The present invention relates to a process for the preparation of barium gluconate.
The present invention particularly relates to the development of a fermentation process for the production of barium D-gluconate from a carbohydrate source in a single step and in high concentrations by the mediation of a strain of Aspergillus niger without the accumulation of any other organic acids or their keto derivatives. The product thus obtained by the fermentation can be used directly without isolation / crystallisation and purification for the preparation of various industrially \ pharmaceutically important salts of gluconicacid.
Gluconic acid and the numerous salts produced thereof is an important industrial product used as acidulant / preservative in food industry and as readily available source of essential elements in pharmaceutical industry. Some of the very important salts and their uses are:
Calcium Gluconate for Calcium therapy, Calcium Borogluconate for the treatment of lactation tetany in cows and for administration by injection in veterinary medicines, Ferrous Gluconate for treatment of iron deficiency anaemia., Potassium Gluconate in all cases of potassium deficiency caused by diabetic acidosis, diarrhoea, vomiting etc. Magnesium Gluconate for the treatment of hypomagnesemic conditions and magnesium supplements, Zinc Gluconate for the treatment of simple zinc deficiency and as active ingredient in lozenges given for the protection against common colds.
Gluconic acid is produced by oxidation of D-glucose, which can be accomplished by electro-chemical catalytic process or by bio-catalytic process using enzymes or whole cell fermentation process. Both chemo-catalytic and bio-catalytic processes are of importance. Sometimes open competition exists between bio- and chemo-methods, e.g. the oxidation of glucose towards gluconic acid; sometimes the choice is obvious because of large differences in selectivity and /or economy, e.g. the glucose / fructose isomerization (enzyme-catalyst) and the glucose-to-sorbitol hydrogenation (metal catalyst).
Both fields of catalysis are still in full development and it is to be expected that the spectrum of catalytic techniques in carbohydrate conversion will broaden considerably in the years to come.Differences between bio- and chemo-catalysis include:- purified bio-catalysts, i.e. (supported) enzymes, are often highly product and reactant selective and the conditions of use (temperature, pH, solvent, susceptibility to cations) of bio-catalysts generally are more limited than those of chemo-catalysts. chemo-catalysts can often be re-activated / regenerated, bio-catalysts generally cannot. However, due to economic reasons the fermentation process is preferred one.
Gluconic acid produced by either of the methods is produced as its sodium or calcium salt due to constant pH control with carbonates of sodium or calcium. However, for the production of its Cu(II), Mg (II), Zn(II), Fe(II) and other such salts, free gluconic acid is generated from sodium or calcium salt by acid treatment or decationisation using ion exchange resins. The free gluconic acid thus generated is converted to its barium salt and
used for production of desired divalant metal salts by simple ion replacement. Barium-D-gluconate is therefore, an excellent choice for the preparation of Fe(II), Cu(II), Mg(n), Zn(II) and related gluconates.
The state of art for the preparation of gluconic acid salts like Ca(II) / Na(I) by fermentation at a production rate of 5-6 kg/m3/h is possible by constant addition of Na2CO3 or NaOH or incorporation of CaCO3 within the medium which lead to formation of Na(I) / Ca(II) gluconate, which in turn is converted to free gluconic acid by sulphuric acid treatment or by ion exchange method followed by its conversion to barium -D-gluconate as an intermediate step to produce other useful divalant salts of gluconate. One of the major consumer of barium gluconate is the pharmaceutical industry for the preparation of pharmaceutical grade ferrous/ magnesium/ zinc/ copper gluconates and the related salts. In the present process barium gluconate can be prepared directly in quantitative yields at a production rate of 6-7 kg/m3/h by fermentation of a broth containing 200-250g/l of glucose with a specially selected strain of Aspergillus niger which was developed through known process of enrichment techniques . In this process of enrichment technique, barium carbonate was introdued in the medium in gradual increments so as make the organisium to adapt to the barium salt in a very high concentarion as given in the example 1 and 2 herewith. In the present process therefore, barium -D-gluconate is directly produced in one step by fermentation thus simplifying the process of production of divalant salts of gluconate.
In literature the Gluconic acid preparation is made by electrolytic method Damodaran et al. (1948) Indian patent no. 39441 as well as by fermentation e.g. calcium gluconate
production by submerged fermentation culture Chopra et al.(1975),Res. and Ind.20,1-3. Calcium gluconate production form glucose by fermentation using Aspergillus niger in which the acid formed is neutralized by the addition CaCOs was reported by Wells, P.A. et al (1937) Chem. and Met. Eng., 44, 188, Wells, P.A. et al (1937) Ind. Eng. Chem., 29, 653. Gluconic acid is also reported to be produced by fermentation of glucose with Aspergillus niger or Gluconobacter suboxydans, Lockwood , L.B. (1979) In microbioal technology, 2nd edit., Vol. 1, H.J. Peppier & D. Perlmann, Eds 1: 355-387. Academic Press N.York. Another fermentation process reported for the production of sodium gluconate from D-glucose by continuous neutralization of gluconic acid with sodium base at pilot scale with Aspergillus niger NRRL-3 Blom et al.(1951) Ind. and Eng.Chem.,44, 435-441. Sakura et al (1992) in their patent WO 9218637A described production of D-gluconic acid by cultivating a microorganism belonging to the genus Bacillus, which is capable of producing gluconic acid from D-glucose mediated by high glucose dehydrogenase activity. In another patent DD 259969 (1988) continuous fermentative production of gluconic acid was described by using substrate-shocked starter culture to increase yields by raising oxidizing ability of Gluconobacter or Acinetobacter species. The oxidation capacity is increased by subjecting the culture to substrate shock for 1-15 hr. at the end of a C-limited growth phase. During the production phase the pH is maintained at 2.5-3.3 in the case of Acetobacter methanolicus or at 5-6.5 in the case of Acinetobacter calcoaceticus. However, some portion of gluconic acid is oxidised further to generate its keto derivatives when Gluconobacter is used for fermentation . This property of Gluconobacter species is considered detrimental for their employment as producers of gluconic acid at an industrial scale [Meiberg & Spa Ant. V.
Leeuwenhoek (1983) 49, 89-90].Continuous gluconic acid production by immobilized Gluconobacter species was reported by Seiskari et al.(1983) 3rd European Congress on Biotechnol. 339-344. The effect of dissolved oxygen tension and contribution of endo-and exocellular glucose oxidase to gluconic acid production at increased oxygen tension in . Pilot scale airlift rector was reported by Trager et al. (1991) J.Chem. Tech. Biotechnol., 50, 1, Traeger et al. (1992), J. Ferment. Bioengg, 274-281.
The first report on the continuous cultivation of Gluconobacter oxydans for the production of gluconic acid has been published by Olijve & Kok, (1979) Arch. Microbiol.121, 291-297. Subsequently Seiskari et al. (1985) Appl. Microbiol. Biotechnol. 21, 350-360, reported the continuous production of D-gluconic acid by the immobilised cells of G. oxydans yielding a productivity of 5g 1-1 h-l(25 mmol. 1-1 h-1).
In another patent DD 253836 (1988) continuous microbial synthesis of partial oxidation products, e.g. gluconic acid, dihydroxyacetone and acetic acid, is effected under chemostatic conditions using a microorganism capable of simultaneous growth and product formation on a single substrate, the ratio between assimilation and product formation being controlled by the flow rate. Specifically, the microorganism is a bacterium of the genus Acetobacter, Gluconobacter, Acinetobacter, Pseudomonas or Thiobacillus. For production of gluconic acid, Acetobacter methanolicus is cultured on glucose as sole C source at a pH of 3-5 and a flow rate of 0.16 - 0.35 h-1.
In yet another patent DD 238067 (1988) Microbiological production of gluconic acid by continuously culturing of Acinetobacter, Acetobacter, Gluconobacter or Thiobacillus strain in a medium containing acetate, MeOF or EtOH as C source is described. In the case of Acinetobacter calcoaceticus EB114 (ZIMET 11089), and acetate medium is used, with the acetate: glucose wt. ratio of 1:15-30, and the bacterium is cultured at 25-35°C and pH 6-8 with a throughput (dilution) rate of 0.1-0.5 h-1..
However, the detailed study on the continuous production of gluconate (sodium or calcium salts) and its keto-derivatives by Gluconobacter oxydans has been reported recently by Buse et al. Enzyme Microb. Technol. (1992) 14, 1001-1006; Trager et al. 1992, Trager et al., J. Chem. Technol. Biotechnol. (1991) 50, 1-11 and Qazi et al. Enzyme Microb. Technol. (1992) 13, 504-507.
The free gluconic acid or its salts like Ca(II)/ Na(I) prepared by fermentation method cannot be employed directly for the preparation of all desired divalent metal-gluconate derivatives.Therefore,the discovery of an alternate method / process for direct production of barium gluconate is useful and simplifies the process of production of all the desired derivatives of the gluconates.
However, no method is available in the litreture for the production of Barium-D-gluconate in a single step using fermentation method.
The main object of the present invention is to provide a single step process for the production of barium-D-gluconate which obviates the drawbacks of the present invention.
The another object of the present invention is the development of a fermentation process for the production of barium salt of gluconic acid in very high concentrations by the mediation of an isolated fungal strain Aspergillus niger RRL BAG 11 from a D-glucose in presence of barium carbonate incorporated in the fermentation medium. The barium salt of gluconic acid accumulation in the fermented broth is more than 25 Og /I. without the accumulation of any other organic acids or their keto derivatives.
Yet another object of the present invention is to develop a process of barium gluconate by which the final fermented broth will be used for the production of various gluconte salts like Fe(II),Cu(II), Mg(II),Mn(II), Zn(II).
Yet another object of the present invention is to evolve an economic method for the production of barium gluconic gluconate for its use in the preparation of various gluconate salts like Fe(II), Cu(II), Mg(II), Mn(II), Zn(II) and the like.
Accordingly the present invention provides a single step process for the preparation of barium gluconate which comprises: fermenting a carbohydrate by mediation of fungal strain which is capable of oxidising the carbohydrates in presence of a conventional fermentation medium containing barium salt at a temperature in the range of 20 to 40 °C
for a period at least 16hr followed by separating cell mass by the known method and recovering the barium gluconate by conventional crystallisation method.
In an embodiment of the present invention the soluble carbohydrate used may be selected from liquid D-glucose, dextrose monohydrate, anhydrous D-glucose . In a feature of the present invention the carbohydrates such as soluble starch,cellobiose can also be used for the production of barium-D- gluconate.
In an another embodiment of the invention the fungal strain used may be any strain which oxidize carbohydrates to gluconic acid such as Aspergillus niger.Penicillium notatum, Rhizopas sp.
In yet another embodiment of the invention the barium salt used may be such as barium carbonate, barium hydroxide, barium oxide.
Separation of the cell mass from the fermented broth may be effected by centrifugation, decantation or filtration.
Barium-D-gluconate may be recovered by keeping the fermented broth at 10 to 40 °C 24hr or by concentration of the fermented broth to its half the volume and keeping at 25 °C or by prcepitation with methanol or ethanol.
The present invention provides a process for the development of a single pot fermentat ion process for the direct production of barium gluconate from a carbohydrate source in one step and in very high concentrations by the mediation of fungal strain designated as Aspergillus niger, RRL BAG-II without the requirement of pH control and without the accumulation of any other organic acids or their keto derivatives.
Aspergillus niger -RRL BAG 11 was isolated from soil using dilution plate method( Waksman 1927) . One gram of soil was weighed on sterilized filter paper it was
transfer. ,ed to a 150 ml flask containing 100ml of sterilized water. Flask was thoroughly shaken to mix the contents well and 10 ml of this suspension was pipetted out with
sterilised pipette into 150 ml flask containing 10 ml of sterilized water. Finally 10 ml of
the dilution was taken and mixed in 20 ml of distilled and sterilized waterthus making of
dilution of 1: 2000, ,one ml soil suspension of 1: 2000 dilution was plated in 6 to 7 sterilized petri dishes. 1 ml of soil suspension was poured in each petri dish followed by addition of potato dextrose agar medium containing streptomycin and rose Bengal. The plates were gently rotated to mix the contents. Finally the plates were incubated at 26 C for 4 to 5 days after which the discrete colonies were picked up
The overall reaction is as follows(Formula Removed)
The process involves the use of a fugal strain of Aspergillus niger which is a known
natural and unaltered strain.The fermentation medium containing 50-300g/l D-glucose as substrate and carbon a source. The medium also contains a source of nitrogen, minerals, vitamins etc. such as cornsteep liquor, yeast extract, malt extract or the like in the concentration range of 2 to 25 g 1-1 . Source of inorganic ions such as potassium and magnesium are added to the medium in the range of 0.1 to 1.0 g I"1 . The fermentation is carried out at a temperature of 20 to 40°C under constant aeration and agitation rate of 0.2 to 2.0 vvm and 200 to 800 revolutions per minute (rpm) respectively. pH of the fermentation broth may range from 7 to 4 during the course of fermentation cycle. At the
end of the fermentation cycle (20 to 40 h) the cell mass is removed by filtration or any
other physical separation methods and the sole product (barium gluconic acid) is
separated from the broth by precipitation at room temperature or may be directly
converted to any desired divalent metal salt by known state of art ion displacement
reaction.
The following examples are given by way of illustrations of the present invention and
should not be construed to limit the scope of the present invention.
Example 1:
Barium Gluconic acid production in 61 scale stirred tank fermentor by Aspereillus nieer using 100g/I glucose.:
The seed medium for the fermentor was prepared form the spores of the Aspergillus
niger.The spore were prepared in Rouex bottles by culturing the Aspirgillus niger RRL BAG -II on a solid medium of the following composition (g /1)
Molases,7.5; Glycerol, 7.5;Nacl, 10; CaSO4,0.25; Yeast ext.,5.0 and trace elements,.MgSO4 7. H20 0.005; Fe ( NH4)2 SO4 0.0016; KH2PO4 0.0016; CuSO4. 7H2O, 0.001; Agar,20.0. The spores were collected from the Rouex culture bottle in 0.1% Tween 80 solution distilled H2O and inoculated to a seed medium in the proportion of 107 spored/ml. The seed medium comprised of (g /I) Sucrose, 140;HH2NO3, 2.5;KH2PO4, 1.0; MgS04 7H20, 0.25; Starch, 1.0; pH : 3.0 with HCL. The seed culture was prepared under shaken condition at 30°C for 24 hrs. 6 1 of the production medium comprising (g/1) Glucose monohydrate 100 g (92% as glucose); (NH^ HPO4, 0.42,; Urea, O.llg; KH2PO4, 0.2; MgS04 , 7H2O ,0.17; corn steep liquor (CSL),4.0; pH 6.5 with Barium hydroxide solution, autoclaved for 15 minutes at 15psig and cooled to
30°C. Barium carbonate (50 g 1) was autoclaved separately and added at the time of inoculation with 6% (v/v) of the above seed-culture and fermentation operated under the following conditions.

Agitation rate
Working volume
Antifoam:
Temperature:
Air
Overhead pressure
pH

600
61
Ground nut oil(3.0ml)
29+ 1°C
Ivvm
0.3kg
:(Medium pH 6 was adjusted with barium
hydroxide before autoclaving.)

Table 1 depicts the results of above example.

(Table Removed)Gluconic acid 95g/l corresponding to 103.3g as barium gluconate was estimated by
HPLC using a Cation column HPX 87, (Biorad make) using 25mM f^SC^ as mobile
phase at UV210nm.
The fermented broth recovered after the fermentation was concentrated to half the
origional volume at 50°C , 60°Hg on keeping overnight at 25°C provided, barium- D-
gluconate101.2g/l(98%).

Example 2:
Barium Gluconic acid production in 61 scale stirred tank fermentor by Aspereillus
nieer using 100g/l glucose at 40°C.:
The seed medium for the fermentor was prepared form the spores of the Aspergillus
niger.The spore were prepared in Rouex bottles by culturing the Aspirgillus niger RRL BAG -II on a solid medium of the following composition (g /1)
Molases,7.5; Glycerol, 7.5;Nacl, 10; CaSO4,0.25; Yeast ext.,5.0 and trace elements,.MgSO4 7. H2O 0.005; Fe ( NH4)2 SO4 0.0016; KH2PO4 0.0016; CuSO4. 7H20, 0.001; Agar,20.0. The spores were collected from the Rouex culture bottle in 0.1% Tween 80 solution distilled H2O and inoculated to a seed medium in the proportion of 107 spored/ml. The seed medium comprised of (g /1) Sucrose, 140;HH2NO3, 2.5;KH2P04, 1.0; MgSO4 7H2O, 0.25; Starch, 1.0; pH : 3.0 with HCL. The seed culture was prepared under shaken contion at 30°C for 24 hrs. 6 1 of the production medium comprising (g/1) Glucose monhydrate 100 g (92% as glucose); (NIttk HPO4, 0.42,; Urea, O.llg; KH2PO4, 0.2; MgSO4 , 7H2O ,0.17; corn steep liquor (CSL),4.0; pH 6.5 with Barium hydroxide solution, autoclaved for 15 minutes at 15psig and cooled to 30°C. Barium carbonate (50 g /1) was autoclaved separately and added at the time of inoculation with 6% (v/v) of the above seed-culture and fermentation operated under the following conditions.

Agitation rate
Working volume
Antifoam:
Temperature:
Air
Overhead pressure
600
61
Ground nut oil(3.0ml)
40± 1°C
1vvm
0.3kg
pH :(Medium pH 6 was adjusted with barium
hydroxide before autoclaving.)

Table 1 depicts the results of above example.

(Table Removed)
Gluconic acid 95g/l corresponding to 103.3g as barium gluconate was estimated by
HPLC using a Cation column HPX 87, (Biorad make) using 25mM H2SO4 as mobile
phase at UV210nm.
Tlie fermented broth recovered after the fermentation was concentrated to half the
origional volume at 50°C , 60°Hg on keeping overnight at 25°C provided, barium- D-
gluconate101.2g/l(98%).
Example 3:
Barium Gluconic acid production in 61 scale stirred tank fermentor by Aspereillus
nieer using 100g/l sucrose.:
The seed medium for the fermentor was prepared form the spores of the Aspergillus niger.The spore were prepared in Rouex bottles by culturing the Aspirgillus niger RRL BAG -II on a solid medium of the following composition (g /1)
Molases,7.5; Glycerol, 7.5;Nacl, 10; CaSO4,0.25; Yeast ext.,5.0 and trace elements,.MgSO4 7. H20 0.005; Fe ( NH^ SO4 0.0016; KH2PO4 0.0016; CuSO4. 7H2O, 0.001; Agar,20.0. The spores were collected from the Rouex culture bottle in 0.1% Tween 80 solution distilled H2O and inoculated to a seed medium in the proportion of 107 spored/ml. The seed medium comprised of (g /I) Sucrose, 140;HH2NO3, 2.5;KH2PO4, 1.0; MgSO4 7H2O, 0.25; Starch, 1.0; pH : 3.0 with HCL. The seed culture
was prepared under shaken contion at 30°C for 24 hrs. 6 1 of the production medium comprising (g/1) Glucose monhydrate 100 g (92% as glucose); (NH4)2 HPO4, 0.42,; Urea, O.1lg; KH2PO4, 0.2; MgS04 , 7H2O ,0.17; corn steep liquor (CSL),4.0; pH 6.5 with Barium hydroxide solution, autoclaved for 15 minutes at 15psig and cooled to 30°C. Barium carbonate (50 g /1) was autoclaved separately and added at the time of inoculation with 6% (v/v) of the above seed-culture and fermentation operated under the following conditions.

Agitation rate
Working volume
Antifoam:
Temperature:
Air
Overhead pressure
PH

600
61
Ground nut oil(3.0ml)
29± 1°C
1vvm
0.3kg
:(Medium pH 6 was adjusted with barium
hydroxide before autoclaving.)

Table 1 depicts the results of above example.

(Table Removed)Gluconic acid 50g/l corresponding to 54.368 as barium gluconate was estimated by HPLC using a Cation column HPX 87, (Biorad make) using 25mM H2SO4 as mobile phase at UV210nm.
The fermented broth recovered after the fermentation was concentrated to half the origional volume at 50°C , 60°Hg on keeping overnight at 25°C provided, barium- D-gluconate 53.28 g/1 (98%).
Eiample:4
Production of Barium Gluconate at 101 scale fermentor using Aspereillus nieer in a
fed batch experiment using 250g/l D-glucose (as monohydrate);
The seed medium for the fermentor was prepared form the spores of the Aspergillus niger.The spore were prepared in Rouex bottles by culturing the Aspirgillus niger RRL BAG -II on a solid medium of the following composition (g 1)
Molases,7.5; Glycerol, 7.5;Nacl, 10; CaSO4,0.25; Yeast ext.,5.0 and trace elements,.MgSO4 7. H2O 0.005; Fe ( NH4)2 SO4 0.0016; KH2PO4 0.0016; CuSO4. 7H2O, 0.001; Agar,20.0. The spores were collected from the Rouex culture bottle in 0.1% Tween 80 solution distilled H20 and inoculated to a seed medium in the proportion of 107 spored/ml. The seed medium comprised of (g /I) Sucrose, 140;HH2NC>3, 2.5;KH2PO4, 1.0; MgSO4 7H2O, 0.25; Starch, 1.0; pH : 3.0 with HCL. The seed culture was prepared under shaken contion at 30°C for 24 hrs. 6 1 of the production medium comprising (g/1) Glucose monhydrate 200 g (92% as glucose); (NH4)2 HPO4, 0.42,; Urea, O.llg; KH2PO4, 0.2; MgSO4, 7H2O ,0.17; corn steep liquor (CSL),4.0; pH 6.5 with Barium hydroxide solution, autoclaved for 15 minutes at 15psig and cooled to 30°C. Barium carbonate (125 g /I) was autoclaved separately and added at the time of
inoculation with 6% (v/v) of the above seed-culture and fermentation operated under
the following conditions.

Agitation rate
Working volume
Antifoam:
Temperature:
Air
Overhead pressure
PH

600rpm 101
Ground nut oil (3.0ml) 29± 1°C 1vvm 0.3kg
(Medium pH 6 was adjusted with barium hydroxide before autoclaving)

Table 2 depicts the results of above example.

(Table Removed)Gluconic acid 245g/1 corresponding to 266.6g/l as barium gluconate was estimated by HPLC using a Cation column HPX 87, (Biorad make) using 25mM H2SO4 as mobile phase at UV210nm.
The fermented broth recovered after the fermentation was concentrated to half the origional volume at 50°C , 600Hg on keeping overnight at 25°C provided, barium- D-gluconate 261.0 g/1 (98%).
Example:5
Production of Barium Gluconate at 101 scale fermentor using Asuereillus nieer in a
fed batch experiment using 250g/l D-glucose (as monohvdrate) and barium hydroxide;

The seed medium for the fermentor was prepared form the spores of the Aspergillus niger.The spore were prepared in Rouex bottles by culturing the Aspirgillus niger RRL BAG -II on a solid medium of the following composition (g 1)
Molases,7.5; Glycerol, 7.5;Nacl, 10; CaS04,0.25; Yeast ext.,5.0 and trace elements,.MgSO4 7. H2O 0.005; Fe ( NH4)2 SO4 0.0016; KH2P04 0.0016; CuSO4. 7H2O, 0.001; Agar,20.0. The spores were collected from the Rouex culture bottle in 0.1% Tween 80 solution distilled H2O and inoculated to a seed medium in the proportion of 107 spored/ml. The seed medium comprised of (g /I) Sucrose, 140;HH2NO3, 2.5;KH2PO4, 1.0; MgS04 7H20, 0.25; Starch, 1.0; pH : 3.0 with HCL. The seed culture was prepared under shaken contion at 30°C for 24 hrs. 61 of the production medium comprising (g/1) Glucose monhydrate 200 g (92% as glucose); (NH4)2 HPO4, 0.42,; Urea, 0.1 Ig; KH2P04, 0.2; MgSO4 , 7H2O , 0.17; corn steep liquor (CSL),4.0; pH 6.5 for 15 minutes at 15psig and cooled to 30°C. Barium hydroxide solution[ Barium hydroxide octahydrate, Ba(OH)2 8H20, MW 315.47] 40g/l (required 197.17g/l final, 1.972kg for 10 1) autoclaved was autoclaved separately and added time to time during the fermentation. Inoculation with 6% (v/v) of the above seed-culture and fermentation operated under the following conditions.

Agitation rate
Working volume
Antifoam:
Temperature:
Air
Overhead pressure
600rpm
101
Ground nut oil( 3.0ml)
29± 1°C
1vvm
0.3kg/ meter 2
pH :(Medium pH 6 was adjusted with barium
hydroxide before autoclaving)
Table 2 depicts the results of above example.

(Table Removed)Gluconic acid 245 g/1 corresponding to 266.6g/l as barium gluconate was estimated by
HPLC using a Cation column HPX 87, (Biorad make) using 25mM H2SO4 as mobile
phase at UV210nm.
The fermented broth recovered after the fermentation was concentrated to half the
origional volume at 50°C , 600Hg on keeping overnight at 25°C provided, barium- D-
gluconate261.0g/l(98%).
Advantages of the present invention:
1. The organism is nonpathogenic stable(GRAS), fast growing and easy to handle and
can be stored as spores for 3-4 months 4°C.
2. Barium D-gluconate is direclty produced in large quantities as white crystalline
material without any need of further purification/crystallization.
3. Barium D-gluconate is direclty produced in large quantities as white crystalline
material without any need of further purification/crystallization at 25-40°C.
4. The fermented broth after removal of the biomass can be directly used as such for the
preparation of various other gluconates like Cu,(II), Mg(II), Mn(II), Zn(II) Fe(II) etc by
known state of art.
5.The biomass botained after or during the fermentation cycle can be used for the isolation of the glucose oxidase enzyme.
6. The over all fermentation cycle is short (50-53hrs.) for obtaining 250 g/1 barium D-
gluconate.
7. Glucose oxidation is complete, as a result not residual glucose is left in the broth at the
end of the fermentation cycle, therefore, making the product isolation process easier.

We Claim:
l.A single step process for the preparation of barium gluconate which comprises:
fermenting a carbohydrate by mediation of a fungal strain which is capable of oxidising
the carbohydrates in presence of a conventional fermentation medium containing barium
salt at a temperature in the range of 20 to 40 °C for a period at least 16hr followed by
separating the cell mass by the known method and recovering the barium gluconate by
conventional crystallisation method.
2. A process as claimed in claim 1 wherein the production of barium D-gluconate using a
wild strain belonging to genus Aspergillus .Penicillium , Rhizopas.
3.A process as claimed in claims 1-2 wherein the carbohydrate used is selected from
starch hyrdolysate ,dextrose monohydrate ,sucrose,anhydrous glucose, cellobiose.
4. A process as claimed in claims 1-3 wherein the barium salts used in the process is
selected from Ba(OH)2 , BaCO3.
5. A process as claimed in claims 1-4 wherein a carbohydrate used is a carbon source
and Yeast extract, Malt extract, com steep liquor and peptone as the sources of nitrogen,
vitamins and other trace requirements.
6. A process as claimed in claim 1-5 above for the production of barium- D-gluconate by
an unmodified strain of Asperigllus species in fermentors in fermentors agitated at 200-
800 rpm and aerated at a rate of 0.2-2 wm at 1-3 bars over pressure.
7. A process as claimed in claims 1-6 above wherein the fermentation is carried out at
29°C to 40°C and at pH values between 4-8.
8. A single step process for the production of barium-D-gluconate substantially as herein
described with reference to the examples.