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WO1992003565A1 - Oligosaccharide mixture, and procedure for its manufacturing - Google Patents

Oligosaccharide mixture, and procedure for its manufacturing Download PDF

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Publication number
WO1992003565A1
WO1992003565A1 PCT/FI1991/000239 FI9100239W WO9203565A1 WO 1992003565 A1 WO1992003565 A1 WO 1992003565A1 FI 9100239 W FI9100239 W FI 9100239W WO 9203565 A1 WO9203565 A1 WO 9203565A1
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WO
WIPO (PCT)
Prior art keywords
starch
cgtase
trehalose
acceptor
oligosaccharides
Prior art date
Application number
PCT/FI1991/000239
Other languages
French (fr)
Inventor
Marianne Rossi
Yu-Yen Linko
Pekka Linko
Timo Vaara
Marja Turunen
Original Assignee
Oy Alko Ab
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Filing date
Publication date
Application filed by Oy Alko Ab filed Critical Oy Alko Ab
Publication of WO1992003565A1 publication Critical patent/WO1992003565A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

Definitions

  • the present invention concerns a starch-based oligosaccharide mixture containing acceptor sugar, and a procedure for manufacturing same.
  • Oligosaccharides containing trehalose and cel ⁇ lobiose are linear molecules in which one or several glucose units are adjoined to acceptor sugar (trehalose or cellobiose) .
  • acceptor sugar trehalose or cellobiose
  • CGTase cyclomaltodextrin-glucanotransferase
  • Oligosaccharides can be applied as new raw materials in the foodstuff, animal feed and medical industry, and in chemical industry. To this purpose certain oligosaccharides have already been launched on the market, e.g. starch-based products such as maltose, maltotriose, maltotetraose, isomaltose, panose, etc. Furthermore, lactose-based oligosaccharides and sugar alcohols have been produced, such as maltitol, and sac ⁇ charose-based products, such as 'coupling sugar', fruc- tooligosaccharides, palatinose, etc., which have been reported in: Alternative sweeteners (1986), edited by L.O. Nabors and R.C.
  • oligosaccharides are often low in calories, their sweetness is milder than that of saccharose, and they are either less cariogenic than saccharose or not cari- ogenic at all. In addition to these properties, oligo ⁇ saccharides have also good technical characteristics and positive effects both physiologically and in view of health.
  • fructooligo- saccharides A procedure for manufacturing fructooligo- saccharides has been disclosed e.g. in the British Patent No. 2,179,946, in which fructosyltransferase is made to act on saccharose. Palatinose, or isomaltulose, is produced from saccharose with glucosyltransferase, lactose-based oligosaccharides such as 6-galactosyl lactose are produced from lactose with ⁇ -galactosidase.
  • K. Ajisaka and H. Fujimoto (1989) Ameri ⁇ can Society Meeting, Sept. 10-15, Miami Beach, a proce- dure for manufacturing trisaccharide containing treha ⁇ lose, from glucose and trehalose with R. niveus gluco- amylase, has been disclosed.
  • Patent No. 2,019,406 in both of which the enzyme is made to act on starch or dextrin in the presence of either saccharose or fructose, whereby as product are formed oligosaccharides of 'coupling sugar* type.
  • J. Biochem., 79 ./ 641-648 J. Biochem., 79 ./ 641-648, is disclosed the manufacturing from dextrin or starch, with CGTase, of oligosaccharides containing xylose and sorbose. Determination of the acceptor specificity of CGTase from various acceptor sugars and from cyclodex- trin is described in scientific magazines (D.
  • starch is used for substrate and cellobiose and/or trehalose for acceptor. No such production method has been described heretofore.
  • a novel, unique oligosaccharide mixture which is usable as a new raw material e.g. in the foodstuff and animal feed indus ⁇ tries.
  • the object of the invention is an oligosaccha ⁇ ride mixture containing trehalose and cellobiose as acceptor sugar, and a procedure for manufacturing same.
  • cyclomaltodextrin-glucanotransferase (CGTase; E.C. 2.4.1.19) is made to act on starch in the presence of acceptor sugar (trehalose or cellobiose), whereby oligosaccharides are formed which in their sugar composition contain trehalose and cellobiose.
  • the basic idea of the invention consists in that CGTase has been found to produce oligosaccharides from a suitable acceptor sugar and starch, in suitable conditions. It is possible in the procedure of the in ⁇ vention, by varying the conditions of reaction, to achieve oligosaccharide mixtures of novel type in which the oligosaccharides have DP numbers starting with 3 and up to 7 at least.
  • the reaction model of the proce ⁇ dure for producing an oligosaccharide mixture, on which the invention reads, can be assumed to be as follows. when cellobiose ( ⁇ -glu-(l-4)- ⁇ -glu) or trehalose ( ⁇ - glu-(l-l)- ⁇ -glu) is used for acceptor.
  • acceptor trehalose or cellobiose (di- saccharide)
  • the trehalose-containing oligosaccharide 0SG3 has the fol ⁇ lowing structure: 0- ⁇ -D-glu-(l-4)-0- ⁇ -D-glu-(l-l)- ⁇ -D- glu, that is, a glucoside molecule is attached to tre ⁇ halose with an ⁇ -(l-4)-glycosidic bond.
  • the oligosac ⁇ charides with DP higher than 3 are hard to analyse by nmr technique, owing to their complex structure. It has, however, been found in enzymatic structural analy- sis that the glucose molecule(s) are linked with the acceptor sugar in conformity with the above model.
  • Oligosaccharides according to the invention can be used e.g. in the foodstuff industry as new raw materials, because ⁇ -amylase breaks up very slowly, or not at all, oligosaccharides with DP between 3 and 5 (OSG3-OSG5) (e.g. in: Starch, Chemistry and Technology (1984) edited by R.L. Whistler, J.N. BeMiller and E.F. Paschall, Academic Press Inc., London, England, p. 93- 102) .
  • the oligosaccharide mixture obtained as product may furthermore contain the acceptor sugars trehalose or cellobiose, which are also used as single sugars for raw materials of the foodstuff industry.
  • the proportion in the product mixture of oligosaccharides having DP 3- 5 can be modified by means of dry matter and enzyme concentration, relative mass proportion of starch and acceptor sugar, and reaction time.
  • compositions of the product mixtures were determined by liquid chromatography.
  • the method enabled the concentrations of oligosaccharides with DP less than 8 to be determined in that the concentration of any given oligosaccharide was calculated in accordance with the concentration of whichever standard (DP from 1 to 7) had the most closely equal retention time. Con ⁇ firmation of the results was made qualitatively with TLC.
  • the CGTase required in the present invention is produced by cultivating a microorganism producing the respective enzyme, e.g. certain bacteria of genus Bacillus, in a culture solution containing a carbon and nitrogen source, minerals and vitamins (M. Makela, Bio- technical production of cyclodextrins (1990), Depart- ment of Biochemistry, University of Turku, Finland) .
  • the CGTase thereby formed is recovered using procedures of prior art, such as centrifuging the culture solu ⁇ tion, or filtering.
  • the crude enzyme saved in this manner may be purified and concentrated e.g. by salting out, gel filtration and/or by ion exchange chromatog ⁇ raphy or affinity chromatography.
  • starch materials of various origins can be used, e.g. those derived from cereals such as barley starch, or from root crops, such as potato starch.
  • the starch may also be pretreated e.g. by acid hydrolysis and/or enzymatically so that the dextrose equivalent of the liquefied starch thus formed is in the range from 0.5 to 20.
  • enzymatic hydroly ⁇ sis of starch one may use e.g. ⁇ -amylase or it is also possible to add the production enzyme, or CGTase, di ⁇ rectly to the starch, in which case pretreatment is unnecessary.
  • the advantageous DE number of the starch is about 1.
  • the total concentration of starch reacting with CGTase and acceptor sugar should be within 5-60%.
  • Advantageous concentration is 30-40% for production of cellobiose saccharides, and 40-50% for production of trehalose saccharides, whereby the relatively greatest amount of short-chain oligosaccharides will be formed in the mixture. Particularly in production of trehalose oligosaccharides the concentration should be high.
  • Sub- strate and acceptor may be dissolved either in water or in buffer, e.g. in 50 mM i idazole buffer pH 6.8, in 50 mM acetate buffer pH 5.5 or in 50 mM glycine-NaOH buffer pH 9.0.
  • Favourable pH range for the reaction is 6.5-7.0.
  • the reaction may be carried out at 50-80°C, with reaction time 2 days at the most.
  • Favourable tem ⁇ perature for the reaction is 60°C and reaction time, 20 to 48 hrs, depending on the CGTase concentration.
  • the relative mass proportion of starch and acceptor sugar should be within 0.5-4 in the solution.
  • Favourable pro- portion of starch and acceptor is 1:1, whereby OSG3- OSG5 oligosaccharides will be formed in the mixture most of all.
  • OSG3- OSG5 oligosaccharides When the starch quantity exceeds that of acceptor, long-chain oligosaccharides are formed most, in proportion.
  • short-chain oligosacchar ⁇ ides among others the OSG3 oligosaccharide, are formed relatively most.
  • the CGTase concentration in the reac ⁇ tion should be within 30-350 U per g of starch.
  • the favourable CGTase concentration is affected, among others, by concentration in that the higher the concen ⁇ tration of the solution the higher should the enzyme concentration be in the reaction. High enzyme concen ⁇ tration also shortens the reaction time.
  • Oligosaccharide-production enzyme or CGTase, (isolated from a Bacillus circulans strain, activity 7600 U/ml, Oy Alko Ab) was added 30 U/g to a mixture containing starch as stated in Table 1, in 50 mM imid- azole buffer pH 6.8, to which had been added 1.5 mM CaCl 2 .
  • the CGTase was allowed to act at 85°C for 30 min. with simultaneous agitation, whereafter in the starch (DE 1) was dissolved cellobiose (Sigma, U.S.A.) as stated in Table 1, and the solution was tempered to 60°C reaction temperature.
  • the reaction was started by adding to the so- lution 50 U CGTase per g of starch, and the reaction was allowed to proceed 48 hrs at 60°C, agitating at the same time.
  • the compositions of the products were deter ⁇ mined at room temperature by liquid chromatography (Zsadon B., Otta K.H., Tudos F. and Szejtli J. (1979), J. Chromatogr., 172, 490-492).
  • the elution rate in car ⁇ bohydrate column was 0.9 ml/min and the standards (e.g.
  • Example 1 (see Example 1) was added 30 U/g to a mixture contain ⁇ ing starch, as stated in Table 2, in buffer as in Example 1.
  • the CGTase was allowed to act for 30 min, at 85°C, with agitation, whereafter in the starch (DE 1) was dissolved ⁇ , ⁇ -trehalose (Sigma, U.S.A.) as stated in Table 2, and the solution was tempered to the reac ⁇ tion temperature, 60°C.
  • the reaction was started by adding CGTase 150 U per g of starch, and the reaction was allowed to proceed 48 hrs at 60°C under agitation.
  • the composi ⁇ tions of the products were determined by liquid chroma- tography (see Example 1) .
  • the concentrations of OSG3-OSG7 oligosaccha ⁇ rides in the products after 48 hrs reaction time are stated in Table 2.
  • CGTase (see Example 1) was added 30 U/g to a mixture containing starch in the mass proportion stated in Table 3, so that the ultimate concentration after acceptor addition in the solution would be 30 g/100 g.
  • the starch had been dissolved in imidazole buffer as in Example 1.
  • the CGTase was allowed to act for 30 min, at 85°C, with agitation, whereafter in the starch (DE 1) was dissolved either trehalose or cellobiose as stated in Table 3 (ultimate concentration 30 g/100 g) , and the solution was tempered to 60°C reaction temperature.
  • the reaction was started by adding CGTase 50 U per g of starch, and the reaction was allowed to pro- ceed 48 hrs at 60°C under agitation.
  • the compositions of the products were determined by liquid chromatogra ⁇ phy (see Example 1).
  • the concentrations of OSG3-OSG7 oligosaccha ⁇ rides in the products after 48 hrs reaction time are stated in Table 3.
  • the total quantities of oligosaccha ⁇ rides OSG3-OSG5 were, with the cellobiose saccharides, 13.1 g/100 g (mass proportion 1:4) and 14.4 g/100 g (1:2), and with trehalose saccharides 6.9 g/100 g in either case (mass proportions 1:2.3 and 1:1.2).
  • the proportion of the OSG3 oligosaccharide was highest when the acceptor concentration in the solution was high.
  • Trehalose oligosaccharides were produced in the manner described in Example 2, in the reaction being used 50 g starch (DE 1) and 50 g trehalose dis ⁇ solved in 200 g imidazole buffer (dry matter content 50%). The reaction was started by adding CGTase (see Example 1) 150 and 210 U per g of starch, and the reac- tion temperature was 60°C.
  • Fig. 2 the yield of oligosaccharides and the consumption of treha ⁇ lose (g/100 g) are plotted over time (48 hrs, CGTase concentration 210 U/g) .
  • the concentrations of OSG3-OSG5 oligosaccha ⁇ rides after 48 hrs reaction time are presented in Table 4.
  • the total of OSG3-OSG5 oligosaccharides in the pro ⁇ duct was measured to be 20.0 g/100 g (40.0% of the dry matter content, CGTase 150 U/g) and 22.7 g/100 g (45.4% of the dry matter content, CGTase 210 U/g).
  • Example 1 The production solution of Example 1, with 30% dry matter content, was diluted with water to 4.72% dry matter content and centrifuged, to remove the dry mat ⁇ ter. To the solution was added 135.5 U/ml ⁇ -amylase (isolated from a Bacillus subtilis strain, activity 1355 U/mg, Sigma, U.S.A.) and the reaction was allowed to proceed 24 hrs at 60°C, with agitation.
  • ⁇ -amylase isolated from a Bacillus subtilis strain, activity 1355 U/mg, Sigma, U.S.A.
  • the composi ⁇ tion of the product was determined by liquid chromatog- raphy (see Example 1), and measurement showed the total content of OSG3-OSG5 oligosaccharides in the product to be 14.2 g/100 g (OSG3: 7.3 OSG4: 4.2 g/100 g, OSG5: 2.7 g/100 g).
  • ⁇ -amylase broke off 16% oligosaccharide and 25% OSG5 oligosaccharide from the product of Example 1, while on the other hand the content of OSG3 oligosac ⁇ charide increased 22%.
  • Trehalose oligosaccharides were produced in the manner described in Example 2, in the reaction being used 15 g starch (DE 1) and 13.6 g trehalose dis- solved in 200 g imidazole buffer (dry matter content 28.6%).
  • the reaction was started by adding CGTase (see Example 1) 50 U per g of starch, and the reaction tem ⁇ perature was 60°C.
  • the reaction was allowed to proceed for 48 hrs, whereafter the composition of the product was determined by liquid chromatography (see Exam ⁇ ple 1).
  • OSG3- OSG5 oligosaccharides were 6.6 g/100 g (OSG3: 1.9 g/100 g, OSG4: 2.3 g/100 g, OSG5: 2.4 g/100 g, total 23.1% of the dry matter content).
  • the production solution was diluted with water to 6% dry matter content and centrifuged, to remove the dry matter.
  • the composition of the product was determined by liquid chromatography (see Example 1), and measurement showed the total quan ⁇ tity of OSG3-OSG5 oligosaccharides in the product to be 10.7 g/100 g (OSG3: 5.8 OSG4: 3.3 g/100 g, OSG5: 1.6 g/100 g) .
  • OSG3 5.8
  • OSG4 3.3 g/100 g
  • OSG5 1.6 g/100 g
  • ⁇ -amylase broke off 33% 0SG5 oligosaccharide from the product, while on the other hand the content of OSG3 oligosaccharide was tripled.
  • Trehalose oligosaccharides were produced in the manner described in Example 2, but the starch was first hydrolyzed with ⁇ -amylase (BAN 120 L, activity 120 KNU/g, Novo, Denmark). Amylase was added 0.3 and 0.9 KNU/g to a mixture containing 16 g starch in imid ⁇ azole buffer (see Example 2). The amylase was allowed to act 30 min. at 85°C, with stirring, whereafter in the starch (DE 5 and DE 16) was dissolved 16 g treha ⁇ lose (dry matter content of solution 32%) and the solu ⁇ tion was tempered to reaction temperature, 60°C.
  • ⁇ -amylase BAN 120 L, activity 120 KNU/g, Novo, Denmark.
  • the reaction was started by adding to the solution CGTase (see Example 1) 120 U per g of starch, and the reaction was allowed to proceed 48 hrs at 60°C, with stirring.
  • the composition of the products was de ⁇ termined by liquid chromatography (see Example 1) and by TLC on silicagel plates (running solution: acetone- 1-butanol-water 11:9:5; staining solution: aniline 2 ml, diphenylamine 2 g, acetone 100 ml, and 80% phos ⁇ phoric acid 15 ml; after plate staining, 30 min. heat ⁇ ing at 105°C) .
  • the total quantity of OSG3-OSG5 oligosaccha- rides in the product was found, in either case, to be 11.5 which was 35.9% of the dry matter content.
  • the product compositions were also analysed by TLC, which differentiates the produced oligosaccharides from the hydrolysis products formed in the pretreatment of the starch, such as maltose, maltotriose, maltotetraose, etc.
  • TLC the product solutions contained very small quantities of hydrolysis products formed in the pretreatmen ; their concentrations varied within 0.1 to 2.5 g/100 g, depending on the DE number of the solution.
  • the oligosaccharide contents of the product were found by measurement to be: OSG3: 4.4 g/100 g, 0SG4: 3.9 g/100 g, 0SG5: 3.2 g/100 g, and with starch having DE 16: 0SG3: 4.6 g/100 g, OSG4: 4.0 g/100 g, OSG5: 2.9 g/100 g.
  • CGTase (see Example 1) was added 30 U/g to a mixture containing 2 g starch in water or in 50 mM ace- tate, imidazole or glycine-NaOH buffer, to which had been added 1.5 mM CaCl 2 .
  • the buffers had pH 5.5, 6.8 and 9.0, respectively.
  • CGTase was allowed to act 30 min, at 85°C, with stirring, whereafter in the starch (DE 1) was dissolved 3 g trehalose (dry matter content of solution 40%) and the solution was tempered to reaction temperature 60°C.
  • CGTase was bonded with covalent bonds to Eupergit C beads (Rohm Pharma, Federal Republic of Germany) in that to 0.5 g of the beads (dry matter mass) was added 2 ml CGTase (isolated from Bacillus circulans strain, activity 265 U/ml, Oy Alko Ab) . The enzyme was left to be bonded for 20 hours at room tem ⁇ perature, shaking at the same time, whereafter the beads were washed with imidazole buffer according to Example 2.
  • soluble CGTase was added 30 U/g to a mixture containing 0.5 g starch and 4 g buffer as in Example 1.
  • the CGTase was allowed to act 30 min. at 85°C, with stirring, whereafter in the starch (DE 1) was dissolved 0.5 g trehalose (dry matter content of solution 20%), and the solution was tempered to reaction temperature 60°C.
  • the reaction was started by adding to the solution immobilized CGTase 0.5 g (dry matter mass, activity 424 U/g) , and the reaction was allowed to pro ⁇ ceed 21 hrs at 60°C, shaking at the same time.
  • the com- position of the product was determined by liquid chro ⁇ matography (see Example 2) . Measurements gave for the total OSG3-OSG5 oligosaccharide quantity in the pro ⁇ duct: 7.5 g/100 g (OSG3: 3.2 OSG4 2.4 g/100 g, and OSG5: 1.9 g/100 g) , which is 37.5% of the dry matter content.

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Abstract

A procedure for manufacturing an oligosaccharide mixture, in which cyclomaltodextrin-glucanotransferase (CGTase; E.C. 2.4.1.19) acts on starch in the presence of acceptor sugar (trehalose or cellobiose), whereby oligosaccharides containing trehalose and cellobiose, and unique of their sugar composition, are produced.

Description

OLIGOSACCHARIDE MIXTURE, AND PROCEDURE FOR ITS MANUFAC¬ TURING
The present invention concerns a starch-based oligosaccharide mixture containing acceptor sugar, and a procedure for manufacturing same.
Oligosaccharides containing trehalose and cel¬ lobiose are linear molecules in which one or several glucose units are adjoined to acceptor sugar (trehalose or cellobiose) . In the procedure of the invention, cyclomaltodextrin-glucanotransferase (CGTase is made to act on starch in the presence of acceptor sugar (treha¬ lose or cellobiose), whereby oligosaccharide mixtures containing trehalose and cellobiose, with unique sugar composition, are formed. Under effect of CGTase on starch alone, cyclodextrins with ring structure are formed, which are not produced in the manufacturing procedure on which the invention reads.
Oligosaccharides can be applied as new raw materials in the foodstuff, animal feed and medical industry, and in chemical industry. To this purpose certain oligosaccharides have already been launched on the market, e.g. starch-based products such as maltose, maltotriose, maltotetraose, isomaltose, panose, etc. Furthermore, lactose-based oligosaccharides and sugar alcohols have been produced, such as maltitol, and sac¬ charose-based products, such as 'coupling sugar', fruc- tooligosaccharides, palatinose, etc., which have been reported in: Alternative sweeteners (1986), edited by L.O. Nabors and R.C. Gelard, Marcel Deccer Inc., New York, U.S.A., p. 165-244 and 309-323, and in: Develop¬ ments in sweeteners - 2 (1983), edited by K.J. Parker and M.G. Lindley, Applied Science Publishers Ltd., Essex, England, p. 1-88. The significance of oligosaccharides is based on the fact that they are often low in calories, their sweetness is milder than that of saccharose, and they are either less cariogenic than saccharose or not cari- ogenic at all. In addition to these properties, oligo¬ saccharides have also good technical characteristics and positive effects both physiologically and in view of health.
A procedure for manufacturing fructooligo- saccharides has been disclosed e.g. in the British Patent No. 2,179,946, in which fructosyltransferase is made to act on saccharose. Palatinose, or isomaltulose, is produced from saccharose with glucosyltransferase, lactose-based oligosaccharides such as 6-galactosyl lactose are produced from lactose with β-galactosidase. In a paper by K. Ajisaka and H. Fujimoto (1989), Ameri¬ can Society Meeting, Sept. 10-15, Miami Beach, a proce- dure for manufacturing trisaccharide containing treha¬ lose, from glucose and trehalose with R. niveus gluco- amylase, has been disclosed.
As has been reported in the Japanese Patents No. 72 20,373, 75 63,189 and 75 88,290 and in a paper by Hans Bender (1977), Arch. Microbiol., Ill, 271-282, the enzyme used in the present work, or CGTase, is pro¬ duced both by bacteria of genus Bacillus, such as Bacillus macerans. Bacillus megaterium. Bacillus circulans. Bacillus polymyxa and Bacillus stearother- mophilus, and by certain bacteria of genus Klebsiella.
The manufacturing of oligosaccharides with the
CGTase employed in the invention has been described in the German Patent No. 2 162,276 and in the British
Patent No. 2,019,406, in both of which the enzyme is made to act on starch or dextrin in the presence of either saccharose or fructose, whereby as product are formed oligosaccharides of 'coupling sugar* type. In the Japanese Patent No. 75 123,832 and in a paper by S. Kitahata and S. Okada (1976), J. Biochem., 79./ 641-648, is disclosed the manufacturing from dextrin or starch, with CGTase, of oligosaccharides containing xylose and sorbose. Determination of the acceptor specificity of CGTase from various acceptor sugars and from cyclodex- trin is described in scientific magazines (D. French et al. (1948), J. Am. Chem. Soc, 7fJ/ 3145; D. French et al. (1954), J. Am. Chem. Soc, 7_6. 2387-2390; S. Kita- hata (1982), Kagatu to Kogyo, 56_, 127-130). French et al. (1948 and 1954) used in their experiments cyclo- dextrin for substrate and e.g. cellobiose for acceptor. In the procedure constituting the object of the present invention, differing from the procedure cited in the foregoing, starch is used for substrate and cellobiose and/or trehalose for acceptor. No such production method has been described heretofore. One obtains by this production method a novel, unique oligosaccharide mixture which is usable as a new raw material e.g. in the foodstuff and animal feed indus¬ tries.
The object of the invention is an oligosaccha¬ ride mixture containing trehalose and cellobiose as acceptor sugar, and a procedure for manufacturing same. In the procedure, cyclomaltodextrin-glucanotransferase (CGTase; E.C. 2.4.1.19) is made to act on starch in the presence of acceptor sugar (trehalose or cellobiose), whereby oligosaccharides are formed which in their sugar composition contain trehalose and cellobiose. For the reason mentioned above, we have arrived at the in¬ vention as disclosed in the application, which is char¬ acterized by the features stated in the claims.
The basic idea of the invention consists in that CGTase has been found to produce oligosaccharides from a suitable acceptor sugar and starch, in suitable conditions. It is possible in the procedure of the in¬ vention, by varying the conditions of reaction, to achieve oligosaccharide mixtures of novel type in which the oligosaccharides have DP numbers starting with 3 and up to 7 at least. The reaction model of the proce¬ dure for producing an oligosaccharide mixture, on which the invention reads, can be assumed to be as follows. when cellobiose (β-glu-(l-4)-β-glu) or trehalose (α- glu-(l-l)-σ-glu) is used for acceptor.
starch + acceptor acceptor glu-acceptor (0SG3) glu-glu-acceptor (OSG4) glu-glu-glu-acceptor (OSG5) etc. where acceptor = trehalose or cellobiose (di- saccharide)
It was found in C-nmr structural analysis that the trehalose-containing oligosaccharide 0SG3 has the fol¬ lowing structure: 0-α-D-glu-(l-4)-0-α-D-glu-(l-l)-α-D- glu, that is, a glucoside molecule is attached to tre¬ halose with an α-(l-4)-glycosidic bond. The oligosac¬ charides with DP higher than 3 are hard to analyse by nmr technique, owing to their complex structure. It has, however, been found in enzymatic structural analy- sis that the glucose molecule(s) are linked with the acceptor sugar in conformity with the above model. Oligosaccharides according to the invention can be used e.g. in the foodstuff industry as new raw materials, because α-amylase breaks up very slowly, or not at all, oligosaccharides with DP between 3 and 5 (OSG3-OSG5) (e.g. in: Starch, Chemistry and Technology (1984) edited by R.L. Whistler, J.N. BeMiller and E.F. Paschall, Academic Press Inc., London, England, p. 93- 102) . The oligosaccharide mixture obtained as product may furthermore contain the acceptor sugars trehalose or cellobiose, which are also used as single sugars for raw materials of the foodstuff industry. The proportion in the product mixture of oligosaccharides having DP 3- 5 can be modified by means of dry matter and enzyme concentration, relative mass proportion of starch and acceptor sugar, and reaction time.
The compositions of the product mixtures were determined by liquid chromatography. The method enabled the concentrations of oligosaccharides with DP less than 8 to be determined in that the concentration of any given oligosaccharide was calculated in accordance with the concentration of whichever standard (DP from 1 to 7) had the most closely equal retention time. Con¬ firmation of the results was made qualitatively with TLC.
The CGTase required in the present invention is produced by cultivating a microorganism producing the respective enzyme, e.g. certain bacteria of genus Bacillus, in a culture solution containing a carbon and nitrogen source, minerals and vitamins (M. Makela, Bio- technical production of cyclodextrins (1990), Depart- ment of Biochemistry, University of Turku, Finland) . The CGTase thereby formed is recovered using procedures of prior art, such as centrifuging the culture solu¬ tion, or filtering. The crude enzyme saved in this manner may be purified and concentrated e.g. by salting out, gel filtration and/or by ion exchange chromatog¬ raphy or affinity chromatography.
In the procedure starch materials of various origins can be used, e.g. those derived from cereals such as barley starch, or from root crops, such as potato starch. The starch may also be pretreated e.g. by acid hydrolysis and/or enzymatically so that the dextrose equivalent of the liquefied starch thus formed is in the range from 0.5 to 20. For enzymatic hydroly¬ sis of starch one may use e.g. α-amylase or it is also possible to add the production enzyme, or CGTase, di¬ rectly to the starch, in which case pretreatment is unnecessary. The advantageous DE number of the starch is about 1.
The total concentration of starch reacting with CGTase and acceptor sugar should be within 5-60%.
Advantageous concentration is 30-40% for production of cellobiose saccharides, and 40-50% for production of trehalose saccharides, whereby the relatively greatest amount of short-chain oligosaccharides will be formed in the mixture. Particularly in production of trehalose oligosaccharides the concentration should be high. Sub- strate and acceptor may be dissolved either in water or in buffer, e.g. in 50 mM i idazole buffer pH 6.8, in 50 mM acetate buffer pH 5.5 or in 50 mM glycine-NaOH buffer pH 9.0. Favourable pH range for the reaction is 6.5-7.0. The reaction may be carried out at 50-80°C, with reaction time 2 days at the most. Favourable tem¬ perature for the reaction is 60°C and reaction time, 20 to 48 hrs, depending on the CGTase concentration. The relative mass proportion of starch and acceptor sugar should be within 0.5-4 in the solution. Favourable pro- portion of starch and acceptor is 1:1, whereby OSG3- OSG5 oligosaccharides will be formed in the mixture most of all. When the starch quantity exceeds that of acceptor, long-chain oligosaccharides are formed most, in proportion. Similarly, when there is more acceptor than starch in the solution, short-chain oligosacchar¬ ides, among others the OSG3 oligosaccharide, are formed relatively most. The CGTase concentration in the reac¬ tion should be within 30-350 U per g of starch. The favourable CGTase concentration is affected, among others, by concentration in that the higher the concen¬ tration of the solution the higher should the enzyme concentration be in the reaction. High enzyme concen¬ tration also shortens the reaction time.
It is thus understood that it is possible in the procedure of the invention, by appropriately modi¬ fying conditions of reaction, to produce in controlled manner, and with good yield, oligosaccharide mixtures containing cellobiose and trehalose, of a novel type. EXAMPLE 1
Manufacturing cellobiose oligosaccharides at various concentrations
Oligosaccharide-production enzyme, or CGTase, (isolated from a Bacillus circulans strain, activity 7600 U/ml, Oy Alko Ab) was added 30 U/g to a mixture containing starch as stated in Table 1, in 50 mM imid- azole buffer pH 6.8, to which had been added 1.5 mM CaCl2. The CGTase was allowed to act at 85°C for 30 min. with simultaneous agitation, whereafter in the starch (DE 1) was dissolved cellobiose (Sigma, U.S.A.) as stated in Table 1, and the solution was tempered to 60°C reaction temperature.
The reaction was started by adding to the so- lution 50 U CGTase per g of starch, and the reaction was allowed to proceed 48 hrs at 60°C, agitating at the same time. The compositions of the products were deter¬ mined at room temperature by liquid chromatography (Zsadon B., Otta K.H., Tudos F. and Szejtli J. (1979), J. Chromatogr., 172, 490-492). The elution rate in car¬ bohydrate column was 0.9 ml/min and the standards (e.g. glucose, maltose, ..., naltoheptaose, cellobiose, tre¬ halose, cyclodextrin) had concentration from 1 to 5 mg/ml. The concentrations of OSG3-OSG7 oligosaccha¬ rides in the products after 48 hrs reaction time are stated in Table 1. The total quantity of oligosaccha¬ rides OSG3-OSG5, at 10% dry matter content, was 4.8 g/100 g (48.0% of the initial dry matter content) and at 30% dry matter content, 14.6 g/100 g (48.7% of initial dry matter content) . In Fig. 1 is presented the yield of OSG3-OSG5 oligosaccharides and the cellobiose consumption (g/100 g) plotted over time (48 hrs) at 30% dry matter content. TABLE I. The effect of substrate solution concentra¬ tion (g/100 g) on composition of the oligosaccharide mixture produced (Example 1). Starch (DE 1) was reacted with CGTase (50 U/g) for 48 hrs in presence of cello¬ biose (mass proportion of starch and acceptor in the substrate, 1:1). The concentrations of oligosaccharides (with DP less than 8) were determined by liquid chroma¬ tography (see Example 1) .
Dry matter content in solution (%) 10% 30% 10% 30%
Concentration (g/100 g)
Product Prior to reaction After reaction
Starch 5.0 15.0
Glucose
Cellobiose 5.0 15.0
OSG3
OSG4
0SG5
OSG6
OSG7
Figure imgf000010_0001
EXAMPLE 2
Manufacturing trehalose oligosaccharides at various concentrations Oligosaccharide producing enzyme, or CGTase
(see Example 1) was added 30 U/g to a mixture contain¬ ing starch, as stated in Table 2, in buffer as in Example 1. The CGTase was allowed to act for 30 min, at 85°C, with agitation, whereafter in the starch (DE 1) was dissolved α,α-trehalose (Sigma, U.S.A.) as stated in Table 2, and the solution was tempered to the reac¬ tion temperature, 60°C.
The reaction was started by adding CGTase 150 U per g of starch, and the reaction was allowed to proceed 48 hrs at 60°C under agitation. The composi¬ tions of the products were determined by liquid chroma- tography (see Example 1) .
The concentrations of OSG3-OSG7 oligosaccha¬ rides in the products after 48 hrs reaction time are stated in Table 2. The total quantity of oligosaccha- rides OSG3-OSG5, at 32% dry matter content, was 12.1 g/100 g (37.8% of the initial dry matter content) and at 50% dry matter content, 20.0 g/100 g (40.0% of initial dry matter content) .
TABLE II. The effect of substrate solution concentra¬ tion (g/100 g) on composition of the oligosaccharide mixture produced (Example 2). Starch (DE 1) was reacted with CGTase (150 U/g) for 48 hrs in presence of treha¬ lose (mass proportion of starch and acceptor in the substrate, 1:1). The concentrations of oligosaccharides (with DP less than 8) were determined by liquid chroma¬ tography (see Example 1).
Dry matter content in solution (%) 32% 50% 32% 50%
Concentration (g/100 g)
Product Prior to reaction After reaction
Starch 16.0 25.0
Glucose 0.2 0.1
Trehalose 16.0 25.0 8.5 11.5
0SG3 4 5 9.1
0SG4 4 8 6.6
OSG5 3, 5 4.3
0SG6 2, 6 2.7
OSG7 2, 4 1.3
EXAMPLE 3
Effect of starch/acceptor mass proportion in oligo¬ saccharide production
CGTase (see Example 1) was added 30 U/g to a mixture containing starch in the mass proportion stated in Table 3, so that the ultimate concentration after acceptor addition in the solution would be 30 g/100 g. The starch had been dissolved in imidazole buffer as in Example 1. The CGTase was allowed to act for 30 min, at 85°C, with agitation, whereafter in the starch (DE 1) was dissolved either trehalose or cellobiose as stated in Table 3 (ultimate concentration 30 g/100 g) , and the solution was tempered to 60°C reaction temperature. The reaction was started by adding CGTase 50 U per g of starch, and the reaction was allowed to pro- ceed 48 hrs at 60°C under agitation. The compositions of the products were determined by liquid chromatogra¬ phy (see Example 1).
The concentrations of OSG3-OSG7 oligosaccha¬ rides in the products after 48 hrs reaction time are stated in Table 3. The total quantities of oligosaccha¬ rides OSG3-OSG5 were, with the cellobiose saccharides, 13.1 g/100 g (mass proportion 1:4) and 14.4 g/100 g (1:2), and with trehalose saccharides 6.9 g/100 g in either case (mass proportions 1:2.3 and 1:1.2). The proportion of the OSG3 oligosaccharide was highest when the acceptor concentration in the solution was high.
TABLE III. The effect of starch/acceptor mass propor¬ tion on composition of the oligosaccharide mixture pro¬ duced (Example 3). Starch (DE 1) was reacted with CGTase (50 U/g) for 48 hrs in presence of cellobiose or trehalose at 30% concentration. The concentrations of oligosaccharides (with DP less than 8) were determined by liquid chromatography (see Example 1).
Figure imgf000013_0001
Trehalose oligosaccharides were produced in the manner described in Example 2, in the reaction being used 50 g starch (DE 1) and 50 g trehalose dis¬ solved in 200 g imidazole buffer (dry matter content 50%). The reaction was started by adding CGTase (see Example 1) 150 and 210 U per g of starch, and the reac- tion temperature was 60°C.
The composition of the product was determined by liquid chromatogrraphy (see Example 1). In Fig. 2 the yield of oligosaccharides and the consumption of treha¬ lose (g/100 g) are plotted over time (48 hrs, CGTase concentration 210 U/g) . Fig. 3 displays the elution chro atogram from liquid chromatography of the oligo- saccharide mixture after 48 hrs reaction time. This chromatogram represents a typical oligosaccharide mix¬ ture, its numbered peaks being: l=glucose, 2=trehalose, 3=OSG3, 4=OSG4, 5=OSG5, 6=OSG6, and 7=OSG7. The concentrations of OSG3-OSG5 oligosaccha¬ rides after 48 hrs reaction time are presented in Table 4. The total of OSG3-OSG5 oligosaccharides in the pro¬ duct was measured to be 20.0 g/100 g (40.0% of the dry matter content, CGTase 150 U/g) and 22.7 g/100 g (45.4% of the dry matter content, CGTase 210 U/g).
TABLE IV. The effect of enzyme concentration on composition of the oligosaccharide mixture produced (Example 3). Starch (DE 1) was reacted with CGTase for 48 hrs in presence of trehalose at (mass proportion of starch and acceptor in the substrate 1:1, dry matter content 30%). The concentrations of oligosaccharides (with DP less than 8) were determined by liquid chroma¬ tography (see Example 1).
Enzyme concentration (U/g) 150 210
Product Concentration (g/100 g)
Glucose Trehalose OSG3 OSG4 OSG5 OSG6 OSG7
Figure imgf000014_0001
EXAMPLE 5
Hydrolyzability with amylase of cellobiose oligo¬ saccharides
The production solution of Example 1, with 30% dry matter content, was diluted with water to 4.72% dry matter content and centrifuged, to remove the dry mat¬ ter. To the solution was added 135.5 U/ml α-amylase (isolated from a Bacillus subtilis strain, activity 1355 U/mg, Sigma, U.S.A.) and the reaction was allowed to proceed 24 hrs at 60°C, with agitation. The composi¬ tion of the product was determined by liquid chromatog- raphy (see Example 1), and measurement showed the total content of OSG3-OSG5 oligosaccharides in the product to be 14.2 g/100 g (OSG3: 7.3 OSG4: 4.2 g/100 g, OSG5: 2.7 g/100 g). α-amylase broke off 16% oligosaccharide and 25% OSG5 oligosaccharide from the product of Example 1, while on the other hand the content of OSG3 oligosac¬ charide increased 22%.
EXAMPLE 6 Manufacturing of trehalose oligosaccharides and their hydrolyzability with amylase
Trehalose oligosaccharides were produced in the manner described in Example 2, in the reaction being used 15 g starch (DE 1) and 13.6 g trehalose dis- solved in 200 g imidazole buffer (dry matter content 28.6%). The reaction was started by adding CGTase (see Example 1) 50 U per g of starch, and the reaction tem¬ perature was 60°C. The reaction was allowed to proceed for 48 hrs, whereafter the composition of the product was determined by liquid chromatography (see Exam¬ ple 1). Measurement showed the total quantity of OSG3- OSG5 oligosaccharides to be 6.6 g/100 g (OSG3: 1.9 g/100 g, OSG4: 2.3 g/100 g, OSG5: 2.4 g/100 g, total 23.1% of the dry matter content). The production solution was diluted with water to 6% dry matter content and centrifuged, to remove the dry matter. To the solution was added 271 U/ml α-amy¬ lase (see Example 5) and the reaction was allowed to proceed 24 hrs at 60°C, with agitation. The composition of the product was determined by liquid chromatography (see Example 1), and measurement showed the total quan¬ tity of OSG3-OSG5 oligosaccharides in the product to be 10.7 g/100 g (OSG3: 5.8 OSG4: 3.3 g/100 g, OSG5: 1.6 g/100 g) . α-amylase broke off 33% 0SG5 oligosaccharide from the product, while on the other hand the content of OSG3 oligosaccharide was tripled.
EXAMPLE 7
Use of starches with various DE numbers in manufacturing oligosaccharides
Trehalose oligosaccharides were produced in the manner described in Example 2, but the starch was first hydrolyzed with α-amylase (BAN 120 L, activity 120 KNU/g, Novo, Denmark). Amylase was added 0.3 and 0.9 KNU/g to a mixture containing 16 g starch in imid¬ azole buffer (see Example 2). The amylase was allowed to act 30 min. at 85°C, with stirring, whereafter in the starch (DE 5 and DE 16) was dissolved 16 g treha¬ lose (dry matter content of solution 32%) and the solu¬ tion was tempered to reaction temperature, 60°C.
The reaction was started by adding to the solution CGTase (see Example 1) 120 U per g of starch, and the reaction was allowed to proceed 48 hrs at 60°C, with stirring. The composition of the products was de¬ termined by liquid chromatography (see Example 1) and by TLC on silicagel plates (running solution: acetone- 1-butanol-water 11:9:5; staining solution: aniline 2 ml, diphenylamine 2 g, acetone 100 ml, and 80% phos¬ phoric acid 15 ml; after plate staining, 30 min. heat¬ ing at 105°C) .
The total quantity of OSG3-OSG5 oligosaccha- rides in the product was found, in either case, to be 11.5 which was 35.9% of the dry matter content. The product compositions were also analysed by TLC, which differentiates the produced oligosaccharides from the hydrolysis products formed in the pretreatment of the starch, such as maltose, maltotriose, maltotetraose, etc. According to TLC the product solutions contained very small quantities of hydrolysis products formed in the pretreatmen ; their concentrations varied within 0.1 to 2.5 g/100 g, depending on the DE number of the solution.
Using starch with DE 5, the oligosaccharide contents of the product were found by measurement to be: OSG3: 4.4 g/100 g, 0SG4: 3.9 g/100 g, 0SG5: 3.2 g/100 g, and with starch having DE 16: 0SG3: 4.6 g/100 g, OSG4: 4.0 g/100 g, OSG5: 2.9 g/100 g.
EXAMPLE 8
Use of various CGTase preparations and buffers in manufacturing trehalose oligosaccharides
CGTase (see Example 1) was added 30 U/g to a mixture containing 2 g starch in water or in 50 mM ace- tate, imidazole or glycine-NaOH buffer, to which had been added 1.5 mM CaCl2. The buffers had pH 5.5, 6.8 and 9.0, respectively. CGTase was allowed to act 30 min, at 85°C, with stirring, whereafter in the starch (DE 1) was dissolved 3 g trehalose (dry matter content of solution 40%) and the solution was tempered to reaction temperature 60°C.
To the reaction mixtures was added, for pro¬ duction enzyme, either purified CGTase (see Example 1, activity 7600 or 6150 U/ml) or equivalent crude prepa- ration (activity 4300 U/ml), 300 U per g of starch. The reaction was allowed to proceed 24 hrs at 60°C, with stirring. The compositions of the products were deter¬ mined by liquid chromatography (see Example 1) and are presented in Table V. TABLE V. Preparation of trehalose oligosaccharides using various CGTase preparations and buffers at 40% dry matter content. Starch (DE 1) was allowed to react with CGTase (300 U/g) during 24 hrs in the presence of trehalose (starch/trehalose mass proportion 1:1). The oligosaccharide concentrations were determined by liquid chromatography (see Example 1) . The yields of oligosaccharides OSG3-OSG5 have been calculated rela¬ tive to dry matter content.
Activities of CGTase preparations: A=7600 U/ml, B=6150 U/ml, and C=4300 U/ml. Test 1. Acetate buffer pH 5.5, CGTase A. Test 2. Acetate buffer pH 5.5, CGTase C. Test 3. Glycine-NaOH buffer pH 9.9, CGTase C, Test 4. Water pH 7.0, CGTase B.
Figure imgf000018_0001
OSG3-OSG5 yield (%)
45.8 47.5 47.8 45.8 39.8
EXAMPLE 9 Production of trehalose oligosaccharides with immobilized CGTase
CGTase was bonded with covalent bonds to Eupergit C beads (Rohm Pharma, Federal Republic of Germany) in that to 0.5 g of the beads (dry matter mass) was added 2 ml CGTase (isolated from Bacillus circulans strain, activity 265 U/ml, Oy Alko Ab) . The enzyme was left to be bonded for 20 hours at room tem¬ perature, shaking at the same time, whereafter the beads were washed with imidazole buffer according to Example 2.
In order to prepare the substrate for the oligosaccharide producing reaction, soluble CGTase was added 30 U/g to a mixture containing 0.5 g starch and 4 g buffer as in Example 1. The CGTase was allowed to act 30 min. at 85°C, with stirring, whereafter in the starch (DE 1) was dissolved 0.5 g trehalose (dry matter content of solution 20%), and the solution was tempered to reaction temperature 60°C.
The reaction was started by adding to the solution immobilized CGTase 0.5 g (dry matter mass, activity 424 U/g) , and the reaction was allowed to pro¬ ceed 21 hrs at 60°C, shaking at the same time. The com- position of the product was determined by liquid chro¬ matography (see Example 2) . Measurements gave for the total OSG3-OSG5 oligosaccharide quantity in the pro¬ duct: 7.5 g/100 g (OSG3: 3.2 OSG4 2.4 g/100 g, and OSG5: 1.9 g/100 g) , which is 37.5% of the dry matter content.

Claims

1. A procedure for manufacturing an oligosac¬ charide mixture, c h a r a c t e r i z e d in that cyclomaltodextrin-glucanotransferase (CGTase; E.C. 2.4.1.19) is made to act on starch in the presence of acceptor sugars trehalose and/or cellobiose.
2. Procedure according to claim 1, c h a r¬ a c t e r i z e d in that CGTase is made to act on starch which has been pre-treated.
3. Procedure according to claim 1, c h a r¬ a c t e r i z e d in that the CGTase is immobilized CGTase.
4. Procedure according to claim 1, c h a r- a c t e r i z e d in that the dextrose equivalent of the starch is within the range 0.5-20.
5. Procedure according to claim 1, c h a r¬ a c t e r i z e d in that the dry matter content of the starch and acceptor sugar is within 5-60%, and that the starch is dissolved either in water or in a buffer having pH in the range 5.5-9.0.
6. Procedure according to claim 1, c h a r¬ a c t e r i z e d in that the reaction is carried out at a temperature between 50 and 80°C, that the mass proportion of starch and acceptor in the solution is in the range 0.5-4, and that the CGTase concentration is in the range 30-350 U per g of starch.
7. A starch-based oligosaccharide mixture manufactured by a procedure according to any one of claims 1-6, c h a r a c t e r i z e d in that it contains glucose 0-5.0%, acceptor sugar 7.0-75%, OSG3 1.8-32%, OSG4 3.0-20%, OSG5 2.5-16%, 0SG6 0.8-12% and 0SG7 0.2-10%, calculated on the dry matter of the solu¬ tion, the acceptor sugar being trehalose and/or cello- biose and the oligosaccharides containing trehalose and/or cellobiose.
8. A starch-based oligosaccharide mixture according to claim 7, c h a r a c t e r i z e d in that it contains glucose 0-1.0 g/100 g, cellobiose 0.7- 35 g/100 g, 0SG3 0.9-15 g/100 g, 0SG4 0,8-9.0 g/100 g, OSG5 0.8-6.0 g/100 g, OSG6 0.2-5.0 g/100 g and 0.05- 3.0 g/100 g, the oligosaccharides containing cellobio¬ se.
9. A starch-based oligosaccharide mixture according to claim 7, c h a r a c t e r i z e d in that it contains glucose 0-2.0 g/100 g, trehalose 7.0- 35 g/100 g, OSG3 0.4-14 g/100 g, OSG4 0,8-10.0 g/100 g, 0SG5 0.8-7.0 g/100 g, OSG6 0.8-6.0 g/100 g and 0.5- 5.0 g/100 g, the oligosaccharides containing trehalose.
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