Sugar reduction of food products

Abstract

A process for reducing the monosaccharide and/or disaccharide content in a food material, the process comprising contacting the food material with a glucosyltransferase that comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 1.

Claims

1. A process for reducing the monosaccharide and/or disaccharide content in a food material, the process comprising reducing the sucrose level in the food material by at least 95% by contacting the food material with calcium chloride having a concentration of 0.8-1.2 mM and a glucosyltransferase having a concentration of 12 to 18 U/g sucrose, at a pH between 3 and 3.5, the glucosyltransferase comprising an amino acid sequence having 100% identity to SEQ ID NO:1, the food material comprises orange juice, and the glucosyltransferase is the only enzyme that caused the reducing of the sucrose level in the food material by at least 95%.

2. The process according to claim 1 comprising the glucosyltransferase converting monosaccharides and/or disaccharides in the food material to oligosaccharides and/or polysaccharides.

3. The process according to claim 2, wherein the oligosaccharides comprise α-1,3 glycosidic bonds and/or α-1,6 glycosidic bonds.

4. The process according to claim 2, wherein the oligosaccharides comprise α-1,2 glycosidic bonds.

5. The process according to claim 1 comprising immobilizing the glucosyltransferase on a support.

6. The process according to claim 1 comprising reducing the total combined monosaccharide and disaccharide content in the food material by at least 5%.

7. The process according to claim 1 comprising reducing the total combined monosaccharide and disaccharide content in the food material by at least 10%.

8. The process according to claim 1, wherein the food material contains at least 5% oligosaccharides based on the dry weight of the food material, after exposure to the glucosyltransferase.

9. A method for reducing the monosaccharide and/or disaccharide content and/or increasing the oligosaccharide content of a food material, the method comprising reducing the sucrose level in the food material by at least 95% by contacting the food material with calcium chloride having a concentration of 0.8-1.2 mM and a glucosyltransferase having a concentration of 12 to 18 U/g sucrose, at a pH between 3 and 3.5, the glucosyltransferase comprising an amino acid sequence having 100% identity to SEQ ID NO:1, the food material comprises orange juice, and the glucosyltransferase is the only enzyme that caused the reducing of the sucrose level in the food material by at least 95%.

10. The process according to claim 1, wherein the process is carried out at a temperature from about 45° C. to about 60° C.

11. The process according to claim 5 further comprising terminating the reduction by removing the immobilized glucosyltransferase from contact with the food material.

12. The process according to claim 1 comprising processing the food material into a food product after the contacting the food material with the calcium chloride and the glucosyltransferase, wherein the food product is selected from the group consisting of a fruit yoghurt, a powdered fruit beverage mix, a breakfast cereal with a fruit filling or inclusion, a dog treat containing berries, a frozen confectionery product, a baked confectionery product, a chocolate confectionery product, a sugar-style confectionery product, and combinations thereof.

13. The method according to claim 9 comprising processing the food material into a food product after the contacting the food material with the calcium chloride and the glucosyltransferase, wherein the food product is selected from the group consisting of a fruit yoghurt, a powdered fruit beverage mix, a breakfast cereal with a fruit filling or inclusion, a dog treat containing berries, a frozen confectionery product, a baked confectionery product, a chocolate confectionery product, a sugar-style confectionery product, and combinations thereof.

14. The process according to claim 1, wherein the calcium chloride is present at a concentration of about 1 mM.

15. The process according to claim 1, wherein the glucosyltransferase is present at a concentration of about 5.8 mg glucosyltransferase/g sucrose.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Further advantages and features of the present invention are apparent from the following Examples and Figures.

(2) FIG. 1 shows the effect of pH on the total enzyme activity of six different glucosyltransferases. Enzymatic activity was measured by the dextran sucrase activity assay according to Example 1.

(3) FIG. 2 shows the effect of pH on the glycosylation activity of six different glucosyltransferases.

(4) FIG. 3 shows the hydrolytic, transferase and total activities of the enzyme C39/3 in 50 mM sucrose, citrate buffer solution with (panel A) and without (panel B) 1 mM CaCl.sub.2.

(5) FIG. 4: The determination of optimal enzyme concentration for enzyme C39/3. Panel A shows the total C39/3 activity for different enzyme concentrations. Panel B shows C39/3 transferase activity for different enzyme concentrations. Panel C shows C39/3 hydrolytic activity for different enzyme concentrations. Panel D shows C39/3 activity at an enzyme concentration of 14.46 U/g.sub.sucrose, with and without 1 mM CaCl.sub.2.

(6) FIG. 5 shows the reduction of mono- and disaccharides catalysed by C39/3 at the optimal enzyme concentration (i.e. 14.46 U/g.sub.sucrose), in the presence of 1 mM CaCl.sub.2.

(7) FIG. 6 shows the activity of C39/3 in apple juice concentrate. Panel A shows the change in concentration of mono-, di- and oligosaccharides in apple juice concentrate over time after the addition of enzyme C39/3 (14.46 U/g.sub.sucrose, in the presence 1 mM CaCl.sub.2). The initial sucrose concentration was reduced by 96.75% after 90 min (open triangles). Panel B shows the change in concentration of leucrose, isomaltose, isomaltose triose, maltose, panose and maltotriose. Panel C shows the percentage reduction of mono- and disaccharides in apple juice concentrate catalysed by C39/3; a reduction of 7.9% was achieved after 90 min.

(8) FIG. 7 shows the activity of C39/3 in orange juice concentrate. Panel A shows the change in concentration of mono-, di- and oligosaccharides in orange juice concentrate over time after the addition of enzyme C39/3 (14.46 U/g.sub.sucrose, in the presence 1 mM CaCl.sub.2). The initial sucrose concentration was reduced by 99.46% after 90 min (open triangles). Panel B shows the change in concentration of leucrose, isomaltose, isomaltose triose, maltose, panose and maltotriose. Panel C shows the percentage reduction of mono- and disaccharides in apple juice concentrate catalysed by C39/3; a reduction of 23.2% was achieved after 90 min.

(9) FIG. 8 shows the activity of C39/1 in orange juice concentrate. Panel A shows the change in concentration of mono-, di- and oligosaccharides in orange juice concentrate over time after the addition of C39/1 (14.46 U/g.sub.sucrose, in the presence 1 mM CaCl.sub.2). Panel B shows the change in concentration of leucrose, isomaltose, isomaltose triose, maltose, panose and maltotriose. Panel C shows the percentage reduction of mono- and disaccharides in orange juice concentrate catalsyed by C39/1.

EXAMPLE 1

Methods

(10) Enzymes

(11) The use of different glucosyltransferases (EC 2.4.1.-) was investigated for enzymatic treatments of fruit juice concentrates to reduce the intrinsic sugars by polymerization/transformation into slowly or non-digestible oligosaccharides.

(12) The glucosyltransferases investigated were supplied by Biocatalysts Ltd, UK are shown in Table 1 below:

(13) TABLE-US-00002 TABLE 1 PDN Accession number Organism C39/1 Q5SBL9 Lactobacillus reuteri 121 C39/2 Q5SBN0 Lactobacillus reuteri ML1 C39/3 Q5SBN3 Lactobacillus reuteri 180 C39/4 Q5SBM3 Lactobacillus sakei Kg15 C39/5 Q5SBM8 Lactobacillus parabuchneri C39/14 Q2I2N5 Leuconostoc mesenteroides

(14) Chemicals

(15) Glucose, fructose, leucrose, isomaltose, sucrose, isomaltotriose, maltose, panose, maltotriose, maltotetraose and calcium chloride were purchased from Sigma Aldrich USA. Apple juice concentrate and orange juice concentrate were supplied by Austria juice; Ybbstaller fruit Austria and Argoterenas S.A-Industrial citrus, respectively.

(16) Glucosyltransferase Activity

(17) Glucosyltransferase activity (μ moles of fructose produced per min per 1 g of enzyme powder) was measured according to the dextran sucrase activity assay. Activities were determined by measuring D-glucose and D-fructose release from sucrose at different conditions. The amount of released fructose corresponds to the total activity (total sucrose conversion). The amount of free glucose represents the hydrolytic activity (hydrolysis of sucrose). The transferase activity is represented by the amount of released fructose minus free glucose (sucrose that has been used for transferase reactions). The assay is described below:

(18) Assay

(19) Absorbance: 575 nm; Temperature: 220 C; pH: 4.5; Incubation time: 30 min

(20) Assay Conditions

(21) TABLE-US-00003 pH 4.5 Temperature 20° C. Substrate 6.5% (w/v) sucrose Incubation time 30 minutes

(22) Unit Definition

(23) One unit of enzyme activity is defined as that amount of enzyme that causes the release of 1 micromole of glucose equivalents per minute at pH 4.5 and 20° C.

(24) Equipment

(25) Waterbath, set at 20° C.

(26) pH meter

(27) Boiling Bath

(28) Spectrophotometer, set at 540 nm

(29) Timer

(30) P1000 and P5000 pipettes

(31) Glass Test Tubes

(32) All equipment is calibrated to the requirements set out in the appropriate EOP, according to the Biocatalysts ISO9001 Manual.

(33) Reagents

(34) Water is RG grade unless otherwise specified.

(35) 1. Phosphate/Citrate/CaCl.sub.2 Buffer, pH 4.5-stable for 1 month at 15-25° C. 6.6 g di-sodium hydrogen orthophosphate-anhydrous, 5.6 g citric acid.H.sub.2O and 0.055 g CaCl.sub.2 is dissolved in approximately 400 ml of RG water. If required, the pH is adjusted to 4.5 with 1M NaOH or 1M citric acid and made up to 500 ml in volumetric flask.

(36) 2. Sucrose/CaCl.sub.2 Solution

(37) 6.5 g of sucrose and 0.011 g CaCl.sub.2 is added to a beaker and dissolved in approximately 80 ml of water. 10 ml buffer (1) is added and the solution is made up to a final volume in a volumetric flask.

(38) 3. 3-5, Dinitrosalicyclic Acid (DNS)

(39) 5 g of DNS is moistened in about 10 ml water. 100 ml 2M sodium hydroxide is added slowly with continuous stirring. 250 ml water is added followed by stirring until completely dissolved. 150 g potassium sodium (+) tartrate is added with stirring until dissolved (slight heating may be required). The solution is made up to a final volume of 500 ml with water in a volumetric flask. Filtration is performed if necessary.

(40) 4. 2M Sodium Hydroxide

(41) 40 g sodium hydroxide is dissolved in 400 ml water and made up to 500 ml in a volumetric flask.

(42) 5. D-Glucose Standard

(43) 0.5 g D-glucose is dissolved in approximately 400 ml deionised water and made up to a final volume of 500 ml with RG water in a volumetric flask.

(44) 6. Enzyme Samples

(45) Liquid and solid enzymes are first inverted to distribute the sample and weighed in an analytical balance (+/−0.001 g). The enzyme sample is diluted in buffer to a concentration which when assayed gives an absorbance change of between 0.095 and 0.2

(46) Procedure

(47) 3 test tubes are labelled for each enzyme sample (2 reactions and a blank). A colour blank and 2 assay standard tubes are also required each time an invertase assay is carried out.

(48) To each of the 3 enzyme analysis tubes 0.5 ml sucrose (2) is added. At this stage the colour blank and standard tubes remain empty. The tubes are placed in a water bath at 20° C. for 5 minutes to equilibrate. For the assay, the following procedure is used:

(49) TABLE-US-00004 Tube Time/mins Reagent Sample 1 Sample 2 Enzyme Blank Glucose Standard 1 Glucose Standard 2 Color Blank 0 Sucrose 0.5 ml 0.5 ml 0.5 ml — — — 5 Enzyme 0.5 ml 0.5 ml — — — — Vortex, and incubate at 20° C. for exactly 30 minutes. 35 DNS   3 ml   3 ml   3 ml   3 ml   3 ml 3 ml Enzyme — — 0.5 ml — — — Glucose — — — 0.5 ml 0.5 ml — Water — — — 0.5 ml 0.5 ml 1 ml Vortex, and incubate at 100° C. in boiling bath for 5 minutes. 40 Remove from boiling bath, and place in 20° C. water bath for 20 mins. 60 Read absorbance of all tubes at 540 nm, zeroing spectrophotometer with Color Blank.

(50) Calculation

(51) $\mathrm{Glucose}\mathrm{equivalents}\left(G\mathrm{mg}\text{/}\mathrm{ml}\right)=\frac{\left[\left(\mathrm{Mean}{A}_{540}\mathrm{Sample}\right)-A_{540}\mathrm{Enzyme}\mathrm{blank}\right]\times 0.5}{\left(\mathrm{Mean}{A}_{540}\mathrm{Glucose}\mathrm{Standard}\right)}$$\mathrm{Where}\text{:}0.5=\mathrm{Amount}\mathrm{of}\mathrm{glucose}\mathrm{present}\mathrm{in}\mathrm{standard},\mathrm{in}\mathrm{mg}\text{/}\mathrm{ml}$$\mathrm{Convert}\mathrm{to}\mathrm{\mu mol}\text{/}\min \text{/}g=\frac{G\times 1000\times 1000\times \mathrm{DF}}{180\times 30\times 0.5\times C}$

(52) Where: 1000=Conversion of glucose equivalents to μg 1000=Conversion of enzyme concentration to μg 0.5=volume of enzyme (ml) 180=Molecular weight of glucose 30=Reaction time (minutes) C=Concentration of enzyme (mg/ml) G =Glucose Equivalents (mg/ml) DF=Dilution Factor

(53) Therefore:

(54) $\begin{array}{cc}\begin{array}{c}U\text{/}g=\frac{G\times 1000\times 1000\times \mathrm{DF}}{180\times 30\times 0.5\times C}\\ =\frac{G\times \mathrm{DF}\times 370.37}{C}\end{array}& \\ \begin{array}{c}U\text{/}\mathrm{ml}=\frac{G\times 1000\times \mathrm{DF}}{180\times 30\times 0.5}\\ =G\times \mathrm{DF}\times 0.370\end{array}& \end{array}$

(55) Quantification of Free D-glucose and D-fructose Using a Megazyme Kit

(56) To quantify the free D-glucose and D-fructose the K-Frugil kit from Megazyme was used. Samples were first diluted by adding 20 μL sample to 2000 μL mQ water. 60 μL of the diluted sample was transferred to a 96 cell microplate and diluted further with 150 μL mQ water. 60 μL of a reference standard containing 0.2 mg/mL of D-glucose and D-fructose was included in the absorbance measurements and diluted with 150 μL mQ water. The assay was performed according the instructions supplied for the Megazyme kit (K-Frugil., 2012). Absorbance was measured at 340 nm using Varioskan flash multireader 5250510 (Thermo Scientific, USA,) at 25° C.

(57) Qualitative Analysis of Samples Using High-Performance Thin-Layer Chromatography

(58) Qualitative analysis of monosaccharides and oligosaccharides in the samples was performed by first diluting the samples 20 times with mQ water and spotting the samples as thin bands with a 1 μL micro syringe (Hamilton) on a HPTLC silica gel 60 plate (20×10 cm, 200 μm) Merck (1.05641.0001) 1 cm above the bottom edge. Standards (1 mg/mL) were also spotted (glucose, fructose, sucrose, isomaltose, panose, isomaltotriose, maltose, maltotriose, maltotetraose, leucrose and hydrolyzed dextran). Two different mobile phases (A and B) were used for different resolutions and contained chloroform, acetic acid and water in different proportions (Vol:Vol:Vol): A (36:42:5, CHCl.sub.3:CH.sub.3COOH:H.sub.2O) B (30:35:11 CHCl.sub.3:CH.sub.3COOH:H.sub.2O).

(59) Quantitative Analysis of oligosaccharides Using HPAEC

(60) Mono and oligosaccharides were analyzed using a Dionex ICS-3000 DC apparatus equipped with an HPLC carbohydrate column.

(61) Screening of glucosyltransferases at Different pHs in Pure sucrose Solutions

(62) Reactions were performed in 1.5 mL Eppendorf tubes by adding 100 μL of citrate-phosphate buffer solution containing 1320 mM sucrose (452 mg/mL) and 100 μL enzyme solution. The final reaction volume was 200 μL containing 660 mM sucrose. Reactions were performed at different pHs (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 and 7.0) incubated in a water bath at 30° C. for 30 min. To deactivate the enzyme 10% (v/v) 1M NaOH solution was added (to reach pH 11.0-11.5). To investigate the effect of Ca.sup.2+ ions on the enzyme, activity reactions were performed in the same way using 100 mM sucrose buffer solutions containing 2 mM CaCl.sub.2. The samples were analyzed by the Megazyme kit.

(63) Time Course in Pure sucrose Solution—C39/3

(64) To follow the enzymatic activity over time reactions were performed at an enzyme concentration of 7.23 U/g.sub.sucrose at an initial substrate concentration of 660 mM sucrose. The enzyme was first diluted in mQ water containing 0.04% (w/w) NaN3 as preservative. 5 mL citrate-phosphate buffer pH 3.5 (same pH as the juice concentrates) containing 1320 mM sucrose (452 mg/mL) was mixed with 5 mL enzyme solution in a 50 mL Falcon tube and incubated in an oil (Thermal M) bath at 50° C. stirred with a magnetic stirrer. Samples (500 μL) were taken out at the following time points: 0, 30, 60, 120, 180, 240, 300, 1200, 1440 min and deactivated by adding 10% (v/v) 1M NaOH solution. Samples were analyzed by HPLTC and HPAEC.

(65) Optimal Temperature—C39/3

(66) To determine the optimal temperature, for the enzyme C39/3, reactions were performed at an enzyme concentration 7.23 U/g.sub.sucrose at an initial substrate concentration of 660 mM sucrose. 200 μL citrate-phosphate buffer containing 1320 mM sucrose (452 mg/mL) pH 4.5 was mixed with 200 μL enzyme solution in 2 mL Eppendorf tubes and incubated in thermo mixers at 45° C., 50° C. and 55° C., 1000 rpm. Samples were taken out of the thermomixers after 60 min and deactivated by adding 10% (v/v) NaOH solution. The samples were analyzed by the Megazyme kit.

(67) Initial Substrate Concentration—C39/3

(68) To evaluate the effect of the initial substrate concentration on the enzyme activity, reactions were performed at an enzyme concentration 7.23 U/g.sub.sucrose at initial substrate concentrations of 660 mM, 330 mM and 165 mM sucrose. 200 μL citrate-phosphate buffer 0.1 M containing 1320 mM, 660 mM and 330 mM sucrose pH 3.5 was mixed with 200 μL enzyme solution in 2 mL Eppendorf tubes and incubated in thermomixers at 50° C., 1000 rpm. Samples were taken out of the thermomixers after 300 min and deactivated by adding 10% (v/v) 1M NaOH solution. The samples were analyzed by HPAEC.

(69) Determination of the Optimal Enzyme Concentration for C39/3

(70) To determine the optimal enzyme concentration the following enzyme concentrations were investigated: 1.45, 3.62, 7.23, 14.46, 36.15 and 72.35 U/g.sub.sucrose in the presence of 1 mM CaCl.sub.2 at an initial substrate concentration of 660 mM sucrose. The enzyme was first diluted in mQ water containing 0.04% (v/v) NaN.sub.3. 200 μL citrate-phosphate buffer containing 1320 mM sucrose (452 mg/mL) pH 4.5 was mixed with 200 μL of each enzyme solution and incubated in a thermomixer at 50° C., 1000 rpm. Samples were taken out of the thermomixer at the following time points: 0, 30, 60, 120 and 180 min and deactivated by adding 10% (v/v) 1M NaOH solution. Samples were analyzed for D-glucose and D-fructose content using the Megazyme kit.

(71) Enzymatic Treatment of Fruit Juice Concentrates

(72) Apple and orange juice concentrates were enzymatically treated with the C39/3 enzyme. Reactions were performed at enzyme concentration 14.46 U/g.sub.sucrose, pH 4.5, 50° C., 1 mM CaCl.sub.2, and 0.02% (w/w) NaN.sub.3. Samples (˜500 μL) were taken at time points t: 0, 30, 60, 90, 120, 150 and 180 min and the enzyme was deactivated by adding 50 μL 10 M NaOH solution. The reactor chambers were 50 mL Schott bottles made of Pyrex glass. Magnetic stirrers were used and the reactor chambers were immersed in temperature controlled oil (Thermal M) baths. The original pH of the juice concentrates was 3.44 for the apple concentrate and 3.55 for the orange concentrate and it was adjusted to 4.5 by adding 2.95% (v/v.sub.concentrate) of 10M NaOH (for apple concentrate) and 4.55% (v/v.sub.concentrate) of 10M NaOH (for orange concentrate) to 20 mL juice concentrate. The orange juice concentrate was diluted with 12.5% (v/v.sub.concentrate) mQ water to enable sufficient stirring. The enzyme concentration (14.46 U/g.sub.sucrose) was selected based on previous optimization of enzyme sucrose, concentration and the quantity of enzyme added was calculated based on the initial sucrose concentrations: 167.5±16.75 g/L in apple concentrate and 297±29.7 g/L in orange concentrate which was measured by HPAEC. The enzyme was diluted in 250 μL mQ water before added to the concentrates. Samples were analyzed by HPAEC.

(73) To evaluate whether the enzyme C39/3 is deactivated during standard fruit juice pasteurisation conditions 95° C. for 15 sec, the enzyme was incubated in citrate-phosphate buffer (to simulate the juice) at pH 3.0 for 0, 0.25, 0.50, 1, 2, 4, 7 and 10 min. First, 27.19 mg enzyme was dissolved in 200 μL mQ water which corresponds to 116.56 U/mL. 10 μL of the enzyme solution was injected through a septum by a syringe into a glass vial containing 500 μL citrate-phosphate buffer pH 3.0 preheated to 95° C. using a heat block (final enzyme concentration 21.89 U/mL). To measure the residual enzyme activity, 15 μL of the “pasteurized” enzyme solution was mixed with 385 μL citrate-phosphate buffer pH 4.56 solution containing 52.08 mM sucrose, 1.039 mM CaCl.sub.2 and 0.02% (w/w) NaN.sub.3 as a preservative and incubated in a thermomixer for 30 min at 50° C., 1000 rpm. The final assay conditions were: enzyme concentration 14.46 U/g.sub.sucrose, sucrose concentration 50 mM and pH 4.5. The samples were deactivated by adding 10% (v/v) 1M NaOH solution and analyzed by Megazyme kit and HPAEC.

(74) Pasteurisation

(75) To simulate a standard fruit juice pasteurisation step which lasts for 15 sec at 95° C. a setup with two oil baths containing two heating coils and one cooling coil were built. First the enzymatically treated orange juice concentrate was diluted with mQ water and the pH was adjusted to 3.0 and 3.5 by addition of 3.2 M citric acid solution, to simulate the pH range of commercial fruit juices. The diluted and pH adjusted juice was pumped at room temperature (23.8° C.) into the system through a silicone tube (4/8 mm internal/outer diameter) by an Ismatec pump (ISM 444) with a flow rate of 96 mL/min (178 rpm). First, the juice passed a preheating coil (volume 68 mL, internal diameter 0.6 cm, length 240 cm) in an thermostat (HAAKE B5/F6) controlled oil bath. The temperature of the oil (Merck S4870800728 1.06900.5000) in the first oil bath were 148° C. The juice entered the second heating coil (volume 24 mL, internal diameter 0.6 cm, Length 85 cm) at 94.6° C. where the actual pasteurisation took place. The temperature of the oil in the second oil bath (Thermo mix BU) was 98.2° C. The juice was cooled in a cooling coil (volume 48 mL, internal diameter 0.45 cm, length 300 cm) directly after the pasteurization to 8.0° C. using an ice bath and tapped on a shott bottle. After the pasteurisation, the juice was reincubated with 50% (w/v) sucrose syrup (filtered 0.2 μm), 31 g/kg.sub.final juice in a 1 L Das gip fermentor for 6 days at 50° C. The pH was adjusted to 4.5 by adding 10M NaOH solution, to measure residual enzyme activity.

(76) Measurement of Free Calcium Ions

(77) The free Ca.sup.2+ concentration was determined using a pH/Ion meter device (Metrohm 692) fitted with a perfect ION™ Ca ISE combination calcium electrode (MettlerToledo). Prior to Ca.sup.2+ measurement, the Ca ISE was calibrated using standard solutions of 1 mmol/L and 10 mmol/L calcium chloride containing 4% (v/v) 2M KCl as ionic strength adjuster.

EXAMPLE 2

Activity at Different pHs

(78) The activity of the enzymes was determined and is shown in Table 2 below.

(79) TABLE-US-00005 TABLE 2 PDN Accession number Organism Activity* (U/g) Protein*** (mg/g) Specific activity (U/mg) Sample size (mg) C39/1 Q5SBL9 Lactobacillus reuteri 121 3860 598.0 6.45 700 C39/2 Q5SBN0 Lactobacillus reuteri ML1 154 550.0 0.28 700 C39/3 Q5SBN3 Lactobacillus reuteri 180 2475 594.0 4.17 700 C39/4 Q5SBM3 Lactobacillus sakei Kg15 90 572.9 0.16 700 C39/5 Q5SBM8 Lactobacillus parabuchneri 42 534.0 0.079 700 C39/14 Q2I2N5 Leuconostoc mesenteroides 1589 570.8 2.78 700 *Activity measured with dextran sucrase assay ***Protein measured with Bradford assay

(80) The six different glucosyltransferases were screened for activity at different pHs in pure sucrose solution. The enzymes concentrations (U/g.sub.sucrose and mg/g .sub.sucrose) are presented in Table 3. The samples were analyzed for D-fructose and D-glucose using the Megazyme kit.

(81) TABLE-US-00006 TABLE 3 Equivalence between U/g.sub.sucrose and mg.sub.protein/g.sub.sucrose for the six different glucosyltransferases. Enzyme U/g.sub.sucrose mg.sub.protein/g.sub.sucrose C39/1 0.129 0.02 C39/2 0.1148 0.41 C39/3 0.0813 0.0195 C39/4 0.0528 0.33 C39/5 0.076 0.97 C39/14 0.0667 0.024

(82) The results are shown in FIGS. 1 and 2.

(83) The enzyme C39/3 was shown to have very high activity (see Table 2), and it was shown to be active even at low pH (FIGS. 1 and 2).

EXAMPLE 3

Effect of Calcium Ions on the Activity of C39/3

(84) The hydrolytic, transferase and total activity of the enzyme C39/3 in 50 mM sucrose, citrate-phosphate buffer solution with and without 1 mM CaCl.sub.2 is shown in FIG. 3. The average transferase activity, hydrolytic activity, and total activity are 17%, 5% and 13% respectively higher in presence of 1 mM CaCl.sub.2 after 30 min.

EXAMPLE 4

Effect of Temperature on the Activity of C39/3

(85) The enzyme was assayed at 45° C., 50° C. and 55° C. The hydrolytic activity at 45° C. and 55° C. is 85% and 90% respectively of the hydrolytic activity at 50° C. The transferase activity at 45° C. and 55° C. is 82% and 76% respectively of the transferase activity at 50° C. in 660 mM sucrose solution. 50° C. is considered as the optimal temperature for the enzyme.

EXAMPLE 5

Effect of Initial Substrate Concentration on the Activity of C39/3

(86) The transferase activity was found to be 68%, 54% and 50% of the total activity in 660 mM, 330 mM and 165 mM sucrose respectively. Thereby the transferase activity is favoured by high substrate concentrations. The % sucrose reduction (approx. 8%) is not affected by the initial sucrose concentration.

EXAMPLE 6

Determination of the Optimal Enzyme Concentration for C39/3

(87) Reactions at different enzyme concentrations were measured. At an enzyme concentration of 72.35 U/g.sub.sucrose, the total activity reached a maximum of approximately 75 mg.sub.fructose/mL after 30 min compared to approximately 80 mg/mL after 60 min at an enzyme concentration of 14.46 U/g.sub.sucrose with 1 mM CaCl.sub.2 (FIG. 4A). The transferase activity was higher at an enzyme concentration of 14.46 U/g.sub.sucrose with 1 mM CaCl.sub.2 compared to 72.35 U/g.sub.sucrose (FIG. 4B). The hydrolytic activity was lower at an enzyme concentration of 14.46 U/g.sub.sucrose with 1 mM CaCl.sub.2 compared to 72.35 U/g.sub.sucrose (FIG. 4C). The average transferase activity, hydrolytic activity, and total activity were 16%, 5% 14% higher, respectively in presence of 1 mM CaCl.sub.2 after 30 min (FIG. 4D). The average transferase activity, hydrolytic activity, and total activity are 17%, 15% 17% higher, respectively in presence of 1 mM CaCl.sub.2 after 60 min (FIG. 4D). An enzyme concentration of 14.46 U/g.sub.sucrose with 1 mM CaCl.sub.2 was considered to be optimal.

(88) The equivalence between U/g.sub.sucrose and mg.sub.protein/g.sub.sucrose for the enzyme C39/3 (Q5SBN3, from Lactobacillus reuteri 180) was determined, see Table 4 below.

(89) TABLE-US-00007 TABLE 4 Equivalence between U/g.sub.sucrose and mg.sub.protein/g.sub.sucrose for the enzyme C39/3 (Q5SBN3, from Lactobacillus reuteri 180). U/g.sub.sucrose mg.sub.protein/g.sub.sucrose 72.3 29.22 36.15 14.61 14.46 5.844 7.23 2.922 3.62 1.46 1.45 0.584

EXAMPLE 7

Quantitative Analysis of Products Created by C39/3 at the Optimal Enzyme Concentration

(90) Reaction samples at the optimal enzyme concentration (14.46 U/g.sub.sucrose with 1 mM CaCl.sub.2) were analyzed by HPAEC. The main products (% mg/mg.sub.sucrose) are fructose (35.12%) and leucrose (10.45%), followed by glucose (3.85%) and isomaltose (1.41%). The sucrose was reduced to 99.56% and a 48.2% reduction of mono and disaccharides was achieved after 60 min (FIG. 5 and Table 5). The activities by the enzyme are much higher in reality than predicted by the Megazyme kit method because of the formation of leucrose by acceptor reactions which is not detected by the method.

(91) TABLE-US-00008 TABLE 5 Concentrations (mg/mL) of mono-, di- and oligosaccharides generated by C39/3 in 660 mM sucrose, citrate-phosphate pH 4.5 buffer solution with 1 mM CaCl.sub.2. time(min) glucose fructose leucrose isomaltose sucrose isomaltotriose maltose panose maltotriose 0 1.38 2.15 0.00 0.00 220.72 0.15 0.00 0.00 0.00 30 14.70 68.21 14.84 2.06 40.91 0.39 0.00 0.00 0.00 60 9.85 79.33 22.95 3.11 0.96 0.56 0.00 0.23 0.00 120 9.41 86.52 22.78 3.46 1.29 1.06 0.00 0.26 0.00 180 9.82 88.56 24.27 3.83 0.63 1.27 0.00 0.26 0.00

EXAMPLE 8

C39/3 Activity in Apple Juice Concentrate

(92) The apple juice concentrate has high initial concentrations of glucose and fructose (249.74±24.97 mg/mL and 384.57±38.46 mg/mL, respectively). The initial sucrose concentration in the apple juice concentrate was measured to be 149.38 mg/mL and was reduced by 96.75% to a concentration of 4.85 mg/mL after 90 minutes. A reduction of mono and disaccharides of 7.9% was achieved after 90 min (FIG. 6A, FIG. 6C and Table 6). The main identified products formed (% mg/mg.sub.sucrose) were leucrose (28.60%), fructose (23.05%), isomaltose (19.09%) and isomaltotriose (7.45%) (FIG. 6B and Table 6). The high production of leucrose can be explained by the high fructose concentration since fructose act as acceptor molecule in the so called acceptor reaction catalyzed by the enzyme

(93) TABLE-US-00009 TABLE 6 Concentrations (mg/mL) of mono-, di- and oligosaccharides generated by C39/3 in apple juice concentrate. time(min) glucose fructose leucrose isomaltose sucrose isomaltotriose maltose panose maltotriose 0 249.74 384.57 0.00 1.80 149.38 0.00 1.67 0.00 0.33 30 239.06 412.61 30.01 23.40 54.57 6.94 2.32 0.00 0.00 60 230.67 417.37 40.27 29.35 16.03 9.32 1.77 0.05 0.11 90 229.32 417.89 41.33 29.40 4.85 10.76 1.79 0.00 0.50 120 234.56 439.32 43.72 30.88 3.03 12.07 2.20 0.13 0.69 150 227.44 439.48 43.16 30.01 2.30 11.63 2.14 0.29 0.88 180 223.94 421.32 42.87 32.34 3.27 10.71 1.98 0.09 0.54

EXAMPLE 9

C39/3 Activity in Orange Juice Concentrate

(94) The initial sucrose concentration in the orange juice concentrate was measured to be 277.39 mg/mL and was reduced by 99.46% to a concentration of 1.51 mg/mL after 90 minutes. A reduction of mono and disaccharides of 23.2% was achieved after 90 min (FIG. 7A, FIG. 7C and Table 7). The main products formed (% mg/mg.sub.sucrose) were fructose (32.90%), leucrose (11.93%) and isomaltose (7.63%) (FIG. 7B and Table 7).

(95) TABLE-US-00010 TABLE 7 Concentrations (mg/mL) of mono-, di- and oligosaccharides generated by C39/3 in orange juice concentrate. time(min) glucose fructose leucrose isomaltose sucrose isomaltotriose maltose panose maltotriose 0 156.75 151.53 1.69 0.67 277.39 0.00 1.52 0.00 0.00 30 147.58 193.59 19.25 14.85 120.34 6.74 4.23 0.00 3.64 60 150.21 231.57 30.97 18.67 12.70 5.95 0.35 0.00 0.00 90 150.93 242.29 34.60 21.73 1.51 11.38 1.70 0.00 0.00 120 149.25 231.86 29.65 19.85 2.38 12.65 1.92 0.00 0.00 150 144.42 233.63 34.96 25.23 2.28 14.61 0.66 0.00 0.00 180 146.31 259.00 39.00 24.03 2.67 11.15 0.65 0.00 0.00

EXAMPLE 10

C39/1 activity in Orange Juice Concentrate

(96) The enzyme GTF121 C39/1 (referred to herein as C39/1) was also used to treat the orange juice concentrate to compare the sugar reduction and products with products created by the enzyme C39/3.

(97) The initial sucrose concentration in the orange juice concentrate was measured to be 296.75 mg/mL and was reduced by 83.60% to a concentration of 48.65 mg/mL after 180 minutes (FIG. 8A and Table 8). A reduction of mono and disaccharides of 19.4% was achieved after 180 min (FIG. 8C). The main products formed (% mg/mg.sub.sucrose) were fructose (27.64%), isomaltose (14.95%) and leucrose (13.46%) (FIG. 8A, FIG. 8B and Table 8).

(98) TABLE-US-00011 TABLE 8 Concentrations (mg/mL) of mono-, di- and oligosaccharides generated by GTF 121 C39/1 in orange juice concentrate. time(min) glucose fructose leucrose isomaltose sucrose Isomaltotriose maltose panose maltotriose 0 179.17 172.45 2.69 1.35 296.75 0.00 1.33 0.00 0.14 30 163.51 177.54 11.42 13.62 236.25 0.27 1.14 0.39 0.12 60 162.95 192.04 20.40 21.56 198.15 0.33 1.37 0.98 0.00 90 176.79 234.37 25.09 27.28 116.84 0.54 1.61 1.66 0.00 180 160.09 241.03 36.09 38.45 48.65 1.80 2.87 4.15 0.00 1440 149.84 239.94 50.17 59.39 3.95 4.32 3.04 5.51 0.00

(99) The use of C39/3 results in a greater reduction in monosaccharides and disaccharides over the using of C39/1 (compare FIG. 7C and FIG. 8C).

EXAMPLE 11

NMR Analysis of Enzymatic Treated Samples

(100) Samples from determination of optimal enzyme concentration and the enzymatically treated orange and apple juice concentrates were sent for external analysis to Spectral Service AG in Germany. The analysis showed that the linkages in the formed products in apple juice concentrate, orange juice concentrate and sucrose solution are different α-1,6 glycosidic linkages are predominant in apple juice, products in orange juice possesses both α-1,6 and α-1,3 glycosidic linkages. Products with α-1,3 glycosidic linkages are predominant in enzymatically treated 660 mM sucrose citrate-phosphate buffer solution. The presence of α-1,2 glycosidic linkages are present in low amounts in juices and absent in sucrose solution. The analysis also showed that the presence of oligomers was lower in the juice concentrate than in the sucrose citrate-phosphate buffer solution (Table 9).

(101) TABLE-US-00012 TABLE 9 Overview of presence of oligomers in samples sent for NMR analysis. Presence of Sample oligomers Orange juice enzymatically treated 60 min C39/3 Yes low Orange juice control No Apple juice enzymatically treated 60 min C39/3 Yes low Apple juice control No Sucrose 660 mM pH 4.5, 1 mM CaCl.sub.2 enzymatically Yes high treated 60 min C39/3

EXAMPLE 12

Free Calcium in Fruit Juice Concentrates

(102) Sucrose reduction by C39/3 was slower in orange juice than in 660 mM sucrose citrate phosphate buffer solution (at pH4.5, 50° C.). One reason for this could be the chelation of Ca.sup.2+ by different agents present in the fruit juice concentrates (e.g. citrates). Since Ca.sup.2+ have a stimulating effect on the enzyme activity, the presence of free Ca.sup.2+ in fruit juice concentrates was investigated.

(103) Measurement in sucrose 660 mM, pH 4.5 citrate-phosphate solution after the addition of 1 mM Ca.sup.2+ added showed that the free Ca.sup.2+ concentration was 0.28 mM. The free Ca.sup.2+ concentration in orange juice concentrate was 0.1 mM after the addition of 1 mM total Ca.sup.2+ and 0.55 mM after 22.23 mM total Ca.sup.2+ addition. This can be attributed to the higher concentration of chelating agents (e.g. citric acid) in the fruit concentrate than in the sucrose solution.

(104) The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O′D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; and E. M. Shevach and W. Strober, 1992 and periodic supplements, Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. Each of these general texts is herein incorporated by reference.