F0RM2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the Invention. - "A PROCESS FOR THE PRODUCTION
OF CALCIUM GLUCONATE"
2. Applicant(s)
(a) NAME : SFPL CROP LIFE SCIENCE PRIVATE LIMITED
(b) NATIONALITY: An Indian Company.
(c) ADDRESS: 302, Royal House, 11/3, Usha Ganj, Indore-452001,
Madhya Pradesh, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:

Field of the Invention:
The present invention relates to the field of fermentation. Particularly, the invention provides a highly efficient and an economical process for the production of a metal gluconate i.e. calcium gluconate through microbial fermentation technology. Said fermentation process produces improved yield of metal gluconate (specifically calcium gluconate) resulting into complete conversion of the raw material into the finished product and the ratio of the raw material i.e. Dextrose Mono Hydrate (DMH) and the finished product (in the form of metal gluconate i.e. calcium gluconate) is achieved around 108% in a short period of about less than 16-18 hours. The process is highly energy saving as the same requires very less aeration and agitation for the complete conversion of raw material into the calcium gluconate.
Background of the invention:
Metal gluconates acts as mineral supplements and thus have wide application in areas like drug industry, food industry, nutritional supplemental formulations, textile industry etc., like sodium gluconate is useful as an environmentally friendly industrial detergent, calcium gluconate can be used for dietary calcium supplementation in nutrient formulation, ferrous gluconate is used for iron supplementation of anemic patient, zinc gluconate is used as a zinc supplement in nutritional diets etc.
Calcium gluconate is the calcium salt of gluconic acid. A significant disadvantage of calcium gluconate is that it is relatively costly to produce. The production of gluconic acid or its salts from glucose may be achieved by the use of processes like enzymatic, microbial fermentation, electrochemical, chemical etc.
For the production of calcium gluconate an enzymatic process is preferred because of its high specificity. However, the solubility of calcium gluconate under the conditions required for optimal enzymatic conversion (pH 5-7, temp 30-35°C.) is only 40 g/L. In order to obtain industrial quantities of the product, large reactors are needed and the concentration of the calcium gluconate crystals requires the evaporation of large amounts of water. Increase of the initial glucose concentration and instant crystallization of the formed calcium gluconate results in an increased viscosity of the reaction mixture and a concomitant decrease in the rates of oxygen transfer and enzymatic conversion. Intensification of agitation to restore oxygen transfer and

overcome viscosity problems, e.g. by using Rushton turbines for stirring will raise equipment costs and heightens the risk of damage to the used equipment and concomitant metal contamination (chromium or nickel) of the final product. These drawbacks have rendered the enzymatic production of calcium gluconate on an industrial scale unattractive.
The enzymatic process farther suffers with the drawback of having low stability of the enzymes (even when immobilized), low conversion efficiency etc. This feature results in incomplete conversion of glucose to gluconic acid leaving residual unconverted glucose as a contaminant in the gluconic acid solution produced thereby and further in order to reduce or eliminate such unconverted glucose from the final product costly downstream processing steps are required, which consequently adds up more cost to the whole process. The process for the production of gluconate is cost inefficient for reasons like use of excess costly starting materials, loss of product through purification procedures etc.
U.S. Pat. No. 4,845,208 describes a process for the production of aldonic acids by aldose oxidation using a palladium-based catalyst. The disadvantage of this method is the use of very expensive and toxic catalysts and the formation of several by-products.
U.S. Pat. No. 3,935,071 describes the conversion of glucose into gluconic acid using enzymes bound to a carrier and separation of the gluconic acid by anion exchange chromatography.
The inefficiencies and costs of current production techniques for gluconates are even more magnified during large scale production. Each reaction vessel or fermentor in the manufacture of gluconic acid may have a capacity of greater than 100,000 liters and multiple fermentors of this size are required to produce millions of pounds of gluconates.
In addition to the cost of materials, existing process for the production of gluconates generates substantial amounts of waste materials. Each production batch can generate millions of gallons of industrial as well as biological wastes.
The microbial fermentation process is also being used for the production of gluconic acid or salts thereof. However, the conventional fermentation process takes very long time for the production of a product i.e. usually it takes several days for the growth of microorganism and the

bioconversion of a raw material into the finished product, which is a major disadvantage of this
process.
US5017485 relates to a process for obtaining gluconic acid or gluconate, by fermentation in the
presence of permeabilized Zymomonas mobilis cells, starting from aqueous glucose/fructose
mixtures.
FR-A-1 590 031 describes a fermentation process for the conversion of glucose in to gluconic
acid comprising the stepwise addition of glucose to the fermentation broth.
However, these reported fermentation process for the production of gluconates usually takes long
duration, which is quite undesirable.
Accordingly, a highly efficient and economical fermentation process for the production of metal gluconate (specifically calcium gluconate), which produces high yield of about 108% calcium gluconate in short time duration of about less than 16-18 hours is needed in the art.
Further, there is also a requirement for a fermentation process which can completely convert a raw material (DMH) into the metal gluconate, particularly calcium gluconate whereby the ratio of raw material and the metal gluconate i.e. calcium gluconate is achieved around 108% in about less than 16-18 hours.
Object of the Invention:
The primary object of the invention is to overcome the drawbacks mentioned in the prior art.
An object of the present invention is to provide a fermentation process for the production of calcium gluconate which yields about 108% calcium gluconate in a short time period of about less than l6-18hrs.
Another object of the present invention is to provide a fermentation process which can cause complete conversion of a raw material into the calcium gluconate, whereby the ratio of raw material and the metal gluconate i.e. calcium gluconate is achieved around 108% in about less than 16-18 hours.
Another object of the present invention is to provide an economical and energy saving process for the production of calcium gluconate wherein very less aeration (0.3 to 0.5 v/v/m) and agitation (50 to 60 rpm) is required for the complete conversion of raw material into calcium

gluconate.
Another object of the present invention is to provide a process for the production of 100% pure calcium gluconate.
Another object of the present invention is to provide a novel strain of Aspergillus niger having AcccessionNo. MTCC 5713.
These and other advantages of the present invention will be more apparent from the foregoing description in conjunction with the accompanying drawings.
Summary of the Invention:
The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.
The invention provides a process for the production of a calcium gluconate, said process steps comprises treatment of a raw material in a fermentation media by a fermenting organism adapted to produce glucose oxidase, thereby enabling conversion of said raw material to said calcium gluconate wherein said raw material is completely converted into said calcium gluconate and the ratio of raw material and metal gluconate is achieved around 108% in a short time period of about less thanl6-18 hrs.
Brief description of the accompanying drawings:
Other features as well as the advantages of the invention will be clear from the following
description.
Figure 1 shows the diagrammatic representation of the process for the production of calcium gluconate.
Detailed description of the invention:
The following description is of exemplary embodiments only and is not intended to limit the

scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.
Accordingly, the present invention provides a process for the production of calcium gluconate using efficient native strain of Aspergillus niger, through microbial fermentation technology which converts 100% organic raw material to the metal gluconate.
In compliance with the requirements of full disclosure, the Aspergillus niger strain referred herein has been deposited in the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India (according to international deposition based on Budapest Treaty) under the Accession number MTCC 5713. The fungal strain has following characteristics:
• Hyphae septate and hyaline,
• conidial heads radiate initially,
• Splitting into columns at maturity,
• Conidia brown to black coloured, very rough, globose,
• Reverse side on culture plate's shows white or no any pigmentation and growth produces radial fissures in agar,
• Conidiophores-long (400-3000 jam), smooth, and hyaline, becoming darker at the apex and terminating in a globose vesicle (30-75 urn in diameter)
The fermentation process of the present invention provides more than 100% yield i.e. about 108% of 100% pure Calcium Gluconate in less than 16-18 hours fermentation cycle. The process is highly efficient and energy saving as there is very less requirement of aeration and agitation in the fermentation process. The aeration during the fermentation period is provided in the range of 0.3-0.5 v/v/m while agitation is provided in the range of 40-60 rpm. The process is highly productive and is stable at variable temperature range 25°C to 42°C.
The fermentation process of the present invention employs Dextrose mono hydrate as the raw

material for production of Calcium gluconate. The fermentation medium for the generation of Aspergillus niger seed inoculum essentially comprises sucrose in an amount of 120.0g/L, starch in an amount of 1.25g/L, Ammonium nitrate in an amount of 2.5g/L, Magnesium sulphate in an amount of 0.3g/L, potassium hydrogen phosphate in an amount of 1.5g/L. The medium is sterilized at 121°C for 10 minutes and is subsequently cooled for the addition of Aspergillus niger spores (lOVmlto lOVmll at 10% v/v.
While, following parameters were maintained throughout the growth of Aspergillus niger
inoculum:
Aeration - 0.3 v/v/m
pH - 3.0
Temperature - 30±1°C
Agitation - 50 rpm
Pressure - 0.25 bar
The A.niger seed inoculum preparation process is given in below (Table 1):

SrNo Biomass of A.niger Time Required
1 20g/liter 23 hours
The optimum generation of biomass is successfully achieved by maintaining the aforesaid parameters throughout the growth of the seed inoculums.
The fermentation for the production of calcium gluconate was carried out at the temperature ranging from 25°C to 42°C. The fermentation media composition in the range for production of calcium gluconate are mentioned below:
Fermentation medium contains (per40 liter water) following constituents:
Medium Preferred Range
Dextrose mono hydrate - 100-150g/L

Di Ammonium Phosphate - 0.3 - 0.6g/L
Urea - 0.1 -2.0g/L
Magnesium Sulphate - 0.15 - 2.5g/L
Vegetable oil (soybean) - 1.0 - 2.0ml/L
KH2P04 - 0.2-0.25g/L
Calcium Carbonate - 15 - 30 g/L
While, the preferred range of the constituents in the fermentation media comprises:
Medium Composition
Dextrose mono hydrate - 480 kg (120.0g/L)
Di Ammonium Phosphate - 20.0 g (0.50g/L)
Urea - 6.00 g (0,15g/L)
Magnesium Sulphate - 8.0 g (0.20g/L)
Vegetable oil (soybean) - 60ml(1.5ml/L)
KH2P04 - 8.8 g (0.22g/L)
Calcium Carbonate - 960 gm (24g/L)
The above fermentation media was sterilized inside the fermenters at 115°C for 10 minutes, cooled and further inoculated with Aspergillus niger seed inoculum at the rate of 10% v/v and Aspergillus niger spore suspension (cfu 10s) at 1% v/v.
Following conditions were maintained throughout the fermentation process for the production of
metal gluconate:
Parameter Preferred Range
Aeration - 0.3-1.0 v/v/m
pH - 6.0-7.2
Temperature - 25- 42°C
Agitation - 50-80 rpm
Pressure - 0.4 - 0.75 bar

The specific value of parameters like aeration, agitation, temperature etc. maintained during the fermentation process are following:
Parameter Value
Aeration - 0.5 v/v/m
pH - 7.0
Temperature - 28°C to 35°C
Agitation - 60.0 rpm
Pressure - 0.5 bar
The fermentation process in the present invention requires very less aeration i.e. around 0.3 - 1.0 v/v/m and very less agitation i.e. around 50 - 80 rpm for the production of gluconate and therefore is an energy saving process and an economical process. The complete conversion of raw material (DMH) into the pure gluconate in very less time makes the process higly efficient and cost effective. The fungal isolate Aspergillus niger having MTCC no. 5713have higher potential to convert raw material (DMH) into gluconic acid/gluconate and therefore faster conversion is achieved when the above mentioned production parameters are maintained throughout the fermentation process.
The broth obtained from the above fermentation process was filtered through 5 micron filtration cloth in order to separate the biomass of A.niger and resultantly a clear solution of Calcium Gluconate was obtained. The calcium gluconate produced by this fermentation process is 100% pure and do not contain any residual sugar or other undesirable material.
The invention will now be explained with the help of following examples. However, the scope of the invention should not be limited to these examples as the person skilled in the art can easily vary the proportion of the ingredients and combinations.
EXAMPLE-1
Production of Calcium Gluconate through Fermentation of Dextrose Mono Hydrate, using

Aspergillus niger having MTCC no.5713( at normal temperature (30°C):
A. Preparation of Aspergillus niger seed inoculum:
Aspergillus niger seed inoculum was prepared on medium containing (/4 liter water):
• Sucrose - 480.0 g (120g/L)
• Starch - 5.0 g(l,25g/L)
• Ammonium nitrate - 10.0 g(2.5g/L)
• Magnesium Sulphate - 1.2 g(0.30g/L)
• Potassium hydrogen phosphate - 6.0g(1.5g/L)
The above medium was sterilized at 121°C for 10 minutes and Aspergillus niger spores were added at 15% v/v. Following parameters were maintained throughout the growth of Aspergillus niger seed inoculum:
• Aeration - 0.3 v/v/m
• pH - 3.0
• Temperature - 30°C
• Agitation - 50 rpm
• Pressure - 0.25 bar
Results of A.niger seed inoculum preparation process are given below in Table 2:

SrNo Biomass of A.niger Time Required
1 20g/liter 23 hours
B. Preparation of Calcium Gluconate: Fermentation was carried out at the temperature of 30°C. The fermentation media composition and parameters maintained for production of calcium gluconate are:
Production medium contains (/40 liter water):
Medium Composition
Dextrose mono hydrate - 480 kg (120.0g/L)

Di Ammonium Phosphate - 20.0 g (0.50g/L)
Urea - 6.00 g (0.15g/L)
Magnesium Sulphate - 8.0 g (0.20g/L)
Vegetable oil (soybean) - 60ml(1.5ml/L)
KH2P04 - 8.8 g (0.22g/L)
Calcium Carbonate - 960 gm (24g/L)
The above production media was sterilized inside the fermenters at 115DC for 10 minutes, cooled and inoculated with above developed Aspergillus niger seed inoculum at 10% v/v and Aspergillus niger spore suspension (cfu 108) at 1% v/v. Following parameters were maintained throughout the fermentation process:
• Aeration - 0.5 v/v/m
• pH - 7.0
• Temperature - 30.0°C
• Agitation - 60.0 rpm
• Pressure - 0.5 bar
The broth obtained from the above fermentation process was filtered through 5 micron filtration cloth in order to separate the biomass of A.niger and a clear solution of Calcium Gluconate was obtained.
Results of above Fermentation process is given in Table 3:

SrNo Parameters Results
Raw material (Dextrose Mono Hydrate) - 12%
1 Calcium Gluconate 12.9% (Yield-107%)
2 pH 4.8
4 Specific Gravity 1.0

5 Solubility in Water 100% soluble
6 Appearance Liquid
7 Colour Light Yellow
8 Odour Sweet Sugary
9 Sedimentation No
EXAMPLE-2
Production of Calcium Gluconate through Fermentation of Dextrose Mono Hydrate, using Aspergillus niger at low temperature (25°C):
A. Preparation of Aspergillus niger seed inoculum;
Aspergillus niger seed inoculum was prepared on medium containing (/4 liter water):
• Sucrose - 480.0 g(120g/L)
• Starch - 5.0 g(1.25g/L)
• Ammonium nitrate - 10.0 g(2.5g/L)
• Magnesium Sulphate - 1.2 g (0.30g/L)
• Potassium hydrogen phosphate - 6.0g(1.5g/L)
The above medium was sterilized at 121°C for 10 minutes and Aspergillus niger spores were
added at 15% v/v. Following parameters were maintained throughout the growth of Aspergillus
niger seed inoculum:
• Aeration - 0.3 v/v/m
• pH - 3.0
• Temperature - 30°C
• Agitation - 50 rpm
• Pressure - 0.25 bar
Results of A.niger seed inoculum preparation process are given below in Table 4;

SrNo Biomass of A.niger (SFPL1) Time Required
1 20g/liter 23 hours
B. Preparation of Calcium Gluconate: Fermentation was carried out at the temperature of 25°C. The fermentation media composition and parameters maintained for production of calcium gluconate are:
Production medium contains (/40 liter water):
Medium Composition
Dextrose mono hydrate - 480 kg (120.0g/L)
Di Ammonium Phosphate - 20.0 g (0.50g/L)
Urea - 6.00 g (0.15g/L)
Magnesium Sulphate - 8.0 g (0.20g/L)
Vegetable oil (soybean) - 60ml(1.5ml/L)
KH2P04 - 8.8 g (0.22g/L)
Calcium Carbonate - 960 gm (24g/L)
The above production media was sterilized inside the fermenters at 115°C for 10 minutes, cooled and inoculated with above developed Aspergillus niger inoculum at 10% v/v and Aspergillus niger spore suspension (cfu 108) at 1% v/v. Following parameters were maintained throughout the fermentation process:
• Aeration - 0.5 v/v/m
• pH - 7.0
• Temperature - 25.0°C
• Agitation - 60.0 rpm
• Pressure - 0.5 bar
The broth obtained from the above fermentation process was filtered through 5 micron filtration

cloth in order to separate the biomass of A.niger and a clear solution of Calcium Gluconate was obtained.
Results of above Fermentation process is given in Table 4:

SrNo Parameters Results
Raw material (Dextrose Mono Hydrate) -12%
1 Calcium Gluconate 12.9% (Yield-107.0%)
2 pH 4.6
4 Appearance Liquid
5 Solubility in Water 100% soluble
6 Specific Gravity 1.0
7 Colour Light Yellow
8 Odour Sweet Sugary
9 Sedimentation No
EXAMPLE 3
Production of Calcium Gluconate through Fermentation of Dextrose Mono Hydrate, using Aspergillus niger at high temperature (42°C):
A. Preparation of Aspergillus niger seed inoculum:
Aspergillus niger seed inoculum was prepared on medium containing (/4 liter water):
• Sucrose - 480.0 g (120g/L)
• Starch - 5.0 g(1.25g/L)
• Ammonium nitrate - 10.0 g(2.5g/L)
• Magnesium Sulphate - 1.2 g (0.30g/L)

• Potassium hydrogen phosphate - 6.0 g (1.5g/L)
The above medium was sterilized at 121°C for 10 minutes and Aspergillus niger spores were
added @ 15% v/v. Following parameters were maintained throughout the growth of Aspergillus
niger seed inoculum:
• Aeration - 0.3 v/v/m
• pH - 3.0
• Temperature - 30°C
• Agitation - 50 rpm
• Pressure - 0.25 bar
Results of A.niger seed inoculum preparation process are given below in Table 6:

SrNo Biomass of A.niger (SFPL1) Time Required
1 21g/liter 23 hours
B. Preparation of Calcium Gluconate: Fermentation was carried out at the temperature of 42°C. The fermentation media composition and parameters maintained for production of calcium gluconate are:
Production medium contains (/40 liter water):
Medium Composition
Dextrose mono hydrate - 480 kg (120.0g/L)
Di Ammonium Phosphate - 20.0 g (0.50g/L)
Urea - 6.00 g (0.15g/L)
Magnesium Sulphate - 8.0 g (0.20g/L)
Vegetable oil (soybean) - 60 ml (1.5ml/L)
KH2P04 - 8.8g(0.22g/L)
Calcium Carbonate - 960 gm (24g/L)

The above production media was sterilized inside the fermenters at 115°C for 10 minutes, cooled and inoculated with Aspergillus niger seed inoculum at 10% v/v and Aspergillus niger spore suspension (cfu 108) at 1% v/v. Following parameters were maintained throughout the fermentation process:
• Aeration - 0.5 v/v/m
• pH - 7.0
• Temperature - 42.0°C
• Agitation - 60.0 rpm
• Pressure - 0.5 bar
The broth obtained from the above fermentation process was filtered through 5 micron filtration cloth in order to separate the biomass of A.niger and a clear solution of Calcium Gluconate was obtained.
Results of above Fermentation process is given in Table 7:

SrNo Parameters Results
Raw material (Dextrose Mono Hydrate) -12%
1 Calcium Gluconate 13.0% (Yield-108.0%)
2 pH 5.1
4 Appearance Liquid
5 Solubility in Water 100% soluble
6 Specific Gravity 1.0
7 Colour Light Yellow
8 Odour Sweet Sugary
9 Sedimentation No
The present invention will be explained further with reference to non-limiting embodiments of

the invention.
In an embodiment of the invention, there is provided a high yielding economical process for the
production of metal gluconates, which produces more than 100% gluconate.
In another embodiment of the invention, there is provided a fermentation process for the
production of gluconates which yields more than 100% in a short fermentation cycle of less than
16-18 hours.
In another embodiment of the invention, there is provided a fermentation medium comprises the
components selected from a group comprising of Dextrose mono hydrate, Di Ammonium
Phosphate, Urea, Magnesium Sulphate, Soyabean oil, KH2P04, Calcium Carbonate or
combinations thereof.
In another embodiment of the invention, there is provided a novel strain of Aspergillus niger
having Acccession No. MTCC 5713.

WE CLAIM:
1. A process for the production of a calcium gluconate, said process steps comprises treatment of a raw material in a fermentation media by a fermenting organism adapted to produce glucose oxidase, thereby enabling conversion of said raw material to said calcium gluconate; wherein said raw material is completely converted into said calcium gluconate and the ratio of raw material to metal gluconate, achieved, is around 108% in a time period of less than 18 hours.
2. The process for the production of calcium gluconate as claimed in claim 1, wherein said fermentation media comprises:
a. Raw material in the range of 100 to 150g/l
b. Di Ammonium Phosphate in the range of 0.3 to 0.6g/l
c. Urea in the range of 0.1 to 2.0g/l
d. Magnesium Sulphate in the range of 0.15 to 2.5g/l
e. Antifoam Agent in the range of 1.0 to 2.0 ml/1
f. KH2P04 in the range of 0.2 to 0.25g/l
g. Calcium Carbonate in the range of 15 to 30g/l
3. The process for the production of calcium gluconate as claimed in claim 2, wherein said
fermentation media comprises:
a) Raw material in an amount of 120.0g/l
b) Di Ammonium Phosphatein an amount of 0.50g/l
c) Urea in an amount of 0.15g/l
d) Magnesium Sulphate in an amount of 0.20g/l
e) Antifoam Agent in an amount of 1.5 ml/1
f) KH2P04 in an amount of 0.22 g/l
g) Calcium Carbonate in an amount of 24g/l

4. The process as claimed in claim 1 or 2, wherein said raw material is Dextrose mono hydrate.
5. The process as claimed in claim 2 or 3, wherein said anti foam agent is a vegetable oil.
6. The process as claimed in claim 1, wherein said fermenting organism is a strain of Aspergillus niger having accession number MTCC 5713.
7. The process as claimed in claim 1, wherein aeration to the media is provided in the range of 0.3 to l.Ov/v/m and more preferably at 0.5v/v/m.
8. The process as claimed in claim 1, wherein agitation to the media is provided in the range of 50 to 80 rpm and more preferably at 60 rpm.
9. The process as claimed in claim 1, wherein temperature ranges from 25°C to 42°C and more preferably from 28 to 35°C
10. The process as claimed in claim 1, wherein the pH of said fermentation media is maintained in the range of 6.0 - 7.2 and more preferably at 7.0.
11. The process as claimed in claim 1, wherein the pressure is maintained in the range of 0.4 to 0.75 bar and more preferably at 0.5 bar.
12. The process as claimed in claim 1, wherein said calcium gluconate is 100% pure.