FORM 2
THE PATENTS ACT 1970
(39 OF 1970)
&
PATENTS RULES, 2006
PROVISIONAL SPECIFICATION (SECTION 10; RULE 13)


A PROCESS FOR THE PRODUCTION OF STABLE FRUCTOOLIGOSACCHARIDES "
ALKEM LABORATORIES LIMITED, A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956, HAVING ITS CORPORATE. OFFICE AT ALKEM HOUSE, DEVASHISH, ADJACENT TO MATULYA CENTRE, S.B.MARG, LOWER PAREL. MUMBAI - 400013. MAHARASHTRA. INDIA.
THE FOLLOWING SPECIFICATION DESCRIBES THE NATURE OF THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED:


■A PROCESS FOR THE PRODUCTION OF STABLE FRUCTOOLIGOSACCHARIDES"
FIELD OF THE INVENTION
The present invention relates to a process for the production of stable fructooligosaccharides by using fructosyltransferase from Aureobasidium species immobilized in sodium alginate.
BACKGROUND OF THE INVENTION
Fnictooligosaccharides (FOS) are short-chain polymers comprised of D-fructose and D-glucose; which are not metabolized by the body like simple sugars are, and are therefore considered non-digestible oligosaccharides. They occur naturally in chicory root bananas, garlic, Jerusalem artichokes and certain other foods, and technically are a soluble fiber. FOS are effective sweeteners, being half as sweet as sucrose, yet are not absorbed and have minima! caloric value. FOS may be considered prebiotic as they selectively support the proliferation of intestinal probiotics, especially the bifidobacteria Fnictooligosaccharides are added to foods to stimulate growth of bifidobacteria that regulate the intestinal flora, alleviating constipation, reducing cholesterol levels, and inhibiting the production of putrefiable compounds and the incidence of colon cancer. They have excellent flavor and texture properties. As per USFDA regulation FOS is generally recognized as safe (GRAS). However, the stability of FOS is poor. The industrial production of purified FOS is a problem and still in the developmental stage.
For commercial use, FOS are produced by the action of a fructosyl transferase (fructose transferase) on sucrose. Microorganisms, from which such enzymes can be derived are the strains of the genus Aspergillus such as A. Niger, A. japonicus etc., those of the genus Penicillium such as P. nigricans etc., those of the genus Fusarium such as F. lini IAM 5008 etc., those of the genus Qoeosporium such as G. kaki IAM
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5011 etc.. and those of the genus Aureobasidium such as A. pullulans and so on. Aside from the above-mentioned microorganisms, the enzymes derived from yeasts and other microorganisms, for example strains of the genus Saccharomyces such as S. cerevisiae etc., those of the genus Rhodotorula such as R. glutinis etc., those of the genus Pichia such as P. miso etc., those of the genus Hansenula such as H. miso etc., and those of the genus Candida such as C. tropicalis etc. can also be used.
Yun et al., Applied Biotechemistry and Biotechnology, 24-25, (1), 299-308, 1990, studied Aureobasidum puUulan cells with fructosyltransferase activity immobilized in calcium alginate beads for the production of fructosyl transferase. By comparison with the system of free cells in batch operation, the total amount of fructo-oligosaccharides produced by immobilized cells was similar although the composition of fructo-oligosaccharides was found to be different. In semibatch operation with immobilized cells, reproducible results up to 60 cycles were obtained at 50 °C and this operation resulted in no loss of activity of immobilized cells.
M. Antosova, M. Polakovic et al., Chemical Papers, 56, (6), 394-399, 2002 studied Aureobasidum puUulan for the production of fructosyl transferase and it was found that maximum of specific activity per dry cell mass was achieved at the same sucrose concentration.. Also the fructosyltransferase production in relation to cell growth and state of the culture, the sucrose conversion, produced amount of fructooligosaccharides and organic acids, and the yield factor of biomass on substrate, were determined.
Aniko Matusek, Peter Meresz et al., European Food Research and Technology, 228, (3), 355-365, 2009 studied degradation process of fructo-oligosaccharides in buffered solution affected by temperature of 60 - 100 °C and pH of 2.7 - 3.3, where the increase of proton concentration of the solution made degradation faster; however, there was a slighter effect of pH on the rate of hydrolysis than of temperature in the studied range. The rate of the changes in the value of average degree of polymerisation (DPa) was significantly depending on the temperature and pH.
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United States Patent No. 5,314,810 relates to a fructose transferring enzyme immobilized by adsorption on a granular carrier having a primary to quaternary amine like an epoxy polymer, a vinyl polymer or a chitosan derivative having a primary, secondary or tertiary amine. The immobilized enzyme without or with a crosslinking agent is used for producing fructooligosaccharides by passing a sucrose solution through a column containing the immobilized enzyme.
United States Patent Nos. 6,972,189 and 7,241,607 relate to Penicillium citrinum KCTC 10225BP of soil origin which produces fructosyl transferase and hydrolyzes sucrose into fructooligo saccharides and neofructooli go saccharides, at the same time using the fructosyl transferase, and thus provides a method for producing simultaneously fructooligosaccharides and neofructooligosaccharides using said microorganism.
United States Patent No. 2005/0069627A1 relates to the preparation of fructooligo saccharide (FOS) from extracellular Fructosyl Transferase (FTase) enzyme obtained from Aspergillus species wherein it provides use of jaggery as carbon source and stevia as to improve the sweetness of fructooligosaccharides.
OBJECT OF THE INVENTION
It is an object of the present invention to provide a process for the production of stable fructooligosaccharides by using fructosyltransferase from Aureobasidium species immobilized in sodium alginate.
It is another object of the present invention to provide a process for the production of stable fructooligosaccharides by using extracellular fructosyltransferase from Aureobasidium pullulans immobilized in sodium alginate.
At least one of the preceding objects is met, in whole or in part, by a process for the production of stable fructooligosaccharides by using extracellular fructosyltransferase from Aureobasidium pullulans immobilized in sodium alginate.
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SUMMARY OF THE INVENTION
The present invention provides a process for the production of stable fructooligosaccharides by using fructosyltransferase from Aureobasidium species immobilized in sodium alginate.
More particularly, the present invention provides a process for the production of stable fructooligosaccharides by using extracellular fructosyltransferase from Aureobasidium pullukms immobilized in sodium alginate.
Accordingly, the present invention provides a process for the production of stable fructooligosaccharides comprising:
(a) Transferring inoculum to growth medium for biomass production and harvesting the cells.
(b) Mixing the cell suspension with sodium alginate and immobilizing using cationic solution.
(c) Incubating the immobilized system of (b) with substrate under continuous stirring.
(d) Filtering the charcoal mixed solution after separating from incubation medium of (c) and concentrating the stable fructooligosaccharides.
DESCRIPTION OF THE INVENTION
Before the present formulations and methods are described, it is to be understood that this invention is not limited to particular compounds, formulas or steps described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values" is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise,
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between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of such compounds and reference to "the step" includes reference to one or more step and equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior inventioa Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
The term "stable" as used herein indicates no change in the percentages of total fhictooligosaccharides on dry basis, when kept at room temperature or 30°C / 65% RH or 40°C / 75% RH or 2°C - 8°C for 3 months or it passes the microbial analysis test.
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For passing the microbial analysis test the fructooligosaccharides should have total bacterial count of NMT 1000CFU per gram and total coli forms of NMT 30CFU per 100 gram and absence of pathogens and total yeast and mold of NMT 10CFU per gram.
The terms "dry basis" and "absolute basis" as used herein indicate "exclusive" and "inclusive" of any moisture content which may be present respectively. In other words, "dry basis" means [(Weight of the wet article - Weight of the dry article) x 100 +- Weight of the dry article] and "absolute basis'" means [(Weight of the wet article -Weight of the dry article) x 100 + Weight of the wet article].
We have surprisingly identified a process of producing stable fructooligosaccharides by using fructosyltransferase from Aureobasidium species and found that stable fructooligosaccharides could be produced by the present immobilized system of Aureobasidium pullulans immobilized in sodium alginate as against the conventional fructooligosaccharides prepared by prior arts.
The present invention provides a process for the production of stable fructooligosaccharides by using fructosyltransferase from Aureobasidium species immobilized in sodium alginate.
More particularly, the present invention provides a process for the production of stable fructooligosaccharides by using extracellular fructosyltransferase from Aureobasidium pullulans immobilized in sodium alginate.
More particularly, the present process for the production of stable fructooligosaccharides comprises:
(a) Transferring inoculum to growth medium for biomass production and harvesting the cells.
(b) Mixing the cell suspension with sodium alginate and immobilizing using cationic solution.
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(c) Incubating the immobilized system of (b) with substrate under continuous stirring.
(d) Filtering the charcoal mixed solution after separating from incubation medium of (c) and concentrating the stable fructooligosaccharides.
Accordingly, the present invention provides a process for the production of stable fructooligosaccharides comprising:
(a) Transferring predetermined concentration of Aureobasidium pullulans to growth medium for biomass production and harvesting the cells by centrifugation method or the like thereof.
(b) Mixing of 10-20% w/w (wet cell) of cells with 2-3 % w/w sodium alginate to prepare cell suspension and immobilizing preferably by the gel entrapment method using 2-4 % w/w calcium chloride solution.
(c) Incubating the immobilized system of (b) with substrate such as 30-70 % sucrose solution with pH 4.5-6.5 at temperature of 40-60°C under continuous stirring.
(d) Filtering the charcoal mixed solution after separating from incubation medium of (c) and the filtrate was concentrated till moisture content was found to be 18-20% using vacuum pump at pressure 750 mm Hg and temperature 45 to 50°C.
Aureobasidium pullulans, the "black yeast," is an environmentaJ fungus that grows on tree leaves and in salt water marshes. The fungus contains multiple life forms (polymorphic) including blastospores, hyphae, chlamydospores, and swollen cells. The chlamydospores and swollen cells are considered resting forms. The fungus produces a green melanin which turns black over time. The fungus also produces the polysaccharide puliulan. The fungus is currently used in industrial applications such as a coating for pill tablets, cosmetics, foods, and dissolvable breath fresheners. As the fructose transferring enzyme producing strain, Aureobasidium pullulans is preferably used.
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In preferred embodiments of the process of the present invention, the growth medium for transferring inoculum comprises of sucrose in the range of 18-22%, yeast extract in the range of 0.5-1.5%, MgSO 4-7H20 in the range of 0.03-0.07%, NaN03 in the range of 0.5-1.5%, K2HP04 in the range of 0.3-0.7%, K2HP04 m the range of 0.8-1.0% and the like or mixtures thereof
Still another embodiment of the present invention relates to a process, wherein the process provides stable fructooligosaccharides of 50-58 % w/w on dry basis (ODB) having thermal stability higher than that of sucrose solutions and are stable within the said temperature and pH ranges. Also the stable FOS produced by the present process contains 20-25 % w/w of glucose, 0.6-2 % w/w of fructose and 11-13 % w/w of sucrose on absolute basis in the concentrated product.
The following examples are intended to illustrate the scope of the present invention in all its aspects but not to limit it thereto.
EXAMPLE 1
The process for the production of stable fructooligosaccharides by using fructosyltransferase from Aureobasidium species immobilized in sodium alginate may be prepared as given in the following steps.
STEP I: PREPARATION OF MASTER SLANTS.
Microorganism used: Aureobasidium pullulans. ATCC No. 20524
Sterile distilled water (0.5-1.0 ml) was taken in a pasture pipette and added to the
culture of freeze- dried Aureobasidium pullulans. The contents were mixed
thoroughly and transferred into the sterile test tube containing 5 ml of sterile distilled
water.
The above mixture was stored in a refrigerator for the future trials.
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Preparation of agar slants of following composition:

Sr. No. Composition Quantity (gm/lit.)
1 Sucrose 4.0 gm
2 Yeast extract 4.0 gm
3 Malt extract 10.0 gm
4 Agar 20.0 gm
5 Dist. water 1000.0 ml
The above ingredients were weighed and dissolved in distilled water while heating and mixing to form a uniform suspension, pH was adjusted to 6.5 with IN sodium hydroxide solution. Medium was transferred into the culture tubes and autoclaved at 121°C and 15 lbs pressure for 15 minutes. After sterilization the culture tubes were kept in slanting position. Slants were kept at 30.0°C for 24 hrs to check for contamination as well as to remove extra moisture. Note: Heating and mixing is done to prevent lump formation of agar. Observation: No growth was observed. Inference: The culture medium was sterile.
Aureobasidium pullulans culture was streaked over these slants with sterile nichrome loop in sterile area. Slants were, incubated at 25°C for 48 -72 hrs.
STEP II: PREPARATION OF SEED CULTURE:

Sr.
no. Composition Quantity (gm/lit.)
1 Sucrose 200.0 gm
2 Yeast extract 10.0 gm
3 K2HP04 5.0 gm
4 NaN03 10.0 gm
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5 MgS04.7H20 0.5 gm
6 Chloramphenicol 0.0001 gm
7 Dist. water 1000.0 ml
The above ingredients were weighed and dissolved in distilled water while heating and mixing to form a uniform suspension. pH was adjusted to 6.50 with IN sodium hydroxide solution. Medium was autoclaved at 121°C and 15 lbs pressure for 15 minutes. The medium was cooled to room temperature. The medium was inoculated with master slants (1 slant sufficient for 100 ml medium) and incubated at 28°C for 72 hrs at 240 rpm.
STEP III: PRODUCTION OF CELLS:

Sr. no. Composition Quantity (gm/lit.)
1 Sucrose 200.0 gm
2 Yeast extract 10.0 gm
3 K2HP04 5.0 gm
4 NaN03 10.0 gm
5 MgS04.7H20 0.5 gm
6 Chloramphenicol 0.0001 gm
7 Dist. water 1000.0 ml
The above ingredients were weighed and dissolved in distilled water while heating and mixing to form a uniform suspension, pH was adjusted to 6.50 with IN sodium hydroxide solutioa Medium was autoclaved at 121°C and 15 lbs pressure for 15 minutes. The medium was cooled to room temperature. The above medium was inoculated with seed culture. 10 ml of seed culture is sufficient for 100 ml production medium. Production medium was incubated at 28°C for 72 hrs under rotating
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condition at 240 rpm. After incubation cells were separated by centrifugation and washed with normal saline (0.85% NaCl).
STEP IV: IMMOBILIZATION OF CELLS:
Cells harvested in STEP III were again centrifuged to remove supernatant.
The cells were mixed well with 2.0% sodium alginate slurry.
3.0% calcium chloride solution was prepared.
The sodium alginate slurry was dropped with the help of syringe into calcium chloride
solution, with continuous stirring.
STEP V: PRODUCTION OF FOS:
60.0% sucrose solution was prepared. The pH of sucrose solution was adjusted to
6.62. The solution was filtered by passing through 0.5 um filter.
Sodium alginate beads were inoculated in sucrose solution (10 gm beads in 100.0 ml
solution) and incubated at 55°C for 24 hrs with continuous stirring.
After incubation beads were allowed to settle down. The beads were separated,
washed with dist. water and again stored in 3.0% calcium chloride solution.
STEP VI: DISTILLATION AND CONCENTRATION OF FOS:
1.0% charcoal was mixed with solution obtained in STEP V.
Hiflow supercell slurry was prepared (1.0%) and spread into the sparkler filter to form
a 5 to 6 mm thick bed. Charcoal mixed solution was passed through sparkler filter and
filtrate was collected. Filtrate was passed through 10.0 and 0.45 micron filtration unit.
Filtrate was concentrated till moisture content was found to be 18-20% using vacuum
pump at pressure 750 mm Hg and temperature 45 to 50°C.
The concentrated product was collected in clean stainless steel container for packing.
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EXAMPLE 2
The stable fructoohgosaccharides of the invention as prepared in example 1 was subjected to analytical studies to determine the percentage yield of FOS and the results are shown in Table 1.
Table 1

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EXAMPLE 3
The stable fructooligosaccharides of the invention as prepared in example 1 was subjected to stability studies for 3 months at room temperature, 30°C / 65% RH, 40°C / 75% RH and 2°C - 8°C and the results are shown in Table 2.
Table 2

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EXAMPLE 4
The stable faictooligosaccharides of the invention as prepared in example 1 was subjected to microbial analysis for 3 months and the results are shown in Table 3.
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Table 3


Although the invention has been described in terms of particular embodiments and applications, one of ordinary skill in the art, in light of this teaching, can generate additional embodiments and modifications without departing from the spirit of or exceeding the scope of the claimed invention. It should be emphasized that the above-described embodiments of the present invention, particularly any "preferred" embodiments, are merely possible examples of the invention of implementations, merely set forth for a clear understanding of the principles of the invention. Accordingly, it is to be understood that the drawings and descriptions herein are proffered by way of example to facilitate comprehension of the invention and should not be construed to limit the scope thereof.
Dated this 30th day of January 2009
SanjayKher
Applicants'Patent Agent.
To:
The Controller of Patents, Patent Office, Mumbai 400 037
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