JP2820279B2 - Method for producing sorbic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention relates to a method for producing sorbic acid by microbiologically oxidizing sorbaldehyde, by maintaining the activity of the microorganism for a long period of time, and The present invention relates to a method for producing the acid in a high yield by accumulating it in a high concentration.

[Prior art] Sorbic acid or a salt thereof has been used as a food preservative because of its excellent antifungal activity. However, as an industrial production method, crotonaldehyde is usually reacted with ketene. In this method, the polyester is produced via an intermediately formed β-lactone, and then the polyester is thermally decomposed, acid-decomposed or ion-exchange resin-decomposed to produce sorbic acid.

However, such a method is not always advantageous in terms of production and economy, for example, the recovery or purification operation of sorbic acid after polyester decomposition is troublesome, the process is long, and complicated process control is required.

As a solution to this problem, a method of producing sorbic acid by treating sorbaldehyde with a specific microorganism has recently been proposed (Japanese Patent Laid-Open No.
No.-152990) has been proposed, and the present applicant has also filed a patent application (Japanese Patent Application No. 63-230534).

[Problems to be Solved by the Invention] In such an oxidation method using microorganisms, it is considered that how high a concentration of sorbic acid can be accumulated in this reaction system is a problem for industrialization. The main consideration was the study of the yield depending on the type of.

However, the present inventors have examined the relationship between the yield of sorbic acid and other factors during oxidation without being bound by the conventional fixed concept of selecting microorganisms. As oxidation conditions, for example, as described in JP-A-63-152990, various factors such as temperature, PH control, additives, reaction atmosphere and the like can be considered.

[Means for Solving the Problems] Accordingly, the present inventors conducted detailed studies to improve the yield of sorbic acid by selecting the reaction conditions during the oxidation reaction, and as a result, determined the PH of the system during the reaction to a specific value. It has been found that the amount of sorbic acid accumulated can be drastically improved by adjusting with an alkali metal compound, and the present invention has been completed.

That is, the present invention provides a method for producing sorbic acid by oxidizing sorbaldehyde with a microorganism capable of oxidizing sorbaldehyde, wherein the sorbic acid produced is neutralized with a sodium compound to reduce the pH of the system to 6%. A method for producing sorbic acid, wherein the reaction is carried out while adjusting as described above. "

A feature of the present invention is that the pH of the system at the time of the reaction is adjusted with a sodium compound. As a result, an unexpected effect was obtained in which the microbial activity was stabilized for a longer period than when other metal compounds were used, and the amount of sorbic acid accumulated was dramatically improved.

First, microorganisms used in the present invention include a genus Alcaligenes and Enterobacter
genus, Proteus, Rhodopseudomonas, Flavobacterium, Nocardia,
Mycobacterium, Bacteridium, Streptomyce
s) genus, Citrobacter, Pseudomonas, Acetobacte
r) genus, genus Arthrobacter, genus Corynebacterium, genus Saccharomycopsis, Klebsiella
ella), Rhodotorula, Bacillus (B
acillus), Microbacterium
Genus, Paracoccus, Serratia
ia), Brevibacterium, Escherichia, Micrococcus
coccus), and any of them can be used as long as they have the ability to oxidize sorbaldehyde.

Further, to give some specific examples, Alkaligenes
Eutrophus (Alcaligenes eutrophus; ATCC 1769)
9), Enterobactor cl
oacae; IFO 12935), Proteus mirabilis (Proteus
mirabilis; IFO 13300), Rhodopseudomonas spheroides; IFO 12203,
Flavobacterium suaveorensu
suaveolens; ATCC 958), Nocardia SP (No
cardia sp.; IFO 14326), Mycobacterium rhodochrous (IFO 13161), Bacteridium subglandi
s; IFO 31322), Streptomyces albidoflavus (IFO 13010), Pseudomonas pseudo
alcaligenes; IFO 14167), Pseudomonas earuginosa (IFO 3445), Acetobacter ascendens; IFO 31
88), Acetobacter pasteurianus subsp lovanien (Acetobacter pasteurianus subsp lov)
anien; IFO 13753), Arthrobactor oxydans (ATCC 14359), Arthrobacter aurescens; IFO 121
36), Corynebacterium glutamicum (Corynebact
erium glutamicum (ATCC 21650), Saccharomycopsis lipolytica (IFO)
1548), Klebsiella pneumonia
umoniae; IFO 3321), Rhodotorul
a minuta; IFO 1435), Bacillus cereus (Bacillus)
cereus; IFO 13690), Microbacterium anmoniaphilum; ATCC 2149
0), Paracoccus denitrificans (Paracoccus
denitrificans; IFO 12442), Serratia marcescens (IFO 3046), Brevibacterium lactofermentum
tum; ATCC 13655), Brevibacterium flavum (Br
evibacterium flavum; ATCC 21269), Brevibacterium ammoniagenes (Brevibacterium anmoniagenes;
IFO 12072), Escherichia col
i; IFO 3301), Micrococcus Roseus
s roseus; IFO 3764).

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

In the reaction, of course, microbial cells,
Alternatively, an oxidase derived from such a microorganism may be used, or an immobilized cell or an immobilized enzyme may be used.

The oxidation reaction is to add solvaldehyde to the culture medium of microorganisms capable of oxidizing solvaldehyde,
The microorganisms are collected from the culture medium, and this is carried out by an arbitrary method such as adding sorbaldehyde to a system in which water, saline, and a buffer are dispersed, but using a sodium compound during the reaction as described above, PH of the system is 6 or more, preferably 6.5
It is necessary to limit to ~ 8.5. The purpose of the use of the sodium compound is to prevent the oxidizing activity of the microorganism from being inactivated by sorbic acid generated in the system as the reaction proceeds, and it is not necessary to completely neutralize sorbic acid. Neutralization may be performed, and in any case, the sodium compound may be added so that the pH becomes 6 or more.

If the pH is less than 6, sorbic acid is hardly neutralized and remains as a free acid, so that the effect of the present invention is hardly obtained.

The method of charging the reaction solution with the sodium compound of the present invention can be arbitrarily changed, such as batch, division, or continuous, as long as the pH of the system is maintained at 6 or more, but a sequential charging method is practically advantageous.

The sodium compound of the present invention is not particularly limited as long as it is a compound containing sodium, and examples thereof include sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium borate, sodium acetate, sodium phosphate and the like. More than one species may be used in combination.

The sodium compound may be used in any form such as a solid such as a powder or a granule or a liquid dissolved in water or an organic solvent, but is usually used in an aqueous solution.

Solvent concentration in the reaction solution is 0.01-
It is added in an amount of 10%, preferably about 0.05 to 5%. The charging method can be arbitrarily changed, such as batch, division, or continuous. However, in practice, the sequential charging method is advantageous.

The cells are used in an amount of about 0.5 to 2.0 g (dry bacteria) per suspension.

At the time of the reaction, stirring is performed and aerobic conditions are adopted. In order to set these conditions, air or oxygen, or a mixed gas obtained by mixing other gases as necessary, is blown into the system. It is desirable that the oxygen concentration in the solution be 1 ppm or more.

The reaction temperature is 10-70 ° C, preferably 20-40 ° C, and the reaction time is
It is selected from the range of about 0.1 to 150 hours.

Further, a coenzyme such as PQQ and NAD (P), a surfactant, an organic solvent, and the like can be appropriately added to the reaction solution as needed.

After completion of the reaction, the method for isolating sorbic acid from the reaction solution is not particularly limited, but usually, first, solids such as cells are removed from the reaction product solution by a method such as centrifugation, and then the supernatant is removed. Add any acid such as hydrochloric acid, sulfuric acid, phosphoric acid, etc.
Sodium sorbate is hereinafter referred to as sorbic acid.
Thereafter, the desired sorbic acid is obtained by purification and isolation according to a conventional method.

[Action] In the present invention, when sorbic acid is produced by oxidizing sorbaldehyde with a microorganism capable of oxidizing sorbaldehyde, sorbic acid produced is neutralized with a sodium compound to reduce the PH of the system to 6%. By performing the reaction while adjusting as described above, the oxidation activity of the microorganism is stabilized for a long time,
The sorbic acid yield is greatly improved.

[Example] Next, the method of the present invention will be described more specifically with reference to examples.

Example 1 Ordinary nutrient medium (potato extract 10% by weight, glucose 1.
0% by weight, ethanol 0.5% by weight, peptone 3% by weight, yeast extract 0.5% by weight, PH7) were inoculated with Acetobacter ascendens (IFO 3188) and cultured with shaking at 30 ° C. for 48 hours. After culturing, collect cells by centrifugation, PH7, 1 / 15M
After washing twice with a phosphate buffer, it was used for the reaction.

Next, PH7, so that the above cells become 12 g / l on a dry basis.
Suspend in 1 / 15M phosphate buffer, 100 ml of this cell suspension
Was placed in a 500 ml round bottom flask, and sorbaldehyde was mixed therein, and an oxidation reaction was carried out at 30 ° C. while stirring.

Solvent was added in 50 mg increments every 30 minutes, and the pH of the reaction solution was constantly maintained at 6.8 to 7.1 with 4N aqueous sodium hydroxide.

The upper part of the reaction vessel was opened, and the reaction solution was constantly brought into contact with fresh air.

This reaction was carried out for 72 hours until the bacteria were inactivated, and sorbic acid was obtained in a yield of 83.6 g / l (100 g / l as sodium sorbate).

Examples 2 to 7 and Control Examples 1 to 5 Instead of the microorganism of Example 1 and the 4N aqueous sodium hydroxide solution, the microorganisms and PH adjustment solutions shown in Table 1 were used.
The experiment was performed in the same manner as in the above example except that the reaction time was changed.

 The yield of the obtained sorbic acid is also shown in Table 1.

[Effect] In the present invention, when sorbic acid is produced by oxidizing sorbaldehyde with a microorganism capable of oxidizing sorbaldehyde, the generated sorbic acid is neutralized with a sodium compound to increase the pH of the system to 6 or more. By carrying out the reaction while adjusting the concentration, the activity of the microorganism can be stabilized for a long time, and sorbic acid can be accumulated to a high concentration, which is extremely useful in industry.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI (C12P 7/40 C12R 1:13) (C12P 7/40 C12R 1:13) (C12P 7/40 C12R 1:15) (C12P 7/40 C12R 1:32)

Claims (1)

(57) [Claims] When sorbic acid is produced by oxidizing sorbaldehyde with a microorganism capable of oxidizing sorbaldehyde, the generated sorbic acid is neutralized with a sodium compound to increase the pH of the system to 6 or more. A method for producing sorbic acid, wherein the reaction is carried out while adjusting.