WO1992007947A1

WO1992007947A1 - Oligosaccharide mixture, and procedure for its after-treatment

Info

Publication number: WO1992007947A1
Application number: PCT/FI1991/000240
Authority: WO
Inventors: Marianne Rossi; Yu-Yen Linko; Pekka Linko; Timo Vaara; Marja Turunen
Original assignee: Oy Alko Ab
Priority date: 1990-11-01
Filing date: 1991-08-08
Publication date: 1992-05-14
Also published as: AU8319491A; FI905402A0; FI905402L; FI905402A7

Abstract

A procedure for after-treatment of an oligosaccharide mixture, wherein glucoamylase and/or yeast is made to act on an oligosaccharide mixture which has been obtained by making cyclomaltodextrin-glucanotransferase (CGTase, E.C.2.4.1.19) act on starch in the presence of acceptor sugar. By this procedure a novel oligosaccharide mixture lower in calories and less cariogenic than before is obtained.

Description

OLIGOSACCHARIDE MIXTURE, AND PROCEDURE FOR ITS AFTER- TREATMENT

The present invention concerns a procedure for after-treatment of an oligosaccharide mixture according to the patent application FI 904124, and a mixture obtained by means of said procedure.

It is known in the art to use a amylase in order to liquefy starch-based, viscous mixtures and to lower their viscosity. Hydrolysis of starch with a amylase gradually leads to production of maltose. In the British Patent GB 2,019,406 commercial α and β amy- lases are employed to lower the viscosity of "coupling sugar' syrups. In the Japanese Patent JP 76 73,140, β amylase is used to decompose oligosaccharides pro¬ duced from invert sugar or glucose isomerization mix¬ tures, maltose being obtained as yield.

It is known in the art to use glucoamylase for hydrolyzing starch, whereby starch breaks up to glu- cose. Commercial glucoamylase preparations are mostly composed of several different amylases (in: Starch, Chemistry and Technology (1984), Eds. R.L. Whistler, J.N. BeMiller and E.F. Paschall, Academic Press Inc., London, England) . As disclosed in the British Patent GB 3,660,236, glucoamylase is produced by moulds of genus Aspergillus, such as Aspergillus niger, Aspergil- lus awamori, and by some moulds of genus Rhizopus and Endomyces. In the Japanese Patent JP 63 216,492, gluco¬ amylase and/or commercial yeast has been used in con- nection with producing neotrehalose and 2'-α-glucosyl- maltose, for removal of the hydrolysis products formed of starch, and free glucose.

In the procedure constituting the object of the present invention, in the after-treatment is used, differing from the procedures cited above, either solu¬ ble or immobilized glucoamylase and/or yeast. Since, moreover, the oligosaccharide mixture after-treatment method of patent application FI 904124, with glucoamy¬ lase and/or yeast, has not been heretofore described, the procedure of the invention is a completely novel after-treatment method. In the after-treatment procedure of the inven¬ tion, glucoamylase is made to act on an oligosaccharide mixture as specified in patent application FI 904124, whereby the long-chain oligosaccharides are broken up to shorter units, and glucose is set free in the solu- tion. The free glucose is eliminated with baking yeast from an oligosaccharide mixture according to patent application FI 904124 or from a glucoamylase-treated mixture. Proceeding in this manner, one obtains a novel oligosaccharide mixture which is lower in calories and less cariogenic. The characteristic features of the invention are presented in the claims and are hereby included in the disclosure section.

The object of the invention is a procedure for after-treatment of an oligosaccharide mixture according to patent application FI 904124, and a mixture obtained by this procedure. In the after-treatment procedure of the invention, glucoamylase is made to act on an oligo¬ saccharide mixture according to patent application FI 904124, whereby the long-chain oligosaccharides, particularly those with DP (glucose polymerizing grade) higher than 5, are broken down to shorter units and glucose is set free in the solution. The glucoamylase- treated oligosaccharide mixture contains in the first place oligosaccharides with DP between 3 and 5 (OSG3- OSG5). The free glucose is eliminated with baking yeast from the oligosaccharide mixture according to patent application FI 904124 or from the glucoamylase-treated mixture, whereby an oligosaccharide mixture lower in calories and less cariogenic than the untreated oligo- saccharide mixture is obtained.

The significance of after-treating the oligo¬ saccharide mixture is based on the fact that it is pos- sible by glucoamylase treatment to increase the yield of oligosaccharides with DP 3 to 5 (OSG3-OSG5). Such oligosaccharides can be used e.g. in the foodstuff industry as new raw materials because oligosaccharides with DP 3 to 5 are broken up very slowly, or not at all, by α amylase (e.g. in: Starch, Chemistry and Tech¬ nology (1984), Eds. R.L. Whistler, J.N. BeMiller and E.F. Paschall, Academic Press Inc., London, England). Furthermore, removal of free glucose from the oligo- saccharide mixture reduces the cariogenicity and calo¬ rie content of the mixture (e.g. in: Alternative sweet¬ eners (1986), Eds. L.O. Nabors and R.C. Gelardi, Marcel Dekker Inc., New York, U.S.A., p. 185-216).

In the after-treatment procedure of the inven- tion, the after-treatment may also be carried out with immobilized glucoamylase and/or yeast. Using the immo¬ bilizing technique it is possible to achieve a continu¬ ous after-treatment procedure, by which 30 to 60% of the cost can be saved, compared with conventional tech- nique. Furthermore, the product is obtained in rather pure condition, which even further lowers the after- treatment cost and, on the other hand, makes possible new applications of the product.

The fundamental idea of the invention consists of the fact that glucoamylase has been found to break up α-(l-4) and α-(1-6)-glycosidic bonds in starch, producing glucose from starch. The oligosaccharide mixture of the patent application FI 904124 is starch- based, and the long glucose chains containing acceptor sugar (trehalose or cellobiose) can be broken up with glucoamylase to shorter units, the broken-up glucose chains decomposing further to glucose. By modifying the reaction conditions in controlled manner the yield of oligosaccharides with DP 3 to 5 can be increased. With prolonged reaction time and high glucoamylase concent¬ ration, the oligosaccharide mixture of patent applica¬ tion FI 904124 will decompose to glucose and acceptor sugar. The glucose that has been set free in the oli¬ gosaccharide mixture can be removed with baking yeast, which is known to use glucose in its metabolism. The product thus obtained consists of acceptor sugar and an oligosaccharide mixture containing, among others, oli¬ gosaccharides with DP 3-5.

The composition of the product mixtures was determined by liquid chromatography. The method enabled the concentrations of oligosaccharides under DP 8 to be determined in that the concentration of any given oligosaccharide was calculated in accordance with the concentration of the reference standard (with DP 1 to 7) having the closest retention time. Confirmation of the results was made qualitatively with TLC. Glucoamylase preparations of various origins can be used in the invention, such as commercial prepa¬ rations, for instance. For yeast, e.g. Saccharomyces cerevisiae baking yeast may be used.

The concentration of the oligosaccharide mix- ture reacting with glucoamylase may be in the range from 5 to 60%. The concentration may be high when solu¬ ble glucoamylase is used, while in the case of immobi¬ lized glucoamylase the favourable concentration range is 5 to 30%. For glucose elimination either free or immobilized yeast cells may be used, and the concentra¬ tion of the oligosaccharide mixture may then be in the range of 5 to 60%. The advantageous concentration range is 5 to 30%. The favourable pH range of the oligosac¬ charide mixture to be after-treated is 5.5 to 7.5. The reactions may be effected with glucoamylase at 10 to 70°C, advantageous temperature is 30 to 50°C. For yeast, the temperature range may be 10 to 60°C, advan¬ tageous temperature is 30 to 45°C. In either case, the reaction time is 24 hrs at the most, advantageous reac- tion time is 10 min. to 8 hrs, depending on the concen¬ tration of the glucoamylase or yeast used. The longer the reaction time with glucoamylase the more of the OSG3-OSG5 oligosaccharides will the solution contain, compared with the untreated mixture. The solution con¬ tains the greatest amount of 0SG3 oligosaccharide, next of OSG4 oligosaccharide, and so on, until at the turn- ing point of the reaction the contents, first of OSG5 saccharide, then of OSG4 decrease as the chains are broken up. Ultimately the concentration of OSG3 oligo¬ saccharide also begins to decline. The greater the quantity of oligosaccharides that are broken up the more glucose and acceptor sugar will the solution con¬ tain.

After-treatment with immobilized glucoamylase and/or yeast can be carried out either in batches or as a continuous column reaction. It is advisable, prior to continuous reaction, to filter the oligosaccharide mixture in order to remove any insoluble and unhydro- lyzable starch. Baking yeast immobilized in alginate binds to itself some of the oligosaccharide contents in the mixture being after-treated, for which reason the continuous system should be allowed to stabilize until the yeast beads are saturated with the oligosaccharide mixture. Using a long retention time in the column and low immobilized yeast concentration will result in favourable conditions for the after-treatment. Following upon the after-treatment of the invention, the oligosaccharide syrup may further be after-treated according to procedures known in the art, such as decolouring with active carbon and desalting with ion exchangers, whereafter the oligosaccharide syrup may be either cold-dried, powdered or concentrat¬ ed in vacuum. Ethanol (e.g. 70%) may be used to pre¬ cipitate the long-chain oligosaccharides and polysac- charides out of the mixture.

It is thus understood that in the after-treat- ment procedure of the invention oligosaccharide mix¬ tures according to patent application FI 904124 can be modified in controlled manner, whereby mixtures are obtained in which the proportion of oligosaccharides with DP 3 to 5 has been advantageously altered. In addition, it is possible with this after-treatment to achieve oligosaccharide mixtures lower in calories and less cariogenic than the untreated mixtures.

EXAMPLE 1

Yeast immobilization

The yeast cells were bonded to sodium alginate in the form of beads (Linko, Y-Y., Weckstrδm, L. and Linko, P. (1980), Food Process Engineering, Vol. 2, p. 81-91). Baking yeast, 16.0 g wet weight, (Oy Alko Ab, Rajamaki) was suspended in 10 ml distilled water and added to 50 g 8% sodium alginate (BDH, Poole, England) . The mixture was extruded with the aid of nitrogen gas to beads of 0.6 mm diameter through needles into 0.5 M calcium chloride solution, in which the beads were left to mix for 60 min. The beads were then washed with dis¬ tilled water, and the excess of water was removed. The activity of the immobilized yeast was de¬ termined using for substrate, 2% glucose solution (in 50 mM imidazole buffer, pH 6.8). 1.0 g of the immobi¬ lized yeast were added to 5 g substrate and the reac¬ tion was allowed to proceed at 40°C for 30 min. The concentration of residual glucose was determined at room temperature by liquid chromatography (Zsadon, B., Otta, K.H., Tudos, F. and Szejtli, J. (1979), J. Chrom- atogr., 172. 490-492). The elution rate in carbohydrate column was 0.9 ml/min, and the concentrations of the standards (with DP 1-7, including glucose, maltose, ..., maltoheptaose, cellobiose, trehalose) were 1-5 mg/1.

One unit of activity of the immobilized yeast beads is equivalent to 1 μmol glucose per min. used up under the above reaction conditions. The activity of the immobilized yeast was found to be 7 U/g and the activity of free yeast cells under equivalent condi- tions of reaction, 33.3 U/g. The activity yield of the immobilized yeast was found to be 100%.

EXAMPLE 2 Glucoamylase immobilization

The glucoamylase (Spezyme, Suomen Sokeri Oy, activity 340 GU/ml) was immobilized on Duolite ES 762 resin (Duolite International, Vitry, France) . For im¬ mobilization, to 50 mM imidazole buffer (pH 6.8) was added suitably diluted glucoamylase 2 ml per g resin, and the enzyme was allowed to become linked to the resin under 16-hr shaking at room temperature. The en¬ zyme solution was filtered off and 2.5% glutaric alde¬ hyde was added 2 ml per g resin for cross-linking. Shaking was continued for another 3 hrs at room temper¬ ature, whereafter the resin was washed with distilled water and filtered to moist constitution.

The activity of the immobilized glucoamylase was determined using for substrate, 2% starch solution (soluble starch in 50 mM imidazole buffer, pH 6.8).Im¬ mobilized glucoamylase was added 0.5 g to a solution containing 4.5 g substrate and 0.5 ml distilled water.

< The reaction was allowed to proceed at 40°C for 30 min.

The concentration of the glucose thus formed was deter- mined by liquid chromatography (see Example 1). The activity of the soluble glucoamylase was determined by adding suitably diluted enzyme 0.5 ml to 4.5 g sub¬ strate under equivalent conditions of reaction as above. The unit of activity (U) of immobilized gluco¬ amylase corresponds to 1 μmol glucose produced in one minute under the above reaction conditions. The activ¬ ity of the immobilized glucoamylase was found to be 3.6 U/g and that of the soluble enzyme, 378 U/ml. The grade of activity of the immobilized glucoamylase was found to be 4.3%. EXAMPLE 3

After-treatment of oligosaccharide mixture with glucoamylase

Oligosaccharide mixtures according to patent application FI 904124 were after-treated with either soluble or immobilized glucoamylase preparation accord¬ ing to Example 2. To the oligosaccharide mixture, of which the composition is presented in Table 1 (treha¬ lose oligosaccharides, dry matter content of the mix- ture 50%), was added soluble glucoamylase, either 1.1 U per g of mixture (Test 1) or 3.3 U per g of mixture (Test 2). Immobilized glucoamylase was added 0.11 U per g of mixture (Test 3). The batch reactions were allowed to proceed at 40°C under shaking for 3 hrs (Test 1), 5 hrs (Test 2) and 1.75 hrs (Test 3). The compositions of the products were determined by liquid chromatography (see Example 1), and they are given in Table 1.

A cellobiose oligosaccharide mixture was simi¬ larly after-treated, its composition being presented in Table 2 (dry matter content 30%). Soluble glucoamylase was added 0.67 U per g of mixture (Test 4) and immobi¬ lized glucoamylase, 0.22 U per g of mixture (Test 5). The batch reactions were allowed to proceed at 40°C under shaking for 3 hrs (Test 4) and 5 hrs (Test 5). The compositions of the products were determined by liquid chromatography (see Example 1), and they are given in Table 2.

The concentration of OSG3-OSG5 oligosacchar¬ ides could be increased by the glucoamylase treatment in all tests. The untreated oligosaccharide mixture presented in Table 1 had OSG3-OSG5 concentration 21.5 g/100 g, while the OSG3-OSG5 concentrations after glucoamylase treatment were 22.0-26.3 g/100 g. Espe¬ cially in Test 2 the concentration of OSG3 oligosaccha- ride increased 85%, compared with the untreated mix¬ ture. Similarly, the untreated oligosaccharide mixture presented in Table 2 had OSG3-OSG5 concentration 11.2 g/100 g, while the OSG3-OSG5 concentrations after glucoamylase treatment were 12.3-14.3 g/100 g.

TABLE I. After-treatment of oligosaccharide mixture with soluble and immobilized glucoamylase (Example 3) . The mixture was reacted with soluble (Test 1: 1.1 U per g of mixture, and Test 2: 3.3 U per g of mixture) or immobilized (Test 3: 0.11 U per g of mixture) gluco¬ amylase at 40°C for 3, 5 and 1.75 hrs, respectively. The concentrations of oligosaccharides (with DP less than 8) were determined by liquid chromatography (see Example 1) .

TABLE II. After-treatment of oligosaccharide mixture with soluble and immobilized glucoamylase (Example 3). The mixture was reacted with soluble (Test 4: 0.67 U per g of mixture) or immobilized (Test 5: 0.22 U per g of mixture) glucoamylase at 40°C for 3 and 5 hrs, re¬ spectively. The concentrations of oligosaccharides (with DP less than 8) were determined by liquid chroma¬ tography (see Example 1) .

Native mixture Test 1 Test 2

Product Concentration (g/100 g)

2.9 5.6

7.5 8.8

7.8 8.1

4.5 2.3

2.0 0.9 0.6

14.3 12.3

EXAMPLE 4

After-treatment of oligosaccharide mixture with yeast An oligosaccharide mixture as in Test 1 pre- sented in Example 3 was after-treated with free yeast cells as in Example 1, in a batch reaction, and an oligosaccharide mixture as in Example 3, with immobi¬ lized yeast cells as in Example 1, in a continuous re¬ action. The aim was to reduce the glucose concentration in the mixture.

To the oligosaccharide mixture of Test 1 free yeast cells were added 3.2 U per g of mixture and the reaction was allowed to proceed under shaking at 40°C for 1 hr, whereafter the yeast cells were filtered off. The composition of the product was determined by liquid chromatography (see Example 1) and is presented in Table 3 .

The oligosaccharide mixture of Test 3 was after-treated with immobilized yeast in a continuous column reaction at 40°C. Prior to glucose removal, the oligosaccharide mixture of Test 3 was diluted 1:10 with water, and filtered, whereafter the dry matter content of the solution was 5%. 20 g immobilized yeast (activ¬ ity 7 U/g) were added into the column (1.8 cm diameter and 14 cm height) , and the flow rate in the column was 0.8 ml/min. Collection of eluate was commenced upon stabilization of the column reaction. The composition of the eluate was determined by liquid chromatography (see Example 1) and is presented in Table 3.

It proved possible, with free yeast cells, to reduce the glucose concentration in the mixture 82%, and with immobilized yeast the glucose could be totally eliminated from the mixture. Fig. 1 displays the liquid chromatographic elution chromatogram of the oligosac¬ charide mixture of Test 3, after immobilized yeast treatment. The peaks numbered 1 to 6 in the chromato¬ gram are: 1= trehalose, 2= OSG3, 3= OSG4, 4= OSG5, 5= OSG6, and 6= OSG7.

TABLE III. After-treatment with free yeast cells (3.2 U per g of mixture, reaction time 1 hr) of the oligosac¬ charide mixture of Test 1, presented in Example 3, and after-treatment with immobilized yeast in a column (column activity 140 U, flow rate 0.8 ml/min) of the oligosaccharide mixture of Test 3, diluted to 5% dry matter content, at 40°C (Example 4). The oligosaccha¬ ride (DP less than 8) concentrations were determined by liquid chromatography (see Example 1).

Free Immobilized yeast cells yeast cells

Product Concentration (g/100 g)

1.07 1.26 0.77 0.38 0.19 +

0.241

Claims

1. A procedure for modifying an oligosaccha¬ ride mixture, c h a r a c t e r i z e d in that free or immobilized glucoamylase and/or yeast is made to act on an oligosaccharide mixture which has been obtained by making cyclomaltodextrin-glucanotransferase (CGTase, E.C. 2.4.1.19) act on starch in the presence of accep¬ tor sugar.

2. Procedure according to claim 1, c h a r¬ a c t e r i z e d in that glucoamylase is made to act on the oligosaccharide mixture, whereby the glucoamyla¬ se breaks up the long-chain oligosaccharides to shorter units, at the same time setting free glucose, whereby the proportion in the mixture of oligosaccharides with DP from 3 to 5 can be modified.

3. Procedure according to claim 1, c h a r¬ a c t e r i z e d in that yeast is made to act on. the oligosaccharide mixture, whereby the yeast will use the free glucose in the mixture towards its metabolism, whereby an oligosaccharide mixture lower in calories and less cariogenic is obtained.

4. Procedure according to claim 1, c h a r¬ a c t e r i z e d in that glucoamylase and yeast are made to act on the oligosaccharide mixture, either in steps or concurrently, whereby the glucoamylase breaks up the long-chain oligosaccharides to shorter units and the yeast uses towards it metabolism the glucose that has been set free in the solution, whereby an oligosac- charide mixture lower in calories and less cariogenic is obtained in which the proportion of oligosaccharides with DP from 3 to 5 has been modified.

5. A starch-based oligosaccharide mixture manufactured by a procedure according to any one of claims 1-4, c h a r a c t e r i z e d in that it contains 0-100%, advantageously 85-100%, less 0SG7 and OSG6, and 0-100%, advantageously 97-100%, less glucose than the untreated oligosaccharide mixture and 0-55% more OSG5, 0-75% more OSG4, 0-90% more OSG3 and 0-15% more acceptor sugar than the untreated oligosaccharide mixture.

6. Starch-based oligosaccharide mixture manu¬ factured by a procedure according to claim 2, c h a r¬ a c t e r i z e d in that it contains glucose 0-35%, acceptor sugar 3-85%, OSG3 1-60%, OSG4 1-35%, OSG5 0.5- 25%, OSG6 0-15% and OSG7 0-10%, calculated on the dry matter of the solution.

7. Starch-based oligosaccharide mixture manu¬ factured by a procedure according to claim 3, c h a r¬ a c t e r i z e d in that it contains glucose 0-3%, acceptor sugar 7-75%, OSG3 1.8-32%, OSG4 3-20%, OSG5 2.5-16%, OSG6 0.8-12% and OSG7 0.2-10%, calculated on the dry matter of the solution.

8. Starch-based oligosaccharide mixture manu¬ factured by a procedure according to claim 4, c h a r¬ a c t e r i z e d in that it contains glucose 0-3%, acceptor sugar 3-85%, 0SG3 1-60%, OSG4 1-35%, 0SG5 0.5- 25%, OSG6 0-15% and OSG7 0-10%, calculated on the dry- matter of the solution.