Alternan derivatives

Abstract

The present invention relates to alternan-carboxylic acid esters, to processes for the preparation of alternan-carboxylic acid esters, and to compositions comprising alternan-carboxylic acid esters and to the use of alternan-carboxylic acid esters. The invention relates to alternan-carboxylic acid esters which are emulsifiers.

Claims

1. An alternan-carboxylic acid ester.

2. A method of preparing an alternan-carboxylic acid ester comprising reacting alternan with a carboxylic acid or its anhydride, a carboxylic acid halide, or a vinyl ester.

3. An emulsifier comprising the alternan-carboxylic acid ester of claim 1.

4. An emulsion comprising the emulsifier of claim 3.

5. A composition comprising the alternan-carboxylic acid ester of claim 1.

6. A cleaning composition comprising the alternan-carboxylic acid ester of claim 1.

7. A method of preparing food, cosmetic, or pharmaceutical composition comprising admixing the composition of claim 5 with at least one food item, cosmetic item, or pharmaceutically active substance.

8. A composition comprising the emulsifier of claim 3.

9. A composition comprising the emulsion of claim 4.

10. The method of claim 7, wherein the alternan-carboxylic acid ester acts as a surfactant.

11. The method of claim 10, wherein the surfactant is a foaming agent.

12. The method of claim 10, wherein the surfactant is a body care substance.

13. An alternan-carboxylic acid ester, wherein said alternan-carboxylic acid ester has emulsifying properties.

14. A modified alternan which is an alternan-carboxylic acid ester.

15. An alternan-carboxylic acid ester depicted by the following formula ##STR00002## where R is a straight-chain or branched alkyl residue having 1 to 11 carbon atoms, which can carry one or more oxo, hydroxy, carboxy residues or a straight-chain or branched alkenyl residue having 1 to 11 carbon atoms, which can carry one or more oxo, hydroxy, carboxy residues.

16. An emulsifier comprising the alternan-carboxylic acid ester of claim 15.

17. An emulsion comprising the emulsifier of claim 16.

18. A composition comprising the alternan-carboxylic acid ester of claim 15.

19. A composition comprising the emulsifier of claim 16.

20. A composition comprising the emulsion of claim 17.

21. An alternan-carboxylic acid ester, depicted by the following formula ##STR00003## where R is a straight-chain or branched alkyl residue having 1 to 11 carbon atoms, which can carry one or more oxo, hydroxy, carboxy residues or a straight-chain or branched alkenyl residue having 1 to 11 carbon atoms, which can carry one or more oxo, hydroxy, carboxy residues, wherein said alternan-carboxylic acid ester has emulsifying properties.

22. The alternan-carboxylic acid ester of claim 1, which is an alternan-acetic acid ester, alternan-succinic acid ester, or an alternan-octenylsuccinic acid ester.

23. An emulsifier comprising the alternan-carboxylic acid ester of claim 22.

24. An emulsion comprising the emulsifier of claim 23.

25. A composition comprising the alternan-carboxylic acid ester of claim 22.

26. A composition comprising the emulsifier of claim 23.

27. A composition comprising the emulsion of claim 24.

28. The alternan-carboxylic acid ester of claim 22, wherein said alternan-carboxylic acid ester has emulsifying properties.

29. A method of preparing an alternan-carboxylic acid ester comprising reacting alternan with straight-chain or branched alkyl carboxylic acid or its anhydride, or a straight-chain or branched alkenyl carboxylic acid or its anhydride, or a straight-chain or branched alkyl or alkenyl carboxylic acid halide, or a straight-chain or branched alkyl or alkenyl vinyl ester.

30. A method of preparing an alternan-carboxylic acid ester comprising reacting alternan with acetic acid or its anhydride or halide, or with succinic acid or its anhydride or halide, or with octenylsuccinic acid or its anhydride or halide or with vinyl acetate or with vinyl succinate or with vinyl octenylsuccinate.

31. A cleaning composition comprising the alternan-carboxylic acid ester of claim 15.

32. A cleaning composition comprising the alternan-carboxylic acid ester of claim 22.

33. A method of preparing food, cosmetic, or pharmaceutical composition comprising admixing the composition of claim 18 with at least one food item, cosmetic item, or pharmaceutically active substance.

34. A method of preparing food, cosmetic, or pharmaceutical composition comprising admixing the composition of claim 25 with at least one food item, cosmetic item, or pharmaceutically active substance.

35. The method of claim 33, wherein the alternan-carboxylic acid ester acts as a surfactant.

36. The method of claim 34, wherein the alternan-carboxylic acid ester acts as a surfactant.

37. The method of claim 35, wherein the surfactant is a foaming agent.

38. The method of claim 36, wherein the surfactant is a foaming agent.

39. The method of claim 35, wherein the surfactant is a body care substance.

40. The method of claim 36, wherein the surfactant is a body care substance.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Molar mass distribution of alternan and alternan-succinic acid esters with different degrees of substitution (DS 0.034, DS 0.088, DS 0.161).

(2) FIG. 2: Comparison of the flow behavior of alternan and alternan-succinic acid esters with different degrees of substitution (DS 0.034, DS 0.088, DS 0.161) at 25 C. The viscosity [Pas] is shown as a function of the shear rate [Hz].

(3) FIG. 3: Frequency sweep of alternan and alternan-succinic acid esters with different degrees of substitution (DS 0.034, DS 0.088, DS 0.161). The storage modulus (G) and the loss modulus (G), measured in Pascals [Pa] are shown as a function of the frequency [Hz] at constant shear stress.

(4) FIG. 4: Gel formation of alternan, alternan-succinic acid esters with different degrees of substitution (DS 0.034, DS 0.088, DS 0.161) and gelatinized corn starch (CST), and mixtures thereof. The storage modulus (G) and the loss modulus (G), measured in Pascals [Pa], are shown as a function of the temperature [ C.].

(5) FIG. 5: Comparison of the gel state of alternan, alternan-succinic acid esters with different degrees of substitution (DS 0.034, DS 0.088, DS 0.161) and gelatinized corn starch (CST), and also of mixtures thereof in the frequency sweep. The storage modulus (G) and the loss modulus (G), measured in Pascals [Pa], are shown as a function of the frequency [Hz] at a measurement temperature of 5 C.

(6) FIG. 6: Comparison of the gel solidity and gel stability of alternan, alternan-succinic acid esters with different degrees of substitution (DS 0.034, DS 0.088, DS 0.161) and gelatinized corn starch (CST), and also of mixtures thereof in the stress sweep. The storage modulus (G) and the loss modulus (G), measured in Pascals [Pa], are shown as a function of the shear stress [Pa] at a measurement temperature of 5 C.

(7) FIG. 7: Photograph of the emulsions which were obtained with different concentrations of alternan-octenylsuccinic acid esters (see also table 10).

(8) FIG. 8: Molar mass distribution of alternan-octenylsuccinic acid esters with a degree of substitution of 0.024.

GENERAL METHODS

1. Preparation of Alternan

(9) Alternan can be prepared with the help of the enzyme alternansucrase. The enzyme alternansucrase can be prepared in various ways by processes known to the person skilled in the art.

(10) The preparation of alternansucrase and alternan with the help of bacterial strains of the species Leuconostoc mesenteroides are described inter alia in Reamakers et al (1997, J. Chem. Tech. Biotechnol. 69, 470-478) or in WO 2006 088884 (see in particular example 1).

(11) However, processes which use Leuconostoc mesenteroides bacterial strains for the preparation of the enzyme alternansucrase have the disadvantage that these strains also produce other sucrases, in particular dextransucrases. These other sucrases have hitherto been unable to be completely separated from alternansucrase. It is therefore a mixture of different enzymes. Consequently, the alternan prepared using such an enzyme mixture has, besides alternan, also dextran, at least in small amounts. To prepare pure alternans, methods for the preparation of alternansucrase by means of recombinant organisms are therefore to be preferred.

(12) Processes for the preparation of alternansucrase prepared by means of recombinant organisms and for the preparation of alternan by means of the enzyme prepared in this way are described, inter alia, in WO 2000 47727, US 2003 229923 (see in particular examples 2, 5 and 8) or Joucla et al (2006, FEBS Letters 580, 763-768).

2. Determination of the Degree of Esterification

(13) The degree of esterification of the various alternan derivatives was ascertained by means of alkaline saponification and subsequent acid-base titration. The percentage fraction of the substitution (mass of the substituent in %, based on the dry substance of the alternan derivatives) was determined. Using the resulting values, the degree of substitution (DS) was ascertained in accordance with the following formula:
DS.sub.x=162% X/(100M.sub.xM% X) % X=Fraction (in %) of the mass of the analytically determined group (substituent) of the mass of the dry substance Mx=Molar mass of the analytically determined group M=MSML MS=Molar mass of the substituent ML=Molar mass of the saponified group

3. Determination of the Molar Mass Distribution by Means of GPC-MALLS

(14) To determine the molar mass distribution by means of gel permeation chromatography, the following instruments were used: Instruments: Alliance 2695 separation module from Waters, DRI detector 2414 from Waters, MALLS detector Dawn-HELEOS from Wyatt Technology Inc., Santa Barbara, USA, wavelength =658 nm and a K5 flow cell Columns: SUPREMA gel column set (PSS Mainz), exclusion limits S30000 with 10.sup.8-10.sup.6, S1000 with 2.Math.10.sup.6-5.Math.10.sup.4, S100 with 10.sup.5-10.sup.3 Eluent: 0.5 m NaNO.sub.3 Temperature: 30 C.
To evaluate the data obtained, Astra Software 5.3.0.18 was used.

4. Rheological Characterization by Means of Rheometer

(15) To determine the rheological properties, the following instruments were used with the stated (adjustable) parameters: Instrument: Rheometer CVO 120HR from Malvern (Bohlin)
Parameters Torque: 0.0001-120 mNm (6 powers of ten) Torque resolution: better than 10.sup.9 Nm Angle resolution: 5.Math.10.sup.5 rad Frequency range: 10.sup.5-150 Hz Speed range: <10.sup.5-3100 min.sup.1
With the help of the specified instrument, the storage modulus (G) and loss modulus (G) were determined as a function of the frequency at constant deformation/shear stress (frequency sweep) and as a function of the shear stress at constant frequency (stress sweep).

5. Viscosity Determination by Means of RVA

(16) The material is dissolved in distilled H.sub.2O and homogenized using an Ultra-Turrax T 25 digital (IKA-Werke GMBH & CO. KG, D-79219 Staufen, Germany) for 1 minute at 9000 rpm. 27 ml of 10% strength solution are used in an RVA beaker in a Rapid Visco Analyzer (Newport Scientific Pty Ltd., Investment Support Group, Warriewod NSW 2102, Australia) to measure the viscosity. The instrument is operated in accordance with the manufacturer's instructions. Here, the viscosity values are stated in centipoise (1 cP=1 mPas) in accordance with the manufacturer's operating instructions. To determine the viscosity of the aqueous solution of the material, the suspension is firstly stirred at 25 C. for 10 seconds at 1200 rpm, then the temperature is kept constant at 25 C. and the mixture is stirred at a stirring speed of 1000 rpm for a further 2 minutes and 50 seconds. During the total time of 3 minutes, the viscosity is determined in centipoise (cP).

EXAMPLES

1. Alternan-Acetic Acid Ester (Alternan-Acetyl Ester)

(17) a) Preparation

(18) Firstly 80 ml of demineralized water were initially introduced in a 250 ml beaker and then 10 g of alternan were dissolved with constant stirring by means of a magnetic stirrer. After dissolving the alternan, a pH of 8.5 was established using 0.5M NaOH (Merck). In two separate mixtures, the reaction was started by adding in each case 1 ml (sample 1) or 2 ml (sample 2) of vinyl acetate (Merck). Throughout the entire reaction time, the pH was kept constant using a 0.5M NaOH solution (Merck) using an automatic titrater (pH-Stat, Metrohm 719 S Titrino). The reaction was carried out at 23 C.

(19) The reaction was ended by neutralization (pH6.3) with the help of 0.5M HCl (Merck), and, to precipitate out the alternan, the reaction mixture was poured into a 500 ml beaker with twice the volume of ethanol (denatured, Monopoly Administration). After stirring for 5 min using a magnetic bar, the alternan derivative was separated off from the mixture with the help of a vacuum suction filter ( 100 mm), then the filter cake was resuspended for washing in ca. 100 ml of ethanol/demineralized water (80:20; v:v). The washing procedure was repeated twice, and the filter cake was then granulated using a laboratory sieve ( 200 mm, mesh width 3 mm) and then the granules were dried in the air for two days. Prior to characterizing the product, the agglomerates were comminuted using a laboratory mill (IKA model A 10).

(20) b) Characterization

(21) The turbidity measurement was carried out using a 0.5% strength solution of alternan or alternan derivatives. 49.75 g of demineralized water were weighed into a 150 ml beaker and 0.25 g of alternan/alternan derivatives were dissolved therein at room temperature and with constant stirring using a magnetic stirrer. The turbidity was measured after stirring for 1 hour with the help of a photometer (PM 200 from Rhle, Berlin) using a filter 525 nm and a cell of 1 cm in thickness.

(22) The acetic acid fraction (acetyl content) of the alternan-carboxylic acid esters was determined in accordance with the method described above under General Methods under point 2.

(23) TABLE-US-00002 TABLE 1 Turbidity measurement (column 4) and amount of acetyl content (column 5) of alternan-acetic acid esters which have been prepared using different amounts of vinyl acetate. Amount of Reaction Turbidity [absor- Acetyl content Sample vinyl acetate time bance 525 nm] (DS value) Reference 0.30 Sample 2 1 ml 1 h 0.24 0.062 Sample 3 1 ml 2 h 0.23 0.053 Sample 3 2 ml 2 h 0.23 0.048 The amount of vinyl acetate used in the esterification reaction and the duration of the reaction are shown in columns 2 and 3, respectively. The reference (column 1) referred to is native alternan which has been used as starting material in the reaction. Samples 1, 2 and 3 (column 1) refer to alternan-acetic acid esters which have been prepared by means of different reaction conditions.

2. Alternan-Succinic Acid Esters (Alternan-Succinate)

(24) a) Preparation

(25) 50 g of alternan (dry weight) were initially introduced in a 1 liter jacketed reactor, dissolved in demineralized water and rendered alkaline using an automatic titrater. Succinic acid anhydride was slowly added. After the reaction had ended, the pH was adjusted to 6.5. The resulting alternan-succinic acid ester was precipitated out with ethanol, washed and dried in vacuo in a drying cabinet.

(26) b) Degree of Substitution

(27) The degree of substitution (DS value) of the resulting alternan-succinic acid esters was determined in accordance with the method described under General Methods, point 2.

(28) TABLE-US-00003 TABLE 2 Degree of substitution of various alternan-succinic acid esters, determined by alkaline saponification and acid-base titration. Sample name AlS001 AlS002 AlS003 DS value 0.034 0.089 0.161
c) Turbidity Measurement of Solutions

(29) For the turbidity measurement, the resulting alternan-acetic acid esters were dissolved in different concentrations (see table 3) in water and measured at 525 nm in a spectral photometer.

(30) TABLE-US-00004 TABLE 3 Measurement of the turbidity of alternan which has been used as starting material in the reaction, and alternan- succinic acid esters (AlS001, AlS002, AlS003) in solutions comprising different concentrations of said substances. Absorbance at 525 nm Concentration Substance 0.1% 0.5% 1.0% Alternan 0.048 0.213 0.399 AlS001 0.038 0.150 0.270 AlS002 0.028 0.104 0.165 AlS003 0.027 0.070 0.089

(31) Consideration of the results obtained in example 2b) reveals that the turbidity of solutions comprising alternan-succinic acid esters decreases compared to alternan. The higher the degree of substitution, the lower the turbidity of the solution at the same concentration of the dissolved substances.

(32) d) Molecular Characterization

(33) The molar mass distribution of the resulting alternan-succinic acid esters was analyzed with the help of GPC-MALLS (Gel Permeation ChromatographyMulti Angle Laser Light Scattering) using the agents described under General Methods, point 3. For this, the various substances (alternan, AIS001, AIS002, AIS003) were dissolved in a concentration of in each case 0.2% in demineralized water firstly at room temperature for 24 hours and then at 120 C. for 20 minutes. For all samples, the same refractive index increment (dn/dc) of 0.146 was used. Using this value, recovery rates of ca. 90% in the GPC were obtained for all derivatives.

(34) For the weight-average molar mass (M.sub.w), the following results were obtained (see also FIG. 1):

(35) TABLE-US-00005 TABLE 2 Weight-average molar mass (M.sub.w) of alternan-succinic acid esters with different degrees of substitution (AlS001, AlS002, AlS003) and of alternan which has been used as starting material in the reaction (alternan). Sample name Reference AlS001 AlS002 AlS003 M.sub.w [10.sup.6 g/mol] 25.71 18.32 12.24 9.24
f) Rheological Properties

(36) To determine the rheological properties, the various substances (alternan, AIS001, AIS002, AIS003) were dissolved in a concentration of in each case 5% in demineralized water with stirring at 95 C. The concentration of 5% in each case was chosen because alternan-succinic acid esters of the samples AIS001 and AIS002 were no longer flowable at a concentration of 5%. They formed stable gels in water.

(37) Viscosity

(38) The analysis was carried out with the help of the agents described under General Methods, point 4. The flow behavior (viscosity) of the 5% strength solutions was investigated at 25 C. as a function of the shear rate in the frequency range from 10.sup.0-10.sup.2 Hz.

(39) FIG. 2 shows a comparison of the flow curves of alternan which has been used as starting material in the esterification reaction and of the flow curves of alternan-succinic acid esters with varying degrees of substitution (AIS001, AIS002, AIS003). With increasing degrees of substitution (DS value), the alternan-succinic acid esters have an increase in the viscosity of the solutions in question. A DS value of 0.161 (AIS003) produced an increase in the viscosity of about 2 orders of magnitude compared to the starting material alternan. Besides the viscosity, the solution state also changed.

(40) The table below gives, by way of example, viscosities of alternan-succinic acid esters which have been measured at different shear rates.

(41) TABLE-US-00006 TABLE 3 Viscosities of alternan which has been used as starting material for the carboxylation reaction and alternan- succinic acid esters with different DS values. Shear rate Alternan AlS001 AlS002 AlS003 DS value 0.034 0.089 0.161 Viscosity 5 s.sup.1 7.8 48.0 102.1 1063.1 [mPas] Viscosity 50 s.sup.1 10.2 30.9 44.2 247.2 [mPas]
Oscillation Measurement by Means of Frequency Sweep

(42) Comparative oscillation measurements of alternan, which was used as starting material in the esterification reaction with the alternan-succinic acid esters with varying degrees of substitution (AIS001, AIS002, AIS003), were established at 25 C. in a frequency range from 10.sup.2 Hz to 10.sup.1 Hz using the agents described under General Methods, point 4.

(43) FIG. 3 shows the results of the comparative frequency sweep between alternan and alternan-succinic acid esters (AIS001, AIS002, AIS003). This shows that alternan has the typical solution character of Newtonian liquids with slight interactions of the dissolved substance. By contrast, alternan-succinic acid esters were gel-like at the same concentration, which is evident from the fact that the elasticity modulus (G) and the loss modulus (G) have a low frequency dependency and that G is larger than G (G>G).

(44) g) Emulsion Behavior

(45) Solutions of different concentration (see table 6) of alternan and alternan-succinic acid esters (AIS001, AIS002) were prepared by homogenizing the substances in question in ultrapure water using an Ultra-Turrax (25 k rpm) for one minute. To in each case 20 ml of these solutions were added in each case 20 ml of sunflower oil. Homogenization for one minute using an Ultra-Turrax (Ultra-Turrax T 25 digital, IKA-Werke GMBH & CO. KG, D-79219 Staufen, Germany) was then carried out at ca. 25 k rpm.

(46) The resulting emulsions were then observed as to whether phase separation occurs.

(47) TABLE-US-00007 TABLE 4 Effect of alternan-succinic acid esters (AlS001, AlS002) and alternan on emulsions. Concentration of the substance in the Onset of visible phase Substance water/oil mixture separation Alternan 0.5% Directly after homogenization 2.5% Directly after homogenization .sup.5% Directly after homogenization AlS001 0.5% After ca. 4 hours 2.5% After ca. 4 hours AlS002 0.5% After ca. 4 hours 2.5% After ca. 4 hours Compared to alternan, alternan-succinic acid esters have a stabilizing effect on emulsions.

(48) This shows that alternan-succinic acid esters have a stabilizing effect on emulsions compared to native alternan.

(49) h) Compatibility with Other Gel Formers

(50) Corn Starch

(51) Corn starch was dissolved in demineralized water in a concentration of 5% by boiling under pressure at 150 C. for 20 minutes. By dissolving, with stirring, alternan and various alternan-succinic acid esters (AIS001, AIS003), various mixtures (compositions) containing alternan and alternan-succinic acid esters were prepared from this starch solution. The concentration of alternan or alternan-succinic acid ester in the mixtures was in each case 1%.

(52) The hot solution was introduced into the measurement system, heated to 80 C., of the rheometer (see General Methods, point 4). At a frequency of 10.sup.2 Hz, the gelation of the various mixtures and of a pure starch solution (CST) was monitored during cooling to 5 C. by recording the storage moduli (G) and loss moduli (G) in question (FIG. 4). Following gelation of the mixtures, a frequency sweep (FIG. 5) for estimating the gel state and a stress sweep (FIG. 6) for assessing the gel solidity and the shear stability were recorded at a temperature of in each case 5 C.

(53) The cooling curves (FIG. 4) reveal for all solutions that, at 80 C., the storage modulus (G) is lower than the loss modulus (G). As cooling increases, the respective values for G and G increase. At about 10 C., G and G for all solutions achieve identical or at least approximately identical values, i.e. in this temperature range, the sol-gel transition takes place in the respective samples (gelation point). Solutions comprising mixtures of corn starch and alternan or alternan-succinic acid ester have approximately the same gelation point. The addition of alternan-succinic acid esters to corn starch solutions increases both elastic, and also viscous, fractions of the starch solution, which in the present experiment is most pronounced in the case of the mixture (composition) of corn starch (CST) and the alternan-succinic acid ester with the name AIS003.

(54) From the frequency sweep (FIG. 5) it can deduced that all of the mixtures formed stable gels at a temperature of 5 C.

(55) From the stress sweep (FIG. 6) it can be deduced that the addition of alternan reduces the gel solidity of corn starch gels, whereas the addition of alternan-succinic acid esters, particularly those with relatively high degrees of substitution (e.g. the substance AIS003), increases the gel solidity of corn starch gels.

(56) Stability in Foods

(57) Solutions of varying concentration (see table 5) of alternan-succinic acid esters (AIS001, AIS002, AIS003) were prepared by homogenizing the substances in question in standard commercial milk using an Ultra-Turrax (Ultra-Turrax T 25 digital, IKA-Werke GMBH & CO. KG, D-79219 Staufen, Germany) at a speed of ca. 25 k rpm for one minute. The resulting solutions were then observed over several hours up to one day as to whether all of the constituents remained in solution, or whether inhomogeneous areas formed and/or whether precipitates arose.

(58) TABLE-US-00008 TABLE 5 Stability of solutions of the alternan-succinic acid esters in milk Concentration of the Substance substance in the solution Observation AlS001 1% Stable, homogeneous mixture 5% Stable, homogeneous mixture AlS002 1% Stable, homogeneous mixture 5% Stable, homogeneous mixture AlS003 1% Stable, homogeneous mixture 5% Stable, homogeneous mixture

3. Alternan-Octenylsuccinic Acid Ester (Alternan-Octenylsuccinate)

(59) a) Preparation

(60) Firstly, 60 ml of demineralized water were introduced into a 250 ml beaker and then 10 g of alternan were dissolved with continuous stirring using a magnetic stirrer. After dissolving the alternan, the pH was adjusted to 8.5 by adding 0.5M NaOH solution (Merck).

(61) In mixtures separate from one another, either 1 ml or 2 ml of octenylsuccinic anhydride (OSA) was then continuously metered in by means of a burette over the course of one hour. The various reaction mixtures were then stirred either for one further hour or for three further hours, resulting in a total reaction time for the individual mixtures of 2 or 4 hours (also see table 8 in this regard). Throughout the entire reaction time, the pH was kept constant by using a 0.5M NaOH solution (Merck) using an automated titrater (pH-Stat, Metrohm 719 S Titrino). The reactions were carried out at 23 C.

(62) The reaction was ended by neutralization (pH 6.3) with the help of 0.5M HCl (Merck), and, to precipitate out the alternan, the reaction mixture was poured into a 500 ml beaker with twice the volume of ethanol (denatured, Monopoly Administration). After stirring for 5 minutes using a magnetic stirrer, the alternan derivative was separated off from the mixture with the help of a vacuum suction filter ( 100 mm), then the filter cake was resuspended for washing in ca. 100 ml of ethanol/demineralized water (80:20; v:v). The washing procedure was repeated twice, then the filter cake was granulated using a laboratory sieve ( 200 mm, mesh width 3 mm) and then the granules were dried in the air for two days. Prior to the characterization of the product, the agglomerates were comminuted using a laboratory mill (IKA model A 10).

(63) b) Characterization

(64) Turbidity Measurement

(65) The turbidity measurement was carried out with in each case a 0.5% strength solution comprising alternan or the various alternan-octenylsuccinic acid esters. For their preparation, 49.75 g of demineralized water were weighed in each case into a 150 ml beaker, and 0.25 g of the corresponding substance was stirred therein at room temperature and with continuos stirring using a magnetic stirrer. The turbidity was measured after stirring for 1 hour with the help of a photometer (PM 200 from Rhle, Berlin) using a 525 nm filter and a cell with a thickness of 1 cm. The absorbance value has been given in each case.

(66) Emulsifying Capacity

(67) The emulsifying capacity of alternan and of the alternan-octenylsuccinic acid esters prepared under various conditions was determined by in each case initially introducing 20 ml of a 1% strength solution (stock solution: 0.5 g+49.5 g of demineralized water) into a 100 ml titration beaker (from Mettler titrators), then adding 20 ml of sunflower oil (standard commercial oil from REWE) and initially homogenizing the mixture using an Ultra-Turrax (T 18) at 14 000 rpm for 1 minute. Then, in each case 10 ml of oil were added stepwise, which had in each case been homogenized for 1 minute (Ultra-Turrax, 14 000 rpm). The addition of oil was carried out until the viscosity of the emulsion decreased and/or the emulsion broke.

(68) The emulsifying capacity was calculated according to the following formula:
Emulsifying capacity [ml of oil/g of alternan100 ml of water]=total volume of oil5

(69) The results shown in the table below were obtained.

(70) TABLE-US-00009 TABLE 6 Turbidity measurement (column 4) and amount emulsifying capacity (column 5) of alternan and alternan-octenylsuccinic acid esters which have been prepared by various processes. Amount of octenylsuc- Reaction Emulsifying Sample cinic anhydride time Turbidity capacity Reference 0.302 125 ml oil/g Sample 2 1 ml 2 h 0.167 300 ml oil/g Sample 1 1 ml 4 h 0.105 300 ml oil/g Sample 3 2 ml 2 h 0.105 350 ml oil/g Sample 4 2 ml 4 h 0.109 350 ml oil/g The amount of octenylsuccinic anhydride used in the esterification reaction and the reaction time are shown in columns 2 and 3, respectively (for further reaction parameters see example 3a)). The reference (column 1) referred to is native alternan which was used as starting material in the reaction. Samples 1, 2, 3 and 4 (column 1) refers to alternan-octenylsuccinic acid esters which have been prepared by means of stated different reaction conditions.

(71) The emulsifying capacity of alternan-octenylsuccinic acid esters is increased compared to alternan. The turbidity of solutions comprising alternan-octenylsuccinic acid ester is reduced compared to alternan at identical concentration.

4. Emulsifying Properties of Alternan-Octenylsuccinic Anhydride

(72) a) Preparation of Alternan-Octenylsuccinic Anhydride

(73) Alternan was reacted with octenylsuccinic anhydride in the ratio 1:0.05 in the alkaline medium and neutralized when the reaction was complete. The resulting alternan-octenylsuccinic acid ester was precipitated out with ethanol, washed and dried.

(74) The degree of substitution (DS) of the alternan-octenylsuccinic acid ester ascertained by means of the method described under General Methods, point 2 was 0.024. These alternan-octenylsuccinic acid esters have been analyzed below.

(75) b) Molecular Characterization

(76) The molar mass distribution of the resulting alternan-succinic acid esters was analyzed with the help of GPC-MALLS (Gel Permeation Chromatography-Multi Angle Laser Light Scattering) using the agents described under General Methods, point 3 (FIG. 8). The resulting average molar mass (M.sub.w) was 21.510.sup.6 g/mol.

(77) c) Rheological Properties

(78) Viscosity Using a Rheometer

(79) The analysis was carried out with the help of the means described under General Methods, point 4. The flow behavior (viscosity) of the 10% strength solutions was investigated at 20 C. as a function of the shear rate in the frequency range shown (FIG. 9).

(80) Alternan-octenylsuccinic acid esters exhibited viscosity of ca. 25 mPas, which was slightly higher than the viscosity of alternan (ca. 15 mPas).

(81) Viscosity Using an RVA

(82) The viscosities of alternan and alternan-octenylsuccinic acid esters were determined compared to the viscosity of gum arabic with the help of an RVA (Rapid Visco Analyzer) using the method given under point 5, General Methods. In each case, 10% strength solutions (w/v) were used. The results are shown in the table below.

(83) TABLE-US-00010 TABLE 9 Viscosities, ascertained with an RVA, of gum arabic, alternan and alternan-octenylsuccinic acid ester. Viscosity [mPa s] Substance after 30 s after 60 s 60 s to 300 s Gum arabic 120 0 0 Alternan 40 120 120 Alternan-octenylsuccinic 250 250 250 acid ester
d) Emulsifying Properties of Alternan-Octenylsuccinic Anhydride

(84) In each case 20 mg, 40 mg, 200 mg and 1 g of alternan-octenylsuccinic acid ester (AI-OSA) were dissolved in 20 ml of demineralized water with the help of an Ultra-Turrax (1 minute, (Ultra-Turrax T 25 digital, IKA-Werke GMBH & CO. KG, D-79219 Staufen, Germany) at ca. 25 k rpm). 20 ml of sunflower oil (commercial product: ja! from REWE) were then added. The resulting mixture was homogenized by treatment for one minute using an Ultra-Turrax (see above). The consistency and the stability of the consistency were then assessed. FIG. 7 shows a diagram of the resulting emulsions. Further results are summarized in the table below.

(85) TABLE-US-00011 TABLE 10 Consistency and stability of oil/water mixtures comprising different amounts of alternan-octenylsuccinic acid ester (Al-OSA). Amount of substance Phase in the mixture separa- Water Oil Al-OSA Consistency/deposition tion after No. in [ml] [ml] [mg] of oil droplets ca. 72 h FIG. 7 20 20 20 Emulsion beaten egg none 1 whites-like/increased 20 20 40 Emulsion, beaten egg none 2 whites-like/slight 20 20 200 Emulsion, beaten egg none 3 whites-like/none 20 20 1000 Emulsion, creamy/none none 4 Column 6 contains the name (No.) of the corresponding mixtures, as stated in FIG. 7.
e) Comparison of the Emulsifiability of Alternan-Octenylsuccinic Acid with Gum Arabic

(86) In each case 3% strength aqueous solutions (w/v) of alternan-octenylsuccinic acid or gum arabic were prepared. To these solutions were added different amounts of standard commercial sunflower oil before a homogenization was carried out using an Ultra-Turrax (Ultra-Turrax T 25 digital, IKA-Werke GMBH & CO. KG, D-79219 Staufen, Germany, 1 minute at 9 k rpm). After a period of six days, the resulting emulsions were assessed as to the stability of the emulsion. The results obtained are shown in the table below.

(87) TABLE-US-00012 TABLE 11 Stability of emulsions with gum arabic and alternan-octenylsuccinic acid ester. Amount of substance in the mixture Phase separation after 5 days Water Oil Emulsifier Alternan-octenylsuccinic [ml] [ml] [mg] Gum arabic acid ester 18 2 600 significant none separation 15 5 600 significant none separation
f) Emulsions with Varying Concentrations of Alternan-Octenylsuccinic Acid Ester

(88) Aqueous solutions with different concentrations of alternan-octenylsuccinic acid ester were prepared. To these solutions were then added different amounts of standard commercial sunflower oil before a homogenization was carried out using an Ultra-Turrax (Ultra-Turrax T 25 digital, IKA-Werke GMBH & CO. KG, D-79219 Staufen, Germany, 1 minute at 9 k rpm). After a period of six days, the resulting emulsions were assessed as to the stability of the emulsion. The results obtained are shown in the table below.

(89) TABLE-US-00013 TABLE 12 Stability of emulsions with varying oil content with different amounts of alternan-octenylsuccinic acid (Al-OSA) after 6 and 18 days. Amount of substance in the mixture Phase separation at the amounts of Al-OSA Water Oil in the mixture stated in each case [ml] [ml] 5 mg 10 mg 30 mg 50 mg 100 mg 500 mg 10.0 0.1 none none none none none none after 9.7 0.3 none none none none none none 18 days 9.5 0.5 complete significant none none none none 9.0 1.0 complete complete none none none none 8.0 2.0 complete none none after 7.0 3.0 complete significant none 6 days
g) Preparation of Creams

(90) Alternan-octenylsuccinic acid esters were added to a mixture of water comprising 30% (v/v) standard commercial sunflower oil up to an end concentration of 15% (w/v) and homogenized using an Ultra-Turrax (9 k rpm) for 1 minute. A stable cream was obtained. Similar results were obtained with a water/oil mixture comprising 36% sunflower oil. In contrast to the cream prepared from the mixture comprising 30% oil, that prepared from the mixture with 36% oil was more creamy.