DE2134826A1 - Process for the production of a citrate isocitrate mixture - Google Patents

Description

HECHrSANWWJf 213482 « DJt. JUR. DlPL-CHEM. WALTER BSI! ALFRED HOEPPENER DR. JUR. DlPL-CHEM. H-J. WOlW DR. JUR. HANS CHR. BEIL ₍ 12th JuW MS FIANKFUtTAMMAiN-HOCHJf

Our no. 17 236

Pfizer Inc.,

New York, M.Y., V.St.A.

Process for the production of a citrate-isocitrate mixture

The present invention relates to a process for the production of technically pure citrate-isocitrate compositions, in particular a process for the production of citric acid in an aqueous, hydrocarbon-containing nutrient medium with a citric acid-enriching strain of Candida yeast, purification and neutralization or partial neutralization
the fermentation broth with an alfcalimetall base or ammonia and concentrating the broth to a concentrate or to pesticides.

Most of the global supply of citric acid and its Derivatives is based on citric acid produced by fermentation processes generally selected strains of Aspergillus niger with carbohydrates such as Molasses and grape sugar are used as the main sources of assimilable carbon. ;

While these femnentation methods using Aspergillus niger are attractive, advice applies to many
Trouble. For example, leans over a certain amount
Aspergillus niger's ability to produce citric acid over a period of time leads to degeneration. Furthermore is a

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a relatively long period of time, usually more than 7 days, required for the production of large quantities of citric acid by such fermentations. The strongly :: efürbten filtered Fennentationsbrülien contain considerable ^f 'enfen of complex Verunreiniganssger.ischen. These factors led to the development of a faster and more stable fermentation process using yeast turner, the genus Candida in place of Aspergillus niger, and normal paraffinic hydrocarbons as the source of assimilable carbon in place of holasses and other carbohydrate materials. The Candida FerraentationsbrÜhen are almost colorless, and, depending on the ^T V. ahl of the tribe, they enthalte'n varying amounts of citric acid, isocitric acid, erythritol, arabitol and mannitol.

In the Beli / gschen patent 71o 2 * 17 a method for Production of citric acid using various yeasts of the Candida genus as ingredients described, which have the ability to fortify citric acid and assimilate hydrocarbons. In the specialist literature the spread of members of the genus Candida in coal. media containing hydrogen, for example in * "agr. Biol. Chem.", Vol. 30, p. 1175 (1966); "Nature," Vol. 215, P. 177 (1967); "Appl. Microbiol., Vol. 15, p. 690 (1967) and in "J. Bacteriol.", Vol. 93 p. 1847 (1967).

A primary goal of the present invention is manufacture an inexpensive, technically pure citrate-isocitrate mixture for use as a builder in detergent compositions. Builders improve the detergency of soaps and detergents through their effect in such areas like softening the water by binding calcium, magnesium and iron ions, generating alkalinity, stabilizing of solid dirt suspensions, emulsification of dirt

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particles, increasing the surface activity of aqueous Detergent solutions, the solubilization of those that are insoluble in water Materials, to increase the foaming or soapy properties the washing solution and peptization; of dirt line rates.

Examples of known builder compounds are alkali metal carbonates, -borates, -citrates, -phosphates, -polyphosphates, -bicarbonates and -silicates. The most common builder compounds are alkali metal tripolyphosphates and other more highly condensed phosphates. With a typical strong one Detergent has a Μθί-iger or higher phosphate content than considered necessary.

There is a growing interest in the remote ones Effectiveness of the phosphate part of food preparation products, dr "it is believed that they are the reason ffSr the accelerated F.utrophizing the Great Lakes and other inland areas are. The more highly condensed phosphates break down quickly into simple phosphates in sewage treatment plants. Those in streams, rivers and Vaschwasser flowing out of lakes lead to an excess of nutrient media, such as phosphates, with it, so that the algae growth takes over in a wet way and other LebVi · - forms, such as fish, because it uses up the oxygen in the water. Sodium citrate and isocitrate are biodegradable and quickly turns into sodium bicarbonate in sewage treatment plants and metabolizes carbon dioxide with minimal stimulatory effect on algae and others Plant life.

Sodium citrate is an excellent builder compound, which takes the place of all or most of the phosphate * step into cash and cleaning agents! can. There is one limiting factor in its use

to this day in the relatively high cost im Compared to the phosphates, thanks to the present invention, citrates can be produced at a much lower cost will.

Sodium isocitrate has been found to be an effective builder compound is, and that mixtures of sodium isocitrate and Sodium citrate the sodium citrate alone in detergent preparations are equivalent. The citrates are chemically stable, biological degradable and excellent chelating agents, and thanks to their high water solubility, they are superior nutrient compounds for weak and strong liquid detergents and cleaning agents and solid mechanical dishwashing detergents. Further Citrates are non-toxic, do not travel the skin, are harmless to paints and fibers, and are non-corrosive and non-toxic flammable.

Another object of the present invention is to produce an inexpensive, technically pure citrate-isocitrate mixture for use as a general chelating agent. The ammonium salts of citrates are effective agents in metalworking and GalvanJ & FungelSsungen. The aranonium citrate-isocitrate composition is particularly valuable as a rust remover with a wide range of applications for cleaning boilers as well as the holds of tankers.

The present invention relates to a process for the preparation of citrate-isoeifcrate compositions, which is characterized is to produce a citric acid-enriching strain of Candida yeast in an aqueous nutrient medium which at least a normal paraffinic hydrocarbon having 9 to about 23 carbon atoms as the major source of assimilable carbon contains.

The Fernentation Broth is refined, ranz or partially with neutralized by an alkali metal base or arrunonia, with active

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treated with charcoal and concentrated into a concentrate or pesticide .

Certain strains of yeast in the Candida genus, especially Candida ipolytica, have the ability to produce citric acid in high yield during the aerobic fermentation of the aqueous nutrient medium contains normal paraffinic hydrocarbons as the primary or sole source of assimilable carbon accumulate, with isocitric acid and small amounts of polyhydric alcohols, such as krythritol, Arabitol and mannitol are produced. Ea has been found that these fermentation properties fully for the production of inexpensive, technically pure citrates can be used.

It is in the initial stages of candida citric acid fermentation necessary to maintain a pH of about 4 to 7 (usually about 5 to 6) to allow cell growth. Has However, if the cell mass develops, the pH value may of course fall off as more citric acid is produced without adding an additional amount of buffering agent or other base must be added to react with the product. Fermentation is usually complete in 5-7 days. Before expiration ' by the second day the pH is already below 4, and it continues to drop to a final range of 2-3, while more Acid is generated.

The initial maintenance of the pH could be done by adding caused by caustic soda or another base. Ee is most convenient, however, simply a limited amount of a carbonate at the beginning to add, so that its buffering capacity through The citric acid produced is depleted around the time when the cell mass is in one for use as an inoculum for the Fermentation ^ has developed sufficient degree. The addition of calcium carbonate to the medium is preferred as a buffer. The CaI

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Calcium carbonate reacts with citric acid, which accumulates during the growth stage, so that the pH of the medium cannot become too acidic. E3 it is known that during the initial stages of yeast growth, when the pH in the medium drops to less than about 4.0, the growth of the yeast cells ceases or is markedly reduced. Fs is further assumed that a p; ev7is3e of Menpe can be metabolized in the reaction of citric acid with Calciuniearbonat generated carbon dioxide by the yeast cells and the ψ scnit promotes growth in the medium. Besides calcium carbonate, barium carbonates can also contain calcium oxide, parium oxide and other ruthenium known

Buffers are used.

The use of n-hexadecane as the main or only one While hydrocarbon in fermentation is preferred, other n-alkanes and n-alkenes can be used. Mixtures of hydrocarbons, including raw and semi-refined Materials can also be used, but at least a portion of a hydrocarbon should also be used a chain length of about 9 to 23 hydrocarbon atoms.

In general, the hydrocarbon is used in a concentration of about 5-9% by weight, based on the Jledium, however, this aspect is not critical, and a lower or higher hydrocarbon concentration can also be used come to use. Of the available inorganic Nitrogen sources, nitrogen salts such as ammonium sulfate, ammonium chloride and ammonium nitrate are preferred. Be of the numerous organic nitrogen sources Wheat bran, soybean meal, urea, the amino acids and peptones preferred. The company's commercial product "IJZ-Amin YTT" Sheffield Chemical Co., Norwich, H.Y. proves to be a useful one Peptone source. It is of course known that vitamins like

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Biotin and inorganic cations and ions, such as sodium, potassium, cobalt, phosphate, and sulfate, are also beneficial for yeast growth. Most of these trace vitamins and minerals are found in mois soaking water and some Nitrogen sources, e.g. soybean meal, are present, and therefore it is usually not necessary to add them individually to the fermentation medium to p-even.

As indicated above, the fermentation medium contains conventional sources of assimilable nitrogen, minerals as well as other growth factors contained in the aqueous phase. A medium that was found to be special is suitable is an anunonium salt, corn steeping water and a hydrocarbon containing medium. The corn steep water can optionally be replaced by potassium dihydrogen phosphate be replaced. The hydrocarbon concentration «* should be at least about 3 weight percent based on the medium to give a significant concentration and yield of citric acid bring; between about 5 and 20 wt. ί is the range which generally leads to optimal results. The main function of water is to act as a medium for the nitrogen sources, To provide minerals and growth factors, although some water is of course required » to carefully maintain the yeast cells. It is true that very high hydrocarbon concentrations, e.g. 50ί or even more, can be used from the practical point of view From the point of view, however, an optimal production of citric acid is achieved when the hydrocarbon concentration is kept within the preferred ranges.

While any form of aerobic incubation is satisfactory, controlled aeration is preferred, such as by stirring the Meiiun under air or bubbling air through the medium. Since the hydrocarbon in the

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aqueous phase is immiscible, it is desirable to have it during fermentation in finely dispersed form in the aqueous To hold medium so that a large surface area of the hydrocarbon is in contact with the aqueous phase. on this way there will be the greatest possible contact between the Yeast cells, the aqueous phase and the hydrocarbon erzeilt. A preferred means of achieving these goals is in submerged fermentation, "rapidly stirring the mixture while passing air through it, e.g. Gush through. To have good hydrocarbon dispersion too a surfactant can also be incorporated into the medium.

The usual temperatures known in the art for yeast growth, for example about 20-37 ° C., can be used, a range of about 25 to 29 ° C. being preferred with fermentation times of 1 to 7 days. The initial period of time for the growth of the yeast cells for the production of the inoculum is preferably 2k to ή8 hours. These general growth and fermentation conditions are well known in the art.

It is obvious that the method of the present invention also encompasses the use of mutants or variants of the Candida strain as indicated by various chemical and physical means are created provided that the citric acid enriching ability of the original Trunk is preserved. Such mutants are treated with X-rays and UV irradiation Nitrogen mustard or organic peroxides as well as others, the Methods well known to those skilled in the art. Furthermore, the use of subcultures, of course mutants, variants and the like provided for carrying out the method according to the invention.

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213A826

The citric acid fermentation broth is clarified by filtration or centrifugation, and the pH can be adjusted to the desired value, usually about 9 to 9, with an alkali metal base such as sodium or potassium hydroxide, or to about 3 to 3% ammonium hydroxide, respectively according to the intended use. Activated charcoal (0.25-10 / S based on solution solids) is added and stirred for about 15 minutes to an hour. The broth is then filtered and at atmospheric pressure or under vacuum at a temperature of about Ί0 to about HO ⁰ C, preferably 50-70 ° C, to a concentration of at least 30 * w / v. Solids, but not above about 502 ow./vol. concentrated because of excessive viscosity and solubility. The clarified fermentation broth can, however, also be neutralized or partially neutralized and, after concentration, treated with carbon dioxide.

The treated concentrated fermentation broth can be stored as such or, preferably by drum drying or spray drying, dried. Spray drying is carried out in a spray drying device which consists of a drying chamber through which a stream of heated drying air or another gas is puffed. A device for separating the dry solids obtained from the drying gas containing moisture is provided either in the drying chamber itself or separately therefrom. The solution is sprayed into the spray dryer, and air having a Lufteinlaßtemperfcur of about 200 35O ° C, and an air outlet of about 80 - 200 ⁰ C supplied.

The following examples are intended to illustrate the present invention:

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-ίο -

example 1

A potato dextrose agar slanted culture medium containing cells of Candida lipolytica ATCC 20, 228 was placed in a liquid amedium which was composed of 3 U of a commercially available source of assimilable nitrogen from peptones from the illustration of casein (commercial product "MZ Amin YTT" from the company The Sheffield Co., Norwich, MY), 3 * 1.7 g ^c iii ~ ^c ₁ β η paraffins (available from The Continental Oil Co., Jew York, NY) and 600 ml of tap water. The medium was first sterilized ^{at 120 ° C. for 30 minutes.} The Candida cells were then incubated under aerobic conditions in the medium with agitation at room temperature (27 ° C.) for 48 hours using a rotary shaker. At the end of this period, a 5 igen inoculum of Candida-VJachs turn was placed in an aqueous sterilized nutrient medium containing 5 »0 g corn steeping water, 4.0 g ammonium sulfate, 15.0 g calcium carbonate and 155 S ^c iij ~ ^c iß η-paraffins contained. The inoculated medium was stirred for 48 hours and with aner rate of _C a ?? liters / titer / hr. (4.0 ft ³ / h / gallon) vented at a temperature of 26 ° C. During this 48 hour growth period, the pH was maintained at about 5-6 to allow optimal cell development, with an additional amount of calcium carbonate buffer being added in small M aliquots as needed. This limited amount of buffer was used up while the cellulose produced citric acid, so that by the end of the 48 hours the pH was already slightly below 4 and dropped fairly quickly.

A part of 5% of this actively growing inoculum was then added to a large amount of a fermenter containing the following components per liter of the sterilized medium: 0.7 g urea, 0.001 g thiamine hydrochloride, 180 g C ^ - C ₁ ^ η -Paraffins and 0.375 g XH ₉ PO ^ (sterilized separately). That

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Fermentation medium was 114 hours (6 days) at 1,725 rpm, stirred and aerated at a rate of about 29 liters / liter / hour at a temperature of 26 ° C. The fermentation yield of citric acid monohydrate was 22 ^ g / l. At the start In this 6 day experiment the pH dropped to about 2-3 and no corrections were necessary. After the 6th Days the solids and the mycelium were centrifuged off.

18 liters of the refined broth, which was ^{heated to about PO 0} C, was adjusted to a pH of 8.6 with a 5OXi-gen sodium hydroxide solution and under vacuum (90 mm Hg) at a temperature of about 50-70 ° C a concentration of about 50? Weight / volume Solids evaporated.

Example 2

The procedure of Example 1 was repeated with the difference that activated carbon ("Nuchar C-115", commercial product of the West Virginia Paper Company) was added to the broth concentrate at a concentration of about 1 g per 30 g of solids as an additional process step; after stirring for about one hour, it was filtered.

Example 3

The procedure of Example 2 was repeated, the filtered, treated with charcoal Konsentrat was passed into a spray-drier equipped with an atomizer with a Lufteinla4emperatur of about 200 ⁰ C and an air outlet temperature of about 80 ° C, and the solids were recovered.

The almost white, free-flowing, colorless and non-flowing ones Solids had the following analysis:

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Part size

20-80 microns, average 40-50 microns density

0.59 g / cem (not dehydrated)
0 * 67 g / ccrn (dehydrated)

Water (Karl Fischer)

8.35 % w / aew.
Gas-liquid chromatography (after silylation)

Citric acid (anhydrous) 53 »1 %

Isocitric acid (anhydrous) 9.6 ί

Erythritol 0.5 %

«C-ketoglutaric acid 0.2 %

Aconitic acid 0.2 %

Traces of malic acid Mannitol traces example 1

•>

The procedure of Example 1 was repeated and the pH of the purified broth was adjusted with potassium hydroxide instead of sodium hydroxide.

Example 5

The process of Example 1 was repeated, but instead of the C ^ -C ^ g η-paraffins in the final fermenter n-Hexadefcan to achieve a comparable yield of citric acid

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used. The fermentation broth was centrifuged, the pH was adjusted to 3 to about ο with ammonium hydroxide, the broth was concentrated and treated with charcoal as described in Example 2.

Example 6

The procedure of Example 5 was repeated with the treated coal concentrate was passed at an air inlet temperature of about 35O ° C and an air outlet temperature of about 200 ⁰ C in a spray-dryer and the solids were recovered.

Example 7

The procedure of Example 1 was repeated with the difference that in the final fermentation stage the ^c i4 "* ^c i5 η-paraffins were replaced by a mixture of 28.9 g each of the following hydrocarbons: n-decane, n-nonane, n- Undecane, n-tridecane, n-pentadecane, n-hexadecane and n-octadecane; comparable results were obtained.

Example 8

A nutrient liquid medium was prepared from 150 g of corn steep water (commercial product "Cereloae", from Pirma Corn Products Sales Co., New York ? NT), 15 g of peptone, 5 g of yeast extract, 1 g of sodium chloride and one liter of water. After the disruption, this medium was inoculated with cells of Candida lipolytica ATCC 20, 228 and incubated with agitation at room temperature under submergenic aerobic conditions for 21 hours. At the end of this period the pH was 6.5. The Candida cell content of the medium (spin down) was determined by centrifuging a 15 ml sample of the medium at 200 g for 15 minutes. As one

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Density of 0.5 ml cells per 15 * nl growth medium is achieved the cell suspension was used to inoculate the respective fermentation medium.

Several milliliters of this 24 hour old inoculum were added to an aqueous, sterilized nutrient medium which contained the following components per liter: H ₃ I g urea, 0.01 g thiamine hydrochloride, 0.1 g MgSO ^ x? H ₂ O, 180 g of C ₁ ^ -C ₁₆ η paraffins (from Continental Oil Company) and 0.75 g of KH ₂ POjJ (sterilized separately).

The fermentation medium was stirred for five days at 1,725 rpm and a temperature of 25 ° C., while it was stirred at a rate of approx. 29 liters / liter / hour. has been ventilated. The pH dropped from about 6.5 to about 2 ₃ 5 within the first two days and stabilized at 2.0-2.5. The solids and mycelium were filtered off after five days. The fermentation broth was treated according to the procedure of Example 3 and the solids recovered.

t Example 9

The process of Example 1 was repeated, the C ^ - ^C 16 ⁿ "" ^Paraf ^ i ^{ne were} replaced by a hydrocarbon mixture , which consisted of η-paraffins with 9 to about 23 carbon atoms (commercial product "NP-200" of the company Continental Oil company, New York, N.Y.); comparable results were obtained.

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Claims (8)

- 15 claims: 1. Process for the production of a citrate-isocitrate gene, characterized in that it contains an isocitric acid Lemon ture broth by remote entation with a citric acid enriching strain of Candida yeast in a wäP.ripen Ilährneniuni produces, the venipstens a normal Paraffinic hydrocarbon has 9 to about 23 carbon atoms Containing this as the main source of assimilable carbon Lemon sour broth refines and completely or partially neutralizes and concentrating or drying the neutralized or partially neutralized citric acid broth. 2. The method according to claim 1, characterized in that the refined and neutralized or partially neutralized broth is treated with activated charcoal. 3. The method according to claim 1 or 2, characterized in that the refined and neutralized or partially neutralized broth to at least about 3O.t wt. / Vol. Solids is concentrated. 4. The method according to any one of the preceding claims, characterized in that "DAFE the drying stage in a spray dryer at an Lufteinlaßtenperatur between about 200" nd 35O ° C and a Luftauslaßt emperature between about 80 and 200 ⁰ C is performed. 5. The method according to any one of the preceding claims, characterized in that the yeast is a strain of Candida lipolytica used. 6. The method according to any one of the preceding claims, characterized in that nan for neutralization or partial Neutralization sodium hydroxide is used. 109884/1747 7. The method according to any one of claims 1-5, characterized in that that one uses potassium hydroxide for neutralization or partial neutralization. 8. The method according to any one of claims 1 to 5 »thereby« indicates that one uses Ammoniuinhy droxi d for neutralization or partial neutralization. For: Pfizer Inc., New York, N.Y., 'V.St .A. (Dr H.JjJKolff) Lawyer 109884/1747