FI81116B - METHOD FOER TILLVERKNING AV CYKLODEXTRINER INNEHAOLLANDE MALTODEXTRINER, STAERKELSE- OCH MALTOSSIRAPER. - Google Patents

Description

1 81116

Process for the preparation of cyclodextrin-containing maltodextrins and starch and maltose syrups

The invention relates to a process for the preparation of maltodextrins and starch and maltose syrups containing cyclodextrins (CD). In the production process according to the invention, the immobilized cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19) is allowed to act on preprocessed (for example digested by acid hydrolysis or enzymes) starch.

CDs are sugars formed from several glucose units with an annular molecular structure. As in Japanese Patent Laid-Open No. J2 20,373. 75 63,189 and 75 88,290 and in Hans Bender (1977) Arch. Microbiol., Ill., 271-282 discloses CGTase-producing as well as bacteria of the genus Bacillus, such as Bacillus macerans. Bacillus megaterium. Bacillus circulans, Bacillus polymyxa and Bacillus stearothermophilus and bacteria of the genus Klebsiella, such as Klebsiella 20 pneumoniae. When the enzyme acts on the starch, CDs of different ring sizes are formed, which are named alpha, beta and gamma forms based on the 6, 7 and 8 glucose units they contain.

The significance of CDs is based on their ability to form so-called inclusion complexes with small molecule compounds. 25 Small molecule compounds thus become "packaged" inside the sugar and protected e.g. from unwanted chemical reactions. Thanks to this special feature, the applications of CDs are spreading to several different fields, covering e.g. food, pharmaceutical, dye, sanitary, paper and agrochemical industries. Of these, the food industry can be considered as one of the most important components, as CDs are suitable, for example, for protecting aromas or vitamins from light, oxygen or heat, preventing evaporation, removing harmful compounds from products (bad smell or taste) or reducing product hygroscopicity. CDs can be acceptably 35 used in food in Japan and legislation is evolving 2 81116 to allow them to be allowed in other countries as well (Cyclodextrin News (1986), 1, (2), 2).

In connection with the production of CDs, sugar mixtures containing CDs are formed, the sweetness and sugar composition of which are different from those of the process according to the invention. The preparation of these is described in J. Szejtli, Proceedings of the First International Symposium on Cyclodextrins (1982), Akademiai Kiadö, Budapest, 25-40. The method is based on the cleavage of starch by soluble CGTase. After crystallization of the beta-CD, a sugar mixture is obtained, containing mainly glucose, maltose and CDs.

The disadvantage of this method is that the enzymes have to be removed from the product by means of activated carbon treatment and ion exchangers, so that the process is quite laborious and uneconomical,

The use of immobilized CGTase for the preparation of CDs has been described both in Patent Publications (Japanese Patent Nos. 71 09,223 and 80,124,494 and German Patent No. 3,330,571) and in scientific journals (Nakamura N. and Horikoshi K. (1977). Biotechnol. Bioeng ., lf), 87_99; Ivony K., Szajäni B. and Seres G. (1983). J. Appl. Biochem., 158-164; Kato T. and Horikoshi K. (1983). Biotechnol. 20 Bioeng., 26, 595-598). Due to technical difficulties such as viscosity and diffusion barriers, relatively low starch concentrations (2-4%) have generally been used and in some cases the viscosity has been reduced by prehydrolysis to a low DE (SP Crump and JD Rozzell, presentation at Congress 4th International Symposium on 25 Cyclodex 22.4.1988, Mlinchen, Federal Republic of Germany).

The use of immobilized CGTase as in the process of the invention, i.e. the use of high DE starch syrups as a reaction starting material, has not been previously described. The treatment results in high DE syrups containing CDs.

30 For the reasons set out above, the invention has been concluded, which is characterized by the elements set out in the main claim.

The basic idea of the invention is that immobilized CGTase has been found to form sugar solutions containing CDs from preprocessed starch (maltodextrins, starch and maltose syrups DE 35-60) under suitable conditions. The process according to the invention has the possibility of a continuous process and can be added as a further processing step to the sugar syrup production processes already used. The method saves on the cost of using the enzyme and is able to increase the number of CDs in the product. Another advantage of the method is that the enzyme protein does not remain in the 5 products, so no additional purification steps are required. By appropriate control of various parameters such as temperature, degree of immobilization, substrate solution and the concentration of ethanol in it, and the flow rate, the composition of the product formed can be controlled. In addition, novel products are obtained which cannot be prepared by adding CDs to starch or maltose syrups afterwards, since CGTase at the same time typically changes the composition of preprocessed starch so that the proportion of polysaccharides higher than DP 10 is reduced and between sugars below DP 10. concentration differences level off. The effects on the sweetness and reducing sugars of the 15 products are minor.

Table I shows the composition of maltodextrin (DE 20) and starch syrup (DE 30) native and after reaction with immobilized CGTase. The compositions of the preparations were determined by liquid chromatography. The method is able to measure the concentrations of sugars and 20 CDs with a DP number of less than 8. The amounts of CD formed in the product (1-10%) are in the same range as those normally used in food (in Cyclodextrins and Their Inclusion Complexes (1982), J. Szejtli (ed.), Akad6miai Kiadö, Budapest, pp. 236-255) ·

The CGTase required in the invention is produced by growing the microorganism which forms the enzyme in question, for example certain bacteria of the genus Bacillus or Klebsiella, in a growth solution containing a source of carbon and nitrogen, minerals and vitamins. The CGTase formed is recovered using known methods such as centrifugation or filtration of the growth medium. The crude enzyme thus stored can be purified and concentrated, for example, by desalting, dialysis, adsorption and desorption to starch, gel filtration and / or ion exchange chromatography or affinity chromatography.

The crude enzyme or, preferably, the purified and concentrated CGTase can be immobilized using existing methods, such as by binding the enzyme to a solid support, for example, covalently, by adsorption, or by ion exchange forces. Suitable solid carriers are 4,81116 various naturally occurring or synthetically prepared organic macromolecules, such as cellulose, agarose, polyacrylamide, polyethylene glycol and polystyrene, or inorganic compounds, such as diatomaceous earth and glass. Immobilization can be performed under conditions in which the effective activity of 5 CGTase is maintained after the reaction, for example, in the pH range of 4-10 and in the temperature range of <90 ° C.

In the process, starch materials of different origins can be used, for example, cereal-derived, such as barley and wheat starch, or root-derived, such as potato starch. The starch 10 is pretreated, for example, by acid hydrolysis and / or enzymatically. TABLE I: Composition of maltodextrin (DE 20) and starch syrup (DE 30) native and after reaction with immobilized CGTase (*) 15 as determined by liquid chromatography. MD 20 * describes the composition of the sugar solution prepared according to Example 4 and TS 30 * according to Example 5 when the substrate solution does not contain ethanol.

MD 20 MD 20 * TS 30 TS 30 * 20 _ _ AMOUNT OF SUGAR {%) glucose 5.2 4.1 3 * 9 6.9 maltose 4.7 5.8 10.4 9-2 25 maltotriose 4.8 6.1 11.4 8.9 maltotetraose 4.6 6.0 7 · 0 8.1 maltopentaose 4.3 5 * 3 10.2 7.0 maltohexaose 3 * 8 4.2 14.4 5 * 5 maltoheptaose 3 * 2 3-7 3 * 2 3-7 30 alpha-CD - 0.8 - 0.4 beta-CD - 3.1 - 1.7 gamma CD - 0.7 “0.3 so that the resulting the dextrose equivalent 35 of the liquefied starch is in the range of 5 to 60.

The concentration of preprocessed starch reactive with immobilized CGTase should be in the range of 5_1 * 0%. Substrate 5 81116 can be dissolved in either water or a buffer, for example 100 mM imidazole buffer pH 6.8 supplemented with 5 ®M CaCl 2. When allowing the substrate to react with the CGTase adsorbed on the ion exchangers, buffers should generally not be used. The reaction can be carried out at 20-70 ° C.

5 By adding ethanol to the substrate solution until its concentration does not exceed 50% by volume, the amount of CDs in the product substantially increases. At the same time, the balance between different CD formats changes. When the amount of ethanol is large enough, the resulting product is sterile, thus being less sensitive to microbial contamination. Starch and maltose syrups containing CDs produced in the presence of ethanol can be added directly to some products, for example liqueurs. The process according to the invention gives a novel maltodextrin, starch or maltose syrup, which preferably contains 2.0-16% of cyclodextrins and 30-60% of short malto-oligosugars.

The compositions of the invention are described in more detail in the following Tables II-VI.

6 81116 TABLE II. Compositions of maltodextrin (DE 20; 100 g / l) and starch syrup (DE 30; 100 g / l) as native (1) and after reaction with Sepharose 4B gel-immobilized CGTase without ethanol (2) and after the addition of 15% by weight of ethanol (3 ). Concentrations of sugars (below DP 5 5) were determined by liquid chromatography (see Examples 4 and 5).

MD 20 TS 30 SUGAR (g / l) _ ___ 10 12 3 12 3 glucose 5 * 2 4.1 4.6 3 * 9 6.9 6.9 maltose 4.7 5-8 6.2 10.4 9-2 10.0 maltotriose 4.8 6.1 6.0 11.4 8.9 9 * 1 15 maltotetraose 4.6 6.0 5 * 5 7 * 0 8.1 7-7 maltopentaose 4.3 5 · 3 4.7 10.2 7 * 0 6.4 maltohexaose 3 * 8 4.2 4.4 14.4 5-5 4.9 maltoheptaose 3 * 2 3 · 7 3 · 2 3 · 2 3 · 7 3 ·! alpha-CD 0.0 0.8 0.2 0.0 0.4 0.1 20 beta-CD 0.0 3-1 9-5 0.0 1.7 4.9 gamma-CD 0.0 0.7 1.0 0.0 0.3 0.3 7 81116 TABLE III. Compositions of maltodextrin (DE 20; 100 g / l) and starch syrup (DE 30; 100 g / l) native (1) and after reacting with Serdolit AW-14 gel immobilized CGTase without ethanol (2) and after adding 15% by weight to the reaction mixture ethanol (3) · Concentrations of sugars (below DP 5 5) were determined by liquid chromatography (see Example 7) · MD 20 TS 30 SUGAR (g / l) _ _ 12 3 12 3 10____ glucose 5 * 2 4.3 4.7 3 * 9 6.2 5.8 maltose 4.7 5 * 6 5 * 8 10.4 10.4 10.0 maltotriose 4.8 5 · 7 5 · 6 11.4 10.0 9-4 maltotetraose 4.6 5 * 3 5 * 1 7-0 9.2 7.8 15 maltopentaose 4.3 4.9 4.5 10.2 8.4 8.1 malttohexaose 3 · 8 3 · 8 3 · 7 14.4 6.6 7-0 maltoheptaosis 3 · 2 3 · 0 2.8 3-2 4.1 3-9 alpha-CD 0.0 0.2 0.1 0.0 0.2 0.0 beta-CD 0.0 2.3 3-3 0.0 1.6 2.2 20 gamma- CD 0.0 0.6 0.4 0.0 0.4 0.2 e 81116 TABLE IV: Effect of maltodextrin (DE 20) concentration on the composition of the cyclodextrin product formed. The substrate solution was reacted with CGTase immobilized on a Sepharose 4B gel (Example 2). Flow rate 60 ml / h, 25 ° C, ratio of reaction column diameter 5 to height 1: 6.5 · Concentrations of sugars (below DP number 8) were determined by liquid chromatography.

SUGAR md 20 (%) {% of total sugar content) 10 10 15 glucose 4.6 4.6 4.7 maltose 6.6 6.9 6.9 maltotriose 6.6 7 · 1 7-3 15 maltotetraose 6.4 6.9 7-4 maltopentaose 5 · 6 5 · 9 6.8 malttohexaose 5 · 0 5 · 5 6.1 Maltoheptaose 3 * 4 4.1 4.5 Alpha-CD 2.0 0.7 0.7 20 Beta-CD 4.2 2.9 2.5 Gamma-CD 1.2 0.6 0.3 9 81116 TABLE V: Composition of cyclodextrin syrup upon administration of maltodextrin (DE 20; 50 g / l) CGTase (Example 2) immobilized on a Sepharose 4B gel at a temperature in the range of 25-70 ° C. Reaction column diameter to height ratio 1: 6.5, flow rate 60 5 ml / h. The concentrations of sugars (below DP number 8) were determined by liquid chromatography.

TEMPERATURE (° C) SUGAR (g / l) _ 10 25 40 50 60 70 glucose 2.5 2.3 2.2 2.3 4.1

maltose 3.3 3.1 3.0 3-1 M

maltotriose 3 * 3 3 * 1 3-1 3.1 4.3 15 maltotetraose 3-5 3 * 5 2.9 2.9 4.2 maltopentaose 3 * 1 3 * 0 3.0 2.8 4.0 maltohexaose 2.6 2.3 2.1 2.1 3 * 6 maltoheptaose 1.8 1.2 1.4 1.5 2.9 alpha-CD 0.4 0.6 0.8 0.8 0.2 20 beta-CD 3.1 3.2 3.1 3.3 2.6 gamma-CD 0.6 0.7 0.7 0.7 0.6 10 81116 TABLE VI: Effect of ethanol concentration on the composition of cyclodextrin syrup consisting of maltodextrin (MD 20; 100 g / l).

The substrate solution was reacted with CGTase immobilized on a Sepharose 4B gel (Example 2). Flow rate 60 ml / h, 5 25 ° C, reaction column diameter to height ratio 1: 6.5 · Concentrations of sugars (below DP number 8) were determined by liquid chromatography.

ETHANOL {t-%) SUGAR _ 10 (g / l) 5 10 15 glucose 4.2 4.6 4.8 maltose 6.2 6.3 6.3 maltotriose 6.4 7 · 7 6.1 15 maltotetraose 6.2 6.1 6.1 maltopentaose 5 * 2 5 * 5 5 * 8 maltohexaose 4.3 4.6 4.6 maltoheptaose 3 * 1 2.9 2.8 alpha-CD 0.0 0.0 0.0 20 beta-CD 5.2 8.0 10.7 gamma-CD 0.6 0.9 1.2 Starch and maltose syrup solutions containing CDs can be concentrated or dried using known methods. The resulting syrup or powder can be used in various products of the food, confectionery and beverage industries.

Example 1.

30 Bacillus circulans var. alkalophilus strain (ATCC 21783) was inoculated into Horikoshi II medium (Horikoshi K. (1971). Agric. Biol. Chem., 25 · 1783 ~ 1791) and grown at 37 ° C, with shaking and aeration for 3 days. The growth solution was centrifuged and the activity of the supernatant was measured to be 28 U / ml maltotriose-methyl orange by 81116 (Mäkelä M.J. and Korpela T.K. (1988), J. Biochem. Biophys. Methods., I5, 307-318).

The CGTase was purified by affinity chromatography (Hungarian Patent Publication No. 175,584) and the enzyme solution was concentrated on an Amicon-5 apparatus (membrane membrane PM 10). The activity of the CGTase concentrate was measured to be 19,400 U / ml with a specific activity of 1205 U / mg.

Example 2.

10 4 g of CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals,

Uppsala, Sweden) the gel was washed with 10'3 M HCl (1000 ml). The washed vehicle was mixed with 0.1 M NaHCO 3 buffer (18 mL) pH 8.3. containing 0.5 M NaCl. To the suspension was added 2 ml of CGTase concentrate prepared according to Example 1 (32.2 mg of ethnzyme). Reaction 15 was allowed to proceed for 2 hours at room temperature while shaking the mixture.

The immobilized enzyme thus obtained was filtered and washed three times in succession with a sufficient amount of 0.1 M acetate buffer pH 4.0 containing 0.5 M NaCl and 0.1 M Tris-HCl buffer pH 8.0, respectively, containing 0.5 M NaCl, respectively. Finally, the immobilized CGTase 20 was rinsed clean with water.

By determining the protein contents of the filtrate and washings, the amount of CGTase bound to agarose was 31-8 mg, so that the degree of binding was 99%.

25 Example 3.

5 g of Serdolit AW-14 anion exchanger (Serva, Heidelberg, Germany; grain size 0.05-0.1 mm) were washed alternately with 1 M HCl and 1 M NH 4 OH three times. The regenerated ion exchanger was rinsed with 200 mM Tris-acetate buffer pH 8.3 and finally equilibrated with 10 mM Tris-acetate buffer pH 8.3 * 5 ml of equilibrated adsorbent and 4.5 ml of 10 mM Tris-acetate buffer pH 8.3 were mixed and added to the suspension. 2.5 ml of CGTase concentrate prepared according to Example 1 (40.2 mg of enzyme). The reaction mixture was transferred to a shaker, room temperature i2 for 81116 days. After the reaction, the immobilized enzyme was filtered and washed with sufficient water.

By measuring the protein contents of the filtrate and the washing solution, the amount of immobilized CGTase was 24.7 mg, so that the degree of binding of the enzyme was 61%.

Example 4.

The immobilized CGTase prepared according to Example 2 was packed in a column with a height to height ratio of 1: 6.5 · 10 Maltodextrin (DE 20; Suomen Sokeri, Jokioinenealaat) was dissolved in water (100 g / l) and the solution (substrate solution) was eluted at room temperature through the column at 60 ml. /B.

The composition of the solution passed through the column was determined by liquid chromatography (Zsadon B., Otta KH, Tudos F. and Szejtli J. 15 (1979). J · Chromatogr., 172. 490-492) and the total amount of CDs in the elution solution was measured at 4.6 g / l ( alpha-CD: 0.8 g / l, beta-CD: 3 [mu] g / l and gamma CD: 0.7 g / l) · Using a substrate solution in which maltodextrin (DE 20; 100 g / l) was added 15% v / v aqueous ethanol solution, the total amount of CDs in the elution solution 20 under the same reaction conditions was measured to be 10.7 g / l (alpha-CD: 0.2 g / l, beta-CD: 9 * 5 g / l and gamma-CD: 1.0 g / l) .

Example 5 ·

The immobilized CGTase prepared according to Example 2 was packed into 25 columns with a diameter to height ratio of 1: 6.5. An aqueous solution of starch syrup (100 g / l; DE 30; Suomen Sokeri, Jokioinen factory) was eluted through the column at a flow rate of 60 ml / h.

The composition of the elution solution was determined as in Example 4 and the total amount of CDs was measured to be 2.4 g / l (alpha-CD: 0.4 g / l, beta-CD »1.7 g / l and gamma CD: 0.3 g / l). Using a substrate solution in which starch syrup (DE 30; 100 g / l) was added to a 15% v / v aqueous ethanol solution, the total amount of CDs in the elution solution under the same reaction conditions was measured to be 5.3 g / l (alpha-CD: 0.1 g / l, beta -CD: 4.9 g / l and gamma-CD: 0-3 g / l) · i3 81116

Example 6.

1 ml of the CGTase concentrate prepared according to Example 1 (16.1 mg of enzyme) was immobilized on 3 * 2 g of CNBr-activated Sepharose 4B gel as in Example 2. The degree of enzyme binding 5 was measured to be 99 * 5% ·

The immobilized CGTase was packed in a column with a height to height ratio of 1:13. A substrate solution consisting of maltodextrin (200 g / l; DE 20; Suomen Sugeri, Jokioinen mills) dissolved in a 10% v / v aqueous solution of ethanol at a flow rate of 10 ml was eluted through the column. / h, at 40 ° C.

The composition of the elution solution was determined as in Example 4 and the total amount of CDs was measured to be 6.6 g / l (alpha-CD: 0.4 g / l, beta-CD: 5.0 g / l and gamma-CD: 1.2 g / l).

15 Example 7.

The immobilized CGTase prepared according to Example 3 was packed in a column with a height to height ratio of 1: 6.5. · An aqueous solution of maltodextrin (100 g / l; DE 20; Suomen Sokeri, Jokioinen mills) was eluted through the column at a flow rate of 10 ml / h.

The composition of the elution solution was determined as in Example 4 and the total amount of CDs was measured to be 2.5 g / l (alpha-CD: 0.2 g / l, beta-CD: 1.7 g / l and gamma-CD: 0.6 g / l). Using a substrate solution in which maltodextrin (DE 20; 100 g / l) was dissolved in a 15% v / v aqueous ethanol solution, the total amount of CDs was measured to be 3 * 7 g / l (alpha-CD: 0 g / l, beta) under corresponding reaction conditions. -CD: 3 * 3 g / l and gamma CD: 0.4 g / l).

By exchanging the aqueous solution of starch syrup (100 g / l; DE 30; Suomen Sokeri, Jokioinenealaat) for the substrate solution, the total amount of CDs in the elution solution was measured to be 2.2 g / l (alpha-CD: 0.2 g / l, beta-CD: 1.6 g) under the corresponding reaction conditions. / 1 and gamma CD: 0.4 g / l). By dissolving the starch syrup (DE 30; 100 g / l) in a 15% v / v aqueous solution of ethanol and using the resulting solution as a substrate solution, the total amount of CDs in the elution solution was measured to be 2.4 g / l (alpha-CD: 0 g / l, beta CD: 2.2 g / l and gamma CD: 0.2 g / l).

Claims (3)

A process for the preparation of syrups containing cyclodextrins (alpha, beta and / or gamma forms), characterized in that 5-40% by weight of? preprocessed starch having a dextrose equivalent of 5 in the range of 5 ~ 60 dissolved in either water, a buffer, or a maximum of 50% by volume of these ?! the ethanol solution is reacted with immobilized cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19) to give a maltodextrin, starch or maltose syrup, preferably containing 2.0-16% of cyclodextrins and 30-60% of short malto-oligosaccharides. Process according to Claim 1, characterized in that by dissolving the starting material in either water or a buffer, maltodextrin, starch or maltose syrup is obtained, which preferably contains glucose 4.1-8.2?, Maltose 5.6-10.4%, maltotriose 5.7_10.0? , maltotetraose 5 · 3 “9 · 2?!, maltopentaose 4.9-8.4?!, maltohexaose 3.7-7-2?!, maltoheptaose 2.9-4.5?!. alpha-cyclodextrin 0.4-2.0?!, beta-cyclodextrin 1.6-6.6?! and gamma-cyclodextrin 0 · 3 “1 · 4?!. 20 Process according to Claim 1, characterized in that by dissolving the starting material in either water or a buffer in a volume of at most 50% by volume. a maltodextrin, starch or maltose syrup is obtained in the ethanol solution, which preferably contains glucose 4.2-6.9?!, maltose 5-3-10.0?!, maltotriose 5-6-9-4?!, maltotetraose 5-1 "7-8? !, maltopentaose 4.5-8.1?!, maltohexaose 2.8-4.6?!, maltoheptaose 2.8-3.9 X. alpha-cyclodextrin 0.0-0.2?!, beta-cyclodextrin 2.2-10.7% and gamma-cyclodextrin 0.2-1.2?!. is 81116