U.S. patent application number 13/948334 was filed with the patent office on 2013-11-21 for protective hydrocolloid for active ingredients.
This patent application is currently assigned to DSM IP Assets B.V.. The applicant listed for this patent is DSM IP Assets B.V.. Invention is credited to Navagnana (Navam) S. HETTIARACHCHY, Bruno H. Leuenberger, Ilankovan Paraman, Christian Schafer.
Application Number | 20130310302 13/948334 |
Document ID | / |
Family ID | 39523300 |
Filed Date | 2013-11-21 |
United States Patent
Application |
20130310302 |
Kind Code |
A1 |
HETTIARACHCHY; Navagnana (Navam) S.
; et al. |
November 21, 2013 |
PROTECTIVE HYDROCOLLOID FOR ACTIVE INGREDIENTS
Abstract
Partially deamidated rice endosperm protein or rice endosperm
protein which is partially conjugated with mono-, di-, oligo- or
polysaccharides is used as novel protective hydrocolloid for
fat-soluble active ingredients and/or fat-soluble colorants. The
present invention further includes compositions comprising that
rice endosperm protein and at least one fat-soluble active
ingredient/colorant, as well as their manufacture, that rice
endosperm protein itself and its manufacture. These compositions
are used for the enrichment, fortification and/or coloration of
food, beverages, animal feed, personal care or pharmaceutical
compositions. The present invention is directed to theses uses and
to food, beverages, animal feed, personal care and pharmaceutical
compositions containing such a rice endosperm protein and such a
composition, respectively.
Inventors: |
HETTIARACHCHY; Navagnana (Navam)
S.; (Fayetteville, AR) ; Leuenberger; Bruno H.;
(Allschwil, CH) ; Paraman; Ilankovan;
(Fayetteville, AR) ; Schafer; Christian;
(Rheinfelden, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DSM IP Assets B.V. |
Te Heerlen |
|
NL |
|
|
Assignee: |
; DSM IP Assets B.V.
Te Heerlen
NL
|
Family ID: |
39523300 |
Appl. No.: |
13/948334 |
Filed: |
July 23, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11729981 |
Mar 30, 2007 |
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13948334 |
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Current U.S.
Class: |
514/1.1 ; 426/18;
426/321; 426/540; 426/541; 426/573; 426/590; 426/656; 426/72;
426/73; 435/99; 530/372 |
Current CPC
Class: |
A23P 10/30 20160801;
A23V 2002/00 20130101; A23L 2/66 20130101; A61P 3/02 20180101; A23J
3/14 20130101; A23L 33/15 20160801; A23L 33/155 20160801; A23V
2200/224 20130101; A23J 1/125 20130101; A23V 2250/712 20130101;
A23V 2250/211 20130101; A23V 2002/00 20130101; A23L 7/152 20160801;
C07K 14/415 20130101; A23L 33/18 20160801; A23L 5/43 20160801; A23K
20/147 20160501; A23V 2200/224 20130101; A23J 3/346 20130101; A23V
2002/00 20130101; A23V 2250/5482 20130101; A23V 2250/5482 20130101;
A23V 2200/30 20130101; A23V 2200/30 20130101 |
Class at
Publication: |
514/1.1 ;
426/656; 426/540; 426/72; 426/73; 426/541; 426/18; 426/573;
426/321; 426/590; 530/372; 435/99 |
International
Class: |
A23J 3/34 20060101
A23J003/34; C07K 14/415 20060101 C07K014/415; A23L 2/66 20060101
A23L002/66; A23L 1/305 20060101 A23L001/305; A23K 1/16 20060101
A23K001/16 |
Claims
1-36. (canceled)
37. A process for the manufacture of a rice endosperm protein which
is partially deamidated starting from milled rice, whereby before
milling the rice bran was removed, comprising the following steps
a) to e): a) preparing an aqueous solution or suspension of milled
rice, whereby the rice bran was removed before milling, whereby the
solution or suspension preferably has a dry mass content of from
0.1 to 30 weight-%, based on the total amount of the aqueous
solution or suspension; b) optionally removing the non-protein part
or the protein part of the milled rice, whereby the rice bran was
removed before milling, to obtain the rice endosperm protein; c)
modifying the protein part of the milled rice, whereby the rice
bran was removed before milling, by partially deamidating the
protein part of the milled rice to obtain rice endosperm protein
which is partially deamidated; d) optionally isolating the rice
endosperm protein which is partially deamidated; and e) optionally
converting the rice endosperm protein which is partially deamidated
into a solid form, wherein the rice endosperm protein has an
emulsion activity of .gtoreq.0.62 as determined turbidimetrically
by emulsifying a solution of a sample rice endosperm protein in
corn oil, homogenized by sonication and measuring absorbance, the
absorbance at a time 0 after homogenization indicating the emulsion
activity of the rice endosperm.
38. The process according to claim 37, wherein the removal of the
non-protein part (step b) is achieved by treating the milled rice
with non-protein degrading enzymes, deactivating the enzymes,
separating and removing the non-protein part from the protein part
of the milled rice.
39. The process according to claim 38, wherein the non-protein
degrading enzymes are starch-degrading enzymes, cellulose-degrading
enzymes or mixtures thereof.
40. The process according to claim 38, wherein the separation of
the non-protein part is achieved by centrifugation followed by
washing off the non-protein part with water.
41. The process according to claim 37, wherein the removal of the
protein part (step b) is achieved by adjusting the pH of the milled
rice solution or suspension to a value of from 7 to 12.
42. The process according to claim 37, wherein step e) is achieved
by drying.
43. The process according to claim 37, wherein the deamidation is
achieved by adjusting the pH value of an aqueous colloidal solution
of the protein part of the rice endosperm protein obtained in step
b) to a value in the range of from 9.0 to 13.0 and at a temperature
in the range of from 25 to 90.degree. C.
44. A rice endosperm protein which is partially deamidated
obtainable by any process according to claim 37.
45. A composition comprising a rice endosperm protein obtained by
the process of claim 37 and a fat-soluble active ingredient and/or
a fat-soluble colorant.
46. The composition according to claim 45, wherein the fat-soluble
active ingredient and/or the fat-soluble colorant is a carotene or
a structurally related polyene compound, a fat soluble vitamin, a
triglyceride rich in polyunsaturated fatty acids, an oil soluble
UV-A filter, an UV-B filter or a mixture thereof.
47. The composition according to claim 46, wherein the carotene or
structurally related polyene compound is a carotenoid such as
.alpha.-carotene, .beta.-carotene, 8'-apo-.beta.-carotenal,
8'-apo-.beta.-carotenoic acid esters, canthaxanthin, astaxanthin,
lycopene, lutein, zeaxanthin, crocetin, .alpha.-zeacarotene,
.beta.-zeacarotene or a mixture thereof.
48. The composition according to claim 47, wherein the carotenoid
is .beta.-carotene.
49. The composition as in claim 46, wherein the fat-soluble vitamin
is Vitamin A or E.
50. The composition as in claim 45, wherein at least one compound
selected from the group consisting of monosaccharides,
disaccharides, oligosaccharides, polysaccharides, glycerol,
triglycerides, water-soluble antioxidants and fat-soluble
antioxidants is additionally present.
51. The composition as in claim 50, wherein the mono- or
disaccharide is sucrose, invert sugar, xylose, glucose, fructose,
lactose, maltose, saccharose and sugar alcohols.
52. The composition as in claim 50, wherein the oligo- or
polysaccharide is a starch, a starch hydrolysate or a modified
starch.
53. The composition as in claim 52, wherein the starch hydrolysate
is a dextrin, a maltodextrin or a glucose syrup.
54. The composition as in claim 46, wherein the triglyceride is a
vegetable oil or fat.
55. The composition as in claim 45, 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.
56. The composition as in claim 45, wherein the rice endosperm
protein has an emulsion activity of .gtoreq.0.6.
57. The composition as in claim 45, wherein the rice endosperm
protein has an emulsion stability of .gtoreq.20 minutes.
58. The composition as in claim 45, wherein the amount of the rice
endosperm protein which is partially deamidated is from about 1 to
about 70 weight-% and/or the amount of the fat-soluble active
ingredient and/or the fat-soluble colorant is from about 0.1 to
about 90 weight-%, based on the total amount of the
composition.
59. The composition as in claim 45 in the form of a powder.
60. A process for the manufacture of a composition as claimed in
claim 45 which comprises the following steps: I) preparing an
aqueous solution or colloidal solution of a rice endosperm protein
which is partially deamidated, II) optionally adding at least a
water-soluble excipient and/or adjuvant to the solution prepared in
step I), III) preparing a solution or dispersion of at least a
fat-soluble active ingredient and/or a fat-soluble colorant, and
optionally at least a fat-soluble adjuvant and/or excipient, IV)
mixing the solutions prepared in step I) to III) with each other,
V) homogenising the thus resulting mixture, VI) optionally adding a
cross-linking agent for (further) cross-linking the rice endosperm
protein which is partially deamidated, VIa) optionally submitting
the mixture resulting after having performed step VI) to enzymatic
treatment or heat treatment to cross-link the modified rice
endosperm protein VII) optionally converting the dispersion
obtained in step V) and/or VI) into a powder, VIII) optionally
drying the powder obtained in step VII), IX) optionally submitting
the powder resulting from step VII or the dry powder resulting from
step VIII to heat treatment or to enzymatic treatment to cross-link
the rice endosperm protein which is partially deamidated, with the
proviso that only step VIa) or step IX) is carried out, but not
both, when step VI) is carried out.
61. The process according to claim 60, wherein the enzymatic
treatment according to step VIa) or step IX) is a treatment with a
cross-linking enzyme.
62. The process according to claim 60 wherein the rice endosperm
protein which is partially deamidated is one having an emulsion
activity of .gtoreq.0.6.
63. Food, beverages, animal feed, personal care and pharmaceutical
compositions containing a composition as claimed in claim 45.
64. Food, beverages, animal feed, personal care and pharmaceutical
compositions containing a rice endosperm protein as claimed in
claim 44.
65. Process for the protection of fat-soluble active ingredients
and/or fat-soluble colorants by whereby the rice endosperm protein
as claimed in claim 44 is added to said fat-soluble active
ingredients and/or said fat-soluble colorants as a protecting
agent.
66. The process according to claim 42, wherein the drying is by
freeze drying or spray drying.
67. The composition as in claim 42, wherein the rice endosperm
protein has an emulsion activity of .gtoreq.0.62.
68. The composition as in claim 45, wherein the rice endosperm
protein has an emulsion activity of .gtoreq.0.7.
69. The composition as in claim 45, wherein the rice endosperm
protein has an emulsion activity of .gtoreq.23 minutes.
70. The composition as in claim 45, wherein the rice endosperm
protein has an emulsion activity of .gtoreq.25 minutes.
71. The process according to claim 61, wherein the cross-linking
enzyme is a transglutaminase.
72. Process for the enrichment, fortification and/or coloration of
food, beverages, animal feed, personal care or pharmaceutical
compositions, whereby a composition as claimed in claim 45 is added
to the food, beverages, animal feed, personal care and
pharmaceutical compositions, respectively.
73. A process for the manufacture of a rice endosperm protein which
is partially deamidated starting from milled rice, whereby before
milling the rice bran was removed, comprising the following steps
a) to e): a) preparing an aqueous solution or suspension of milled
rice, whereby the rice bran was removed before milling, whereby the
solution or suspension preferably has a dry mass content of from
0.1 to 30 weight-%, based on the total amount of the aqueous
solution or suspension; b) optionally removing the non-protein part
or the protein part of the milled rice, whereby the rice bran was
removed before milling, to obtain the rice endosperm protein; c)
modifying the protein part of the milled rice, whereby the rice
bran was removed before milling, by partially deamidating the
protein part of the milled rice to obtain rice endosperm protein
which is partially deamidated; d) optionally isolating the rice
endosperm protein which is partially deamidated; and e) optionally
converting the rice endosperm protein which is partially deamidated
into a solid form wherein the rice endosperm protein has an
emulsion stability of .gtoreq.23 minutes where emulsion stability
is calculated by the formula: To.times..DELTA./.DELTA., where
.DELTA.T is the decrease in turbidity (absorbance) of an initial
absorbance (To) over/after a time interval of .DELTA.t 10 minutes.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a Continuation of application Ser. No.
11/729,981, filed Mar. 30, 2007, the specification of which is
incorporated herein by reference.
PROTECTIVE HYDROCOLLOID FOR ACTIVE INGREDIENTS
[0002] The present invention is directed to the use of rice
endosperm protein as novel protective hydrocolloid for fat-soluble
active ingredients and/or fat-soluble colorants, whereby the rice
endosperm protein is partially conjugated with mono-, di-, oligo-
or polysaccharides (especially with mono- or polysaccharides) or
partially deamidated. Moreover, the present invention is directed
to compositions comprising that partially conjugated or partially
deamidated rice endosperm protein and at least one fat-soluble
active ingredient and/or fat-soluble colorant and to their
manufacture, as well as to that partially conjugated or partially
deamidated rice endosperm protein itself and its manufacture. The
present invention is further directed to the use of such
compositions for the enrichment, fortification and/or coloration of
food, beverages, animal feed, personal care or pharmaceutical
compositions, and to food, beverages, animal feed, personal care
and pharmaceutical compositions containing such a partially
conjugated or partially deamidated rice endosperm protein and such
a composition, respectively.
[0003] Active ingredients, especially fat-soluble active
ingredients or fat-soluble colorants, are often not added as such
to food, beverages, animal feed, personal care and pharmaceutical
compositions, but in form of formulations of the active ingredient
in a hydroprotective colloid for reasons of enhancing properties
such as chemical stability, (water-)solubility, free-flowing and
controlled release etc. Known hydroprotective colloids are e.g.
gelatine of different origin (poultry, bovine, pork, fish) and
starch. Since hydroprotective colloids of animal origin are often
not desired for religious or allergenic reasons and starch-based
hydroprotective colloids might have low preference for consumers
who are interested in gluten and corn-free products there is an
on-going need for alternative hydroprotective colloids.
[0004] Rice endosperm proteins are recognized as nutritional and
hypoallergenic and can, thus, be a suitable alternative source of
protective hydrocolloid for formulations of active ingredients.
However, high insolubility and poor functionality of rice endosperm
protein at neutral pH limits its industrial application as a
functional ingredient in food and pharmaceuticals products. The
present invention overcomes these limitations and incorporates the
rice endosperm protein which is partially conjugated with mono-,
di-, oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated as a protective
hydrocolloid for formulations of fat-soluble active ingredients
and/or fat-soluble colorants.
[0005] Rice proteins rank high in nutritional quality in comparison
to other cereals including corn and wheat, and are therefore
perceived to have immense potential uses as food ingredients.
Cereal grain proteins are rich in the essential amino acids
cysteine and methionine. Lysine is the primary limiting amino acid
in cereal proteins, but rice contains more lysine (3.8 g/16 g N)
than other cereal proteins (wheat 2.3, corn 2.5 g/16 g N) (see
reference 4 cited below). Although rice is generally regarded as
having the lowest protein content (7.3%) among the common grains
(wheat 10.6%, corn 9.8%, barley 11.0%, millet 11.5%), the net
protein utilization of rice protein (73.8%) is the highest among
the cereal grains (wheat 53.0%, corn 58.0%, barley 62.0%, millet
56.0%).
[0006] Compared with other cereal proteins, isolation of rice
protein is difficult and therefore costly. The predominant rice
protein, glutelin, is hydrophobic and is cross-linked with
disulfide bonds. The extracted proteins are highly insoluble in
nature and the conditions used in protein isolation further
decrease their solubility, and thus have limited application as a
functional ingredient. High-protein rice products can be obtained
from rice flour by alkali extraction followed by precipitation at
the isoelectric pH of the protein. Starch-hydrolyzing enzymes such
as alpha-amylase, glucoamylase, and pullulanase are often used to
separate proteins in rice flour by solubilizing and removing
starch. In addition to starch hydrolyzing enzymes, cellulase and
hemicellulase enzymes have been used to further increase the
protein content in rice protein concentrate. However, information
on suitable extraction methods and functionalities of such isolates
is limited. Efficient extraction methods using approved food grade
enzymes and chemicals are essential for commercial production and
application of rice protein.
[0007] This need is fulfilled by the compositions of the present
invention which comprise a rice endosperm protein which is
partially conjugated with mono-, di-, oligo- or polysaccharides
(especially with mono- or polysaccharides) or partially deamidated
and a fat-soluble active ingredient and/or fat-soluble
colorant.
BACKGROUND INFORMATION
[0008] 1. Approved Methods of the American Association of Cereal
Chemists, 8th Ed. AACC methods 1990, 44-16 and 46-12. The
Association: St. Paul, Minn. [0009] 2. Achouri, A., Joyce Irene
Boye, J. I., Yaylayan V. A., And Yeboah, F. K.: Functional
Properties of Glycated Soy 11S Glycinin. J Food Sci. 2005, 70 (4),
p. 269. [0010] 3. Baniel. A., Caer. D., Colas, B. and Gueguen, J.:
Functional Properties of Glycosylated Derivatives of the 11s
Storage Protein from Pea (Pisum sativum L.). J. Agric. Food Chem.
1992, 40, p. 200-205. [0011] 4. Bera M B, Mukherjee R K.:
Solubility, emulsifying, and foaming properties of rice bran
protein concentrates. J. Food Sci. 1989, 54(1), p. 142-145. [0012]
5. Cabra, V. Arreguin, R. Vazquez-Duhalt, R. Farres, A.: Effect of
Alkaline Deamidation on the Structure, Surface Hydrophobicity, and
Emulsifying Properties of the Z19 alpha-Zein J. Agric. Food Chem
2007, 55, p. 439-445. [0013] 6. Kato, A., Sasaki, Y., Furuta, R.,
and Kobayashi, K.: Functional protein polysaccharide conjugate
prepared by controlled dry-heating of ovalbumin-dextran mixtures.
Agric. Biol. Chem. 1990, 54, p. 107-112. [0014] 7. Kato, A.,
Shimokawa, K., and Kobayashi, K.: Improvement of the functional
properties of insoluble gluten by Pronase digestion followed by
dextran conjugation. J. Agric. Food Chem. 1991, 39, p. 1053-1056.
[0015] 8. Kato, Y., Aoki, T., Kato, N., Nakamura, R., and Matsuda,
T.: Modification of ovalbumin with glucose-6-phosphate by
amino-carbonyl reaction. Improvement of protein heat stability and
emulsifying activity. J. Agric. Food Chem. 1995, 43, p. 301-305.
[0016] 9. Kinsella, J. E.: Functional properties of proteins in
foods: a survey. Crit. Rev Food Sci. Nutr. 1976, 8(4), p. 19-80.
[0017] 10. Nakamura S, Kato A, Kobayashi K.: Bifunctional
lysozyme-galactomannan conjugate having excellent emulsifying
properties and antibacterial effects. J Agric Food Chem 1992, 40,
p. 735-9. [0018] 11. Nakamura, S., Saito, M., Goto, T., Saeki, H.,
Ogawa, M., Gotoh, G., Gohya, Y., and Hwang, J.-K.: Rapid formation
of biologically active neoglycoprotein from lysozyme and xyloglucan
hydrosylates through naturally occurring Maillard reaction. J. Food
Sci. Nutr. 2000, 5, p. 65-69. [0019] 12. Nielsen, P. M., Petersen,
D., and Dambmann, C.: Improved method for determining food protein
degree of hydrolysis. J. Food Sci. 2001, 66 (5), 642-646. [0020]
13. Nielson, P. M.: Functionality of protein hydrolysates. In:
Damodaran S, Paraf A, editors. Food proteins and their
applications. 1st ed. New York: Marcel Dekker Inc. 1997, p. 443-72.
[0021] 14. Oliver, C. M., Melton, L. D., & Stanley, R. A.:
Creating proteins with novel functionality via the Maillard
reaction: A review. Critical Reviews in Food Science and Nutrition
2006, 46, p. 337-350. [0022] 15. Paraman, I., Hettiarachchy, N. S.,
Schaefer, C., and Beck. M. I.: Hydrophobicity, Solubility, and
Emulsifying Properties of Enzyme modified Rice Endosperm Protein.
Submitted to Cereal chem. 2007, Manuscript ID. CC 10-06-0125 [0023]
16. Pearce K N, Kinsella J E.: Emulsifying properties of proteins:
evaluation of a turbidimetric technique. J Agric Food Chem 1978,
26, p. 716-722. [0024] 17. SAS (Statistical Analysis System). 2002.
JMP.RTM. User's Guide, Version 5. SAS Institute Inc. Cary, N.C.
[0025] 18. Schwenke K. D.: Enzyme and Chemical Modification of
Proteins. Chapter 13 in "Food Proteins and their Applications"
edited by A. Damodaran and A Paraf, 1997, Marcel Dekker, Inc., New
York, USA. [0026] 19. Wen, T. N., and Luthe, D. S.: Biochemical
characterization of rice glutelin. Plant Physiol. 1985, 78, p.
172-177.
DETAILED DESCRIPTION OF THE INVENTION
Compositions of the Present Invention
[0027] The compositions of the present invention may be solid
compositions, i.e. stable, water-soluble or water-dispersible
powders, or they may be liquid compositions, i.e. aqueous colloidal
solutions or oil-in-water dispersions of the aforementioned
powders. The stabilised oil-in-water dispersions, which may be
oil-in-water emulsions or may feature a mixture of suspended, i.e.
solid, particles and emulsified, i.e. liquid, droplets, may be
prepared by the methods described below or by an analogous
manner.
[0028] More specifically, the present invention is concerned with
stable compositions in powder form comprising one or more
fat-soluble active ingredient(s) and/or one or more fat-soluble
colorant(s) in a matrix of a rice endosperm protein which is
partially conjugated with mono-, di-, oligo- or polysaccharides
(especially with mono- or polysaccharides) or partially
deamidated.
[0029] Preferably the amount of that rice endosperm protein which
is partially conjugated with mono-, di-, oligo- or polysaccharides
(especially with mono- or polysaccharides) or partially deamidated
is from 1 to 70 weight-%, more preferably from 5 to 50 weight-%,
even more preferably from 10 to 40 weight-%, most preferably from
10 to 20 weight-% (with 20 weight-% being the most preferred one)
and/or the amount of the fat-soluble active ingredient and/or
fat-soluble colorant is from 0.1 to 90 weight-%, preferably from 1
to 80 weight-%, more preferably from 1 to 20 weight-%, based on the
total amount of the composition. If additional adjuvants and/or
excipients such as tocopherol and/or ascorbyl palmitate are
present, they are present in an amount of from 0.01 to 50 weight-%,
preferably in an amount of from 0.1 to 30 weight-%, more preferably
in an amount of from 0.5 to 10 weight-%, based on the total amount
of the composition.
Rice Endosperm Protein which is Partially Conjugated with Mono-,
Di-, Oligo- or Polysaccharides (Especially with Mono- or
Polysaccharides) or Partially Deamidated
[0030] In preferred embodiments of the present invention the rice
endosperm protein is a modified rice endosperm protein whose
manufacture is described below.
[0031] An especially preferred rice protein is one obtained by the
following steps: alkaline extraction, (enzymatically modification,
especially with Alkalase), partial cross-linking with at least one
compound selected from the group consisting of mono-, di-, oligo-
and polysaccharides (especially from the group consisting of mono-
and polysaccharides), centrifugation and ultra-filtration. A
further especially preferred rice protein is one obtained by the
following steps: alkaline extraction, (enzymatically modification,
especially with Alkalase), partial deamidation, centrifugation and
ultra-filtration.
[0032] If needed for the further use the thus obtained modified
rice endosperm protein may also be dried.
[0033] In preferred embodiments of the invention the used rice
endosperm proteins which are partially conjugated with mono-, di-,
oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated have an emulsion activity
of .gtoreq.0.2, preferably of .gtoreq.0.45, more preferably of
.gtoreq.0.5, even more preferably of from 0.5 to 1.0. The
determination of the emulsion activity is described in example 1.
The present invention refers also to these rice endosperm proteins
which are partially conjugated with mono-, di-, oligo- or
polysaccharides (especially with mono- or polysaccharides) or
partially deamidated themselves.
[0034] The amount of the rice endosperm protein which is partially
conjugated with mono-, di-, oligo- or polysaccharides (especially
with mono- or polysaccharides) or partially deamidated may be in
the range of from 1 to 70 weight-%, preferably in the range of from
5 to 50 weight-%, more preferably in the range of from 10 to 40
weight-%, most preferably in the range of from 10 to 20 weight-%,
based on the total weight of the composition as disclosed
below.
Fat-Soluble Active Ingredient and/or Fat-Soluble Colorant
[0035] The fat-soluble active ingredients are preferably those
ingredients with a pharmacological effect or those providing health
benefits to the human or animal body in general. "Fat-soluble"
(fat-soluble active ingredient/fat-soluble colorant) in the context
of the present invention means that the compound is hardly soluble
in water at room temperature and at atmospheric pressure.
[0036] The fat-soluble active ingredient and/or the fat-soluble
colorant is preferably selected from the group consisting of
carotenes and structurally related polyene compounds, fat-soluble
vitamins, coenzyme Q10, polyunsaturated fatty acids such as
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and
esters thereof (such as the ethyl esters or the triglycerides
(containing the same or different fatty acids)), mono-, di-,
triglycerides rich in polyunsaturated fatty acids, fat-soluble UV-A
filters, UV-B filters, as well as their physiologically acceptable
derivatives such as their esters, especially with C.sub.1-20
carbonic acids, and any mixtures of them.
[0037] The most preferred fat-soluble vitamins are Vitamin A or
E.
[0038] Preferred examples of the carotenes and structurally related
polyene compounds are carotenoids such as .alpha.-carotene,
.beta.-carotene, 8'-apo-.beta.-carotenal, 8'-apo-.beta.-carotenoic
acid esters such as the ethyl ester, canthaxanthin, astaxanthin,
lycopene, lutein, zeaxanthin, crocetin, .alpha.-zeacarotene,
.beta.-zeacarotene, as well as their physiologically acceptable
derivatives such as their esters, especially with C.sub.1-20
carbonic acids, and any mixtures of them.
[0039] The most preferred carotenoid is .beta.-carotene.
[0040] The term ".beta.-carotene" encompasses the all-cis as well
as the all-trans isomers and all possible mixed cis-trans-isomers.
The same applies for the other carotenoids.
[0041] The term "zeaxanthin" encompasses the natural
R,R-zeaxanthin, as well as S,S-zeaxanthin, meso-zeaxanthin and any
mixture of them. The same applies for lutein.
[0042] The fat-soluble active ingredients may be of natural origin,
i.e. isolated/extracted from plants, purified and/or concentrated,
as well as those synthesized by chemical and/or microbiological
(fermentative) routes.
[0043] The amount of the fat-soluble active ingredient and/or the
fat-soluble colorant may be in the range of from 0.1 to 90
weight-%, preferably in the range of from 1 to 80 weight-%, more
preferably in the range of from 1 to 20 weight-%, based on the
total weight of the composition as disclosed below.
Further Components
[0044] Beside the active ingredient and the rice endosperm protein
which is partially conjugated with mono-, di-, oligo- or
polysaccharides (especially with mono- or polysaccharides) or
partially deamidated the compositions of the present invention may
preferably additionally contain at least one water-soluble
antioxidant and/or fat-soluble antioxidant. The amount of the
water-soluble antioxidant and/or fat-soluble antioxidant may be in
the range of from 0.1 to 10.0 weight-%, preferably in the range of
from 0.5 to 5.0 weight-%, more preferably in the range of from 0.5
to 3.0 weight-%, based on the total weight of the composition.
[0045] The water-soluble antioxidant may be for example ascorbic
acid or a salt thereof, preferably sodium ascorbate, watersoluble
polyphenols such as hydroxytyrosol and oleuropein aglycon;
epigallocatechingallate (EGCG) or extracts of rosemary or
olives.
[0046] The fat-soluble antioxidant may be for example a tocopherol,
e.g. dl-.alpha.-tocopherol (i.e. synthetic tocopherol),
d-.alpha.-tocopherol (i.e. natural tocopherol), .beta.- or
.gamma.-tocopherol, or a mixture of two or more of these; butylated
hydroxytoluene (BHT); butylated hydroxyanisole (BHA); ethoxyquin,
propyl gallate; tert. butyl hydroxyquinoline; or
6-ethoxy-1,2-di-hydroxy-2,2,4-trimethylquinoline (EMQ), or an
ascorbic acid ester of a fatty acid, preferably ascorbyl palmitate
or stearate.
[0047] The compositions of the present invention may further
contain a co-emulgator selected from the group consisting of mono-
and diglycerides of fatty acids, polyglycerol esters of fatty
acids, lecithins; N-acylated amino acids and derivatives thereof,
N-acylated peptides with an alkyl or alkenyl radical, and salts
thereof; alkyl or alkenyl ether or ester sulfates, and derivatives
and salts thereof; polyoxyethylenated alkyl or alkenyl fatty ethers
or esters; polyoxyethylenated alkyl or alkenyl carboxylic acids and
salts thereof; N-alkyl or N-alkenyl betaines;
alkyltrimethylammonium or alkenyltrimethylammonium and salts
thereof; polyol alkyl or alkenyl ether or ester; and mixtures
thereof.
[0048] Preferred examples of polyol alkyl or alkenyl ethers or
esters are sorbitan alkyl or alkenyl esters polyoxyethylenated with
at least 20 units of ethylene oxide, such as sorbitan palmitate 20
EO or Polysorbate 40 marketed under the tradename Montanox 40 DF by
the company Seppic, sorbitan laurate 20 EO or Polysorbate 20
marketed under the tradename Tween 20 by the company ICI, and
sorbitan monostearate.
[0049] The amount of the co-emulgator may be in the range of from 0
to 90 weight-%, preferably in the range of from 0 to 50 weight-%,
more preferably in the range of from 0 to 20 weight-%, based on the
total weight of the composition.
[0050] The formulations according to the present invention may
further be pressed into tablets, whereby one or more excipients
and/or adjuvants selected from the group consisting of
monosaccharides, disaccharides, oligosaccharides and
polysaccharides, glycerol, and triglycerides, may be added.
[0051] Preferred examples of mono- and disaccharides which may be
present in the compositions of the present invention are sucrose,
invert sugar, xylose, glucose, fructose, lactose, maltose,
saccharose and sugar alcohols.
[0052] Preferred examples of the oligo- and polysaccharides are
starch, modified starch and starch hydrolysates. Preferred examples
of starch hydrolysates are dextrins and maltodextrins, especially
those having the range of 5 to 65 dextrose equivalents (DE), and
glucose syrup, especially such having the range of 20 to 95 DE. The
term "dextrose equivalent" (DE) denotes the degree of hydrolysis
and is a measure of the amount of reducing sugar calculated as
D-glucose based on dry weight; the scale is based on native starch
having a DE close to 0 and glucose having a DE of 100.
[0053] The triglyceride is suitably a vegetable oil or fat,
preferably corn oil, sunflower oil, soybean oil, safflower oil,
rapeseed oil, peanut oil, palm oil, palm kernel oil, cotton seed
oil, olive oil or coconut oil.
[0054] The amount of the excipient(s) and/or adjuvant(s) may be in
the range of from 0.1 to 50 weight-%, preferably in the range of
from 0.1 to 30 weight-%, more preferably in the range of from 0.5
to 10 weight-%, based on the total weight of the composition.
[0055] Solid compositions may in addition contain an anti-caking
agent, such as silicic acid or tricalcium phosphate and the like,
and up to 10 weight-%, as a rule 2 to 5 weight-%, of water.
[0056] The amount of the anti-caking agent may be in the range of
from 0 to 5 weight-%, preferably in the range of from 0 to 3
weight-%, more preferably in the range of from 0.2 to 3.0 weight-%,
based on the total weight of the composition.
Manufacture of the Composition
[0057] An object of the present invention is also a process for the
manufacture of the composition of the present invention which
comprises the following steps:
I) preparing an aqueous solution or colloidal solution of a rice
endosperm protein which is partially conjugated with mono-, di-,
oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated, (manufacture of such a
rice endosperm protein is described below), II) optionally adding
at least a water-soluble excipient and/or adjuvant to the solution
prepared in step I), III) preparing a solution or dispersion of at
least a fat-soluble active ingredient and/or fat-soluble colorant,
and optionally at least a fat-soluble adjuvant and/or excipient,
IV) mixing the solutions prepared in step I) to III) with each
other, V) homogenising the thus resulting mixture, VI) optionally
adding a cross-linking agent for partially cross-linking said rice
endosperm protein which is partially conjugated with mono-, di-,
oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated, VIa) optionally
submitting the mixture resulting after having performed step VI) to
enzymatic treatment or heat treatment to partially cross-link the
rice endosperm protein which is partially conjugated with mono-,
di-, oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated; VII) optionally
converting the dispersion obtained in step V) and/or VI) into a
powder, VIII) optionally drying the powder obtained in step VII),
IX) optionally submitting the dry powder to heat treatment or to
enzymatic treatment to cross-link the (modified) rice endosperm
protein, with the proviso that only step VIa) or step IX) is
carried out, but not both, when step VI) is carried out.
Step I
[0058] This step is simply performed by adding water to the rice
endosperm protein which is partially conjugated with mono-, di-,
oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated (manufacture see below) or
vice versa, optionally under stirring. Alternatively homogenization
may be possible via ultrasonication.
[0059] Preferably the rice endosperm protein which is partially
conjugated with mono-, di-, oligo- or polysaccharides (especially
with mono- or polysaccharides) or partially deamidated is used with
the preferences as described above and below.
Step II
[0060] Water-soluble excipients and/or adjuvants that may be added
are e.g. monosaccharides, disaccharides, oligosaccharides and
polysaccharides, glycerol and water-soluble antioxidants. Examples
of them are given above.
Step III
[0061] Fat-soluble active ingredients and fat-soluble colorants are
those as described above.
[0062] The (fat-soluble) active ingredient and/or fat-soluble
colorant and optional fat-soluble excipients and adjuvents are
either used as such or dissolved or suspended in a triglyceride
and/or an (organic) solvent.
[0063] Suitable organic solvents are halogenated aliphatic
hydrocarbons, aliphatic ethers, aliphatic and cyclic carbonates,
aliphatic esters and cyclic esters (lactones), aliphatic and cyclic
ketones, aliphatic alcohols and mixtures thereof.
[0064] Examples of halogenated aliphatic hydrocarbons are mono- or
polyhalogenated linear, branched or cyclic C1- to C15-alkanes.
Especially preferred examples are mono- or polychlorinated or
-brominated linear, branched or cyclic C1- to C15-alkanes. More
preferred are mono- or polychlorinated linear, branched or cyclic
C1- to C15-alkanes. Most preferred are methylene chloride and
chloroform.
[0065] Examples of aliphatic esters and cyclic esters (lactones)
are ethyl acetate, isopropyl acetate and n-butyl acetate; and
.gamma.-butyrolactone.
[0066] Examples of aliphatic and cyclic ketones are acetone,
diethyl ketone and isobutyl methyl ketone; and cyclopentanone and
isophorone.
[0067] Examples of cyclic carbonates are especially ethylene
carbonate and propylene carbonate and mixtures thereof.
[0068] Examples of aliphatic ethers are dialkyl ethers, where the
alkyl moiety has 1 to 4 carbon atoms. One preferred example is
dimethyl ether.
[0069] Examples of aliphatic alcohols are ethanol, iso-propanol,
propanol and butanol.
[0070] Furthermore any oil (triglycerides), orange oil, limonen or
the like and water can be used as a solvent.
[0071] Fat-soluble excipients and/or adjuvants that may be added
are e.g. corn oil, mono- or di-glycerides of fatty acids,
polyglycerol fatty acids, and middle chain triglycerides
("MCT").
Step IV
[0072] In an alternative process of the present invention step III)
is not carried out, but the fat-soluble active ingredient and/or
fat-soluble colorant and the optional fat-soluble excipient and/or
adjuvant is directly added to the solution of step I) or II).
Step V
[0073] For the homogenisation conventional technologies, such as
high-pressure homogenisation, high shear emulsification
(rotor-stator systems), micronisation, wet milling, microchanel
emulsification, membrane emulsification or ultrasonification can be
applied. Other techniques used for the preparation of compositions
containing fat-soluble active ingredient(s) and/or fat-soluble
colorant(s) for enrichment fortification and/or coloration of food,
beverages, animal feed, cosmetics or pharmaceutical compositions
are disclosed in EP-A 0 937 412 (especially paragraphs [0008],
[0014], [0015], [0022] to [0028]), EP-A 1 008 380 (especially
paragraphs [0005], [0007], [0008], [0012], [0022], [0023] to
[0039]) and in U.S. Pat. No. 6,093,348 (especially column 2, line
24 to column 3, line 32; column 3, line 48 to 65; column 4, line 53
to column 6, line 60), the contents of which are incorporated
herein by reference.
Step VI
[0074] The cross-linking agent is preferably selected from the
group consisting of reducing sugars, glycoproteins, and
glycopeptides. Thus an intermolecular cross-linking between the
(modified) rice endosperm protein and the sugar or sugar part of
the glycoprotein/glycopeptide is formed. Preferred examples of the
cross-linking agent are the monosaccharides (fructose glucose,
galactose, xylose), disaccharides (saccharose, lactose),
oligosaccharides (dextrin) and polysaccharides (Xanthan gum,
pectin), most preferred are fructose, glucose and Xanthan gum.
[0075] Glycoprotein is a compound containing carbohydrate (or
glycan) covalently linked to protein. The carbohydrate may be in
the form of a monosaccharide, disaccharide, oligosaccharide,
polysaccharide, or their derivatives (e.g. sulfo- or
phospho-substituted).
[0076] Preferred examples of glycoproteins are egg albumin, milk
casein.
[0077] A glycopeptide is a compound consisting of carbohydrate
linked to an oligopeptide composed of L- and/or D-amino acids. A
glyco-amino-acid is a saccharide attached to a single amino acid by
any kind of covalent bond.
[0078] A preferred example of glycopeptides is milk lactoferrin, an
iron-binding glycopeptide.
[0079] Thus, in contrast to co-pending PCT/EP2006/011873 here the
rice endosperm protein which is partially conjugated with mono-,
di-, oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated may further be partially
cross-linked with at least one compound selected from the group
consisting of reducing sugars, glycoproteins or glycopeptides.
Step VIa
[0080] The cross-linking can be achieved by submitting mixtures
additionally containing a cross-linking agent as described above to
heat-treatment to cause cross-linking of the sugar with the protein
in a Maillard type reaction, i.e. by thermally treatment,
preferably at temperatures from about 30 to about 160.degree. C.,
more preferably at temperatures from about 70 to about 100.degree.
C., most preferably at temperatures from about 80 to about
90.degree. C.
[0081] Cross linking is an enzymatic or a non-enzymatic reaction
resulting from the initial condensation between an available amino
group of protein and a carbonyl-group of mono-, di-, oligo- or
polysaccharides (especially of mono- or polysaccharides). Cross
linking is a specific type of modification which is being used to
alter protein physicochemical and functional performance such as
improving emulsification, encapsulation.
[0082] Further partial cross-linking of the rice endosperm protein
which is partially conjugated with mono-, di-, oligo- or
polysaccharides (especially with mono- or polysaccharides) or
partially deamidated with the cross-linking agent can also be
achieved by treatment with cross-linking enzymes (acyltransferases,
EC 2.3, e.g. transglutaminase, EC 2.3.2.13,
protein-glutamine:.gamma.-glutamyltransferase), i.e. by
enzymatically treatment, conveniently carried out at temperatures
from about 0 to about 70.degree. C., preferably at temperatures
from about 20 to about 40.degree. C. Preferably the enzymatic
treatment according to step VIa) is a treatment with a
cross-linking enzyme, particularly with a transglutaminase.
[0083] Enzymatic cross-linking results in stable protein-containing
polysaccharide networks, in the case of a transglutaminase by the
formation of .epsilon.-(.gamma.-glutamyl)-lysine isopeptide bonds.
The use of glycoproteins or glycopeptides is preferred for the
enzymatic cross-linking.
[0084] Both techniques, heat-treatment to cause cross-linking of
the sugar with the protein in a Maillard type reaction and
enzymatic cross-linking can be used for the incorporation of
lipophilic moieties and can be carried out either in a dried form
of the composition (step IX), or in an aqueous solution or
suspension (step VIa). The enzymatic cross-linking is preferably
carried out in an aqueous solution or suspension.
Step VII
[0085] The so-obtained dispersion, which is an oil-in-water
dispersion, can be converted after removal of the organic solvent
(if present) into a solid composition, e.g. a dry powder, using any
conventional technology such as spray drying, spray drying in
combination with fluidised bed granulation (the latter technique
commonly known as fluidised spray drying or FSD), or by a
powder-catch technique whereby sprayed emulsion droplets are caught
in a bed of an absorbent, such as starch, calcium silicate and
silicon dioxide, and subsequently dried.
[0086] Spray-drying may be performed at an inlet-temperature of
from about 100 to about 250.degree. C., preferably of from about
150.degree. C. to about 200.degree. C., more preferably of from
about 160 to about 190.degree. C., and/or at an outlet-temperature
(product temperature) of from about 45 to about 160.degree. C.,
preferably of from about 55 to about 110.degree. C., more
preferably of from about 65 to about 95.degree. C.
Step VIII
[0087] The drying of the powder obtained in step VII is preferably
carried out at a temperature of .ltoreq.100.degree. C., preferably
at a temperature of from 20 to 100.degree. C., more preferably at a
temperature of from 60 to 70.degree. C. If the drying is performed
in vacuum the temperature is lower.
Step IX
[0088] The cross-linking via heat-treatment is carried out as
already described above for step VIa. The same applies for the
enzymatic treatment, which is, however, preferably carried out in
solution/suspension.
Manufacture of the Rice Endosperm Protein which is Partially
Conjugated with Mono-, Di-, Oligo- or Polysaccharides (Especially
with Mono- or Polysaccharides) or Partially Deamidated
[0089] The present invention is also directed to a process for the
manufacture of a rice endosperm protein which is partially
conjugated with mono-, di-, oligo- or polysaccharides (especially
with mono- or polysaccharides) or partially deamidated starting
from milled rice, whereby the rice bran was removed before milling,
comprising the following steps a) to e):
a) preparing an aqueous solution or suspension of milled rice,
whereby the rice bran was removed before milling, whereby the
solution or suspension preferably has a dry mass content of from
0.1 to 30 weight-%, preferably from 10 to 15 weight-%, based on the
total amount of the aqueous solution or suspension; b) removing the
non-protein part or the protein part of the milled rice, whereby
the rice bran was removed before milling, to obtain the rice
endosperm protein; c) modifying the protein part of the milled
rice, whereby the rice bran was removed before milling, by reacting
the protein part of the milled rice partially with mono-, di-,
oligo- or polysaccharides (especially with mono- or
polysaccharides) in a Maillard-type reaction or by partially
deamidating the protein part of the milled rice to obtain rice
endosperm protein which is partially conjugated with mono-, di-,
oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated; d) optionally isolating
the rice endosperm protein which is partially conjugated with
mono-, di-, oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated; e) optionally converting
the rice endosperm protein which is partially conjugated with
mono-, di-, oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated into a solid form.
Step a)
[0090] Milled rice, where the rice bran was removed before milling,
is also known under the expression "rice flour".
[0091] This step is simply performed by adding water to the rice
flour or vice versa, optionally by stirring vigorously (with a
mechanical stirrer) until the rice flour is completely dispersed,
or by homogenizing the rice flour suspension with a homogenizer,
e.g. for 5 minutes at room temperature.
Step b)
[0092] Step b) may be performed as described by Paraman, I.,
Hettiarachchy, N. S., Schaefer, C., and Beck. M. I. in Cereal Chem.
2006, 83(6), 663-667: "Physicochemical properties of rice endosperm
proteins extracted by chemical and enzymatic methods".
Removing of the Non-Protein Part
[0093] Step b) may preferably be achieved by treating the rice
flour with non-protein degrading enzymes, e.g. with a 0.5% aqueous
suspension of Termamyl.RTM. at a temperature of 90.degree. C. for 2
hours and then with a 0.1% aqueous suspension of a cellulase at a
temperature of 50.degree. C. for 30 minutes--without any pH
adjustment (pH 6-7), deactivating the enzymes, separating and
removing the non-protein part from the protein part of the rice
flour.
[0094] Preferred examples of non-protein degrading enzymes are
starch-degrading enzymes such as .alpha.-amylases and cellulases,
i.e. cellulose-degrading enzymes, and mixtures thereof. A preferred
example of an .alpha.-amylase is Termamyl.RTM. 120, Type L,
commercially available from Novo Nordisk Biochem, North America,
Inc., USA. Other preferred examples are Liquzyme.RTM. Supra,
commercially available from Novo Nordisk Biochem, North America,
Inc., USA, Amylase S "Amano" 35 G, commercially available from
Amano Pharmaceutical Co. Ltd., Nagoya, Japan, Multifect Cellulase,
commercially available from Genencor International, Inc., USA, and
Cellulase T "Amano" 4, commercially available from Amano
Pharmaceutical Co. Ltd., Nagoya, Japan.
[0095] The reaction of the enzymes can be stopped by neutralising
the solution or suspension if an inorganic acid (e.g. hydrochloric
acid) or an organic acid (e.g. citric acid) or base is used or by
heating to denature the enzymes.
[0096] The denaturation may be achieved by heating the solution to
a temperature of from 80 to 95.degree. C., preferably to a
temperature of from 80 to 85.degree. C. (especially at a low pH of
from 3.5 to 4.5) for 10 to 15 minutes. Afterwards the solution may
be cooled to 50.degree. C.
[0097] The separation of the non-protein part may be achieved by
centrifugation (5000 g for 15 minutes) (whereby the non-protein
part is in the water phase), followed by washing with deionized
water. The rice endosperm protein remains in pellets.
Removing of the Protein Part
[0098] Alternatively a so-called "alkaline extraction" or a
so-called "salt-extraction" may be performed before the
centrifugation or filtration.
[0099] "Alkaline extraction" means that first the pH of the
solution or suspension of the rice flour is adjusted to a value of
from 7 to 12, preferably to a value of from 8 to 10, more
preferably to a value of about 9, with an alkali solution (e.g. an
aqueous NaOH solution) at 40 to 60.degree. C. for 3 hours.
[0100] In cases where the protein yield is more important than the
protein functionality it may be advantageous to adjust the pH
preferably to a value of from 8 to 12, more preferably of from 9 to
12, even more preferably from 10 to 12.
[0101] Preferably such a base has a concentration of about 0.1 to 5
M, preferably of about 0.5 to about 2 M. The base may be an
inorganic base. Examples of inorganic bases are (earth) alkali
hydroxides such as sodium hydroxide (preferred), potassium
hydroxide and calcium hydroxide.
[0102] A "salt-extraction" is similar to an "alkali-extraction",
but in addition to the base a salt such as sodium chloride is used.
In a preferred embodiment of the invention an aqueous 0.08 M sodium
chloride solution (adjusted to pH 11 with NaOH) is used as the
extracting solvent.
[0103] In both cases (alkaline or salt extraction) the protein part
is transferred to the water phase. The protein part may be
separated then by centrifugation or filtration from the non-protein
part.
Further Modification of the Protein Part
[0104] A further modification of the rice flour may be achieved by
treating it(s protein part) with (commercially available) food
grade alkaline, neutral and/or acid proteases. For some proteases
the enzyme specifications and the optimum conditions are given in
the tables below.
TABLE-US-00001 TABLE 1a Enzyme specification I Type of Enzyme
protease Source Preferential specificity Protex 6L Serine Protease
Bacillus Hydrolysis of proteins with broad specificity
licheniformis for peptide bonds Bromelain Cysteine Pineapple Broad
specificity, but strong preference Protease stem for Arg-Arg in
peptides Alkalase Serine Protease Bacillus Broad specificity, and a
preference for a licheniformis large uncharged residue's carboxyl
sites Liquipanol Cysteine Concentrated Broad specificity Protease
papain Alkaline Serine Protease Bacterial protease Hydrolysis of
proteins with broad specificity protease for peptide bonds Pepsin
Aspartic Porcine stomach The C-terminal side of tyrosine, Protease
phenylalanine, and tryptophan residues
TABLE-US-00002 TABLE 1b Enzyme specification II Enzymes pH-range
Activity/g Company Protex 6L 6-10 580 000 Genencor International,
Inc., Rochester, NY 14618, USA Bromelain 5-8 150 000 Enzyme
Development Corporation, New York, NY 10001, USA. Alkalase 6-9 2.4
AU Novo Nordisk Biochem, Franklinton, NC 27525, USA Liquipanol 5-8
125 000 Enzyme Development Corporation, New York, NY 10001, USA.
Alkaline 6-9 175 000 Enzyme Development Corporation, protease New
York, NY 10001, USA.
TABLE-US-00003 TABLE 2 The optimum enzyme conditions used in
protein hydrolysis Amount of Enzyme [weight-%, based on Temperature
Time Enzyme protein weight] pH [.degree. C.] [minutes] Liquipanol
1.0 8.0 50 60 Bromelain 1.0 7.0 50 60 Alkalase 1.0 9.0 60 60 Protex
6L 1.0 10.0 60 60 Pepsin 0.5 3.0 37 30
[0105] The proteases may be from bacteria or fungi, as well as from
fruit or may have animal origin.
[0106] Examples of alkaline proteases are the commercially
available Alkalase.RTM. (Novo Nordisk Biochem, Franklinton, N.C.,
USA), Alkaline Protease.RTM. (Enzyme Development Corporation, New
York, N.Y., USA), Protex 6L.RTM. (Genencor.RTM. Bacterial Alkaline
Protease, Genencor International, Inc., Rochester, N.Y., USA) and
Genencor.RTM. Protease 899 (Genencor International, Inc.,
Rochester, N.Y., USA).
[0107] Examples of neutral proteases are the commercially available
Bromelain.RTM. (Enzyme Development Corporation, New York, N.Y.,
USA), Liquipanol.RTM. (Enzyme Development Corporation, New York,
N.Y., USA) and bacterial neutral-protease (Genencor International,
Inc., Rochester, N.Y., USA). A further example of a neutral
protease is the commercially available Collupilin.RTM. of DSM Food
Beverages, Delft, Netherlands, produced from Carica papaya, a
plant, i.e. an enzyme of fruit origin.
[0108] Examples of acid proteases are pepsin (Sigma, USA) and Acid
protease (Amano Pharmaceutical Co. Ltd., Nagoya, Japan).
[0109] In a preferred embodiment of the process of the present
invention the protein part of the rice flour is treated
subsequently by two different alkaline proteases at a pH range of
from 7 to 10 for 10 to 80 minutes at 40 to 60.degree. C.
[0110] Preferably one of these proteases is a serine specific
protease such as Alkalase.RTM., Protex 6L.RTM. or Alkaline
Protease.RTM. and the other is a cysteine specific protease such as
Liquipanol.RTM. or Bromelaina
[0111] This modification step may also be modified by not adding
the enzyme(s) at once but by adding them (subsequently or
simultaneously) portion wise.
Step c)
[0112] The protein part obtained in step b) is used as starting
material for performing step c), i.e. either a partial reaction
with mono- (di- or oligo-) or polysaccharides or a partial
deamidation.
Partial Reaction of the Protein Part of the Milled Rice with Mono-,
Di-, Oligo- or Polysaccharides (Especially with Mono- or
Polysaccharides)
[0113] The reaction of the protein part with the mono-, di-, oligo-
or polysaccharides proceeds in a Maillard-type reaction. Aqueous
dispersions of the protein part and aqueous solutions/dispersions
of the mono-, di-, oligo- or polysaccharide (especially of the
mono- or polysaccharide), respectively, may be prepared separately
and then added together under stirring.
[0114] Usually the aqueous solution of the mono- or polysaccharide
(or di- or oligosaccharide), most preferably having a concentration
of 10 weight-%, was slowly added to the aqueous solution of the
protein part, most preferably having a concentration of 10
weight-%, under stirring to obtain a mixture of both
solutions/dispersions. The mixing is preferably carried out at a
temperature in the range of from 20 to 60.degree. C., preferably in
the range of from 30 to 50.degree. C., more preferably in the range
of from 35 to 45.degree. C., and for a time in the range of from 10
minutes to 2 hours, preferably in the range of from 30 minutes to
90 minutes, more preferably in the range of from 45 minutes to 60
minutes.
[0115] Then the pH value of the mixture is adjusted to a value in
the range of from 6.0 to 9.0, preferably to a value in the range of
from 6.5 to 8.5, preferably to a value in the range of from 7.0 to
8.0 by adding a base, preferably in form of an aqueous solution.
Preferably such a base has a concentration of about 0.1 to 3 M,
preferably of about 0.5 to about 2 M.
[0116] The base may be an inorganic base. Examples of inorganic
bases are (earth) alkali hydroxyides such as sodium hydroxide (most
preferred), potassium hydroxide (preferred) and calcium
hydroxide.
[0117] The thus obtained alkaline mixture is then dried, preferably
by spray-drying. The spray-drying is preferably carried out at an
inlet-temperature in the range of from 50.degree. C. to 250.degree.
C., more preferably in the range of from 60.degree. C. to
100.degree. C., most preferably in the range of from 60 to
90.degree. C.
[0118] The reaction (cross-linking) may then be performed by
incubating the dried protein and mono- or polysaccharide (or di- or
oligosaccharide) mixture to a temperature in the range of from 30
to 70.degree. C., preferably in the range of from 40 to 60.degree.
C., more preferably in the range of from 45 to 55.degree. C. in a
humidity chamber. Depending on the reaction temperature the
duration of the reaction is in a time range from 2 hours to 60
hours, preferably from 4 hours to 40 hours, more preferably from 6
hours to 30 hours. The relative humidity may vary in a range of
from 39 to 85%, more preferably in the range of from 44 to 79%,
most preferably in the range of from 54 to 69%, to maintain the
water activity at a range of from 0.5 to 0.8.
[0119] The cross-linking may also be carried out by enzymatic
treatment as already described above for step VIa.
[0120] Preferred examples of monosaccharides are pentoses and
hexoses (fructose, glucose, galactose, xylose, especially fructose
and glucose).
[0121] Preferred examples of polysaccharides are Xanthan gum and
pectin.
[0122] Instead of mono- or polysaccharides also disaccharides
(saccharose, lactose) and oligosaccharides (dextrin) may be
used.
[0123] The weight ratio of the protein part to the monosaccharide
lies in the range of from 0.5 to 12% (w/w), preferably in the range
of from 0.1 to 8% (w,w), more preferably in the range of from 0.5
to 4% (w/w).
[0124] The weight ratio of the protein part to the polysaccharide
(or disaccharide or oligosaccharide) lies in the range of from 0.1
to 20% (w,w), preferably in the range of from 0.5 to 20% (w/w).
[0125] If glucose is used as monosaccharide the weight ratio of
glucose to the protein part obtained in step b) is preferably in
the range of from 0.1 to 8% (w/w), more preferably in the range of
from 0.5 to 4% (w/w).
[0126] If Xanthan gum is used as polysaccharide the weight ratio of
Xanthan gum to the protein part obtained in step b) is preferably
in the range of from 0.1 to 20% (w/w), more preferably in the range
of from 0.5 to 10% (w/w).
Partial Deamidation of the Protein Part of the Milled Rice
[0127] The deamidation is performed by adjusting the pH value of an
aqueous colloidal solution of the protein part of the rice
endosperm protein obtained in step b) to a value in the range of
from 9.0 to 13.0, preferably to a value in the range of from 9.5 to
12.5, preferably to a value in the range of from 10.5 to 12 by
adding a base, preferably in form of an aqueous solution.
Preferably such a base has a concentration of about 0.1 to 3 M,
preferably of about 0.5 to about 2 M. The base may be an inorganic
base. Examples of inorganic bases are (earth) alkali hydroxides
such as sodium hydroxide (most preferred), potassium hydroxide
(preferred) and calcium hydroxide.
[0128] The thus resulting alkaline mixture is then brought to a
temperature in the range of from 25 to 90.degree. C., preferably in
the range of from 30 to 80.degree. C., more preferably in the range
of from 40 to 70.degree. C.
[0129] Depending on the deamidation temperature the duration of the
deamidation is in a time range from 0.5 hours to 24 hours,
preferably from 0.5 to 12 hours, more preferably from 0.5 to 6
hours.
Step d)
[0130] Step d) may be performed by any method known to the person
skilled in the art for isolating proteins.
[0131] Step d) may e.g. be carried out by centrifugation and/or
filtration.
[0132] Usually it is not necessary to isolate the protein partially
conjugated with mono-, di-, oligo- or polysaccharides or partially
deamidated.
Step e)
[0133] The conversion into a solid form, e.g. a dry powder, can be
achieved by any drying method known to the person skilled in the
art. Preferred are spray drying or freeze-drying. Spray drying is
preferably performed at an inlet temperature of 200.degree. C. to
240.degree. C. and at an outlet temperature of 80 to 100.degree. C.
The freeze-drying is preferably performed at a temperature of from
about -20.degree. C. to about -50.degree. C. for 10 to 48
hours.
[0134] An object of the present invention is also the rice
endosperm protein which is partially conjugated with mono-, di-,
oligo- or polysaccharides (especially with mono- or
polysaccharides) or partially deamidated itself, especially the one
as obtainable by any process as described above. Even more
preferred is the rice endosperm protein which is partially
conjugated with mono-, di-, oligo- or polysaccharides (especially
with mono- or polysaccharides) or partially deamidated itself as
obtained by any process as described above.
INDUSTRIAL APPLICABILITY
[0135] The present invention is directed to the use of a
composition as described above for the enrichment, fortification
and/or coloration of food, beverages, animal feed, personal care or
pharmaceutical compositions, as well as to the food, beverages,
animal feed, personal care and pharmaceutical compositions
containing such a composition as described above themselves.
[0136] The present invention is also directed to food, beverages,
animal feed, personal care and pharmaceutical compositions
containing a rice endosperm protein which is partially conjugated
with mono-, di-, oligo- or polysaccharides (especially with mono-
or polysaccharides) or partially deamidated as described above, as
well as to the use of such a rice endosperm protein (with the
preferences as described above) as protective hydrocolloid for
fat-soluble active ingredients and/or fat-soluble colorants.
[0137] Animals including humans in the context of the present
invention encompass besides humans especially farm animals such as
sheep, cow, horses, poultry (broiler and egg pigmentation), shrimps
and fish (especially salmon and rainbow trout) as well as pets such
as cat, dogs, birds (e.g. flamingos) and fish.
[0138] Beverages wherein the compositions of the present invention
can be used, especially as a colorant or a functional ingredient,
can be carbonated beverages e.g., flavoured seltzer waters, soft
drinks or mineral drinks, as well as non-carbonated beverages e.g.
flavoured waters, fruit juices, fruit punches and concentrated
forms of these beverages. They may be based on natural fruit or
vegetable juices or on artificial flavours. Also included are
alcoholic beverages and instant beverage powders. Besides, sugar
containing beverages, diet beverages with non-caloric and
artificial sweeteners are also included.
[0139] Further, dairy products, obtained from natural sources or
synthetic, are within the scope of the food products wherein the
compositions of the present invention can be used, especially as a
colorant or as a functional ingredient. Typical examples of such
products are milk drinks, ice cream, cheese, yoghurt and the like.
Milk replacing products such as soymilk drinks and tofu products
are also comprised within this range of application.
[0140] Also included are sweets which contain the compositions of
the present invention as a colorant or as a functional ingredient,
such as confectionery products, candies, gums, desserts, e.g. ice
cream, jellies, puddings, instant pudding powders and the like.
[0141] Also included are cereals, snacks, cookies, pasta, soups and
sauces, mayonnaise, salad dressings and the like which contain the
compositions of the present invention as a colorant or a functional
ingredient. Furthermore, fruit preparations used for dairy and
cereals are also included.
[0142] The final concentration of the fat-soluble active ingredient
and/or the fat-soluble colorant which is added via the compositions
of the present invention to the food products may be from 0.1 to
500 ppm, particularly from 1 to 50 ppm, based on the total weight
of the food composition and depending on the particular food
product to be coloured or fortified and the intended grade of
coloration or fortification.
[0143] The food compositions of this invention are preferably
obtained by adding to a food product the fat-soluble active
ingredient and/or the fat-soluble colorant in the form of a
composition of this invention. For coloration or fortification of a
food or a pharmaceutical product a composition of this invention
can be used according to methods per se known for the application
of water dispersible solid compositions of the present
invention.
[0144] In general the composition may be added either as an aqueous
stock solution, a dry powder mix or a pre-blend with other suitable
food ingredients according to the specific application. Mixing can
be done e.g. using a dry powder blender, a low shear mixer, a
high-pressure homogeniser or a high shear mixer depending on the
formulation of the final application. As will be readily apparent
such technicalities are within the skill of the expert.
[0145] Pharmaceutical compositions such as tablets or capsules
wherein the compositions are used as a colorant are also within the
scope of the present invention. The coloration of tablets can be
accomplished by adding the compositions of the present invention in
form of a liquid or solid colorant composition separately to the
tablet coating mixture or by adding a colorant composition to one
of the components of the tablet coating mixture. Coloured hard or
soft-shell capsules can be prepared by incorporating a colorant
composition in the aqueous solution of the capsule mass.
[0146] Pharmaceutical compositions such as tablets such as chewable
tablets, effervescent tablets or filmcoated tablets or capsules
such as hard shell capsules wherein the compositions are used as an
active ingredient are also within the scope of the present
invention. The compositions of the present invention are typically
added as powders to the tableting mixture or filled into the
capsules in a manner per se known for the production of
capsules.
[0147] Animal feed products such as premixes of nutritional
ingredients, compound feeds, milk replacers, liquid diets or feed
preparations wherein the compositions are either used as a colorant
for pigmentation e.g. for egg yolks, table poultry, broilers or
aquatic animals (especially shrimps, salmon, rainbow trout) or as
an active ingredient are also within the scope of the present
invention.
[0148] Personal care compositions: Cosmetics, toiletries and derma
products i.e. skin and hair care products such as creams, lotions,
baths, lipsticks, shampoos, conditioners, sprays or gels wherein
the compositions are used as a colorant or as an active ingredient
are also within the scope of the present invention.
[0149] The present invention is further illustrated by the
following examples.
EXAMPLES
[0150] The following abbreviations are used:
DH=degree of hydrolysis DI water=deionized water dw=dry weight
basis RH=relative humidity rpm=rounds per minute SDS=sodium dodecyl
sulfate w/v=weight/volume
[0151] Rice flour made from long grain rice was provided by
Riceland Foods (Stuttgart, Ark.). Whey protein isolate was obtained
from Biozate.RTM., Davisco Foods International, INC., Minnesota as
benchmark standard protein containing 88.6% protein (N.times.6.25)
on dry weight basis. Alcalase 2.4 L, bacterial serine protease from
Bacillus lieheniformis, was provided by Novo Nordisk Biochem.,
(Franklinton, N.C., 2.4 AU units/g). Analytical reagents were
purchased from Fisher Scientific (Pittsburgh, Pa.) and Sigma
chemical Co. (St. Louis, Mo.).
(A) Analytical Methods: Examples 1-5
Example 1
Determination of Emulsion Activity and Emulsion Stability
[0152] The emulsion activity and stability was determined by the
turbidimetric method of Pearce and Kinsella, Journal of Agric Food
Chem. 1978, 26, 716-722. A mixture of 6 mL of a 0.1% solution of
the rice endosperm protein in 10 mM phosphate buffer of a pH of 7.0
and 2 mL of corn oil was homogenized for 1 minute with a sonicator
at setting 6 (Virtishear Tempest, The Virtis Co., Gardiner, N.Y.,
U.S.A.). 50 microliters of the mixture were transferred into 5 mL
of an 0.1% aqueous solution of SDS (w/v) 0 and 10 minutes after the
homogenization. The absorbance of the solution at 500 nm was
determined with a spectrometer (Shimadzu Model UV-1601, Kyoto,
Japan). The absorbance at the time 0 after homogenization is the
emulsion activity of the rice endosperm protein. The decrease in
turbidity (absorbance) of the initial absorbance during the time
interval (10 min) was used to calculate the emulsion stability.
(ES) was calculated as follows:
[0153] Emulsion Stability=To.times..DELTA.t/.DELTA.T--where,
.DELTA.T is the decrease in turbidity (absorbance) of the initial
absorbance (To) during the time interval of .DELTA.t (10 min).
Example 2
Determination of the Degree of Hydrolysis
[0154] The DH was determined by the method of Nielsen and others
(Nielsen, P. M., Petersen, D. & Dambmann, C.: Improved method
for determining food protein degree of hydrolysis. Journal of Food
Science 2001, 66 (5), 642-646). The o-phthaldialdehyde (OPA)
reagent was prepared as follow: 7.620 g of di-sodium tetraborate
decahydrate (Na.sub.2B.sub.4O.sub.7.10H.sub.2O) and 200 mg sodium
dodecyl sulfate (SDS) were dissolved in 150 mL of deionized water
and then mixed with 160 mg of OPA (97% OPA pre-dissolved in 4 mL of
ethanol) and 176 mg of 99% dithiothreitol (DTT). The final solution
was made up to 200 mL with deionized water. Freeze dried protein
sample of 0.1 g was solubilized in 10 mL deionized water. To
measure the absorbance, 3 mL of OPA reagents was added to 10 mL
tubes and then 400 .mu.L of sample solution, serine standard (10
mg/l 00 mL) and deionized water was added in four tubes for each
sample, standard and blank, respectively. This was followed by
mixing for 5 s and held for exactly 2 min. Absorbance was read at
340 nm with a spectrophotometer (Shimadzu Model UV-1601, Kyoto,
Japan). The DH was calculated as follows.
[0155] DH=h/h.sub.total*100%; where h is the number of hydrolyzed
bonds and h.sub.total is the total number of peptide bonds per
protein equivalent; h=(Serine-NH2-.beta.)/.alpha. equiv/g protein;
where h is the number of hydrolyzed bonds and h.sub.total is the
total number of peptide bonds per protein equivalent; for cereal
protein .alpha. is 1.00, .beta. is 0.40, and h.sub.total is
8.0.
[0156]
Serine-NH2=[(A.sub.340sample-A.sub.340blank)/(A.sub.340standard-A.s-
ub.340blank)]*0.9516 meqv/L*0.01*100/(X*P); where serine-NH2=meqv
serine NH2/g protein; X=g sample; P=% protein in sample; 0.01 is
the sample volume in liter (L).
Example 3
Determination of the Protein and Total Solubility
[0157] Protein solubility (also N-solubility) was determined by the
method of Bera and Mukherjee (Bera, M. B., Mukherjee, R. K.:
Solubility, emulsifying, and foaming properties of rice bran
protein concentrates. J Food Sci 1989, 54(1), 142-145) with some
modifications. 200 mg of protein sample was dispersed in 10 mL of
deionized water, the pH was adjusted to 7.0 by 1 N HCl or 1 N NaOH.
The dispersion was stirred continuously for 30 min and centrifuged
at 5000 rpm for 15 min. (model J2-21, Beckman, Fullerton, Calif.,
U.S.A.). The supernatant was recovered, and the protein content in
the supernatant was determined by the Automatic Kjeldahl method
(AACC 1990). The percentage of protein solubility was calculated by
following equation:
Protein Solubility ( % ) = Protein content of the supernatant
Protein in 200 mg protein - isolate .times. 100 ##EQU00001##
[0158] The protein solubility was calculated as the percent ratio
of protein in the supernatant to that of the total protein in the
initial sample.
[0159] The total solubility was determined by oven drying method,
and expressed as the percent ratio of total soluble portion of the
supernatant to that of the total weight of the protein isolate.
Total Solubility ( % ) = Soluble portion of the supernatant 200 mg
protein - isolate .times. 100 ##EQU00002##
Example 4
Determination of Viscosity
[0160] Viscosity of the protein isolates was determined by a
rotational rheometer (Haake VT 550, Germany) equipped with a MVDIN
measuring spindle (radius=19.36 mm, height=58.08 mm) at room
temperature (26.degree. C.). The protein isolates were mixed with
deionized water to form slurry of 10%, and the slurry was left for
60 min for equilibrium before analysis. The samples (30 ml) were
loaded into the cylindrical cup (radius=21.0 mm) and were subjected
to a shear rate that changed from 0 to 400 l/s over 3 min using a
computer-controlled program. The data were analyzed by Rheowin Pro
Data manager version 2.84 (Haake Mess Tech, Germany).
Example 5
Determination of the Protein Content
[0161] The protein contents were determined by an Automatic
Kjeldahl method (AACC, 1990). The Kjeldahl 2006 Digester (Foss
Tecator, Hoganas, Sweden) was used for digesting the samples in
concentrated sulfuric acid with Kjeldahl.RTM. tablet as catalyst at
420.degree. C. for 1 hour, and a Kjeltec.RTM. 2300 Analyzer Unit
(Foss Tecator, Hoganas, Sweden) was used to determine the nitrogen
content of the products. The protein contents were automatically
calculated by multiplying the nitrogen content by a factor of
5.95.
(B) Manufacture of Rice Endosperm Protein Isolates (RP) (Starting
Material and Comparison Example)
Example 6
[0162] One kilogram of rice flour was homogenized with 8 L
deionized water (1:8, w/v) in a homogenizer (Virtishear Tempest,
The Virtis Co., Gardiner, N.Y., USA) for 1 minute. The pH of the
slurry was adjusted to 11.0 by 1M NaOH, and the suspension was
stirred for 3 hours at 40.degree. C. The soluble protein in the
solution was separated by centrifugation (5,000 rpm, 15 minutes).
This procedure was repeated once to extract additional protein from
the residue. Proteins in the combined supernatants of first and
second extractions were isoelectrically precipitated at pH 4.5 and
kept at 4.degree. C. for 1 hour. The precipitate was recovered by
centrifugation at 5,000 rpm for 20 minutes, washed with deionized
water (1:4, w/v, pH 4.5), adjusted to pH 7.0. The extracted
proteins (RP) were used as a starting material for the preparation
of the following modifications (C) and (D).
Experimental Design of Examples (C), (D) and (E)
[0163] The present study includes two sets of experiments: (1)
optimization rice protein glycosylation and, (2) comparison of
controlled glycosylation, deamidation, and alcalase modification
methods (comparison examples) on rice protein physicochemical
properties. The optimization of glycosylation experiment was
conducted in a 2.times.7 two factor factorial design (glucose and
Xanthan gum) with repeated measurements (7 reaction-times) with 3
replicates. The comparison of glycosylation, deamidation, and
alcalase modification methods were conducted in one factor
completely randomized design, which included control (unmodified)
rice protein and bench mark whey protein to evaluate and compare
the effectiveness of the selected modification methods.
[0164] Thus, the rice endosperm protein was modified by the
following methods: (1) Glycosylation of rice endosperm protein with
D-glucose (RP.sub.Glu); (2) Glycosylation of rice endosperm protein
with Xanthan gum (RP.sub.XG); (3) Deamidation of rice endosperm
protein using alkali treatment (RP.sub.DA); (4) Treatment of rice
endosperm protein with alcalase to 1.8% DH (RP.sub.Alc). The
physicochemical and functional properties of the protein
derivatives were evaluated and compared with those of unmodified
rice endosperm protein (RP) and as bench mark compared with whey
protein isolates.
(C) Manufacture of a Partially Hydrolyzed Rice Endosperm
Protein
Example 7 (Comparison Example)
Treatment of Rice Endosperm Protein with Alcalase to 1.8%
DH(RP.sub.Alc)
[0165] The conditions for alcalase treatment were chosen based on a
previous study (Paraman et al 2007) and our preliminary data. The
rice endosperm protein isolate (RP) was homogenized with DI water
(8% w/v) and, adjusted to pH 6.5. The protein colloidal solution
was treated with 0.1% alcalase at 40.degree. C. for 8.5 minutes.
The enzyme was inactivated at 85.degree. C. for 7 minutes. The
hydrolysate was cooled immediately to 30.degree. C. by adding ice,
and the pH was readjusted to 7.0. The protein hydrolysate was spray
dried and stored at 5.degree. C. in air tight containers until they
were used (RP.sub.Alc).
(D) Manufacture of a Rice Endosperm Protein Partially Conjugated
with Mono- or Polysaccharides
Example 8
Evaluation of Optimal Reaction Conditions for the Partial
Glycosylation of Rice Endosperm Protein
[0166] The extracted rice protein RP (20 g on dw) was dissolved in
deionized water to give a 10% (w/v) protein colloidal solution.
D-glucose (0.465 g) dissolved in 10 mL DI water was added into the
protein solution while stirring the protein solution at 37.degree.
C. The protein-glucose mixture was adjusted to pH 8.0 and stirred
for 1 h at 37.degree. C. The mixture was freeze-dried and the dried
protein-sugar mixture was placed in aluminum plate, and incubated
at 50.degree. C. in an incubator maintained at 65% relative
humidity. Approximately, 2.5 g of the protein-glucose mixture were
drawn as samples at 4 hours intervals for 24 hours. The
glycosylated protein was stored at 5.degree. C. in air tight
containers until they were used.
[0167] Rice protein-Xanthan gum optimization was conducted
essentially similar to the method of protein-glucose optimization
described above except for the following changes; the
protein-Xanthan gum ration was 100:1, and the pH of the
protein-Xanthan gum solution was adjusted to a value of 7.0.
Example 9
Glycosylation of Rice Endosperm Protein with D-Glucose
(RP.sub.Glu)
[0168] Glycosylation was conducted based on above optimization and
the conditions chosen from literature (Kato at al 1990, Kato at al
1991, Achouri et al 2005, Oliver et al 2006) with some
modifications as described below. The alkali extracted rice protein
isolates RP (200 g on dw) were dissolved in water to give a 10%
(w/v) protein colloidal solution. D-glucose (4.65 g) dissolved in
100 mL DI water was added to the protein solution while stirring
the protein solution at 37.degree. C. The protein-glucose mixture
was adjusted to pH 8.0 and stirred for 1 hour at 37.degree. C. The
mixture was spray-dried and stored at 5.degree. C. in air tight
containers until they were used. The spray-dried protein-sugar
mixture was placed in aluminum plate and incubated for 8 hours at
50.degree. C. and 65% RH. The glycosylated protein (RPGlu) was
stored at 5.degree. C. until its use.
Example 10
Glycosylation of Rice Endosperm Protein with Xanthan Gum
(RP.sub.XG)
[0169] The rice endosperm protein isolate (RP) 200 g, on dw basis,
was dissolved in water to give a 10% (w/v) protein colloidal
solution. Xanthan gum (2 g) was dissolved separately in water to
give 1% (w/v) xanthan-gum solution. The xanthan gum solution was
added into the protein solution while stirring the protein solution
at 37.degree. C. The protein-xanthan gum mixture was spray-dried,
and stored at 5.degree. C. in air tight containers until they were
used. The spray-dried protein-Xanthan gum mixture was placed in
aluminum plate and incubated for 20 hours at 50.degree. C. and 65%
RH. The glycosylated protein (RP.sub.XG) was stored at 5.degree. C.
until analyzed.
Example 11
Glycosylation of Rice Endosperm Protein with Potato Dextrin
(RP.sub.PD)
[0170] The rice endosperm protein isolate (RP) 200 g, on dw basis,
was dissolved in water to give a 10% (w/v) protein colloidal
solution. Potato dextrin (2 g) was dissolved separately in water to
give 1% (w/v) potato dextrin solution. The potato dextrin solution
was added into the protein solution while stirring the protein
solution at 37.degree. C. The protein-dextrin mixture was adjusted
to pH 7.0, stirred for 1 hour at 37.degree. C., spray-dried, and
stored at 5.degree. C. in air tight containers until they were
used. The spray-dried protein-dextrin mixture was placed in
aluminum plate and incubated for 20 hours at 50.degree. C. and 65%
RH. The glycosylated protein (RP.sub.PD) was stored at 5.degree. C.
until analyzed.
Example 12
Glycosylation of Rice Endosperm Protein with Cyclodextrin
(RP.sub.CD)
[0171] The rice endosperm protein isolate (RP) 200 g, on dw basis,
was dissolved in water to give a 10% (w/v) protein colloidal
solution. Cyclodextrin (2 g) was dissolved separately in water to
give 1% (w/v) cyclodextrin solution. The cyclodextrin solution was
added into the protein solution while stirring the protein solution
at 37.degree. C. The protein-cyclodextrin mixture was adjusted to
pH 7.0, stirred for 1 hour at 37.degree. C., spray-dried, and
stored at 5.degree. C. in air tight containers until they were
used. The spray-dried protein-cyclodextrin mixture was placed in
aluminum plate and incubated for 20 hours at 50.degree. C. and 65%
RH. The glycosylated protein (RP.sub.CD) was stored at 5.degree. C.
until analyzed.
Example 13
Glycosylation of Rice Endosperm Protein with Pectin (RP.sub.P)
[0172] The rice endosperm protein isolate (RP) 200 g, on dw basis,
was dissolved in water to give a 10% (w/v) protein colloidal
solution. Pectin (2 g) was dissolved separately in water to give 1%
(w/v) pectin solution. The pectin solution was added into the
protein solution while stirring the protein solution at 37.degree.
C. The protein-pectin mixture was adjusted to pH 7.0, stirred for 1
hour at 37.degree. C., spray-dried, and stored at 5.degree. C. in
air tight containers until they were used. The spray-dried
protein-pectin mixture was placed in aluminum plate and incubated
for 20 hours at 50.degree. C. and 65% RH. The glycosylated protein
(RP.sub.P) was stored at 5.degree. C. until analyzed.
(E) Manufacture of a Partially Deamidated Rice Endosperm
Protein
Example 14
Deamidation of Rice Endosperm Protein Using Alkali Treatment
(RP.sub.DA)
[0173] The deamidation conditions were chosen based on literature
information (Schwenke 1997, Cabra et al 2007) with some
modifications as described below. Alkali extracted rice protein
isolate was mixed with DI water (8% w/v). The protein colloidal
solution was adjusted to pH 11.0 and stirred at 25.degree. C. for
12 hours. Then, the temperature of the protein solution was
increased under stirring for 30 min at 70.degree. C. The solution
was cooled immediately to 30.degree. C. by adding ice, and the pH
was readjusted to 7.0. The deamidated protein solution was spray
dried, and stored at 5.degree. C. (RP.sub.DA).
(F) Results
Statistical Analysis
[0174] All the experiments were conducted in duplicate. Data were
analyzed for variance and multiple mean comparisons with JMP 6
software (SAS Inst 2002). The significance of difference between
means was determined by the Tukey HSD procedure at the 5%
significance level (P<0.05).
Optimization of the Glycosylation of Rice Endosperm Protein
[0175] The glycosylation of rice protein with D-glucose and Xanthan
gum were optimized as described above. The solubility and
emulsifying properties of the glycosylated proteins are presented
in Table 3.
TABLE-US-00004 TABLE 3 Solubility and emulsifying properties of
D-glucose and Xanthan gum glycosylated rice protein as a function
of incubation time at 50.degree. C. and 65% relative humidity
Glycosylation D-glucose glycosylated Xanthan gum glycosylated
treatments: rice protein rice protein Reaction Emulsion Emulsion
Emulsion Emulsion time (h = Solubility activity stability
Solubility activity stability hour(s)) (%) (A.sub.500) (min) (%)
(A.sub.500) (min) RP-control 18.0 0.266 14.7 18.0 0.266 14.7 RP-0 h
23.6 0.371 15.4 26.1 0.479 18.1 RP-4 h 28.9 0.707 20.4 27.0 0.497
20.8 RP-8 h 34.4 0.712 23.4 30.0 0.515 24.5 RP-12 h 33.5 0.627 25.4
32.3 0.523 24.0 RP-16 h 29.7 0.601 26.8 33.1 0.583 25.5 RP-20 h
28.3 0.462 27.2 31.8 0.629 26.4 RP-24 h 24.3 0.350 28.0 33.0 0.632
27.0
[0176] Glycosylation of rice protein D-glucose (2.25%, w/w) and
rice protein-Xanthan gum (1%, w/w) conjugates were prepared at
50.degree. C. and 65% relative humidity for varying incubation time
(0-24 hours). Since the reaction starts very fast, even at the
beginning a difference compared to untreated rice endosperm protein
isolate was recognized.
[0177] The functional properties of the glycosylated proteins
differed significantly as a function of Maillard reaction time and
depended on the type of sugar used to glycosylate the protein. For
D-glucose, the optimum Maillard reaction time was 8 hours at
50.degree. C. and 65% RH. The glycosylated proteins demonstrated a
gradual improvement in solubility and emulsifying properties as a
function of Maillard reaction up to 8 hours. This trend was
illustrated by an increase in solubility from 23% to 34%, and the
emulsion activity from 0.371 to 0.712 as reaction time progressed
from 0 hours to 8 hours. These properties decreased beyond 8 hours
of incubation. However, the emulsion stability of the glycosylated
proteins gradually increased from 15.4 to 28 minutes, as the
function of incubation time from 0 to 24 hours. The increment of
emulsion stability was higher at the early stage of incubation
(0-12 hours) than that of the late stage (12-24 hours), which might
be due to decreasing availability of amino groups with progressing
Maillard reaction.
[0178] In Xanthan gum glycosylated proteins, the solubility and
emulsifying properties gradually increased up to 16 and 20 hours,
respectively. The solubility increased from 26% to 33% in 16 hours
incubation; the emulsifying activity increased from 0.479 to 0.629
in 20 hours of incubation. Compared to glucose, Xanthan gum
mediated glycosylation improved the functional properties at a
slower rate and required more time for Maillard reaction. A 18-20
hour period of incubation was the optimum reaction time for Xanthan
gum at 50.degree. C., 65% RH, and 1:100 Xanthan gum to protein
ratio. The optimum reaction time varied and depended on the
reactants and reaction conditions (Oliver 2006). Simple sugars can
react to faster and required shorter duration than
polysaccharides.
[0179] In the present study, the optimal reaction times were much
shorter than the studies published previously. Lysozyme
glycosylated with Xanthan gum demonstrated superior emulsifying
properties after 7 days of Maillard reaction (Nakamura et al 2000).
Similarly, Ovalbumin conjugated with glucose reached maximum
emulsion activity in 1 day and emulsion stability in 14 days of
reaction time (Kato et al 1995). The differences in time
requirement might be due to the differences in pre-incubation
conditions used in these experiments. For instance, in these two
previous studies, protein-sugar mixture was adjusted to pH 7.0 and
immediately freeze-dried. However, in the present study, the
protein-sugar mixture was stirred for 1 h before drying. The pH of
the sugar-rice protein mixture was maintained at pH 8.0. The slight
alkali condition (pH 8.0) and longer mixing condition (1 hour)
might have facilitated Maillard reaction in the liquid stage.
[0180] The solubility and emulsifying data of native and
glycosylated protein with 0 h incubation supported this presumption
(Table 3). The solubility and emulsifying properties of
glycosylated protein with 0 hour incubation showed higher
solubility and emulsifying properties than that of native rice
protein controls. For glucose-rice protein conjugate, the
solubility improved from 18 to 24% and emulsion activity improved
from 0.266 to 0.371 without any dry-stage reaction (Table 3); for
Xanthan gum-rice protein conjugate, the solubility increased from
18 to 26%, emulsion activity and stability increased from 0.266 to
0.479 and from 14.7 to 18.1 min, respectively.
In Glycosylation Cross-Linking Study, Preliminary Experiments were
Conducted with D-Glucose, Potato-Dextrin, Cyclodextrin, Pectin,
Xanthan Gum.
[0181] Based on our preliminary data presented in (Table 4),
D-glucose and Xanthan gum conjugation significantly improved both
solubility and emulsifying properties compared to rice endosperm
protein and potato-dextrin, cyclodextrin, or pectin conjugated rice
protein. Thus, D-glucose and Xanthan gum were selected for further
process optimization of glycosylation.
[0182] Potato-dextrin, cyclodextrin conjugation did not improve the
endosperm protein solubility, but slightly improved the emulsifying
properties compared to control rice endosperm protein. Pectin
conjugation did not improve the solubility or emulsifying
properties.
TABLE-US-00005 TABLE 4 Solubility and emulsifying properties of
rice endosperm protein glycosylated with various carbohydrates at
50.degree. C. and 65% relative humidity for 12 and 24 hours
Incubation Emulsion Emulsion Type of time activity stability
carbohydrate (h) Solubility (%) (A.sub.500) (min) Control rice
protein 17.4 0.259 15.8 D-glucose 12 31.4 0.596 23.1 D-glucose 24
26.7 0.417 26.2 Potato-dextrin 12 16.1 0.367 18.6 Potato-dextrin 24
17.9 0.339 20.4 Cyclodextrin 12 14.8 0.317 24.3 Cyclodextrin 24
11.8 0.294 22.5 Pectin 12 14.1 0.266 15.8 Pectin 24 13.7 0.386 17.9
Xanthan gum 12 29.7 0.509 22.6 Xanthan gum 24 35.7 0.577 28.3
[0183] The protein:carbohydrate ratios used in this experiment were
100:2.265 for glucose, and 100:1 for potato-dextrin, cyclodextrin,
pectin, and Xanthan gum.
[0184] The optimized glycosylated rice proteins were further
compared and evaluated for physicochemical and functional
properties of the proteins modified by controlled enzymatic
hydrolysis and alkali deamidation. The treatments and brief methods
of modification are summarized in Table 5.
TABLE-US-00006 TABLE 5 Summary of rice protein modification methods
Rice protein isolate Brief out line type of rice endosperm protein
preparation RP.sub.Control RP Rice endosperm protein with no
further modification RP.sub.Alcalase RP.sub.Alc Alcalase treated
rice endosperm protein to 1.8% DH RP.sub.Glucose RP.sub.Glu
Glycosylated rice endosperm protein with D-Glucose RP.sub.Xanthan
Gum RP.sub.XG Glycosylated rice endosperm protein with Xanthan gum
RP.sub.Deamidation RP.sub.DA Alkali deamidated rice endosperm
protein
[0185] The rice endosperm protein was modified by the above
methods: (1) Glycosylation of rice endosperm protein with D-glucose
(RP.sub.Glu); (2) Glycosylation of rice endosperm protein with
Xanthan gum (RP.sub.XG); (3) Deamidation of rice endosperm protein
using alkali treatment (RP.sub.DA); (4) Treatment of rice endosperm
protein with alcalase to 1.8% DH(RP.sub.Alc) to improve the
solubility and emulsifying properties
Influence of Controlled Glycosylation, Deamidation, and Alcalase
Modifications on Rice Protein Physicochemical and Functional
Properties
Physicochemical Properties
[0186] Protein and moisture content of the rice protein products
are presented in Table 6.
TABLE-US-00007 TABLE 6 Moisture, protein content, and viscosity of
rice protein isolates modified by control enzymatic hydrolysis,
glycosylation and deamidation Protein Protein (%) Rice protein pH
(as Moisture (as (Dry weight Viscosity isolate type is) % is, %)
basis) (mPas)* RP.sub.Control 7.1 2.7 84.6 86.7 8.7 RP.sub.Alcalase
7.4 3.3 83.7 86.5 7.0 RP.sub.Glucose 6.9 8.8 77.4 84.9 12.4
RP.sub.Xanthan Gum 6.8 9.0 75.9 83.4 31.6 RP.sub.Deamidation 6.9
4.5 74.2 77.6 14.0 Whey protein 8.4 3.9 85.2 88.6 5.6 Values are
means of duplicates and expressed on dry weight basis. Mean values
with different letters in the same column are significantly
different (P < 0.05). *Viscosity at 26.degree. C., at constant
shear rate of 400 s.sup.-1; 1 Pas (Pascal second) = 1000 cP
(centipoises = mPas); Viscosity was measured on `as is` basis.
[0187] The glycosylated proteins, RP.sub.Glu, RP.sub.XG, contained
higher moisture content (8.8-9.0%) than that of other proteins.
These two products, RP.sub.Glu, RP.sub.XG, absorbed moisture during
the glycosylation process that was carried out at 65% RH for 8 and
18 hours, respectively. Protein content of the glycosylated
proteins (RP.sub.Glu, RP.sub.XG) did not differ from the control
rice protein isolates (RP) as low percent of glucose (2.25%, w/w)
and Xanthan gum (1%, w/w) were used to glycosylate the
proteins.
[0188] The viscosity of the protein proteins are presented in Table
6 and FIG. 1.
[0189] FIG. 1 shows the relationship between shear stress (y-axis;
in Pa) and shear rate .alpha.-axis; in sec.sup.-1) for rice protein
isolates (--RP.sub.control), hydrolysate
(.diamond-solid.P.sub.Alc), glycosylated (.largecircle.RP.sub.Glu
and .box-solid.RP.sub.XG), and deamidated
(.tangle-solidup.RP.sub.DA) rice protein at 10% w/v concentration;
1 Pas (Pascal second)=1000 cP (centipoises=mPas). The viscosity was
the slope of shear stress and shear rate of the linear line.
[0190] Glycosylated (RP.sub.Glu, RP.sub.XG) and deamidated
(RP.sub.DA) proteins showed much higher viscosity than the protease
treated (RP.sub.Alc) and control rice proteins (RP). The increased
viscosity can be an indication of increasing hydrophilicity of the
glycosylated. Particularly, glycosylation with Xanthan gum
increased the viscosity of the product to 31.6 mPas, which was
significantly higher than that of glucose mediated glycosylation
(12.4 mPas) and deamidation (14.0 mPas) methods. The higher
viscosity of Xanthan gum conjugated protein might be due the
complex size of the glycosyl residue. In general, increasing
viscosity of glycosylated proteins indicated the structural changes
in protein. Glycosylated proteins have greater hydrodynamic volume
and increases hydration properties due to the increase of surface
hydrophilicity and partial unfolding of quaternary structure of
protein (Baniel et al 1992).
[0191] to The deamidation also increased the viscosity from 8.7 to
14.0 mPas. This indicated the improved hydration properties of the
deamidated proteins. Deamidation of amide groups into carboxyl
groups might possibly alter the charge content of the protein and
improved the interaction of protein with water molecules. However,
considering the condition use to perform the deamidation, it is
obvious that the viscosity of the deamidated protein was the net
result of deamidation, denaturation, and peptide bond cleavage.
[0192] The alcalase treatment reduced the viscosity slightly from
8.7 to 7.0 mPas in RP.sub.Alc. The reduction in viscosity was due
to the reduction of protein molecular size. However, the viscosity
change was small, since the protein hydrolysis was controlled to
1.8% DH.
Protein Solubility
[0193] The solubility and emulsifying properties of the proteins
are presented in Table 7.
TABLE-US-00008 TABLE 7 Nitrogen solubility and emulsifying
properties of the rice protein isolates Degree Emulsion Emulsion
Rice protein of hydrolysis N solubility activity stability isolates
(DH)* (%) (A500) (min) RP.sub.Control -- 18.0 0.266 14.7
RP.sub.Alcalase 1.8 33.3 0.468 17.5 RP.sub.Glucose -- 39.7 0.721
26.8 RP.sub.Xanthan Gum -- 38.6 0.661 26.1 RP.sub.Deamidation --
68.3 0.776 24.0 Whey protein -- 97.2 0.952 18.8 Values are means of
duplicates and expressed on dry weight basis. Mean values with
different letters in the same column are significantly different (P
< 0.05). *DH values are presented only with the samples that
were subjected to protease treatments.
[0194] Glycosylation with glucose and Xanthan gum increased the
solubility of rice protein to .about.40%, however, the increment of
solubility by glycosylation was not substantial to the level
expected.
[0195] Alcalase treatment (1.8%, DH) improved the solubility from
18% to 33%. The increasing solubility was due to decreasing
molecular size and increasing number of polar groups (Nielsen
1997). A previous study reported that a high percent of the rice
protein remained insoluble even after 13.5% DH by alcalase enzyme
(Paraman et al 2007), which suggested that the either peptides
cleaved by proteases were associated with unhydrolyzed protein via
inter molecular hydrophobic or sulfhydryl interactions or the
amorphous regions of the protein only accessible to hydrolysis.
[0196] The deamidated protein showed higher solubility (68%) than
any other treatments (28-39%). The deamidated protein showed 68.3%
solubility. The deamidation in alkali condition might have
deamidated the side chain amide group of the aspartic and glutamic
amino acids and possibly alerted the protein polarity. Deamidation
increase the negative charge and can disrupt hydrophobic and
hydrogen bonds (Schwenke 1997). These structural changes can
contribute to an increase in solubility. Particularly, the most
abundant amino acids in rice glutelin are glutamine, asparagine,
arginine, glycine, and alanine (Wen and Luthe 1985). Deamidation of
these amino acid residues can facilitate protein solubility, and
contributed to an improvement in emulsifying properties. Further,
partial cleavage of peptide bonds could be possible in the
deamidation process. The combined effect of hydrolysis along with
deamidation might have contributed to the great improvement of
solubility.
Emulsifying Properties
[0197] The emulsion activity and stability of the glycosylated and
deamidated proteins were significantly higher than that of protease
treated rice protein isolates. The glycosylated protein with
glucose showed 0.721 emulsion activity and 26.8 min emulsion
stability. The deamidated protein showed 0.776 emulsion activity
and 24.0 min emulsion stability. The emulsifying properties of
these two protein isolates are higher than that of unmodified and
protease modified rice protein isolates. Glycosylation makes the
proteins more hydrophilic (Kato et at 1991). Therefore, controlled
and limited glycation improves emulsifying properties. Increasing
hydrophilic nature improves the hydration and solubility and thus
possibly opens up the globular structure of protein. The partial
unfolding could have improved the ability of the glycosylated
protein to form stable interfacial film at the oil-water
interface.
[0198] In the case of deamidated protein, improvement in emulsion
properties may be attributed by increasing protein solubility and
polarity (Schwenke 1997). In addition to deamidation, as discussed
earlier, the partial cleavage of peptide bond also might have
contributed to the improvement of solubility and emulsifying
properties. Increased solubility of the deamidated protein might
help produce stable interaction at the oil-water interface and thus
improved the emulsifying properties. The proper balances of
deamidation and peptide bond cleavage might be the key in improving
the emulsion properties rice protein by deamidation.
[0199] The alcalase treatment to 1.8% DH(RP.sub.Alc) improved
emulsion activity from 0.266 to 0.468, and emulsion stability from
17.7 to 17.5 min compared to unmodified rice protein (RP). In
general, a low degree of hydrolysis is recommended to improve the
functionality of food proteins, especially emulsion stability.
Limited proteolysis could improve molecular flexibility and
hydrophobic-hydrophilic balance of protein which resulting better
emulsification (Nielsen 1997; Schwenke 1997). However, the limited
improvement of solubility and emulsifying properties might be due
to (1) high hydrophobic and sulfhydryl interactions, and exposure
of buried hydrophobic regions of protein that accompanied high
temperature enzyme inactivation may have promoted aggregation and
cross linking of partially hydrolyzed proteins (Paraman et al
2007). Further, due to the compact nature of the protein, the
enzyme alcalase might have a limited access to interior peptide
bonds, which could possibly lead to the formation of uneven size of
peptide and decrease the emulsifying properties of the resulting
hydrolysate.
CONCLUSION
[0200] To improve the solubility and emulsifying properties of rice
endosperm protein, glycosylation and deamidation type modifications
were more effective than proteolysis modification by alcalase to
1.8% DN. In rice endosperm protein glycosylation, the optimum
Maillard reaction time was 8 h and 20 H at 50.degree. C. and 65% RH
for D-glucose and Xanthan gum, respectively. The deamidated protein
showed the highest solubility (68%) and emulsifying properties
(0.776-emulsion activity, 24 min emulsion stability) among the
proteins evaluated in this study. The controlled enzymatic
hydrolysis by alcalase 1.8% DH only improved emulsion activity from
0.266 to 0.468, and emulsion stability from 14.7 to 17.5 min
compared to unmodified rice protein (RP). The highest solubility
and emulsifying properties of deamidated protein were the net
result of deamidation, peptide bond cleavage, and protein
denaturation that took place in the process of deamidation.
(G) Manufacture of a Composition of a Fat-Soluble Active Ingredient
or a Fat-Soluble Colorant According to the Present Invention
Example 15
Manufacture of a Formulation of Vitamin E (Acetate)
[0201] A formulation comprising a rice endosperm protein and
vitamin E may be prepared as follows:
a) Preparation of an Emulsion:
[0202] 20 g of RP.sub.Glu (Glycosylation of rice endosperm protein
with D-glucose) according to Example 9 were mixed with 60 g of
sucrose and re-dissolved in 140 ml of water by stirring at
75.degree. C. for 15 minutes. 88.3 g of dl-.alpha. tocopherol
acetate were heated to 70.degree. C. and under vigorous stirring
added to the aqueous solution. The dispersion was vigorously
stirred for another 15 minutes. Under gently stirring further 225
mL of water were added and the so-obtained emulsion was
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 1.86 am as measured by laser diffraction
(Malvern Masersizer). After storage for 12 hours, the emulsion was
again 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 after storage was 1.81 .mu.m as
measured by laser diffraction (Malvern Masersizer).
b) Preparation of a Solid Formulation from the Emulsion:
[0203] The emulsion may be sprayed into a pre-cooled fluidised bed
of cornstarch. Excess cornstarch can be removed by sieving and the
powder obtained can be dried in an air stream at room temperature.
The powder particle fraction in the range of 0.16 to 0.63 mm can be
collected by sieving Alternatively, the emulsion can be converted
into a solid form by using further well known drying technologies
such spray drying.
Example 16
Manufacture of a Formulation of Vitamin E (Acetate)
[0204] A formulation comprising a rice endosperm protein and
vitamin E may be prepared as follows:
a) Preparation of an Emulsion:
[0205] 20 g of RP.sub.XG (Glycosylation of rice endosperm protein
with Xanthan gum) according to Example 10 were mixed with 60 g of
sucrose and re-dissolved in 140 ml of water by stirring at
75.degree. C. for 15 minutes. 83.2 g of dl-.alpha. tocopherol
acetate were heated to 70.degree. C. and under vigorous stirring
added to the aqueous solution. The dispersion was vigorously
stirred for another 15 minutes. Under gently stirring further 225
mL of water were added and the so-obtained emulsion was
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 1.65 am as measured by laser diffraction
(Malvern Masersizer). After storage for 12 hours, the emulsion was
again 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 after storage was 1.64 .mu.m as
measured by laser diffraction (Malvern Masersizer).
b) Preparation of a Solid Formulation from the Emulsion:
[0206] The emulsion may be sprayed into a pre-cooled fluidised bed
of cornstarch. Excess cornstarch can be removed by sieving and the
powder obtained can be dried in an air stream at room temperature.
The powder particle fraction in the range of 0.16 to 0.63 mm can be
collected by sieving alternatively; the emulsion can be converted
into a solid form by using further well known drying technologies
such spray drying.
Example 17
Manufacture of a Formulation of Vitamin E (Acetate)
[0207] A formulation comprising a rice endosperm protein and
vitamin E may be prepared as follows:
a) Preparation of an Emulsion:
[0208] 20 g of RP.sub.DA (Deamidation of rice endosperm protein
using alkali treatment) according to Example 14 were mixed with 60
g of sucrose and re-dissolved in 140 ml of water by stirring at
75.degree. C. for 15 minutes. 81.9 g of dl-.alpha. tocopherol
acetate were heated to 70.degree. C. and under vigorous stirring
added to the aqueous solution. The dispersion was vigorously
stirred for another 15 minutes. Under gently stirring further 225
mL of water were added and the so-obtained emulsion was
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 1.77 .mu.m as measured by laser
diffraction (Malvern Masersizer). After storage for 12 hours, the
emulsion was again 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 after storage was 1.68 .mu.m
as measured by laser diffraction (Malvern Masersizer).
b) Preparation of a Solid Formulation from the Emulsion:
[0209] The emulsion may be sprayed into a pre-cooled fluidised bed
of cornstarch. Excess cornstarch can be removed by sieving and the
powder obtained can be dried in an air stream at room temperature.
The powder particle fraction in the range of 0.16 to 0.63 mm can be
collected by sieving Alternatively, the emulsion can be converted
into a solid form by using further well known drying technologies
such spray drying.
Example 18
Manufacture of a Formulation of .beta.-Carotene
[0210] A formulation comprising a rice endosperm protein and
.beta.-carotene may be prepared as follows:
a) Preparation of a(n Oil-Based) Solution 1:
[0211] 6.6 g of corn oil and 1.2 g of dl-.alpha.-tocopherol were
mixed. 13.8 g of crystalline .beta.-carotene were dispersed in 180
ml of chloroform (trichloromethane) and the resulting dispersion to
was added to the mixture of corn oil and tocopherol. By gently
stirring and simultaneous heating the mixture to about 60.degree.
C. a solution was obtained.
b) Preparation of a(n Aqueous) Solution 2:
[0212] 30 g of RP.sub.Glu (Glycosylation of rice endosperm protein
with D-glucose) according to Example 9 was re-dissolved in 120 ml
of water by stirring at 60.degree. C.
c) Preparation of an Emulsion from the Solutions 1 and 2:
[0213] Under vigorous stirring solution 1 was added to solution 2
at 53.degree. C. and the dispersion was vigorously stirred for
another 30 minutes. The stirred dispersion was kept at 50 to
55.degree. C. for 30 minutes. Residual trichloromethane was removed
at 50 to 55.degree. C. After removing entrapped air bubbles by
centrifugation the emulsion was gently stirred at 50 to 55.degree.
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 440 nm as
measured by laser diffraction (Malvern Masersizer).
d) Preparation of a Solid Formulation from the Emulsion:
[0214] The emulsion may be sprayed into a pre-cooled fluidised bed
of cornstarch. Excess cornstarch can be removed by sieving and the
powder obtained can be dried in an air stream at room temperature.
The powder particle fraction in the range of 0.16 to 0.63 mm can be
collected by sieving 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.
TABLE-US-00009 TABLE 8 Calculated composition of the dried
formulation Amount [weight-%, based on the total Compound dry
weight] RP.sub.Glu: Rice endosperm 25.0 protein according to
example 9 sucrose 30.5 ascorbyl palmitate 1.5 .beta.-carotene 11.5
corn oil 5.5 dl-.alpha.-tocopherol 1.0 Corn starch fluid 25.0
Example 19
Manufacture of a Formulation of .beta.-Carotene
[0215] A formulation comprising a rice endosperm protein and
(3-carotene may be prepared as follows:
a) Preparation of a(n Oil-Based) Solution 1:
[0216] 6.6 g of corn oil and 1.2 g of dl-.alpha.-tocopherol were
mixed. 13.8 g of crystalline (3-carotene were dispersed in 180 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 60.degree. C. a
solution was obtained.
b) Preparation of a(n Aqueous) Solution 2:
[0217] 30 g of RP.sub.XG (Glycosylation of rice endosperm protein
with Xanthan gum) according to Example 10 was re-dissolved in 120
ml of water by stirring at 60.degree. C. Additionally 1.8 g of
ascorbyl palmitate and 36.6 g of sucrose were added. 0.5 ml of
aqueous 1 N NaOH were used to adjust the pH to a value of 7.3.
e) Preparation of an Emulsion from the Solutions 1 and 2:
[0218] Under vigorous stirring solution 1 was added to solution 2
at 53.degree. C. and the dispersion was vigorously stirred for
another 30 minutes. The stirred dispersion was kept at 50 to
55.degree. C. for 30 minutes. Residual trichloromethane was removed
at 50 to 55.degree. C. After removing entrapped air bubbles by
centrifugation the emulsion was gently stirred at 50 to 55.degree.
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 380 nm as
measured by laser diffraction (Malvern Masersizer).
d) Preparation of a Solid Formulation from the Emulsion:
[0219] The emulsion may be sprayed into a pre-cooled fluidised bed
of cornstarch. Excess cornstarch can be removed by sieving and the
powder obtained can be dried in an air stream at room temperature.
The powder particle fraction in the range of 0.16 to 0.63 mm can be
collected by sieving 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.
TABLE-US-00010 TABLE 9 Calculated composition of the dried
formulation Amount [weight-%, based on the total Compound dry
weight] RP.sub.XG: Rice endosperm 25.0 protein according to example
10 sucrose 30.5 ascorbyl palmitate 1.5 .beta.-carotene 11.5 corn
oil 5.5 dl-.alpha.-tocopherol 1.0 Corn starch fluid 25.0
Example 20
Manufacture of a Formulation of .beta.-Carotene
[0220] A formulation comprising a rice endosperm protein and
.beta.-carotene may be prepared as follows:
a) Preparation of a(n Oil-Based) Solution 1:
[0221] 6.6 g of corn oil and 1.2 g of dl-.alpha.-tocopherol were
mixed. 13.8 g of crystalline .beta.-carotene were dispersed in 180
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 60.degree.
C. a solution was obtained.
b) Preparation of a(n Aqueous) Solution 2:
[0222] 30 g of RP.sub.DA (Deamidation of rice endosperm protein
using alkali treatment) according to Example 16 was re-dissolved in
120 ml of water by stirring at 60.degree. C. Additionally 1.8 g of
ascorbyl palmitate and 36.6 g of sucrose were added. 0.4 ml of
aqueous 1 N NaOH were used to adjust the pH to a value of 7.0.
c) Preparation of an Emulsion from the Solutions 1 and 2:
[0223] Under vigorous stirring solution 1 was added to solution 2
at 53.degree. C. and the dispersion was vigorously stirred for
another 30 minutes. The stirred dispersion was kept at 50 to
55.degree. C. for 30 minutes. Residual trichloromethane was removed
at 50 to 55.degree. C. After removing entrapped air bubbles by
centrifugation the emulsion was gently stirred at 50 to 55.degree.
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 500 nm as
measured by laser diffraction (Malvern Masersizer).
d) Preparation of a Solid Formulation from the Emulsion:
[0224] The emulsion may be sprayed into a pre-cooled fluidised bed
of cornstarch. Excess cornstarch can be removed by sieving and the
powder obtained can be dried in an air stream at room temperature.
The powder particle fraction in the range of 0.16 to 0.63 mm can be
collected by sieving 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.
TABLE-US-00011 TABLE 10 Calculated composition of the dried
formulation Amount [weight-%, based on the total Compound dry
weight] RP.sub.DA: Rice endosperm 25.0 protein according to example
14 sucrose 30.5 ascorbyl palmitate 1.5 .beta.-carotene 11.5 corn
oil 5.5 dl-.alpha.-tocopherol 1.0 Corn starch fluid 25.0
* * * * *