JPH0475588A - Production of sorbic acid - Google Patents

Abstract

PURPOSE:To obtain sorbic acid in higher yield by oxidation of sorbaldehyde with microorganisms in the presence of specific mercapto group-protective agent to stabilize the oxidative activity of the microorganisms. CONSTITUTION:Sorbaldehyde is oxidized with microorganisms in the presence of at least one kind of SH group-protective agent selected from EDTA, ascorbic acid, SH group-carrying alcohols, amino acids and peptides. As a result, protective action of said SH group-protective group from SH enzyme-inhibitory mechanism will stabilize the microorganism activity for a long period of time leading to improving the yield of the objective sorbic acid. Said microorganisms is e.g. Alcaligenes sp., Enterobacter sp., however, may be any kind so long as it can oxidize sorbaldehyde. The concentration of the sorbaldehyde in the reaction system is 0.5-50mmol or so, while the concentration of said SH group- protective agent in the system be pref. 0.06-10mmol or so. The amount of the microbial cells to be used is 0.5-20g or so (on a dry basis) per l of the reaction liquor.

Description

[Detailed Description of the Invention] [Industrial Field of Application The present invention: ethylenediaminetetraacetic acid; alcohols, amino acids, etc. having yascorhinic acid or mercapto groups;
The present invention relates to a method for producing sorbic acid by allowing at least one mercapto group protecting agent selected from peptides to coexist in a system in which sorbaldehyde is oxidized by microorganisms.

U Prior Art Sorbic acid has excellent anti-cajicity properties and is therefore used as a food preservative.The industrial method for producing it is usually made by reacting crotonaldehyde and ketone to form an intermediate β- A method has been practiced in which the polyester is produced through lactone, and then the polyester is decomposed by thermal decomposition, acid decomposition, or exchange resin decomposition to produce sorbic acid.

However, in such a method, recovery and purification of sorbic acid after decomposition of polynistil is troublesome, the process is long, and complicated process control is required, so it is difficult to say that it is completely viable from a production and economic perspective. .

As a solution to this problem, a method has been proposed in which sorbic acid is produced by oxidizing sorbaldehyde using specific microorganisms.
The present applicant is also in the process of filing a patent application.

However, in the method for producing sorbic acid using microorganisms, the produced sorbic acid accumulates in the reaction system, but the nucleic acid is absorbed by the microorganism that oxidizes sorbaldehyde. The yield of sorbic acid decreases because the oxidative activity of microorganisms is suppressed by inhibiting mercapto group enzymes. Therefore, there are still some problems in implementing it on an industrial scale, and there are ingenious ways to improve the yield of sorbic acid by stabilizing microbial activity even if sorbic acid accumulates in the reaction system using a simple operation method. It is needed.

[Means for Solving the Problems] As a result of intensive research to solve the above problems, the present inventors found that when producing sorbic acid by oxidizing sorbaldehyde with microorganisms, ethylenediaminetetraacetic acid (hereinafter referred to as SH selected from alcohols, amino acids, or peptides having an ascorbic acid or mercapto group (hereinafter abbreviated as SH group);
The present inventors have discovered that the yield of sorbic acid can be improved by producing sorbic acid while avoiding the coexistence of at least one type of group protecting agent, and have completed the present invention.

Does sorbic acid cut the nosulfite bonds of mercapto enzymes (hereinafter abbreviated as SH enzymes) in microorganisms, destroy the tertiary structure, and reduce the activity of SH enzymes?The present invention; ED T
, A or ascorbic acid or S selected from alcohols, amino acids, or peptides having a S H group
By allowing at least one type of H group protecting agent to coexist, the SH enzyme inhibition mechanism described above is prevented by the S H group protecting agent, thereby stabilizing microbial activity over a long period of time and improving the yield of sorbic acid. .

The present invention will be explained in detail below.

First, the microorganisms used in the present invention include the genus Alcaligenes, the genus Enterobacter, and the genus Proteus.
oteus), Rhodoseudomonas (Rhodops)
eudomonas) genus, Corynebacterium (Fl
avobacterium genus, Nocardia (Noc.
ardia), Mycobacterium
erium), Bacteridi
um) genus, Streptomyce
s) genus, Notrobacter (C1trobacter)
Genus, PseudomonaS, Acetobacter, Arthrobacter, Corynebacterium, Saccharomycopsl
s), genus Klebsiella,
Rhodotorula genus, Bacillus (
Bacillus S), Microbacterium (Micr
genus obacterium, Paracoccus
occus ) genus, Serratia (5erratia) genus,
Brevibacterium
Examples include the genus Escherichia, the genus Escherichia, and the genus Micrococcus, and any of them can be put to practical use as long as they have the ability to oxidize sorbaldehyde.

Furthermore, to give some specific examples, Alcaligenes eutropus (Alcaligenes eutropus)
hus AT CCI 7699), Enterobacter cloac
aa, IFO12935), Proteus mirabilis (
Proteus ff1irabilis; I F
O13300), Rhodopseudomonas spheroids (Rhodopseudomonas 5pher)
oides ; IFO12203), Flavobacterium aheolenos
um 5uaveolens ATCC958), Nocardia sp.
+4326), Mycobacterium rotochus (Myc
obacterium rhodochrous:
I F Ol 3161), Bacterium subgIanc
lis; IFO31322), Streptomyces alhydroflavus
albidoflavus IF 013010),
Pseud
ononas pseudoalcaligenes
, l F 014167), Pseudomonas earuginosa
; IFO3445), Acetobacter ascendens:
l F O3188), Acetobacter pasteurianus subsp.
er pasteurianus 5ubsp Io
vanienIFO! 3753), 7-7, Cybacter ox
ydans; ATCCl 4359), Arthrobacter au
IFOI 2136), Corynebacterium daltamicum
um glutamicum AT CC2+650
), sub-power romykopnos lipolyti-power (Sacch
allomycopsis 1ipolytica;
IF 01548), Talebnoella pneumoniae (
Klebsiella pneumoniae.

IFO3321), Rhodotorula minuta (Rhodot
orulaminuta; IFO1435), Bacillus cereus (I
F 0 13690), Microbacterium ammoniaphyllum fJicrobacterium amm
oniaphilum; ATCC21490), Paracoccus denitrificans (Paracoccu
s denitrificans ; r P 0 1
2442), Serrati marcescens
a marcescens; IFO3046), Brevibacterium lactofamentum (Breviba
cterium lactofermentum A
TCC13655, ), Brevibacterium flavum (A
T CC21269), Brevibacterium ammoniagenes (Brevibacterium+IIo
niagenes; IFOI 2072)
, Escherichia col
i; I F O3301), Micrococcus roseus I F
0 3764) etc.

Of course, the present invention is not limited to these.

As the medium for culturing the microorganisms mentioned above, any medium may be used as long as it contains a carbon source, a nitrogen source, etc. and allows the microorganisms to grow.

As a carbon source, any compound that does not inhibit the sorbic acid production activity of microorganisms can be used, such as glucose, nuclose, ethanol, ethylene glycol, propylene glycol, 4-butanediol,
Glycerin, acetaldehyde, acetic acid, propionic acid t
Examples include. Further, as the nitrogen source, meat extract, peptone, cornstarch liquor, urea, ammonium sulfate, ammonium chloride, sodium nitrate, etc. can be used. Furthermore, inorganic salts such as phosphates, magnesium salts, calcium salts, iron salts, copper salts, zinc salts, and other nutritional substances necessary for the growth of microorganisms can be added to the medium as necessary.

In oxidizing sorbaldehyde, the microorganism obtained as described above is used, and at least one SH group protecting agent selected from EDTA, ascorbic acid, or SH group-containing alcohols, amino acids, or peptides is added to the reaction system. This is done by coexisting with the Among SH group protecting agents, examples of alcohols having an SH group include notiothreitol, 2-mercaptoethanol, etc., examples of amino acids having an SH group include nostino, and examples of peptides having an SH group include nostino, etc. Examples include reduced glutathione. The SH group-protecting agent can be used together with a medium containing the grown microorganisms described above, or it can be collected from the medium and suspended in water, physiological saline, or buffer for one day. It is okay to let things coexist.

The concentration of sorbaldehyde in the system is OI ~ 100 mmol,
It is preferably added in a range of about 05 to 50 mmol,
The concentration of the SH group protecting agent in the system is suitably in the range of about 0.003 to 30 mmol, preferably about 0.006 to IO mmol. The preparation method can be arbitrarily changed such as batch, divided, continuous, etc., but batch is advantageous in practice.

Approximately 0.5 to 209 (dry bacteria) cells are used per 1 ρ of the reaction solution.

During the reaction, stir and use aerobic conditions. In order to set such conditions, it is preferable to blow air, oxygen, or a mixed gas containing other gases as necessary into the system, or to set the oxygen concentration in the solution to I ppm or more.

The reaction temperature is preferably 10 to 70°C, preferably 20 to 40°C, and the reaction time is advantageously about 01 to 200 hours.

Furthermore, it is also possible to appropriately add coenzymes such as PQQ and SAD (P), surfactants, organic solvents, etc. to the reaction solution as necessary.

Furthermore, the reaction can be carried out by combining a culture method using microorganisms in the growth phase with a reaction using resting microorganisms, or by using other immobilized microorganisms or a reaction using a microorganism extract, either alone or in combination with the above methods. etc., various aspects are possible.

After the reaction is completed, sorbic acid is purified according to a conventional method to obtain the desired product.

[Function] In producing sorbic acid by oxidizing sorbaldehyde with microorganisms, the present invention uses ethylenediaminetetraacetic acid, ascorbic acid, or mercapto selected from alcohols, amino acids, or peptides having a mercapto group in the system. By coexisting at least one type of group protecting agent, it is possible to reduce the adverse effect of sorbic acid on the mercapto enzyme in the microorganism, stabilize the oxidation activity of the microorganism over a long period of time, and improve the yield of sorbic acid.

"Example] Next, the present invention will be explained in more detail with reference to Examples.

Examples 1 to 5 Ordinary nutrient medium (meat extract 39, peptone 109, salt 5
water, 1σ, PH7) 50m (!) was placed in a Sakalo flask, and after sterilization, Acetobacter ascendens (IFO3
188) was inoculated into one platinum loop and cultured at 37°C for 24 hours with reciprocal shaking.

After culturing, collect the bacteria by centrifugation and adjust the pH to 7,0,0,1.
After washing with M phosphate buffer, 901 bacterial cells were obtained in dry terms.

Next, put 601 bacterial cells on a dry basis into a large test tube and adjust the pH.
After adding IOm of 7,0,0,1M phosphate buffer and suspending the bacterial cells, a predetermined amount of the mercapto group protecting agent shown in Table 1 was added, and then 10m9 of sorbaldehyde was added to start the reaction. .

The reaction was carried out at 30°C for 20 minutes and I min M? The reaction solution was stirred by shaking back and forth 200 times. After the reaction is complete, separate the supernatant of the reaction solution by centrifugation, and pH it with hydrochloric acid.
2 and 1 Thereafter, the yield of sorbic acid relative to the consumed sorbaldehyde was measured by gas chromad analysis.

The results are summarized in Table 1.

The conditions for Gas Chromado 9 leaves are as follows.

Equipment, Hikorenoto Paso Cart Model 5890
Ram, Methyl Noricon wide bore column 0.53mm
x I 5 m x 8.0 μm column concentration, 80°C, 8 minutes ~ temperature increase (15°C/min) ~ 210°C, 4 minutes Helium flow rate +20m (+/min detection; FID Examples 6 to II Example 1 The same operations as in Example 1 were performed except that the strain and mercapto group protecting agent shown in Table 2 were changed.

The results are shown in Table 2.

Control Example 1 In Example I, the same operation as in Example 1 was carried out without the coexistence of a mercapto group protecting agent, and as a result, the yield of sorbic acid per 1 consumed nisorbaldehyde decreased to 47%.

[Effect] In the present invention, in producing sorbic acid by oxidizing sorbaldehyde with microorganisms, sorbic acid selected from ethylenediaminetetraacetic acid, ascorbic acid, or alcohols, amino acids, or peptides having a mercapto group in the system is used. By carrying out the reaction in the presence of at least one type of mercapto group protecting agent, it is possible to reduce the adverse effect of sorbic acid on the mercapto enzyme in microorganisms, stabilize the oxidation activity of microorganisms over a long period of time, and improve the yield of sorbic acid.
It is extremely useful in industry.

Claims (1)

[Claims] 1. In producing sorbic acid by oxidizing sorbaldehyde with microorganisms, ethylenediaminetetraacetic acid or ascorbic acid or mercapto selected from alcohols, amino acids, or peptides having a mercapto group in the system is used. A method for producing sorbic acid, which comprises coexisting at least one type of group protecting agent. 2. The manufacturing method according to claim 1, characterized in that dithiothreitol and 2-mercaptoethanol are used as the alcohol having a mercapto group. 3. The production method according to claim 1, characterized in that cysteine is used as the amino acid having a mercapto group. 4. The production method according to claim 1, characterized in that reduced glutathione is used as the peptide having a mercapto group.