch).

(5-2): Ethanol fermentation by yeast in the presence of each herbal extract A YPD medium (glucose 15 g, peptone 2 g, yeast extract 1 g, water 100 mL) was prepared, and each herbal extract was added to a solid concentration of 350 mg/L, followed by autoclave sterilization. To the sterilized YPD medium, a sake yeast liquid was added in an amount to give a yeast (optimum temperature 35°C) of 2.0 x 10 ⁸ cells/L, and an air-permeable silicone stopper was attached to prepare samples for ethanol fermentation testing. Each sample was used for the reaction at 35°C.

(5-3): Ethanol fermentation using yeast in the presence of extraction residues of each crude drug A YPD medium (glucose 15 g, peptone 2 g, yeast extract 1 g, water 100 mL) was prepared and sterilized in an autoclave. To the YPD medium after sterilization, add sake yeast solution in an amount that makes yeast (optimum temperature 35°C) 2.0 × 10 ⁸ cells/L and 350 mg/L of extraction residue of each crude drug that was separately sterilized in an autoclave. , prepared samples for ethanol fermentation tests. In addition, in order to consider the influence of solid components, a control was also prepared in which powdered filter paper (cellulose powder C, 350 mg/L) manufactured by Advantech Toyo Co., Ltd. was added. A reaction was performed at 35°C using each sample.

(5-4): Evaluation (5-2) and (5-3) were evaluated using the methods described in Experimental Example 2 (2-5) and (2-6). Additionally, in order to compare the fermentation status under different reaction conditions, the following parameters were defined.

Here, the α values ( _αs value, _αe value, _αr value, _αEtOH value) represent the effect of adding an extract when no fermentation promoter was added (none) was used as a control, and the β values ( _βs value, _βe value, _βr value, _βEtOH value) represent the effect of adding an extraction residue when the addition of cellulose powder (cellulose) was used as a control.

FIG. 13 shows the effects of each crude drug on the fermentation delay time, with the horizontal axis representing the α _s value, which is the effect of adding the extract, and the β _s value, which is the effect of adding the residue, on the vertical axis.
Here, for the fermentation reaction start time (t _s ), a value on the horizontal axis (α _s ) of less than 1 indicates that the extract has a promoting effect, and a value on the vertical axis (β _s ) of less than 1 This indicates that there is a promoting effect on the extraction residue. For example, crude drugs plotted in the region of (α _s <1) and (β _s <1) indicate that both the extract and the residue exert a promoting effect on the fermentation delay time.

FIG. 14 shows the effects of each crude drug on the fermentation rate, with the horizontal axis representing the α _r value, which is the effect of adding the extract, and the β _r value, which is the effect of adding the residue, on the vertical axis.
Here, regarding the fermentation rate (r), when the horizontal axis (α _r ) exceeds 1, it indicates that the extract has a promoting effect, and when the vertical axis (β _r ) exceeds 1, it indicates that the extract has a promoting effect. This shows that there is a promoting effect on the residue. For example, herbal medicines plotted in the region of (1<α _r ) and (1<β _r ) indicate that both the extract and the residue exert a promoting effect on the fermentation rate.

FIG. 15 shows the effect of adding extract ( _αe value) on the horizontal axis and residue ( _βe value) on the vertical axis, showing the effect of adding each herb on the completion time of fermentation.
Here, for the fermentation completion time (t _e ), a horizontal axis (α _e ) of less than 1 indicates that there is a promoting effect in the extract, and a vertical axis (β _e ) of less than 1 indicates that there is a promoting effect in the extraction residue. For example, a crude drug plotted in the region of (α _e < 1) and (β _e < 1) indicates that both the extract and the residue have a promoting effect on the fermentation completion time.

FIG. 16 shows the effect of adding the extract, which is the α _EtOH value, on the horizontal axis, and the effect of adding the residue, which is the β _EtOH value, on the vertical axis, showing the effect of each herb on the amount of ethanol produced.
Here, the amount of ethanol produced indicates that the extract has a stimulating effect when the horizontal axis (α _EtOH ) exceeds 1, and indicates that the extraction residue has a stimulating effect when the vertical axis (β _EtOH ) exceeds 1. For example, herbs plotted in the (1 < α _EtOH ) and (1 < β _EtOH ) regions indicate that both the extract and the residue have a stimulating effect on the amount of ethanol produced.

Furthermore, the correspondence between each crude drug and the numbers in the graphs of FIGS. 13 to 16 is as shown in Table 3.

As shown in FIGS. 13 to 16, the promotion and inhibition of ethanol fermentation varied depending on the type of crude drug, as well as the extract and extraction residue.

As shown in FIG. 13, regarding the fermentation delay time, the ephedra herbal medicine showed a promoting effect in the extract, and in the extraction residue, it was equivalent to the control to which powdered filter paper was added. In addition, the extraction residues of many herbal medicines showed inhibition tendencies. However, the overall fermentation process is dominated by the fermentation rate, which will be offset if the fermentation rate is accelerated.

As shown in FIG. 14, regarding the fermentation rate, the ephedra herbal medicine showed a promoting effect on both the extract and the extraction residue.

The fermentation completion time shown in FIG. 15 can be considered as a comprehensive result of fermentation delay time and fermentation rate. Both the extract and the extraction residue of the ephedra herbal medicine showed a fermentation promoting effect, and the fermentation completion time was shortened to about 3/4. Note that the results for the extraction residue of ephedra herbal medicine are compared with those obtained by adding cellulose powder. Compared to the case without solid components (no addition), the fermentation completion time was significantly shortened to about 1/2.

As shown in Figure 16, α _EtOH, which represents the effect of addition of extract on the amount of ethanol produced, and β _EtOH , which represents the effect of addition of extraction residue on the amount of ethanol produced, are approximately 1 for all crude drugs, and regardless of the crude drug, ethanol The effect on production was small. Ephedra herbal medicine had (α _EtOH , β _EtOH ) = (1, 0.8), which was equivalent to each control, and no fermentation inhibition was observed.

[Experiment example 6]
A total of 15.5 g of 5 g of ephedra, 5 g of Japanese radish, 4 g of cinnamon bark, and 1.5 g of licorice (all manufactured by Tochimoto Tenkaido) were decocted in 600 mL of ion-exchanged water for 30 minutes. Thereafter, the solution and residue were separated using a 30-mesh wire mesh, the solution was centrifuged at 15,000 rpm for 5 minutes, and the supernatant was further filtered through a 0.45 μm membrane filter to obtain an extract of maoto. When the effect on ethanol fermentation was similarly evaluated using Maoto extract, the fermentation rate was 1.4 times that of the additive-free control.

[Experiment Example 7]
Samples for ethanol fermentation test were prepared using blackstrap molasses concentration 350 g/L, peptone 20 g/L, yeast extract 8 g/L, initial yeast concentration 2.0 x 10 ⁸ cells/L, and ephedra crude drug extraction residue 7 g/L. and reacted at 35°C. The ephedra crude drug extraction residue prepared in Experimental Example 5 (5-1) was used.
Ethanol fermentation was evaluated in the same manner as in Experimental Example 2. The results are shown in FIG.
As shown in FIG. 17, ethanol fermentation was possible even with a high concentration of blackstrap molasses of 350 g/L. In addition, blackstrap molasses contains aldehydes, which are fermentation inhibitors, so fermentation is difficult at high concentrations, and substrate inhibition occurs at high sugar concentrations, but the addition of ephedra can increase concentrations. Ethanol fermentation was possible even with blackstrap molasses, and any inhibition of fermentation could be overcome.

[Experiment example 8]
Lactic acid fermentation was evaluated using Ephedra extract extracted from Ephedra herbal medicine as a fermentation promoter of the present invention. Mix 40 g of Meiji Bulgaria Yogurt (registered trademark) (manufactured by Meiji Dairies Co., Ltd.), 400 g of Meiji Delicious Milk (registered trademark) (manufactured by Meiji Dairies Co., Ltd.), and 60 g of ephedra extract (final extract concentration 5 g/L), The reaction was carried out at ℃ for 8 hours.
Furthermore, instead of the ephedra extract, 5 g of the ephedra crude drug extraction residue and 60 g of water were mixed and reacted in the same manner.
Note that Meiji Bulgaria yogurt contains Lactobacillus bulgaricus and Streptococcus thermophilus as lactic acid bacteria. As the ephedra extract, the ephedra extract prepared in (1-1) of Experimental Example 1 was used. The ephedra crude drug extraction residue prepared in Experimental Example 5 (5-1) was used.
In addition, a control (no addition) was prepared by mixing 60 g of water in place of the ephedra extract and reacting in the same manner.
Lactic acid fermentation was monitored by the amount of organic acid produced by alkaline titration. FIG. 18 shows the results of plotting the amount of organic acid produced versus fermentation time. As shown in FIG. 18, the addition effect of both ephedra extract and ephedra extraction residue was also seen on lactic acid fermentation, and the amount of organic acid produced increased by 1.2 to 1.3 times.

The method for producing a fermented product of the present invention is useful because it can produce ethanol or a lactic acid fermented product in a short time. In addition, the new fermentation accelerator not only promotes alcoholic fermentation, but can also be applied to lactic acid fermentation, and enables the effective use of crude drug residues and Chinese herbal medicine residues that were conventionally discarded.
The method for producing ethanol of the present invention is an advantageous method because the fermentation time in simultaneous saccharification and fermentation can be shortened by mixing the ephedra-derived composition. In addition, the new alcohol fermentation accelerator not only promotes alcohol fermentation, but also makes it possible to effectively utilize crude drug residues and herbal medicine residues that were conventionally discarded.

Claims (10)

An ethanol production method comprising an ethanol production step of producing ethanol by alcoholic fermentation of sugars with yeast in the presence of an ephedra- derived composition ,
A method for producing ethanol, wherein the ephedra-derived composition is an extraction residue of a substance containing an ephedra crude drug . The method for producing ethanol according to claim 1 , wherein the saccharide is obtained by saccharifying cellulosic biomass. 3. The method for producing ethanol according to claim 2, wherein the ethanol production process is a saccharification and fermentation process that includes a saccharification treatment of saccharifying the cellulosic biomass with a saccharification enzyme and a fermentation treatment of alcoholically fermenting the sugars obtained by the saccharification treatment with the yeast to produce ethanol. 4. The method for producing ethanol according to claim 3, wherein in the saccharification and fermentation step , a raw material saccharification and fermentation liquid containing the cellulosic biomass, the saccharification enzyme, the yeast, and the ephedra-derived composition is prepared, and then the saccharification treatment and the fermentation treatment are carried out while maintaining a liquid temperature of the raw material saccharification and fermentation liquid at a temperature higher than an optimal temperature for the yeast. In the saccharification and fermentation step, after preparing a raw material saccharification and fermentation liquid containing the cellulosic biomass, the saccharification enzyme, the yeast, and the ephedra-derived composition, the temperature of the raw material saccharification and fermentation liquid is adjusted to the optimal temperature for the yeast. The method for producing ethanol according to claim 3 or 4, wherein the saccharification treatment and the fermentation treatment are performed at a temperature that is +1°C or higher than the yeast temperature and +5°C or lower than the optimum temperature for the yeast. In the saccharification and fermentation step, the amount (g) of the ephedra-derived composition relative to the volume (L) of the raw material saccharification and fermentation liquid containing the cellulosic biomass, the saccharifying enzyme, the yeast, and the ephedra-derived composition is 0.5 g. The method for producing ethanol according to any one of claims 3 to 5, wherein the raw material saccharification and fermentation liquid is prepared and reacted so that the amount of saccharification and fermentation is equal to or higher than /L. 3. The method for producing ethanol according to claim 2 , further comprising a saccharification step of saccharifying the cellulosic biomass into saccharides using a saccharifying enzyme before the ethanol production step. The method for producing ethanol according to any one of claims 2 to 7 , wherein the cellulosic biomass is pretreated to remove lignin. The method for producing a lactic acid fermentation product includes a lactic acid fermentation step of producing a lactic acid fermentation product by lactic acid fermentation of sugars with lactic acid bacteria in the presence of a composition derived from Ephedra ,
The method for producing a lactic acid fermentation product, wherein the composition derived from Ephedra is an extraction residue of Ephedra herbal medicine-containing material . It is a fermentation accelerator containing the extraction residue of ephedra herbal medicine .
Fermentation accelerator for the production of ethanol or lactic acid ferments .