DD140415A5 - METHOD FOR THE PRODUCTION OF SYRUP WITH HIGH FRUIT SUGAR CONTENT FROM CANE SUGAR - Google Patents

Description

The invention relates to a method for the manufacture of syrup _β The invention generally relates to the ensymatischo transfructosylation of sucrose ,, the invention iriötteoondere relates to an exceptional process for the Herntelltmg of Pruotofjo from sucrose by means of a Fruotoeepolymer containing substrate * This process provides a novel en2yrn.atisch.es process for the preparation of syrups with high Fruotosegeha.lt _f represents the Pruotosegehalt is substantially higher than it is achievable by Gluooseißoraerisierung of Stärkehydrolysaton sur time, with a physical separation of entst8.nde.nc5n Pruotose end product is not necessary The invention also relates to a novel trane-fruotosylation enzyme from cultures of yeast,. Pallularia pallulansj, which has proved useful in such a production *,

By the. This may include a variety of products. These include the finished fructose or a high fructose syrup, some numerous intermediates such as Fr.uo'tosepolymor. <3bs substrate containing the initial fructose polymer (or pulp saccharide) from the .that Polymer was removed and substrate (with or without polymer) after isomerization, Each of these products is intrinsically valuable in itself.

2 - Berlin, d.8.2.197.9 APA 23G / 206.028

ci

Each of these products has the properties of conventional Zuokerarten and syrups, and can be used for the usual purposes "These include the use as a sweetener and as raw materials for the usual representation of Pharinazeutika _e Moreover, these products can in industrial use for sugars and syrups used Therefore, you can use _$ 3 for the production of adhesives, 3? ₉ tannins? electrical insulators foundry ^ core binder, inaektiaido, dyes and the like or in general ale plasticizer _for thickening agent, etc. "find application

In the short term, they are used in a wide range of applications where analogue products have already been used. "

Recently, those skilled in the art have discovered methods of immobilizing iBomerasee.iisyra on a water-insoluble inert carrier. The immobilized enzyme is then suitable for use in the continuous conversion of glucose into a high-fructose syrup. Examples of such US-PS 3 7O8 _e 397s 3 * 788 * 945? 3 * 850.751? 3 * 868304? BB-FS 8.19.859 and US-PS 3'96O-663 (BE-PS Ir * 810.480).

The aim of the invention is to provide a simple and economical process for the preparation of syrup with over 55 % fructose «.

Before the nature of its invention is explained in more detail _s , the terms used in the description of the invention should be construed.

Berlin, dV8.2.'1979

AP A 23G / 206 028

W will be. And are common in your area of expertise. * * -., - ^! , .- -

The terms "glucose" and "dextrose" are in this application are used interchangeably ₉ to this mono s acc ha "rid in, any form, in solution or dry au _s label".

The term "sucrose" shall look to a disaccharides in purified or in crude formulas solution or dry, from any sucrose raw material source, 2 * B _S sugar cane or sugar beet * In the practice of the invention, the Saocharose ~ source-material © rial generally in used in an aqueous medium,

Fructose and levulose

The terms "fructose" and "levulose" are generally used interchangeably in the field and refer to the isomer of Destro.se _y which is more egg than dextrose ", fructose occurs in honey and, in. Invert sugar together with dextrose and is due to its Sweet Values The terms "levulose" and "fructose" are used interchangeably throughout this specification to refer to this monosaccharide in any form, solution or dry. "

Dio term "enzyme preparation ^t! Is used here as a term for any composition of matter has he '·' wish-enzymatic activity" The designation is for example in relation to living whole inches ,, ^r I'reckende 1-lon, cell extracts ₉ purified lime concentrated-from the cells

4 - Berlin, 8.2.1979 AP A 23G / 2O6 028

and cells derived from currency liquids. The enzyme preparations may be used either as a solution or in an immobilized form in the practice of the invention. -

Isomeraseens

Each enzyme preparation $ the dextrose too. Lava isomerization site ₉ is referred to herein as "isomerase enzyme". These enzymes are well known in the art and are referred to as dextrose isomerase xylose isoiaerase and glucose isomerase. Such enzymes can be obtained from a variety of suitable microorganisms. Examples of such microorganisms are those of the genus Streptomyces, Bacillus, Artnrobac "ter _s. Aotinoplanes j Curtobaoeterium and other"

A preferred isomerase enzyme which is suitable for the practice of the invention was olivochromogenes of Streptomyoes ATCC No. ₆ 21 713 _S ATCC Kr · 21.714 or ATCC Hr · 21.715 (of which the latter is a single 'colony isolate of ATCC No. _s 21.713) obtained, as disclosed in US Pat. No. 3,813,318 and US Pat. No. 3,957,587, especially, when disclosed in US Pat. No. 3,770,0589 or US Pat. No. 3,813,318 described process was prepared _ß

Isoiaerase unit.

The Auamaß the enzyme activity is ^iS than ⁿ isomerase unit called "the will gebrauohlt to generate a micromoles fructose per minute under the Isomerisierungs'bedingungen which then * ^t under the ^heading? Analysis of the leomerase "activity" can be described _β

The term refers to the meaning used here

~ _5 -. · Berlin, cU8.2.1979

AP A 23G / 2O6 028

the method of analysis which involves a spectrophotometric determination of ketose produced from a glucose solution under a range of `standardized conditions»

A 'Stannnlösuhg' is made in the following way %

component

O ₅ 1M MgSO ₄ , 7H ₂ O '1 ml

O ₉ OOiM CuCl ₂₀ GH ₂ O ^: 1 ml

1M sodium phosphate buffer, pH 7 _S 5 0 _? S ml

Anhydrous D-Glncο bq ' 1 _$ 44 g

Add distilled water to a total volume of 7j5 ml *

The KU analyzing enzyme preparation is first diluted _f so that it contains 1 to 6 isomorase units per ml «

'A ^ enzymatically isomerization is carried out in the form of _s that 1 ml of enzyme preparation to. 3 ml stock solution 'is added, and is inlcubiert at 60 ⁰ C for 30 minutes * laugh waste' course of the incubation period, an aliquot of 1 ml entnoMTien and in 9 ml of _0? 5% perchloric acid quenched. The quenched aliquot is then diluted to a total volume of 250 ml. As a control, for comparison purposes, a glucosoflinase sample is also examined by using 1 ml of water instead of 1 ml of enzyme preparation in the solution at the beginning of the incubation period 'becomes"

The ketosis is then determined by means of an oyetein-sulfuric acid method

- 6 - Berlin, <U8.2.1979 AP Ä 23G / 206 028

defines an isomerase unit as the extent of enzyme activity? used to produce one micromole of levulose per minute under the described isomerization conditions. "

This term as used herein refers to the transition of a fructosyl substituent from the donor _. S ₀ B ₀ sucrose _s to an acceptor z * B *. polysaccharides

As intended, does this term refer to any enzyme _? catalyzes the transfructosylation and contains the enzyme preparation obtained from Pullularia pullulans ATCC 9348 (synonym for aureobacteric pullulane)

A s'ructosyltransferase unit is specified herein as the amount of enzyme activity required to produce a micromole of reducing sugars 5, calculated as ron glucose, per minute under the following conditions: (a) pH 5, 5 _S (b) temperature 55 ° C and (c) substrate concentration. 60 g of food grade sucrose per 100 ml of an aqueous reaction mixture.

Determinations of reducing sugars (calculated in the form of glucose) are carried out using a "Technicon Autoanalyzer II" (Te-chnicon, Ine *, Tarrytown, Uew York). The analysis värd according to · the usual alkali ^ Perrocyanid method Ana * - lytical Biochemistry ·, 45 * # 2 _S S _e 517-524 (1972), guided by = "which is suitable for use in the ^'s ilutoanalyser II". Unless stated otherwise, the destination will become

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the Ensymaktivität executed _f aas has the following composition by the continuous monitoring of a Reaktionsgemisohes? ,

7 _S 5 in! 80% (w / v) aqueous solution of sucrose in

Food quality. ,

2 _r 3 ml O _? 1M citrate buffer with a pH of 3% 5

0J2 ml Enzymprqbe _s containing the amount Fructosyltransferaseenzym which 5 to 25 micrograms of reducing sugar (be "counted in the form of glucose) per minute per ml of reaction rates

mieoh errseugen vri.rd) ·.

Pr irm s ub.s t r a t

The term "primary substrate" is here used to designate such saccharides _? suitable for transfructosylation and having a pructosyl portion available for participation in transfruotosylation, such as, for example, aqueous solutions of sucrose

The term "secondary substrate ^ίΓ is used here to denote the Reaktioneproduktes _f arises when the primary ·« · eu.bstrat the effect of a Pructosyltransferase-Enzympräpa "Board naoh is exposed to defined herein _e

Parts and Proprietary Parts

In this patent specification, all parts are given by weight and all percentages by weight to volume (parts by weight), unless otherwise expressly stated.

With this name the procedure is called _¥ with its

8 »Berlin, d; 8.2.1379 'AP A 23G / 206 028

Help the syrups according to the invention using the high-pressure liquid chromatography according to the following technique be agreed «. The. Ingredients are going through. Eluting with water from a cation exchange resin in the calcium form chromatographed. The eluted components are detected by differential refractometer. "Non-dextrose carbohydrates are quantified using an electronic integrator and the dextrose is determined from the difference. the general procedure is as in "Analysis of Carbohydrate Mixtures by Liquid Chromatography", Anu Soc "Brew * Chem. Proc <, _{s S} S _e described in 1373 43 to 46. The resin used is "Aminex -Q" 15-5 in the calcium form, Bio-Bad Laboratories, Richmond. California «·

The invention has for its object _t find suitable conditions for the conversion of sucrose into a pro, which consists of dextrose and fructose polysaccharide _β

 According to the invention, the sucrose is the action of an effective amount of a Fruotoeyltransferase-Snzympräparates to produce a fructose polysaccharide and dextrose best ». suspended reaction product

It is appropriate _? that the sucrose which is exposed to the action of an enzyme has a concentration of at least 20 % , and the pull-up pullulans fructosyltransferase enzyme preparation comes from

According to the invention, the dextrose of the reaction product is isomerized to produce fructose, for example using an immobilized glucose isomerase enzyme or

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in the presence of polysaccharides «

According to the invention, the polysaccharide is separated physically from the dextrose, for example by ultrafiltration.

Bas ~ polysaccharide of the reaction product is hydrolyzed according to the invention to produce fructose, preferably after physical separation from the dextrose and preferably without active isomerase enzyme

According to the invention a process for the preparation of syrups is provided, which consists in that a primary substrate, _e B ', sucrose, to the action of a fructosyl transferase enzyme preparation is suspended', capable of converting the sucrose to a product of " a monosaccharide fraction containing a major amount of glucose and a small amount of fructose, and polysaccharides containing at least 66 wt '% Fructosylkomponenten, is "such a product includes an inventive secondary substrates These polysaccharides' of the secondary substrate include all fructosehalt strength η polymers (except Sucrose) having two or more mono-saccharide moieties. These polymers can be further characterized as comprising polysaccharides containing fructosyl caffeine moieties linked by (2 "" M) -beta bonds. As will be described later, dia from the given conditions of transfructosylation ge obtained polysaccharides within a predeterminable range ». For example, secondary substrate can be produced in which most (z _e B * at least 60 moles) of the polysaccharide oligomers with 2 to 10 (d _e h "DP 2-10) usually. But 3 to 6 (see DP 3-6) monosaccharide components _e . Subsequently, the glucose is isomerized in the presence of the polysaccharides (via the action of the isomerase enzyme) to fructose,

10 '' Berlin, d.8.2.1979 AP A 23Q / 2O6 028

followed by hydrolysis of the polysaccharides without an active isomerase enzyme. "The hydrolysis can be carried out enzymatically using invertase or by acid hydrolysis under mild conditions."

In another embodiment of the invention, the polysaccharides of the glucose and fructose can be separated and subsequently hydrolyzed as described above ^ an ultra-high Fmctosesirup, z ^ B "with a Fr uotosegehalt of over 66 wt _e #, and preferably from about 90 Qqvt to produce ₉ % directly from the secondary substrate of the present invention without the need for isomerization of the dextrose in the substrate. Such separation may be carried out in a conventional manner by conventional physical separation techniques based on molecular size, such as by conventional membrane technique (z <> B * ultrafiltration, dialysis)., solvent precipitation. Carbon Absorption and the like "Examples of this membrane technique include U.S. Patent Nos. 3,173,867; Re 26097s' 3.54.1.006 and 3 * 691 "068,

A preferred embodiment is a process for the production of high-grade fructose sir upe, which is that the sucrose of the action of a Fructosy transferase-Ensym preparation j such as that of Pullularia pullulejas such as ATCC 9348? ATCC 12535? IiERL 1673? NRRLY2311; IREL ΪΒ3892; ' and HERL YB3861 UERL 3937 and ATCC 15223. The resulting product or secondary substrate is exposed to the influence of an isomerase enzyme. Thereafter, the isomerized product is hydrolyzed without active isomerase enzyme. "

The secondary substrate prepared by the process of the present invention is believed to be novel

* Λ - 'Berlin, do8.2.1979 AP A 23G / 206 028

Substrate is uniquely suited for isomerization and subsequent hydrolysis to produce a syrup containing more than 55 % fructose and is prepared by exposing sucrose to the action of a fructosyltransferase enzyme preparation. Therefore, another embodiment of the invention relates to substrates suitable for enzymatically ^ isomerization and subsequent hydrolysis to syrups containing from about · 55% fructose syrup and consist of (1) about 20 to about 60 wt% _s monosaccharides _s which contain a major amount of glucose and a minor amount of fructose, and (2) about 70 to about 40 % polysaccharides containing more than 66% by weight fructosyl components. "

Especially preferred v / ground secondary substrates derived from sucrose by Transfruotosylierung in the presence of an effective amount of obtained from a strain of Pullularia pullulans ATCC · 3348 FruGtosyltransferase-enzyme preparation at a between about 25 C and about 65 C, and preferably between about 5O ⁰ C and about 6O ⁰ C lying temperature and in a range of about 4 $ 5 to about 6.5, preferably between about 5j4 and 5§6 lying pH were obtained "The applied output concentration of sucrose needs only 10 g per 100 ml of water." However, it is preferred; To use as high a concentration / dry matter as possible, preferably between about 30 g to about 60 g per 100 ml (for maximum reaction rate) to sum saturation. .suspension of sucrose (and 'more ₉ ' as will be described in more detail later)

A minimum of 0 $ 5 units of fructoeyltransferase per gram of sucrose can be used to produce the novel substrate of the present invention. Generally, the amount of enzyme used will not exceed 50 units per gram

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Sucrose are for economic reasons. Particularly preferred for the recovery of the intended secondary substrate in an economically feasible time and in the context of the process parameters described above is an amount of about 2 to about 30 units per gram of sucrose.

For the "process for manufacturing the new fiction, contemporary ** fructosyltransferase conventional fermentation techniques can be applied for _s' B _e see US Patent Ur. 3" 565 * 756? 3 * 806 * 4191 3e535 "123i S. Uedati.a. Applied Microbiology, 11, 211 - 215 (1963) "Particular new methods of separation and purification are used, which will be described in more detail below: The following example" is a typical fermentation process for the preparation of the enzyme from Pullularia pullulans ATOC 9348 "

embodiment

The invention will be explained in more detail below with reference to some embodiments.

carrier

method

As a medium for vaccine development and fermentation cure generation of the Snaym the following was used;

Oy5 % dibasic potassium phosphate 0 * 1 % üTatri.um chloride, 0.02 % magnesium-sulfate heptahydrate 0.06 % diammonium sulfate 0 _? 3 % yeast extract (Difco 'Laboratories) 7.5 % sucrose (food grade) from 6 ₉ 8 adjusted pH of the medium.

«73 -

The vaccine flasks (500 ml Erlenmeyer) containing 3.00 ml sterile medium * are inoculated with a slant culture of the black yeast, Pullularia ptsllulans * The respective strain of the yeast used is in the catalog of the American Type Culture Collection (Rockville, Maryland) ATCC 9348: "Irnpfkolberf are after a developmental period of 48 hours at 32 ⁰ C on a Vibrafcionsschüttler for the inoculation of!" - ^ Erlenmeyer ^ fermentation flasks, each containing 200 ml of the medium described above; used »The concentration of the vaccine used is 0 ^ 5 The fermentation is carried out on a vibration debris« ·

Continue for 7 days at 32 ⁰ C. Remove de? en? .yras from the Fertnentationsbröhe

The fermentation broths from forty I-L shake flasks are taken together, - and the flasks are rinsed with water * of is also added to the pooled broth · The final volume oer broth after dilution is 12 liters _e Dos original volume of the broth was about 8 Liter »The 12 liter fermentation broth is passed through a continuously running Sharples centrifuge to remove UQP yeast cells and cell pellets. The supernatant, which is a black viscous solution, is then treated with calcium chloride to a concentration of 0 _f 5%. v _e % / vol _{e is} metered, and the pH of the resulting solution is adjusted to 7.0 with sodium hydroxide. "This is followed by a second pass through the Sharples centrifuge to produce a viscous supernatant liquid which is slightly colored pH uev decoloured supernatant liquid is adjusted to 5.5 with hydrochloric acid falls, after which the dose of 1000 units (as defined in the US "PS 3,806,419) Pullulanase'folgt" The resulting broth is preserved with toluene (addition to saturation), and the pullulanase to let at ambient temperature overnight respond " After uev digestion with pullulanase overnight, add 1 % Grefco Celit Np. 503 (Oohno-Manville Products Corporation,

Lonipoc, California) in the broth. "Followed by the addition of 2 volumes (24 liters) of acetone." A precipitate forms which is collected by filtration , the filter cake is washed with acetone and dried at ambient temperature. The collected filter cake the insolubilized fructosyltransferase enzyme _e

In Example 1, it should be noted that the addition of calcium chloride to the overnight broth results in the pH of the broth being adjusted to eliminate the black pigment and the acidic polysaccharides present; fuhrtV ^ f For a discussion of these acidic polysaccharides see Acta Chem _c "Scand x 16 / 615-622 (1962) J7 _O The final. Enzyme product is thus in the form of a relatively pure ^ color-imparting "loose preparation before _e This cleaning process represents a preferred embodiment of the invention and enables the use of simple purification method d" h _e centrifugation, filtration or precipitation, to obtain the final product, "In This Ausföhrüngs.form is therefore a process for the separation of acidic polysaccharides and black colored by-products of the finished fermentation broth or black yeast. Pullularia pullulans, asked at your disposal By this method a finished enzyme preparation that is free of unwanted dye and acidic polysaccharide by-products becoming formed during the fermentation process occurs These by-products if they are not removed with the enzyme after the solvent addition to Fermentation chest precipitated »

As a further improvement to obtain purified erfindungsgeraäßer Enzyrapräparete it is advisable to remove the inevitably in the Ferraentationsbröhe existing Piillulan polysaccharide, because it would also precipitated with the Fructosyltransferaseenzym after solvent added to the fermentation broth "Therefore, by the treatment with calcium chloride and the setting of pH-recovered supernatant fluid can be further treated with the well-known hydrolysis-type pullulanase pullulan

nose-enzyme hydrolyses randomly the pullulan and produces a polymer with lower molecular weight * whereby the coprecipitation and consequent contamination of the fructosyl transferase Enzyrapräparates during treatment suit solvent (e.g. _e B _e acetone * alcohol and the like) is avoided "The purification of the intended Fructosyltransferase enzyme in this way represents another novel embodiment of the present invention.

The fructosyltransferase enzyme preparations of the present invention can be used without removal of the pullulan. Therefore, the invention can be practiced without hydrolysis of the pullulan by pulluianase * in which case the pullulan would serve as the carrier for the fructosyltransferase enzyme.

The following example illustrates the use of pullulan as a vehicle «,

Example 2?

Secondary substrate using Fructo = *

Buffered to a 20% sucrose solution with 0.5 M ^ citrate buffer at a pH of 5 * 5 _f is a quantity of 1% dry pullulan added * prepared according to the procedure of Example 1 * however, the pullulan not vmrde hydrolyzed with pullulanase and thus serves as a carrier so that no celite was used. The fructosyltransferase activity is 677 units / graratr) pullulan * The reaction is carried out at ambient temperature for as long as possible. leads * until the mixture becomes turbid »A sample of this material is analyzed by means of high-pressure liquid chromatography and shows the following results:

Saccharide separation by HPLC analysis

Fructose 'Dextrose DP ₂ DP ₃ ^Di> 4 $ 6.9 40.6 6.2 H ₁ I 35.2

The following examples further illustrate the enzyme produced in Example 1,

In with screw caps provided reaction bottle 60 g of sucrose in food quality and a Fructosyltrans "are * transferase ~ enzyme preparation given _e The enzyme preparation is obtained from the Enzyraprodukt of Example 1 by dispersing suitable aliquots of the solid Celite-Enzyrnproduktes in metered Wasserniengen _# to a suitable concentration of an enzyme solution The celite is later removed by filtration. The filtrate is used to dose the reaction mixture to 10, 20 and 30 units of enzyme per gram of sucrose substrate. These mixtures are then each diluted to a final volume of 100 ml water-diluted conversions for a period of 66 hours. pH values of 5 _s -5 and 55 ⁰ C or _e 60 ⁰ C After 24 _f 43 and 66 hours, samples are taken for the determination of reducing sugars in order to demonstrate the presence of enzymatic activity worden'sind determined * the rest of the reaction mixture is frozen * the enzymatiache activity shortstop ^ and the samples are subjected to determine the carbohydrate composition of the high-pressure · FIüssigkeits chromatography "the following results were obtained;

Analyzes reduz i ng th e sugar *

In the case of IWIi-IIi iBiBiiiBiWi »ini il iii

Temp "

Units Jl

reducing

sugar

mg / ml

pH reducing

Sugar mg / ml

pH reducing

sugar

pg / Rax

24 hours

43 hours

66 hours

0 - , 7 200 5.80 231 " 5.80 268 10 191 219 5.40 270. 5.25 301 20 192 282 5.40 334 5.25 390 30 246 5, 35 1.5 5.15 2.1 0 5.75 243 5.80 270 10 .5,10 · 288 4.70 346 20 5.15 360 4.90 420 30 5.20 4.95

The method used was in the definition of fructosyl transferase unit

explains, ·.

Units of enzyme per gram of sucrose

Carbohydrate '-'Zusamtne.neetzunc; .. a nhand

Reaction Toner 55 C Dextrose \ / o) levulose DP DP α) DP / (JK) Enzyme dose one-nil / g sucrose Reaction time hours 31.7 2.3 10.0 25.0 31.0 24 34.5 3.2 9.4 17.9 35.0 10 43 36.7 3.9 8.6 15.0 35.8 66 33.9 2.8 8.8 7.19 34.8 24 37.4 4.2 7.9 12.1 38.4 20 43 40.5 4.9 7.4 10.8 36.4 66 37.4 4.0 7.1 12.5 39.0 24 41, 8 5.9 6.8 11.4 34.1 30 43 46.1 7.5 7.0 27.9 66

Reactive temperature 60 ° C

Enzyme dose one-nil / g sucrose Reaction time hours Dextrose (%) Levulose GS) DP \ 7oJ DP (%) DP ⁺ (%) 24 "2,8 ' 6.1 24.3 30.6 10 43 35.0 4.0 9.8 18.2 33.0 66 36.7 , 5 * 4 9.4 16.0 32.5 24 35.5 3.8 8.4 17.1 35.2 20 * 43 38.4. 5.0 8.0 12.3 36.3 66 41.3 6.6 7.9 11.1 33.1 24 39.0 5.5 7.1 12.1 36.3 30 43 43.7 7.1 7.3 11.0 30.9 66 47.8 9.2 7.4 1.11 24.5

Example 3 demonstrates the thermal stability in the presence of enzyme substrate at 55 ⁰ C and 60 ⁰ C for a period of 66 hours at a pH value of 5.5, thus demonstrating the commercial potential of the enzyme · Dar "überninaus is assisted by the enzymatic Carbohydrate analysis of sucrose with the novel fructosyltransferase preparation of the present invention demonstrated that it is a particularly valuable product for the production of high-quality fructose syrups from sucrose / because the main constituents are dextrose and polymers which are the predominant fructose in the subsequent hydrolysis Monosaccharide. This experiment also shows that the novel enzyme according to the invention is effective at a sucrose concentration of 60 % (% by weight). The functionality of the enzyme in the presence of high glucose concentrations is also demonstrated.

Example 4i

JEinf lufi SSSR _T Ten ^ erature on Enzymaktiyitat

The influence of temperature on the reaction rate of the fructosyltransferase enzyme is determined using the "Technicon Autoanalyzer II" as follows:

The reaction mixture consists of 7.5 ml of 80% (w / v) sucrose solution, 2/3 ml of an O, IM citrate buffer with a pH of 5.5 and 0.2 ml of a 2% (w / v) pullulan Enzyrolösung. (Enzyme preparation of Example 2) _β is the final sucrose content of 60% (w / v) _ö Samples -Be at the following temperatures maintained for 10 minutes in the "Technicon Autoanalyzer II" analyzed The results show that the rate of the enzymatic reaction at 40 ⁰ C, the l, 89 ~ times the amounts at 30 ⁰ C, at 50 ° C, the l / 29 "fold over 40 ⁰ C, and at 60 ⁰ C, the l _f 48 times the at 50 ° c _e As a result, an increasing reaction rate with increasing temperature becomes clear.

Demonstration of the Michaelie-Menten-Kpnstanten (K) of the

The K of an enzyme indicates the substrate concentration at which the rate of product formation is one-half of V * The following procedure was used to determine the K of the fructosyltransferase enzyme.

Using a 90% (w / v) sucrose strain, the pH of which is adjusted to 5.5 with O, 1M citrate buffer, the correct concentrations of saccharose rose in 9.8 ml aliquots are prepared in that the following concentrations of sucrose are obtained in a final volume of 10 ml: 5, 10, 30, 40, 50, 60 and 70 % _e. To the cooled samples is added 0.2 ml of enzyme solution containing 1.1 Einhe'iten Fructosyltransferaseenzym given "Then who" aie the samples immediately at 55 ⁰ C in the above described "Technicon auto Analyzer II" analyzes (see fructosyl unit) · As a control, a glucose Standsrd (in ug / ml calibrated) with included _o

Subsequently, the rate of glucose formation, expressed in μg / ml / minute, at the various substrate concentrations using a constant dose (1.1 units) of the fructylsyltransferase enzyme preparation of Example 1 is shown.

% Substrate / Ug / ral / niin 5 8.0 10 11.8 20 15.7 30 18.0 40 18.6 50 19.2 60 8.17 70 15.6

8.0

11.6 15 _f 2 17.6 18.2 17.2 18.2

a) Another try on the next day with a 60%

Sucrose stock solution,

The K value is 0.27 mol of sucrose.

m. ,

At a substrate concentration of 1.374 moles of sucrose at a pH of 5.5 at 55 ° C.

There rs division, up.d leomerisierunc! from

Sacchjros ^

Y9 ⁿ

600 g of food grade sucrose are dissolved in water to a volume of 800 ml. The pH of this solution is adjusted to 5.5 with dilute hydrochloric acid. 11 g of a dry celite enzyme preparation with an activity of 500 units / g prepared in Example, are slurried in 100 ml of water. The slurry is filtered under vacuum onto Whatman filter paper # 1 on a Buchner funnel. The filter cake is washed with an additional 100 ml of water. The 200 ml filtrate is then added to the sucrose solution contained in a 0.5-gallon screw cap bottle. The bottle is then placed in a water bath at a temperature of 58 ⁰ C, and the reaction is allowed to run for 20 hours, after which a sample of the reaction "product by the high pressure ^ Flössigkeits chromatography to determine the KohlehydratztJsammensetzung follow" the results was analyzed:

Fructose 2.4 %

Dextrose 32.8%

OP ₂ 10.6 %

22.9 % . 31.3 %

To the remaining reaction product (i.e., the secondary substrate) is added magnesium chloride to a level of 5 millimoles, and the pH is adjusted to 8.4 with dilute sodium hydroxide.

V ° ⁿ Strep torn yc it oliyochromogenes / "ATCC 21.715 (See US 'Patent No. Re. 29..152) gained glucose is (on porous alumina a regulated pore carrier manufactured by Corning Glass Co _8, Corning, New York, for example, see U.S. Patent No. 3,992,329) as follows:

Der The carrier is washed twice with water;

2 * The carrier is incubated for 1 hour with stirring with O, IM sodium citrate;

The sodium citrate is washed off the carrier until the conductivity of the wash solution is 1000 micro ohms;

4. Incubate the carrier for one hour with 0.05M magnesium chloride and decant the magnesium chloride solution;

Then, an amount of 0.05M magnesium chloride is added to obtain a final enzyme concentration of 400 units / ml;

6. The isomerase enzyme concentrate is added to the carrier in an amount of 14 million units per cubic foot;

7. The carrier and enzyme are kept in contact for 22 to 24 hours, after which the unbound enzyme is washed off the carrier with distilled water.

A jacketed glass column (3 cm χ 18 cm) equipped with a pump connected to a feed tank is then filled with the immobilized enzyme thus prepared. The bed volume of the column after filling is 45 ml. In all isomerizations, the column is operated at 60.degree. The filled column is treated with a dextrose syrup (50% by weight / volume concentration) with 5 millimoles of magnesium chloride and a pH adjusted to 8.4.

starts to prove that the column is active. "The flow rate of the column is 292 ml / h. _e The column is then emptied to bed height. * The secondary substrate is filled by hand until 20 ml of effluent is collected, and then the throughflow running speed for the secondary substrate set to 292 ml / h. The first 100 ml of collected syrup are poured away to account for any change in the substrate. " The remaining secondary substrate (about 850 ml) is then passed through the column. * After one hour, the flow rate increases to 570 ml / h. The flow rate is set to 300 ml / h and kept at that value until the end of the experiment. Upon completion of the secondary substrate experiment, the column is again switched to dextrose to show that the isomerase activity is still present. The following table compares the high pressure liquid chromatographic analyzes of the starting secondary substrate and the final product from the isomerization column.

Secondary substrate (A) final product (B)

fructose 2.4 15.7 dextrose 32.8 18.9 DP ₂ 10.6 11.0 DP ₃ 22.9 22.9 ,, DP , 31.3 31.5

These results show that 42.38 % of the free dextrose in the secondary substrate in the column was isomerized to fructose. "Also, the fructo-sepolyraers present in the secondary substrate did not appear to be affected or to have affected the isomerization of dextrose to fructose." It is worth mentioning in that the total composition of the monosaccharide has about 45 % fructose and 55 % dextrose,

The following example shows two ways for Spal · be tung is in the secondary substrate polysaccharides present and also the consequential Isoraerisationsproduktes applied 'One Vtleg is enzymatic and is in the other

a weak acid hydrolysis applied »

Example 7:

--in the. inn "f '' ΊΐΐΜΐίιιιι

100 (TiI product B of Example 6 are mixed with 10 mg of a purified, obtained from Caindjuja H ^ lJ ₁ Q invertase dosed "This mixture (preserved with toluene) is allowed to react overnight at ambient temperature, after which a sample taken and with the aid of The remaining product B is allowed to react for a further 6 days and then a second sample is analyzed by the same method with the following results:

Starting material After In = «After further (product B) littering effect 6 days

fructose 15.7% 4.1, -9 % 62.4 0 / / 0 dextrose 18.9 % 29.4% 36.2 0 / / Ü ^DP 2 11.0 ^ j 1 # 9 % O ^DP 3 22.9 % 12.9% 0.5 Cj / / O ^DP 4, 13.9% 0.9 %

When used invertase is an enzyme preparation manufactured by Siekagaku Kogyo Co _e, Ltd. "Tokyo, Oapan, Kiit an activity of 123 units / rag, wherein 1 unit of invertase, the cleavage of sucrose to form 1 tnMol glucose and 1 mfviol catalysed fructose per minute under specified conditions

B _e acid hydrolysis of product B

^ lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll / mm \ ! !! Ι Λ !! ''I'll * -! IIJM! BI fcll 'If! M ^- J ^ l I ^ l TlI 1 l 1I ¹ IlII

The acidic hydrolysis of a sample of product B from Example 6 was carried out by the addition of 0.05N sulfuric acid and heating at 75 to 80 ° C. After a hydrolyzing time of one and two hours, samples were taken and washed with

Aid of high-pressure liquid chromatography analyzed »The following results were obtained:

Starting material (product B) 1 hour 2 hours fructose 15.7 60.3 59.1 dextrose 18.9 38.7 37.9 ^DP 2 11.0 1.9 2.6 ^DP 3 22.9 0.4 Q, 3 4+ 31.5 0 0.2

The above results of step A show that there was a significant increase in fructose yield and a lower increase in glucose yield with a corresponding decrease in the DPo * DPo and ΟΡ ^ ,, fractions, thus confirming the presence of a fructose polymer.

The results of the acidic hydrolysis in step B are in agreement with those of step A above and thus show the cleavage of fructose polymers ».

The above discussion concerns transfructosylation using a primary substrate wherein the dry matter content of the starting sucrose does not exceed the saturation point under certain reaction conditions. The following example shows the application of a Primärsubstra "tes with a temperature above the saturation initial sucrose concentration which yields under the action of Fructosyltransferaseenzyms a product that increased amounts DP" material (i.e., "h _e fructosyl-sucrose) and a decreased concentration of DP It has turned out

4+

also show an increase in the dry matter concentration (w / w) of the secondary substrate over the tartar substance concentration, which has arisen without the action of the fructosyltransferase enzyme. It is also shown that as the dry matter concentration of the primary substrate increases, the degree of polymerisation of the fructose polymer in the

Secondary substrate decreases and the D? ₄₊ material is present in minor amounts.

This is in stark contrast to the results obtained in the previous examples where sucrose substrates were used below the saturation level. In the examples, the DP. -Material the primary products

Example 8; '

ÜHH ι "mm ι me min ι in ι ι n mli <

Representation of secondary substrate from fructose sludge

Food grade sucrose is added to I-pint glass flasks with screw caps in amounts of 200 g. As a control, 50 ml of water are poured into a glass flask and into the other 50 ml of water containing increasing amounts of fructosyltransferase enzyme concentrate (prepared as described in US Pat example 1), as the following table indicates ,, Oeder flask is sealed and placed in a to 55 ⁰ C set at 54 Schüttelvvasserbad "the flasks are shaken for 24 hours with occasional manual mixing" a sample of the supernatant liquid from each flask These samples are then analyzed by means of high-pressure liquid chromatography and the determinations of the excess dry matter (according to the K ₀ Fischer method) are carried out with the following Results made:

Piston ί Sucrose enzyme * (g) units 100 Dry matter in the above (w / w) Composition of in solution by HPLC Dex * ^DP 2 Carbohydrates (JK). ^DP 4 * 200 200 74.5 fruc, 9.6 80.9 ND ² 1 200 300 75.6 0.6 12.7 72, 2 8.9 0.7 2 200 400 76.3 0.7 14.5 66.8 13.7 1.1 3 200 500 77.1 1> 15.7 62.6 16.6 1.8 4 200 600 77.7 , 0.9 17.3 • 58.5 19.0 2.1 5 200 700 78, 1 1.1 18.0 56.0 21.0 2.8 6 200 800 78.1 iI3 18.8 53.9 21.9 3.0 7 200 900 80.0 1.1 19.3 52.2 23.1 3.4 8th. 200 1000 79.0 1.0 19.9 50.0 24.1 3.7 9 200 none 79.6 1.3 20.6 48.6 25.0 4.1 10 200 72.7 3.1 ND ² 99.7 25.4 ND2 · control 0.3 ND ²

Total units of Fructosyltransferaseenzytn in 50 ml of water ND β not detected

It is worth noting the fact that in the above example, the control sample crystallized on cooling to the ambient temperature (about 25 ⁰ C), while the enzymatically generated secondary substrates remain in solution and show excellent storage stability without crystallization.

Although in Example 8 200 g of sucrose per 50 ml of water are given. ie _e 80% Ge \ v '/ w _{e /} be yet, the dry matter concentration of sucrose feedstock increases "

The novel secondary substrate of this embodiment can be used as a very durable non-syrupy foodstuff syrup. It also has a unique high dry matter content that is resistant to microbial contamination and color formation, making it suitable for shipment or storage of sugar in concentrations previously unavailable in a form with the properties described above. _E In addition, this novel secondary solid substrate having the high dry matter content can undergo hydrolysis as described in Example 7 to yield an invert sugar "blend with a desirable sweetness level." The novel secondary substrate of this embodiment has a dry matter content (wt / wt) of between about 70 % and about 82 % and contains a f.ionosaccharide portion consisting essentially of dextrose and polysaccharide polymers except DP ₂ composed mainly of DP ^ product * It is also a small amount (4.1% and less) DP ₄ ^ PoIyHere available «

Although the Transfructosylierungsachritt invention has been described in terms of batch operation method, it will be understood by those skilled that a continuous process can be used equally "When performing continuous uer fructosylation the Transfructolyseenzym is best immobilized using the method,

which was explained in the context of the definition of the immobilized enzyme and the continuous procedure described here.

Claims (15)

Invention Request · A process for the preparation of sirap, characterized in that sucrose is exposed to the action of an effective amount of a fructosyltransferase enzyme preparation to produce a fructose polysaccharide and dextrose ligation product. 2 "Method according to item 1", characterized in that the sucrose exposed to the action of an enzyme has a concentration of at least 20% «, · _; 3 # Method according to item 1, characterized in that the fructosyltransferase enzyme preparation is from Pullularia pullu- lans, 4 · A method according to item 1, characterized in that the sucrose to the action of a Pructosyltransferaseenzyms at a temperature of about 25 ⁰ C to about 65 ° C, a pH value of about ~ 4 »5 to about 6.5 and with an initial sucrose concentration is subjected to at least $ 10 _(e Vol wt "/). ' 5. The method according to any one of the items 1 to 4, characterized in that the dextrose of the reaction product is for the production of fructose isomerized. 6, method according to point 5 », characterized in that the isomerization using an immobilized. Glucose isomerase enzyme is carried out * 7 · Method according to point 5 », characterized in that the. Dextrose isomerized to fructose in the presence of the polysaccharide ", 3a- Berlin, d.8.2.1979 JIP A 23G / 206 023 8, method according to any one of items 1 to 6, characterized in that the polysaccharide is physically separated from the dextrin. 9 «method according to item 8, characterized in that the separation takes place with the aid of ultrafiltration, 10 "method according to one of the items 1 to 9 $ characterized by ^ that the polysaccharide of the reaction product is hydrolyzed to produce fructose. 11 ". Process according to item 10, characterized in that the polysaccharide is hydrolyzed without active isomerase enzyme * 12 »Process according to item 10, characterized in that the polysaccharide is hydrolyzed by the physical separation of the dextrose * Substrate prepared according to any one of items 1 to 4 and suitable for enzymatic isomerization and hydrolysis to syrups containing more than 55 % fructose, characterized in that it contains (1) from about 20 % to about 60 % monosaccharide, the mainly consisting of dextrose and fructose, and (2) of about 70 %. to about 40% polysaccharides consisting predominantly of eructosyl components, which are by weight percent of the substrate. 14 "Substrate according to item 13", characterized in that the polysaccharide contains at least 66% by weight of Fructosyl components. Berlin, d.8.2.1979 AP-A 23G / 206 028 15 »Substrate, characterized by the preparation according to one of the items 1 to 4 with a dry matter content (wt / wt) of about 70% to about 82 % and a monosaccharide fraction contained therein, which consists predominantly of dextrose, and a polysaccharide fraction (except sucrose) mainly consisting of DP ^ product,.