FI64642C - FRAMEWORK FOR FRUIT PROCESSING IN ENCLOSURE FRUIT INSIDE PRODUCTS - Google Patents

Description

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Patent Office of the United States of America Patent Application No. 31-0O-Ö3 (32) (33) (31) Privilege — Begird Prlorltet 16.06.77 U.S. Pat. No. 807,289 (71) CPC International, Inc., International Plaza, Englewood Cliffs,

New Jersey 07632, USA (72) Robert E. Heady, Argo, Illinois, USA (7 * 0 Oy Kolster Ab (5 * 0 Method for preparing a fructose-rich saccharide product - Förrarande för framställning av en rikligt fruktos in This invention relates to a process for preparing a saccharide product containing at least 55% by weight of fructose, which process comprises the following steps: 1) treating sucrose to form a mixture containing glucose, fructose and polysaccharides, 2) optionally treating the mixture formed in 1) with an isomerase enzyme, glucose isomerizes to fructose, and 3) hydrolysis of the polysaccharides formed in 1) under conditions in which no active isomerase enzyme is present.

The process according to the invention is characterized in that in step 1) sucrose with an initial content of at least 10% (w / v) is treated with the enzyme fructosyltransferase obtained from the microorganism Pullularia pullulans at a temperature of 25 to 65 ° C

2,64642 and at pH 4f5 to 6.5 to obtain a product comprising glucose, fructose and polysaccharides, wherein the polysaccharides contain at least 66% by weight of fructosyl moieties and wherein the fructosyl moieties are linked by {2 -1) - / 2> bonds.

This method provides a new enzymatic way to prepare fructose-rich syrups with a significantly higher fructose content than is achievable by glucose isomerization of starch hydrolysates without having to physically separate the resulting fructose end product. This method is particularly suitable for the preparation of fructose-rich fructose syrups.

Numerous products can be prepared by the method according to the invention. These include predominantly or high fructose syrup and various intermediates such as fructose polymer, primary fructose polymer (or polysaccharide) -containing substrate, polymer-depleted substrate, and substrate (with or without polymer) after isomerization. Each of these products has the properties of conventional sugars and syrups and can be used in their conventional applications. These include, for example, use as food sweeteners and raw materials in the manufacture of pharmaceuticals. They can also be used in the manufacture of binders, wetting agents, polished parchment paper, tanning agents, electrical insulators, foundry core binders, insecticides, dyes and the like, or most commonly plasticizers, thickeners, etc.

Definitions

Due to the varying terminology commonly used in the art, the following definitions of the meaning of the terms used are given.

Glucose and dextrose

The terms "glucose" and "dextrose" are both used in this application to include this monosaccharide in any form in solution or dry.

sucrose

The term "sucrose" refers to this disaccharide, in purified or untreated form, in solution or dry, which disaccharide is derived from any source of sucrose raw material, 3,646,442 e.g. sugar cane or sugar beet. In the practice of this invention, the sucrose starting material is typically used in an aqueous medium.

Fructose and levulose

The terms "fructose" and "levulose" are both commonly used in the art to refer to an isomer that is sweeter than glucose. Fructose is found in honey and invert sugar with glucose, and fructose is valuable because of its sweetness. The terms levulose and fructose are both used in this publication to denote this monosaccharide in any form, solution or dry.

The enzyme preparation

The term "enzyme preparation" is used herein to mean any composition of matter that exhibits the desired enzymatic activity. The term is used to denote, e.g., living whole cells, dry cells, cell extracts, and purified and concentrated preparations derived from cells and culture solutions. Enzyme preparations can be used either in solution or in immobilized form in the practice of this invention.

isomerase enzymes

Any enzyme preparation that isomerizes glucose to fructose is referred to herein as an "isomerase enzyme." These enzymes are well known in the art and are referred to as dextrose isomerase, xylose isomerase, and glucose isomerase. These enzymes can be derived from a variety of suitable microorganisms, examples of which are the genera Streptomyces, Bacillus, Arthrobacter, Actinoplanes, and Curtobacterium.

A preferred isomerase enzyme useful in the practice of this invention is derived from Streptomyces Olivo-chromogenes strain ATCC No. 21,713, ATCC No. 21,714, or ATCC No. 21,715 (the latter being an isolate of a single colony of ATCC No. 21,713). , as disclosed in U.S. Patent No. 3,813,318 and U.S. Patent No. 3,957,587, especially as described in U.S. Patent No. 3,770,589 or U.S. Patent No. 3,813,318.

Recently, methods for immobilizing an isomerase enzyme on a water-insoluble 64642 4 inert carrier have begun to be valued in the art. The immobilized enzyme is then suitable for use in the continuous conversion of glucose to a fructose-rich syrup. Examples of such methods are described in U.S. Patent Nos. 3,708,397, 3,788,945, 3,850,751, 3,868,304, BE Patent No. 819,859, and U.S. Patent No. 3,960,663 (BE Patent No. 810,480).

Isomerase Unit An "isomerase unit" is defined as the amount of enzyme activity required to produce one micromole of fructose per minute under isomerization conditions, hereinafter described under the heading "Determination of Isomerase Activity".

Determination of Isomerase Activity As used herein, this term refers to an assay procedure involving the spectrophotometric determination of ketose prepared from a glucose solution under standardized conditions. The stock solution is prepared as follows: Stock solution for analysis

Component Amount 0.1 -m MgSO 4. 7 H 2 O 1 mL 0.001-m CoCl 2. 6 H 2 O 1 ml 1-m sodium phosphate buffer, pH 7,5 0,5 ml anhydrous D-glucose 1.44 g of distilled water is added to a total volume of 7,5 ml. The enzyme preparation to be determined is first diluted to 1 to 6 isomerase units per ml.

Enzymatic isomerization is performed by adding 1 ml of enzyme preparation to 3 ml of stock solution and incubating for 30 minutes at 60 ° C. At the end of the incubation period, an aliquot of 1 mol is taken and stored in a volume of 9 ml of 0.5% perchloric acid. The stored aliquot is then diluted to a total volume of 250 ml. As a check for comparison, a blank test for glucose is also performed by replacing the incubation of the enzyme preparation in solution with 1 ml of water with 1 ml at the beginning of the period.

5,64642

Ketose is then determined by the cysteine sulfuric acid method. For purposes of this assay, one unit of isomerase is defined as the amount of enzyme activity required to produce one micromole of levulose under the isomerization conditions described.

Transfructosylation As used herein, the term refers to the transfer of a fructosyl moiety from a donor, e.g., sucrose, to a recipient, e.g., a polysaccharide,

Fructosyltransferase Enzyme As used herein, the term refers to any enzyme that catalyzes transfructosylation and includes an enzyme preparation derived from the strain Pullularia pullulans ATCC 9348 (synonymous with the strain Aureobasidium pullulans).

Fructosyltransferase unit As used herein, one fructosyltransferase unit is defined as the amount of enzyme activity required to produce one micromole of reducing sugar, calculated as glucose, per minute under the following conditions: (a) pH 5.5, (b) temperature 55 ° C and (c) substrate concentration 60 g per ml of aqueous reaction mixture,

Reducing sugar assays (calculated as glucose) are performed using a Technicon Autoanalyzer II (Tech-Nicon, Inc., Tarrytown, NY). The analysis is performed by a conventional alkaline-ferricyanide method modified for use with this instrument, Analytical Biochemistry 45, n: o 2, pp. 517-524 (1972). Unless otherwise stated, determinations of enzyme activity are carried out by continuous monitoring of a reaction mixture having the following composition: 7.5 ml of 8C% (w / v) food grade sucrose-aqueous solution 2.3 ml of 0.1- m citrate buffer, pH 5.5 A 0.2 ml enzyme sample containing the amount of fructosyltransferase enzyme that produces 5-25 ug of reducing sugar (calculated as glucose) per minute per 1 ml of reaction mixture per- -f- -L-_ ·

Primary Substrate As used herein, the term "primary substrate" refers to those saccharides that are in a suitable form and whose fructosyl moiety is available to participate in transfructosylation, namely, aqueous solutions of sucrose.

Secondary Substrate As used herein, the term “secondary substrate” is a reaction product that results from the action of a fructosyltransferase enzyme preparation as defined above on the primary substrate.

Parts and percentages In this application, all parts are by weight and all percentages are by weight (w / v), unless explicitly stated otherwise,

high pressure

The term as used herein defines a procedure in which the syrups of the invention are analyzed using high performance liquid chromatography according to the following procedure. The components are isolated by chromatography eluting with water from a cation exchange resin in the form of calcium. The eluted components are detected by a differential refractometer. Carbohydrates other than dextrose are quantified using an electronic integrator, and dextrose is obtained by separation. The general procedure is described in "Analysis of Carbohydrate Mixtures by Liquid Chromatography", Am, Soc, Brew. Chem. Proc., 1973, pp. 43 - * + 6. The resin used is "Aminex Q" in the form of calcium 15-5 , edited by Bio-Rad Laboratories, Richmond, California.

The process for preparing syrups according to the invention comprises reacting a primary substrate, namely sucrose, with a fructosyltransferase enzyme preparation capable of converting sucrose into a product consisting of a monosaccharide fraction containing a higher amount of glucose and a lower amount of fructose fructose and at least 66%. The product is a secondary substrate of this invention. Secondary substrate polysaccharides are all fructose-containing polymers (other than sucrose) having two or more monosaccharide moieties. These polymers can be further characterized as polysaccharides containing (7-4) -β-linked fructose groups. As described below, trans f ruktosy '! By adjusting the conditions of oinn i., the desired polysaccharides can be prepared. Thus, for example, a secondary substrate can be prepared in which 7,64642 most (e.g. at least 60% in molar ratio) of the polysaccharides are 2-10 (i.e., OP 2--10), most usually 3-6 (i.e., DP 3-6) monosnk carid moieties containing oligomers. Said glucose is then isomerized (by the action of an isomerase enzyme) to fructose in the presence of said polysaccharide, followed by hydrolysis of said polysaccharides under a condition in which no active isomerase enzyme is present. The hydrolysis can be carried out enzymatically using ir.

In a further embodiment of the invention, the polysaccharides can be separated from glucose and fructose and then hydrolyzed, as described above, to prepare a syrup with a high fructose content, e.g. more than 66% by weight and preferably more than 90% by weight, directly from the secondary substrate of this invention without isomerization. glucose in the substrate.

The separation can be conveniently performed by conventional molecular size-based physical separation techniques, such as membrane techniques (e.g., ultrafiltration, dialysis), solvent precipitation, carbon absorption, and the like. Examples of the use of membrane technology include U.S. Patent Nos. 3,177,867; 3,691,068,

A preferred embodiment is a process for the preparation of fructose-rich syrups, which process comprises reacting sucrose with a preparation of the enzyme fructosyltransferase derived from the microorganism Pullularia pullulans, - such as ATCC 9348, ATCC 12 53 5, NRRL 1573, NRRL Y2311 , NRRL YB3892, NRRL YB3861, NRRL 3937 and ATCC 15223. The resulting product or secondary substrate is allowed to act on the isomerase enzyme. The isomerized product is then hydrolyzed so that no active isomerase enzyme is present. The secondary substrate prepared by the process of this invention is a novel product. This substrate is uniquely suited for isomerization and subsequent hydrolysis to produce a syrup containing more than 55% fructose, and the substrate is prepared by reacting a preparation of fructosyltransferase enzyme 8,64642 with sucrose. Substrates are suitable for contain (1) from about 20% to about 60% by weight of monosaccharides containing a higher proportion of glucose and a lower amount of fructose, and (2) from about 70% to about 40% of polysaccharides containing more than 66% by weight of fructosyl moieties.

Particularly preferred are secondary cesubstrate4- derived from sucrose by transfructosylation in the presence of an effective amount of a fructosyltransferase enzyme preparation derived from Pullularia pullulans ATCC 9348 at a temperature ranging from about 25 ° C to about 60 ° C and at a pH of ranges from about 4.5 to about 5.5, and preferably from about 5.4 to 5.6. The concentrations of sucrose initially used can be as low as 10 g per 100 ml of water. However, it is preferred to use the highest possible dry matter content, preferably about 30 to 60 g per 100 ml.

A minimum amount of 0.5 fructosyltransferase units per 1 g of sucrose can be used to produce new substrate. In general, the amount of enzyme used does not exceed 50 units per 1 g of sucrose, due to the cost of the enzyme. In particular, it is preferred to obtain the desired secondary substrate in a sufficiently short time, so that in practice the amount of enzyme is 30 units. 1 g per sucrose.

Fructosyltransferase is prepared by conventional fermentation techniques, cf. e.g., U.S. Patent Nos. 3,565,756, 3,806,419, and 3,535,123, and S, Ueda et al., Applied Microbiology, 11, 211-215 (1963). cleaning procedures described in more detail below. The following example is a typical fermentation procedure used to prepare an enzyme from Pullularia pullulans strain ATCC 9348.

Example 1

Production of the enzyme fructosyltransferase

Preparation - Celite-kantoalne A. Fermentation method used to prepare the enzyme ΰχ

The medium used for the inoculation and fermentation of the inoculum required for the preparation of the enzyme is as follows; 0.5% dibasic potassium phosphate 0.1% sodium chloride 0.02% magnesium sulphate heptahydrate 0.05% diammonium sulphate 0.3% yeast extract (Difco Laboratories) 7.5% sucrose (food grade) The pH of the medium is adjusted to 6.8 per 100 ml of sterile medium 500 ml Erlen-Meyer flasks are inoculated with an oblique culture of black yeast Pullularia pulluians. The yeast strain used is designated ATCC 9348 in the American Type Culture Collection (Rockville, Maryland). The inoculum content used is 0.5% w / v. The fermentation is carried out on a reciprocating shaker at 32 ° C for seven days, B, Enzyme recovery from the fermentation broth The fermentation broths from the forty 1 liter shake flasks are combined and the flasks are rinsed with water, which is also added to the combined broth. The final volume of the broth after dilution is 12 liters, the initial volume of the broth is about 8 liters. 12 liters of fermentation broth are passed through a continuous Sharples centrifuge to remove yeast cells and cellular debris. The supernatant, which is a black viscous solution, is then adjusted to 0.5% (w / v) with calcium chloride and the pH of the resulting solution is adjusted to 7.0 with sodium hydroxide. The second step is then performed with a Sharples centrifuge to obtain a viscous solution on the precipitate with a faint color. The pH of the solution on the decolorized precipitate is adjusted to 5.5 with hydrochloric acid, and 1,000 units of pullulanase (as defined in U.S. Patent 3,806,419) are added. The broth thus obtained is stored with toluene (added until saturation) and the pullulanase is reacted at room temperature. overnight. After overnight digestion with 10 64 64 2 pullulanase, 1% filtration aid Grefco J & fc 503 Celite diatomaceous earth, manufactured by Johns-'Manville Products Corporation, Lompoc, California, is slurried in broth, followed by the addition of acetone 2 (24 liters). A precipitate forms and is collected by filtration, the filter cake is washed with acetone and dried at ambient temperature. The collected filter cake contains immobilized fructosyltransferase enzyme.

In Example 1, it is significant that the addition of calcium chloride to the fermentation broth and the adjustment of the pH of the broth result in the removal of the black pigment and the acidic polysaccharides present (see Acta Chem. Scan, 16 (1962)). The final enzyme product is thus obtained in the form of a relatively pure, colorless preparation. This cleaning method allows simple cleaning procedures, i. centrifugation, filtration and precipitation to obtain the final product.

; It is also desirable to remove the pullulan polysaccharide naturally present in the fermentation broth because, like the black pigment and acidic polysaccharides, it co-precipitates with the enzyme fructosyltransferase when the solution is added to the fermentation broth; unit. Therefore, the supernatant solution obtained by treatment with calcium chloride and pH adjustment can be further treated with a well-known hydrolysis enzyme, pullulanase. The pullulanase enzyme randomly hydrolyzes pullulan to produce a low molecular weight polymer, and thus avoids fructosyltransferase with the preparation of the fructosyltransferase enzyme during solvent treatment (e.g., acetone, alcohol, and the like); precipitation and related contamination.

The resulting fructosyltransferase enzyme can be used without removing pullulan. Accordingly, the present invention can be practiced without the hydrolysis of pullulanase by pullulanase, in which case pullulan acts as a carrier for the enzyme fructyltransferase,

The following example illustrates the use of pullulan as a carrier.

Example 2

Preparation of secondary substrate using fructosyltransferase enzyme on pullulan carrier 11,64642 in 20 μl of sucrose solution buffered with 0.05 m citrate buffer to ρΗτ 5.5. a 1% concentration of dry pullulan prepared according to the procedure of Example 1 is added, except that the pullulan is not hydrolyzed by pullulanase and therefore acts as a carrier and Ce-lite is not used. The fructosyltransferase activity is 577 units / g pullulan. The reaction is carried out at ambient temperature until the mixture becomes cloudy. A sample of this mixture was analyzed using high performance liquid chromatography to give the following results:

Saccharide distribution determined by high performance liquid chromatographic analysis

Fructose Glucose DP ^ DP ^ DP ^ + 6.9% 40.6% 6.2% 11.1% 35.2%

The following examples further characterize the enzyme prepared in Example 1.

Example 3

Enzymatic products and thermal stability of the enzyme

To the reaction flasks with screw caps are added 60 g of food grade sucrose and the fructosyltransferase enzyme preparation. The enzyme preparation is obtained from the enzyme product of Example 1 by dispersing appropriate aliquots of the solid enzyme product in measured amounts of water to prepare a suitable concentrated enzyme solution. The filter aid is then removed by filtration. The filtrate is added to the reaction mixture in an amount of 10, 20 and 30 units of enzyme per 1 g of sucrose substrate. Each of the mixtures is then diluted to a final volume of 100 ml with water. The conversion reactions are performed at pH 5.5 at 55 ° C or 60 ° C, respectively. Samples are taken after 24, 43 and 66 hours for reducing sugar determinations to ensure the presence of enzymatic activity. After the reducing sugar determinations of the samples are made, the remaining reaction mixtures are cooled to stop the enzymatic action, and the samples are assayed for the carbohydrate composition by high performance liquid chromatography. The following results were obtained:

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Example 3 shows the thermal stability of the enzyme in the presence of a substrate at 55 ° C and 60 ° C for a period of 66 hours at pH 5.5; In addition, the new fructosyltransferase preparation by enzymatic modification of sucrose shows in hydrolysis give fructose as the major monosaccharide. This example also demonstrates that the novel enzyme used in the method of the invention is effective at a sucrose content of 60% (w / v). The activity of the enzyme in the presence of high glucose concentrations is also demonstrated.

Example 4 Effect of temperature on enzyme activity The effect of temperature on the reaction rate of the fructosyltransferase enzyme is determined using a Technicon Autcanalyzer as follows:

The reaction mixture consists of 7.5 ml of 80% (w / v) sucrose solution, 2.3 ml of 0.1 M citrate buffer at pH 5.5 and 0.2 ml of 2% (w / v) / volume) of pullulan enzyme solution (enzyme preparation of Example 2). The final sucrose content is 60% (w / v). Samples are maintained at the following temperatures and analyzed for 10 minutes on a Technicon Auto-Analyzer II. The results show that at 40 ° C the enzymatic rate of the reaction is 1.89 times the rate at 30 ° C, at 50 ° C 1.29 times higher than at 40 ° C and 60 ° C 1, 48 times higher than at 50 ° C. This indicates an increasing reaction rate with increasing temperature.

Example 5

Illustration of the Michaelis-Menten constant (K) of the fructosyltransferase enzyme m

The Kjr of the enzyme denotes the concentration of the substrate at which the Turr 'snopous form of the product is half the value of V.

Ly'i ty-1 i! r, fruk toayy litrancferase enzyme constant K ash - :; nkn i.

15 64642 Using a 90% (m / v) stock solution of sucrose adjusted to pH 5,5 with 0,1 m citrate buffer, prepare appropriate concentrations of sucrose in 9.8 ml aliquots to give 5, 10, 30, 40, 50, 50 and 70% sucrose contents in a final volume of 10 ml. A 0.2 ml enzyme solution containing 1.1 units of fructosyltransferase enzyme is added to the separated samples. The samples are then immediately analyzed at 55 ° C on a Technicon Autoanalyzer II as previously described (see fructosyltransferase unit). The glucose standard (calibrated in units / μg / ml) is included as a control.

The following is the rate of glucose formation expressed in units> μg / ml / minute at various substrate concentrations using the standard dosage (1.1 units) of the fructosyltransferase enzyme preparation of Example 1. ·% substrate μg / ml / min pg / ml / min ^ 5 8 .0 8.0 10 11.8 11.6 20 15.7 15.2 30 18.0 17.6 40 18.6 18.2 50 19.2 17.2 60 17.8 18.2 70 15, 6 a) Run again the next day using 60% sucrose stock solution.

K is the 0.27 m sucrose content. The maximum reaction rate occurred at a substrate concentration of 1.374 molar with respect to sucrose, at pH 5.5 and 55 ° C.

Example 6

Preparation and isomerization of secondary substrate from sucrose.

A. Preparation of Secondary Substrate

Dissolve 600 g of food grade sucrose in water to a volume of 800 ml. The pH of this solution is adjusted to 5.5 with dilute is 6 4 64 2 - 1 Ί-Ί I "i * i '“' --- p. 1 ig dry C e 1 it e - enzyme preparation with ak- 'iVitgs -J .; CC'- I, cn y ^ sxkkoa / g and prepared as exemplified by ^ J. o S ^ ^ ^ ^ # # · «* is read as JOG per ml of water The slurry is filtered off with suction λ atmg - > 1 η '· o 1 using sucdat paper in a Bdchnsr funnel. Add a further IQo ml of water to the filtrate. Add 200 ml of the filtrate to a solution of sit-, en -9 \ carcose in a 1.9 l screw-capped 5 · Pu.lo is then placed in a water bath at 58 ° C, allowed to continue for 20 hours, after which the reaction product Γ. 3 V t P -¾,, -Pöiy is used to determine KcrkRaoa i n en es 12 kr chromatograph 1 la carbohydrate- * 0- s -Umj and to obtain the results obtained.

Carbohydrate composition

Fructose 2.4%

Glucose 32.8% 0p2 10.6% 0P3 22.9% 0P4 31.3% Magnesium chloride is added to the remaining reaction product (i.e., secondary substrate) to a concentration of 5 millimolar, and the pH is adjusted to 8.4 with dilute sodium hydroxide. .

B. Continuous isomerization of the secondary substrate

Glucose isomerase derived from Streptomyces oivochromogenes ATCC 21,715 (see U.S. Pat. No. Pe 29,152) is immobilized on porous alumina by Corning Glass Co., Corning, Dew York, (see U.S. Patent No. 3,992,329). as follows: 1. The carrier is washed twice with water. 2. The carrier is incubated with 0.1 M sodium citrate for one hour with stirring.

4. Incubate the vehicle with U, 05-m magnesium chloride for one hour and pellet the magnesium chloride solution.

5. 1 t .i lunnusa Π .0B-m magn os Iumklorid ia, wherein un t; y ym! n I «nu 1! ! n ti kr. : p to ί nootinko i inlet 4ΡΠ yku: kkö / ml, 17 64 64 2 6. The isomerase enzyme concentrate is added to the support at a concentration of 494 units / ml 7. The carrier and the enzyme are kept in contact for 22-24 hours and the unbound enzyme is washed with distilled water from the support.

A jacketed glass column (3 cm x 18 cm) equipped with a pump connected to the feed tank is charged with the immobilized enzyme thus prepared. After loading, the volume of the enzyme layer is 45 ml. The column operates at B0 ° C for all isomerizations. The charged column is started with glucose syrup [50% w / w) and 0.005 M magnesium chloride, pH 8.4, and the column is found to be active. The flow rate is adjusted to 292 ml / hour. The solution is then removed from the column to the bed level. The introduction of the secondary substrate is performed manually until 20 ml of effluent has accumulated, and then the flow rate of the secondary substrate is adjusted to 292 ml / hour. The first 100 ml of accumulated syrup is discarded to allow the substrate to change. The remaining secondary substrate (about Θ50 ml) is then passed through the column. After one hour, the flow rate rises to 570 ml / hour. The flow rate is adjusted and maintained at 300 ml / hour until the end of the run. After the run on the secondary substrate, the column is switched back to glucose and it is found that the isomerase activity is maintained. The following table compares the high pressure liquid chromatography analysis of the starting secondary substrate and the final product of the isomerization column:

Secondary substrate (A) End product (B)

Fructose 2.4% 15.7%

Glucose 32.8% 18.9% DP2 10.6% 11.0% DP3 22.9% 22.9% DP4 + 31.3% 31.5% 18,64642 These results show that 42.38% of the free substrate of the secondary substrate from glucose isomerized in the column to fructose. Also, the fructose polymer preform present in the secondary substrate did not appear to be affected by the isomerization of glucose to fructose and these polymers did not affect the isomerization. It should be noted that the total distribution of monosaccharides consists of about 45% fructose and 55% glucose.

In the following example, two methods are used to decompose the polysaccharides present in the secondary substrate and also in the isomerization product prepared therefrom. One tao is enzymatic and the other is mild acid hydrolysis.

Example 7

Preparation of a fructose-rich syrup by hydrolysis A. Enzymatic hydrolysis 100 ml of the product from Example 5 are mixed with purified invertase from Candida utilis. This mixture (preserved with toluene) is allowed to react overnight at ambient temperature, after which a sample is taken and analyzed by high performance liquid chromatography. The remaining sample 3 is allowed to react for a further six days and the second sample is analyzed by the same procedure with the following results: Starting material After inversion of the invertase after six days (product B).

Fructose 15.7% 41.9% 62.4%

Glucose 18.9% 29.4% 36.2% 0p2 11.0% 1.9% 0 DP3 22.9% 12.9% 0.5% ΠΡ4 + 31, S% 13.9% 0.9% Used invertase is an enzyme preparation manufactured by Siekagu Kogyo Co., Ltd., Tokyo, Jaoani, with a specific activity of 123 units / mg protein, and one invertase unit catalyzes the cleavage of sucrose to form 1 micromole of glucose and 1 micromole of fructose per minute under standardized conditions.

19 64642 B. Acid hydrolysis of product B.

Acid hydrolysis of a sample of the product of Example 6 was performed by adding sulfuric acid to a concentration of 0.05 n and heating to 75-8 ° C. Samples are taken after one and two hours of hydrolysis and analyzed by high pressure liquid chromatography. The results are as follows: Starting material (product B) 1 hour 2 hours

Fructose 15.7% 60.3% 59.1%

Glucose 18.9% 38.7% 37.9% DP2 11.0% 1.9% 2.6% DP3 22.9% 0.4% 0.3% DP4 + 31.5% 0% 0.2%

The above results of step A show an increase in the prevailing fructose yield and a smaller increase in glucose yield, which occurs with a corresponding decrease in the fractions of DP2, DP1 and DP4 +, thus indicating the presence of the fructose polymer.

The results of the acid hydrolysis of step B are in agreement with the results of step A, thus also showing the fragmentation of the fructose polymers.

The above discussion is directed to transfructosylation using a primary substrate in which the dry matter content of the starting sucrose does not exceed the boiling point under the given reaction conditions. The following example demonstrates the use of a primary substrate which has an initial sucrose content above saturation and which, under the influence of the enzyme fructosyltransferase, results in a product containing improved amounts of DP4 material (i.e. fructosyl sucrose) and reduced DP4 + products. An increase in the dry matter content (w / v) of the secondary substrate compared to the dry matter content obtained in the absence of the enzyme fructosyltransferase is also shown. In addition, it is shown that as the dry matter content of the primary substrate increases, the degree of polymerization of the fructose polymer of the secondary substrate decreases and the DP 2+ material is present in smaller amounts.

20 64642 This is a significant contrast to the results obtained in the previous examples using sucrose substrates under saturation. In those examples, DP ^ + ^ material is the main product. Example 8

Preparation of secondary substrate from sucrose slurries with a concentration above saturation point

Protein-grade sucrose (200 g aliquots are placed in 0.5 l containers with screw caps. For comparison, 50 ml of water is added to one vessel and 50 ml of water containing increasing amounts of fructosyltransferase enzyme (prepared according to Example 1) is added to each other. ), followed by the following table: Each vessel is sealed and placed in a stirring water bath set at 54 to 55 ° C, shaken for 24 hours with occasional agitation, and each bottle is sampled with the supernatant and placed in a boiling test tube with a screw cap. in a water bath to inactivate the enzyme. The samples are then analyzed by high pressure liquid chromatography and the dry matter of the liquid on the precipitate is determined using the method of K. Fischer / (see Kirk-Othmer, Encyclopedia of Chemical Technology, Vol. 2, 2nd ed. pages 673 and 674, and Angewandte Chemie 48, (1935), page 394) · Results obtained are shown in the following table.

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It should be noted that in the above example, the reference substance crystallizes on cooling to ambient temperature (about 25 ° C), while the enzymatically prepared secondary substrates remain in solution and have excellent storage stability (do not crystallize).

The secondary substrate can be used as a very stable, non-crystallizing syrup in food applications. It is a uniquely high-dry composition that is resistant to microbial contamination and dye formation.

The new very dry secondary secondary substrate can be hydrolyzed as shown in Example 7 to give i. An overt sugar mixture with the desired level of sweetness; this substrate has a dry matter content of about 70-82% (w / w) and the substrate consists essentially of glucose and polysaccharide dipolymers, D 2 and D 2. A smaller amount (4.1% and less) of DP 2 polymers is also present.

Although the transfructosylation step of this invention has been described in the form of batch unit processes, it will be apparent to those skilled in the art that continuous unit processes may be used as well. In carrying out such continuous transfrucosylation, the transfructosylase enzyme is conventionally immobilized using the technique previously considered for the definition of immobilized enzyme and using the continuous method described therein.

Claims (5)

23 64642 A process for the preparation of a saccharide product containing at least 55% by weight of fructose, which process comprises the following steps: 1. treating sucrose to form a mixture containing glucose, fructose and polysaccharides, 2. optionally treating the mixture formed in 1) with an isomerase enzyme to isomerize glucose fructose, and 3. hydrolysis of the polysaccharides formed in 1) under conditions in the absence of an active isomerase enzyme, characterized in that in 1) sucrose with an initial content of at least 10% (w / v) is treated with Pullularia from pullulans fructosyltransferase enzyme at a temperature of 25 to 65 ° C and a pH of 4.5 to 6.5 to obtain a product comprising glucose, fructose and polysaccharides, wherein the polysaccharides contain at least 66% by weight of fructosyl moiety and the fructosyl moieties are bound ( 2-1) - / 5-linkages. Process according to Claim 1, characterized in that the sucrose content to be treated is at least about 20% (w / v). Process according to Claim 1 or 2, characterized in that the isomerization is carried out using an immobilized glucose isomerase enzyme. Process according to Claims 1 to 3, characterized in that the polysaccharides are physically separated from glucose and fructose before hydrolysis. Process according to Claim 4, characterized in that the polysaccharides are separated by ultrafiltration.