NZ603719B - Method for preparing high purity gentiooligosaccharides, high purity gentiooligosaccharides obtained therefrom, and uses thereof - Google Patents

Abstract

603719 Disclosed is a method for preparing a high purity gentiooligosaccharide comprising: adding a low purity gentiooligosaccharide to a liquid medium; subjecting the liquid medium to inoculation with a microorganism wherein the microorganism is a strain selected from the group consisting of Saccharomyces sp., Bacillus sp., Lactobacillus sp., and Candida sp., followed by incubation and fermentation to consume glucose that is contained in the low purity gentiooligosaccharide; removing the microorganism from the obtained fermentation broth; and subjecting the obtained broth to purification. accharomyces sp., Bacillus sp., Lactobacillus sp., and Candida sp., followed by incubation and fermentation to consume glucose that is contained in the low purity gentiooligosaccharide; removing the microorganism from the obtained fermentation broth; and subjecting the obtained broth to purification.

Description

FORM 5 NEW ZEALAND Reg.19(4) Fee: $250.00 PATENTS ACT 1953 METHOD FOR PREPARING HIGH PURITY GENTIOOLIGOSACCHARIDES, HIGH PURITY GENTIOOLIGOSACCHARIDES ED THEREFROM, AND USES THEREOF COMPLETE SPECIFICATION WE, CORN PRODUCTS DEVELOPMENT, |NC., a corporation ' incorporated in the state of Delaware, U.S.A., of 5 ook, , Corporate Center, Westchester, IL 60154, United States of America hereby declare the invention for which we pray that a patent may be d to us and the method by which it is to be performed, to be particularly described in and by the following statement:- INTELLECTUAL PROPERTY OFFICE OF NZ. 2 I lréAR 2W: ' 3i l'-*'~‘ 6"? r: if ‘x i 1'; kw...w“-_—__~_-..........

The present invention relates to methods for preparing high purity gentiooligosaccharides, high purity oligosaccharides obtained therefrom and uses thereof, and more particularly to methods for preparing high purity gentiooligosaccharides that can be beneficially used as alternatives to foods such as cocoa, chocolate, coffee, beer, tea, bread or confectionery ts, ges, etc., and main ingredients thereof without producing unnecessary by-products by ting low purity gentiooligosaccharides containing large amounts of glucose with microorganisms to remove glucose therefrom.

BACKGROUND OF THE INVENTION Gentiooligosaccharides are sweeteners composed of mainly biose and cellobiose that are disaccharides synthesized by enzymes from glucose, and are the generic name of oligosaccharides having [3-1,6-glucoside bonds. Gentiooligosaccharides are composed of gentiobiose, cellobiose, gentiotn'ose, tetraose, etc., and their main ingredient is gentiobiose which is a haride having a strong bitter taste.

Gentiooligosaccharides are found in the roots or stems of plants and in natural honey and are prepared by an enzymatic reaction using starch as raw material.

Gentiooligosaccharides are known as low digestible sugars that aid in mineral absorption, are selectively utilized by Bifidobacterium and Lactobacillus to help improve intestinal microbial flora, and have an intestinal regulation effect similar to that of galactooligosaccharide which has an excellent inal regulation effect.

Gentiooligosaccharides are characterized by having a softer and fresher bitterness in comparison with substances having a bitter taste, such as Naringin. The bitter taste of gentiooligosaccharides, in harmony with the bitter taste of various foods such as chocolate, cocoa, coffee, beer, tea, etc.,, lessens the astringency of fruits and vegetables and softens the bitterness of foods having a bitter taste and increases ences to food by the on of small amounts. Gentiooligosaccharides can be applied to various foods, for example, fruit juices such as orange juice and pineapple juice, coffee, vegetable drinks, pumpkin congee, etc.

Such gentiooligosaccharides can be ed industrially from a high concentration of glucose by condensation and transglucosylation reactions using a fungal B-glucosidase (see [Umino Takehiro: Study of Industrial Production of Gentiooligosaccharide, Journal of Applied Glycoscience (Japan), 42, 83 (1995)]).

Currently, gentiooligosaccharides having a purity as high as 45% are produced by enzymatic sis, and those having a purity as high as 90% or higher are produced by using cation exchange resins. Here, the remaining components other than gentiooligosaccharides are ily glucose, and for example, gentiooligosaccharides having a purity of 45% n about 50 to 55% by weight of glucose, based on the total weight of saccharide components, while gentiooligosaccharides having a purity of 90% contain about 0.5 to 5% by weight of glucose based on the total weight of saccharide components.

Glucose has about 60% of the sweetness of e. Sometimes, glucose contained in gentiooligosaccharides plays a role in providing sweetness, but there are instances where it inhibits the manifestation of bitterness depending on the subjects to which it is applied. In addition, it is lt to apply gentiooligosaccharides to subjects due to the e contained therein. For e, in the case of chocolates, the addition of gentiooligosaccharides containing a high content of glucose may cause difficulties in molding. Accordingly, there may be a need to use gentiooligosaccharides having high purities depending on the ts to which they are applied. Further, the higher the purity of oligosaccharides, one can obtain the effect with less amount, thereby making it le to achieve the same effects with small amounts. Thus, the preparation of high purity gentiooligosaccharides is essential.

However, practically there are a number of limitations in preparing high purity gentiooligosaccharides. Previously, a case has been reported where the purity of gentiooligosaccharides was raised to about 90% or higher by columns utilizing ion exchange resins (see [Umino Takehiro: Study of Industrial Production of Gentiooligosaccharide, Journal of Applied Glycoscience (Japan), 42, 85-91 (1995)]). r, according to column chromatography, the purity is determined depending on the degree of separation during the ation of high purity oligosaccharides, but there are disadvantages in that when separating gentiooligosaccharides with high purity, the amount of the by—product fractions increases and gentiooligosaccharides are eluted with the glucose fractions, resulting in a low yield. Furthermore, in cases where by- product fractions are sought to be used for other purposes, the by-product ons need to be t to retreatment such as decomposition of gentiooligosaccharides into glucose by low concentrations of enzyme, since large amounts of glucose and small amounts of gentiooligosaccharide are contained therein.

SURE OF THE INVENTION MS TO BE SOLVED BY THE INVENTION It is an object of the present invention to provide methods for preparing high purity gentiooligosaccharides having a purity of at least 90% without the production of unnecessary by—products.

It is another object of the present invention to provide high purity gentiooligosaccharides having a purity of at least 90% prepared by the above methods.

It is yet another object of the present ion to provide methods of improving food taste by adding the above high purity gentiooligosaccharides having a purity of at least 90% to foods.

It is still another object of the t ion to provide methods of using the above high purity gentiooligosaccharides having a purity of at least 90% as alternatives to foods or food additives.

MEANS TO SOLVE THE PROBLEMS In order to achieve the above objectives, there are provided methods for preparing high purity gentiooligosaccharides comprising: adding a low purity gentiooligosaccharide to a liquid medium; subjecting the liquid medium to inoculation with a microorganism, followed by incubation and fermentation to consume glucose that is contained in the low purity oligosaccharide; removing the microorganism from the obtained fermentation broth e.g., by filtration and centrifugation; and subjecting the obtained broth to purification.

Also, the present invention provides high purity gentiooligosaccharides having a purity of at least 90% prepared by the above methods.

In addition, the present invention provides methods of improving food taste by adding the above high purity gentiooligosaccharides having a purity of at least 90% to foods.

Further, the present invention provides methods of using the above high purity oligosaccharides having a purity of at least 90% as alternatives to foods or food additives.

EFFECT OF THE ION According to the methods of the present invention where low purity gentiooligosaccharides containing large amounts of glucose are subjected to inoculation with microorganisms that consume glucose as carbon s but do not e gentiooligosaccharides and fermentation, and then the obtained fermentation broth is subjected to filtration and purification, high purity gentiooligosaccharides having a purity of at least 90% can be prepared without producing unnecessary ducts. Also, the high purity gentiooligosaccharides having a purity of at least 90% obtained according to the t invention can improve the flavor or taste of foods such as cocoa, chocolate, coffee, beer, tea, bread or confectionery products, beverages, etc.., by the addition of small amounts and can be beneficially used as alternatives to foods or their main ingredients.

DETAILED PTION FOR PRACTICING THE INVENTION The methods for preparing high purity gentiooligosaccharides according to the present invention comprise adding a low purity oligosaccharide to a liquid medium; ting the liquid medium to inoculation with a microorganism, followed by incubation and fermentation to consume glucose that is contained in the low purity gentiooligosaccharide; removing the microorganism from the obtained tation broth; and subjecting the broth to purification.

The low purity gentiooligosaccharides that are used in the t invention as raw material are not particularly limited, and any gentiooligosaccharides that are obtained by the conventional methods of preparing gentiooligosaccharide, e.g., by condensation and lucosylation reactions using a fungal beta-glucosidase from a high concentration of glucose (see [Umino Takehiro: Study of Industrial Production of Gentiooligosaccharide, Journal of Applied Glycoscience (Japan), 42, 83 (1995)]), or gentiooligosaccharides obtained by conventional gentiooligosaccharide preparation processes and then initially purified with columns utilizing ion exchange resins to enhance purity may be used. The low purity gentiooligosaccharides that are used in the present invention contain about 15 to 65% by weight of glucose and can be added in solid concentrations of 5 to 70% (w/v), based on the volume of the e medium.

In addition, the e medium for fermentation of the present invention may n 0.1 to 1.5% (w/v) of yeast extract, 0.1 to 0.5% (w/v) of malt extract, and 0.1 to 0.8% (w/v) of peptone as culture medium components.

Among the above culture medium components, those such as yeast extract, malt extract, and peptone are added for the growth of microorganisms for removing the glucose contained in the gentiooligosaccharides, and it is desirable that such components are added in amounts within the above ranges for the efficient culture of microorganisms.

The yeast extracts ed in the liquid medium for the fermentation of the present invention are soluble exudates that are ted from yeasts and are well known in the art. They are superior stimuli for the growth of microorganisms, and play a role in supplying the incubation medium with vitamins, nitrogen, amino acids, carbon, etc. It is desirable that the yeast ts are used in the concentration of 0.1 to 1.5% (w/v) based on the culture medium.

The malt extracts are obtained by growing barley grain with yeast and mold.

Further, since they contain high concentrations of carbohydrates, maltose in particular, malt extracts are suitable for the culture of microorganisms such as yeast and fungi and play a role in supplying the medium with nutrients such as carbon and ns. It is desirable that the malt ts are used in the concentration of 0.1 to 0.5% (w/v) based on the culture medium.

Peptones are yellow powders of small molecules containing trace amounts of proteose, which are obtained by the digestion of proteins such as animal meat and casein using enzymes such as pepsin, trypsin, pancreatine, and are used as nitrogen sources in the culture medium. It is desirable that peptones are used in the concentration of 0.1 to 0.8% (w/v) based on the culture medium.

In addition, the liquid medium for the fermentation of the present invention may contain small amounts of onal components, such as an antifoaming agent, for the efficient culture when foam is generated during microorganism fermentation for the consumption of glucose contained in the gentiooligosaccharides.

As the microorganisms used in the fermentation for obtaining high purity gentiooligosaccharides according to the present invention, those that consume glucose as carbon sources while not consuming gentiooligosaccharides, for example, fermentation fungi such as yeast, bacteria and mold may be used. Representative examples of microorganisms include yeast strains, for example, those belonging to Saccharomyces sp. such as Saccharomyces cerevisiae, romyces carlsbergensis, Saccharomyces uvarum, Saccharomyces ellipsoideus, Saccharomyces bayanus. Besides these, those belonging to Bacillus sp., Lactobacillus sp., and Candida sp. may be used. In particular, yeasts are desirable in that they can be easily separated and removed from the liquid medium afier the tation is terminated, and the removed yeast may be sold as by- product goods such as animal feed.

In the present invention, it is desirable that microorganisms that are used for the fermentation for removing glucose contained in low purity gentiooligosaccharides are added at a concentration of about 10% (v/v) relative to the amount of the culture medium.

Adding microorganisms in amounts exceeding the above amount is problematic in that efficiency is d due to over fermentation, whereas using microorganisms in amounts less than the above amount have problems in that the tion time is prolonged.

In the present invention, fermentation may be carried out under ions of a temperature ranging from 25 °C to 35 °C, an aeration ranging fiom 0.5 to 2 v/v/m (air volume/working volume/minute) and a stirring rate of 200 rpm to 500 rpm for 24 hours to 72 hours.

As described above, if a culture medium containing low purity oligosaccharides is subjected to inoculation with rganisms, incubation and tation, glucose that is ned in the low purity gentiooligosaccharides are consumed by microorganisms and removed, thereby making it possible to obtain high purity gentiooligosaccharides.

After the fermentation is terminated, the microorganisms that are used in the fermentation may be removed from the fermentation broth e.g., by using centrifugation or ion, and then the obtained broth may be subjected to a purification process including decolorization using active carbon and demineralization using ion exchange resins and evaporated under reduced pressure, thereby making it possible to obtain high purity oligosaccharides having a purity of at least 90%.

The high purity gentiooligosaccharides having a purity of at least 90% obtained according to the methods of the present invention have ride compositions as shown in Table 1 below (based on the total weight of the residual saccharides). <Table 1> As such, according to the methods for preparing high purity gentiooligosaccharides by fermentation of the present invention, the glucose content in gentiooligosaccharides may be reduced to 0.5% by weight and gentiooligosaccharides having a purity of at least 90% may be easily obtained without the production of unnecessary by—products.

In addition, during the preparation of high purity gentiooligosaccharides by fermentation according to the present invention, glucose is metabolized by microorganisms, such as yeast and yeast meal, to produce ethanol. Among the ucts of such tation process, yeast meal may be used as animal feed, while ethanol is expected to be in the spotlight as bio-ethanol, a new future fuel.

Further, high purity gentiooligosaccharides having a purity of at least 90% obtained according to the t invention can solve problems associated with conventional oligosaccharides, where their use was limited due to the lties in molding or shaping of the products that are caused by the physical properties of glucose and the detrimental effects of the high glucose content thereof on sweetness. Also, they may improve the flavor and taste of s foods, such as chocolate, cocoa, beer, tea, coffee and red ginseng by the addition of small amounts. Further, they can be beneficially used as alternatives to foods such as cocoa, , chocolate, tea, bread or confectionery products, and their main ingredients.

For example, in preparing bread products of cocoa, chocolate, green tea or the like, substituting high purity gentiooligosaccharides having a purity of at least 90% obtained according to the present invention for 0.1 to 40% by weight of the main ingredients of the bread ts, i.e., cocoa (e.g., Sugarless 100% Cocoa Powder (Valrhona), HERSHEY'S COCOA (HERSHEY), etc..), dark chocolate (e.g., Real Choco Chip Dark (Belcolade), Le Noir 68% Baking Bar (Valrhona), etc.. ), or green tea powder, etc. may exhibit a ness and taste similar to those of conventional products to which no gentiooligosaccharide is added.

In addition, in the case of brownies using cocoa and dark chocolate, tuting high purity gentiooligosaccharides having a purity of at least 90% obtained according to the present invention for 0.1 to 40% by weight of dark chocolate and 0.1 to 40% by weight of cocoa, which are the main ingredients of brownies may also exhibit a bitterness and taste similar to those of conventional products to which no gentiooligosaccharide is added.

For red ginseng beverages, the proportion of high purity gentiooligosaccharides to be used varies ing on the tration of the red g concentrate to be added therein. In the case of red ginseng beverages that contain 1.5 to 2% by weight of red ginseng concentrate, substituting high purity gentiooligosaccharides having a purity of at least 90% for 0.1 to 40% by weight of red ginseng concentrate can exhibit a bitterness similar to that of the conventional beverage; whereas in the case of red ginseng beverages that contain 0.4 to 0.5% by weight of red ginseng concentrate, tuting high purity gentiooligosaccharides having a purity of at least 90% for 0.1 to % by weight of red ginseng concentrate can exhibit a bitterness and taste similar to those of the conventional beverages to which no oligosaccharide is added. In addition, when high purity gentiooligosaccharides having a purity of at least 90% are used instead of red ginseng concentrates as described above, high purity gentiooligosaccharides may be added by one— to ten-fold the amount of red ginseng trate to be substituted.

For coffee beverages, although the substituted amounts vary depending on the characteristics of the coffee beans, such as the place of origin for the coffee plant, high purity gentiooligosaccharides of the present invention may be substituted for 0.1 to 10% by weight ofthe coffee beans.

The present invention is further described and illustrated according to the examples provided below. r, it should be noted that the following examples are presented only for illustrative purposes and are not intended to limit the scope of the present invention.

E 1: (1) Preparation of seed culture 30 mL of YM broth (Difco) was ated with 0.5 mL of the Saccharomyces cerevisiae Y1135 strain, followed by incubation at 30 °C for 24 hours while stirring at 180 rpm. (2) Incubation The crude gentiooligosaccharide (purity 45%) was added to 50 mL of medium containing 0.3% (w/v) of yeast extract (Difco), 0.3% (w/v) of malt extract (Difco), 0.5% (w/v) of peptone (Difco), and water at the solid concentrations of 5% (w/v) and 10% (w/v), tively, based on the volume of the medium, and was inoculated with 1 mL of the Saccharomyces cerevisiae Y1135 seed culture obtained in step (1), and ed by incubation for 48 hours under conditions of a culture temperature of 300C, aeration of 1.0 v/v/m and stirring rate of 300 rpm in an incubator.

The culture broth was analyzed using high performance liquid tography (HPLC) (Waters, HPX-42A column) and BIO-LC (DIONEX, ICS-SOOO Bio-LC Chromatography System, PA detector, PA—l column). As a result, in the case of performing incubation with the addition of crude gentiooligosaccharide (purity 45%) at the solid concentration of 5% (w/v) based on the volume of the liquid medium, gentiooligosaccharides having a oligosaccharide content of 92.8% by weight based on the residual saccharides were obtained after fermentation, while in the case of performing incubation with the addition of crude gentiooligosaccharide y 45%) at the solid concentration of 10% (w/v), gentiooligosaccharides having a gentiooligosaccharide t of 92.0% by weight based on the residual saccharides were obtained after fermentation.

E 2: The crude gentiooligosaccharide (purity 45%) was added to 3 L 'of medium containing 0.3% (w/v) of yeast t (Difco), 0.3% (w/v) of malt extract (Difco), 0.5% (w/v) of peptone (Difco), 1 mL of anti-foaming agent (Dow Corning, LS-300), and water at the solid concentrations of 30% (w/v), 40% (w/v) and 50% (w/v), respectively, based on the volume of the medium, and was inoculated with 300 mL of the Saccharomyces cerevisiae Y1135 seed culture obtained in step (1), followed by incubation for 48 hours under conditions of a culture temperature of 300C, aeration of 1.0 v/v/m and stirring rate of 300 rpm in a 5 L incubator. The gentiooligosaccharide containing culture broth obtained after incubation comprises, besides gentiooligosaccharides and glucose, glycerol and monosaccharide ingredients (e.g., glucose, fructose, etc.) resulting from the liquefaction and glycosylation of starch. The culture broth was subject to filtration to remove the microorganisms, followed by rization with activated carbon and demineralization with ion ge , and subsequently trated to obtain gentiooligosaccharide powder by using a spray dryer. The contents of the ingredients in the powder (based on the total weight of residual saccharides) were analyzed by a similar manner as in Example 1. The results are shown in Table 2 below. <Table 2> Amount of crude Incubation time Amount of Amount of gentiooligosaccharide oligosaccharide monosaccharide added [% (w/v)] contained in the including glucose powder (wt. %) in the powder (wt.

As shown in Table 2, high purity gentiooligosaccharides having a purity of 90% or higher can be obtained by fermentation regardless of what concentration of crude gentiooligosaccharides is added.

EXAMPLE 3: In preparing brownies having dark chocolate (Real Chocochip Dark from Belcolade um), ingredients: cocoa weight 45.8%, cocoa butter 9.9%, sugar, lecithin, natural a flavor, etc..), and cocoa powder (Sugarless 100% Cocoa Powder from Valrhona (France)) as the main ingredients, Experimental Group 1 where 12 g of high purity gentiooligosaccharide (purity 95.2%) obtained in Example 2 was substituted for 7.7% by weight of the dark chocolate and 33.3% by weight of the cocoa ; mental Group 2 where 10 g of high purity gentiooligosaccharide (purity 95.2%) was substituted for 6.1% by weight of the dark chocolate and 33.3% by weight of the cocoa powder; or Control Group that is made up according to tional combination ratios were employed to prepare brownies. The respective combination ratios of each sample from Experimental Groups 1 and 2 and Control Group were shown in Table 3 below. <TABLE 3> Dark chocolate; High purity entiooli _osaccharide ; n __E_ _m_.:— (*1) Protein t: 8% The bitterness, softness and preference for the prepared control group and experimental groups 1 and 2 were assessed and evaluated h a sensory test.

In the sensory test, a 9-scale test where the attributes (bitterness and sofiness) and preference were evaluated using classification grades ranging from 1 to 9 was used. In the case of bitterness, the closer the score is to 9, it is more bitter, while in the case of sofiness, the closer the score is to 9, it was softer. With respect to taste preference, the closer the score is to 9, the better is the preference. The selected panelists were 6 s and 6 males of ages ranging from 25 to 40 and were educated and trained as to the sensory test for two months. The average values of bitterness, softness and preference for brownies made from the Control Group and Experimental Groups 1 and 2 are shown in Table 4 below. The Sigmaplot 11.0 program from Systat Software as used for the statistical data regarding the sensory test results, where statistically significant differences among the above three groups were examined with respect to the e values by the one-way layout design ANOVA at a significance level of 0.05, and the differences between the groups were tically analyzed using the Tukey Test.

E 4> —__- As a result of the sensory test on bitterness, softness and preference, the brownies of Experimental Group 1 where the high purity oligosaccharide was substituted for 7.7% by weight of the dark chocolate and 33.3% by weight of the cocoa powder showed a statistically significant higher score for bitterness, but showed no tical differences for softness "and preference, in comparison to the brownies of the Control Group that contain no high purity gentiooligosaccharide. Further, the brownies of Experimental Group 2 where the high purity gentiooligosaccharide was included in lesser amounts, i.e., substituted for 6.1% by weight of the dark chocolate and the 33.3% by weight of the cocoa powder, showed no statistical significant differences for bitterness and sofiness, while exhibiting a tically significant higher preference, when compared to the brownies of the Control Group.

That is to say, it can be confirmed from the results of mental Groups 1 and 2 that substituting high purity gentiooligosaccharides having the purity of 90% or higher of the present invention for not more than 40% by weight of the main ingredients of brownies, i.e., cocoa powder and dark chocolate, showed no sensory differences, while ting ed overall taste and preference, in comparison to the conventional brownies that contain no gentiooligosaccharide.

E 4: In preparing red ginseng beverages containing 2% by weight (6 g) of red g concentrate, the high purity gentiooligosaccharide (purity 92.5%) obtained in Example 2 was substituted for 25% by weight (1.5 g) of the red ginseng concentrate, while the high purity gentiooligosaccharide was added in an amount three-fold (4.5 g) the amount of the red ginseng concentrate to be substituted (1.5 g). The main ingredients and combination ratios of the red ginseng beverages are shown in Table 5 below. <Table 5> Ingredients Red __‘nsen concentrate ; Hi 1 ' Glucose - owder The red ginseng beverages of the Control Group and Experimental Group were evaluated with respect to bitterness, softness, and preference by a y test and the results are shown in Table 6 below. The y test and the statistical analysis were ted in the same manner as described in Example 3. <Table 6> __—— Control goup 5.833 3.167 4.583 EXoerimental :4 cm 5.917 3.083 4.833 As a result, the red ginseng beverages of the Control Group and Experimental Group ted no statistically significant differences at the significance level of 0.05, and showed no large differences in the total average values for bitterness, softness and preference. Accordingly, substituting high purity gentiooligosaccharide of the present invention for a portion of red g trate that is the main ingredient of red ginseng beverages resulted in no sensory differences in bitterness, softness and preference, in comparison with the original beverages that contain no gentiooligosaccharide.

Claims (12)

WHAT WE CLAIM IS:

1. A method for preparing a high purity gentiooligosaccharide comprising: adding a low purity gentiooligosaccharide to a liquid medium; ting the liquid medium to inoculation with a microorganism wherein the microorganism is a strain selected from the group consisting ofSaccharomyces sp., Bacillus sp., Lactobacillus sp., and Candida sp., followed by incubation and fermentation to consume glucose that is contained in the low purity oligosaccharide; removing the microorganism from the obtained fermentation broth; and subjecting the obtained broth to purification.

2. The method of claim 1, wherein the low purity gentiooligosaccharide contains 15 to 65% by weight of glucose and 35 to 85% by weight of gentiooligosaccharide.

3. The method of claim 1, wherein the low purity gentiooligosaccharide is added to the liquid medium at a concentration of 5 to 70% (w/v).

4. The method of claim 1, wherein the microorganism consumes glucose as a carbon source while not consuming gentiooligosaccharide.

5. The method of claim 4, wherein the microorganism is yeast, bacteria or mold.

6. A method as claimed in claim 1 n the purification is d out by decolorization by an ted carbon, demineralization by an ion exchange resin, and evaporation.

7. The method of claim 6, wherein the high purity gentiooligosaccharide has a purity of at least 90%.

8. The high purity gentiooligosaccharide having a purity of at least 90% that is obtained by the method according to claim 6.

9. A method of improving food flavor or taste by adding the high purity gentiooligosaccharide having a purity of at least 90% according to claim 8 to food.

10. A method of using the high purity gentiooligosaccharide having a purity of at least 90% according to claim 8 as alternatives to food or main ingredient f.

11. The method of claim 10, wherein the high purity gentiooligosaccharide having a purity of at least 90% is added in an amount ranging from 0.1 to 40% by weight of the food or main ingredients thereof.

12. The method of claim 10, wherein the food is one or more selected from the group consisting of cocoa, chocolate, coffee, bread or tionery product, and beverage.