WO2020039231A1

WO2020039231A1 - Bioreactor and process for producing a low-calorie sugar composition

Info

Publication number: WO2020039231A1
Application number: PCT/IB2018/056309
Authority: WO
Inventors: Banibrata Pandey; Sibnath RAY
Original assignee: Petiva Private Limited
Priority date: 2018-08-21
Filing date: 2018-08-21
Publication date: 2020-02-27

Abstract

The present invention provides a process for producing a low-calorie sugar composition in a bioreactor. The bioreactor comprises a pre-treatment reactor, a buffered tank, plurality of product storage tanks and columns having immobilized enzymes, wherein the enzyme columns are coupled in such a manner that each enzyme column is operable individually or in combination on a sugar source and the enzymes are selected from a group comprising hydrolases, isomerases and epimerases.

Description

BIOREACTOR AND PROCESS FOR PRODUCING A LOW-CALORIE

SUGAR COMPOSITION

TECHNICAL FIELD

The present invention relates to sugar compositions. Particularly, the invention relates to a process for producing a low-calorie sugar composition in a bioreactor.

BACKGROUND

Rare sugars such as isomaltulose, trehalulose, D-psicose (D-allulose) and D-allose are those that exist in extremely small amounts in nature because usually they are the products of intermediary cellular metabolism. Rare sugars are converted within the cell to another form as soon as they are formed. Despite their low natural occurrence, rare sugars have enormous potential in several important applications, including as components for antiviral drugs, low-calorie sweeteners with low glycemic indexes, anti-inflammatory agents with immunosuppressive properties and chiral building blocks in natural products synthesis.

Attention has been focused recently on rare sugars because rare sugars have various physiological effects. Therefore, research works have been done intensively for wide industrial application. Furthermore, efficient production of these rare sugars is essential. Known processes currently use costly and complex biochemical processes to transform easily found abundant sugars into rare sugars. These biochemical processes use three main classes of enzymes, isomerases, oxidoreductases and epimerases, as they offer high specificity for products and are capable of selectively modifying sugars at multiple carbon positions. However, due to the process constraint such as active biocatalyst, enzyme recovery or reutilization, enzyme substrate specificity, raw material cost etc. especially on a large scale, the cost of these rare sugars for daily applications is still far too high.

Other known processes for producing compositions of rare sugars include simple admixing of individual purified sugars in the required amount. However, sugars in their pure form may be quite expensive, and the purity and therefore quality for each sugar may vary from source to source, resulting in variability of the end composition.

Therefore, there is a desire to develop an economic production process of a low- calorie sugar composition that includes rare sugars. SUMMARY

Accordingly, the present invention provides a process for producing a low-calorie sugar composition from a sugar source in a bioreactor. In some instances, the bioreactor comprises a pre-treatment reactor, a buffered tank, plurality of product storage tanks and columns having immobilized enzymes, wherein the enzyme columns are coupled in such a manner that each enzyme column is operable individually or in combination on a sugar source and the enzymes are selected from a group comprising hydrolases, isomerases and epimerases.

In some instances, the bioreactor comprises:

a pre-treatment reactor (R-801) for the treatment of biomass to obtain cellulose and xylose; a first column (C-801) connected to the pre-treatment reactor (R-801) via tank (T- 802) for receiving the cellulose from the pre-treatment reactor (R-801) and configured to treat the cellulose to obtain a mixture of glucose and cellobiose;

a second column (C-701) connected to the first column (C-801) via tank (T-701) for receiving the glucose from the first column (C-801) and configured to treat the glucose to obtain a mixture of glucose and fructose;

a third column (C-702) connected to the second column (C-701) via tank (T-702) for receiving the fructose from the second column (C701) and configured to treat the fructose to obtain a mixture comprising fructose and psicose;

a fourth column (C-703) connected to the third column (C-702) via tank (T-703) for receiving the psicose from the third column (C702) and configured to treat the psicose to obtain a mixture comprising psicose and allose;

a fifth column (C-301) connected to the pre-treatment reactor (R-801) via tank (T- 301) for receiving the xylose and configured to treat the xylose to obtain a mixture comprising xylose and xylulose;

a sixth column (C-401) connected to the first column (C-801) via tank (T-401) for receiving the cellobiose from the first column (C-801) and configured treat the cellobiose to obtain a mixture of cellobiose and glucose; wherein the second column (C-701) is connected to the sixth column (C-401) via tank (T-701) to receive glucose from the sixth column (C-401); a seventh column (C-601) configured to receive sucrose, glucose and fructose in form of juices and configured to treat the juices to obtain a mixture of glucose and fructose; wherein the second column (C-701) is connected to the seventh column (C-601) to receive glucose from the seventh column (C-601);

an eighth column (C-901) configured to receive a mixture of sucrose, glucose and fructose in form of juices and configured to treat the juices to obtain a mixture comprising sucrose and isomaltulose;

a ninth column (C-902) configured to receive sucrose, glucose and fructose in form of juices and configured to treat the juices to obtain a mixture comprising sucrose and trehaluose; and a product collection tank (T-1003) connected to and receive output from the fourth column (C-703), the fifth column (C-301), the eighth column (C-901) and the ninth column (C-902).

In another aspect, the present invention provides a process for producing a low- calorie sugar composition comprising:

a) feeding a sugar source to the bioreactor as claimed in claim 1 ;

b) processing the sugar source to get a sugar substrate;

c) reacting the sugar substrate with a suitable enzyme immobilized on a suitable matrix in a column under pre-determined reaction conditions to obtain a corresponding sugar product; and

d) feeding the sugar product obtained from one column to another pre determined column and withdrawing the desired low-calorie sugar composition from final product collection tank;

wherein, the reaction of sugar substrate in each column is optionally repeated one or more times to get desired ratio of low-calorie sugar composition.

BRIEF DESCRIPTION OF THE FIGURES

A complete understanding of the exemplary embodiments of the present invention and the advantages thereof may be acquired by referring to the following description in consideration of the accompanying figures.

FIG. 1 illustrates a representative bioreactor according to one embodiment of the present invention; FIG. 2 illustrates a representative chromatogram of bio-conversion of hexose with residual cellobiose from cellulosic saccharified sugars according to Example 1 ;

FIG. 3 illustrates a representative chromatogram of bio-conversion of glucose to a low-calorie sugar composition according to Example 2;

FIG. 4 illustrates a representative chromatogram of bio-conversion of sucrose to a low-calorie sugar composition containing mono- and di-saccharides according to Example 3 ;

FIG. 5 illustrates a representative chromatogram of bio-conversion of sucrose to a low-calorie sugar composition containing mono- and di-saccharides according to Example 4;

FIG. 6 illustrates a representative chromatogram of bio-conversion of sucrose to a low-calorie sugar composition containing mono- and di-saccharides according to Example 5 ; and

FIG. 7 illustrates a representative chromatogram of bio-conversion of cellobiose to a low-calorie sugar composition containing mono- and di-saccharides according to Example 5.

FIGS. 1-7 should be understood to present an illustration of the disclosure and/or principles involved. Details including valves, instrumentation, and other equipment and systems not essential to the understanding of the disclosure are not shown.

DETAILED DESCRIPTION

Before the methods of the present disclosure are described in greater detail, it is to be understood that the methods are not limited to particular embodiments 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 methods 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, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods.

Certain ranges are presented herein with numerical values being preceded by the term“about.” The term“about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

In certain embodiments, the present invention provides a bioreactor for converting a sugar source into a pre-determined low-calorie sugar composition. In some instances, the bioreactor includes a reactor, a buffered tank, plurality of product storage tanks and plurality of enzyme columns, wherein the enzyme columns are coupled in such a manner that each enzyme column is operable individually or in combination on a sugar source. In some instances, the enzymes are selected from a group comprising hydrolases, isomerases and epimerases. In certain embodiments, the enzymes are immobilized on a suitable matrix. Examples of matrices for immobilization of the enzyme include, but are not limited to, natural polymers, synthetic polymers and inorganic materials. In further embodiments, the natural polymer is selected from a group comprising alginate, chitosan, chitin, collagen, carrageenan, gelatin, cellulose, starch and pectin. In certain embodiments, the synthetic polymers are ion exchange resins or polymers and are insoluble supports with porous surface. Examples of synthetic polymers include, but are not limited to, diethylaminomethyl cellulose (DEAE cellulose), polyvinyl chloride (PVC), polyethylene glycol (PEG). In certain embodiments, the inorganic material is selected from a group comprising zeolites, ceramics, diatomaceous earth, silica, glass, activated carbon, and charcoal.

In certain embodiments, the matrix used for the immobilization of the enzyme is alginate. In further embodiments, the matrix is sodium alginate particles.

In an embodiment, the present invention provides a process for producing low- calorie sugar composition from a sugar source in a bioreactor. FIG. 1 depicts a bioreactor according to an embodiment of the invention for producing low-calorie sugar composition. The bioreactor comprises:

a pre-treatment reactor (R-801) for treatment of biomass to obtain cellulose and xylose;

a first column (C-801) connected to the pre-treatment reactor (R-801) via tank (T- 802) for receiving the cellulose from the pre-treatment reactor (R-801) and configured to treat the cellulose to obtain a mixture of glucose and cellobiose;

a sixth column (C-401) connected to the first column (C-801) via tank (T-401) for receiving the cellobiose from the first column (C-801) and configured treat the cellobiose to obtain a mixture of cellobiose and glucose; wherein the second column (C-701) is connected to the sixth column (C-401) via tank (T-701) to receive glucose from the sixth column (C-401);

a seventh column (C-601) configured to receive sucrose, glucose and fructose in form of juices and configured to treat the juices to obtain a mixture of glucose and fructose; wherein the second column (C-701) is connected to the seventh column (C-601) to receive glucose from the seventh column (C-601);

an eighth column (C-901) configured to receive a mixture of sucrose, glucose and fructose in the form of juices and configured to treat the juices to obtain a mixture comprising sucrose and isomaltulose; a ninth column (C-902) configured to receive sucrose, glucose and fructose in form of juices and configured to treat the juices to obtain a mixture comprising sucrose and trehaluose; and

a product collection tank (T-1003) connected to and configured to receive output from the fourth column (C-703), the fifth column (C-301), the eighth column (C-901) and the ninth column (C-902).

In another embodiment, the bioreactor comprises a first downstream separation unit (DSP 801) provided between the first column (C-801) and the second column (C- 701). The first downstream separation unit (DSP 801) is configured to control the flow from the first column (C-801) towards the first column (C-801), second column (C-701) or the sixth column (C-401) based on the composition of mixture of glucose and cellobiose.

In yet another embodiment, the bioreactor comprises a second downstream separation unit (DSP 701) provided between the second column (C-701) and the third column (C-702). The second downstream separation unit (DSP 701) is configured to allow flow from the second column (C-701) towards the second column (C-701) or the third column (C-702) based on the composition of mixture of glucose and fructose received from the second column (C-701).

In yet another embodiment, the bioreactor comprises a third downstream separation unit (DSP 702) provided between the third column (C-702) and the fourth column (C-703). The third downstream separation unit (DSP 702) is configured to allow flow from the third column (C-702) towards the third column (C-702) or the fourth column (C-703) based on the composition of mixture of psicose and fructose received from the third column (C-702).

In yet another embodiment, the bioreactor comprises a fourth downstream separation unit (DSP 703) provided between the fourth column (C-703) and the product collection tank (T-1003). The fourth downstream separation unit (DSP 703) is configured to allow flow from the fourth column (C-703) towards the fourth column (C-703) or the product collection tank based on the composition of mixture of psicose and allose received from the fourth column (C-703). In yet another embodiment, the bioreactor comprises a fifth downstream separation unit (DSP 301) provided between the fifth column (C-301) and the product collection tank (T-1003). The fifth downstream separation unit (DSP 301) is configured to allow flow from the fifth column (C-301) towards the fifth column (C-301) or the product collection tank (T1003) based on the composition of mixture of xylose and xylulose received from the fifth column (C-301).

In yet another embodiment, the bioreactor comprises a sixth downstream separation unit (DSP 401) provided between the sixth column (C-401) and the second column (C-701). The sixth downstream separation unit (DSP 401) is configured to allow flow from the sixth column (C-401) towards the sixth column (C-401) or the second column (C-701) based on the composition of mixture of glucose and cellobiose received from the sixth column (C-401).

In yet another embodiment, the bioreactor comprises a seventh downstream separation unit (DSP 601) provided between the seventh column (C-601) and the second column (C-701). The seventh downstream separation unit (DSP 601) is configured to allow flow from the seventh column (C-701) towards the seventh column (C-701) or the second column (C-701) based on the composition of mixture of glucose and fructose received from the seventh column (C-601).

In yet another embodiment, the bioreactor comprises an eighth downstream separation unit (DSP 901) provided between the eighth column (C901) and the product collection tank (T-1003). The eighth downstream separation unit (DSP 901) is configured to allow flow from the eighth column (C-901) towards the eighth column (C-901) or the product collection tank (T-1003) based on the composition of mixture of sucrose and isomaltulose received from the eighth column (C-901).

In yet another embodiment, the bioreactor comprises a ninth downstream separation unit (DSP 902) provided between the ninth column (C-902) and the product collection tank (T-1003). The ninth downstream separation unit (DSP 902) is configured to allow flow from the ninth column (C-902) towards the ninth column (C-902) or the product collection tank (T-1003) based on the composition of mixture of sucrose and trehaluose received from the ninth column (C-902). The enzymatic reactor is designed in such a manner that each individual column can operate separately or in combination with another enzymatic column in tandem. Each enzyme column is provided with jacket for maintaining temperature conducive for individual enzyme optimum for conversion of the substrate to desired products. Further, each enzyme column is connected to a downstream processing unit or downstream separation unit for separating the desired products. In some instances, the downstream processing unit may be an ion-exchange column or a simulated moving bed column. In certain embodiments, the sugar stream withdrawn from each column is fed to the other column with or without recycling of the enzyme based on the desired sugar composition. The product withdrawn from each column is stored in a storage tank and concentrated or fed from storage tank to the other column, and then collected in a final tank. The reaction of sugar substrate in each column is optionally repeated one or more times to get desired low-calorie sugar composition.

In another embodiment, the present invention provides a process for producing a low-calorie sugar composition. In some instances, the process comprises:

a) feeding a sugar source to the bioreactor, e.g., described above;

b) processing the sugar source to get a sugar substrate;

Lignocelluloses and their sugar content are the largest sources of naturally available sugar. The lignocellulose material may be selected from any convenient source, such as a group comprising hardwood, soft wood, and agricultural residue. In certain embodiments, the lignocellulosic biomass is obtained from the fibrous remains of sugarcane and sorghum or any such material of organic origin or a mixture thereof. Sugar containing juice source such as sweet sorghum, sugar cane and sugar beet contain substantial quantity of sugar. However, the composition of sugar in the juice changes according to the feedstock used. Sugar cane and sugar beet juice contain mostly sucrose whereas sweet sorghum contains sucrose, fructose and glucose.

In certain embodiments, the starting material used for the process is a juice containing plant material, wherein the juice contains substantial amounts of sucrose, glucose and fructose. After extraction of juice, the leftover of plant fiber, commonly referred as bagasse, may be used for the extraction of sugar which is utilized in the process. As an example, the juice containing plant material is sweet sorghum (sorghum bicolor) which is a hybrid for higher sugar content.

Sweet sorghum is a plant with C4 photosynthetic pathway, so its photosynthetic rate and dry matter production in g/m²/day per unit of inputs are more than those of other sugar producing crops like sugar cane and sugar beet. The freshly harvested sweet sorghum stalks are passed through 3 roller crushers. The collected juice is passed through a sieve to remove any particulate object and pre -heated for about 20 min at 70°C. The juice is cooled down to 40°C. The sugar stream is clarified using the combination of lime, magna flock and carbon dioxide (C0₂) at 80°C. After removing the press mud, the sugar stream is concentrated and utilized for the composite sugar composition production.

The bagasse obtained after extracting the juice, is then fractionated for separation of cellulose, hemicellulose and lignin. Several options are available for the fractionation of the cellulosic material. However, it has been observed that a single stage process using an organoslov process containing a metal salt provides the separate fractions of cellulose, lignin and pentosans which are easy for recovery. Moreover, the cellulose obtained in the process is amenable to nearly 95% saccharification with commercial cellulase enzyme for efficient glucose, cellobiose and cello-oligosaccharide production in a specialized packed bed enzyme column in which cellulase enzyme immobilized on the cellulose and continuously feeding cellulose in the column. The saccharification column optionally contains b-glucosidase enzyme, for converting cellobiose or cello-oligosaccharide to monomeric sugars depending on the requirement of the final product composition.

Employing lignocellulosic biomass as a starting material, different compositions are prepared. As an example, the composite sugar may contain sucrose, isomatulose or sucrose and trehalulose or sucrose, isomaltulose and trehalulose. In some instances, the composition includes monosaccharide sugars which may include glucose, fructose, D- xylose, D-xylulose, D-psicose, D-allose, and cellobiose, individually or in combination. In some instances, the composition contains both monosaccharide and disaccharides.

In certain embodiments, the enzyme used in the present process is selected from a group comprising xylose isomerase, D-tagatose-3-epimerase, rhamnose isomerase, isomaltulose synthase, sucrose isomerase, glucose isomerase, invertase and b-glucosidase. To achieve the composite sugars, enzyme plays vital part that needs to be stable to be industrially applicable. The present invention incorporates the enzyme disclosed in PCT/IB2013/053039 and PCT/IB2013/053038 (the disclosures of which are herein incorporated by reference) for the enzymes isomaltulose synthase, sucrose isomerase, tagatose 3-epimerase and rhamnose isomerase.

In certain embodiments, the present invention provides a process for preparing a sugar composition comprising natural sugars and rare sugars.

In certain embodiments, the present invention provides a process for production of composite low-calorie sugar compositions from a cellulosic route. Selection of plant for the specific purpose such as low-calorie sugar production is an important parameter for the present disclosure. Majority of the sugar producing plants are either sugar cane or sugar beet. Sweet sorghum (Sorghum bicolor) juice comprises substantial amounts of sucrose followed by glucose and fructose. Moreover, the bagasse that is generated after the extraction of juice also acts as a source of cellulosic sugar.

In certain embodiments, the present invention provides a process for producing a low-calorie sugar from sweet sorghum juice and bagasse. It is customary to clarify and concentrate the juice after extraction from plants having sugar containing juices, such as sweet sorghum, sugar beet, sugar cane etc. The uniqueness of the sweet sorghum juice is the composition of sugar which mostly comprises sucrose, glucose, and fructose, where the concentration of sucrose is much higher than the glucose and fructose.

In certain embodiments, the sugar substrate is selected from a group comprising aldose or ketose sugars. In some instances, pentose and hexose sugars are employed. In certain embodiments, the pentose sugar is selected from a group comprising ribose, arabinose and xylose; and the hexose sugar is selected from a group comprising glucose, fructose, galactose, mannose, allulose and rhamnose. The process parameters like temperature, reaction time, and pH depend on certain factors like the sugar substrate and the enzyme being used in the process.

In certain embodiments, the pentose is acted upon by an enzyme xylose isomerase at a temperature of about 60-80°C and at a pH of about 6 to about 7.

In certain embodiments, the hexose is acted upon by an enzyme xylose isomerase, tagatose 3-epimerase and rhamnose isomerase at a temperature of about 30-80°C and at a pH of about 6 to about 9.5.

In certain embodiments, the sucrose is acted upon by an enzyme isomaltulose synthase and trehalulase at a temperature of about 5-50°C and at a pH of about 6 to about 7.

In certain embodiments, the operating temperatures of isomaltulase synthetase and trehalulase are in the range of about 10 to 50°C.

In certain embodiments, the present invention provides a low-calorie sugar composition comprising rare sugars, natural sugars and optionally at least one prebiotic component.

In certain embodiments, the rare sugar is selected from a group comprising isomaltulose, trehalulose and D-psicose.

In certain embodiments, the natural sugar is selected from a group comprising glucose, fructose, sucrose and xylose.

In certain embodiments, the prebiotic component is either monomeric or dimeric.

In certain embodiments, the monomeric prebiotic component is xylulose obtained from bioconversion of xylose by xylose isomerase and the dimeric prebiotic component is cellobiose obtained by hydrolyzing the natural sugar source.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the term "comprises" or "comprising" is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

Downstream processing refers to the recovery and purification of biosynthetic products.

As used herein, the term“invention” or“present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification.

A prebiotic is defined as‘a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or more bacteria in the colon and thus improves host health’ and that stimulated bacteria should be beneficial in nature, namely bifidobacteria and lactobacilli. Lower molecular weight oligosaccharides have been subject of recent interest because, apart from the non-starch polysaccharides, they are the most potential source of carbon for colonic bacteria. It is also reported that cellobiose contributed to a larger increase in bifdobacteria counts than fructo-oligosaccharide (FOS).

EXAMPLES

The following examples are given by way of illustration, which should not be construed to limit the scope of the invention.

EXAMPLE 1

Sweet sorghum bagasse after removal of juice pretreated for separating lignocellulosic composition and the cellulose recovered was stored in T-802. The cellulose was continuously fed into the continuous packed bed reactor containing immobilized cellulase enzyme C-801. Saccharified cellulosic sugar, mainly comprising of glucose about 95- 98% and 3-5% cellobiose, passed through DSP-801. Once the sugar composition attained 2% (as beyond this concentration, the enzyme activity reduced drastically) concentration, the sugar obtained from the process is concentrated by any suitable means and passed through the column C-701 containing immobilized recombinant xylose isomerase for the conversion of glucose to fructose. Said enzyme column was connected to DSP-701. The formed product is either stored in the storage tank or transferred to additional column C- 702 containing immobilized D-tagatose 3-epimerase for the conversion of fructose to D- psicose. The D-tagatose 3-epimerase was immobilized on suitable matrix, such as on sodium or calcium alginate that may contain at least 5 mm of MnCk for optimal functioning. The formed D-psicose is separated through DSP-702 and either collected in storage tank T-703 and concentrated or fed from the storage tank to column C-703 which contains immobilized rh am nose isomerase for the conversion of D-psicose to D-allose and finally the sugar stream collected in T-704. The present process integration is designed in such a way that all the enzyme optimum temperature is 60°C. The process parameters are depicted in Table 1 below.

TABLE 1: PROCESS PARAMETERS

The following Table 2 shows the conversion of the 20% hexose sugar along with residual 3% cellobiose (see FIG. 2) present in the saccharified broth. The table shows that when all the columns are operated for a single cycle, the resultant sugar composition contains nearly 50% glucose, 38% fructose, 9% psicose, and 3% allose with a residual amount of 3% cellobiose. It is also possible to operate the enzyme reactor for different cycles depending on the sugar composition and thereby it is possible to generate different sugar compositions from the single source of hexose and cellobiose where glucose is present in the range of 3-50%, fructose 31-73%, D-psicose 9-50% and allose 3-45% along with 3% residual cellobiose. TABLE 2

EXAMPLE 2

This example is an illustration of the disclosure to produce desired low-calorie sugar composition containing glucose, fructose, psicose and allose in the mixture. Commercial glucose solution was passed through enzyme columns of xylose isomerase, tagatose-3- epimerase and rhamnose isomerase for different time cycles. 20% glucose solution was fed through the immobilized enzyme column as described in Example 1. The conversion of glucose to fructose, followed by fructose to psicose and psicose to allose was done using immobilized DTEase and Rhlase column. The composition obtained by this process was depicted in Table 3 below. A representative chromatogram of this bio-conversion is shown in FIG. 3.

TABLE 3

EXAMPLE 3

This example is an illustration of the disclosure to produce a desired low-calorie sugar composition containing sucrose, glucose, fructose, psicose and allose in the mixture. Sugar juice, preferably sugar containing C12 sugars, is passed through enzyme columns of invertase, xylose isomerase, tagatose-3-epimerase and rhamnose isomerase for different time cycles. The fed sugar comprises 15% sucrose which is fed through the column C- 601 containing invertase for the conversion of sucrose to glucose and fructose and the final feed solution passed through the column(s) as described in Example 1. The process parameters and sugar compositions are depicted in Table 4 and Table 5 respectively. A representative chromatogram of this bio-conversion is shown in FIG. 4.

TABLE 4

It is possible to operate the enzyme reactor for different cycles depending on the sugar composition and thereby it is possible to generate different sugar compositions from the single source of hexose and cellobiose where glucose is present in the range of about 28% sucrose 1-18% glucose, 23-53% fructose, 10-36% D-psicose and 3-33% allose.

TABLE 5: SUGAR COMPOSITION

5

EXAMPLE 4

This example is an illustration of the disclosure to produce a desired low-calorie sugar composition containing sucrose, isomaltulose, glucose, fructose, psicose and allose in the mixture. The process is developed using calcium alginate immobilized enzymes invertase, 10 isomaltulose synthase (ISase), glucose isomerase (Glase), D-tagatose-3-pimerase (DTEase) and rhamnose isomerase (Rhlase). The substrate (sucrose) used was commercially available or can be isolated from sugar cane and sweet sorghum juice. 15% sucrose solution was passed through immobilized isomaltulose synthase C-901 as well as through the configuration as described herein. The process parameters and sugar 15 compositions are depicted in Table 6 and Table 7 respectively. A representative chromatogram of this bio-conversion is shown in FIG. 5. TABLE 6: PROCESS PARAMETERS

TABLE 7: SUGAR COMPOSITION

5

EXAMPLE 5

This example is an illustration of the disclosure to produce a desired low-calorie sugar composition containing sucrose, trehalulose, glucose, fructose, psicose and allose in the mixture. The process was developed using calcium alginate immobilized enzymes 10 invertase, sucrose Isomerase (Slase) and xylose isomerase. The process was developed using calcium alginate immobilized enzymes. The substrate used was either commercial sucrose or sucrose isolated from sugar cane and sweet sorghum juice. 15% sucrose solution passed through immobilized isomaltulose synthase C-902 as well as through the configuration as described herein. The process parameters and sugar compositions are depicted in Table 8 and Table 9 respectively. A representative chromatogram of this bio- 5 conversion is shown in FIG. 6.

TABLE 8: PROCESS PARAMETERS

TABLE 9: SUGAR COMPOSITION

10 EXAMPLE 6

This example is an illustration of the disclosure to produce a desired low-calorie sugar composition containing cellobiose, glucose, fructose, psicose and allose in the mixture. The process was developed using calcium alginate immobilized enzymes beta- glucosidase, xylose isomerase (XIase/GIase), D-tagatose epimerase (DTEase) and rhamnose isomerase (Rhlase). The substrate used was either commercial cellobiose or cellobiose generated from sugar cane and sweet sorghum biomass. 1.7 % cellobiose solution was passed through an immobilized beta-glucosidase column as well as through the configuration as mentioned herein. The process parameters and sugar compositions 5 were depicted in Table 10 and Table 11 respectively. A representative chromatogram of this bio-conversion is shown in FIG. 7.

TABLE 10: PROCESS PARAMETERS

0 TABLE 11: SUGAR COMPOSITION

EXAMPLE 7

This example is an illustration of the disclosure to produce a desired low-calorie component containing xylose, xylulose in the mixture. The process was developed using calcium alginate immobilized enzymes xylose isomerase (XIase/GIase). The substrate was either commercial xylose or xylose generated from sugar cane and sweet sorghum biomass. 10 % xylose solution was passed through immobilized XIase column. The process parameters are depicted in Table 12. TABLE 12

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

THE CLAIMS:

1. A bioreactor for preparing a low-calorie sugar composition, the bioreactor comprising:

a first column (C-801) connected to the pre-treatment reactor (R-801) via tank (T-802) for receiving the cellulose from the pre-treatment reactor (R-801) and configured to treat the cellulose to obtain a mixture of glucose and cellobiose; a second column (C-701) connected to the first column (C-801) via tank (T-701) for receiving the glucose from the first column (C-801) and configured to treat the glucose to obtain a mixture of glucose and fructose;

a fifth column (C-301) connected to the pre-treatment reactor (R-801) via tank (T-301) for receiving the xylose and configured to treat the xylose to obtain a mixture comprising xylose and xylulose;

a sixth column (C-401) connected to the first column (C-801) via tank (T- 401) for receiving the cellobiose from the first column (C-801) and configured treat the cellobiose to obtain a mixture of cellobiose and glucose, wherein the second column (C-701) is connected to the sixth column (C-401) via tank (T-701) to receive glucose from the sixth column (C-401);

a seventh column (C-601) configured to receive sucrose, glucose and fructose in the form of juices and configured to treat the juices to obtain a mixture of glucose and fructose, wherein the second column (C-701) is connected to the seventh column (C-601) to receive glucose from the seventh column (C-601); an eighth column (C-901) configured to receive a mixture of sucrose, glucose and fructose in the form of juices and configured to treat the juices to obtain a mixture comprising sucrose and isomaltulose;

a ninth column (C-902) configured to receive sucrose, glucose and fructose in the form of juices and configured to treat the juices to obtain a mixture comprising sucrose and trehaluose; and

2. The bioreactor as claimed in claim 1, wherein a first downstream separation unit (DSP 801) is provided between the first column (C-801) and the second column (C-701), the first downstream separation unit (DSP 801) configured to control the flow from the first column (C-801) towards the first column (C-801), second column (C-701) or the sixth column (C-401) based on the composition of mixture of glucose and cellobiose.

3. The bioreactor as claimed in claim 1, wherein a second downstream separation unit (DSP 701) is provided between the second column (C-701) and the third column (C-702), the second downstream separation unit (DSP 701) configured to allow flow from the second column (C-701) towards the second column (C-701) or the third column (C-702) based on the composition of mixture of glucose and fructose received from the second column (C-701).

4. The bioreactor as claimed in claim 1, wherein a third downstream separation unit (DSP 702) is provided between the third column (C-702) and the fourth column (C-703), the third downstream separation unit (DSP 702) configured to allow flow from the third column (C-702) towards the third column (C-702) or the fourth column (C-703) based on the composition of mixture of psicose and fmctose received from the third column (C-702).

5. The bioreactor as claimed in claim 1, wherein a fourth downstream separation unit (DSP 703) is provided between the fourth column (C-703) and the product collection tank (T-1003), the fourth downstream separation unit (DSP 703) configured to allow flow from the fourth column (C-703) towards the fourth column (C-703) or the product collection tank based on the composition of mixture of psicose and allose received from the fourth column (C-703).

6. The bioreactor as claimed in claim 1, wherein a fifth downstream separation unit (DSP 301) is provided between the fifth column (C-301) and the product collection tank (T-1003), the fifth downstream separation unit (DSP 301) configured to allow flow from the fifth column (C-301) towards the fifth column (C-301) or the product collection tank (T1003) based on the composition of mixture of xylose and xylulose received from the fifth column (C-301).

7. The bioreactor as claimed in claim 1, wherein a sixth downstream separation unit (DSP 401) is provided between the sixth column (C-401) and the second column (C-701), the sixth downstream separation unit (DSP 401) configured to allow flow from the sixth column (C-401) towards the sixth column (C-401) or the second column (C-701) based on the composition of mixture of glucose and cellobiose received from the sixth column (C-401).

8. The bioreactor as claimed in claim 1, wherein a seventh downstream separation unit (DSP 601) is provided between the seventh column (C-601) and the second column (C-701), the seventh downstream separation unit (DSP 601) configured to allow flow from the seventh column (C-701) towards the seventh column (C-701) or the second column (C-701) based on the composition of mixture of glucose and fructose received from the seventh column (C-601).

9. The bioreactor as claimed in claim 1, wherein an eighth downstream separation unit (DSP 901) is provided between the eighth column (C901) and the product collection tank (T-1003), the eighth downstream separation unit (DSP 901) configured to allow flow from the eighth column (C-901) towards the eighth column (C-901) or the product collection tank (T-1003) based on the composition of mixture of sucrose and isomaltulose received from the eighth column (C-901).

10. The bioreactor as claimed in claim 1, wherein a ninth downstream separation unit (DSP 902) is provided between the ninth column (C-902) and the product collection tank (T-1003), the ninth downstream separation unit (DSP 902) configured to allow flow from the ninth column (C-902) towards the ninth column (C-902) or the product collection tank (T-1003) based on the composition of mixture of sucrose and trehaluose received from the ninth column (C-902).

11. The bioreactor as claimed in claim 1 , wherein each of the first column to the ninth column are operable individually or in combination and the reaction of sugar substrate in each column is optionally repeated one or more times to get desired low-calorie sugar composition.

12. A process for preparing a low-calorie sugar composition comprising:

a) feeding a sugar source to the bioreactor as claimed in claim 1 ;

b) processing the sugar source to get a sugar substrate;

wherein the reaction of sugar substrate in each column is optionally repeated one or more times to get desired ratio of low-calorie sugar composition.

13. The process as claimed in claim 12, wherein the enzyme is selected from a group comprising xylose isomerase, D-tagatose-3-epimerase, rhamnose isomerase, isomaltulose synthase, sucrose isomerase, glucose isomerase, invertase and b- glucosidase.

14. The process as claimed in claim 12, wherein the low-calorie sugar composition comprises: rare sugar, natural sugar and optionally at least one prebiotic component.

15. The process as claimed in claim 14, wherein,

rare sugar is selected from a group comprising isomaltulose, trehalulose and D-psicose;

natural sugar is selected from a group comprising glucose, fructose, sucrose and xylose; and

prebiotic component is monomeric or dimeric.

16. The process as claimed in claim 15, wherein the monomeric prebiotic component is xylulose.

17. The process as claimed in claim 15, wherein the dimeric prebiotic component is cellobiose.