MXPA98003606A - h. METHOD FOR THE PRODUCTION OF RICH JARS IN ISOMALTO-OLIGOSACAR - Google Patents

Abstract

The present invention relates to a method for the production of isomalto-oligosaccharide syrups, the method comprises the use of enzymes immobilized on a reusable vehicle, the vehicle is preferably an anion exchanger, the enzymes used for the conversion of starch hydrolysates they are transglucosidase and pullulanase and these enzymes are preferably co-immobilized, the vehicle / enzyme conjugate is further strengthened by interlacing.

Description

METHOD FOR THE PRODUCTION OF RICH JARS IN ISOMALTO- OLIGOSACARIDO TECHNICAL FIELD The present invention describes a method for the production of isomalto-oligosaccharide syrups. The method comprises the use of enzymes immobilized on a reusable vehicle. The invention also relates to syrups obtained by the use of said enzymes immobilized on reusable vehicles, and to the use of said syrups.

BACKGROUND OF THE INVENTION The isomalto-oligosaccharide syrups contain a substantial amount of branched oligosaccharides, such as isomaltose, panose, isomaltotriose, isomaltotetraose, nigerose, kojibiose, isopanoses and higher branched oligosaccharides. The products are sold in powder or liquid form, depending on the desired application. The potential applications are in the food area, for example: - condiments (mayonnaise, vinegar, soup base, etc.) confectionery (candy, chewing gum, chocolate, ice cream, sorbet, syrup, pastry) - processed foods from fruits and vegetables (preserves, marmalade, fruit compote, pickles), meat or fish foods (ham, sausage, etc.) - bakery products (bread, cake, biscuit) - pre-cooked foods (salad, cooked beans, etc.) ) - canned and bottled foods and beverages (coffee, juice, nectar, soft drinks, lemonade, cola) convenience foods (instant coffee, instant cake base, etc.). Isomalto-oligosaccharide syrups can also be applied as ingredients in food. animals and pet food. The non-food application areas are in cosmetics and medicine (cigarettes, lipstick, toothpaste, internal medicine, etc.). It has been known for some years that isomalto-oligosaccharides are related to the increase in the general well-being of humans and animals when taken orally on a regular daily basis. The main action of oligosaccharides is to increase the number of Bifidobacteria and Lactobacilli in the small intestine, and reduce the concentration of putrefactive bacteria. Bifidobacteria are associated with some health-promoting properties, such as the inhibition of pathogen growth, either by acid formation or by antimicrobial activity. They are also associated with various effects such as the modulation of the immune system (anti-tumor properties), the reduction of triglyceride and cholesterol levels, the production of vitamins (group B), the reduction of concentrations of ammonia in the blood, the Transposition prevention, restoration of the normal flora of the intestines after antimicrobial therapy, production of digestive enzymes, reduction of side effects associated with antibiotics (Kohmoto T., Fukui F., Takaku H., Machida Y., and others, Bifidobacteria Microflora, 7 (2) (1988), 61-69; Kohmoto K., Tsuji K., Kaneko T., Shiota M., and others, Biosc. Biotech, Biochem 56 (6) (1992), 937-940; Kaneko T, Kohmoto T., Kikuchi H., Fukui F. and others, Nippon Nógeikagaku Kaishi, 66 (8) (1992), 1211-1220, Park JH, Jin-Young Y., Ok-Ho S ., Hyun-Kyung S., et al., Kor. J. Appl. Microbiol. Biotechnol. 20 (3) (1992), 237-242). Isomalto-oligosaccharides are synthesized by a transglycosylation reaction using a D-glucosyl transferase (E.C. 2.4.1.24, transglycosidase, alpha-glucosidase). This enzyme catalyzes both hydrolytic and transfer reactions, by incubation with alpha-D-glyco-oligosaccharides. The transfer occurs more frequently to 6-OH (hydroxyl group 6 of the glucose molecule), producing isomaltose from D-glucose, or panose from maltose. The enzyme can also transfer to 2-OH or 3-OH from D-glucose to form kojibiosa or nigerosa, or back to 4-OH to reform maltose. As a result of the transglucosidase reactions, the malto-oligosaccharides are converted to isomalto-oligosaccharides, resulting in an oligosaccharide class containing a higher proportion of glucose portions linked by alpha-D-1, 6-glucosidic bonds. The transglucosidase of A. niger acts only on oligosaccharides with a low DP (McCleary B.V., Gibson T.S., Carbohydrate Research 185 (1989) 147-162, Pazur J.H., To inaga Y., DeBrosse C.W., Jackman L.M. Carbohydrate Research, 61 (1978) 279-290). Isomalto-oligosaccharides can be obtained in different ways. For example, glucose syrups at high concentration of dry solids, ie, 60-80%, are treated with glucoamylase, resulting in the formation of isomalto-oligosaccharides, mainly DP2. Other examples are maltose transfer achieved by the addition of pullulanase to liquefied starch, branching of maltose syrups and treatment of sucrose with dextran sucrase. It is reported that the commercial production of isomalto-oligosaccharides is carried out in a non-continuous manner. A normal production method (JP 61-212296, Sho to Sangyo Co. Ltd.) begins with the liquefaction of a suspension of corn, potato or tapioca starch of 30% dry solids with a thermostable alpha-amylase at a DE of liquefied product from 6 to 10. This liquefied product is brought to pH 5 and 60 ° C, and beta-amylase and transglucosidase are added, and saccharification is continued for 48 to 72 hours. At the end of the saccharification period, the syrup is filtered and refined with activated charcoal and ion exchangers. The pure product is finally concentrated to about 80% dry solids (Takaku H., Handbook of Amylases and related enzymes, Ed. The Amylase Research Society of Japan, Pergamon Press, Oxford (1988), 215-217). The beta-amylase used originates from soy or wheat, the transglucosidase comes mainly from a fungal source, preferably Aspergillus niger. Other methods of production are known; these include the conversion of hydrolyzed product of starch with a mixture of alpha-amylase and transglucosidase (JP 41-48693, Nippon Corn Starch KK) and the conversion of starch into a high DP3 or DP4 syrup with alpha-amylases forming DP3 or DP4, in conjunction with ransglucosidase to produce branched oligosaccharides (JP 31-87390, Gunei, Kagaku Kogyo). Much attention has been paid to soluble enzyme systems for the production of isomalto-oligosaccharides; such activity can not be found in the field of immobilized enzymes. Japanese Patent Application JP63-109790 (assigned to Sho a Sangyo Co.) describes the use of an immobilized ransglycosidase conjugate for the production of isomalto-oligosaccharide syrups. The conjugate is made by cross-linking gelatin with glutaric dialdehyde in the presence of transglucosidase. The conjugate obtained has a high mechanical stability, and has to be ground before transporting it to the retention columns. Due to the rather heterogeneous reaction, the enzyme is not homogeneously distributed within the carrier material, which leads to disturbed kinetics, producing a final product that does not have the maximum obtainable amount of isomalto-oligosaccharides.

A further disadvantage is that the vehicle is not reusable. After depletion of the enzyme activity, all the conjugate has to be discarded, which is not an economically or ecologically preferred solution. European patent application EP 301522 relates to the production of isomaltulose starting from a mixture of glucose and fructose. The description does not disclose the use of reusable vehicles for this mentioned procedure; moreover, the examples show only the use of non-immobilized enzymes to effect the conversion. The US patent No. 3,935,070 refers to the isomerization of a starch hydrolyzate to convert at least a portion of the dextrose to levulose. This conversion can be followed by a treatment with ransglucosidase on bentonite. Bentonite is not a reusable vehicle; In addition, the starting material is a dextrose mother liquor. Japanese Patent Application JP04 051899 (assigned to NGK Insulators Ltd.) describes the use of immobilized enzymes on porous ceramic particles composed of Si 2 and MgO. These ceramic particles are not reusable. Japanese Patent Application JP62 278984 (assigned to Daikin Kogyo KK) describes the use of a co-immobilized compound of cells and enzymes. Neither is this product reusable.

BRIEF DESCRIPTION OF THE INVENTION The present invention describes a method for producing isomalto-oligosaccharide syrups, wherein a starch hydrolyzate is converted enzymatically by means of a transglucosidase using a reusable vehicle for the immobilization of transglucosidase. The starch hydrolyzate is a syrup having an ED between 4 and 70, preferably between 20 and 60. Preferably, the carrier is an anion exchange resin and the transglucosidase is immobilized thereon by adsorption. A further aspect of the invention is that other enzymes are co-immobilized with the transglucosidase, these other enzymes are a pullulanase or an alpha-amylase. The enzymes can be immobilized together on the same vehicle, but it is also possible to immobilize the enzymes separately. This makes it possible to separate the two steps of enzymatic conversion. In another aspect of the invention, the vehicle / enzyme conjugate is further reinforced by reaction with one or more entanglement agents. The continuous production of an isomalto-oligosaccharide syrup containing more than 40% isomalto-oligosaccharides, preferably more than 45% is described. These values are attained with a flow velocity of at least 3 bed volumes per hour and for a period of at least 25 days. In another aspect, the isomalto-oligosaccharide syrup is refined, i.e., further processed by chromatographic means or by filtration. Another part of the invention is that during the production of the isomalto-oligosaccharide syrup or after the same, the sweetness is increased. This can be done by the addition of a sweetener or by an additional enzymatic conversion with glucose isomerase or a hydrolase, whereby the glucose is converted to fructose. This enzymatic conversion can be carried out simultaneously with, or successively, transglycosylation.

DESCRIPTION OF THE FIGURES Figure 1 shows the production of isomalto-oligosaccharides (-% isomaltose +% nigerose +% isomaltotriose +% panose +% isomaltotetraose) as a function of the lifetime of the conjugate and the amount of glutaric dialdehyde used for entanglement.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes a method for producing isomalto-oligosaccharide syrups, wherein a starch hydrolyzate is converted enzymatically by means of a transglucosidase or an enzyme having a comparable activity, using a reusable vehicle for immobilization of the enzyme. The starch hydrolyzate has an ED of between 4 and 70, preferably between 20 and 60. The starch hydrolyzate is obtained for example by enzymatic or acid hydrolysis, by methods known in the art. As a carrier, "any reusable material can be used." For the present purpose, reusable means that the vehicle can be released from the enzyme or enzymatic activity in such a way that the carrier material remains intact.The carrier material can then be reloaded with enzyme The cleaning of the vehicle can be done for example by washing with acid or basic solution, this can be done by batch or in the column.It can be advantageous to add a salt.Another possibility is the use of protein degrading enzymes. preferably, materials having anion exchange groups are used, such materials may be in the cellulose base, Other preferred vehicles are polyacrylate or polystyrene-based vehicles having weakly basic groups, preferably phenol-formaldehyde-based vehicles. such as Duolite ™ A 568 (Rohm and Haas), preferably the vehicle it is an anion exchange resin and the transglucosidase is immobilized thereon by adsorption, that is, in a non-covalent manner. This allows the easy removal of the inactive enzyme and the subsequent recharge with enzyme new. It is shown in the examples presented (notably in Example 11) that the enzyme can be easily removed from the vehicle. Reloading the vehicle results in complete recovery of conjugate activity. This means that the vehicle material can be used for a considerable period before being replaced. Example 1 illustrates the immobilization of transglucosidase on an anion exchange resin. The application of this catalyst in a column for the conversion of a 30% maltose syrup into dry solids shows that at a constant flow rate of approximately 3 bed volumes per hour, the total amount of isomalto-oligosaccharides formed is about 40% and the process is stable for at least about 30 days. A small change in the amount of individual isomalto-oligosaccharides is observed. The activity of the enzyme is mainly on the DP2-DP6 scale. The entanglement of the immobilized transglucosidase with glutaric dialdehyde is described in example 2. The dialdehyde was used at a final concentration of 1%. The results (example 2) show that the enzyme relaxes the formation of a syrup having a composition different from that obtained without entanglement. Specifically, the amount of glucose increases. The total amount of isomalto-oligosaccharides is about 35%, the decrease of 5% can be attributed almost completely to a decrease in panose. A further aspect of the invention is that other enzymes are co-immobilized with the transglucosidase, said other enzymes being pullulanases or alpha-amylases. Pullulanase or alpha-amylase will degrade higher fractions of DPn into smaller fragments, which are accessible for the action of transglucosidase. The enzymes can be immobilized together on the same vehicle, but it is also possible to immobilize the enzymes separately. This makes it possible to separate the two steps of enzymatic conversion. The alpha-amylase and / or pullulanase can in this case be physically separated from the ransglucosidase. It can be placed, for example, in front of the transglucosidase. The advantage of this procedure is that when one of the enzymes is consumed, it is not necessary to replace all the enzymes, instead, it would be possible to replace only one of the enzymes and continue with the other enzyme not consumed. Said separate immobilization is described in example 10. Also, a glutaric dialdehyde treatment can be carried out on the conjugate produced to stabilize the immobilized enzymes. The co-immobilization of transglucosidase with pullulanase (1: 7.5 (w / w)) resulted in a conjugate that when applied to a maltose syrup of 30% dry solids, gave an isomalto-oligosaccharide syrup having a much lower glucose content and an increased DP3 content. The content of DPn was divided into two equal parts due to the pullulanase activity (example 3). The amount of isomalto-oligosaccharides started at approximately 48%, however, this value decreased considerably with time. Therefore, the catalyst is clearly not stable. The amount of panose was around 18%. A similar experiment was performed with half the amount of pullulanase giving a different spectrum of isomalto-oligosaccharides (example 4). To stabilize the conjugate, the coinmobilized enzyme / vehicle product was treated with glutaric dialdehyde at different concentrations. The use of more than about 0.1% of the dialdehyde resulted in considerably increased stability. The total amount of isomalto-oligosaccharides was more than 45%, and remained above this value, even though the flow velocity was increased to 6 bed volumes per hour (example 6). Examples 6 and 7 also show that the increased stability was achieved with 0.25 and 1% glutaric dialdehyde. In this way, the present invention shows the continuous production of an isomalto-oligosaccharide syrup containing more than 40% isomalto-oligosaccharides, preferably more than 45%. These values are achieved with a flow velocity of at least 3 bed volumes per hour for a period of at least 25 days. In another aspect, the isomalto-oligosaccharide syrup is refined, i.e., further processed by chromatographic means or by filtration. The produced isomalto-oligosaccharide syrup can be further fractionated by means of a chromatographic technique or by ultra- or nano-filtration to remove the glucose fraction and thus obtain a syrup enriched in isomalto-oligosaccharide content. Another part of the invention is that during the production of the isomalto-oligosaccharide syrup or after, the sweetness increases; this can be done with the addition of a sweetener or by an additional enzymatic conversion with glucose isomerase or a hydrolaea. In example 8, an isomalto-oligosaccharide syrup was converted onto a glucose isomerase column giving a conversion of glucose to fructose without significantly affecting the other oligosaccharides. Another route to increase the sweetness or isomalto-oligosaccharide content is to treat the isomalto-oligosaccharide syrup produced with a hydrolase (in soluble or immobilized form) that hydrolyzes preferentially, or almost exclusively, malto-oligosaccharides, and has only a small affinity, or none, for isomalto-oligosaccharides. Examples of said enzyme is glucoamylase from A. niger, or other sources such as Aspergillus sp. or Rhizopus sp. which preferentially hydrolyzes malto-oligosaccharides (Manjunath P., Shenoy BC, Raghavendra Rao MR, Journal of Applied Biochemistry, 5 (1983), 235-260; Meagher MM, et al., Biotechnology and Bioengineering, 34 (1989), 681-693 Pazur JH, Kleppe K., The Journal of Biological Chemistry, 237 (4) (1962), 1002-1006, Hiromi K., Nitta Y., and others, Biochimica et Biophysica Acta, 302 (1973), 362-37 ). The same was done using glucoamylase. In this case, there was a considerable production of dextrose at the expense of all the other oligosaccharides (example 9). An enzyme such as Bacillus stearothermophilus alpha-D-glucopyropylase can also be applied. This enzyme is not able to hydrolyze isomalto-oligosaccharides and will only degrade the malto-oligosaccharides present in the isomalto-oligosaccharide rich syrup (Suzuki Y., Shinji M., Nobuyuki E., Biochimica et Biophysica Acta, 787 (1984), 281 -289). Other alpha-D-glucosidases that are called maltases can also be used. Yeast maltase, for example, will only hydrolyze maltose and to a lesser extent (Kelly C.T., Fogarty W.M., Process Biochemistry, May / June (1983), 6-12). After hydrolysis of the malto-oligosaccharides into glucose, the syrup can be enriched in iso-high oligosaccharides by a chromatographic technique or by nano- or ultrafiltration.

The following examples serve to illustrate the main aspects of this invention.

Experimental part Determination of the Enzymatic Activity The activity of transglucosidase is measured by the method of McCleary et al. (McCleary B.V., Gibson T.S., Carbohydrate Research, 185 (1989), 147-162). Methyl-alpha-D-glucopyroposide is allowed to react in the presence of transglucosidase at pH 5.0 and 60 ° C for 10 minutes, and is thereby converted to D-glucose. D-glucose is measured and the activity is expressed in international units. An international unit (U) is the amount of enzyme required to break a micromole of glycosidic bond per minute. The pullulanase activity is measured by a modified method of Lappalainen et al. (Lappalainen A., Niku-paavola M.-L., Suortti T., Puntanen K., Starch, 43 (12) (1991), 477-482) . The pullulanase activity is measured by allowing pullulan to react with the pullulanase at pH 5 and 50 ° C for 15 minutes. Pullulan is hydrolyzed to oligosaccharides, which are quantified colorimetrically by the DNS reagent. Enzymatic activity is calculated from a standard curve expressing the relationship between maltose concentration and absorption. One unit is the amount of enzyme required to produce a micromole of reducing groups, expressed as maltose equivalents per minute.

Substrates The syrups used as substrates in all examples, except examples 7 and 8, have the following approximate composition: DPI DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP9 DP10 DP > 11 3.4 48.1 20.8 1.2 1.1 1.6 2.7 3.2 2.4 1.0 14.5 These substrates contain a very low amount of isomalto-oligosaccharides: BPl talose istmaltasa nigerosa -.alotrosa panosa soialtotrosa isaialtatetracsa iso total Dpn 3.4 47.0 1.1 0.0 20.3 0.5 0.1 1.2 2.8 26.5 The use of these substrates as described in the examples, does not exclude the use of other substrates such as bad todex fights (products of hydrolysis of starch with equivalent dextrose <20), or syrups composed of different amounts of DP1-DP20 and higher DPn .

Analysis of Oligosaccharides The analytical characterization of the substrates and products was carried out in an HPLC equipped with a column Shodex KS-801, in the Na * form, and IR detection. This method gives the composition of DPI, DP2, DP3, DP4 and DPn. The composition of DP1-DP10 was determined with a Bio-Rad HPX 42-A column. The quantification of the individual isomalto-oligosaccharides was carried out by high performance anion exchange chromatography, that is, using a Dionex Carbopac PA-1 column with pulse perometric detection.

EXAMPLE I IMMOBILIZED TRANSGLUCOSIDASE ml of Duolite A568 ™ was washed with demineralised water to remove the fine particles. The resin was subsequently conditioned with HCl to a pH of 3.5. 2 g of transglucosidase L "Amano" (liquid preparation, 11.2 mg of protein / g of enzyme solution, 103 UTG / g of enzyme solution) were added, and the mixture was stirred for 1 night at room temperature. The conjugate produced was rinsed with demineralized water and placed on a double-jacketed glass column at 50 ° C. A 30% maltose syrup was pumped into dry solids (3.3% DPI, 49.7% DP2, 21.9% DP3, 0.9% DP4, 24.2% DP> 5), brought to pH 4.2, through the column at a flow rate of 3 VL / h (bed volumes / hour). The temperature of the column was maintained at 50 ° C. The product at the exit of the column was analyzed by HPLC. Table 1 illustrates the change in composition of saccharides by the addition of the immobilized transglucosidase: TABLE 1 Table 2 describes the different isomalto-oligosaccharides produced.

TABLE 2 In addition to the total amount of isomalto-oligosaccharides of about 40%, also the DPn fraction contains a significant amount of branched oligosaccharides with a DP > 5. Although the total isomalto-oligosaccharide content remains constant over time, a small change in the production of the individual isomalto-oligosaccharides can be noted. Table 3 illustrates the change in peripil of oligosaccharides after the conversion of the substrate by the conjugate.

TABLE 3 From Table 3 it is evident that the action of the immobilized transglucosidase is on the DP2-6 scale. In general, molecules DP2 and DP3 are converted to molecules of glucose and DP4-6. The DP10 + fraction did not change substantially.

EXAMPLE 2 IMMOBILIZED TRANSGLUCOSIDASE TREATED WITH GLUTARY LDEHYDE DI ml of Duolite A568 ™ was washed with demineralised water to remove the fine particles. The resin was subsequently conditioned with 0.3 ml of a2C? 3 1M. 2 g of transglucosidase L "Amano" (liquid preparation, 11.2 mg of protein / g of enzyme solution, 103 UTG / g of enzyme solution) were added and the mixture was stirred for 4 hours at room temperature. 5 ml of a 5% glutaric dialdehyde solution (final concentration 1%) was added and the resin was stirred for 1 night at room temperature. The conjugate produced was rinsed with demined water and placed on a double-jacketed glass column at 50 ° C. A 30% maltose syrup was pumped into dry solids (3.3% DPI, 49.7% DP2, 21.9% DP3, 0.9% DP4, 24.2% DP> 5), brought to pH 4 through the column at a rate of flow of 3 VL / h (bed volumes / hour). The temperature of the column was maintained at 50 ° C. The product at the exit of the column was analyzed by HPLC. Table 4 illustrates the change in composition of saccharides by the action of immobilized transglucosidase: TABLE 4 When the results are compared with those presented in Table 1, it is evident that glutaric dialdehyde changes the pattern of action of the immobilized transglucosidase. The conjugate treated with glutaraldehyde produces more glucose than the conjugate described in Example 1. Table 5 describes the different isomalto-oligosaccharides produced.

TABLE 5 Days BV / h Dextrose Maltose Haltotriose Isoaltose Isoyaltotriose Nigerose Panosa Isoaaltotetraose Total Iso DPn 4 2.9 39.2 2.7 1.5 16.7 8.3 2.7 1.0 6.8 35.6 21.0 8 3 38.4 2.8 1.0 15.6 8.7 3.3 0.9 6.9 35.5 22.3 12 3 39.0 3.1 1.8 15.9 8.1 3.0 0.9 7.0 34.8 21.3 2.9 36.8 3.3 1.8 15.3 8.1 3.4 1.0 7.0 34.8 21.3 19 3 38.1 3.2 1.9 15.9 8.2 3.1 1.0 7.0 35.2 21.6 A decrease in the amount of isomalto-oligosaccharides is observed in comparison with the untreated TG conjugate (Table 2). This is directly related to the lower production of panose in the glutaric dialdehyde conjugate.

EXAMPLE 3 Co-immobilized transglucosidase / pullulanase (1) ml of Duolite A568 ™ was washed with demineralised water to remove the fine particles. The resin was subsequently conditioned with HCl at a pH of 3.5. 2 g of Transglucosidase L "Amano" (liquid preparation, 11.2 mg of protein / gram of enzyme solution, 103 TGU / gram of enzyme solution) was added and the mixture was stirred for 4 hours at room temperature. 15 g of pullulanase (Optimax L300 ™ from Genencor Int., 2.6 mg protein / g enzyme solution, 400 PU / g enzyme solution) was added and immobilization was continued overnight. The conjugate produced was rinsed with demineralized water and placed in a double cover glass column at 50 ° C. A maltose syrup was pumped with 30% dry solids (3.3% DPI, 49.7% DP2, 21.9% DP3, 0.9% DP4, 24.2 DP> 5), adjusted to a pH of 4.2, through the column at a flow rate of 3 BV / h (bed volumes / hour). The temperature of the column was maintained at 50 ° C. The product was analyzed at the exit of the column by HPLC. Table 6 illustrates the change in saccharide composition by the action of immobilized transglucosidase / pullulanase conjugate.

TABLE 6 In comparison with the immobilized transglucosidase conjugate prepared in Example 1, less glucose is produced, while especially DP3 is much higher.

In addition, the DPn fraction is reduced by half due to the action of the immobilized pullulanase. A decrease in dextrose formation is noted over time, which indicates that this conjugate does not possess high stability. Table 7 describes different isomalto-oligosaccharides produced.

TABLE 7 For Table 7 it is evident that the content of isomalto-oligosaccharides decreases with time.

EXAMPLE 4 Co-mobilized transglucosidase / pullulanase (2) The difference with the conjugate described in the Example 3, is the modified ratio of the activity of transglucosidase / pullulanase activity, offered to the enzyme vehicle. 10 ml of Duolite A568 ™ was washed with demineralised water to remove the fine particles. The resin was subsequently conditioned with HCl at a pH of 3.5. 2 g of Transglucosidase L "Amano" (liquid preparation, 11.2 mg of proein / gram of enzyme solution, 103 TGU / gram of enzyme solution) was added and the mixture was stirred for 4 hours at room temperature. 7.5 g of pullulanase (Optimax L300 ™ from Genencor Int., 2.6 mg protein / g enzyme solution, 400 PU / g enzyme solution) was added and the immobilization was continued overnight. The conjugate produced was rinsed with demineralized water and placed in a double cover glass column at 50 ° C. A maltose syrup was pumped with 30% dry solids (3.3% DPI, 49.7% DP2, 21.9% DP3, 0.9% DP4, 24.2 DP> 5), adjusted to a pH of 4.2, through the column at a flow rate of 3 BV / h (bed volumes / hour). The temperature of the column was maintained at 50 ° C. The product was analyzed at the exit of the column by HPLC. Table 8 illustrates the change of the saccharide composition by the action of the immobilized ransglucosidase / pullulanase conjugate.

TABLE 8 It is evident that also this conjugated transglucosidase / pullulanase is losing effectiveness over time as the conjugate described in Example 3. Table 9 describes the different isomalto-oligosaccharides produced TABLE 9 The results shown in Table 9 show that this conjugate produces a decreasing amount of isomalto-oligosaccharides over time. It can be concluded that this conjugate is not stable.

EXAMPLE 5 Co-mobilized transglucosidase / pullulanase treated with glutaric dialdehyde in different concentrations ml of Duolite A568 ™ was washed with demineralised water to remove the fine particles. The resin was subsequently conditioned with 0.3 ml of 1M Na 2 C 3 - 2 g of Transglucosidase L "Amano" (liquid preparation, 11.2 mg of protein / enzyme solution branch, 103 TGU / gram of enzyme solution) was added and stirred the mixture for 4 hours at room temperature. Subsequently 15 g of pullulanase (Optimax L300 ™ from Genencor Int., 2.6 mg protein / g enzyme solution, 400 PU / g enzyme solution) and immobilization was continued for another 4 hours. Then 5 ml of a 5%, 2.5%, 1.0%, 0.5% or 0.1% glutaric dialdehyde solution was added to give respectively 1%, 0.5%, 0.2%, 0.1% and 0.02% glutaric dialdehyde solution, and the resin was stirred overnight at room temperature. The conjugate produced was rinsed with demineralized water and placed in a double cover glass column at 50 ° C. A maltose syrup was pumped with 30% dry solids (3.3% DPI, 49.7% DP2, 21.9% DP3, 0.9% DP4, 24.2 DP> 5), adjusted to a pH of 4.2, through the column at a flow rate of 3 VL / h (bed volumes / hour). The temperature of the column was maintained at 50 ° C. The product was analyzed at the exit of the column by HPLC. Figure 1 shows the production of isomalto-oligosaccharides (-alpha isomaltose +% nigerose +% isomaltotriose + palnosa% + isomaltotetraose%) as a function of the life time of the conjugate. It is clear from FIG. 1 that the glutaric dialdehyde treatment has a stabilizing effect on the effectiveness of the transglucosidase / pullulanase conjugates.

EXAMPLE 6 Transglycosidase / polyphenases co-stabilized treated with glutaraldehyde in a final concentration of 0.2% The transglucosidase / pullulanase conjugate was made according to Example 5. The entanglement was performed with glutaric dialdehyde at a final concentration of 0.2%. A maltose syrup with 30% dry solids was pumped, adjusted to a pH of 4.2, through the column at a flow rate of 3VL / h (bed volumes / hour). The temperature of the column was maintained at 50 ° C. The product was analyzed at the exit of the column by HPLC. Table 10 illustrates the change in the composition of saccharides by the action of immobilized transglucosidase: TABLE 10 As indicated, only an original decrease in dextrose was observed over time. Table 11 describes the different isomaltos-oligosaccharides produced.

TABLE 11 The results shown in Table 11 show that stable production of isomalto-oligocasarides at 46% at 3VL / h can be obtained with this conjugate. An increase in flow velocity at approximately 6VL / h decreases the dextrose content produced, while the amount of isomalto-oligosaccharides produced remains constant. A further increase in flow velocity at approximately 9VL / h decreases the amount of isomalto-oligosaccharides produced. Table 12 gives the variation of the oligosaccharide distribution of the syrup produced over time.

TABLE 12 Substrate Days 14 15 16 19 20 21 22 23 26 27 28 BV / h 3.0 3.0 3.1 3.0 3.0 3.0 3.0 3.0 2.9 3.0 5.2 3.3 DPI 37.3 37.4 37.5 36.7 36.7 36.8 36.6 36.1 36.2 36.4 28.4 48.4 DP2 28.3 28.3 28.3 28.5 28.1 28.1 28.3 28.3 28.3 28.2 25.9 20.8 DP3 17.1 17.2 17.1 17.1 17.4 17.4 17.6 17.8 17.8 17.4 20.8 1.3 DP4 8.2 8.2 8.2 8.3 8.2 8.2 8.4 8.4 8.3 8.4 9.8 1 DP5 3.7 3.6 3.7 3.6 3.7 3.7 3.5 3.5 3.7 3.8 4.5 1.6 DP6 1.3 1.3 1.3 1.3 1.4 1.4 1.3 1.4 1.5 1.5 2.1 2.7 DP7 0.7 0.7 0.7 0.7 0.8 0.8 0.7 0.7 0.8 0.8 1.4 3 DP8 0.4 0.4 0.4 0.4 0.4 0.4 0.5 0.5 0.4 0.4 0.9 2.4 DP9 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.7 15.4 DPlOt 2.6 2.5 2.4 2.7 2.8 2.8 2.7 2.9 2.7 2.8 5.1 Table 12 shows that the majority of the oligosaccharides in the syrup produced are located under the DP6-7, unlike the substrate in which a substantial part (23.5%) of the oligosaccharides have a DP higher than 6. When compared to the conjugate described in Example 1 (Table 3), it is directly evident that the action of the coinmobilized polylanase decreases significantly with the DP10 + fraction.

EXAMPLE 7 Co-mobilized transglucosidase / pullulanase treated with glutaric dialdehyde at a final concentration of 1.0% and with different flow rates. ml of Duolite A568 ™ was washed with demineralised water to remove the fine particles. The resin was subsequently conditioned with 0.3 ml of 1M a2C? 3. 2 g of Transglucosidase L "Amano" (liquid preparation, 11.2 mg of protein / gram of enzyme solution, 103 TGU / gram of enzyme solution) was added and the mixture was stirred for 4 hours at room temperature. Subsequently, 15 g of pullulanase (Opti ax L300 ™ from Genencor Int., 2.6 mg protein / g enzyme solution, 400 PU / g enzyme solution) and immobilization was continued for another 4 hours. Then 5 ml of a 5.0% glutaric dialdehyde solution was added to give a 1% glutaric dialdehyde solution and the resin was stirred overnight at room temperature. The conjugate produced was rinsed with demineralized water and placed in a double cover glass column at 50 ° C. A maltose syrup was pumped with 30% dry solids (3.3% DPI, 49.7% DP2, 21.9% DP3, 0.9% DP4, 24.2 DP> 5), adjusted to a pH of 4.2, through the column at a flow rate of 3 VL / h (bed volumes / hour). The temperature of the column was maintained at 50 ° C. The product was analyzed at the exit of the column by HPLC. Table 13 shows the change in the composition of saccharides by the action of immobilized ransglycosidase: TABLE 13 For Table 13 it is evident that a large amount of dextrose is produced at 3VL / h while the portion of DPn is greatly reduced compared to the substrate. Increasing the flow rate to 6-9VL / h decreases glucose production and at the same time increases the residual content of DPn. Table 14 illustrates the change in the composition of saccharides by the action of immobilized transglucosidase: TABLE 14 The data shown in Table 11 show that around 45-46% of iso-oligosaccharides can be obtained at 3VL / h. When the flow rate is increased to 6VL / h, the amount of isomalto-oligosaccharides does not decrease. At 9VL / h, a decrease in the production of isomalto-oligosaccharides is noted.

EXAMPLE 8 Increase in the sweetness of isomalto-oligosaccharide syrup by conversion by glucose isomerase This example illustrates the process for increasing the sweetness of an isomalto-oligosaccharide syrup by converting part of the available glucose to fructose. A syrup rich in isomalto-oligosaccharides (pH 7.8, 60% dry solids, 200 ppm Mg +) was sent through an immobilized glucose isomerase conjugate (thermally regulated at 50 ° C) at 3-4.5% VL / h . The results of isomerization are given in Table 15.

TABLE 15 The results in Table 15 show that a 9% fluctuant version of an isomalto-oligosaccharide syrup is easily made. Of course, it is also possible to obtain isomalto-oligosaccharide syrups with different fructose percentages of 9%. Table 16 shows that the isomalto-oligosaccharide compositions remain almost unchanged during the isomerization process.

TABLE 16 EXAMPLE 9 Increase in the sweetness of isomalto-oligosaccharide syrup by hydrolase conversion This example illustrates the action of a hydrolase, in this case glucomilasa of A. niger, on a syrup of isomalto-oligosaccharides. A syrup rich in isomalto-oligosaccharides (80% dry solids, pH 4) is sent through a conjugate of immobilized glucomylase at approximately VL / h. Table 17 shows the change of the oligosaccharide spectrum.

BOX _¡ L7 DPI DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP9 DP10 ÜP11 + Substrate 22.1 23.7 23.3 9.6 4.2 2.3 1.9 1.7 1.1 0.6 9.4 Product 39.7 20.4 15.6 7.2 3.3 1.9 1.5 1.0 0.8 0.5 7.8 Dextrose is clearly produced, while decreasing the oligosaccharides. Table 18 shows the change in content of isomalto-oligosaccharides.

TABLE 18 Table 18 shows that a significant amount of amaltose, maltatrose, maltatetraose and DPn fraction has been reduced to glucose. Of course, the hydrolytic reaction can also be conducted with less dry solids, for example with 30% dry solids, as shown in Table 19.

TABLE 19 Days Substrate 1 2 3 4 5 6 7 8 9 10 11 BV / h No syrup 8.3 8.7 9.5 9.5 11.2 11.8 13 14.2 15.2 15.6 16.4 DPI 27.3 41.8 40.8 39.8 39.1 39.2 38.7 37.7 36.6 36.5 35.8 35.6 DP2 26.2 24.8 25.1 25.3 35.4 25.4 25.5 25.5 26.0 25.8 26.0 26.0 DP3 22.3 19.7 19.9 20.1 20.3 20.2 20.3 20.5 20.6 20.7 20.6 20.6 DP4 10.5 7 7.3 7.6 7.7 6.7 7.7 8.0 8.2 8.3 8.5 8.6 DP5 4.5 2.5 2.6 2.7 2.7 3.2 2.9 3.1 3.1 3.2 3.3 3.4 DP6 2.0 0.9 1 1.0 1.1 1.3 1.1 1.2 1.3 1.2 1.3 1.3 DP7 1.3 0.4 0.4 0.5 0.5 0.6 0.5 0.6 0.6 0.6 0.6 0.7 DP8 0.9 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.4 0.4 0.4 0.4 DP9 0.6 0.2 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 DP10 0.3 0.1 0.1 0.1 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.2 DP11 + 4.0 2.3 2.4 2.3 2.5 2.6 2.7 2.7 2.7 2.8 3.0 2.9 Days 12 13 14 15 18 19 20 21 22 25 26 27 BV / h 1.3 2.3 2.3 2.4 2.3 recirrecirreir 1.3 1.3 0.9 culation culation culation DPI 49.1 48.9 49.1 48.9 48.6 59.9 64.9 67.9 69.4 50.9 54.4 54.5 DP2 24.3 24.4 24.3 24.4 24.4 24.8 24.4 23.6 22.7 24.4 24.5 24.5 DP3 16.9 16.9 16.9 17.0 17.0 9.4 7.5 6.3 5.7 15.7 13.6 13.5 DP4 5.2 5.3 5.2 5.3 5.3 2.8 1.8 1.4 1.2 4.7 4.1 4.0 DP5 2.0 1.9 1.8 1.8 1.9 1.5 0.8 0.6 0.8 1.9 1.5 1.5 DP6 0.5 0.5 0.5 0.5 0.6 0.4 0.2 0.1 0.3 0.5 0.4 0.4 DP7 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.0 0.2 0.2 0.2 DP8 0.1 0.1 0.1 0.1 0.1 O.l 0.0 0.0 0.0 0.1 0.1 0.1 DP9 0.1 0.1 0.1 0.1 C.l 0.0 0 0 0.0 0.0 0.0 0.1 0.1 DP10 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 DP11 + 1.6 1.7 1.7 1.7 1.7 0.0 0.3 0.0 0.0 1.6 1.0 1.2 Table 19 clearly shows that a decrease in flow velocity leads to a greater release of the DPn fraction. This is also exemplified in Table 20.

TABLE 20 By adjusting the flow rate, a maximum amount of DPn fraction can be degraded, without too much hydrolysis of the isomalto-oligosaccharides (Table 21).

TABLE 21 i -o -a- r-. co - * a - co s * - o ^ ^ - co co t • r-i cn o > -a * co Or co co so so 0 0 - »a- - ** - * a * co co co s or r-. EXAMPLE 10 Immobilized pullulanase followed by an immobilized ransglucosidase A) Preparation of immobilized pulylanase 5 ml of Duolite A568 was washed with demineralized water to remove the fine particles. The resin was subsequently conditioned with 0.15 ml of Na2CO3 to 1M. 7.5 grams of Otpimax 300L- was added, followed by the addition of 0.008 ml of glutaric dialdehyde solution (25% w / v) ml of supernatant. The mixture was stirred gently overnight at room temperature. The conjugate produced was rinsed with demineralized water and placed in a double cover glass column at 50 ° C. A maltose syrup with 30% dry solids, adjusted to a pH of 4.2, was pumped through the column at an initial flow rate of 6 VL / h (bed volumes / hour). The temperature of the column was maintained at 50 ° C. Table 22 clearly shows the debranching activity of the conjugate. The debranched maltose syrup was pumped through a transglucosidase conjugate in distilled form, as described in B).

TABLE 22 B) Preparation of immobilized transglucosidase 5 ml of Duolite A568 were washed with demineralized water to remove the fine particles. The resin was subsequently conditioned with 0.15 ml of N 2 C 3 to 1 M. 1 g of Transglucosidase L "Amano" was added, followed by the addition of 0.08 ml of glutaric dialdehyde solution (25% w / v). The mixture was stirred gently overnight at room temperature, the conjugate produced was rinsed with demineralized water and introduced into a double cover glass column at 50 ° C. The syrup produced by the pulolanase conjugate was pumped through the column at an initial flow rate of 6 VL / h. (bed volumes / hour). The temperature of the column was maintained at 50 ° C. Table 23 shows the production of isomalto-oligosaccharide syrup.

TABLE 23 EXAMPLE 11 Regeneration of the resin and recharge with the enzyme A) 10 ml of Duolite A568 ™ was washed with demineralized water to remove the fine particles. The resin was subsequently conditioned with 0.3 ml of 1M Na2CO3. 2 g of Transglucosidase L "Amano" (liquid preparation, 11.2 mg of protein / gram of enzyme solution, 103 TGU / gram of enzyme solution) was added and the mixture was stirred for 4 hours at room temperature. Subsequently, 15 g of pullulanase (Opti ax L300 ™ from Genencor Int., 2.6 mg protein / g enzyme solution, 400 PU / g enzyme solution) and immobilization was continued for another 4 hours. 5 ml of a 1.0% glutaric dialdehyde solution was then added to give a 0.2% glutaric dialdehyde solution and the resin was stirred overnight at room temperature. The conjugate produced was rinsed with demineralized water and placed in a double cover glass column at 50 ° C. A maltose syrup with 30% dry solids, adjusted to a pH of 4.2, was pumped through the column at a flow rate of 10 VL / h (bed volumes / hour). The temperature of the column was maintained at 50 ° C. The Iconjugado was treated for 30 days producing the 43/45% of isomato-oligosaccharides at 3 VL / h. B) The conjugate was subsequently transported to a beaker and washed with water to remove sugars and fine particles. The pH of the resin was adjusted to 1.5 and the resin was stirred for 1 hour at 60 ° C. The pH was then raised to 12.5 with NaOH and stirring was continued for 1 hour while maintaining the pH at 12.5. The conjugate was then washed with preliminary dismeralized water, the fine particles. C) Procedure A) was repeated on the regenerated resin. The same amount of enzyme was mobilized and the new conjugate produced a 43-45% isomalto-oligosaccharide syrup at the same flow rate described in A.

Claims (9)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for producing a syrup containing isomalto-oligosaccharide characterized in that a starch hydrolyzate is converted by a transglucosidase enzymatically and a reusable vehicle is used for the immobilization of the glucosidase.

2. A method according to claim 1, further characterized in that the starch hydrolyzate has an ED of between 4 and 70, preferably between 20 and 60.

3. A method according to claim 1, further characterized in that the vehicle is an anion exchange resin and the transglucosidase is immobilized thereon by adsorption.

4. A method according to claim 1, further characterized by immobilizing other enzymes with transglucosidase, so that other enzymes are selected from the group consisting of pullulanases and alpha-amylases and because such co-immobilization is performed by one or more separate vehicles.

5. A method according to claim 1, further characterized in that the vehicle / enzyme conjugate is further reinforced by reaction with an entanglement agent.

6. A method according to claim 5, further characterized in that the entanglement agent is glutaric dialdehyde.

7. A method according to claim 1, further characterized in that the isomalto-oligosaccharide contains more than 40% isomalto-oligosaccharides, preferably more than 45% and because these values are achieved with a flow velocity of at least 30. bed volumes per hour and at least for a period of 25 days.

8. A method according to any of the preceding claims, further characterized in that the isomalto-oligosaccharide syrup is further refined, ie it is treated by chromatographic means or by filtration.

9. A method according to claim 1, further characterized in that during the production of the isomalto-oligosaccharide syrup or then the sweetness is increased by the addition of a sweetener or by an additional enzymatic conversion with glucose isomerase or a hydrolase.