WO1991009963A1

WO1991009963A1 - Procedure for producing maltodextrins and starch and maltose syrups containing cyclodextrins

Info

Publication number: WO1991009963A1
Application number: PCT/FI1990/000006
Authority: WO
Inventors: Pekka T. Mattson; Mauri J. MÄKELÄ; Timo K. Korpela
Original assignee: Oy Alko Ab
Priority date: 1988-09-19
Filing date: 1990-01-04
Publication date: 1991-07-11
Also published as: FI81116B; FI884287A0; FI884287A; FI81116C

Abstract

A procedure for producing maltodextrins and starch and maltose syrups containing cyclodextrins and to be used primarily in the foodstuff, sweets and beverage industries, in which pre-processed starch, dissolved either in water or buffer or an aqueous ethanol solution of these is reacted with immobilized cyclomaltodextrin-glucanotransferase (E.C. 2.4.1.19).

Description

PROCEDURE FOR PRODUCING MALTODEXTRINS AND STARCH AND MALTOSE SYRUPS CONTAINING CYCLODEXTRINS

The present invention concerns a procedure for producing altodextrins and starch and maltose syrups containing cyclodextrins (CD's). In the manufacturing procedure of the invention, immobilized cyclomaltodex- trin-glucanotransferase (CGTase; E.C. 2.4.1.19) is made to act on starch that has been pre-processed (e.g. by acid hydrolysis or split with enzymes) , possibly in the presence of ethanol, whereby maltodextrin, starch or maltose syrup, with unique sugar composition and con¬ taining CD's is formed.

CD's are sugars with annular molecular struc- ture composed of a plurality of glucose units. As pre¬ sented in the Japanese Patents 72 20,373, 75 63.189 and 75 88,290 and in: Hans Bender (1977) Arch. Microbiol., Ill, 271-282, CGTase is produce both by bacteria of genus Bacillus, such as Bacillus macerans. Bacillus megaterium, Bacillus circulans, Bacillus polvmvxa and Bacillus stearothermophilus, and by bacteria of genus Klebsiella, such as Klebsiella pneumoniae. When the enzyme acts on starch, CD's with various ring sizes are produced, which are referred to as alpha, beta and ga - a forms according to their content of six, seven or eight glucose units, respectively.

The significance of CD's is based on their ability to form so-called inclusion complexes with low- molecular compounds. In this way the low-molecular co - pounds become "packaged" and the sugar and are thus protected e,g, against undesired chemical reactions. Owing to this particular property of CD's their appli¬ cations are spreading into new fields, encompassing e.g. the foodstuff, drug, dye, sanitation, paper and agroσhemical industries. Among these, the foodstuff industry can pe considered one of the most significant partial domains: CD's are suitable in protecting fla- vours or vitamins against the effects of heat and light, inhibiting evaporation, eliminating harmful compounds from the products (objectionable taste or smell) , or reducing the hygroscopiσity of the product. CD's are approvedly usable in Japan, and the legisla¬ tion is developing in the direction that they are like¬ ly to become allowed in other countries as well (Cyclo- dextrin News (1986), ___- (2), 2).

In connection with manufacturing CD's sugar mixtures containing CD's are formed which present a sweetness and sugar composition different from those in the procedure of the invention. Their preparation has been presented in: J. Szejtli (Ed.), Proceedings of the First International Symposium on Cyclodextrins (1982) , Akademiai Kiadό, Budapest, 25-40. The procedure is based on splitting starch with soluble CGTase. After crystallizing the beta-CD, a sugar mixture is obtained which in the first place contains glucose, maltose and CD's. The drawback of this procedure is that the en- zymes have to be removed from the product with the aid of active carbon treatment and ion exchangers and the process is therefore rather cumbersome and uneconomi¬ cal.

The use of immobilized CGTase in producing CD^rs is described in patents (Japanese Patents No. 71 09,223 and 80 124,494, and German Patent No. 3,330,571) as well as scientific papers (Naka ura N. and Horikoshi K. (1977), Biotechnol. Bioeng. , 19, 87- 99; Ivony K. , Szajani B. and Seres G., (1983), J. Appl. Biochem., _5_ 158-164; Kato T. and Horikoshi K. (1983), Biotechnol. Bioeng., 26, 595-598). Owing to technical difficulties, such as viscosity and diffusion obsta¬ cles, comparatively low starch concentrations (2 to 4%) have mostly been applied, and the viscosity has been lowered in some cases by pre-hydrolysis to a low DE number (S.P. Crump and J.D. Rozzell, Paper read at the 4th International Symposium on Cyclodextrins, April 20- 24 1988, Mϋnchen, German Federal Republic) . The use of immobilized CGTase in the way of the procedure consti¬ tuting the object of the present invention, i.e., using as starting material in the reaction starch syrups hav- ing a high DE number, has not been heretofore describ¬ ed. High DE number syrups containing CD's are obtained as a result of the treatment.

The reasons mentioned above have led to the invention of the present application, which is mainly characterized by the features stated in the main claim. The fundamental idea of the invention consists of the observation that immobilized CGTase produces from pre-processed starch (maltodextrins, starch and maltose syrups with DE number 5 to 60) under suitable conditions, sugar solutions containing CD's. The proce¬ dure of the invention offers a possibility to run a continuous process, and it can be adjoined as a further treatment step to sugar syrup manufacturing processes used heretofore. Savings are achieved with the aid of the procedure in enzyme consumption cost, and it be¬ comes possible to increase the quantity of CD's in the product. It is also an advantage of the procedure that the enzyme protein does not remain in the product; no extra purifying steps are therefore required. The com- position of the product that is being formed can be controlled by appropriate control of various parame¬ ters, such as temperature, degree of immobilization, concentration of the substrate solution and of the ethanol therein, DE number, and flow rate. In addition, novel products are obtained which cannot be produced by afterwards adding CD's to starch or maltose syrups be¬ cause CGTase at the same time changes the composition of pre-processed starch typically so that the propor¬ tion of polysaccharides larger than DP 10 goes down and, on the other hand, the differences in concentra¬ tion between sugars under DP 10 are levelled out. The effects on viscosity of the product and on the quantity of reducing sugars are minor. In Table I is shown the composition of maltodextrin (DE 20) and of starch syrup (DE 30) in native state and after reaction with immo¬ bilized CGTase. The compositions of the preparations were determined by liquid chromatography. The method enables concentrations of sugars and CD's with DP less than 8 to be determined. The quantities forming in the product (1 to 10%) are on the same order of magnitude as those normally used in connection with foodstuffs (in: Cyclodextrins and Their Inclusion Complexes (1982), J. Szejtli (Ed.), Akade iai Kiadό, Budapest, p. 236-255) .

The CGTase needed in the invention is produced by cultivating the respective enzyme-forming micro- organism, e.g. certain bacteria of genus Bacillus or Klebsiella in a culture solution containing a carbon and nitrogen source, minerals and vitamins. The CGTase which is formed is recovered using methods known in the art, such as centrifuging or filtering the culture sol- ution. The crude enzyme thus recovered may be purified and concentrated e.g by salting out, dialysis, adsorp¬ tion and desorption on starch, gel filtering and/or by ion exchange chromatography or affinity chromatography. The crude enzyme, or preferably the purified and concentrated CGTase, can be immobilized using ex¬ isting methods, e.g. by binding the enzyme to a solid carrier, e.g. by covalency, adsorption or ion exchange forces. Various organic macromolecules occurring in nature or prepared synthetically are appropriate for solid carriers, e.g. cellulose, agarose, polyacryl- amide, polyethylene glycol and polystyrene, or organic compounds, e.g. diatomite and glass. Immobilization can be carried out in conditions in which the effective activity of the CGTase is preserved after the reaction, e.g. within the pH range from 4 to 10 and in the tem¬ perature range <90°C. In the procedure it is possible to use starch materials of different origins, e.g. those derived from cereals, such as barley and wheat starch, or those derived from root crops, such as potato starch. The starch is pre- processed e.g. by acid hydrolysis and/or enzymatically so that the dextrose equivalent of the liquefied starch is within 5-60.

The concentration of the pre-processed starch reacting with immobilized CGTase should be in the range from 5 to 40%. The substrate may be dissolved either in water or in a buffer, e.g. 100 mM imidazole buffer pH

6.8, to which 5 mM CaCl_> have been added.

When the substrate is reacted with CGTase ad¬ sorbed on ion exchangers, buffers should not be used as a rule. The reaction may be carried out at 20 to 70⁵C. Adding to the substrate solution ethanol until its concentration is 50% by vol. at the most causes the quantity of CD's in the product to increase substan¬ tially. The equilibrium between the different CD forms changes at the same time. When the ethanol quantity is large enough, the product that is formed will be ste¬ rile and therefore less sensitive to icrobial contami¬ nation. Starch and maltose syrups containing CD's pro¬ duced in the presence of ethanol can be directly added to some products, e.g. to liqueurs. By the procedure of the invention a novel maltodextrin, starch or maltose syrup is obtained which advantageously contains cyclo¬ dextrins 2.0 to 16% and short malto-oligosugars 30 to 60%.

The compositions according to the invention are more closely described in Tables II to VI, follow¬ ing below. TABLE I: Composition of maltodextrin (DE 20) and starch syrup (DE 30) in native state and after reaction with immobilized CGTase (*) , determined by liquid chromatography. MD 20* represents the composition of a sugar solution prepared as in Example 4, and TS 30* as in Example 5, when the substrate solution contains no ethanol.

TABLE II: Compositions of maltodextrin (DE 20; 100 g/1) and starch syrup (DE 30; 100 g/1) in native state (1) and after reaction with CGTase immobilized on Sepharose 4B gel without ethanol (2) , and when ethanol 15% by wt. had been added to the reaction mix. The con¬ tents of sugars (under DP 8) were determined by liquid chromatography (see Examples 4 and 5) .

TABLE III: Compositions of maltodextrin (DE 20; 100 g/1) and starch syrup (DE 30; 100 g/1) in native state (1) and after reaction with CGTase immobilized on Ser- dolit AW 14 gel without ethanol (2), and when ethanol 15% by wt. had been added to the reaction mix. The con¬ tents of sugars (under DP 8) were determined by liquid chromatography (see Example 7) .

TABLE IV: Effect of maltodextrin (DE 20) concentration on the composition of the cyclodextrin product that is being formed. The substrate solution was reacted with CGTase immobilized on Sepharose 4B gel (Example 2) . Flow rate 60 ml/h, 25^βC, diameter/height ratio of reac¬ tion column 1:6.5. The contents of sugars (under DP 8) were determined by liquid chromatography.

TABLE V: Composition of cyclodextrin syrup when maltodextrin (DE 20; 50 g/1) was reacted with CGTase immobilized on Sepharose 4B gel (Example 2) within tem¬ perature range 25 to 70°C. Diameter/height ratio of reaction column 1:6.5, flow rate 60 ml/h. The contents of sugars (under DP 8) were determined by liquid chromatography.

TABLE VI: Effect of ethanol concentration on composi¬ tion of cyclodextrin syrup formed from maltodextrin (MD 20; 100 g/1) . The substrate solution was reacted with CGTase immobilized on Sepharose 4B gel (Example 2) . Flow rate 50 ml/h, 25°C, diameter/height ratio of reac¬ tion column 1:6.5. The contents of sugars were deter¬ mined by liquid chromatography.

The starch and maltose syrup solutions con¬ taining CD's can be concentrated or dried using methods known in the art. The syrup or powder thus obtained may be used in various products of the foodstuff, sweets and beverage industry.

Example 1

A Horikoshi II medium (Horikoshi K. (1971) , Agric. Biol. Chem. , _35_ 1783-1781) was inoculated with a Bacillus circulans var. alkalophilus strain (ATCC 21783) , which was cultivated at 37°C, with shaking and aeration for 3 days. The growth solution was centri- fuged and the activity of the supernatant was found by measurement to be 28 U/ml by the maltotriose-methyl orange method (Makela M. . and Korpela T.K. (1988) , J. Biochem. Biophys. Methods, 15, 307-318) .

The CGTase was purified ny affinity chromato¬ graphy (Hungarian Patent No. 175,584) and the enzyme solution was concentrated with an A icon apparatus (membrane film PM 10) . Measurement showed the activity of the CGTase concentrate to be 19400 U/ml, the spe¬ cific activity being 1205 U/mg.

Example 2 4 g CNBr-activated Sepharose 4B (Pharmacia

Fine Chemicals, Uppsala, Sweden) gel were washed with 10-³ M HC1 (1000 ml) . The washed carrier was admixed to 0.1 M NaHCOa buffer (18 ml) pH 8.3, containing 0.5 M NaCl. To the suspension were added 2 ml CGTase concen- trate prepared as in Example l (32.2 ml enzyme). The reaction was allowed to proceed 2 hrs at room tempera¬ ture, while shaking the mixture. The immobilized enzyme thus obtained was filtered and washed consecutively three times with a sufficient quantity of 0.1 M acetate buffer pH 4.0, which contained respectively 0.5 M NaCl and of 0.1 M tris-HCl buffer pH 8.0, which contained respectively 0.5 M NaCl. The immobilized CGTase was finally flushed clean with water.

By determining the protein contents of fil¬ trate and washing waters, the quantity of CGTase bound to agarose was found to be 31.8 mg, and therefore the degree of binding was 99 »-_•

Example 3

5 g Serdolit AW-14 anion exchanger (Serva, Heidelberg, German Federal Republic; grain size +.+5- 0.1 mm) were alternatingly washed with 1 M HCl and 1 M NHΛOH, three times. The regenerated ion exchanger was flushed with 200 mM tris-acetate buffer pH 8.3 and ultimately balanced with 10 mM tris-acetate buffer pH 8.3. 5 ml of the balanced adsorbent and 47.5 ml 10 mM tris-acetate buffer pH 8.3 were mixed together and to the suspension were added 2.5 ml CGTase concentrate prepared as in Example 1 (40.2 g enzyme). The reaction mix was transferred to a shaker at room temperature for 24 hrs. After the reaction, the immobilized enzyme was filtered and washed with an adequate quantity of water.

By measuring the protein contents of filtrate and washing solution, the quantity of immobilized

CGTase was found to be 24.7 mg, and therefore the de- gree of binding was 61%.

Example 4

Immobilized CGTase prepared as in Example 2 was packed in a column having diameter/height ratio 1:6.5. Maltodextrin (DE 20; Suomen Sokeri, Jokioinen Works) was dissolved in water (100 g/1) and the solu¬ tion (substrate solution) was eluted at room tempera¬ ture through the column with flow rate 60 ml/h.

The composition of the solution that had passed through the column was determined by liquid chromatography (Zsadon B. , Otta K.H., Tudos F. and Szejtli J. (1979), J, Chromatogr., 172, 490-492) and the total quantity of CD's in the eluate solution was found to be 4.6 g/1 (alpha-CD: 0.8 g/1, beta-CD: 3.1 g/1 and gamma-CD: 0.7 g/1). Using a substrate solution in which maltodextrin (DE 20; 100 g/1) was added to a 15% v/v aqueous ethanol solution, measurement yielded under identical reaction conditions in the eluate solu¬ tion the total CD quantity 10.7 g/1 (alpha-CD: 0.2 g/1, beta-CD: 9.5 g/1 and gamma-CD: 1.0 g/1).

Example 5

Immobilized CGTase prepared as in Example 2 was packed in a column having diameter/height ratio 1:6.5. Aqueous solution of starch syrup (100 g/1; DE 30; Suo en Sokeri, Jokioinen Works) was eluted through the column at room temperature, at flow rate 60 ml/h.

The composition of the eluate solution was determined as in Example 4 and the total quantity of CD's was found to be 2.4 g/1 (alpha-CD: 0.4 g/1, beta- CD: 1.7 g/1 and gamma-CD: 0.3 g/1). Using a substrate solution in which starch syrup (DE 30; 100 g/1) was added to a 15% v/v aqueous ethanol solution, measure¬ ment yielded under identical reaction conditions in the eluate solution the total CD quantity 5.3 g/1 (alpha- CD: 0.1 g/1, beta-CD: 4.9 g/1 and gamma-CD: 0.3 g/1). Example 6

1 ml of CGTase concentrate prepared as in Example 1 (16.1 mg enzyme) was immobilized on 3.2 g CNBr-activated Sepharose 4B gel in equivalent manner as in Example 2. Measurement revealed 99,5% degree of binding of the enzyme.

The immobilized CGTase was packed in a column having diameter/height ratio 1:13. A substrate solution was eluted through the column, consisting of maltodex¬ trin (200 g/1; DE 20; Suomen Sokeri, Jokioinen Works) dissolved in 10% v/v aqueous ethanol solution, at flow rate 10 ml/h at 40^βC.

The composition of the eluate solution was determined as in Example 4 and the total quantity of CD's was found to be 6.6 g/1 (alpha-CD: 0.4 g/1, beta- CD: 5.0 g/1 and gamma-CD: 1.2 g/1). Example 7 Immobilized CGTase prepared as in Example 3 was packed in a column having diameter/height ratio 1:6.5. Aqueous solution of maltodextrin (100 g/1; DE 20; Suomen Sokeri, Jokioinen Works) was eluted through the column at room temperature, at flow rate 10 ml/h. The composition of the eluate solution was determined as in Example 4 and the total quantity of CD's was found to be 2.5 g/1 (alpha-CD: 0.2 g/1, beta- CD: 1.7 g/1 and gamma-CD: 0.6 g/1). Using a substrate solution in which maltodextrin (DE 20; 100 g/1) was dissolved in 15% v/v aqueous ethanol solution, in equivalent reaction conditions the total quantity of CD's was found to be 3.7 g/1 (alpha-CD: 0 g/1, beta-CD: 3.3 g/1 and gamma-CD: 0.4 g/1).

Replacing the substrate solution with an aque- ous solution of starch syrup (100 g/1; DE 30; Suomen Sokeri, Jokioinen works) yielded measurements in equiv¬ alent conditions of reaction indicating total quantity of CD's in the eluate solution 2.2 g/1 (alpha-CD: 0.2 g/1, beta-CD: 1.6 g/1 and gamma-CD: 0.4 g/1). Dissolv- ing starch syrup (DE 30; 100 g/1) in a 15% v/v aqueous ethanol solution and using the solution thus obtained for substrate solution yielded measurements in equival¬ ent conditions of reaction indicating total quantity of CD's in the eluate solution 2.4 g/1 (alpha-CD: 0 g/1, beta-CD: 2.2 g/1 and gamma-CD: 0.2 g/1).

Claims

1. A procedure for producing syrups contain¬ ing cyclodextrins (alpha, beta and/or gamma forms) , characterized in that 5 to 40% by weight of pre-pro¬ cessed starch having a dextrose equivalent in the range from 5 to 60, dissolved either in water or buffer or an ethanol solution of these of 50% by volume at the most is reacted with immobilized σyclomaltodextrin- glucanotransferase (E.C. 2.4.1.19), whereby maltodex¬ trin, starch or maltose syrup is obtained which con¬ tains advantageously cyclodextrins 2.o to 16% and short maltooligosugars 30 to 60%.

2. Procedure according to claim 1, character- ized in that by dissolving the starting material either in water or in a buffer maltodextrin, starch or maltose syrup is obtained which contains advantageously glucose

4.1 to 8.2%, maltose 5.6 to 10,4%, maltotriose 5.7 to 10.0%, maltotetraose 5.3 to 9.2%, maltopentaose 4.9 to 9.4%, maltohexaose 3.7 to 7.2%, maltoheptaose 2.9 to 4.5%, alpha-cyclodextrin 0.4 to 2.0%, beta-cyclodextrin 1.6 to 6.6% and gamma-cyclodextrin 0.3 to 1.4%.

3. Procedure according to claim 1, character¬ ized in that by dissolving the starting material in an ethanol solution of 50% by volume at the most either of water or of a buffer maltodextrin, starch or maltose syrup is obtained which contains advantageously glucose

4.2 to 6.9%, maltose 5.8 to 10.0%, maltotriose 5.6 to 9.4%, maltotetraose 5.1 to 7.8%, maltopentaose 4.5 to 8.1%, maltohexaose 2.8 to 4.6%, maltoheptaose 2.8 to 3.9%, alpha-cyclodextrin 0.0 to 0.2%, beta-cyclodextrin 2.2 to 10.7% and gamma-cyclodextrin 0.2 to 1.2%.