DE2215141A1 - Process for the production of citric acid by fermentation - Google Patents

Description

Ajinomoto Co., Inc. Tokyo / Japan

Process for the production of citric acid by

Fermentation

The invention relates to a process for the production of citric acid by fermentation and deals in particular with a process for the production of citric acid, wherein in an aqueous Medium new species of yeast, namely Candida fibrae and Candida subtropicalis, can be cultivated and the citric acid accumulated in the fermentation broth is recovered.

Citric acid is widely used in the food industry and the pharmaceutical industry, for example as an acidulant for beverages or in used in the production of jams. Citric acid is also used as an antioxidant or as a stabilizer used in various food products.

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So far, citric acid has been made from carbohydrates such as molasses by cultivating yeasts. Recently, it has been reported that certain strains of Candida contain citric acid from carbohydrates or produce n-alkanes.

New species of yeasts have now been isolated, namely Candida fibrae and Candida subtropicalis, from natural occurrences and found that the new species made from citric acid in high yields Different types of assimilable carbon sources can form when aerobically in an aqueous Medium to be cultivated.

The invention provides a process for the production of citric acid, wherein a from Candida fibrae or Candida subtropicalis selected microorganism is cultivated aerobically in an aqueous medium and is used in Citric acid accumulated in the fermentation broth is obtained will-

The new species according to the invention can contain citric acid from various types of carbon sources form in high yield, while the known citric acid-forming yeasts only form citric acid from one or at most two types of carbon sources, for example η-paraffin and carbohydrates im Case of Candida lipolytica, form. Therefore it is possible without any change in the microorganisms that to choose the most preferred carbon sources or those available on the market at lower cost Use carbon source.

The microorganisms used in the context of the invention are new species of the genus Candida, viz Candida fibrae and Candida subtropicalis and have the following toxanomic properties:

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Candida fibrae NAKASE sp. nov.

Growth in malt extract: after 3 days at 25 ⁰ C, the cells are round, oval, cylindrical or oblong (2.5-7) x (3-7-60), and occur singly, in pairs or as Pseudomycele on. Numerous Eyphal fragments are observed. A sediment and a trace of a ring are found. After a month at 17 ^° C., a thin ring and a sediment are present.

Growth on malt agar: after 3 days at 25 ⁰ C only real mycelia are formed. After one month at 17 ⁰ C the streak culture is yellowish-white white hair-like, albeit angled up over the entire surface "part and has a ciliierten edge.

Bevel culture: real mycelium development is abundant. Papilated hyphae are often observed. Blastospores are round to oval in chains or in clusters,

Fermentation: DG-lucose, B-galaetose (slow) ₉ sucrose, maltose (slow and weak), Raffinos® (slow) and trehalose (hidden) are fermented. Melibiose, melezitose, inulin, D-arabinose, L-rhamnose, dulcit, DL-lactic acid and inositol are not assimilated.

Potassium nitrate is not used as the sole source of nitrogen.

Arbutin is split.

Gelatin is liquefied.

No starch-like joints are formed.

The maximum growth temperature is 37-38 ⁰ C.

Vitamins are not required for growth at 25 ^{° C.}

Urease is not detected on the Christensen medium.

Origin: bananas from the food market, Tokyo, May 1962.

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This species forms abundant, well-developed true mycelia and ferments significant numbers of sugars and is similar to Candida tropicalis in these respects, but differs from this one, since it assimilates raffinose, D-ribose, erythritol, ferments raffinose, liquefies gelatin, no vitamin requires and a lower maximum growth temperature than Candida tropicalis.

Candida suptropicalis NAKASE, FUKAZAWA et TSUCHIYA sp. nov.

Growth in MY broth: after 3 days at 25 ⁰ C the cells are round, short-oval, oval or oblong, (2.5-9) x (3-11-30) / U and occur singly, in pairs or in chains.

A pellet is formed.

Growth on MY agar: after one month at 17 ^° C., the colony is white to pale yellowish white, flat to raised, smooth, soft and has a complete or ciliated margin.

Bevel culture on tomato dextrose agar: pseudomycele and real mycelia are abundantly produced. Blastospores are round to short-oval or oblong and occur singly in chains and sometimes in clusters.

Formation of ascospores: not observed.

Fermentation: D-glucose, D-mannose, D-galactose, Sucrose, maltose (sometimes weak) are fermented. Lactose, raffinose and melibiose are not fermented.

Assimilation of carbon compounds: D-glucose, D-mannose, D-galactose, sucrose, I-sorbose (weak), Ellobiose (varying), trehalose, melezitose, D-xylose, Ethanol, glycerine (sometimes latent), adonitol, D-mannitol, D-sorbitol, 2-methyl-D-glucoside, salicin (varying), Potassium gluconate (varying), calcium 2-ketogluconate, succinic acid and citric acid are assimilated. Lactose,

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Melibiose, raffinose, inulin, soluble starch, L-arabinose, D-arabinose, D-ribose, L-rhamnose, erythritol, dulcitol and inositol are not assimilated.

Assimilation of potassium nitrate as the only nitrogen source: negative.

Splitting fat: positive,

Hydrolysis of urea: negative (Christensen medium), Liquefaction of gelatine: negative, production of the predominant carotenoid pigment:

negative,

Production of excess amounts of acids: negative, formation of starch-like compounds: negative, required vitamin: biotin, maximum growth temperature: 40-41 ⁰ C, optimal pH value for growth: 4-7.

This species differs from Candida tropicalis in that it does not assimilate soluble starch and not a fine one Hair forms while an aged colony of Candida tropicalis is covered with fine hair.

The microorganisms were mainly identified according to J. Lodder and N.J.W. Kreger-van Rij, "The Yeasts, a Toxanomic Study ", North Holland Publ. Co., Amsterdam, 1952.

Candida fibrae and Candida subtropicalis are in the Fermentation Research Institute, Agency of Industrial Science and Technology., No. 1-8-5, Inage-higashi, Chiba-3hi, Chiba-ken, Japan, laid down as FERM-P No. 889 and FERM-P No. 888.

The aqueous media used according to the invention contain assimilable carbon sources Sources of nitrogen and inorganic salts and, if necessary, small amounts of organic nutrients.

Carbohydrates, alcohols, organic acids, n-alkanes, glycerides can be used as assimilable carbon sources and other fats or oils and also raw materials,

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containing these carbon materials can be used. These carbon sources can be used singly or in the form of mixtures of several. The carbon sources, in particular the alcohols or acids, are preferably added to the aqueous medium during the cultivation period in accordance with the consumption of the carbon sources.

Assimilable carbohydrates include glucose, fructose, cane sugar and starch. Hydrolysates of starches or molasses can also be used as carbon sources.

Suitable n-alkanes are those with carbon numbers from 9 to 20. They are taken individually or in the form of mixtures used. These carbon sources are made up of carbohydrates, Normal alkanes, ethyl alcohol, and acetic acid are the most preferred carbon sources as they are commercially available at a reasonable cost are available.

Suitable nitrogen sources are inorganic and organic ammonium salts, gaseous ammonia, aqueous Ammonia, amino acids, and other common sources of nitrogen.

The inorganic salts are completely common and examples include phosphates, sulfates, chlorides, as well Potassium, sodium, magnesium, iron (II), iron (III) or manganese salts.

Al3 organic auxiliary nutrient, biotin is required for the growth of Candida subtropicalis. In some Cases int it to increase the yield of citric acid effective, to the medium a yeast extract, malt extract, corn swelling liquid, Peptone, meat extract, soybean hydrolyzate, Adding casein hydrolyzate or other raw materials, which contain organic auxiliary nutrients.

The fermentation is carried out aerobically. The fermentation is allowed to proceed normally to 37 ⁰ C for two to seven days at temperatures of 24th The pH of the medium is usually in the range of 5 to 9 by

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Addition of alkalis or acids kept.

The citric acid accumulated in the permentation broth is obtained by conventional methods, for example after removing the cells, the citric acid is separated from the filtrate by ion exchange resins or the filtrate is concentrated and the calcium citrate from the concentrated piltrate obtained after the addition of calcium hydroxide.

example 1

An aqueous medium was prepared which contained 5 g / dl glucose, 0.4 g / dl yeast extract, 0.1 g / dl KH ₂ PO ₄ , 0.1 g / dl MgSO ₄ -7H ₂ O, 3 g / dl CaCO - (sterilized separately) and adjusted its pH to 6.0. Each 20 ml of the aqueous medium was placed in a 500 ml flask and then heated in a steam autoclave.

The sterilized media were fibrae with Candida or Candida subtropicalis and cultured at 3O ⁰ C for 72 hours with shaking.

After the cultivation, a content of 2.0 g / dl or 2.5 g / dl of citric acid was found in the permentation broths found from Candida fibrae and Candida subtropicalis.

1 liter of the Candida fibrae permentation broth was filtered and acidified with sulfuric acid. After removing the precipitated calcium sulphate by piltration the piltrate was neutralized with calcium hydroxide and heated. 31.5 g of crystals of calcium citrate were obtained.

Example 2

An aqueous medium was prepared which

2 g / dl ethyl alcohol, 0.4 g / dl yeast extract, 0.05 g / dl KH ₂ PO ₄ , 0.05 g / dl MgSO ₄ -7H ₂ O and 3 g / dl CaCo ₅ (separately

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sterilized) and adjusted its pH to 6.0. A number of 500 ml flasks were each mixed with 20 ml of the aqueous medium and then heated in a steam autoclave.

The sterilized media were inoculated with Candida subtropicalis fibrae or Candida and cultured at 30 ⁰ C for 72 hours with shaking.

After cultivation, 0.8 g / dl and 0.9 / dl of citric acid were found in the permentation broths of Candida fibrae or Candida subtropicalis collected.

Example 3

An aqueous medium was prepared containing 5 g / dl η-paraffin (commercially available), 0.4 g / dl yeast extract, 0.1 g / dl KH ₂ PO ₄ , 0.1 g / dl MgSO ₄ -7H ₂ O, Contained 3 g / dl CaCO ₃ (sterilized separately), and the pH was adjusted to 6.0. 20 ml of the aqueous medium were added to a series of 500 ml flasks and then heated in a steam autoclave.

The sterilized media were inoculated with Candida subtropicalis fibrae or Candida and cultured at 30 ⁰ C for 72 hours. The Candida fibrae and Candida subtropicalis permentation broths obtained contained 2.5 g / dl and 2.8 g / dl of citric acid, respectively.

Example 4

An aqueous medium was prepared containing 2.94 g / dl calcium acetate, 0.3 g / dl yeast extract, 0.05 g / dl KH ₂ PO ₄ , 0.05 g / dl MgSO ₄ * 7H ₂ O, and the pH adjusted to 6.0. 20 ml of the aqueous medium was added to a series of 500 ml flasks and thrown through Er-.

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heat sterilized.

The sterilized media were inoculated with Candida subtropicalis fibrae or Candida and cultured at 30 ⁰ C for 72 hours. The resulting fermentation broths of Candida fibrae and Candida subtropicalis contained 0.8 g / dl and 1.0 g / dl of citric acid, respectively.

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Claims (5)

2215H1 - ίο - claims 1. Process for the production of citric acid, characterized in that one of the microorganisms Candida fibrae or Candida subtropicalis is cultivated aerobically in an aqueous medium and the Citric acid accumulated in the fermentation broth is recovered. 2. The method according to claim 1, characterized in that an aqueous medium which carbohydrates as a carbon source is used. 3. The method according to claim 1, characterized in that an aqueous medium which n-alkanes with Containing carbon numbers from 9 to 20 or mixtures of such alkanes as the carbon source is used. 4. The method according to claim 1, characterized in that an aqueous medium which is an alcohol, in particular ethyl alcohol is used as a carbon source. 5. The method according to claim 1, characterized in that an aqueous medium which is an organic Acid, especially acetic acid, as a carbon source contains is used. 209843/0684