FORM 2


THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION [See section 10, Rule 13]
A PROCESS FOR PRODUCING A CHROMIUM ENRICHED YEAST SUPPLEMENT;
UNI-SANKYO LTD., A COMPANY
ORGANIZED AND EXISTING UNDER
THE LAWS OF INDIA, WHOSE
ADDRESS IS GAGANPAHAD,
MAHABOOBNAGAR ROAD,
HYDERABAD 501 323, INDIA

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 a process for producing chromium enriched yeast supplement.
BACKGROUND OF THE INVENTION
Chromium is a mineral and micronutrient that is known to enhance the action of insulin, thereby facilitating the transport of glucose into the cells and preventing the rise of blood glucose levels. An inadequate amount of chromium leads to a deficiency of chromium that may cause glucose intolerance, which can lead to diabetes, inadequate metabolism of carbohydrate, fat, and protein, increased risk of arteriosclerosis, obesity etc. The deficiency of chromium can be prevented and or cured by the chromium supplement. Hence, chromium is one of the essential minerals that mammals require in trace amount for good health.
The metal chromium exists in several valence (reactivity) or "ionic" states. Amongst the various oxidation states of Chromium ranging from 2" to 6+ the most common and stable form of chromium are hexavalent chromium (Cr VI) and trivalent chromium (Cr III). Cr (III), is biologically active and found in food, whereas Cr (VI) is a toxic form but highly stable.
Trivalent chromium (Cr III) was found as biologically important. According to recent reports, dietary supplementation with trivalent chromium was found to improve insulin effectiveness and sensitivity of the target cells. It has been found to be an essential trace element involved in protein structure stabilization and lipid and glucose metabolism. In case of rabbit as a model system, chromium (III) has also been shown to be a protective factor against heart disease, achieving a regression of cholesterol-induced arteriosclerosis.
There are many forms of chromium available in the market as chromium supplements including chromium enriched yeast and inorganic chromium salts such as, chromium chloride, chromium acetate and chromium citrate. Additional experiments indicated that dietary supplements with the inorganic chromium salts

did not achieve ah efficient effect. Dietary Cr supplements for humans as well as animals have to be the non-toxic and stable organically bound Cr complexes.
US Patent 4,348,483 discloses a method of producing a chromium yeast product having high intracellular chromium content useful as a dietary chromium supplement. The method comprises treating an aqueous suspension of live yeast cells with trivalent chromium followed by growing the cells in a nutrient medium to dilute the intracellular concentration of the chromium. In this method the growth of the pretreated seed yeast is severely restricted if the intracellular concentration of the chromium exceeds about 1000ug/g. Thus, more than 1000ug/g of chromium per gram of yeast solids absorbed by the seed yeast will lead to reduced growth of the yeast. Further, the said process requires higher concentration of chromium for treatment, more time and hence can be uneconomical.
U S Patent 6,248,323 provides dried yeast products containing chromium as well as method for producing the same comprising: admixing and incubating live yeast culture with an organochromium solution comprising about 500 ppm to about 25,000 ppm, preferably 7,500 ppm to 20,000ppm chromium glycinate dinicotinate to form a yeast-organochromium mixture, adding growth media to it to form a yeast growing mixture and grown for atleast 10 hours, isolating, washing, pasteurizing and drying the yeast cells to yield the chromium yeast product having intracellular organically complexed chromium content of from 1000 ppm to 6000 ppm, preferably 4066 ppm to 4924 ppm. However, the process is much lengthy, uses very high concentrations of chromium salt solution despite accumulates less concentration of chromium, requires supplementation of trivalent organochromium compounds as source of Chromium and; will not be economically feasible.
Thus, there is need to provide a process to produce a yeast having a enhanced content of stable organically bound chromium, wherein the process utilizes less concentration of chromium. Also, the process should be such that it requires less time, is economical, and is eco-friendly.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a chromium enriched yeast supplement having enhanced stable organically bound intracellular chromium content.

Other object of the present invention is to provide a chromium enriched yeast supplement that is edible and can be used as a dietary supplement.
Yet another object of the present invention is to provide a process for producing said chromium enriched yeast supplement.
One aspect of the present invention is to provide a process for producing a chromium enriched yeast supplement comprising: obtaining the yeast biomass from the live yeast culture, reducing the moisture content of the yeast biomass, treating the yeast biomass with a toxic chromium salt solution at a concentration that is non¬toxic and non-inhibitory to the yeast biomass followed by providing aeration and agitation to the yeast biomass to permit an intracellular accumulation and transformation of the toxic chromium ion to a non-toxic chromium ion.
The chromium enriched yeast biomass produced by the said process is edible, has high content of stable organically bound chromium, and can be used as a dietary supplement.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be described in conjunction with reference to the following drawings however, the same should not be considered as limiting to the scope of the invention.
Figure 1A shows the effect of reduction in moisture content of the yeast biomass and transformation of of Cr (VI) to Cr (III) in the absence of glucose, wherein the cells were dried for 30, 60, 120 min and exposed to Cr (VI) for 4 hr.
Figure 1B shows the effect of reduction in moisture content of the yeast biomass and transformation of of Cr (VI) to Cr (III) in the presence of glucose, wherein the cells were dried for 30, 60, 120 min and exposed to Cr (VI) for 4 hr in the presence of 2% glucose.
Figure 2A shows the Electron paramagnetic resonance (EPR) spectrum of chromium chloride tetrahydrate (trivalent chromium).

Figure 2B shows the EPR spectrum of chromium in chromium enriched Saccharomyces cerevisiae
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a chromium enriched yeast supplement having enhanced stable intracellular chromium content. Moreover the present invention provides a process for producing said chromium enriched yeast supplement using a toxic chromium salt solution, wherein the bioconversion of toxic chromium to edible form of chromium takes place during the process.
One embodiment of the present invention provides a process for producing a chromium enriched yeast supplement comprising; obtaining a yeast biomass from live yeast culture, reducing the moisture content of the yeast biomass, treating the yeast biomass with a toxic chromium salt solution at a concentration that is non-toxic and non-inhibitory to the yeast biomass followed by providing aeration and agitation to the yeast biomass to permit an intracellular accumulation and transformation of the toxic chromium ion to a non-toxic chromium ion. The chromium enriched yeast biomass is further subjected to drying to obtain the chromium enriched yeast supplement.
the chromium enriched yeast supplement as produced by the process of the present invention is edible, has high content of stable organically bound intracellular chromium, and suitable for use as a nutritional supplement.
The yeast cells that can be used for the purpose of the present invention include wet bakers yeast, brewers yeast, and other edible and food grade yeast. However, in the present invention it is preferable to use yeast selected from the group consisting of wet bakers yeast. Moreover it is most preferable to use the yeast Saccharomyces cerevisiae.
The yeast biomass may be obtained by culturing the yeast cells in a medium devoid of chromium and comprising nutrients including nitrogen source, carbon source, potassium source, phosphorus source, vitamins, co-factors, trace minerals including magnesium, cobalt, zinc and any other suitable nutrient and/or trace element that may support the growth of the yeast cells.

Numerous other growth media devoid of chromium and that can support the growth of yeast Saccharomyces cerevisiae may be used in the present invention including peptone, corn, whey, potatoes, rice, molasses, yeast extract and the likes either alone or in combinations for culturing the yeast cells. In a preferred embodiment, molasses devoid of chromium may be used as a medium for culturing the yeast cells. It is: a cheap source and is available easily in plenty.
The yeast cells can be cultured by conventionally used processes such as batch culture, single stage continuous culture, multiple stage continuous culture, fed batch process and the likes at such pH and temperature that may allow the yeast cells to form the yeast biomass.
For the purpose of the present invention in one embodiment, the yeast cells may be cultured in molasses as a growth medium devoid of chromium at pH 5 to 7 for 18 hrs to 24 hrs to obtain the yeast biomass. In a preferred embodiment the yeast cells may be cultured at pH 5.5.
In the present invention the step of culturing of the yeast cells in a nutrient rich
medium devoid of chromium provides a yeast biomass devoid of intracellular
chromium.
After culturing, the yeast biomass may be harvested by suitable means. In a
preferred embodiment centrifugation may be carried out in order to obtain yeast
biomass.
The moisture content of the yeast biomass is reduced so as to get the yeast biomass with 85% to 96% moisture content preferably 94%. The moisture content of the yeast cells may be reduced by any suitable process such as by recirculating sterile dry air at a suitable temperature and period. For the purpose of the present invention, the moisture content of the yeast biomass may be reduce by circulating sterile dry air at a temperature ranging from about 35°C to 40°C for a period of about 20 minutes to about 60 minutes. The temperature and period required for reducing the moisture content of the yeast biomass cake may be varied depending upon the characteristics, moisture content of the yeast biomass and other conditions.
In the present invention the step of reducing the moisture content of the yeast biomass enables the cells to imbibe more chromium salt.

The yeast biomass having reduced moisture content is treated with a toxic chromium salt solution at a non-toxic and non-inhibitory concentration followed by providing aeration and agitation to provide the yeast biomass with enhanced content of intracellular stable non-toxic chromium ions suitable for human consumption.
The step of treating the yeast biomass having "reduced moisture content with a toxic chromium salt solution at a non-toxic and non-inhibitory concentration followed by providing aeration and agitation results in the intracellular accumulation and transformation of the toxic chromium salt to the non-toxic chromium ion.
The toxic chromium salt that may be used for treating the yeast biomass is hexavalent chromium (Cr6+ or Or VI) including potassium chromate (K2Cr04), potassium dichromate (K2Cr207), sodium chromate (NaCr04) or any such suitable source of hexavalent chromium.
Hexavalent chromium is the second most stable form and a strong oxidizing agent, especially in acidic media. In water, hexavalent chromium for example K2Cr04, K2Cr207, NaCr04 dissociates as anion Cr04~. This anion is structurally similar to sulfate (S04~) and can be transported readily into the cell via sulfate transport pathway. Thus, the uptake of hexavalent chromium by yeast cells is better compared to the other forms of the chromium. Intracellular^ these toxic anions are deoxygenated to organo-trivalent chromium Cr (III) complexes by reduction with intracellular reductants such as glutathione, NADH, NADPH etc. Unused Cr (VI) is water soluble and can be easily removed by centrifugation
The toxic chromium salt solution of the present invention is prepared in atleast 0.5% of glucose, as glucose assists the metabolism of yeast. The yeast cells actively detoxify the added toxic chromium salt such as hexavalent chromium thereby converting it to a bioavailable form of organically bound non-toxic chromium such as trivalent chromium. In the most preferred embodiment the hexavalent chromium salt solution is prepared in at least 1% of glucose. The pH of the solution is adjusted to about 4 to about 6.5 with the help of hydrochloric acid. In the most preferred embodiments the pH of the solution is adjusted to about 4.5 to about 5.

For the purpose of the present the yeast biomass with reduced moisture content may be treated with a solution comprising about 5 ppm to 20 ppm of the toxic chromium salt, prepared as mentioned herein above. In the.preferred embodiments the yeast biomass with reduced moisture content may be treated with a solution comprising about 10 ppm to about 12 ppm of the toxic chromium salt.
The yeast biomass with the reduced moisture content is treated with the toxic chromium salt solution for atleast 4 hrs. In the preferred embodiment the yeast biomass with the reduced moisture content is treated with the toxic chromium salt solution for atleast 6 hrs.
After treating the yeast biomass with the toxic chromium salt solution it is preferable to provide aeration at about 0.5 wm to 5 wm and agitation at around 50 rpm to 150 rpm. However, the specific time and the speed for agitation may be varied depending upon the intracellular accumulation and transformation of toxic chromium ions into non-toxic chromium ions by the yeast biomass.
The yeast biomass with accumulated intracellular chromium may be recovered from the medium by centrifugation followed by washing to remove extracellular chromium salts.
The yeast biomass containing a substantial amount of non-toxic intracellular chromium is dried to obtain the chromium enriched yeast supplement. The drying also enhances the shelf life, prevents the contamination, reduces the weight thereby facilitating the transportation, ease of use for blending with commercially available feeds such as starch, CMC, corn based feed for poultry, cereal flours or combinations thereof and preparing a formulation.
The intracellular chromium content of the yeast obtained by the said process of the present invention is atleast 1200ug/g of the intracellular trivalent chromium.
The said process of the present invention is much simpler, faster, economical, eco-friendly, uses a novel source of chromium salt for the treatment of yeast biomass which can be transported readily into the cell and hence there is a better uptake and more intracellular accumulation of chromium salts.

The chromium enriched yeast supplement prepared as per the process of the present invention is suitable for use in nutritional supplements.
The invention is further illustrated in the following non-limiting examples.

EXAMPLES
Example 1
Culture of Saccharomyces cerevisiae to obtain yeast biomass:
The yeast Saccharomyces cerevisiae was cultured in molasses medium devoid of
chromium at pH 5.5 for about 24 hours. From the culture yeast biomass was
i harvested by centrifugation at 8000 rpm.

Example 2
The yeast Saccharomyces cerevisiae biomass as obtained in Example 1 and having
initial cell count of 1 X 106 CFU/ml (was subjected to sterile dry air at temperature of

about 37°C for 30 mins. to obtain the yeast biomass with reduced moisture content of about 94%. The yeast biomass with reduced moisture content was then treated with a solution comprising 20 ppm of potassium chromate and 1% of the glucose for 4 hrs by exposing the yeast biomass to the chromium salt solution. Aeration at 1wm and agitation at lOOrpm was then provided to the yeast biomass in the chromium
salt solution for the intracellular accumulation and transformation of the hexavalent

chromium to trivalent chromium. The yeast biomass was washed with water to

remove the extracellular chromium salts. Followed by washing the yeast biomass was concentrated and dried to obta n the chromium enriched yeast supplement. The final count of the yeast cell was 1 X 105 CFU/ml comprising 1236 ug/g of the intracellular trivalent chromium.
Example 3
The yeast Saccharomyces cerevisiae biomass as obtained in Example 1 and having
initial cell count of 1 X 106 CFU/ml was subjected to sterile dry air at temperature of

about 37°C for 60 mins. to obtain the yeast biomass with reduced moisture content
of about 94%. The yeast biomass With reduced moisture content was then treated
with a solution comprising 20 ppm of potassium chromate and 0.5% glucose for
about 6 hrs by exposing the yeast biomass to the chromium salt solution. Aeration at
1 vvm and agitation at 100 rpm was then provided to the yeast biomass for the

intracellular accumulation and transformation of the hexavalent chromium to trivalent chromium. The yeast biomass was washed with water to remove the extracellular chromium salts. Followed by washing the yeast biomass was concentrated and dried to obtain the chromium enriched yeast supplement. The final count of the yeast cells was 2.5 X 105 CFU/ml comprising 1375 ug/g of the intracellular trivalent chromium.
Example 4
The yeast Saccharomyces cerevisiae biomass as obtained in Example 1 having initial cell count of 1 X 106 CFU/ml was subjected to sterile dry air at temperature of about 37°C for 120'min. to obtain the yeast biomass with reduced moisture content of the about 92%. The yeast biomass with reduced moisture content was then treated with a solution comprising 10 ppm of potassium dichromate, 1% glucose and having pH 5.5 for about 6 hrs by exposing the yeast biomass to the chromium salt solution. Aeration at 1wm and agitation at 120 rpm was then provided to the yeast biomass for the intracellular accumulation and transformation of the hexavalent chromium to trivalent chromium. The yeast biomass was washed with water to remove the extracellular chromium salts. Followed by washing the yeast biomass was concentrated, and dried to obtain the chromium enriched yeast supplement. The final count of the yeast cells was 3.7 X 105 CFU/ml comprising 2189 ug/g of the intracellular trivalent chromium.
Example 5:
An experiment was conducted to demonstrate the effect of reduction in moisture content of the yeast biomass and transformation of of Cr(VI) to Gr (III) in absence and presence of glucose. For the prupose of better visibility in this experiment, yeast cells were exposed to higher concentration that is 52 ppm Cr(VI). Different batches of cells were used, where cells were exposed to'dry air at 37° C for 30, 60 and 120 mins. The cells were allowed to remain in contact with chromium for about 4 hrs. The results shown in Figure 1A indicate no change in peak in absence of glucose with increase in dehydration time. Whereas, as shown in Figure 1B in the presence of 2% of glucose the chromium peaks diminish. A shift is observed in the peak from about 372 nm which is for Cr (VI) to about 340 nm which indicates formation of Cr(lll).

Example 6:
The presence of trivalent chromium in the chromium enriched yeast supplement was confirmed by the Electron paramagnetic resonance (EPR) analysis on the basis of the characteristic of EPR peaks. The Chromium yeast supplement was dried at 80°C and a fine powder was made by grinding. EPR spectroscopy was carried out using Bruker EMX EPR spectrometer for spectral characterization of solid/liquid paramagnetic materials in temperature range of 77-773 K at X and Q band frequencies. Chromium chloride hexahydrate powder was used as standard for obtaining the spectra of trivalent chromium.
The EPR spectrum was obtained for Saccharomyces cerevisiae after,chromium uptake. The peaks obtained as shown in Figure 2B corresponding to the g-values at 2805 and 3794 are in agreement with those obtained for standard trivalent chromium as shown in Figure 2A, which exhibits a paramagnetic character. The additional feature observed at 3329 and 3352 G was due to Cr (V) which was an intermediate often formed during byconversion of Cr (VI) to Cr (III). It may be noted that Cr (V) which is an unstable species gets readily converted into the stable form viz., Cr (III). Besides, Cr (VI) due to absence of unpaired electron gives no EPR signal. The absence of Cr (VI) is confirmed by a spectrophotometry assay,, using diphenyl carbazide reagent. This reagent reacts only with Cr(VI) giving a pink colored end product estimated at 540 nm. EPR spectroscopy is for confirmation of Cr(lll) since this species is paramagnetic due to presence of unpaired electron. The presence of trivalent chromium in the chromium enriched yeast supplement was thus confirmed, on the basis of the characteristic EPR peaks.

WE CLAIM:
1) A process for producing a chromium enriched yeast supplement comprising
steps of:
obtaining a yeast biomass from live yeast culture;
reducing the moisture content of the yeast biomass;
treating the yeast biomass with a toxic chromium salt solution for a specific
period;
providing aeration and agitation to the yeast biomass to permit an
intracellular accumulation and transformation of the toxic chromium ion to a
non-toxic chromium ion.
2) The process as claimed in claim 1, wherein said yeast cells are selected from the group of food grade yeast preferably wet baker's yeast.
3) The process as claimed in claim 2, wherein said yeast cells are Saccharomyces cerevisiae.
4) The process as claimed claim 1, wherein said moisture content of the yeast biomass is reduced so as to obtain the yeast biomass with moisture content of about 85% to 96%, preferably 94%.
5) The process as claimed in claim 1, wherein said toxic chromium salt is a hexavalent chromium salt.
6) The process as claimed in claim 1, wherein treatment of the yeast biomass with a toxic chromium salt solution is carried out at a concentration that is non-toxic and non-inhibitory to the yeast biomass.
7) The process as claimed in claim 6, wherein said concentration of the toxic chromium salt solution for treating the yeast biomass is from about 5ppm to 20ppm.
8) The process as claimed in claim 7, wherein said concentration of the toxic chromium salt solution for treating the yeast biomass is preferably from about 10ppm to 12ppm

9) The process as claimed in claim 1, wherein said toxic chromium salt solution is prepared in atleast 0.5% of glucose solution.
10) The process as claimed in claim 9, wherein said toxic chromium salt.solution is preferably prepared in atleast 1% of glucose solution.
11) The process as claimed in claim 1, wherein the pH of the toxic chromium salt solution is from about 4 to 6.5.
12) The process as claimed in claim 11, wherein pH of the toxic chromium salt solution is preferably from about 4.5 to 5.
13) The process as claimed in claim 1, wherein the yeast biomass with a toxic chromium salt solution is treated for atleast 4 hours.
14) The process as claimed in claim 13, wherein the yeast biomass with a toxic chromium salt solution is treated preferably for atleast 6 hours.
15) The process as claimed in claim 1, wherein said non-toxic.chromium ion is trivalent chromium.
16) The process as claimed in claim 1, further comprises drying the yeast biomass.
17) A chromium enriched yeast supplement produced by the process as claimed in claim 1 to 16, wherein the intracellular non-toxic chromium ion content is atleast 1200 ug/g of the intracellular trivalent chromium.
18) A process for producing a chromium enriched yeast supplement and a chromium enriched yeast supplement as described and illustrated.
Dated this 11th day of June, 2009
FOR UNI-SANKYQ LTD. By their Agent
(MADHURI RAMESHCHANDRA TAWRI) KRISHNA & SAURASTRI