WO2002082923A1

WO2002082923A1 - Modified pectic substance

Info

Publication number: WO2002082923A1
Application number: PCT/DK2002/000240
Authority: WO
Inventors: Annette Kristensen Strarup; Karin Meyer Hansen; Anders Vagnø PEDERSEN; Morten Mohr Hansen; Susanne Lund Madsen; Nina Musaeus Jensen; Carsten Lynggaard Hansen
Original assignee: Cp Kelco Aps; Basf Health & Nutrition A/S
Priority date: 2001-04-10
Filing date: 2002-04-10
Publication date: 2002-10-24

Abstract

Modified pectic substance having emulsifying, microencapsulating, foam stabilizing and/or film forming properties and obtainable by treating a pectic substance with one or more enzymes selected from the group consisting of esterases (E.C.3.1.), glucosidases (E.C.3.2.) and lyases (E.C.4.) and products consisting of or containing such modified substance.

Description

Modified pectic substance

FIELD OF INVENTION

The present invention relates to a modified pectic substance, a process of modifying a pectic substance, products containing a modified pectic substance, an emulsifier, a foam stabilizer, a film forming agent and a microencapsulating agent.

BACKGROUND OF THE INVENTION

Emulsions, i.e. disperse systems consisting of two or more mutually insoluble or sparingly soluble liquids and in which one liquid - the continuous or external phase - usually is present in excess relative to the second liquid - the dispersed or internal phase - find wide spread use in the food, beverage and pharmaceutical industries.

The manufacture of such products normally involve two problems, i.e. droplet formation to form an emulsion and stabilization of the emulsion formed, and it is well-known to use agents, viz. emulsifiers, emulsion promoters and emulsion stabilizers to boost the emulsifying process.

The emulsifiers presently used may be divided into three groups, viz. (1 ) synthetic surface-active emulsifiers, (2), emulsifiers of natural origin, and (3) inorganic emulsifiers which generally have a low surface activity.

Most synthetic surface-active emulsifiers are unacceptable for use in products for human intake such as food products and beverages, and, therefore, natural emulsifiers are preferable for use in such products.

Some of the natural emulsifiers such as whey proteins, e.g. casein, are of animal origin and are unacceptable for use in vegetarian foods, kosher foods and halal foods, and others such as gum arabic can only be obtained in limited amounts. Therefore, it has been attempted to find other natural non-animal emulsifiers such as pectin for use in emulsified food products, beverages and similar products for human intake.

EP 332.027 A1 discloses a modified starch emulsifier, which is obtained by enzymatic treatment of a starch molecule with an amylase.

US patent No. 5 900 268 (Mazoyer et al.) discloses depolymerised citrus fruit, sugar beet pectin and apple pectins and their use as emulsifiers and emulsion stabilizers.

WO 95/34223 A1 discloses a method of producing cloud stable extracts from plant material by using one or more enzymes that attack the hairy regions of pectin from vegetables or fruit, such as apple.

The article "Acetylated pectic polysaccharides of sugar beet", Dea et al., Food Hydrocolioids, Vol. 1, No. 1 , pp. 71-88, 1986, presents a study of the chemical composition and the surface-active and emulsifying properties of sugar beet pectin.

EP 426.434 A1 discloses the use of unmodified sugar beet pectin in food or drug comestibles, e.g. whipped products, emulsions, or gels.

Danish Patent Application 1991 01060 discloses unbranched arabans, e.g. from sugar beet, obtained by treatment of sugar beet araban with α-L- arabinofuranosidase, and their use as gelling agents, as emulsifiers and as encapsulating material. The sugar beet arabans used as enzyme substrate in that process are obtained by an alkaline treatment of sugar beet pulp, and the raw sugar beet arabans comprises typical about 70-85 % arabinose, 5-10 % uronic acid, 8-15 % D-galactose, and a few % rhamnose and other monosaccharides. WO 00/70967 A1 discloses colouring compositions comprising colouring substance bodies which are at least partially coated with a pectin selected from the group consisting of beet pectin, chicory pectin and Jerusalem artichoke pectin which act as a wetting and/or dispersing agent. WO 97/10726 A1 discloses a process for increasing the viscosity or gel strength of food products by subjecting a pertinacious homogenate or slurry from fruit or vegetables, e.g. orange, broccoli or tomato, to an enzymatically treatment.

WO 00/58367 A1 discloses pectin lyase modified pectin, such as lemon pectin, suitable for increasing the storage stability of comestible compositions comprising proteins.

US patent No. 5 998 176 (Budolfsen et al.) discloses a method for causing gelling or increase of viscosity of an aqueous medium containing a pectic material such as sugar beet pectin with pectin esterase (PE) (E.G.3.1.1.1 ), rhamnogalacturonan-acetylesterase (RGAE) or α-arabinofuranosidase (α- ARA) alone or mixtures of PE (E.C.3.1.1.11.) and RGAE or of α-ARA and PE (E.C.3.1.1.11.).

WO 00/17368 discloses an orange fruit acetyl esterase (ACE) and a process wherein the ACE is contacted with a substrate, such as pectin from a fruit or a vegetable. Improved gelling properties are obtained by deacetylating sugar beet pectin with the disclosed ACE.

US patent no. 5.639.494 discloses modified pectin obtained by enzymatic treatment of fruit or vegetables, with pectyl esterase and their use for gelling purposes.

The object of the present invention is to modify a pectic substance from beetroot to improve its emulsifying properties and make it suitable for use in emulsified products for human intake or external use.

SUMMARY OF THE INVENTION The modified pectic substance according to the invention is characterized in that it is obtainable by treating a pectic substance from beetroot with one or more enzymes selected from the group consisting of esterases (E.C.3.1.), glucosidases (E.C.3.2.) and lyases (E.G.4.) with the exception of PE (E.C.3.1.1.11.), ACE, RGAE or α-ARA alone and mixtures of PE (E.C.3.1.1.11.) and RGAE and of α-ARA and PE (E.C.3.1.1.11.).

Surprisingly it has been found that such a modified pectic substance exhibit significantly improved emulsifying properties compared to unmodified pectic substances.

Pectins are high-molecular weight polygalacturonic acids joined by (1=>4)-α- glycosidic links in which some of the carboxylic acid groups are esterified with methanol and they are composed of flexible regions in which the polymer backbone is rich in rhamnose and have side chains, "hairy regions", of a complex nature.

Pectins also comprise "smooth regions" which are rigid regions in which the backbone consists essentially of galacturonic acid or galacturonic acid residues having very small side chains such as methyl or ethyl groups.

As used in connection with the present invention, the term "pectic substance" encompasses pectin, pectic acid and salts and esters of pectic acid (pectates), whereby the pectic substance has a galacturonic acid content of above 40 %.

The galacturonic acid content of the pectic substance is preferable above 50 % and more preferred above 65 %.

US Patent No. 5 929 051 contains a more detailed discussion of the general structure of pectin and pectic substances as presently understood.

The modified pectic substance of the invention is preferably obtainable by treating a pectic substance from beetroot comprising hairy regions and a backbone of smooth regions with one or more enzymes capable of modifying the hairy regions, viz. the side chains of the rhamnogalacturonan backbone of the pectin.

Preferred examples of such enzymes are rhamnogalacturonase, rhamnogalacturonan acetyl esterase, β-galactosidase, arabinanase, galactanase and α-arabinofuranosidase.

Another preferred modified pectic substance is a pectin which is obtainable by treating with one or more enzymes capable of modifying the backbone of the smooth regions of pectin so as to form separate elements comprising hairy regions.

Examples of such enzymes are pectin lyase and combinations of polygalacturonase and pectin methyl esterase.

In another preferred embodiment the modified pectic substance is obtainable by treating a pectic substance from beetroot with one or more enzymes selected from the group consisting of esterases, glucosidases, and lyases followed by additional treating with a proteolytic enzyme, such as papain.

The pectic material to be modified according to the invention is derived from beetroot (Beta vulgaris L. Chenopodiaceae), including sugar beet, garden beet (red beets), chard, mangel, spinach beet, silver beet, and fodder beet. Sugar beet pectin is a particularly useful pectic substance.

The enzyme classification system used in the definition of enzymes for use for modifying pectin substances according to the present invention is described in Enzyme Nomenclature 1992, Academic Press, San Diego, California, with supplements.

Esterases (E.C.3.1) constituting a subclass to hydrolases (E.G.3) are enzymes that catalyse the hydrolysis of ester groups of pectin. Preferred esterases for use for the modification of pectic substances are deacetylating enzymes, such as rhamnogalacturonan-acetyl esterase, pectin acetyl esterase, and pectin methyl esterases.

Glucosidases (E.C.3.2), which also constitutes a subclass to hydrolases (E.G.3) are enzymes that catalyse the hydrolysis of glycosidic bonds in pectin. Preferred glucosidases are debranching enzymes, such as α- arabinofuranosidase, galactanase, arabinanase and endo- and exo- polygalacturonases. A mixture of α-arabinofuranosidase, galactanase, rhamnogalacturonase and arabinanase is particularly useful.

Lyases (E.C.4) are enzymes, which catalyse addition to double bonds.

The term "deacetylating enzyme" means an enzyme, which is capable of removing acetyl groups, which are covalently bonded to galacturonic acid residues in the backbone of pectin.

The terms "shaving enzyme" or "debranching enzyme" mean enzymes which are capable of reducing the length of the side chains of the hairy regions of pectin.

The present invention also relates to a process of modifying a pectic substance, said method comprising the steps of providing an aqueous medium of a pectic substance from beetroot and adding to said medium one or more enzymes selected from the group consisting of esterases (E.C.3.1.), glucosidases (E.C.3.2.) and lyases (E.C.4.) with the exception of PE (E.C.3.1.1.11.), ACE, RGAE or α-ARA alone and mixtures of PE (E.C.3.1.1.11.) and RGAE and of α-ARA and PE.

In practice, the enzymatic modification of pectic substances may be carried out as follows:

Temperature and pH of the pectin preparation is adjusted to working temperature and pH of the enzyme to be used. The enzyme or mixture of enzymes is dissolved/diluted in ion exchanged water and added to the pectin preparation. Reaction is carried out while stirring continuously, and if necessary pH is controlled by titration. After a certain time reaction is terminated by lowering pH. In order to irreversibly inactivate the enzyme, temperature is raised to 80°C for 10 min. Temperature of the solution is lowered and the pectin is precipitated (1 :3) in 80% 2-propanol. The precipitated pectin is drained on a belt press and put in a drying cabinet at 70°C for at least 24 hours. After drying the pectin is ground and sieved (DIN 24).

The enzymatic modification may include an additional treatment with a proteolytic enzyme, such as papain. In this case the process is repeated with the proteolytic enzyme after inactivation of the first enzyme or mixture of enzymes.

The pectin preparation to be used as substrate for enzyme treatment can be an extract directly obtained from the raw material e.g. sugar beet peel or it can be a solution of a refined pectin product. An extract of sugar beet pectin may be prepared as follows:

1 ) Mixing dry granular beet pulp with an aqueous solution of a strong, mineral acid, preferably nitric acid

2) Extracting the pulp with rigorous agitation for about one to five hours at 60-80°C and pH ranging from 1.5 to 2.5.

3) Separating the resulting mixture into waste solids and a liquid containing pectin

4) Treating the liquid containing pectin with enzyme as described above.

A pectin solution is made by adding a pectin powder to hot (70°C) ion- exchanged water. The preparation is stirred continuously, until the pectin is completely dissolved. The invention also relates to an emulsifier obtainable by modifying a pectic substance with one or more enzymes selected from a group consisting of esterases (E.C.3.1.), glucosidases (E.C.3.2.) and lyases (E.C.4.).

The terms "emulsifier" and "emulsifying agent" as used herein are intended to mean products which exhibit emulsion forming or emulsion stabilizing properties or both.

In addition to its excellent emulsifying properties such modified pectic substances exhibit foam stabilizing and film forming properties.

The modified pectic substances according to the invention are suitable for use in a vast number of products such as food emulsions, e.g. soft drinks, margarine, ice cream, organic coffee milk, mayonnaise, salad dressings, bread, confectionary, pharmaceutical products and cosmetics and health care products such as lotions and cremes.

The modified pectic substance is also suitable for use as a microencapsulating agent. The term "microencapsulating" as used herein means a process of providing particles, each comprising a matrix of a microencapsulating agent having embedded therein a plurality of solid or liquid micro particles.

For further information concerning the use of the modified pectic substance as an encapsulating agent reference is made to the co-pending WO patent application No. (Our reference P200100040 WO).

The invention will be described in further detail with reference to the following examples.

EXAMPLES

In the examples the molecular weight is measured by the Capillary Tube Method principle as follows: The outlet time is measured for a pectin/hexametaphosphate solution and the molecular weight is thereafter calculated after a well-known formula (see WO 00/58367 Pectin having reduced calcium sensitivity, page 12).

The outlet time is measured on two outlets. If the difference between the times is more than 0.4 seconds the measuring is repeated until the difference is less than the appropriate value. The outlet time used for the molecular determination is the mean value of the above-mentioned identical or substantially identical measuring results.

Preparation of enzvmaticallv modified pectic substances

Example 1

In this example the pectic substance was derived from sugar beets (GENU beta pectin, lot 82899, produced by CP Kelco, Lille Skensved, Denmark). The enzymes used were α-arabinofuranosidase (batch sp 580, PPJ 4494), arabinanase (batch sp 564, PPJ 4381 ) and galactanase (batch sp 518 PPJ 4368), all produced by Novo Nordisk, Bagsvaerd, Denmark.

1000 I of ion-exchanged water was heated to 70 °C and 10 kg of pectic substance was added, while stirring continuously. After the pectin was completely dissolved, the temperature was lowered to 45 °C, and pH was adjusted to 4.50 by titration with a 2% (w/v) NH₃ solution. 5 grams of arabinanase, 35 grams of α-arabinofuranosidase and 35 grams of galactanase were diluted in approximately 11 ion exchanged water at ambient temperature and added to the pectin solution. After 4 hours, reaction was stopped by addition of a 10% (w/v) HNO₃ solution till pH 3.00. In order to irreversibly inactivate the enzyme, the temperature was raised to 80 °C. After 10 minutes at 80 °C, the solution was cooled to 20 °C, and the modified pectin was precipitated (1 :3) in 80% 2-propanol. The precipitated pectin was drained on a belt press and put into a drying cabinet at 70 °C for 24 hours. After drying the pectin was ground and sieved (DIN 24). Degree of acetylation (%D(Ac)), degree of esterification (%DE), galacturonic acid content (%GA), molecular weight (MW) and neutral sugar content of the enzyme treated pectin sample were determined, and the results obtained appear from Table 1.

TABLE 1

Example 2

In this example the pectic substance was derived from sugar beets (GENU beta pectin, lot 92455 produced by CP Kelco, Lille Skensved, Denmark) and the enzyme was rhamnogalacturonan acetyl esterase (batch PPJ 4456, produced by Novo Nordisk, Bagsvaerd, Denmark).

50 I of ion-exchanged water was heated to 70 °C, and 1 kg of pectic substance was added, while stirring continuously. The temperature was lowered to 50 °C, and pH was adjusted to 4.50 by titration with a 2% (w/v) NH₃ solution. 10 grams of rhamnogalacturonan acetyl esterase was added to the pectin solution. After 24 hours at 50 °C, while stirring continuously, the reaction was stopped by addition of a 10% (w/v) HNO₃ solution till pH 2.50. In order to irreversibly inactivate the enzyme, the temperature was raised to 80 °C. After 10 minutes at 80°C, the solution was cooled to 50 °C, and the modified pectin was precipitated (1 :3) in 80% 2-propanol. The precipitated pectin was drained on a belt press and put into a drying cabinet at 70 °C for 24 hours. After drying the pectin was ground and sieved (DIN 24). Degree of acetylation (%D(Ac)), degree of esterification (%DE), galacturonic acid content (%GA) and molecular weight (MW) were determined and the results obtained appear from Table 2.

TABLE 2

Example 3

In this example the pectic substance was derived from sugar beets (GENU beta pectin, lot 82899 produced by CP Kelco, Lille Skensved, Denmark) and the enzyme was rhamnogalacturonase (Batch PPJ 4478, produced by Novo Nordisk, Bagsvaerd, Denmark).

50 I of ion-exchanged water was heated to 70 °C, and 1 kg of pectic substance was added, while stirring continuously. The temperature was lowered to 50 °C, and pH is adjusted to 4.50 by titration with a 2% (w/v) NH₃ solution. 3.125 grams of rhamnogalacturonase was dissolved in approximately 50 ml of ion-exchanged water and added to the pectin solution. After 4 hours at 50 °C, the reaction was stopped by addition of a 10% (w/v) HNO₃ solution till pH 2.50. In order to irreversibly inactivate the enzyme, the temperature was raised to 80°C. After 10 minutes at 80 °C, the solution was cooled to 50 °C, and the modified pectin was precipitated (1 :3) in 80% 2-propanol. The precipitated pectin was drained on a belt press and put into a drying cabinet at 70 °C for 24 hours. After drying the pectin was ground, and finally the pectin was sieved (DIN 24). Degree of acetylation (%D(Ac)), degree of esterification (%DE), galacturonic acid content (%GA) and molecular weight (MW) were determined and the results obtained appear from Table 3.

TABLE 3

Example 4

In this example the pectic substance was derived from sugar beet (Batch no. 30003, Type SF H-25, produced by CPKelco, Pomosin, Germany) and the enzyme was Enzeco Pectinase PL lyase from Enzyme Development Corporation, Batch no. S-11677, Activity 26 U/ml. 55 I of ion-exchanged water was heated to 70 ° C and 2,75 kg of pectic substance was added, while stirring continuously. After the pectin was completely dissolved, the temperature was lowered to 45 °C; pH was adjusted to 4.50 by titration with a 10% (^w/v) soda solution. 1 ,63 ml Enzeco Pectinase PL lyase was added to the pectin solution. During the experiment the pH was kept constant at 5,5 by titration with 5% (^w/v) soda. After 6 hours the reaction was stopped by addition of a 10% ( /v) soda solution till pH 2,50. In order to irreversibly inactivate the enzyme, the temperature was raised to 80 ° C. After 10 minutes at 80 ° C, the solution was cooled to 50 ° C, evaporated to half amount by the following procedure: The solution was transferred to the evaporator. Heat was applied under vacuum at 0.8 bar, and the solution reached the boiling point (around 60 ° C). The solution was cooled to 50 ° C and the modified pectin was precipitated (1 :3) in 80% 2- propanol. The precipitated pectin was drained on a belt press and put into a drying cabinet at 70 ° C for 24 hours. After drying, the pectin was ground and sieved (DIN 24). Degree of acetylation (% D(Ac)), degree of esterification (% DE), galacturonic acid content (%GA) and molecular weight (MW) of the enzyme treated pectin were determined and the results obtained appear from Table 4.

Table 4

Example 5 In this example the pectic substance V derived from sugar beet (Batch no. 30003, Type SF H-25, produced by CP Kelco, Pomosin, Germany) and the enzymes was Polygalacturonase Rohament PL, Batch no. 97.01.001 , 5270 U/ml produced by Rohm and Polymethylesterase (PME) Rheozyme, Batch no. 0114510L, 8992 μ/ml produced by Novozymes, Denmark.

55 I of ion-exchanged water was heated to 70 ° C and 2,75 kg of pectic substance was added, while stirring continuously. After the pectin was completely dissolved, the temperature was lowered to 45 ° C; pH was adjusted to 4.50 by titration with a 10% (^w/v) soda solution. 0,55 ml

Polygalacturonase Rohament PL was added to the pectin solution. 8,8 ml Polymethylesterase Rheozyme was added to the solution. During the experiment the pH was kept constant at 4,5 by titration with 5% (^w/v) soda. After 2 hours the reaction was stopped by addition of a 10% (^w/v) soda solution till pH 2,50. In order to irreversibly inactivate the enzyme, the temperature was raised to 80 ° C. After 10 minutes at 80 ° C, the solution was cooled to 50 ° C, evaporated to half amount by the following procedure: The solution was transferred to the evaporator. Heat was applied under vacuum at 0.8 bar, and the solution reached the boiling point (around 60 ° C). The solution was cooled to 50 ° C and the modified pectin was precipitated (1 :3) in 80% 2-propanol. The precipitated pectin was drained on a belt press and put into a drying cabinet at 70 ° C for 24 hours. After drying, the pectin was ground and sieved (DIN 24). Degree of acetylation (% D(Ac)), degree of esterification (% DE), content of galacturonic acid (%GA) and molecular weight (MW) of the enzyme treated pectin were determined and the results obtained appear from Table 5:

TABLE 5

Example 6

In this example the pectic substance was derived from sugar beet (Batch no. 30003, Type SF H-25, produced by CP Kelco, Pomosin, Germany) and the enzymes was Polygalacturonase Rohament PL, Batch no. 97.01.001 , 5270 U/ml produced by Rohm, Polymethylesterase (PME) Rheozyme , Batch no. 0114510L, 8992 μ/ml produced by Novozymes, Denmark and Collupulin, Batch R 9741 , Activity 2214 U/g(18.4.2000) and 2186 U/g (19.3.2001) produced by Gist-Brocades in Netherlands.

45 I of ion-exchanged water was heated to 70 ° C and 2,25 kg of pectic substance was added, while stirring continuously. After the pectin was completely dissolved, the temperature was lowered to 45 ° C; pH was adjusted to 4.50 by titration with a 10% (^w/v) soda solution. 0,45 ml

Polygalacturonase Rohament PL was added to the pectin solution. 7,2 ml Polymethylesterase Rheozyme was added to the solution. During the experiment the pH was kept constant at 4,5 by titration with 5% (^w/v) soda. After 2 hours (720 ml 10% (^w/v) soda solution consumed) the reaction was stopped by addition of a 10% (^w/v) soda solution till pH 2,50. In order to irreversibly inactivate the enzyme, the temperature was raised to 80 °C. After 10 minutes at 80 ° C 1052 g NaCI was added (0.4 M NaCI), the temperature was lowered to 45 ° C and pH was adjusted to 5.50 by titration with a 10% soda solution. 27 grams of Collupulin was added to 100 ml of demineralised water and added to the solution for an additional enzymatic modification of the pectin solution. During the additional enzymatic modification step, the pH was kept constant at 5.50 by titration with 5% ammonia. After 20 minutes, the reaction was stopped (703 ml 2 % ammonia). PH was lowered to 2.50 by diluted HNO₃. In order to irreversibly inactivate the Collupulin, the temperature was raised to 80 °C.

After 10 minutes at 80 ° C, the solution was cooled to 50 °C, evaporated to half amount by the following procedure: The solution was transferred to the evaporator. Heat was applied under vacuum at 0.8 bar, and the solution reached the boiling point (around 60 ° C). The solution was cooled to 50 ° C and the modified pectin was precipitated (1 :3) in 80% 2-propanol. The precipitated pectin was drained on a belt press and put into a drying cabinet at 70 ° C for 24 hours. After drying, the pectin was ground and sieved (DIN 24). Degree of acetylation (% D(Ac)), degree of esterification (% DE), content of galacturonic acid (%GA) and molecular weight (MW) of the enzyme treated pectin were determined, and the results obtained appear from Table 6:

TABLE 6

Example 7

In this example the pectic substance was derived from sugar beet pectin (Batch no. 30003, Type SF H-25, produced by CP Kelco, Pomosin, Germany) and the enzymes was Polygalacturonase Rohament PL, Batch no. 97.01.001 , 5270 U/ml produced by Rohm and Polymethylesterase (PME) Rheozyme, Batch no. 0594507L, 6208 μ/ml produced by Novozymes, Denmark.

500 I of ion-exchanged water was heated to 70 ° C and 35 kg of pectic substance was added, while stirring continuously. After the pectin was completely dissolved, the temperature was lowered to 45 ° C; pH was adjusted to 4.50 by titration with a 10% (^w/v) soda solution. 0,55 ml Rohament PL was added to the pectin solution. 8,8 ml Rheozyme was added to the solution. During the experiment the pH was kept constant at 4,5 by titration with 5% (^w/v) soda. After 2 hours (720 ml 10% soda solution consumed) the reaction was stopped by addition of a 10% soda solution till pH 2,50. In order to irreversibly inactivate the enzyme, the temperature was raised to 90 ° C. After 15 minutes at 90 ° C, the solution was cooled to 15 ° C, and left overnight, with weak stirring, in order to precipitate any not bound protein material. The solution was then filtrated (Celite 545). The cleared solution was concentrated and diafiltrated by ultrafiltration.

The ultrafiltration concentration step took place at 50°C and as semi-batch concentration process. The diafiltration was made with a molecular weight cut off approx. 25000. The feed volume was approximately 1 ,0 m³ and the retentate volume is 60 I. The resulting concentration ratio was roughly 8X. Sample was then freeze dried (Freeze dryer DW6-55 & DW6-85, Heto-Holten A/S, Allerød Denmark).

TABLE 7

Emulsion Test: Vitamin emulsions

In order to compare the emulsifying properties of various modified beta pectins an emulsion test was conducted and oil droplet size was measured. The emulsion contained 20% dry matter and 80 % demineralised water.

Comparative Example 1

A 600 ml beaker with 365 ml boiling demineralised water was placed in a water bath at 80 °C. 22.8 grams of sugar beet pectin (GENU beta pectin type BETA, lot 92455, from CP Kelco, Denmark) was dispersed in the water and mixed with Ultra Turrax (Ultra Turrax T50 with a R50 stirring shaft) for 10 minutes at 3000 rpm. 37.3 grams saccharose was added and mixed with Ultra Turrax for 10 minutes at 3000 rpm. 20.0 g hot Vitamin E acetate oil (DL- α-tocopheryl acetate from BASF AG) was added and mixed with Ultra Turrax for 20 minutes at 10,000 rpm. Viscosity (Brookfield Viscometer LVT spindle 4 factor 100 or spindle 3 factor 20 or spindle 2 factor 5, 60 rpm for 1 minute) of the freshly made emulsion was measured at 75 °C and found to be 252 cP. Particle size distribution (Malvern Mastersizer E, focal length 45 mm, presentation 0606, model independent, beam length 2.2 mm, specifications ±5μm) was measured. Mean oil droplet size, d(0.5), was found to be 1.70 μm and d(0.9) to be 3.78 μm.

To verify the emulsion stability the oil droplet size was also measured after 24 hours at 70 °C. The oil droplet size d(0.9) was found to be 3.74 μm.

Example 8

A 600 ml beaker with 365 ml boiling demineralised water was placed in a water bath at 80 °C. 22.8 grams of α-ARA, arabinanase and galactanase modified sugar beet pectin as described in Example 1 was dispersed in the water and mixed with Ultra Turrax (Ultra Turrax T50 with a R50 stirring shaft) for 10 minutes at 3000 rpm. 37.3 grams saccharose was added and mixed with Ultra Turrax for 10 minutes at 3000 rpm. 20.0 grams hot Vitamin E acetate oil (DL-α-tocopheryl acetate from BASF AG) was added and mixed with Ultra Turrax for 20 minutes at 10,000 rpm. Viscosity (Brookfield Viscometer LVT spindle 4 factor 100 or spindle 3 factor 20 or spindle 2 factor 5, 60 rpm for 1 minute) of the freshly made emulsion was measured at 75 °C and found to be 290 cP. Particle size distribution (Malvern Mastersizer E, focal length 45 mm, presentation 0606, model independent, beam length 2.2 mm, specifications +5μm) was measured. Mean oil droplet size, d(0.5), was found to be 1.25 μm and d(0.9) to be 2.65 μm.

To verify the emulsion stability the oil droplet size was also measured after 24 hours at 70 °C. The oil droplet size d(0.9) was found to be 2.62 μm.

As will appear from the results the oil droplet size was smaller and hence the emulsifying properties (including the emulsion stability result) of the composition of the invention were far superior to those of the composition of Comparative Example 1.

Example 9

A 600 ml beaker with 365 ml boiling demineralised water was placed in a water bath at 80 °C. 22.8 grams of rhamnogalacturonan acetyl esterase modified sugar beet pectin as described in Example 2 was dispersed in the water and mixed with Ultra Turrax (Ultra Turrax T50 with a R50 stirring shaft) for 10 minutes at 3000 rpm. 37.3 grams saccharose was added and mixed with Ultra Turrax for 10 minutes at 3000 rpm. 20.0 grams hot Vitamin E acetate oil (DL-α-tocopheryl acetate from BASF AG) was added and mixed with Ultra Turrax for 20 minutes at 10,000 rpm. Viscosity (Brookfield Viscometer LVT spindle 4 factor 100 or spindle 3 factor 20 or spindle 2 factor 5, 60 rpm for 1 minute) of the freshly made emulsion was measured at 75 °C and found to be 173 cP. Particle size distribution (Malvern Mastersizer E, focal length 45 mm, presentation 0606, model independent, beam length 2.2 mm, specifications ±5μm) was measured. Mean oil droplet size, d(0.5), was found to be 1.40 μm and d(0.9) to be 3.41 μm.

As will appear from the results the oil droplet size was smaller and hence the emulsifying properties of the composition of the invention were superior to those of the composition of Comparative Example 1.

Example 10

A 600 ml beaker with 365 ml boiling demineralised water was placed in a water bath at 80 °C. 22.8 grams of rhamnogalacturonase modified sugar beet pectin as described in Example 3 was dispersed in the water and mixed with Ultra Turrax (Ultra Turrax T50 with a R50 stirring shaft) for 10 minutes at 3000 rpm. 37.3 grams saccharose was added and mixed with Ultra Turrax for 10 minutes at 3000 rpm. 20.0 grams hot Vitamin E acetate oil (DL-α- tocopheryl acetate from BASF AG) was added and mixed with Ultra Turrax for 20 minutes at 10,000 rpm. Viscosity (Brookfield Viscometer LVT spindle 4 factor 100 or spindle 3 factor 20 or spindle 2 factor 5, 60 rpm for 1 minute) of the freshly made emulsion was measured at 75 °C and found to be 250 cP. Oil droplet size (Malvern Mastersizer E, focal length 45 mm, presentation 0606, model independent, beam length 2.2 mm, specifications ±5μm) was measured. Oil droplet size, d(0.5), was found to be 1.45 μm and d(0.9) to be 3.31 μm.

As will appear from the results the oil droplet size was smaller and hence the emulsifying properties of the composition of the invention were far superior to those of the composition of Comparative Example 1.

Comparative Example 2

The ratio of pectin:sugar:oil in the following recipe is 3:3:2.

A 600 ml beaker with 350 ml boiling water was placed in a water bath at 80 C. 23 grams of sugar beet pectin (lot 30003, produced by CP Kelco) was dispersed in the water and mixed with Ultra Turrax (Ultra Turrax T50 with a R50 stirring shaft) for 10 minutes at 3000 rpm. 23 grams saccharose was added and mixed with Ultra Turrax for 10 minutes at 3000 rpm. The pectin content in the water/saccharose solution is 6,2 %. Viscosity (Brookfield Viscometer LVT spindle 4 factor 100 or spindle 3 factor 20 or spindle 2 factor 5, 60 rpm for 1 minute) of the sugar/water solution was measured at 75 ° C and found to be 1181 cP. 15.3 gram hot vitamin E acetate oil (DL-alfa- tocopheryl acetate from BASF AG) was added and mixed with Ultra Turrax for 20 minutes at 10.000 rpm. Particle size and distribution was measured by Malvern Mastersizer 2000, Accessory name: hydro 200 G (A), particle Rl: 1 ,494, Adsorption: 0,3; Analysis model: general purpose (spherical). Mean droplet size d(0,5) was found to be 2,25 micron and droplet size distribution d(0.9) to be 5,54 micron.

Example 11

The recipe was formulated after the same guidelines as in comparative Example 2; recipe: pectin:sugar:oil is 3:3:2

A 600 ml beaker with 350 ml boiling water was placed in a water bath at 80 °

C. 70 grams of Pectinase PL lyase modified sugar beet pectin as described in Example 4 was dispersed in the water and mixed with Ultra Turrax (Ultra Turrax T50 with a R50 stirring shaft) for 10 minutes at 3000 rpm. 70 grams saccharose was added and mixed with Ultra Turrax for 10 minutes at 3000 rpm. The gum content in the water/saccharose solution was 15%. Viscosity (Brookfield Viscometer LVT spindle 4 factor 100 or spindle 3 factor 20 or spindle 2 factor 5, 60 rpm for 1 minute) of the water/saccharose solution was measured at 75 ° C and found to be 304 cP. 47 gram hot vitamin E acetate oil (DL-alfa-tocopheryl acetate from BASF AG) was added and mixed with Ultra Turrax for 20 minutes at 10.000 rpm. Particle size and distribution was measured by Malvern Mastersizer 2000, Accessory name: hydro 200 G (A), particle Rl: 1 ,494, Adsorption: 0,3; Analysis model: general purpose (spherical). Mean droplet size d(0.5) was found to be 0.97 micron and droplet size distribution d(0.9) to be 1 ,67 micron.

As will appear from the results the oil droplet size was smaller and hence the emulsifying properties of the composition of the invention were far superior to those of the composition of Comparative Example 2.

Example 12

A 600 ml beaker with 350 mi boiling water was placed in a water bath at 80 ° C. 80 grams of PG/PME modified sugar beet pectin as described in Example 5 was dispersed in the water and mixed with Ultra Turrax (Ultra Turrax T50 with a R50 stirring shaft) for 10 minutes at 3000 rpm. 80 grams saccharose was added and mixed with Ultra Turrax for 10 minutes at 3000 rpm. The pectin content in the water/saccharose solution was 18,6%. Viscosity (Brookfield Viscometer LVT spindle 4 factor 100 or spindle 3 factor 20 or spindle 2 factor 5, 60 rpm for 1 minute) of the water/saccharose solution was measured at 75 ° C and found to be 350 cP. 53 gram hot vitamin E acetate oil (DL-alfa-tocopheryl acetate from BASF AG) was added and mixed with Ultra Turrax for 20 minutes at 10.000 rpm. Particle size and distribution was measured by Malvern Mastersizer 2000, Accessory name: hydro 200 G (A), particle Rl: 1 ,494, Adsorption: 0,3; Analysis model: general purpose

(spherical). Mean droplet size d(0.5) was found to be 0.91 micron and droplet size distribution d(0.9) to be 1.49 micron.

As will appear from the results the oil droplet size was smaller and hence the emulsifying properties of the composition of the invention were far superior to those of the composition of Comparative Example 2. Example 13

A 600 ml beaker with 350 ml boiling water was placed in a water bath at 80 ° C. 92 grams of PG/PME + Collupulin modified sugar beet pectin as described in Example 6 was dispersed in the water and mixed with Ultra Turrax (Ultra Turrax T50 with a R50 stirring shaft) for 10 minutes at 3000 rpm. 92 grams saccharose was added and mixed with Ultra Turrax for 10 minutes at 3000 rpm. The pectin content in the water/saccharose solution was 21%. Viscosity (Brookfield Viscometer LVT spindle 4 factor 100 or spindle 3 factor 20 or spindle 2 factor 5, 60 rpm for 1 minute) of the water/saccharose solution was measured at 75 ° C and found to be 378 cP. 61 gram hot vitamin E acetate oil (DL-alfa-tocopheryl acetate from BASF AG) was added and mixed with Ultra Turrax for 20 minutes at 10.000 rpm. Particle size and distribution was measured by Malvern Mastersizer 2000, Accessory name: hydro 200 G (A), particle Rl: 1 ,494, Adsorption: 0,3; Analysis model: general purpose (spherical). Mean droplet size d(0.5) was found to be 1.06 micron and droplet size distribution d(0.9) to be 1 ,78 micron.

Emulsion test: Beverage emulsions

Comparative Example 3 33.0 grams of Ester Gum 8 BG (from Hercules Incorporated) was dissolved in 67.0 grams of orange oil 8380 NAT. (H.N. Fusgaard A/S), by agitating gently for approx. two hours.

22.5 g of Genu® pectin type BETA batch 92455 (CP Kelco ApS) was dissolved in 910.0 ml of water by means of the high-speed mixer Silverson L4R, adding the pectin slowly to very hot water (70-80 °C). The solution was mixed for 5 minutes to ensure complete dispersion and hydration.

The orange oil phase was added to the pectin solution while continuing mixing with the high-speed mixer. While still mixing, 50 % sol. w/v of citric acid solution was added until a pH of approx. 3.25 (corresponding to approx. 2-10 ml of citric acid solution).

0.5 ml of sodium benzoate (20 % sol. w/v) was added and pH was adjusted to 3.25 (at 25 ° C) with citric acid and mixed for 15 minutes at full speed in a homogenizer APV (Rannie A/S) in two steps: 200 bar first, 50 bar last.

The emulsifying properties of the orange oil emulsions was investigated as follows:

The emulsions were stored 4 days at 40 °C. After storing the mean droplet size was measured to be 0.94 microns (Malvern MASTERSIZER 2000, particle Rl = 1.494, Dispertant Rl = 1.331 , Adsorption 0.3 and Analysis Model: general purpose (spherical)).

Example 15

33.0 grams of Ester Gum 8 BG (from Hercules Incorporated) was dissolved in 67.0 grams of orange oil 8380 NAT. (H.N. Fusgaard A/S), by agitating gently for approx. two hours.

22.5 grams of the sugar beet pectin modified with arabinanase, galactanase and arabinofuranosidase as described in Example 1 was dissolved in 910.0 ml of water by means of the high speed mixer Silverson L4R, adding the pectin slowly to very hot water (70-80 °C). The solution was mixed for 5 minutes to ensure complete dispersion and hydration.

The emulsifying properties of the orange oil emulsions were investigated as follows:

The emulsions were stored 4 days at 40 ° C. After storing the mean droplet size was measured to be 0.79 microns (Malvern MASTERSIZER 2000, particle Rl = 1.494, Dispertant Rl = 1.331 , Adsorption 0.3 and Analysis Model: general purpose (spherical)).

As will appear from the results the oil droplet size was significantly smaller and hence the emulsifying properties of the composition of the inventions were far superior to those of prior art composition of Comparative Example 3.

Example 16

33.0 grams of Ester Gum 8 BG (from Hercules Incorporated) was dissolved in 67.0 grams of orange oil 8380 NAT (H.N. Fusgaard A/S), by agitating gently for approx. two hours.

22.5 grams of sugar beet pectin modified with rhamnogalacturonan acetylesterase as described in Example 2, was dissolved in 910,0 ml of water by means of the high speed mixer Silverson L4R, adding the pectin slowly to very hot water (70-80 ° C). The solution was mixed for 5 minutes to ensure complete dispersion and hydration.

The emulsions were stored 4 days at 40 ° C. After storing the mean droplet size was measured to be 0,88 microns (Malvern MASTERSIZER 2000, particle Rl =1.494, Dispertant Rl = 1.331 , Adsorption 0.3 and analysis model: general purpose (spherical)).

As will appear from the results the oil droplet size was significantly smaller and hence the emulsifying properties, of the composition of the inventions were improved compared to those of prior art composition of Comparative Example 3.

Claims

Patent Claims

1. Modified pectic substance, characterized in that it is obtainable by treating a pectic substance from beetroot (Beta vulgaris) with one or more enzymes selected from the group consisting of esterases (E.C.3.1), glucosidases (E.G.3.2.), and lyases (E.C.4.) with the exception of PE (E.C.3.1.1.11), ACE, RGAE, and α-ARA alone, and mixtures of PE (E.C.3.1.1.11.) and RGAE and of PE (E.C.3.1.1.11.) and α-ARA.

2. Modified pectin according to claim 1 , characterized in that it is obtainable by treating a pectic substance from beetroot comprising hairy regions and a backbone of smooth regions with one or more enzymes capable of modifying the hairy regions.

3. Modified pectic substance according to claim 1 , characterized in that it is obtainable by treating a pectic substance from beetroot with one or more enzymes capable of modifying the backbone of the smooth regions so as to form separate elements comprising hairy regions.

4. Modified pectic substance according to claims 1-3, characterized in that it is obtainable by treating a pectic substance from beetroot with a deacetylating enzyme.

5. Modified pectic substance according to claims 1 -3, characterized in that it is obtainable by treating a pectic substance from beetroot with a debranching enzyme.

6. Modified pectic substance according to claims 1 -3, characterized in that it is obtainable by treating a pectic substance from beetroot with a mixture of α- arabinofuranosidase, galactanase, and arabinanase.

7. Modified pectic substance according to claims 1-3, characterized in that it is obtainable by treating a pectic substance from beetroot with a mixture of polygalacturonase and pectin methyl esterase.

8. Modified pectic substance according to claims 1-3, characterized in that it is obtainable by treating a pectic substance from beetroot (Beta vulgaris) with one or more enzymes selected from the group consisting of esterases (E.C.3.1), glucosidases (E.C.3.2.), and lyases (E.C.4.) followed by additional treating with a proteolytic enzyme, such as papain.

9. Modified pectic substance according to claims 1-8, wherein the pectic substance is derived from the group consisting of sugar beet, garden beet (red beet), chard, mangel, spinach beet, silver beet, and fodder beet.

10. A process of preparing a modified pectic substance comprising the steps of providing an aqueous medium of a pectic substance from beetroot and adding thereto one or more enzymes selected from the group consisting of esterases (E.C.3.1), glucosidases (E.C.3.2.), and lyases (E.C.4.) except for PE (E.G.3.11.11), ACE, RGAE. and α-ARA alone and mixtures of PE (E.C.3.1.1.11.) and RGAE and of PE (E.C.3.1.1.11) and α-ARA.

11 A product comprising a modified pectic substance according to any of claims 1-9.

12. Emulsifying agent, characterized in that it is obtainable by treating a pectic substance from beetroot with one or more enzymes selected from the group consisting of esterases (E.C.3.1), glucosidases (E.G.3.2.), and lyases (E.C.4.).

13. Microencapsulating agent, characterized in that it is obtainable by treating a pectic substance from beetroot with one or more enzymes selected from the group consisting of esterases (E.C.3.1), glucosidases (E.G.3.2.), and lyases (E.G.4.).

14. Foam stabilizer, characterized in that it is obtainable by treating a pectic substance from beetroot with one or more enzymes selected from the group consisting of esterases (E.C.3.1), glucosidases (E.C.3.2.), and lyases (E.C.4.).

15. A film-forming agent, characterized in that it is obtainable by treating a pectic substance from beetroot with one or more enzymes selected from the group consisting of esterases (E.C.3.1), glucosidases (E.C.3.2.), and lyases (E.C.4.).