JPH0476676B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a method for producing citric acid in high yields by aerating oxygen under aerobic culture conditions. Prior Art A method for producing citric acid using yeast of the genus Candida using normal paraffin (paraffin having 10 to 20 carbon atoms) as a fermentation raw material has been known for some time. Furthermore, a method for producing citric acid by aerobically cultivating microorganisms belonging to Candida tropicalis using a fermentation medium containing olefins as a carbon source was published by the present applicant and the same applicant. A patent has been applied for (Patent No. 1096815, etc.). In citric acid fermentation from a fermentation medium containing paraffins and olefins as carbon sources, it varies depending on the culture conditions, but
In addition to citric acid, considerable amounts of isocitric acid are always produced. The amount may be the same as or greater than the amount of citric acid. Problems to be Solved by the Invention Conventionally, in the production of citric acid by petroleum fermentation and oil/fat fermentation, multiple mutations have been developed in order to suppress the production of isocitric acid, which is a by-product, and make the production of citric acid dominant. Methods using stocks have been adopted. However, this method requires a lot of effort and expense, such as searching for mutant strains and determining suitable culture conditions. Furthermore, even when using the obtained mutant strain, production of isocitrate may occur due to the almost unavoidable influence of iron ions, resulting in a decrease in the yield of citric acid. This is because the enzyme aconitase, which converts citric acid to isocitrate, which is retained in the bacterial cells, is reactivated by the presence of iron ions. Therefore, even if a mutant strain that produces less isocitrate is obtained, careful attention must be paid to its maintenance and management. According to the method of the present invention, the production of isocitrate can be suppressed without using mutant strains, so the industrial significance of the present invention is extremely large. Means for Solving the Problems In producing citric acid for the purpose of improving the yield of citric acid, the present inventors discovered that by aerating oxygen during culture under aerobic conditions, The present invention was achieved by discovering that by increasing the oxygen concentration, the production of isocitric acid can be suppressed and the yield of citric acid can be improved. The present invention involves culturing yeast selected from the genus Candeida aerobically in a medium containing fats and oils, fatty acids, their esters, linear paraffins, and sugars as carbon sources, and adding citric acid to the medium. In the citric acid manufacturing method, which involves accumulating and collecting citric acid, the production of isocitric acid is suppressed by aerating oxygen during cultivation under aerobic conditions, and citric acid is obtained in high yield. The present invention relates to a method for producing citric acid. The amount of dissolved oxygen is at least 10 ppm or more, preferably 20 ppm or more, and more preferably 30 ppm or more. If the amount of dissolved oxygen is 10 ppm or more, the effect of suppressing isocitric acid production can be obtained. The microorganism used in the present invention is a yeast selected from the genus Candeida that has the ability to assimilate various carbon sources and accumulate citric acid in the medium. Furthermore, auxotrophic mutants and variants of these yeasts are also included. Oils and fats used as carbon sources in the present invention,
There are no particular restrictions on fatty acids obtained from these fats and oils, natural or synthetic fatty acids, and esters thereof, and they can be used individually or as a mixture as appropriate. Also, as a normal paraffin, the number of carbon atoms is 10.
to 20 are used, but usually n-C ₁₂
~n-C ₁₃ is often used. Furthermore, various sugars are also good carbon sources. The amount of fermentation raw material added to the medium is 1 to 20% (by weight), preferably 5 to 15% (by weight). Inorganic ammonium salts, nitrates and ammonia can also be used as nitrogen sources. The various nitrogen sources mentioned above may be used alone or in combination of two or more. As the inorganic salt, common inorganic salts such as primary phosphate, secondary phosphate, sulfate, hydrochloride, potassium salt, sodium salt, magnesium salt, manganese salt, copper salt, zinc salt, etc. can be used. Furthermore, calcium carbonate, ammonia, sodium hydroxide, etc. are used to adjust the pH. As the organic micronutrients, pyotin and thiamine alone or a mixture thereof, and natural products containing these, such as yeast extract and constable liquor, may be used. When using thiamine and piotin individually, 1000μg/or less is sufficient;
Even if it is used more than that, there is no change in the amount of citric acid produced. Preferably, it is appropriate to use 50 to 100 μg/. When thiamine and biotin are used together, it is sufficient to use a total of 1000 μg/or less. In the fermentation medium used in the method of the present invention, it is of course possible to use a surfactant that does not have an inhibitory effect in order to improve the dispersibility of the fermentation raw material in the medium, but it is also possible to maintain the fermentation raw material in a liquid state under the culture conditions. Therefore, there is no need to use surfactants. Therefore, there is no need to consider the inhibitory effect of surfactants on yeast. Fermentation conditions are preferably aerobic conditions with oxygen aeration. The culture temperature ranges from 25°C to 40°C, preferably from 25°C to 38°C. PH during culture is 3 or more
A range of 10, preferably 4 to 6 is appropriate. Adjustment of pH during culture can be performed using acid, alkali, calcium carbonate, sodium carbonate, or ammonia. Cultivation is usually carried out for 50 to 150 hours, preferably 60 to 100 hours. The citric acid accumulated in the fermentation medium is removed by separating the bacterial cells using methods normally used to separate organic acid salts produced by culturing microorganisms, such as filtration or centrifugation. After that,
It is separated and collected as citric acid or its salts by column chromatography, ion exchange resin treatment, or concentration treatment. Citric acid and isocitric acid were analyzed using high performance liquid chromatography. Examples The present invention will be explained below with reference to Examples, but the present invention is not limited thereto. Example 1 Palm olein was used as a carbon source. The strain used is Candeida tropicalis IFO-
It was 0589 (Candia tropicalis). The basic composition of the medium used in the method of the present invention is as follows. For seed mother Palm Olein 5 (wt/vol%) NH ₄ Cl 0.50 (wt/vol%) KH ₂ PO ₄ 0.05 (wt/vol%) MgSO ₄・7H ₂ O 0.05 (wt/vol%) FlSO ₄・7H ₂ O 1 (mg/) MnSO ₄・7H ₂ O 0.1 (mg/) ZnSO ₄・nH ₂ O 0.1 (mg/) CuSO ₄・5H ₂ O 5 (μg/) Biotin 100 (μg/) Thiamine hydrochloride 100 (μg/) PH 5.0 For production Palm Olein 10wt% NH ₄ Cl 0.3 (wt/vol%) KH ₂ PO ₄ 0.05 (wt/vol%) MgSO ₄・7H ₂ O 0.03 (wt /vol%) FlSO ₄・7H ₂ O 5 (mg/) MnSO ₄・7H ₂ O 0.06 (mg/) ZnSO ₄・nH ₂ O 0.05 (mg/) CuSO ₄・5H ₂ O 5 (μg/) Biotin 50 (μg/) Thiamine hydrochloride 100 (μg/) PH 5.0 Seed culture was carried out using a flask shaking culture machine under the following conditions. As a seed culture, 4 platinum loops of the bacterial strain were inoculated from the agar slant medium with the above composition after slant culture into a liquid medium containing 30 ml of the medium with the above composition in a 500 ml shoulder shake flask, and then shaken 120 times. round trip/minute,
The cells were cultured at 30°C for 3 days using a reciprocating shaker with a shaking width of 7 cm. In addition, when the pH of the medium decreased to 5.0 or less, separately sterilized CaCO ₃ 5wt/vol% was added to adjust the pH. Oxygen can be aerated when culturing seedlings using a jar fermentor. Production culture was carried out using this seed mother. Put 1258 ml of the above culture solution, 166 ml of palm olein, and 75 ml of seed mother into the jar fermenter described in Contents 3, and heat at 34℃.
Rotation speed: 1200 r.pm, oxygen aeration rate: 1/2 V/V/min. (dissolved oxygen amount: 10 ppm or more)
It is. After the cultivation was completed, the culture was brought to pH 2.5 with hydrochloric acid, and diatomaceous earth was used as an aid to suction the mixture, and solid materials, mainly yeast, were washed with a small amount of water. Next, the solution and the washing solution were combined, neutralized with caustic soda, heated to precipitate, cooled and filtered under suction to obtain calcium citrate. The obtained calcium citrate was suspended in about 10 times the amount of water, and a 50% aqueous sulfuric acid solution was added dropwise while stirring until the solution showed the slight presence of sulfate groups due to the barium chloride solution. After that, heat it in boiling water for about 30 minutes. If necessary, this is subjected to a decoloring operation, and then heated, and the liquid is concentrated under reduced pressure at 50 to 80°C. If gypsum precipitates are deposited during the process, this is separated, and further separated, Continue concentrating it until it becomes a thin syrup. This syrup-like concentrate was allowed to stand below freezing to precipitate citric acid. In a similar manner, culture was performed using air aeration instead of oxygen. The results are shown in Table 1.

[Table] It can be seen that both the production amount and the citric acid production ratio in total citric acid significantly improved near the final stage of culture. Example 2 Normal paraffin having 10 to 20 carbon atoms was used as a carbon source. The amount of normal paraffin mixture added to the medium was 10 wt/vol%. The basic composition of the production medium and culture conditions are as in Example 1.
It was the same. However, the bacterium used was Candida lipolyteica ATCC-20346 (Candida
lipolytica). The experimental results are shown in Table 2.

[Table] Example 3 Glucose was used as the carbon source, and the culture conditions were as in Example 1.
It was the same. The bacterial species used was Candida lipolytica ATCC-20346 (Candida lipolytica).
It was hot. The results of Example 3 are shown in Table 3.

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Claims (1)

[Claims] 1 Candeida spp., which has the ability to produce citric acid by assimilating oils and fats, fatty acids, esters of these fatty acids, linear paraffins, and saccharides, singly or in a mixture, as carbon sources. A method for producing citric acid in which yeast selected from Candida (Candida) is aerobically cultured, citric acid is accumulated in the medium, and citric acid is collected. A method for producing citric acid, which is characterized in that production of isocitric acid is suppressed and citric acid is obtained at a high yield by controlling the oxygen concentration in the medium to a dissolved oxygen amount of 30 ppm or more.