THE PATENTS ACT, 1970
COMPLETE SPECIFICATION
Section 10
"Method for Producing and Optimizing the Production ofXylitol"
Tata Chemicals Limited, a corporation organized and existing under the laws of India, of Leela Business Park, Andheri Khuria Road, Andheri (E) Mumbai, India.
And University of Delhi, of University of Delhi, South Campus, Delhi- 110021 India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:
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TITLE OF INVENTION:
"Method for Producing and Optimizing the Production of Xylitol." FIELD OF INVENTION:
The present invention relates to a method for producing and optimizing the production of xylitol from xylose. More particularly, the present invention is directed to a method for optimizing the production of xylitol using Candida species.
BACKGROUND OF THE INVENTION:
Xylitol is a five-carbon low calorie sugar with potential medicinal importance. Xylitol was first manufactured in 1891 by a German chemist Emil Fischer. Xylitol has been used as a sweetening agent in human food since the 1960s. It is a white crystalline powder that is odorless, with a pleasant, sweet taste and is gaining increasing international acceptance as an alternative sweetener due to its role in reducing the development of dental caries (cavities). Xylitol occurs naturally in many fruits and vegetables and is also produced by the human body during normal metabolism. It is a natural, intermediate product, which regularly occurs in the glucose metabolism of man and other animals, as well as in the metabolism of several plants and microorganisms. Produced commercially from plants such as birch and other hard wood trees and fibrous vegetation, xylitol has the same sweetness and bulk as sucrose with one-third fewer calories and no unpleasant aftertaste. Xylitol metabolizes easily and independently from insulin in humans and produces the same amount of energy (4 Kcal/gm), which highlights its application in all diabetic foods. Because xylitol is only slowly absorbed and partially utilized, a reduced calorie claim is allowed: 2.4 calories per gram or 40% less than other carbohydrates. It quickly dissolves and produces a cooling sensation in the mouth.
It is also currently approved for use in foods, pharmaceuticals and oral health products in more than 35 countries. Researchers also discovered xylitol's insulin-independent nature. It metabolizes in the body without using insulin resulting in its use as a preferred sweetener in diabetic diets and as an energy source for infusion therapy in patients with impaired glucose tolerance and insulin resistance.
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Apart from the above, the adhesive properties of xylitol have been reported adequate to replace phenolic resin for plywood bonding.
Xylitol is used in chewing gum, gumdrops, hard candy, pharmaceuticals and oral health products such as throat lozenges, cough syrups, children's chewable multivitamins, toothpastes and mouthwashes.
Xylitol is an alternative to sugar having properties similar to sugar. While sugar wreaks havoc on the body, xylitol heals and repairs. It also builds immunity, protects against chronic degenerative disease, and has anti-aging benefits. While sugar is acid-forming, xylitol is alkaline enhancing. All other fonns of sugar, including sorbitol, another popular alternative sweetener, are six-carbon sugars, which feed dangerous bacteria and fungi.
Xylitol has no known toxic levels. The only discomfort that some sensitive people may notice initially when taking large amounts is mild diarrhea or slight cramping.
Since the body makes xylitol daily, as well as the enzymes to break it down, any discomfort usually disappears within a few days as the body's enzymatic activity adjusts to a higher intake. Xylitol has 40% fewer calories and 75% fewer carbohydrates than sugar and is slowly absorbed and metabolized, resulting in very negligible changes in insulin. About one-third of the xylitol that is consumed is absorbed in the liver. The other two-thirds travels to the intestinal tract, where gut bacteria into short-chain fatty acids break it down.
Thus xylitol is now being considered as an alternative to sugar for the following reasons:
• Good taste with no unpleasant aftertaste
• Helps reduce the development of dental caries
• Reduces plaque formation
• Increases salivary flow to aid in the repair of damaged tooth enamel
• Provides one-third fewer calories than sugar - about 2.4 calories per gram
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• Useful as an alternative to sugar for people with diabetes on the advice of their health care providers
The synthesis of xylitol from natural products is based on the chemistry of pentosans occurring in many plants. Xylan, a constituent of pentosan, is a polysaccharide that can be hydrolyzed into D-xylose, which is also known as wood sugar. Xylitol can be synthesized by hydrogenation of xylose.
Traditionally, xylitol has been produced by chemical reduction (hydrogenation) of D-xylose in the presence of Raney nickel catalyst (Mi/AbO;,) at 135 °C and 40 atm pressure for 2.5 hours. The product cost of the chemical process is high due to difficulty of purification and separation of xylitol, removal of by-products from Hernicellulose hydrolysate and a low yield of 40-50% based on xylan. Another chemical method reported in literature is hydrogenation of xylonic acid in presence of ruthenium carbon catalyst.
These method possesses lacunas as it is either capital intensive or / and result in the production of hazardous and toxic byproducts. Thus, chemical methods cannot be used for routine production of xylitol.
Realizing the importance of xylitol and its application in different industries it was therefore desirable to develop methods for maximum production of this sugar alcohol.
Sud-Chemie AG, Munich, Germany in their patents granted in 1976 (US Patent Number 3980719) had described a process for preparing xylitol by acid hydrolysis of xylan.
Another US Patent (Number 4008285), applied from Finland and granted in 1977, describes a method of producing xylitol on a commercial scale by acid hydrolysis of pentosan-containing raw materials such as wood, corncobs, straw, bran, and cotton-seed hulls.
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United States Patent Number 6911565 discloses a process utilizing ribulose for the preparation of xylitol and involves several different conversion reactions, such as reduction, epimerization and/or isomerisation.
Also, biotechnological processes for xylitol production using natural xylose-fermenting yeasts, which reduce xylose to xylitol by NAD(P)H- dependent xylose reductase(XR), have several advantages such as selective conversion of xylose to xylitol and a high yield. Xylitol can also be produced by microbial transformation reactions, such as from D-xylose by yeast or from D-glucose by yeast and bacteria. Xylitol has also been produced from D-xylulose using Mycobacterium smegmatis and from D-xylose using the commercial immobilized D-xylose isomerase of Bacillus coagulans or immobilized cells of M. smegmatis. D- xylulose could be converted to xylitol by fermentation with Mycobacterium smagematis.
Several biological processes using microorganisms for the production of xylitol from xylose are known. US patent No. 5686277 discloses a fermentation process for preparing xylitol with high productivity using novel mutant cells of Candida parapsilosis. In this process the composition of the medium containing xylose and environmental conditions of culture such as pH temperature and dissolve oxygen concentration are optimized. The fermentation medium comprises of a xylose medium comprising 5-12% weight by volume of xylose; 0.2-2% weight by volume of yeast extract; 0.2-2% weight by volume of ammonium sulphate; 0.2-2% of KH2P04 and .01-.2% of MgS04.7H20. The DO concentration in the medium is 0.1-5% w/v and redox potential in the medium is 50-150mV with a pH of fermentation medium being 4.5-5.5%).
Japanese Publication No. 11192095A is directed to a process for producing xylitol by culturing Candida magnoliae under microaerophilic condition at a dissolved oxygen concentration of .1-lppm. This patent application discloses the use of additional elements in the fermentation medium such as vitamins, minor constituents of carbon sources at a pH of 3.9-4.1 with an agitation speed of 500 rpm for 24 hrs.
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US Patent No. 6271007 is directed to the use of novel yeast strains for the production of xylitol. This patent however, subjects the xylitol producing microorganism to chemical treatment during the fermentation process.
International application no. WO 05/010171 is also directed towards the process for the manufacture of xylitol using a novel Candida tropicali species at a temperature of 30°C and agitation rate of 200 rpm.
US Patent no. 5866382 is also directed to a process for producing xylitol from yeast strain selected from the group of Saccharomyces spp., a Kluyveromyces spp. and a Schizosaccharomyces spp., transformed with a DNA molecule encoding a xylose reductase enzyme of a yeast or a mold.
The aforesaid disclosures although relate to the process for producing high yields of xylitol, however, none of these disclosures relate to the substantial increase in performance in the production of xylitol and more particularly increase in the performance using large scale production.
SUMMARY AND OBJECTS:
In order to enhance the yield of xylitol, in the present invention the physiological and nutritional factors are further optimized for further increasing the production of xylitol.
The present invention was carried out as an eco-friendly and cost-effective procedure for production of xylitol and also help in harnessing the undoubted commercial potential of Candida sp., which produces this important sugar alcohol.
To achieve the said objectives, the present invention provides a....
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS:
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The features of this invention are set forth with particularly in the appended claims. The invention, together with its objects and advantages thereof may be best understood by reference to the following description taken in conjunction with the accompanying drawing, in which:
Figure 1 illustrates the effect of pH alone of the fermentation medium on the production of
xylitol;
Figure 2: illustrates the effect of temperature alone of the fermentation medium on the
production of xylitol;
Figure 3: illustrates the effect of agitation rate alone of the fermentation medium on the
production of xylitol;
Figure 4: illustrates the effect of inoculum density on the production of xylitol.
Figures 5 and 6: illustrates the effect of organic and inorganic nitrogen source on the
production of xylitol
Figure 7: illustrates the effect of concentration of yeast extracts on the production of xylitol
Figure 8: illustrates the effect of the concentration of the carbon on the production of xylitol
Figure 9 illustrates the effect of the concentration of xylose, yeast extract; agitation rate;
inoculum density and incubation period according to the present invention on the
production of xylitol.
DETAILED DESCRIPTION OF THE INVENTION:
In order to determine the identified microorganisms which will be most optimal for the production of xylitol more than 300 microorganism were screened for their ability to produce xylitol Screening was carried out in two steps:
• Qualitative screening on Saborou's medium supplemented with 4.0% xylose
• Quantitative screening in broth
Qualitative screening
Qualitative evaluation of xylitol production from different microorganisms was carried out by point inoculating these cultures on Saborou's medium supplemented with 4.0% xylose.
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Plates were incubated for 72 hours at 30 ± 1 ° C. Observations were recorded at regular intervals of 24 hours up to 72 hours and the cultures able to grow in presence of 4.0% xylose were further evaluated quantitatively.
Quantitative screening
Microorganisms obtained after the qualitative screening were evaluated quantitatively for xylitol production in 5.0 % xylose. The amount of xylitol produced in the culture broth was estimated using HPLC.
Once having identified the yeast strain, the Candida sp. and in particular isolate no SP-7 appeared to be the best producer of xylitol under unoptimized conditions yielding 1 lgms/ltr. Process optimization was carried out to obtain maximum xylitol production by optimizing different physiological and nutritional factors including
(a) Physiological factors such as pH, temperature, agitation rate, inoculum density and incubation period of the fermentation medium.
(b) Nutritional factors such as substrate concentration, type of carbon source and its concentration, nitrogen source and its concentration, etc.
The effect of physiological factors was studied and can be illustrated as follows:
Effect of pH (Figure 1)
In order to study the effect of pH on xylitol production, the pH of the medium was adjusted in the range of 4.0- 8.0 using IN HC1/ NaOH. Maximum xylitol yield of 20.63 g/L was obtained at pH 4.5 after 60 h of incubation.
Effect of temperature (Figure 2):
Xylitol production was carried out at different temperatures ranging from 20°C to 45 °C. Optimal xylitol production of 20.70 g/L was observed at 30° C after 60 h of incubation.
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Effect of agitation rate (Figure 3):
Xylitol production was evaluated at different agitation rates i.e. 100,150, 200 and 250 rpm. An agitation rate of 200 rpm supported maximum xylitol production of 22.78 g/L.
Effect of inoculum density (Figure 4):
Effect of inoculum density (2.0%, 4.0%, 6.0%>, 8.0% and 10.0%) was tested for xylitol production. Maximum xylitol yield of 23.6 g/L was observed at 4.0 % inoculum density.
Effect of different nitrogen sources (Figures 5 and 6):
The following organic and inorganic nitrogenous compounds were tested for their effect on xylitol production:
Organic nitrogenous compounds: Casein hydrolysate, peptone, beef extract, yeast extract, tryptone, asparagine, soybean meal and corn steep liquor.
Inorganic nitrogenous compounds: ammonium chloride, ammonium nitrate, ammonium dihydrogen orthophosphate, sodium nitrate and ammonium sulphate.
Initially, the medium containing a mixture of three nitrogen sources was replaced by other organic and inorganic nitrogen sources on equal nitrogen basis. Maximum xylitol production of 24.9 g/L was observed in the yeast extract.
Concentrations of yeast extract (Figure 7):
Different concentrations of yeast extract viz. 0.2 % to 5.0 % were tried in the medium. Result showed that 0.6 % yeast extract gave maximum xylitol yield of 28.76 g/L after 60 h of incubation.
Concentration of Carbon source (Figure 8):
Different concentration of xylose viz. 10.0 to 200.0 g/L was evaluated for maximum xylitol production. Result showed that xylose in the concentration of 100.0 g/L gave a maximum xylitol yield of 59.5 g/L at 30°C in 96 h of incubation.
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Although each of the aforementioned physiological and nutritional factors known as me one at a time approach did lead to a significant enhancement in the xylitol production, there was a need to further enhance the production keeping in view the interactions between the most influential physiological and nutritional factors of the "one variable at a time" which further optimized xylitol production using a statistical design approach - Central Composite Design (CCD), falling under the response surface methodology (RSM).
The CCD incorporates replication of medial point (000). In this method, the concentration of the components - xylose, yeast extract, agitation rate, inoculum density found significant by "one variable at a time" approach, were further optimized. The ranges of these variables were decided according to the optimum value determined by the "one variable at a time" method. Other variables were set at their optimum levels. The minimum and maximum ranges of the medium components is selected and the design matrix of 30 experiments were generated using Design Expert software 6.0 (Stat Ease Inc., Minneapolis, USA).
Table 1: Experimental range and levels of the five independent variables used in RSM in terms of actual and coded factors
Variables Actual Coded Actual Actual Coded
(zero)

Xylose (%) 5.0 I 10.0 15.0 +1
Yeast extract (%) 0.4 I 0.6 0.8 +1
Inoculum density (%) 2.0 I 4.0 6.0 + 1
Agitation rate (rpm) 150 -1 I 200 250 +1
Incubation period (h) 84 -1 96 120 +1
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Table 2: Experimental design matrix of face centered central composite design for xylitol production
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Thus, the optimum composition of the medium obtained by the response surface methodology was xylose-10.0 %, yeast extract- 0.5 %, agitation rate - 200 rpm, inoculum density - 5.0 % and incubation period of 84 h. This resulted in the production of 64.93 g/L of xylitol, resulting in approximately 5.7-fold increase in the sugar alcohol production from the un-optimized condition (11.5 g/L). Subsequently, the production was carried out in 5 L flasks with 1250 ml under conditions optimized by using RSM. This resulted in the production of 63.8 g/L in 84h (Figure 9).
Large-scale production of xylitol in a 10 1 fermentor
Large-scale production of xylitol production by Candida sp. was carried out in a 10 L fermentor (New Brunswick Scientific, USA) with 7.0 L of the optimized production medium. The optimized medium and production conditions as obtained by "Response surface Methodology" in 250 ml flasks were translated identically in a 10 L bioreactor. Other operating parameters such as were pH 4.5; agitation rate, 200 rpm; temperature 30° C, air flow rate at 0.5 vvm were kept at their optimum level. All the medium components were steam sterilized at 121° C for 15 minutes in situ.
The results showed that a maximum of 63.2 g/L of xylitol was produced in 84 h. The xylose concentration decreased linearly with the increase in the growth and xylitol production.
All documents cited in the description are incorporated herein by reference. The present invention is not to be limited in scope by the specific embodiments, which are intended as illustrations of a number of aspects of the invention and any embodiments, which are functionally equivalent, are within the scope of this invention. Those skilled in the art will know, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.
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We claim:
1. A method for the production of xylitol comprising the steps of:
i) Fermenting a xylose containing medium with a yeast strain that has an ability to reduce xylose in the said medium to xylitol, wherein the said medium is optimized by controlling the following:
a. the concentration of the nitrogenous source ranging between 0.2-5
%.
b. an incubation period of between 36-120 hours.
c. agitation rate between 100-250 rpm.
d. inoculum density of fermentation medium between 2-10 %.
e. the concentration of carbon source substrate ranging between 1-20
% and
ii) Separating and recovering the xylitol from said fermentation medium.
2. A process as claimed in claim 1 wherein the at least one of the following conditions
are further optimized
a. pH of the fermentation medium being between 4- 8.
b. temperature of fermentation medium being between 20-45 C.
c. concentration of the carbon source in the medium being between
10-200 g/L
3. The process as claimed in claim 1 or 2, wherein the yeast strain is a Candida species.
4. The process as claimed in claim lor 2, wherein the yeast strain is Candida species (strain SP-7).
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5. A process as claimed in claim 1 wherein said fermentation medium includes organic and inorganic nitrogenous compound.
6. A process as claimed in claim 5 wherein said organic and inorganic nitrogenous compound is selected from any of the following:
casein hydrolysate; peptone; beef extract; yeast extract; tryptone; asparagines; soybean meal; corn steep liquor; ammonium chloride; ammonium nitrate ; ammonium dihydrogen; orthophosphate; sodium nitrate and ammonium sulphate.
7. A process as claimed in claim 1 or 2 or 6 wherein the concentration of the yeast extract is 0.5% or 0.6 %
8. A process as claimed in claim 2 wherein the pH of the fermentation medium is adjusted using mineral acid and an alkali.
9. A process as claimed in claim 8 wherein the mineral acid is hydrochloric acid and the alkali is sodium hydroxide.
10. A process as claimed in claim 9 wherein the pH of the fermentation medium is 4.5.
11. A process as claimed in claim 1 wherein the inoculum density of the fermentation medium of 4-6 %.
12. A process as claimed in claim 2 wherein the temperature of the fermentation medium is 30-35 C
13. A process as claimed in claim 1 or 2 wherein the agitation rate of the fermentation medium is 200-250 rpm.
14. A method for the production of xylitol comprising the steps of:
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i) Fermenting a xylose containing medium with a yeast strain that has an ability to reduce the said medium to xylitol, wherein the said medium is optimized by the following factors:
a. the concentration of the nitrogenous source being 0.5 %;
b. incubation period being 84 hours
c. agitation rate being 200 rpm
d. inoculum density of fermentation medium being 5 %
e. concentration of the carbon source in the medium is 10 % and
ii) Separating and recovering xylitol from said fermentation medium.
15. A process as claimed in claim 14 wherein the
pH of the fermentation medium is 4.5 and temperature of fermentation medium is 30°C


Dated this 18m day of May 2007

Of Anand and Anand AdAievateS""* Attorney for the Applicant

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Abstract
Method for producing and optimizing the production of xylitol.
The present invention relates to a method for producing and optimizing the production of xylitol from xylose using Candida species by optimizing the following conditions concentration of the nitrogenous source ; incubation period ; agitation rate ; inoculum density of fermentation medium and the concentration of carbon source substrate ranging between 1 -20 %
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