JP7351513B2 - Fermentation bacteria aggregation inhibitor and method for producing useful substances - Google Patents

Description

The present invention relates to an aggregation inhibitor for fermentation bacteria and a method for producing a useful substance.

Tween 80 {polyoxyethylene sorbitan monooleate (20 moles of EO added), Tween is a registered trademark of Croda International PLC. }, using a surfactant such as Tween 40 {polyoxyethylene sorbitan monopalmitate (added with 20 moles of EO)}, the cellulase (useful substance) produced in Trichoderma reesei (fermenting bacteria) can be efficiently removed from the bacteria. Although it has been known for a long time that fermentation bacteria are secreted by aggregation inhibitors (Non-Patent Document 1), there is no known method for suppressing the aggregation of fermentation bacteria using a fermentation bacteria aggregation inhibitor and improving the productivity of useful substances. Ta.

Fermentation Handbook, edited by Bioindustry Association Fermentation and Metabolism Study Group, Kyoritsu Publishing Co., Ltd., published July 2001, 253 pages.

The present invention provides an aggregation inhibitor that can improve the productivity of useful substances (amino acids, proteins, alcohols, carboxylic acids, amines, etc.).

The feature of the aggregation inhibitor for fermentation bacteria of the present invention is that it is an aggregation inhibitor for fermentation bacteria used in the fermentation process for producing useful substances, and is characterized by containing a polyoxyalkylene compound (A) represented by formula (1). The main points include the following points.

R1[(-AO)n-(EO)m-R2]p (1)

R1 is a reactive residue of trehalose, sucrose, sorbitol, arabinose, erythritol, glycerin, galactose or xylitol , AO is an oxyalkylene group having 3 or 4 carbon atoms, EO is an oxyethylene group, R2 is a hydrogen atom or a methyl group , and n is a m is an integer of 0 to 20 ; p is an integer of 3 to 8 ;

The feature of the method for producing a useful substance of the present invention is that in the fermentation process for producing a useful substance selected from industrial raw materials, edible raw materials, or pharmaceutical raw materials, the above-mentioned aggregation inhibitor is added to the fermentation solution. do.

The aggregation inhibitor for fermentation bacteria of the present invention can improve the productivity of useful substances (amino acids, proteins, alcohols, carboxylic acids, amines, etc.).

By using the production method of the present invention, useful substances can be produced with high production efficiency.
In addition, by using the aggregation inhibitor of fermentation bacteria of the present invention, aggregation of fermentation bacteria is suppressed, the contact area of fermentation bacteria with raw materials, culture solution, etc. increases, and the reaction efficiency to useful substances and the production of useful substances are increased. It is thought that the secretion efficiency is improved and, as a result, the productivity of useful substances is improved.

Useful substances include, but are not particularly limited to, amino acids, proteins, alcohols, carboxylic acids, hydroxycarboxylic acids, amines, and hydrocarbons.

Examples of amino acids include valine, leucine, aspartic acid, glutamic acid, and histidine.

Examples of proteins include enzymes (oxidoreductases, hydrolases, synthetic enzymes, etc.), hormone proteins (interferon α, growth hormone, insulin, glucagon, etc.), photoproteins (luciferase, etc.), and peptides.

Alcohols include monools (methanol, ethanol, propanol, butanol, dodecanol, isooctadecanol, phenol, octadecenol, etc.); polyols (ethylene glycol, propylene glycol, diglycerin, trimethylolethane, trimethylolpropane, etc.); Sugars {monosaccharides (glycerin, erythrulose, erythritol, arabinose, xylulose, xylitol, fructose, sorbose, glucose, mannose, lactose, sorbitol, etc.), disaccharides (sucrose, lactose, trehalose, etc.), trisaccharides (raffinose and maltotriose) ), tetrasaccharides (such as acarbose and stachyose), and polysaccharides (such as starch, cellulose, dextrin, and chitin).

Examples of the carboxylic acid include ethanoic acid, propanoic acid, decanoic acid, octadecenoic acid, ethanedioic acid, butanedioic acid, butenedioic acid, 2-methylidenebutanedioic acid, benzoic acid, and benzenedicarboxylic acid.

Examples of hydroxycarboxylic acids include 2-hydroxyethanoic acid, 2-hydroxypropanoic acid, 2,3-dihydroxypropanoic acid, 2-hydroxybutanedioic acid, 2-dihydroxybutanedioic acid, 2-hydroxypropane-1,2,3 -tricarboxylic acid and 2-hydroxybenzoic acid.

Examples of the amine include ammonia, methylamine, triethylamine, ethanolamine, triethanolamine, ethylenediamine, and triethylenetetramine.

Hydrocarbons include methane, ethane, propane, decane, paraffin, and the like.

The reactive residue (R1) of an active hydrogen-containing compound having 1 to 30 carbon atoms means a reactive residue obtained by removing an active hydrogen atom from an active hydrogen compound having 1 to 30 carbon atoms. Examples of active hydrogen-containing compounds having 1 to 25 carbon atoms include compounds containing at least one hydroxyl group (-OH), imino group (-NH-), amino group (-NH ₂ ), and/or carboxyl group (-COOH). Included are alcohols, amides, amines, carboxylic acids, hydroxycarboxylic acids and amino acids.

Alcohols include monools (methanol, butanol, stearyl alcohol, oleyl alcohol, isostearyl alcohol, etc.), polyols (ethylene glycol, propylene glycol, butylene glycol, isobutylene glycol, sorbitan, diglycerin, tetraglycerin, trimethylolethane, and triglycerin). methylolpropane, etc.) and sugars {monosaccharides (dihydroxyacetone, glycerin, erythrulose, erythritol, arabinose, xylulose, xylitol, fructose, sorbose, glucose, mannose, lactose, sorbitol, etc.), disaccharides (sucrose, lactose, trehalose, etc.), Trisaccharides (such as raffinose and maltotriose), tetrasaccharides (such as acarbose and stachyose), pentasaccharides (such as pentaose), and hexasaccharides (such as hexaose), and the like.

Examples of amides include monoamides (such as formic acid amide, propionic acid amide and stearyl amide) and polyamides (such as malonic acid diamide and ethylene bisoctyl amide).

Examples of amines include monoamines (dimethylamine, ethylamine, aniline, stearylamine, etc.) and polyamines (ethylenediamine, diethylenetriamine, triethylenetetramine, etc.).

Examples of the carboxylic acid include the same carboxylic acids as explained in the section of useful substances.

Examples of hydroxycarboxylic acids include 2-hydroxyethanoic acid, 2-hydroxypropanoic acid, 2,3-dihydroxypropanoic acid, 2-hydroxybutanedioic acid, 2-dihydroxybutanedioic acid, 2-hydroxypropane-1,2,3 -tricarboxylic acid, 2-hydroxybenzenic acid and 12-hydroxyoctadecanoic acid.

Examples of amino acids include glycine, 4-aminobutanoic acid, 6-aminohexanoic acid, and 12-aminododecanoic acid.

Among these, alcohols are preferred from the viewpoint of productivity, and more preferably sugars, particularly monosaccharides (dihydroxyacetone, glycerin, erythrulose, erythritol, arabinose, xylulose, xylitol, fructose, sorbose, glucose, mannose, lactose, sorbitol, etc.), disaccharides (sucrose, lactose, trehalose, etc.) and trisaccharides (raffinose, maltotriose, etc.), most preferably monosaccharides and disaccharides.

Examples of the oxyalkylene group (AO) having 3 to 18 carbon atoms include oxypropylene, oxybutylene, oxyisobutylene, oxytetramethylene, oxy(1- or 2-phenylethylene), oxy-1,2-decylene, oxy-1 , 12-dodecylene, oxy-1,2-dodecylene and oxy-1,2-octadecylene. When using multiple types of oxyalkylene groups (AO) having 2 to 18 carbon atoms, these may be added by block addition or random addition.

Among these, from the viewpoint of productivity, oxypropylene, oxybutylene, oxytetramethylene and oxy(1- or 2-phenylethylene) are preferred, and oxypropylene and oxybutylene are more preferred.

Among hydrogen atoms or monovalent organic groups having 1 to 24 carbon atoms (R2), monovalent organic groups having 1 to 24 carbon atoms include alkyl groups (R), alkenyl groups (R'), acyl groups ( -COR) and aroyl groups (-COR'). In the chemical formula written in parentheses, R and R' correspond to the above-mentioned alkyl group (R) and alkenyl group (R'), respectively.

Examples of the alkyl group (R) include methyl, butyl, t-butyl, octyl, 2-ethylhexyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and tetracosyl (lignoceryl).

Examples of the alkenyl group (R') include butenyl, octenyl, isooctenyl, dodecenyl, octadecenyl, and the like.

Among the hydrogen atom or the monovalent organic group having 1 to 24 carbon atoms (R2), a hydrogen atom and an alkyl group are preferred from the viewpoint of productivity.

n is an integer of 2 to 100, preferably an integer of 3 to 70, more preferably an integer of 6 to 40.

m is an integer of 0 to 100, preferably 0 to 40, more preferably 0 to 20.

p is an integer of 1 to 20, preferably an integer of 2 to 15, more preferably an integer of 3 to 8.

The polyoxyalkylene compound (A) can be produced by known organic chemical reactions (polyoxyalkylene addition reaction, esterification reaction, Williamson synthesis method, etc.).

The polyoxyalkylene compound (A) represented by formula (1) may be composed only of the same type of polyoxyalkylene compound, or may be composed of multiple types of polyoxyalkylene compounds (for example, R1, AO, n, , m , R2 and/or p are different). Furthermore, in the polyoxyalkylene compound (A) represented by formula (1), when p is 2 to 20, p [(-AO)n-(EO)m-R2] groups are n, m , AO and/or R2 may be a combination of p different parts or all.

The aggregation inhibitor for fermentation bacteria of the present invention may contain other constituents in addition to the polyoxyalkylene compound (A), as long as they do not inhibit the effects of the present invention. Other constituents include antifoaming agents, thickeners, dispersants, preservatives, antifreeze agents, and diluting solvents (water, mineral oil, organic solvents, etc.). When other constituents are included, there are no limitations on the method for preparing the fermentative bacteria aggregation inhibitor of the present invention as long as the polyoxyalkylene compound (A) and the other constituents can be mixed uniformly.

The amount of the fermentation bacteria aggregation inhibitor of the present invention added is not particularly limited, but is preferably about 0.0001 to 10% by weight based on the weight of the fermentation liquid.

There is no particular restriction on the method of using the aggregation inhibitor for fermentation bacteria of the present invention, and it can be added to and mixed with the raw material of the useful substance in advance, or continuously dripped during the fermentation process.

In the method for producing useful substances of the present invention, there are no particular restrictions on the fermentation bacteria that can be used, and known microorganisms (for example, those described in paragraph 0023 of JP-A No. 2013-39085 and paragraph 0012 of JP-A No. 2011-92188) microorganisms) can be used.

As a method for producing the useful substance of the present invention, known methods can be applied. In addition, there are no particular restrictions on the raw materials that can be used, as long as they do not kill or inhibit the growth of fermentation bacteria and provide useful substances. ), cellulose raw materials (wood such as coniferous or broad-leaved trees, kenaf, mulberry, mulberry, reed, bamboo, etc.), livestock human waste, waste water, microbial culture medium, etc. can be used.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. In addition, unless otherwise specified, parts mean parts by weight.

<Examples 1 to 9>
By a known method, the following polyoxyalkylene compounds (a1) to (a9) {the values in <> are values corresponding to formula (1)} were synthesized, and each of them was in turn synthesized as the aggregation inhibitor for fermentation bacteria of the present invention ( 1) to (9).

(a1) Block adduct of trehalose with 160 moles of propylene oxide and 80 moles of ethylene oxide <n=20, m=10, p=8>
(a2) 88 mol propylene oxide adduct of sucrose <n=11, m=0, p=8>
(a3) Block adduct of sorbitol with 30 moles of butylene oxide and 100 moles of ethylene oxide <n=6, m=20, p=5>
(a4) Block adduct of arabinose with 40 moles of butylene oxide and 16 moles of ethylene oxide <n=10, m=4, p=4>
(a5) Erythritol block adduct of 15 moles of propylene oxide, 30 moles of butylene oxide, and 15 moles of ethylene oxide <n=15, m=5, p=3>
(a6) Block adduct of 42 moles of propylene oxide and 15 moles of ethylene oxide of glycerin <n=14, m=5, p=3>
(a7) Block adduct of 24 moles of propylene oxide and 8 moles of butylene oxide of sucrose <n=4, m=0, p=8>
(a8) Pentamethyl ether of 25 mol propylene oxide adduct of galactose <n=5, m=0, p=5>
(a9) Block adduct of xylitol with 160 moles of propylene oxide and 12 moles of ethylene oxide <n=40, m=3, p=4>

<Comparative example>
TWEEN40 {Polyoxyethylene sorbitan monopalmitate (20 mole ethylene oxide adduct), "TWEEN" is a registered trademark of Croda International PLC. } was used as a fermentation bacteria aggregation inhibitor (h) for comparison.

<Evaluation of flocculation of fermentation bacteria and quantification of useful substance (ethanol)>
A fermentation liquid was prepared by uniformly mixing 200 parts of commercially available sugarcane molasses (purchased from Marukyo Nosan Co., Ltd.), 50 parts of commercially available dry yeast (purchased from Nissin Foods Co., Ltd., Super Camellia), and 750 parts of ion-exchanged water. , put 300 mL of this fermentation liquid into a glass container with an inner diameter of 100 mm and a height of 200 mm, and add evaluation samples {aggregation inhibitors (1) to (9) and (h)} to this fermentation liquid in the amounts shown in the table below. The glass container was placed in a constant-temperature water tank whose temperature was controlled at 40°C, and after 30 minutes of stirring, the presence or absence of aggregation in the fermentation liquid was visually confirmed, and the amount of useful substances (ethanol) was determined, as shown in the table below. Indicated.
A blank was evaluated in the same manner as above except that the evaluation sample was changed to 40 μL of deionized water, and the results are shown in the table below.

Note that the useful substance (ethanol) was quantified by gas chromatography (under the following conditions). A calibration curve was created using special reagent grade ethanol, and the concentration (% by weight) was calculated based on the weight of the fermentation liquid.

Equipment: Agilent 7890A (Agilent Technologies, Inc.)
Column: Agilent J&W GC column-HP-5 (inner diameter 0.25 mm, length 30 m, film thickness 0.25 μm, Agilent Technologies, Inc.)
Detector: Agilent 5977A (Agilent Technologies, Inc.)
Carrier gas and flow rate: He, 2mL/min
Sample injection volume: 1μL

The fermentation bacteria aggregation inhibitor of the present invention was able to suppress aggregation compared to conventional surfactants. When the fermentation bacteria aggregation inhibitor of the present invention was used, the productivity of useful substances (ethanol) was higher than that of conventional surfactants.

It should be noted that aggregation of fermentation bacteria is thought to occur when hydrophobic proteins present in the cell membrane on the surface of the fermentation bacteria coalesce together through hydrophobic interaction, and the aggregation inhibitor of the present invention It is thought that it adsorbs to the hydrophobic protein moiety), suppresses the occurrence of hydrophobic interactions, and suppresses aggregation. As shown in the above results, the polyoxyalkylene compound (A) with a specific chemical structure adsorbs to the cell membrane (hydrophobic protein part) on the surface of fermentation bacteria and suppresses the occurrence of hydrophobic interactions, thereby preventing aggregation. It is thought that it was suppressed.
Therefore, if the hydrophobic protein is present in the cell membrane on the surface of the fermenting bacteria, it will similarly adsorb to the cell membrane (hydrophobic protein part) on the surface of the fermenting bacteria and prevent aggregation by suppressing the occurrence of hydrophobic interactions. It is thought that this can be suppressed.

The aggregation inhibitor for fermentation bacteria of the present invention is suitable as an additive used in the production of useful substances using fermentation bacteria.

Claims (2)

An aggregation inhibitor for fermentation bacteria used in a fermentation process for producing useful substances, characterized by containing a polyoxyalkylene compound (A) represented by formula (1). agent.

R1[(-AO)n-(EO)m-R2]p (1)

R1 is a reactive residue of trehalose, sucrose, sorbitol, arabinose, erythritol, glycerin, galactose or xylitol , AO is an oxyalkylene group having 3 or 4 carbon atoms, EO is an oxyethylene group, R2 is a hydrogen atom or a methyl group , and n is a m is an integer of 0 to 20 ; p is an integer of 3 to 8 ; A method for producing a useful substance, which comprises adding the aggregation inhibitor according to claim 1 to a fermentation solution during fermentation to produce a useful substance selected from industrial raw materials, edible raw materials, or pharmaceutical raw materials.