SU1230469A3 - Method of producing saccharose product containing fructose - Google Patents

Abstract

This invention relates generally to (1) processes for the production and isolation of a novel fructosyl transferase enzyme from the fermentation broth of Pulluaria pullulans, (2) enzymatic transfructosylation of sucrose to produce a novel fructose-polymer containing substrate, and (3) production of fructose syrups containing greater than 55% fructose from said novel substrate.

Description

one

This invention relates to a process for the preparation of a product containing fructose from sucrose, which is used in the food and pharmaceutical industry.

The purpose of the invention is to increase the content of fructose in the target product.

The proposed method provides a half of fruit syrups containing more than 55% fructose.

Preparation 1 is an illustration of a typical fermentation procedure carried out to obtain the desired enzyme from the pullularia pullulans yeast strain. ATCC 9348.

Preparation of the fructosyltransferase enzyme preparation on a celite carrier. Fermentation procedure used to produce the indicated enzyme.

The medium used to develop the seed culture (inoculum) and for fermentation in order to produce the desired enzyme has the following composition,%: potassium dihydrogen phosphate 0.5; sodium chloride 0.1; sulphate magnesium sulfate 0.02; ammonium sulfate 0.06; yeast extract (manufactured by Difco Laboratories) 0.3; sucrose (food grade) 7.5. The pH of the medium is adjusted to 6.8.

Seed flasks (500 ml Erlemeyer flasks containing too ml of sterile medium of the specified composition) are inoculated with seed culture of black yeast, pullularia pullulans, on a slanted agar. For this purpose, these yeasts are used, which is indicated in the catalog under the name US Type Culture Collections (Rockville, MD, American Type Tours Cneture collection) as ATCC strain 93A8. These seeding flasks (after being held in a reciprocating shaker for 48 hours of 32 ° C gf) are used to inoculate 1-liter Erlenmeyer fermentation flasks, each containing 200 ml of medium of the indicated composition. In this case, the inoculum concentration (inoculum) equal to 0.5% (weight / volume) is used. Fermentation is carried out on a rocking chair of a return-pos1 upatel type for 7 days. at 32 s ;,

Vl3B.ne4eHne enzyme from fermentation bupion.

304692

The fermentation broths from forty 1-liter rocking flasks are combined together, the empty flasks are rinsed with water and these

5, the washings are also attached to the combined fermentation broth. The final volume of broth after dilution is 12 liters. The initial volume of fermentation brood 10 is about 8 liters. 12 l of fermentation broth is passed through a Sharpless continuous centrifuge to remove yeast cells and cell debris. Calcium chloride is then added to the resulting supernatant, which is a black viscous solution, to a concentration of 0.5% (w / v) and the pH of the resulting solution is added to 20 ° C. 7.0 with sodium hydroxide. This system is then re-passed through a Sharpless 5 centrifuge, resulting in a viscous supernatant having a minor

25 by intensity of color. The pH of this discolored supernatant is adjusted to 5.5 with hydrochloric acid and then 1000 units of pullulanase are added. The resulting broth is conserved using toluene (added before saturation) and the pulllulase is left to react at ambient temperature overnight. After fermentolysis by pull) olanase overnight

35 Suspended in broth 1% (i.e., sufficient to form a suspension of 1% concentration) Celite Grafco No. 503, Gretco No. 503 Celite manufactured by Dzons Manville Products

40 Corporation, Lompoc, California and then add 2 volumes (24 L) of acetone there too. A precipitate is formed, which is collected by filtration, the filter cake is washed on

45 filter with acetone and dry at ambient temperature. The precipitate collected on the filter (filter cake cake) contains translated into non-growing / :. impaired condition (immobilized)

5Q enzyme fructosyltransferase.

In preparation 1, it should be noted that the addition of calcium chloride to the fermentation broth and bringing the pH of the broth to 7.0 leads to the removal of the black pigment and the polysaccharides present in the broth.

By this treatment, the target enzyme product is obtained in the form of a relatively pure colorless preparation. Using this method, a target enzyme preparation is provided that does not contain undesirable pigment and acidic polysaccharides, which are formed as by-products during the fermentation process and, if not removed in time, are precipitated with the enzyme when added to the fermentation broth (acetone) .

As an additional purification measure, it is desirable to remove the pullulanic polysaccharide that is inevitably present in the fermentation broth, when the purified enzyme preparations are present, because the polysaccharide will also co-precipitate with the fructosil-transferrase enzyme when the solvent is added to the fermentation broth. Therefore, the supernatant obtained by treating the fermentation broth with calcium chloride and adjusting the pH to 7.0 with sodium hydroxide may be further processed by the known hydrolyzing enzyme pullulanase. The enzyme pullu lugase randomly hydrolyzes the Pullulan to form a low molecular weight polymer, thus avoiding coprecipitation and, as a consequence, contamination of the enzyme preparation fructosyltransferase during the processing of the broth with organic

solvent (for example, acetone, ethyl alcohol, etc.).

Fructosyltransferase enzyme preparations can be used without removing Pullulan.

Experiment 1. Preparation of secondary substrate when used. enzyme fructosiltransferase on Pullulan media.

To a 20% sucrose solution, buffered with 0.05 M citrate buffer with a pH of 5.5, 1 wt.% Of dry, pulllulace prepared according to the procedure of preparation 1 is added with the difference that the pulllulase does not hydrolyze. , and remain in the system as the carrier of the enzyme ructosyl transferase. For this reason, E is used as celite, which is added to the broth after being treated with pullulanase as a carrier in Example 1. The activity of the enzyme preparation of fructosyltransferase, obtained in accordance with this modified technique, is 677 e per 1 g of Pullulan. The reaction is carried out at ambient temperature until the mixture becomes cloudy. A sample of this material is analyzed by high pressure liquid chromatography and the following results are obtained.

The carbohydrate (saccharide) composition of the mixture, calculated from the results of analysis by high pressure liquid chromatography,%: fructose 6.9; dextrose 40.6; DR 6.2;

polymerization

flPj P, 1; DR 35.2, DR means the degree of polysaccharide.

Experiment 2 additionally characterizes the enzyme obtained according to preparation I,

Experiment 2. Experiment 1 relates to transfructization using a primary substrate in which the dry matter content of the starting sucrose does not exceed the saturation point under the given reaction conditions. Experiment 2 shows the use of a primary substrate with an initial concentration of sucrose above saturation, which, under the influence of the enzyme fructosyltransferase, yields a product containing elevated levels of DS material (i.e., fructosylsarose) and a reduced concentration of DS products. In addition, an increase in the concentration of dry matter (in May. Ratio) is shown. The secondary: substrate compared to the concentration of dry matter obtained in the absence of fructosyltransferase enzymes. In addition, it was shown that with a decrease in the dry matter concentration of the fructose polymer in the secondary substrate, the DS material is present in insignificant amounts. This differs from the results of experiment 1 with the use of sucrose substrates at a level below the saturation point. In these examples, DR is the primary product.

Preparation of secondary substrate from sucrose sludge at the top of the site.

Food sucrose in the form of 200 g aliquots is placed in 1 pint flasks with a screw cap. As a control, 50 ml of water was added to one bottle, and 50 ml of water containing increasing amounts of the enzyme fructosil-transferase — Selit (prepared according to Preparation 1) —to others. Each vial is capped and placed in a shaking water bath at 54-55 ° C. Sklinki shake for 24 h with periodic manual stirring. Sample

Although in experiment 2 200 g of sucrose were used per: 50 ml of water, i.e. 80 wt.%, The dry matter concentration of the sucrose starting material can be increased. ,

In examples 1 and 2, isomerization of dextrose to fructose is used by it304696

the supernatant is removed from each flask and placed in a test tube with a screw cap in a boiling water bath for 5 inactivation of the enzyme. These samples are then analyzed by high pressure liquid chromatography and the dry matter in the supernatant is determined (Fisher method).

The research results are presented in Table. .

Table 1

the mobilized glucose isomerase and which is mobilized by the following method.

The porous alumina carrier is washed twice with water and incubated for 1 hour with stirring with Ojl M sodium citrate, which is removed from the carrier until then.

 7

until the conductivity of the wash water reaches 1000. The carrier is incubated with 0.05 M magnesium chloride for 1 h, after which the magnesium chloride solution is decanted.

This volume of 0.05 M chloride, magnesium is added to obtain a resulting enzyme concentration of 400 units / ml. The glucose isomerase concentrate (xylose isomerase 5,3.1.5), obtained using Streptomices olivochromogenes ATS 21715, is added to the carrier at a concentration of 14 ml. units per f (about 28.4x10 cm), the Carrier and the enzyme are held in contact for 22-24 hours, after which the unbound enzyme is washed from the carrier with distilled water.

The immobilized enzyme thus obtained is loaded into a jacketed glass column (3 x 18 cm) equipped with a pump connected to a feed supply tank. The main volume of the column after loading is 45 ml.

Example 1. Food grade purity sucrose (600 g), dissolved in water to a total volume of 800 ml, the pH of this solution was adjusted to 5.5 with diluted hydrochloric acid. The dry formulation of zeolitic enzyme (dnulpsucrase 2.4.1.9), 11 g, which has an activity of 550 units / g and according to experiment 1, suspended Fructose 2.4

ETC.

Dr,

C

30469B

dirut in 100 ml of water. The suspension is filtered under vacuum on a Buchner funnel using filter paper Whatman number 1. Filter cake

5 additionally washed with 100 ml of water. Thereafter, 200 ml of the filtrate is added to the sucrose solution, which is placed in a 1.89 liter vessel equipped with a screw cap.

10 Then the vessel is placed in a water bath with a temperature of 58 ° C and the reaction is carried out for 20 hours. The reaction product contains a mixture of dextrose, fructose and polysaccharides containing at least

15 to 66 wt.% Fructosyl residues, the fructosyl residues being bonded by (2-) -linked.

The composition of carbohydrates is as follows,%: fructose 2,4j dextrose 32,8;

20 PD 10.6; Dr 22.9; DR, 31.3.

Thereafter, magnesium chloride with a concentration of 5 mmol / L is added to the reaction product and the pH is adjusted to 8.4 by the addition of a dilute solution.

25 sodium hydroxide. Then this mixture is exposed to glucose isomerase (xylose isomerase 5.3.1.5) immobilized on porous aluminum oxide in a column (immobilized isomerase is obtained according to the indicated procedure) at 60 ° С and pH 8.4. The carbohydrate composition of the product collected from the column is determined.

The data presented in Table. 2

table 2

62.4

 36.2

ABOUT

0.5

0.9

Thereafter, the product is subjected to a purified invertase enzyme obtained from Candida utilis at a ratio of 10 mg of enzyme per 100 ml of product, and is reacted overnight at room temperature. A sample of the product was analyzed by high pressure liquid chromatography, and the remaining product was reacted for another 6 days. after analysis. The final product contains 62.4% fructose, while the usual sucrose inversion treatment with an invertase enzyme yields a product containing 50% fructose.

I Example 2 Food grade purity sucrose (600 g) is dissolved in water. To obtain a total volume of 800 ml, the pH of this solution is adjusted to 5.5 with dilute hydrochloric acid. Zeolit enzyme dry formulation (dnulosucrase 2.4.), 9). 11 g, having an activity of 550. units / g and obtained from experiment 1, are suspended in 100 ml of water. The suspension is filtered under vacuum on a Buchner funnel using Whatman paper filter. 1. The filter cake is washed with additional 100 ml of water. Thereafter, 200 ml of the filtrate is added to the sucrose solution, which is in a 1.89 liter vessel with a screw cap. Then the vessel is placed in a water bath with

Fructose2,4

Dextrose32.8

DR 10.6

.OPj22,9

Dr ..

31.3

69; o

temperature and conduct the reaction for 20 hours. The product of the reaction contains a mixture of dextrose, fructose and polysaccharides containing at least 66% by weight of fructosyl residues, the fructosyl residues being bound by () p. bonds. The composition of the reaction product is determined.

Then, magnesium chloride at a concentration of 5 mmol / L is added to the reaction product, the pH is adjusted to 8.4 by the addition of a dilute sodium hydroxide solution. Then this mixture is exposed to glucose isomerase (xylose isomerase 5.8.1.5) immobilized on porous alumina in a column (immobilized isomerase is obtained according to the indicated procedure) at a temperature of 60 ° С and pH 3.4. The carbohydrate composition of the product, 1, collected from the column is determined. Sulfuric acid with a concentration of 0.05 and was added to this product. The mixture is heated to 75-80 ° C and is reacted for 2 hours. After 1-2 hours the reaction is carried out and samples are taken for analysis of the carbohydrate composition using high-pressure liquid chromatography. The resulting product is 1 | ykit approx. to 60% fructose. Normally | | ; Versus sucrose by inverting with enzyme enzyme gives product 5 containing 50% fructose.

The research results are presented in Table. 3,

T a b, l and c a 3

1-1 -123046912

tl p and me r 3. The product obtained is Example 4. The product obtained after treatment with xylose isomeric after treatment with xylose isomerase 5.3.1.5 of example 1, subjected to 5.3.1.5 of example 2, is subjected to hydrolysis invertase (obtained by hydrolysis of dilute hydrochloric acid from Pfansteihl Laboratories, Wauke-5 lot at 70 - 80 C. Hydrolysis of gan, Illifnois) using fructose and glucose complete with 0.1 ml of invertase per 10 ml of solution. Approximately through hydrolysis for fructose and glucose for i-4, done after 48 h.

Claims (1)

A METHOD FOR PRODUCING A FRUCTOSE-CONTAINING PRODUCT FROM SUGAROSE, comprising treating sucrose with an enzyme at a temperature of 58 ° C and pH 5.5 to obtain a mixture containing dextrose and fructose, which differs in that, in order to increase the fructose content in the target product, sucrose is treated with the fructosyltransferase enzyme dnulosucrase 2.4.1.9 to obtain a product containing dextrose, fructose and polysaccharides containing at least 66 wt.% fructosyl residues, and fructosyl residues are linked (2— * 1) by p-bond, followed by sequential processing an isomerase enzyme immobilized on porous alumina at 60 ° C and pH 8.4 and an invertase enzyme for 2-6 days or an acid such as hydrochloric or sulfuric acid for 1 hour and in the absence of an active isomerase enzyme. SU 1230469 A.3>. Magpie fermentation broths 1 liter shaken on a rocking chair ’flasks are combined together, the empty flasks are rinsed with water and these washings are also connected to the combined fermentation broth. The final volume of the broth after dilution is 12 liters. The initial volume of fermentation broth 10 is approximately 8 l. 12 L of fermentation broth is passed through a continuous Sharpless centrifuge to remove yeast cells and cell debris. Calcium chloride is then added to the obtained supernatant, which is a black viscous solution, to a concentration of 0.5% (mass / volume) and the pH of the resulting solution is brought to 20 up to. 7.0 with sodium hydroxide. Then this system is again passed through a Sharpless centrifuge, resulting in a viscous supernatant having a negligible 25 color intensity. The pH value of this discolored supernatant is adjusted with hydrochloric acid to 5.5 and then 1000 units are added. pullulanase. The resulting broth is preserved30 with toluene (added to saturation) and the pullulanase is allowed to react at ambient temperature overnight. After fermentolysis with pullschlanase overnight 35, 1% (i.e., sufficient to form a suspension of 1% concentration) of Celite Grafco No. 503, Gretco No. 503 Celite manufactured by Dzons Manville Product 40 Corporation, Lompoc, California is suspended in the broth and then added there are 2 volumes (24 l) of acetone. This forms a precipitate, which is collected by filtration, the filter cake is washed on 45 filter with acetone and dried at ambient temperature. The precipitate collected on the filter (filter cake) contains converted to non-rust-4. stealing condition (immobilized) 50 enzyme fructosyl transferase. In preparation 1, one should pay attention to the fact that adding calcium chloride to the fermentation broth55 and adjusting the pH of the broth to 7.0 removes the black pigment and acid polysaccharides present in the broth.