Modified plant gums for preparations of active ingredients

Abstract

The present invention relates to compositions containing (fat-soluble) active ingredients and/or colorants in a matrix based on modified plant gums, i.e. plant gums that have been submitted to hydrolysis to degrade either the protein portion and/or where appropriate, the polysaccharide, and to a process for preparing these compositions as well as to modified plant gums, whose protein part is hydrolyzed up to a degree of about 30%, preferably to a degree of from about 0.05 to about 30%, and/or whose polysaccharide part is hydrolyzed up to a degree of about 50%, a process for the manufacture thereof and such modified plant gums themselves. The present invention further relates to the use of the compositions of this invention for the enrichment, fortification and/or for the coloration of food, beverages, animal feed, cosmetics and pharmaceutical compositions and to such food, beverages, animal feed, cosmetics and pharmaceutical compositions themselves.

Claims

1. A composition comprising i) at least one modified plant gum, ii) at least one fat-soluble active ingredient and/or a colorant, and iii) at least one adjuvant and/or excipient, wherein the modified plant gum has a protein content of 10 weight-%, based on the total weight of the modified plant gum, wherein the fat-soluble active ingredient and/or a colorant ii) is a carotenoid selected from the group consisting of -carotene, -carotene, 8-apo--carotenal, 8-apo--carotenoic acid esters such as the ethyl ester, canthaxanthin, astaxanthin, lycopene, lutein, zeaxanthin or crocetin, -zeacarotene, -zeacarotene, or a mixture thereof, wherein the amount of the modified plant gum i) is from 0.5 weight-% to 60.0 weight-%, the amount of the fat-soluble active ingredient and/or colorant ii) is from 3.0 weight-% to 80.0 weight-%, and the amount of the adjuvant and/or excipient iii) is from 10 to 95 weight-%, based on the total amount of the composition, wherein the fat-soluble active ingredient and/or a colorant ii) is in a matrix of the modified plant gum, wherein said modified plant gum is produced by hydrolysis comprising one or more of a) treatment of a plant gum from Acacia senegal or Acacia seyal with a hydrolase, and/or b) adjustment of the pH of the plant gum from Acacia senegal or Acacia seyal to a pH value of between about 2 and about 9 by addition of an organic and/or inorganic acid or base, to provide the modified plant gum, wherein the protein of the modified plant gum is hydrolysed to a degree of from 1% to 30%, wherein the adjuvant and/or excipient is an oligosaccharide, a polysaccharide, or a combination thereof, and wherein the composition is in the form of a powder.

2. The composition according to claim 1, wherein the carotenoid is -carotene.

3. The composition according to claim 1, further comprising a mono- or disaccharide selected from sucrose, invert sugar, xylose, glucose, fructose, lactose, maltose, saccharose and/or a sugar alcohol.

4. The composition according to claim 1, wherein the oligo- or polysaccharide is selected from a starch, a starch hydrolysate or a modified starch.

5. The composition according to claim 4, wherein the starch hydrolysate is present and is selected from a dextrin, a maltodextrin or a glucose syrup.

6. The composition according to claim 1, further comprising a triglyceride selected from a vegetable oil or fat.

7. The composition according to claim 1, wherein a co-emulgator selected from the group consisting of mono- and diglycerides of fatty acids, polyglycerol esters of fatty acids, lecithins, and sorbitan monostearate is additionally present.

8. The composition according to claim 1, wherein the hydrolysis is an enzymatic hydrolysis comprising treatment of the plant gum from Acacia senegal or Acacia seyal with a hydrolase.

9. The composition according to claim 8, wherein the hydrolase is a protease, a glycosylase, or a mixture thereof.

10. The composition according to claim 8, wherein the enzymatic hydrolysis is carried out at a temperature of from about 30 C. to about 40 C.

11. The composition according to claim 8, further comprising inactivation of the hydrolase at a temperature of from about 80 C. to about 85 C.

12. A food, beverage, animal feed, cosmetic and/or pharmaceutical composition comprising the composition according to claim 1.

Description

EXAMPLES

Example 1

(1) An aqueous suspension of the plant gum from Acacia senegal having a dry matter content of 30 weight-%, based on the total amount of the aqueous suspension, is adjusted to pH 6 with 0.5 N NaOH and to a temperature of 35 C. Thereafter, 1.0 weight-%based on the total amount of the aqueous suspensionof a protease of Aspergillus oryzae, e.g the enzyme preparation Flavourzyme 500 L of Novozymes, Bagsvaerd, Denmark with an activity of 500 Leucine Amino Peptidase Units per gram (500 LAPU/g), is added. During the enzymatic hydrolysis the pH is maintained constant by the addition of 0.5 N NaOH solution and the temperature is maintained at 35 C. After a reaction time of 5 to 10 minutes the protease is inactivated by heating the suspension to 85 C. for 10 minutes. The solution is purified by ultrafiltration using membranes with nominal molecular weight cut-offs from 10 to 1000 kDa. The solution is spray-dried at a product temperature of 70 to 80 C., whereby the inlet temperature is 160 to 190 C.

Example 2

(2) An aqueous suspension of the plant gum from Acacia senegal having a dry matter content of 10 weight-%, based on the total amount of the aqueous suspension, is adjusted to pH 7.4 with 0.5 N NaOH and to a temperature of 20 C. Thereafter, 0.2 weight-%based on the total amount of the aqueous suspensionof a proteolytic enzyme produced from Bacillus licheniformis e.g. Subtilisin Carlsberg, commercially available as Alcalase 2.4 L of Novozymes, Bagsvaerd, Denmark, is added. During the enzymatic hydrolysis the pH is maintained constant by the addition of 0.5 N NaOH solution and the temperature is maintained at 20 C. After a reaction time of 10 minutes the protease is inactivated by heating the suspension to 85 C. for 10 minutes. The solution is purified by ultrafiltration using a polysulfon-membrane with a nominal molecular weight cut-off of 100 kDa. The solution is spray-dried at a product temperature of 68 to 75 C., whereby the inlet temperature is 160 to 190 C.

Example 3

(3) An aqueous suspension of the plant gum from Acacia senegal having a dry matter content of 20 weight-%, based on the total amount of the aqueous suspension, is adjusted to pH 6 with 0.5 N NaOH and to a temperature of 35 C. After 10 to 30 minutes the solution is purified by ultrafiltration using membranes with nominal molecular weight cut-offs from 10 to 100 kDa. The solution is spray-dried at a product temperature of 70 to 80 C., whereby the inlet temperature is 160 to 190 C.

Example 4

(4) A composition comprising a modified plant gum and -carotene is prepared as follows:

(5) a) Preparation of a(n Oil-Based) Solution A:

(6) 9.7 g of corn oil and 2.7 g of dl--tocopherol were mixed. 8.9 g of crystalline -carotene were dispersed in 120 ml of chloroform (trichloromethane) and the resulting dispersion was added to the mixture of corn oil and tocopherol. By gently stirring and simultaneous heating the mixture to about 65 C. a solution was obtained.

(7) b) Preparation of a(n Aqueous) Solution B:

(8) A dry powder of 111.0 g of the plant gum Quick Gum of Alfred L. Wolff (Hamburg, Germany), prepared from Acacia senegal, was subsequently mixed with 220 ml of deionised water at room temperature. Under stirring the mixture was heated to 35 C. and by adding 18.2 ml of a 0.5 N sodium hydroxide solution the pH of the mixture was brought to a value of 6.0. 1.0 ml of the protease Flavourzyme 500 L of Novozymes was added and the mixture was stirred for 10 minutes in order to achieve a homogeneous hydrolysis of the solution and the pH was maintained by adding 0.5 N sodium hydroxide solution (pH-stat-method). The enzyme was deactivated by heating the solution to 85 C. for 10 minutes. The solution was then cooled to 35 C. By adding 4.1 ml of a 0.5 N hydrochloric acid the pH of the mixture was brought to a value of 5.0 and kept at 35 C. In a second step of the enzymatic treatment 1.0 ml of a mixture of the pectinases Pectinex Ultra SP-L and Shearzyme at a volume ratio of 80:20, both of Novozymes, Bagsvaerd, Denmark, was added and the mixture was stirred for 30 minutes in order to achieve a homogeneous hydrolysis of the solution. The enzymes were deactivated by heating the solution to 85 C. for 10 minutes. The solution was then cooled to 50 C.

(9) c) Preparation of an Emulsion from the Solutions A and B:

(10) Under vigorous stirring solution A was added to solution B at 50 C. and the dispersion was vigorously stirred for another 30 minutes. The stirred dispersion was kept at 50 to 55 C. for 30 minutes. Residual trichloromethane was removed at 50 to 55 C. After removing entrapped air bubbles by centrifugation the emulsion was gently stirred at 50 to 55 C. for some minutes and then characterised with respect to the particle size of the inner phase. The mean particle size (Sauter diameter, D[3, 2]) of the inner phase of the emulsion was 430 to 480 nm as measured by laser diffraction (Malvern Mastersizer).

Example 5

(11) A composition comprising a modified plant gum and -carotene is prepared as follows:

(12) a) Preparation of a(n Oil-Based) Solution A:

(13) The preparation was carried out as described in Example 4a).

(14) b) Preparation of (Aqueous) Solution B:

(15) A dry powder of 110 g of the modified plant gum prepared from plant gum according to Example 2 was subsequently mixed with 220 ml of deionised water at room temperature. The suspension was stirred for 120 min.

(16) c) Preparation of an Emulsion from the Solutions A and B:

(17) Under vigorous stirring solution A was added to solution B at 50 C. and the dispersion was vigorously stirred for another 30 minutes. The stirred dispersion was kept at 50 to 55 C. for 60 minutes. Residual trichloromethane was removed at 50 to 55 C. After removing entrapped air bubbles by centrifugation the emulsion was gently stirred at 50 to 55 C. for some minutes and then characterised with respect to the particle size of the inner phase. The mean particle size (D[3, 2]) of the inner phase of the emulsion was 40 to 410 nm as measured by laser diffraction (Malvern Mastersizer).

(18) d) Preparation of a Solid Composition from the Emulsion:

(19) The emulsion was sprayed into a pre-cooled fluidised bed of cornstarch. Excess cornstarch was removed by sieving and the powder obtained was dried in an air stream at room temperature for about 2 hours. Sieving collected the powder particle fraction (approximately 250 g) in the range of 0.16 to 0.50 mm and characterised with respect to the carotenoid content, the colour intensity and the colour hue in an aqueous dispersion, the content of the corn starch and residual humidity.

(20) The obtained powder had a -carotene content of 4.9 weight-% (3.9 weight-%), as measured by UV/VIS-spectroscopy (HPLC), a content of 37.0 weight-% of corn starch and a residual water content of 8.2 weight-%. The powder was dispersed in deionised water and the extinction was measured in a 1 cm quartz precision cell against deionised water. For a 5 ppm dispersion of -carotene an extinction of 314 at a wavelength of 536 nm was calculated [E (1%, 1 cm)=314]. The colour values L*=87.1, a*=12.9 and b*=7.4 were measured according to the CIE-system (D. L. MacAdam, Color Measurement, second edition, Springer Verlag, Berlin Heidelberg, N.Y., Tokyo, 1985, pages 160 to 221) for a 5 ppm dispersion of -carotene with a Hunterlab Ultrascan Spectrocolorimeter (1 cm, TTRAN). Based upon the values of a* and b* a colour hue angle h*=30.0 at a saturation (chroma value) C*=14.9 can be calculated.

Example 6

(21) A dry premix of 238 g of a modified plant gum obtained according to example 1, 2 or 3, 50 g of sucrose and 282 g of a maltodextrin (DE 20 to 23) was prepared. The dry premix was dissolved in 700 ml of deionized water at 60 C. After complete dissolution of the solids 7.2 g of sodium ascorbate was added to the mixture (=solution A).

(22) 110 g of a triglyceride (MCT) and 0.7 g of dl--tocopherol were mixed and heated to 170 C. Subsequently 31 g of -carotene were suspended in the mixture of the triglyceride and tocopherol. By stirring for about 10 minutes at 170 C. a clear solution of -carotene was obtained (=solution B).

(23) Solution A was heated to about 80 C. and a crude emulsion was prepared by adding solution B to solution A while gently stirring. A fine emulsion was obtained by high pressure homogenizing treatment of the preemulsion at 300 bar within two passages (APV Lab Homogenizer Type Gaulin Lab 40-10 RBFI of APV Switzerland AG, CH-3076 Worb). The emulsion was spray-dried in a laboratory spray dryer (Mobile Minor of GEA Niro A/S, DK-2860 Sborg) at an inlet temperature of 200 C. to 210 C. and an outlet temperature of 70 to 75 C. The spray-dried powder was dried in a vacuum oven at room temperature over night. A fine powder was obtained.

Example 7: Determination of the Correction Factors and for Gum Arabic

(24) $\begin{array}{cc}DH=\frac{h}{h_{\mathrm{tot}}}*100\%& \left(\mathrm{Formula}I\right)\end{array}$

(25) DH=degree of hydrolysis; h=number of hydrolyzed peptide bonds; h.sub.tot=total number of peptide bonds.

(26) h.sub.tot [milli equivalents Serin-NH.sub.2 per g; meqv Serin-NH.sub.2/g] refers to the amino acid composition of the protein and is calculated as sum of the amounts of the amino acids (mmol) of the individual amino acid composition per g protein (J. Adler-Nissen, Enzymic Hydrolysis of food proteins; Elsevier Allied Science Publishers, London 1986, p. 115-131).

(27) A calculation with the amino acid compositions given in R. C. Randall, G. O. Phillips, P. A. Williams, Food Hydrocolloids 1989, 3(1), p. 65-75, Table III, entries this study and reference 19, results in a h.sub.tot of gum arabic Senegal of 8.128 meqv/g protein and 8.258 meqv/g protein, respectively. If the amino acid composition given in M. E. Osman et al., Phytochemistry 1995, 38(2), p. 409-417, is taken as basis for the calculation a h.sub.tot of 8.292 meqv/g protein results. The average h.sub.tot (of these three data) is 8.226 meqv/g protein for gum arabic Senegal. Therefore, the further calculation is based on this numerical value.

(28) It is also possible to determine the amino acid composition via analysis of the total hydrolysate and calculate h.sub.tot on basis of these experimentally determined data.

(29) $\begin{array}{cc}h=\frac{\mathrm{Serin}\text{-}{\mathrm{NH}}_2\text{-}}{}\mathrm{meqv}\text{/}g\mathrm{protein}& \left(\mathrm{Formula}\mathrm{II}\right)\end{array}$

(30) To get more exact results the correction factors and were determined experimentally. For this the following values have to be known: h.sub.tot, the absorption of native gum arabic (the native plant gum with a low protein content) and the absorption of totally hydrolysed gum arabic (the totally hydrolysed plant gum with a low protein content).

(31) Serin-NH.sub.2=meqv Serin-NH.sub.2/g protein, i.e. the number of free amino groups per g protein, is calculated according to D. Petersen, P. M. Nielsen, C. Dambmann, Determination of the Degree of Hydrolysis based on OPA reaction, ED-9512723, Novo Nordisk A/S, 1995; Petersen, D., Nielsen, P. M., Dambmann C., Journal of Food Science 2001, 66 (5). 642-646 (Improved method for determining food protein degree of hydrolysis.) as follows:

(32) $\begin{array}{cc}\mathrm{Serin}\text{-}{\mathrm{NH}}_2=\frac{{\mathrm{Abs}}_{\mathrm{sample}}-{\mathrm{Abs}}_{\mathrm{control}}}{{\mathrm{Abs}}_{\mathrm{standard}}-{\mathrm{Abs}}_{\mathrm{control}}}*0.9516\mathrm{meqv}\text{/}l*\frac{V*100}{X*P}*l\text{/}g\mathrm{protein}& \left(\mathrm{Formula}\mathrm{III}\right)\end{array}$
Abs.sub.sample=absorption of the sample to be measured
Abs.sub.control=absorption of a control sample
Abs.sub.standard=absorption of a standard solution of serine
X=weight of the sample [g]
P=Protein content of the sample
V=volume of the sample solution [l]; in our examples: 0.1 l

(33) The determination of DH was based on a modified OPA reaction as disclosed by H. Frister, H. Meisel, E. Schlimme, Anal. Chem. 1988, 330, 631-633.

(34) a) Preparation of the OPA Solution

(35) Solution 1: 7.620 g of Borax were dissolved in 100 g of demineralised water.

(36) Solution 2: 0.160 g of ortho-phthaldialdehyde (OPA) were dissolved in 4 ml of ethanol.

(37) Solution 3: 0.400 g of N,N-dimethyl-2-mercaptoethylammonium chloride (=DMMAC) were dissolved in a small amount of demineralised water.

(38) All solutions 1 to 3 were put together in a 200 ml flask. 0.200 g of sodium dodecyl sulphate (=SDS) were added and demineralised water was added to get a total of 200 ml of the OPA solution. Under exclusion of light the OPA solution can be stored for one week.

(39) b) Preparation of the Samples the Control Sample and the Standard Solution of Serine and Measurement of their Absorption

(40) Native gum arabic as well as totally hydrolysed gum arabic were tested. (Other plant gums with a low protein content may also be measured accordingly.) To 0.1 mg of powderous gum arabic (It is recommended that in case of plant gums with a low protein content the 10-fold amount is used in comparison to plant gums with a high protein content.) was added an aqueous 10 weight-% SDS solution and the mixtures were stirred for 10 minutes at 90 C. Afterwards the solution was filled up with demineralised water to give a total of 100 ml, it was then centrifugated for 10 minutes at 10750 g and filtered over a 0.45 m filter. To 400 l of this resulting solution 3 ml of the OPA solution were added. The mixture was stirred and measured then in a spectral photometer (Lambda 25, Perkin Elmer Instruments, Boston, USA) at 340 nm exactly two minutes after preparation of the mixture. The sample of the totally hydrolysed gum arabic was prepared and measured accordingly. The control sample was demineralised water. To 400 l of demineralised water 3 ml of the OPA solution were added. The mixture was stirred and measured then in a spectral photometer (Lambda 25, Perkin Elmer Instruments, Boston, USA) at 340 nm exactly two minutes after preparation of the mixture.

(41) 0.100 g of L-serine were dissolved in 500 ml of demineralised water to give the standard solution of serine which is used for the purpose of calibration. Standard solutions of serine with concentrations ranging between 10 and 200 mg/l were measured therefore. To each of 400 l of the standard solution of serine of a certain concentration 3 ml of the OPA solution were added. The mixture was stirred and measured then in a spectral photometer (Lambda 25, Perkin Elmer Instruments, Boston, USA) at 340 nm exactly two minutes after preparation of the mixture.

(42) c) Calculation of and

(43) With the measured values for Abs.sub.sample, Abs.sub.control, and Abs.sub.standard (each measured twice and the average value taken) and with further taken into account the coloration of the sample of the totally hydrolyzed gum arabic Ser-NH.sub.2 could be calculated according to formula III given above.

(44) Under the assumptions that the number of hydrolysed peptide bonds in native gum arabic is zero (=Ser-NH.sub.2) and that the degree of hydrolysis is 100% for totally hydrolyzed gum arabic (i.e. h=h.sub.tot), and can be calculated:

(45) For gum arabic Senegal is 1.999 meqv/g protein and is 0.588 meqv/g protein.

(46) The method as described above for gum arabic Senegal may be applied accordingly to other plant gums with a low protein content, especially with a protein content5 weight-%, based on the total weight of the plant gum.

Example 8: Determination of the Degree of Hydrolysis (DH) of Gum Arabic

(47) With the now known correction factors and for gum arabic Senegal (=1.999 meqv/g protein; =0.588 meqv/g protein) it is now possible via measuring the absorption at 340 nm (to determine Ser-NH.sub.2 via formula III) to calculate the number of hydrolysed peptide bonds h via formula II and thus also the DH via formula I (see above).

(48) $\begin{array}{cc}h=\frac{\mathrm{Serin}\text{-}{\mathrm{NH}}_2\text{-}}{}\mathrm{meqv}\text{/}g\mathrm{protein}& \left(\mathrm{Formula}\mathrm{II}\right)\end{array}$

(49) The accuracy of such a DH is ca.0.05%.

(50) If for other plant gums with a low protein content, especially with a protein content5 weight-%, based on the total weight of the plant gum, the correction factors and were also determined according to example 7, their DH can also be calculated accordingly to the method described above.

Example 9: Determination of the Degree of Hydrolysis of the Polysaccharide Part of Gum Acacia

(51) To an aqueous suspension of the plant gum from Acacia senegal having a dry matter content of 10 weight-%, based on the total amount of the aqueous suspension, is added 1 weight-%based on the amount of the plant gumof an enzyme produced from Aspergillus aculeateus e.g. commercially available as Peelzym of Novozymes, Bagsvaerd, Denmark. The solution is then heated up at 50 C. during 10 min without agitation and an aliquot of 0.15 mL is used to determine the relative degree of hydrolysis of sugar part by Osmolality (micro Osmometer, KNAUER). The read value is converted into relative degree of hydrolysis using the next formula, and taking as presumption that the osmotic coefficient for peptides (w) and the total number of peptides bonds (h tot) are constant during this hydrolysis:

(52) $DH=\frac{DC}{S\%}\frac{1}{\mathrm{fosm}}\frac{1}{w}\frac{1}{h\mathrm{tot}}*100$
DH: degree of hydrolysis;
DC: Osmolality (mOsm.Math.kg.sup.1)
S %: substrate concentration
f osm: conversion factor calculated on the basis of percentage content of dry matter in substrate (D %)

(53) $\mathrm{fosm}=\frac{1000}{\left(100-D\%\right)}$
w: osmotic coefficient for peptides
h.sub.tot: total number of peptides bonds (mM g proteins.sup.1)

Example 10

(54) A composition comprising a modified plant gum and -carotene is prepared as follows:

(55) a) Preparation of a(n Oil-Based) Solution A:

(56) 9.7 g of corn oil and 2.7 g of dl--tocopherol were mixed. 8.9 g of crystalline -carotene were dispersed in 120 ml of chloroform (trichloromethane) and the resulting dispersion was added to the mixture of corn oil and tocopherol. By gently stirring and simultaneous heating the mixture to about 65 C. a solution was obtained.

(57) b) Preparation of a(n Aqueous) Solution B:

(58) A dry powder of 111.0 g of the plant gum Quick Gum of Alfred L. Wolff (Hamburg, Germany), prepared from Acacia senegal, was subsequently mixed with 220 ml of deionised water at room temperature. Under stirring the mixture was heated to 50 C. and the pH was brought to a value of 7.0.

(59) c) Preparation of an Emulsion from the Solutions A and B:

(60) Under vigorous stirring solution A was added to solution B at 50 C. and the dispersion was vigorously stirred for another 30 minutes. The stirred dispersion was kept at 50 to 55 C. for 30 minutes. Residual trichloromethane was removed at 50 to 55 C. After removing entrapped air bubbles by centrifugation the emulsion was gently stirred at 50 to 55 C. for some minutes and then characterised with respect to the particle size of the inner phase. The mean particle size (Sauter diameter, D[3, 2]) of the inner phase of the emulsion was 350 nm as measured by laser diffraction (Malvern Mastersizer).

Example 11

(61) An aqueous suspension of the plant gum from Acacia senegal having a dry matter content of 10 weight-%, based on the total amount of the aqueous suspension, is adjusted to pH 7.0 with 0.5 N NaOH and to a temperature of 20 C. Thereafter, 0.5 weight-%based on the total amount of the proteinof a proteolytic enzyme produced from Carica papaya, commercially available as Collupilin L of DSM food beverages, Delft, Netherlands, is added. During the enzymatic hydrolysis the pH is maintained constant by the addition of 0.5 N NaOH solution and the temperature is maintained at 20 C. After a reaction time of 5 minutes the protease is inactivated by heating the suspension to 85 C. for 10 minutes. The solution is spray-dried at a product temperature of 68 to 75 C., whereby the inlet temperature is 160 to 190 C.

Example 12

(62) A composition comprising a modified plant gum and -carotene is prepared as follows:

(63) a) Preparation of a(n Oil-Based) Solution A:

(64) The preparation was carried out as described in Example 4a).

(65) b) Preparation of (Aqueous) Solution B:

(66) A dry powder of 110 g of the modified plant gum prepared from plant gum according to Example 9 was subsequently mixed with 220 ml of deionised water at room temperature. The suspension was stirred for 120 min.

(67) c) Preparation of an Emulsion from the Solutions A and B:

(68) Under vigorous stirring solution A was added to solution B at 50 C. and the dispersion was vigorously stirred for another 30 minutes. The stirred dispersion was kept at 50 to 55 C. for 60 minutes. Residual trichloromethane was removed at 50 to 55 C. After removing entrapped air bubbles by centrifugation the emulsion was gently stirred at 50 to 55 C. for some minutes and then characterised with respect to the particle size of the inner phase. The mean particle size (D[3, 2]) of the inner phase of the emulsion was 40 to 410 nm as measured by laser diffraction (Malvern Mastersizer).

(69) The resulting emulsion was characterised with respect to the carotenoid content, the colour intensity and the colour hue in an aqueous dispersion.

(70) The obtained emulsion had a -carotene content of 3.0 weight-% (3.9 weight-%), as measured by UV/VIS-spectroscopy. The extinction coefficient in deionised water was measured in a 1 cm quartz precision cell against deionised water. For a 5 ppm dispersion of -carotene an extinction of 283 at a wavelength of 518 nm was calculated [E (1%, 1 cm)=283]. The colour values L*=90.2, a*=9.4 and b*=8.5 were measured according to the CIE-system (D. L. MacAdam, Color Measurement, second edition, Springer Verlag, Berlin Heidelberg, N.Y., Tokyo, 1985, pages 160 to 221) for a 5 ppm dispersion of -carotene with a Hunterlab Ultrascan Spectrocolorimeter (1 cm, TTRAN). Based upon the values of a* and b* a colour hue angle h*=42.0 at a saturation (chroma value) C*=12.7 can be calculated.