FORM - 2 THE PATENTS ACT, 1970
{39 of 1970}
THE PATENTS RULES, 2003
COMPLETE
Specification
(See section 10; rule 13)
PROCESS FOR MANUFACTURING TAGATOSE USING SOY
OLIGOSACCHARIDE.
CJ CHEILJEDANG CORPORATION.
a Republic of Korea Company
of 500, Namdaemunro 5-ga,
Jung-gu, Seoul 100-095,
Republic of Korea,
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.

TECHNICAL FIELD
The present invention relates to a method for manufacturing tagatose by using a soy oligosaccharide existed in a soybean or a water-soluble sugar solution containing the soy oligosaccharide, in which galactose existed in the oligosccharide as a component existed in the soybean in a water-soluble type is selectively recovered through a selective hydrolysis, and then the tagatose is produced using the galactose.
BACKGROUND ART
Tagatose has a natural sweetness that cannot be distinguished from the sweetness of sucrose, and also is similar in physical properties to sucrose. However, tagatose doesn't affect the blood-sugar level because the ingested tagatose cannot be well absorbed in a small intestine, and it is a low caloric sweetener which has only about 30% calorie as compared to sucrose's calorie. In addition, tagatose has a prebiotic effect that promotes a proliferation of useful lactobacillus due to the fermentation by intestinal microorganism.
However, such tagatose does not commonly exist in the natural world and it is a .scarce and trace sugar that is included in dairy products or some plants. Therefore,

the technology which can produce in bulk from a low-priced raw material, should be developed in order to use as a functional sweetener having a low calorie. US Patent Nos. 5002612 and 5078796 disclose the method for manufacturing D-tagatose, in which the method includes hydrolyzing the lactose-containing material, or lactose to the mixture of galactose and glucose by using lactase, randomly removing the glucose, and then chemically isomerizing the galactose to the tagatose. In addition, US Patent No. 6057135 discloses the method for manufacturing tagatose, in which the method includes yielding galactose and glucose by hydrolyzing cheese whey or milk, isomerizing the resulted galactose with L-arabinose isomerase, and re-circulating non-converted compounds to the process by separating the products and the non-converted compounds using chromatography.
Until now, the major raw material used for manufacturing tagatose is a by-products of dairy products such as lactose or lactose -containing materials. For byconversion process using the lactose or lactose-containing materials as mentioned above as a start raw material, the method for manufacturing tagatose should be basically performed as a two-steps process (lactose —► galactose —► tagatose).

DISCLOSURE Technical Problem
The inventors finished the present invention by performing the research about the raw materials plentifully containing galactose in a by-product of process and wood that can be possible to be supplied from the natural in a low-priced except a lactose,
confirming that the by-product of isolated soy protein has a high contents of galactose as compared to other materials, selscting the raw materials that can economically use a chromatography, and then founding that a soy oligosaccharide resulted from a whey of isolated soy protein is practically the raw material, which can be possible to industrialize.
Therefore, an object of the present invention is to provide a method for manufacturing galactose by selectively hydrolysing a soy oligosaccharide, or a water-soluble sugar solution containing the soy oligosaccharide.
In addition, another object of the present invention is to provide a method for manufacturing tagatose by yielding the isomarized products prepared by isomerizing galactose in the hydrolyzed sugar solution through the enzymatic isomerization, and manufacturing tagatose using a chromatograghpy.

Technical Solution
To achieve the above object, the present invention provides a method for
manufacturing galactose by selectively hydrolyzing oligosacchride originated from a
soybean, or a water-soluble sugar solution containing the oligosacchride as a major
raw material with a alpha-galatosidase.
The present invention also provides a method for manufacturing tagatose including as
follows:
1) selectively hydrolyzing oligosacchride originated from a soybean, or a water-soluble sugar solution containing the oligosaccharide as a major raw material with a alpha-galactosidase;
2) isomerizing galactose in the hydrolyzed sugar solution with an arabinose isomerase; and
3) continuously seperating the obtained isomerized product by using a chromatography, and then re-circulating the galactose;
The present inventon also provides a method for performing the step 1) and 3) in one reactor at the same time.

Advantageous Effects
As described above, the inventors developed the effective method for manufacturing tagatose considering the materials balance according to each unit process and facility investment, and more specifically, the new and economical process that apply a method for selectively hydrolyzing in order to easily perform chromatography separation. Therefore, for the present invention can be economically produce the tagatose by yielding galactose by selectively hydrolyzing oligosaccharide originated from a soybean which is a vegetable raw material and a water-soluble sugar solution containing the oligosaccharide but not lactose in the pior art and separating the sugar solution which is mixed with at least 3 of sucrose, galactose, and tagatose, that are yielded from the isomerization of galactose resulted from the above.
DESCRIPTION OF DRAWINGS
The above and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments given in conjunction with the accompanying drawings, in which:

FIG. 1 and FIG. 2 are mimetic diagrams of pure chromatography separation process of
mixed monosaccharide solution;
FIG. 3 is a graph showing HPLC analysis profile for analyzing sugar content in a soy
oligosaccharide;
FIG. 4 is a hydrolyzed result and components analysis of a soy oligosaccharide;
FIG. 5 and FIG. 6 are analyzed results of sugar components in a soy oligosaccharide - treated with alpha-glycosidase without invertase;
FIG. 7 and FIG. 8 are pattern analysis of chromatography separation of mixed
monosaccharide that is hydrolyzed; 'FIG. 9 and FIG. 10 are the microscopy pictures that the obtained tagatose crystal is
compared with sucrose on the market in a size and a shape by examining using a
microscopy (a: sucrose, b: tagatose);
FIG. 11 is a process for manufacturing tagatose by using a soy oligosaccharide or a
soybean whey by-product as a raw material; _FIG. 12 is an elusion curve obtained from 1 pass test;
FIG. 13 is a drawing detail of a simulation result of case 9 (three-phase separation);
FIG. 14, FIG. 15, and FIG. 16 are results analyzed by using HPLC a sugar

concentration in the isomerization reactant in the mixed monosaccharide composition solution;
FIG. 17 is a flowchart roughly showing a production process of tagatose using a single chromatography; and
FIG. 18 is a flowchart roughly showing a production process of tagatose by a single ' chromatography including a pre-treating of crystallization.
Best Mode
Hereinafter, the present invention will be described in more detail with reference to the following Examples, but the scope of the present invention is not limited thereto. The present invention includes as follows:
1) selectively hydrolyzing a oligosaccharide originated from a soybean, or a water-soluble containing the oligosaccharide as a major raw material with an alpha-galactosidase;
2) isomerizing galactose in the hydrolyzed sugar solution with arabinose isomerase; and
3) continuously separating the obtained reactant by using a chromatography,

and then re-circulating galactose. The start material in the present invention may includes soybean whey, which is a by-product of isolated soy protein, and a soy oligosaccharide originated from the same, or a water-soluble sugar solution containing the same as a major raw material, and preferably may be a soy oligosaccharide. The sugar included in the soybean whey mostly includes sucrose, raffinose, and
stachyose as a major component, and raffinose and stachyose are composed of polymer which sucrose and galactose combined by alpha-1,4 linkage. The carbohydrate components of the sugars as mentioned above are as following Table 1.
'" The basic components included in the soybean whey or the soy oligosaccharide may be classified into sucrose and galactose, and if the components are subdivided, may be interpreted to the mixed monosaccharide, such as glucose, fructose, and galactose. Table 1
Name of Number of sugar The content of Mole ratio of sugar
Carbohydrate source galactose contents of
components
Raffinose 3 sugars 33.3 Suc:D-Gal=l:l

Stachyose 4 sugars 50 Suc:D-Gal=l:2
Sucrose 2 sugars 0
Likewise, like the soybean whey used in the present invention, the sugar solution obtained through the hydrolysis is also composed of the mixed monosaccharide that is mixed with at least three sugars because most of the galactose in vestetable raw material existed in the.nature is exists as a carbohydrate or olicosaccharide form together with glucose or fructose.
For the by-product originated from milk as the conventional method, most sugars are composed of only lactose, so that the mixed monosaccharide of glucose and galactose can be easily and firstly obtained when hydrolyzing the raw materials, and then it can
. be economically separated through the developed chromatography. However, seperating the sugar solution containing at least three sugars according to the present invention by using the chromatography is difficult to economically
perform without the satisfaction of the selective conditions. For this reason, the economical production of galactose or tagatose by using other raw materials except lactose is very difficult. In generally, the sugar separation according to the chromatography is performed by

using a difference of weak binding force between the sugar that is required to separate and the metal ion that is attached on the ion resin, and especially the metal ion residue that can be use in the chromatography separation and in the food application is limited to K, Na, Ca, Mg, and the like.
Glucose and galactose have physically a very similar structure that is composed of aldose form, and fructose is composed of ketose form. In generally, the separation of
-"mixed sugar composed of aldose form and ketose form can be economically performed by using the chromatography with a resin having cation residue, and has been actually and widely used in a starch sugar process. However, glucose and galactose, which are same aldose form have a physically similar shape, so that glucose and galactose can be economically separated only using a special resin having the residue, such as K, and Na. It means that, basically when two separate chromatography facilities are only sequentially combined, galactose can be purely separated. It illustrated in FIG. 1. In addition, the chromatography separation of
the mixed sugars generally uses a lot of water, so that the cost for concentration in the process becomes high, as well as facility investment requires a relatively high cost. Enzymes used in the hydrolysis according to the present invention may be an

ilpha-galactosidase, and more preferably invertase free alpha-galactosidase.
The alpha-galactosidase is a glycolytic enzyme existed in the nature, and selectively
- cuts alpha-1,4 linkage between sucrose and galactose that constitute raffinose and slachyos, thereby preparing a sugar solution having sucrose and galactose as a major component from the soybean whey or the soy oligosaccharide.
However, when preparing enzymes that can be generally and industrially used, a supernatant which produced by culturing a microorganism or a hypha that produce the enzymes are used as a raw material of the enzyme preparation, so that most enzymes include invertase that is a representative glycolytic enzyme. The selective hydrolysis of mixed sugar into sucrose and galactose is essential for availability of the
*: chromatography in the present invention, so that using invertase free alpha-galactosidase is to facilitate the separation using the chromatography. The alpha-galactosidase is originated from the strain that does not include invertase gene therein. More preferably, the alpha-galactosidase may be originated from Mortierella vinacease var. raffinose utilizer ATCC 20034 or Absidia griseola ATCC 20431. In the preferable embodiment of the present invention, the present invention was

.obtained the sugar solution having reduced sucrose content which is obtained by hydrolyzing the soy oligosaccharide with the alpha-galactosidase and then further selectively sucrose crystallization pre-treating of the resulted sugar solution containing sucrose and galactose. Due to the soy oligosaccharide includes an excess sucrose, there are undesired effects that the increase of the sugar solution volume, the increase of a facility scale, and the increase of loss purified water, and the like. For this
treason, the utility of the progress is decreased by pre-treating of sucrose crystallization.
The present invention completed the technology for economically manufacturing tagatose or galactose using the sugar solution that is selectively hydrolyzed by using the alpha-galactosidase as mentioned above, and arabinose isomerase can be used for converting galactose obtained from the present invention to tagatose. Preferabley, the thermophilic arabinose isomerase originated from Sulfurobus sp., Thermotoga sp., or Geobacillus sp. can be used. More preferably, the thermophilic
- arabinose isomerase originated from Thermotoga neapolitana DSM5068 can be used. For the present invention, the sugar solution composed of the mixed sugars containing sucrose, galactose, and tagatose obtained from the hydrolysis and the isomerization is

separated by the chromatography. More preferably, the three-phase separation of
chromatography is used.
For the reversible reaction such as the isomerization, the phase equilibration state
generally exists under the economic operation condition in theory, but in theory all of
the substances are impossible to convert to product. Therefore, for the such sugar
process, the economic separation and re-circulating of non-reactants using the
chromatography is needed. Another point of such production technology is the
possibility of the economic performance of the chromatography.
A resin for the separation of sugar solution in the chromatography separation may be a
resin having Ca residue. _;The re-circulating of non-reactants in the step of chromatography separation can be
optionally selected by a person skilled in the art as occasion demands according to the
kinds of the sugar solutions as follows. ""When the sugar solution contains tagatose(>90%) as a major component, the
non-reactants can be crystallized under the necessity after the concentration of
tagatose.
When the sugar solution contains galactose as a major component, the non-reactants

can be selectively recycled before the isomerization or before the crystallization according to the content ratio of components in the sugar solution. When the sugar solution includes sucrose as a major component, the non-reactants can be used as a sugar solution or other raw material for production by processing through the concentration or crystallization.
Hereinafter, the present invention will be described in more detailed with reference to .the example. However, the examples are only for the explanation of the present invention, but the range of the present invention will not be limited thereto.
EXAMPLES
In the examples of the present invention, analysis and evaluation of a sugar component in industrial by-products were performed in order to secure the raw material that can be economically obtain tagatose or galactose through the chromatography separation. The hydrolysates were prepared through the selective .hydrolysis using the alpha-galactosidase of the selected soybean whey or the soy oligosaccharide, and the economic production technology that can purely separate galactose or tagatose from the hydrolysates has developed. Especially, by

introducing the continuous re-circulating process in the process, the technique for economic production has a high yield developed to take them up step further was
: completed. For this reason, by-product of the material processing can easily be obtained in the laboratory, the raw material composed of carbohydrate as a major component was firstly selected as a raw material for manufacturing tagatose, the component and content of the water-soluble sugar component (includes mono-, di-, ologosacchraide) contained in the water solution state were analyzed in details, and the use possibility in a easy composition type for the chromatography separation was determined. From the results of the research as mentioned above, we have found that the soybean whey which is a by-product of isolated soy protein, and the soy oligosaccharide and the water-soluble sugar solution originated from the same have an excellent composition for separating economically galactose or tagatose as compared to other
'raw material. And, we have completed technically the process for manufacturing tagatose using it.
Example 1. Analysis of Sugar Composition in the Industrial By-product The soy oligosaccharide available on the market was collected from the Chinese

market and was used as the standard sample. HPLC was performed in order to analysis carbohydrate component in the obtained soy oligosaccharide. Supelco-PB suagr separation column (SUPELCO) was used as the column, and analysis condition used purified water 0.5ml/min as a mobile phase. HPLC grade reagent (sigma) was used respectively as the standard solution. The resulted results were shown in Table 2. -Table 2 Composition of Soy Oligosaccharide
Sugar Residence time [min] Composition
[dry solid basis, wt%]
Stachyose 12.549 51.7
Raffinose 13.441 21.8
Sucrose 14.384 11.9
Glucose 16.919 3.7
Galactose 19.783 6.3
Fructose 23.206 4.6
For the composition of carbohydrate in the soy oligosaccharide, the content ratio of

raffinose and stachyose constitutes 73.5% of total carbohydrate, and the content ratio of sucrose and fructose constitutes 26.5% as other rest sugar. FIG. 2 is a HPLC •analysis chromatography as the ratios.
Example 2. Analysis of Sugar Content in the Soy Oligosaccharide and Analysis after Enzyme Hydrolysis Reaction
The soy oligosaccharide in the Example 1 was treated with the alpha-galactosidase and used as galactose source. The used enzyme was validase AGS (Valley Research, US), and the amount of use of enzyme to the soy oligosaccharide products as a substance were 0.15% (w/w), respectively, and were hydrolyzed at 150rpm at 50°C. ; Galactose production and the change of sugar components by hydrolysis using the alpha-galactosidase according to time were shown in Table 3.
Validase allow manufacturing raffinose and galactose by degrading of stachyose which is soy oligosaccaride, and tend to further degrades the raffinose to sucrose and galactose. A major component of validase is the alpha-galactosidase, and validase is a product which is added in feed, for reducing side effects, such as diarrhea, stomachache, and the like that are caused due to an ingestion of the soy

oligosaccharide by hydrolysis of the soy oligosaccharide
As depicted in FIG. 3, the alpha-galactosidase in product has a function of selectively hydrolyzing galactose-galactose bonding and galactose-sucrose bonding in the soy oligosaccharide, but also there is a function of inverta.se that produces glucose and fructose by degrading sucrose.
There is an unprecedented phenomenon that sucrose does not remain after 24hours, J! but glucose and fructose are continuously produced. It could be inferred that sucrose produced by degrading stachyose and raffinose is degraded by the function of invertase in validase, so that glucose and fructose are produced. 50hours after starting the hydrolysis, there is a phenomenon that galactose, fructose, and glucose were not produced no longer. At this time, the sugar composition was shown in Table 3.
Table 3 .... Composition of Soy Oligosaccharide Solution according to validase
Sugar Composition [dry solid base, %]
Stachyose 0

Raffinose 3.8
Sucrose 0
Glucose 28.4
Galactose 45
Fructose 22.8
Example 3. Invertase free alpha-galactosidase
As disclosed above, the selective hydrolysis of sucrose and galactose using the enzyme to be easy to use the chromatography is essential, so that there is necessary for technology that selectively cuts alpha-1,4 bonding between sucrose and galactose --which compose raffinose and stachyos using the alpha-galactosidase. However, the production of enzyme that can be industrially used in generally can be performed by selecting the microorganism or the hypha that can extracellularly secrete the excess required enzyme, culturing the microorganism or the hypha in a liquid or solid, and using its supernatants as the raw material for the enzyme production. Like the alpha-galactosidase, a type of enzyme that is sold and distributed for an industrial purpose has mostly used a hypha culture supernatant, such as Aspergillus niger as a

raw material for preparing a enzyme, and most culture supernatant include invertase in their enzyme solution.
Most organisms existing in the nature have invertase that is a representative glycolytic enzyme. Invertase has a enzymatic feature that easily hydrolyzes sucrose, and the like. Therefore, invertase included in the enzyme solution quickly hydrolyzes sucrose existed in the sugar solution to glucose and fructose, so that this function is a "major factor that interrupts the economic production for tagatose.
Therefore, the inventors minutely researched whether the organism that can produce
the alpha-galactosidase in the microorganism or the hypha can produce invertase or
not. The results were shown in Table 4.
Table 4
Whether or not invertase can be produced.
Alpha-galactosidase Invertase
Aspergillus niger O O
Aspergillus oryzae O
Aspergillus oryzae d-Aspergillusniger O
Mortierelialla vinaceavar. Rafflnose O

utilize
A bs idia griseo la O
Saccharomyces carlsbergensis O O
Saccharomyces cerevisiae d-Guar seed O O
Bacillus subtilis O
Kluyveromyces fragilis O
In the present invention, Culture hypha of Mortierelialla vinaceavar Raffinose utilizer or Absidia griseola that only produce the alpha-galactosidase, but not invertase was used as the hydrolysis enzyme.
The invertase free alpha-galactosidase that is used for the present invention was obtained from a mold, i.e., Mortierella vinaceae var. raffinose utilizer ATCC20034 or Absidia griseola ATCC20431 received from ATCC.
The culture medium composition for the strains is 0.1M phosphorus buffer (pH6.0), 10g/l lactose, 3g/l Pepton, 3g/l yeast extract, 0.5g/l potassium chloride, 0.5g/l magnesium sulfate, and a small amount of ferrous sulfate. After culture for 3 days at ,30°C, each strains was only collected from the culture medium by using filtration system (CORNING, BOTTLE and BOTTLE TOP FILTERS). The collected strains

were driven into the ice, and then were homogenized by using a homogenizer for 1 Omins. Then, the final enzyme solution was obtained from the supernatant by using a centrifuge at lOOOOrpm for 1 Omins.
.The enzyme hydrolysis was performed with the soy oligosaccharide by using the enzyme solution obtained. The reaction condition of the hydrolysis was 7 hours at 40°C. HPLC analysis (SUPELCO, SUPELCOGEL Pb HPLC Column) was """performed to confirm the sugar content. The results shown that the soy oligosaccharide was selectively hydrolyzed to sucrose and galactose by selective hydrolysis using the invertase free alpha-galactosidase, respectively. The results were shown in FIG. 5 and FIG. 6.
Example 4. Isomerization Conversion Reaction of galactose to Tagatose by using L-arabinose isomerase
The isomerization reaction from galactose to tagatose according to the present
.-invention was achieved by using a thermophilic arabinose isomerase originated from
Thermotoga neapolitana DSM5068 that is a high thermophilic microorganism and its
arabinose isomerase originated from the hyperthermophlies is expressed in the

corynebacterium host.
The gene encoding the arabinose isomerase was inserted into E.coli-corynebacterium
" shuttle vector, pCJ-1 and pCJ-7 (KR Laid-Open Patent No. 10-2006-0068505), respectively, and then finally Corynebacterium glutamicum KCTC 13031 as a host was obtained. 20% (w/v) of the strains expressing the thermostable enzyme was mixted to 2.0% of sodium alginate solution, and then strried to prepare the suspened solution. The hardening reaction was induced by dropping the suspened solution in a natural drop way to 0.1M CaCl2 solution. Cell Capture Arginate Cure Bead prepared from the process was used in a stablization. 20mM Tris-HCl pH 7.5 buffer was added to the sugar solution obtained as the resultant to adjust pH, and then the resultant was
"used as the reaction substance solution. The arabinose isomerase obtained from the process was used to obtain the isomerized product.
Example 5. Analysis of Chromatography Separation Pattern of the hvdrolvzed mixed monosaccharide
Analysis of chromatography separation pattern was performed in order to plan a

chromatography separation way of galactose using the isomerized product (mixed monosaccharide) obtained according to the example of the present invention. For the chromatography separation of the mixed monosaccharide, the experiment was -; performed using as the following materials and facility. FINEX MFG220 Ca++(FINEX) was used as the resin for separation sugar, and it was performed at 60°C and llml/min flow rate using the purified water as the mobile phase. Two XK50/100 columns (Ammersham Bioscience, USA) connected in series was used, and 1900 ml of the resin in each column was filled to use. 50 Bx concentration of the mixed sugar injected for separation was used 160ml per one step. The configuration of the mixed monosaccharide used was firstly sucrose:galactose=80:20, and was secondly randomly adjusted to glactose:tagatose=50:50. And then the separation pattern was analyzed using it as a test sample. The test result shown that sucrose, galactose, and tagatose can be easily separated at regular intervals, respectively. The results were shown in Table 7 and Table 8.
Example 6. Tagatose Crystallization
The crystallization of tagatose was attempted using the recovered solution from the

chromatography separation in Example 5. fraction part which the final content of tagatose is 90% was recovered, and then it was vacuumed, heated and concentrated to be 70Bx sugar concentration in a crystallizer. Temperature was decreased up to 50°C, 2°C per hour was slowly cooled, and tagatose seed was added at 40°C to be crystallized.
The phenomenon, in which the sugar concentration in the supernatant was relatively diluted according to the processing of the tagatose crystallization, was observed. -And the vacuum concentration was continuously performed during the crystallization in order to constantly maintain the concentration of the supernatant to be diluted. The concentration of the supernatant was constantly maintained at least 65Bx during the crystallization to maintain the crystal production and crystallization. The crystallization was stopped after 20hours, and then the supernatant and the crystal were separated using a centrifuge. 71% tagatose in the initial crystal solution was recovered; the purity of tagatose was from 90% to at least 98%. Tagatose separated by the centrifuge was further dried at 50°C for 1 hour using the vacuum drying oven. Spectroscopy picture of tagatose crystal produced from the process is FIG. 9 and FIG. 10. And, the size and shape as compared to sucrose available on the market.

Example 7. Continuous Re-circulating Manufacturing Plan of Tagatose from Soybean Whey hydrolysate using Chromatography Separation
The production process using the soybean whey by-product or soy oligosaccharide as the raw material through the examples was planed as follows, and it was roughly described in FIG. 11. The process for improving the product quality, such as a dicolorization, desalinization, and the like among the following steps was not separately inserted, and can be selectively inserted/excluded according to the change of quality during the process.
1. separating the soy oligosaccharide after the pre-treating through ultra-filtration, micro-filtration, and the like from the soybean whey by-product;
2. hydrolyzing the soy oligosaccharide solution (concentration 10-50%) at a high or low temperature (when using the enzyme originated from a hyperthermophilies or molds);
3. separating using the chromatography of the hydrolyzed sucrose and galactose. The concentration process may be selectively inserted or excluded according to the sugar concentration during the process;

4. manufacturing tagatose by passing through the chromatography separation solution containing galactose (at least 50%) a.s the major component in the L-arabinose isomerase (or L-galactose isomerase) reactor;
5. separating using the chromatography of the isomerized product that is composed of galactose, tagatose, and the small amount of mixed sugar. The concentration process may be selectively inserted or excluded according to the sugar concentration during the process;
6. crystallizing and concentrating (if needed) the chromatography separation solution containing tagatose as the major component (at least 70 %);
7. re-circulating the chromatography remaining solution containing galactose as the major component to the ealier step than isomerizaiton reactor;
8. re-circulating an amorphous working solution that is obtained from the crystallization of tagatose to the ealier step of crystallization or isomerization according to the composition in the sugar solution.
Example 8. Three Phases Separation of Mixed Monosaccharide
1 pass test was performed with all of the sugar included for the material balance of the

whole process in a mixed type, and the obtained result was used for the continuous re-circulating production plan simulation using the chromatography separation technology.
- 1 pass test was performed with the mixed sugar, and a basic parameter was calculated from the obtained result. The concentration profile of each component was simulated on the computer using a new simulated moving-bed system (Oragno, Japan), and the purity of component and recovery rate about each fraction were calculated. An effluent liquid was separated into each fraction. HPLC analysis was performed with each fraction, and an elusion curve of each component was made from the analyzed result. Each elusion pattern was tested, and basic parameters of each component were calculated. 1 pass test condition was shown in Table 5. The - analysis method of the fraction was shown in Table 6. HPLC analysis condition was shown in Table 7. Table 5
1 pass test condition
Sample Mixed Monosaccharide Sample
Sucrose: 73.93%, other-2: 0.51%

Galactose: 12.62%, other-3: 0.39%
Tagatose: 12.56%
Concentration of syrup to be Bx. 60%
supplied.
Absorbent Amberlite CR-1310: Ca-type, New resin
Column Size 20 mm x 1000 mm (314 ml)
Desorption solvent H20
" 'Supply Volume 15 ml
Flow rate 26 ml/min (LV5m/h)
Temperature 60 °C
Table 6
Analysis Method Brix: Concentration
Brix: (g/lOOg) Refractometer: RA-50 (Kyoto Electronics)
Monosaccharide Composition HPLC: VP series (Shimadzu Corp.)

Table 7
HPLC Analysis Condition
Column TSK-GEL SCX-Ca(6.0xL150X5
cloumn)
Desorption solvent H20
Flow Rate 0.8 ml/min
Supply Volume 10 ul
Temperature 70 °C
„ Concentration Measure Device Separate Refraction Index meter
The elusion curve obtained from 1 pass test was shown in FIG. 12.
The basic parameter was calculated from the result of the elusion curve, and computer "simulation was performed for calculating a separation performance. The simulation
condition was shown in Table 8. In order to achieve a optimum separation
performance value, the inventors varied the ratio of the sugar solution sample and the
water that is the mobile phase (the ratio of desorption solvent/supply volume), and
then the sugar component in the whole solution was analyzed according to the
variation. And then the purity and the recovery rate of each sugar component in the

section recovered from the mentioned above were calculated. Case 6, 7, 8, and 9 in Table 9 are mass balance sheets obtained from the simulation results. In each simulation, the tagatose having a preferable 90% purity was obtained. The detailed diagram of the simulation result of Case 9 was shown in FIG. 13 as the representative drawing. Table 8
Simulation Condition
" Condition Three-phase Separation
Concentration of Syrup to be supplied. Bx 60
Goal Purity Tagatose Purity: at least 90%
Result Case 6 Case 7 Case 8 Case 9
.. Table 9 Summary of Simulation Result
Condition Case 6 Case 7 Case 8 Case 9
Flow 0.0464 0.0469 0.0469 0.0446
Rate(L/L-R/hr)

Rate of 4.93 4.91 5.31 5.13
Desorption
solvent/supply
volume
Tagatose purity 95.2 93.6 96.0 97.2
(%)
Tagatose 87.8 91.1 81.7 93.0
recovery rate

Galactose 74.3 76.3 63.7 77.8
purity (%)
~ Galactose 88.5 86.0 98.3 92.7
recovery rate
(%)
The performance disclosed in this Example is the result calculated on the organo
computer simulation.

Example 9. Reaction Stability of L-arabinose isomerase in the mixed monosaccharide
For the economical method for manufacturing tagatose using three-phase chromatography suggested in the present invention, a major part of the substance
„ solution composition is occupied with sugar (glucose, fructose, sucrose) besides galactose, not like the isomerization from galactose to tagatose that has been performed until now. Therefore, the important precondition for introducing the technology of the present invention is whether or not other sugars included in the composition interfere the isomerization using enzyme. For the use of the isomerase that is used in Examples of the present invention, the inventors were tried to discJose a substance specificity of galactose isomerization in the mixed monosaccharide composition solution.
..Each substance solution was prepared to add sucrose, glucose, and fructose at a certain ratio to 100 g/L concentration of galactose solution. The composition ratio of each sugar component is (a) Suc:GIu:Gal:Fru=0:0:l:0, (b) Suc:Glu:Gal;Fru=3:l:l:l,
* or (c) Suc:Glu:Gal:Fru-0:3:l:2. A component ratio of each sugar was randomly prepared to use at the similar level with the result ratio of sugar component (a) when

containing only galactose as Control, (b) when selectively hydrolyzing 1,4-linkage between galactoses in the oligosaccharide, (c) when separating completely by the complete hydrolysis.
10% (w/v) concentration of a deposited strain(CJ-l-TNAI, KCCM10786P) pellet growth and developed in the induction medium was added to the substance mixed solution, and galactose isomerization was performed at 70°C for 1 hr. After the reaction, the mixed solution was cooled at 4°C for 15 min, and then the supernatant was selected after a centrifugation at 12,000rpm for 15 min. The result supernatant
^ was diluted 5 times with the purified water, and passed through 0.45 um syringe filter (Millipore, USA) and then analyzed using HPLC. The analyzed results of the sugar concentration in the isomerization reactant in the
" mixed monosaccharide composition solution using HPLC were shown in FIG. 14, FIG. 15, and FIG. 16. The result quantified the standard solution of each sugar was shown in FIG. 10. Table 10
Class Sugar Composition
Sucrose Glucose Galactose Fructose Tagatose

(g/L) (g/L) (g/L) (g/L) (g/L)
(a) Control 0 0 13.4 0 5.9
(b) 54.7 21.3 13.5 0 6.0
Condition 1
(c) 0 60.4 13.7 37.2 6.0
Condition 2
According to the result, it can be known that galactose isomerization is not effected from the reaction rate; and the conversion rate having same level (reached at equivalent level, i.e., 30.8% for the present invention) is achieved in the mixed sugar solution which is mixed much sucrose, glucose, and fructose. Therefore, it can be known that glucose, fructose, and sucrose obtained from the raw material hydrolysate originated from at least soy oligosaccharide are not functioned as the reaction ^..interruption factor for the performance of the galactose isomerization using enzyme.
Example 10. Continuous Re-circulating Production Process Plan of Tagatose from Soybean Whey Hydrolysate by using Three-phase Separation Technology
The production process of tagatose as disclosed in Example 6 use two times of

chromatography, and this is the need for a lot of purified water and concentration. For the present invention, in order to efficiently overcome the problem, the inventors have planned the method for manufacturing tagatose by using the chromatography once through the advanced chromatography plan, and it was specifically described in the Examples.
The process for manufacturing tagatose using single chromatography was planned as follows, and it was roughly depicted in FIG. 17. The process for improving the product quality, such as a decolorization, desalinization, and the like among the '•following steps was not separately inserted, and can be selectively inserted/excluded according to the change of quality during the following process.
1. separating the soy oligosaccharide after the pre-treating through ultra-filtration, micro-filtration, and the like from the by-product of soybean whey;
2. hydrolyzing the soy oligosaccharide solution (concentration 10-50%) at a high or low temperature (when using the enzyme originated from a hyperthermophilies or molds);
3. isomerizing from galactose in the hydrolyzed sugar solution containing

sucrose and galactose as the major component (total at least 90%) to tagatose through L-arabinose isomerase (or L-galactose isomerase) reactor; 4. separating the isomerization reactant consisting of the mixed sugar that has sucrose, galactose, and tagatose as the major component(total at least 90%) to the three-phase using the chromatography. The concentration process may be selectively inserted or excluded according to the sugar concentration during the process. The sugar separation solution by the chromatography originated from the process as mentioned above is three kinds, as follows;
A. The chromatography separation solution containing tagatose as the
major component (at least 90%) may be concentrated (if needed) and
may be crystallized;
B. The chromatography residue containing galactose as the major
component can be selectively re-circulated to the previous step of
isomerization reactor or crystallization according to the content ratio of
component in the sugar;
C. The chromatography residue containing sucrose as the major

component may be processed or may be used as other advanced production raw material through the post-step of concentration and crystallization for using as the sucrose solution; 5. re-circulating the amorphous mother liquor that is obtained from the
crystallization of tagatose to the pre-crystallization or pre-isomerization
according to the composition in the sugar solution.
Example 11, Continuous Re-circulating Production Process Plan of Tagatose from Soybean Whey Hvdrolysate by using sucrose crystallization pre-treating
As a great forward of the method for manufacturing tagatose by using single chromatography planned as mentioned above, the inventor planned that the processing utility is reduced by reducing the content of sucrose in the sugar solution through the sucrose crystallization process.
The soy oligosaccharide includes an excess sucrose, so that there are undesired effects that the increase of the sugar solution volume, the increase of a facility scale, and the increase of loss purified water, and the like. - The process for manufacturing tagatose using single chromatography that includes

sucrose crystallization as the pre-treating was planned as follows, and it was roughly /depicted in FIG. 18. The process for improving the product quality, such as a decolorization, desalinization, and the like among the following steps was not separately inserted, and can be selectively inserted/excluded according to the change of quality during the following process.
1. separating the soy oligosaccharide after the pre-treating through ultra-filtration, micro-filtration, and the like from the by-product of soybean whey;
2. hydrolyzing the soy oligosaccharide solution (concentration 10-50%) at a high or low temperature (when using enzyme originated from a hyperfhermophilies or molds);
3. obtaining the sugar solution containing the more increased content of galactose through the crystallization of sucrose in the hydrolyzed oligosaccharide solution;
4. isomerizing from galactose in the hydrolyzed sugar solution containing sucrose and galactose as the major component (total at least 90%) to tagatose through L-arabinose isomerase (or L-galactose isomerase) reactor;

5. separating the isomerized reaction solution consisting of the mixed sugar that has sucrose, galactose, and tagatose as the major component to the three-phase using the chromatography. The concentration process may be selectively inserted or excluded according to the sugar concentration during the process. The sugar separation solution by the chromatography originated from the process is three kinds, as follows;
A. The chromatography separation solution containing tagatose as the
major component (at least 90%) may be concentrated (if needed) and
may be crystallized;
B. The chromatography residue containing galactose as the major
component can be selectively re-circulated to the previous step of
isomerization reactor or crystallization according to the content ratio of
component in the sugar;
C. The chromatography residue containing sucrose as the major
component may be processed or may be used as other advanced
production raw material through the post-step of concentration and
crystallization for using as the sucrose solution;

6. re-circulating the amorphous mother liquor that is obtained from the crystallization of tagatose to the pre-crystaliization or pre-isomerization according to the composition in the sugar solution.

WE CLAIM
1. A method for manufacturing tagatose, comprising the steps of:
a. separating a soy oligosaccharide from by-product of soybean whey;
b. hydrolyzing the soy oligosaccharide with an invertase free a-glactosidase;
c. obtaining an jsomerized product consisting of a mixed sugar that has sucrose,
galactose, and tagatose as a major component, by isomerizating the hydrolyzed sugar
solution with an arabinose isomerase; and
d. separating the isomerized reaction solution by using a chromatography.
2. The method according to claim 1, further comprising the step of obtaining the sugar solution that has an reduced content of sucrose through a sucrose crystallization from the hydrolyzed soy oligosaccharide after the step b).
3. The method according to claim 1 or 2, further comprising the step of re-circulating non-reacted galactose during the process
-after the step d).
4. The method according to claim 3, wherein the non-reacted galactose is separated by
the chromatography, and is re-circulated to step b) or step c).

5. The method according to claim 1, wherein the by-product of soybean whey is
pretreated by ultrafiltration or micro filtration.
6. The method according to claim 1, wherein the concentraton of the soy oligosaccharide is 10-50%.
7. The method according to claim 1, wherein the hydrolysis is performed at 40°C~80°C.
8. The method according to claim 1, wherein the a-galactosidase is originated from Mortierella vinaceae var. rafinoseutilizer ATCC 20034 or Absidia griseola ATCC 20431.
.:. 9. The method according to claim 1, wherein the a-galactosidase selectively cuts a-1,4 linkage between sucrose and galactose.
10. The method according to claim 1, wherein sucrose and galactose are obtained by the hydrolysis of step b).
11. The method according to claim 1, wherein the arabinose isomerase is originated from a thermophilic thermotoga neapolitana DSM 5068.
12. The method according to claim 1, wherein the isomerized reaction solution
has total at least 90% of sucrose, galactose, and tagatose.

13. The method according to claim 1, wherein the chromatography in step d) is a
. single chromatography.
14. The method according to claim 13, wherein the chromatography in step d) uses
a resin having Ca residue.
15. The method according to claim 1, wherein the reactions of step b) to c) are performed in one reactor at the same time. 16. A method for manufacturing tagatose, comprising the steps of:
a. separating a soy oligosaccharide from by-product of a soybean whey;
b. hydrolyzing the soy oligosaccharide with an invertase free a-glactosidase;
c. separating sucrose and galactose from the hydrolyzed sugar solution by a
chromatography;
d. isomerizing a chromatography separation solution containing galactose as a
,:,major component with an arabinose isomerase; and
e. obtaining tagatose by separating the isomerized product by using a
chromatograpy.

17. The method according to claim 16, wherein after the step d),
further comprising the step of re-circulating non-reacted galactose to the step d).