JPH047194B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing citric acid from various palm oils using a fermentation method. Until now, no method has been found to ferment various palm oils, accumulate citric acid in a medium at a high concentration, and then collect the citric acid. Palm oil is produced in large quantities in Southeast Asia (4.5 million tons/year, as of 1980), and we believe that increasing the added value of these resources is important for effective resource utilization. From this point of view, the present inventors discovered a yeast that can easily assimilate palm oil,
Produces and accumulates citric acid in high concentration in the fermentation medium,
I have perfected the method to collect this. That is, the present invention involves aerobically cultivating specific yeasts of the genus Candeida and Debaryomyces in a medium containing palm oil as a carbon source, accumulating citric acid in the medium, and collecting the citric acid. This article relates to the characteristic method for producing citric acid. The microorganism used in the present invention is Candida tropicalis, which has the ability to assimilate various palm oils and accumulate citric acid in the medium.
Candida Intermedia
intermedia), Candida brumptii, Candida guilliermondii and Debaryomyces
hansenii). Furthermore, the yeast used in the present invention includes auxotrophic mutants and variants. The medium used in the present invention is selected depending on the properties of the yeast used, and the carbon source is crude palm oil,
Refined palm oil, palm olein and palm stearin alone or in mixture. It is necessary to appropriately use the above-mentioned various palm oils alone or in a mixture in order to improve the dispersibility in the medium. The amount of each type of palm oil as a raw material added to the medium is 1 to 20% by weight, preferably 5 to 15% by weight. Inorganic ammonium salts such as nitrates, sulfates, organic ammonium salts such as ammonium acetate, amino acids, urea and ammonia can be used as nitrogen sources. The various nitrogen sources mentioned above may be used alone or in combination of two or more. Common inorganic salts used include primary phosphates, secondary phosphates, sulfates, hydrochlorides, potassium salts, sodium salts, magnesium salts, iron salts, manganese salts, copper salts, zinc salts, etc. can. As organic micronutrients, natural products such as yeast extract containing vitamins such as biotin and thiamine, and constabilized liquor may be used. Aerobic conditions are preferable for fermentation. Culture temperature is 25℃
The temperature range is from 40°C to 40°C, preferably 35°C. The pH during culturing may range from 3 to 10, preferably from 4 to 7. PH adjustment during culture can be performed using calcium carbonate, caustic soda, ammonia, or the like. Cultivation is usually carried out for 50 to 150 hours, preferably 80 to 100 hours. The citric acid accumulated in the medium is separated from the solids by means normally used to separate organic acids obtained by culturing microorganisms from the culture, such as filtration or centrifugation, and then passed through a column. It can be separated and collected as citric acid or its salt by chromatography, ion exchange resin treatment, concentration treatment, etc. The present invention will be explained below with reference to Examples.
It is not limited to this. Example 1 Medium composition Refined palm oil 6.0wt% NH ₃ Cl 3.0 (g/) KH ₂ O ₄ 0.5 (g/) MgSO ₄・7H ₂ O 0.5 (g/) F ₄ SO ₄・7H ₂ O 5.0 (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/) Thiamin/hydrochloride 100 (μg/) Yeast extract 1.0 (g/) PH 5.0 As a seed culture, 30 ml of the culture solution with the above composition was dispensed into each of five 500 ml Sakaguchi flasks, then sterilized at 115°C for 10 minutes and allowed to cool to about 35°C. rear,
Candida tropicalis
tropicalis) (IFO−0589), Candida intermedia (IFO−
0761), Candida brumptii (IFO−0744), Candida guilliermondii
(IFO-0838) and Debaryomyces hansenii (IFO-0019) were transplanted in three platinum loops each. The culture conditions were a temperature of 35° C., a shaking frequency of 120 cycles/min, and a shaking width of 7 cm. The culture time is 3 days, but after confirming the growth of yeast approximately 20 hours after the start of culture, to adjust the pH, separately sterilized CaCO ₃ was added to the culture solution at 5 wt/vol%, and culture was continued. This was used as a seed culture solution. Add 30 ml of a medium with the same composition as the seed medium to the production culture.
Pour into five 500ml Sakaguchi flasks, heat sterilize at 115℃ for 10 minutes, cool to 35℃, and add the above seed culture solution.
1.5 ml (5 vol%) was inoculated each. This is 120
The cells were cultured for 7 days under the conditions of reciprocation/min, shaking width of 7 cm, and 35°C. Note that 5 wt% of separately sterilized CaCO ₃ was added to the culture solution for PH adjustment 15 hours after the start of culture. After the cultivation was completed, the culture was adjusted to pH 2.5 with hydrochloric acid, and diatomaceous earth was used as a support agent to suction it, and solid materials, mainly yeast, were washed away with a small amount of water.
Next, the liquid and the washing liquid were combined, and the combined liquid was neutralized with caustic soda and then heated to precipitate out, which was cooled and filtered by suction to obtain calcium citrate. This calcium citrate was suspended in about 10 times the amount of water, and a 50% aqueous sulfuric acid solution was added dropwise with stirring until the solution showed a slight presence of sulfate groups due to the barium chloride solution, and then boiled. About 30 underwater
Heat for a minute. If necessary, this is subjected to a decoloring operation, and then heated, and the liquid is concentrated under reduced pressure at 50 to 60°C. If gypsum precipitates are deposited during the process, they are separated, and the concentration is continued. It will turn into a slightly thin syrup. When this syrupy concentrate is left at sub-zero temperatures, citric acid crystals will precipitate. The amount of citric acid produced per fermentation liquid (g/) is as follows. Candida tropicalis
tropicalis) 52.1 Candida intermedia (Candida
intermedia) 30.4 Candida Burumptei (Candida
brumptii) 34.5 Candida gear Monday (Candida
guilliermondii) 18.9 Debaryomyces hanzenii (Debaryomyces
hansenii) 15.0 Example 2 The same method as in Example 1 was used, but the carbon source was various palm oils, and the bacterial species used was Candida tropicalis (IFO-0589).
It was hot. The results are shown in Table 1.

[Table] Example 3 An experiment was conducted using the jar fermenter of Content 3. The carbon source was refined palm oil, and the amount added to the medium was 10 wt%. The medium composition was the same as in Example 1. The yeast used was Candida tropicalis (IFO
-0589), and 5 vol% of the seed cultured in Example 1 was added to the medium. The fermentation conditions were: medium charge amount 1.5, temperature 35° C., stirring number 1000 rpm, air supply rate 1 V/V min., and fermentation time 96 Hrs. During fermentation, pH is controlled by automatically supplying 10N sodium hydroxide aqueous solution.
I kept it at 5.0. The obtained citric acid production concentration is
It was 78.7g/. The seed culture was performed in the same manner as in Example 1, and the culture time was 60 hours.

Claims (1)

[Scope of Claims] 1. In a medium containing palm oil as a carbon source, one or two selected yeasts belonging to the genus Candida or the genus Debaryomyces and capable of assimilating palm oil are added. A method for producing citric acid by a fermentation method, which comprises aerobically cultivating the above yeast, accumulating citric acid at a high concentration in the culture solution, and collecting the citric acid. 2. The method for producing citric acid according to claim 1, wherein the palm oil is one or a mixture of crude palm oil, refined palm oil, palm olein, and palm stearin. 3 Yeasts of the genus Candida are Candida tropicalis, Candida intermedia,
Candida Brumputei
brumptii), Candida guilliermondii, and the yeast belonging to the genus Debaryomyces is Debaryomyces hansenii.