AU596103B2 - Microbiological method for preparation of citric acid - Google Patents

Description

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FORM 10 SPRUSON FERGUSON COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: U Class Int, Class Complete Specification Lodged: b'0 00 o 4 4 4* 4 0r 04t Accepted: Published: Priority: Related Art: Name of Applicant: 0 4o 4 4 o 4 0 *1

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Address of Applicant: Actual Inventor(s): Address for Service: EXPERIMENTALNY ZAVOD BIOKHIMICHESKIKH PREPARATOV INSTITUTA MIKROBIOLOGII IMENI AVGUSTA KIRKHENSHTEINA AKADEMII NAUK LATVIISKOI SSR USSR, Riga, ulitsa Lenina, 222 ALMA ALBERTOVNA RUMBA, INGEMARA EDVARDOVNA SKRASTYNYA, ROMAN YANOVICH KARKLIN and VIA KARLOVNA AZANDA Spruson Ferguson, Patent Attorneys, Level 33 St Martins Tower, 31 Market Street, Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: "MICROBIOLOGICAL METHOD FOR PREPARATION OF CITRIC ACID" The following statement is a full description of this invention, including the best method of performing it known to us SBR/as/087U rsrcZ :I MICROBIOLOGICAL METHOD FOR PREPARATION OF CITRIC ACID

ABSTRACT

A microbiological method for preparation of citric acid comprising surface fermentation on a nutrient medium containing the sources of carbon, nitrogen, mineral salts, growth stimulants, of the microorganism citric acid producer whose function fulfilled by the strain Aspergillus niger R-4 obtained by selection from the strain Aspergillus niger oo R-3 by adapting it to the media with various concentrations 0 0 o0" of cane molasses, exposing it to mutagenous factors nitrosomethyl urea and UV rays, and by natural selection of active cultures, deposited in the collection of the Central Museum of Industrial Microorganisms of the All-Union Research Institute of Genetics and Breeding of Industrial Microorganisms, file No. LMIIIM F-307, then, on completion of fermentation of said strain, the biomass is separated and the deo0 a sired product is extracted from the cultural liquid.

9 9 9 9- 99S 1A MICROBIOLOGICAL METHOD FOR PREPARATION OF CITRIC ACID The present invention relates to microbiological industry and more particularly it relates to a microbiological method for preparation of citric acid used in food industry for the production of confectionery products, soft drinks, canned foods, also in pharmaceutical and perfumery industries.

o 10 Known in the prior art are various microbiological methods for the preparation of citric acid, for example a method wherein the function of the producer is played by the strain of a mould fungus Aspergillus niger R-3.

The method consists in that said strain is subjected to surface fermentation of a nutrient medium containe t I ing the sources of carbon, nitrogen, mineral salts, and growth stimulants. Fermentation proceeds on a nutrient medium with the source of carbon in the form of beet molasses at a temperature of 32°C in the course of 9 20 or 10 days (Inventor's Certificate of the USSR No.

939549, IPC C 12 P7/48, publ. 1982).

Said method ensures a high yield of the desired product when the source of carbon is constituted by beet molasses and cannot be used with cane molasses since the producer in the method, i.e. the strain of Asoergillus niger R-3, grows extremely slowly on the nutrient medium based on cane molasses, which involves the risk of development of foreign microflora.

2 As compared with beet molasses, the cane molasses contains a larger number of inhibitors of growth and biochemical activity of producers, therefore the known strains either fail to grow at all, or demonstrate a low biochemical activity on the media based on cane molasses.

Another prior-art microbiological method for preparation of citric acid consists in surface fermentation of the citric acid producer, strain of Aspergillus 10 niger T55 on a nutrient medium whose carbon source is .o cane molasses. Before preparation of the nutrient medium the cane molasses goes through extensive treatment (repeated boiling, centrifuging, autoclaving, fractionating). The inoculating material is prepared in the t 15 form of suspension of spores.

4.

After such a complex treatment of molasses this producer yields citric acid in the amount of 65 wt.-% of the sugar of the source nutrient medium. (Biotechnology and Bioengineering, Vol. XXVI, pp 1114 1121, S" 20 1984).

Said method is feasible in laboratory conditions alone. It is noted for sophisticated technology since cane molasses passes through long preliminary preparation for making the nutrient medium; besides, the inoculating material in the form of spore suspension is spread irregularly over the surface of the fermentation medium. Said method features a low yield of citric acid (up to 65 of the sugar of the source nutrient

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medium).

The main object of the invention consists in developing a new producer strain for evolving a method based on g cheap source material for the nutrient medium, i.e. cane molasses, increasing the yield of citric acid and simplifying its production techniques.

This problem has been solved owing to the fact that in the herein-disclosed microbiological method for preparation of citric acid by surface fermentation of a 10 microorganism, citric acid producer, strain Aspergillus niger, on the nutrient medium containing the sources of carbon and nitrogen, mineral salts and growth stimulants followed by separation of biomass and extraction of desired product from the cultural liquid, ac- 15 cording to the invention, the function of the microorganism, citric acid producer, is fulfilled by the strain Aspergillus niger R-4, obtained by selection from the strain Aspergillus niger R-3 by adapting it to the media with various concentrations of cane molasses, exposing it to mutagenous factors nitrosomethyl urea and UV rays, and by natural selection of active cultures, said strain being deposited in the collection of the Central Museum of Industrial Microorganisms of the All- Union Research Institute of Genetics and Breeding; file numberLMIIM F-307.

Owing to the use of a new producer strain Aspergillus niger R-4, the disclosed method permits the utilization of a cheap source material, i.e. cane molass- -4 I 't SI 4 $I 4 4i 4, -4es, increases the yield of citric acid up to 75 of the sugar of the source nutrient medium. The disclosed method also simplifies the process of preparation of citric acid since it dispenses with long preliminary treatment of cane molasses for the preparation of the nutrient medium.

Besides, the use of inoculation material in the form of dry spores permits its uniform distribution over the surface of the nutrient medium which is conducive to normal and rapid development of the mycelium of the microorganisms.

The disclosed method is accomplished as follows.

The citric acid is prepared with the aid of a new strain Aspergillus niger R-4.

The strain Aspergillus niger R-4 has been obtained by selection. The source strain was the industrial citric acid producer, strain Aspergillus niger R-3. The strain R-3 was deposited at the Central Museum of 15 Industrial Microorganisms of the All-Union Research Institute of Genetics on April 4, 1980 under No. F-132.

The strain R-3 has the following characteristics.

Morphological features.

The 5-day culture on Tchapek Dox medium has round conidium heads, 200-220m in diameter. The vesicles are slightly elongated, 34 x 37 46 x 50pm. The sterigmas are of a single-layer type. The length of sterigmas varies from 9 to 15pm. The conidia are dark brown, round, 5 7Rm in diameter. The conidiophores are 1 3 mm long.

Cultural features.

25 On the fifth day a gigantic colony of the strain R-3 is grown on Tchapek Dox medium in Petri dish at 32°C, has a round shape, 40 45 mm in diameter, the asporogenic zone is 4 8 mm.

The colonies on agar wort (5 days, at 32°C) have a round shape, 46 48 mm in diameter. Asporogenic zone is 6 10 mm. The centre of a 30 colony is convex, dark brown, rarely sporiferous, highly resistant to antagonist bacteria present in the process of citric acid fermentation.

Biochemical features.

The strain effectively ferments the beet molasses. The citric acid yield of the said beet molasses sugar is about 100%, In the sum of synthesized acids the amount of citric acid is 95-99% and that of oxalic acid, 0 1%.

9444r~ 0 9 '9 44~ 9 #41 4444r 9 444$ r TLH/38T 4A Relation to carbon sources.

Assimilates saccharose, glucose, fructose, maltose, vegetal resources hydrolysates. Does not assimilate acetic acid.

Relation to nitrogen source.

Assimilates organic and mineral nitrogen.

The strain Aspergillus niger R-3 has been adapted to the media with various concentrations of cane mo 1 .sses. The cultures received by adaptation have been exposed to the effect of mutagenous factors: nitrosomethyl urea and UV rays followed by natural selection of active cultures.

The strain Aspergillus niger R-4 is prepared from R-3 as follows.

The strain R-3 was adapted to media containing cane molasses, and the adapted culture was exposed to nitrosomethyl urea and UV illumination.

For purposes of adaptation use was made of a nutrient medium ':15 containing 20% cane iolasses. The culture was reseeded 10 times, and the adapted culture was treated with a 1% solution of N-nitrosomethyl urea with an exposure time of 2 hours, exposure to a quartz mercury vapor lamp for 2 minutes from the distance of 20 cm. Irradiation intensity 170 erg/mm per sec.

The selection of strain colonies was realized after the culture has been grown in Petri dishes according to morphological features, and the selected and isolated colonies were tested for the activity of the S l biosynthesis of the citric acid; the most active culture was selected and 4* propagated. This strain was R-4.

9 25 The new producer Aspergillus niger R-4 is deposited in the collection '6 of the Central Museum of Industrial Microorganisms of the All-Union Research Institute of Genetics and Breeding, file number IIMIIM F-307.

The strain Aspergillus niger R-4 has the following characteristics.

t* TLHI/38T i -i a~ Morphological features The 5-day culture on Capek-Dox medium has round conidium heads, 120-200jum in diameter. The vesicles are slightly elongated, 18-25 x 30-35ju- in size. The sterigmas are of the two-layer type: first order fsterigmas 9-16/um long and second order sterigmas, 5-9,/um long. Two second order sterigmas are formed on each first order sterigma. The conidia are greyish-black, round, in diameter. The conidiophores are straight, 10 colourless, 1.5-2.5 mm long, 6.0-6.5jum in diameter.

a 09 a Cultural features O" On the fifth day a gigantic colony on Capek-Dox medium at 32°C has a round shape, 52-55 mm in diameter.

The colour of the colony is greyish-black with asporo- S 15 genic zone of 14 mam. The conidial heads in the centre of the colony are of a medium size, uncompacted, and form a small elevation. The conidiai heads become smaller towards the edge of the colony and acquire a yellowgreenish hue.

a* 20 The 5-day colonies on agar wort grown at 3200 are round, of greyish-black colour, 40 mm in diameter.The air mycelium is medium-developed with sparsely arranged condial heads in the centre, decreasing in size from the centre to the edge of the colony. Within a radius of 10 mm along the edge of the colony there is a light zone without conidial heads or with immature white conidial heads tightly pressed against the substrate mycelium. The back side of the colony is white, with raw-n 6 t 4 4 t 4 S dial folds.

Biochemical features.

The strain effectively ferments the cane molasses.

In laboratory conditions during surface fermentation (the layer 9 cm high) the yield of citric acid after 9 days is 70-75 of the sugar in the source nutrient medium (initial sugar content in the nutrient medium being 13-15%). In the sum of synthesized acids the amount of citric acid is 95-98 and that of oxa- 0 lic acid, 0.5-1.0 Relation to carbon sources Assimilates saccharose, glucose, fructose, maltose, peat bydrolysate. Does not assimilate acetic acid.

Relation to nitrogen source.

5 Assimilates organic and mineral nitrogen.

The activity of biosaythesis of ctric acid on the media based on beet and cane molasses has been determined for the initial strain Aspergillus niger R-3 and the new producer Aspergillus niger R-4.

0 The comparative data appear in the Table below.

Table Citric acid yield,wt.-% of sugar beet molasses cane molasses t

S.

t* t, t tt 44 4 e t 4 Aspergillus niger-3 Aspergillus niger-4 up to 100 80-85 45-50 70-75 After 2L4 hours of cultivation on the medium with -7cane molasses the strain Aspergillus niger R-4 forms a developed mycelium.

The strain Aspergillus niger R-3 starts forming mycelium only on the 3rd day of cultivation.

Under industrial conditions the slower the development of mycelium, the higher is the danger of development of a fore n culture.

The inoculation material of the strain Aspergillus niger R-4 is obtained by growing it on the nutrient medium containing the sources of carbon, nitrogen and mineral salts. The source of carbon and nitrogen may be ft either malt extract or brewing wort (5 9% sugar, dry matter). The strain is grown on the surface, adjusting the temperature (320C) and humidity. The growing time is 9 10 days.

1 dm 2 of the area of inoculation nutrient medium 4 40 produces 0.5 0.7 g of spores. The spores are collected by vacuum. The inoculation material is prepared by mixing dry spores with active carbon in a proportion 44 of 1:1, carefully stirring the mixture. 12he following step is fermentation. The source of carbon for the preparation of fermentation medium is cane molasses of various origins, with varying content of sugar, e.g.

Indian, Brazilian and Cuban species. The preferable sugar content in the fermentation medium is 13-15 wt.- B esdes, if necessary, a source of nitrogen and mineral salts are introduced.

rThe inoculation material is sown on the surface -8- 4*

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*1 4 Lt of the fermentation medium. Duration of fermentation is 9 10 days at 320C.

On completion of fermentation the biomass is separated and citric acid is extracted from the cultural liquid by commonly-used techniques. The yield of citric acid is 70-75 with relation to the sugar of the source nutrient medium.

The disclosed method, compared with the known method, permits the use of a cheap source material, i.e.

0 cane molasses, for the preparation of the nutrient medium, simplifies the production of citric acid and steps up its output.

For better understanding of the invention, it will be illustrated by the examples of its realization.

Example 1 The inoculation material is produced from the strain Aspergillus niger R-4. The nutrient medium is prepared from brewing wort containing 7% sugar (dry matter), adding the following components, Urea NaC1 CuSO4 5H 2 0 0.0001 Agar pH of the medium is maintained at 5.6 The medium prepared in this manner is sterilized in autoclave in the course of 20-30 minutes at 1200C.

The culture is grown in trays, in a layer 1 cm high.

I P *4 44 4, 9 -9- The trays are placed into a thermal chamber aerated by sterile air and held at 3200 within 10 days. The spores are collected by vacuum. dm 2 of the medium produces 0.68 g of spores. The dry spores are mixed with activated carbon at a ratio of 1:1.

The fermentation medium is prepared on the basis of Indian cane molasses containing 49.2 sugar, 82 dry matter and 14.4 ash.

300 g of molasses is diluted with hot water in 10 a proportion of 1:1, then 19 ml of 10% solution of sodium carbonate are added, followed by heating to the boiling point, then 60 ml of 10% solution of potassium ferrocyanide is added and the mixture is boiled for minutes for sterilization. The solution is coolea to 75°C, then 19 ml of 1% solution of monosubstituted 1 potassium phosphate is added, the mixture is cooled to 500 and 0.4 ml of 1% zinc sulphate is added. Then m* 4 sterile water is added to a volume of 1 1. The pH of the prepared medium is 6.1 and the sugar content\ 14.7wt.-%.

The medium prepared in this manner is poured ito sterile chemical beakers with a bottom area of 0.48d 2 440 ml into each, layer height 9 cm. The inoculation material of the strain Aspergillus niger R-4 in the form of a mixture of spores with activated oarbon io sown on the surface of the medium. Duration of ftrettation is 9 days at T 'he yield of citric acid is 721.

tion to sugar of the source nutrient medium. In the sum of synthetized acids the proportion of citric acid is 95.7wt.-%, that of oxalic acid,0.6wt.-%.

Example 2 The inoculation material of the strain Aspergillus niger R-4 is produced by the techniques similar to those described in Example 1.

The fermentation medium is prepared on the basis of Brazilian cane molasses containing 54.0 sugar, 78.5 dry matter, 19.08 ash.

o 250 g of molasses is diluted with hot water in a proportion of 1:1, then 12.5 g of sodium carbonate is added, brought to the boiling point, then 50 ml of solution of potassium ferrocyanide is introduced and o. 15 boiled for 20 minutes for sterilization. Then 25 ml of i 1% solution of monosubstituted potassium phosphate and ml of 10% solution of ammonium nitrate are added into the solution cooled to 75° 0 C. Now the solution if cooled to 50C and mixed with 1.25 ml of 1% solution of zinc sulphate, and sterile water is added to a vo- |luae of 1 1. The pH of the prepared medium is 5.8.The "sugar content is 15.5 Then fermentation is carried out by the method similar to that described in Example 1.

The yield of citric acid is 74.5 of sugar in the source nutrient medium. Ta the sum of synthetiz- ©ed acids the proportion of citric acid is 96.2 wt.-% and that of oxalic acid, 0.5 417

Claims (1)

1. A microbiological pethod for preparation of cit- ric acid by surface fermentation of the microorganism, citric acid producer, species Aspergillus niger on a nut- rient medium containing the sources of carbon, nitrogen, mineral salts and growth stimulants followed by separa- tion of the biomass and extraction of the desired product from the cultural liquid c h ar a c t e r i z e d i n t h a t the function of microorganism citric acid producer is fulfilled by the strain Aspergillus niger R-4 obtained by selection from the strain Asper- gillus niger R-3 by adapting it to the media with vari- ous concentrations of cane molasses, by exposing it to mutagenous factors nitrosomethyl urea and UV rays, and by natural selection of active cultures, deposited in the collection of the Central Museum of Industrial Mioroorganisms of the All-Union Research Institute of Genetics and Breeding of Industrial Microorganisms, file No. IMIIM F-507. DATED this TENTH day of JUNE 1986 EXPERIMENTALNY ZAVOD BIOKHIMICHESKIKH PREPARATOV INSTITUTA MIKROBIOLOGII IMENI AVGUSTA KIRKHENSHTEINA AKADEMII NAUK LATVIISKOI SSR Patent Attorneys for the Applicant SPRUSO FERGUSON