U.S. patent application number 13/780975 was filed with the patent office on 2013-07-04 for whey protein micelles.
This patent application is currently assigned to NESTEC S.A.. The applicant listed for this patent is NESTEC S.A.. Invention is credited to Lionel Jean Rene Bovetto, Matthieu Pouzot, Frederic Robin, Christophe Schmitt.
Application Number | 20130171318 13/780975 |
Document ID | / |
Family ID | 36691358 |
Filed Date | 2013-07-04 |
United States Patent
Application |
20130171318 |
Kind Code |
A1 |
Bovetto; Lionel Jean Rene ;
et al. |
July 4, 2013 |
WHEY PROTEIN MICELLES
Abstract
The present invention relates to whey protein micelles,
particularly to whey protein micelle concentrates or powders
thereof and to a method for producing them. The present invention
also pertains to the use of these micelles concentrates or powders
thereof in nutrition and/or cosmetics and/or pharmaceutics.
Inventors: |
Bovetto; Lionel Jean Rene;
(Larringes, FR) ; Schmitt; Christophe; (Servion,
CH) ; Robin; Frederic; (Chailly/Lausanne, CH)
; Pouzot; Matthieu; (Lausanne, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NESTEC S.A.; |
Vevey |
|
CH |
|
|
Assignee: |
NESTEC S.A.
Vevey
CH
|
Family ID: |
36691358 |
Appl. No.: |
13/780975 |
Filed: |
February 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12280244 |
Aug 21, 2008 |
8399043 |
|
|
PCT/EP07/52900 |
Mar 27, 2007 |
|
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13780975 |
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Current U.S.
Class: |
426/583 |
Current CPC
Class: |
A23P 10/40 20160801;
A23C 21/026 20130101; A23V 2002/00 20130101; A23V 2200/25 20130101;
A23G 9/40 20130101; A23L 23/00 20160801; A23G 1/46 20130101; A23C
21/08 20130101; A23J 1/205 20130101; A23V 2002/00 20130101; A23C
2210/30 20130101; A23L 27/72 20160801; A23L 33/19 20160801; A23V
2002/00 20130101; Y10T 428/2984 20150115; A23C 11/04 20130101; A23P
10/30 20160801; A23J 3/08 20130101; A23G 1/44 20130101; A23V
2002/00 20130101; A23G 9/38 20130101; A23V 2200/226 20130101; A23V
2250/54252 20130101; A23V 2250/54252 20130101; A23V 2200/254
20130101; A23V 2250/1842 20130101; A23V 2250/54244 20130101; A23V
2250/54252 20130101 |
Class at
Publication: |
426/583 |
International
Class: |
A23J 1/20 20060101
A23J001/20 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2006 |
EP |
06006295.7 |
Claims
1. A powder comprising: whey protein in which at least 20 wt % of
the whey protein is whey protein micelles, and greater than 80% of
the whey protein micelles have an average size between 100 nm and
700 nm.
2. The powder of claim 1, wherein the powder comprises the whey
protein in an amount of at least 85 wt % of the powder.
3. The powder of claim 1, wherein the whey protein micelles
comprise more than 50 wt % of the powder.
4. The powder of claim 1, wherein the whey protein micelles
comprise more than 80 wt % of the powder.
5. The powder of claim 1, wherein the powder has a moisture content
of less than 10 wt % of the powder.
6. The powder of claim 1, wherein the powder has a moisture content
of less than 4 wt % of the powder.
7. The powder of claim 1, wherein the powder has a water binding
capacity of at least 50%.
8. The powder of claim 1, wherein the powder has a water binding
capacity of at least 90%.
9. The powder of claim 1, wherein the powder has a water binding
capacity of 100%.
10. The powder of claim 1, wherein the powder has a glycerol
binding capacity of at least 50%.
11. The powder of claim 1, wherein the powder has an ethanol
binding capacity of at least 50%.
12. The powder of claim 1, wherein the powder has an oil binding
capacity of at least 30%.
13. The powder of claim 1, wherein the powder contains functional
ingredients in an amount of 0.1-50 wt % of the powder.
14. The powder of claim 13, wherein the functional ingredients are
selected from the group consisting of coffee, caffeine, green tea
extracts, plant extracts, vitamins, minerals, bioactive agents,
salt, sugar, sweeteners, aroma, oils, fatty acid, protein
hydrolysates, peptides and combinations thereof.
15. The powder of claim 1, wherein the powder has an angle of
repose that is less than 35.degree..
16. The powder of claim 1, wherein the powder has an angle of
repose that is less than 30.degree..
17. A whey protein micelle concentrate comprising at least 12 wt %
of the concentrate as protein, at least 50 wt % of the protein is
micelles, and greater than 80% of the micelles have an average size
between 100 nm and 700 nm.
18. The whey protein micelle concentrate of claim 17, wherein
greater than 80% of the micelles have an average size between 200
nm and 400 nm.
19. The whey protein micelle concentrate of claim 17, wherein the
micelles have a coating.
20. The whey protein micelle concentrate of claim 19, wherein the
coating is selected from the group consisting of an emulsifier, a
protein, a peptide, a protein hydrolysate and a gum.
21. The whey protein micelle concentrate of claim 19, wherein the
coating is an emulsifier selected from the group consisting of
sulphated butyl oleate, diacetyltartaric acid esters of mono- and
diglycerides, citric acid esters of monoglycerides, stearoyl
lactylates and combinations thereof.
Description
PRIORITY CLAIM
[0001] The present application is a divisional of U.S. Ser. No.
12/280,244, filed Aug. 21, 2008, which is a National Stage of
International Application No. PCT/EP07/052,900, filed on Mar. 27,
2007 which claims priority to European Patent Application No.
06006295.7, filed on Mar. 27, 2006, the entire contents of which
are incorporated herein by reference
FIELD OF THE INVENTION
[0002] The present invention relates to whey protein micelles,
particularly to whey protein micelle concentrates and powders
thereof and to a method for producing them. The present invention
also pertains to the use of these micelles concentrates and powders
thereof in a wide range of applications.
BACKGROUND
[0003] Protein constitutes an indispensable part of the diets of
many people. It is not only used for its nutritional value but also
imparts desirable texture and stabilisation to foods. For instance,
in fat-containing products, the fat must remain stabilized over the
entire shelf life of the product, so that no phase separation
occurs.
[0004] To this end, emulsifying agents are utilised, that provide a
stabilization of the emulsion once formed, based on their inherent
property of a lipophilic or hydrophobic part being soluble in the
non-aqueous phase and a polar or hydrophilic part being soluble in
water such that said molecules facilitate emulsifying one phase in
the other phase. Additionally, the emulsifying agents also protect
the once formed droplets from aggregation and coalescence. As
emulsifying agents naturally occurring substances are used, such as
hydrocolloids, phospholipids (lecithin) or glycolipids and on the
other hand synthetic agents like stearyl-2-lactylate or mono-,
diacylglycerides, etc. may also be used.
[0005] One of the major drawbacks of the agents resides in that
they sometimes substantially add to the costs of the final product,
and do not add to the nutritional value of the product. Sometimes,
such kinds of materials also do not show adequate stabilising
properties because of an interfacial competition with proteins.
[0006] Increasingly, therefore, protein is also being used as an
emulsifier and as a partial substitute for fat.
[0007] U.S. Pat. No. 6,767,575 B1 discloses a preparation of an
aggregate whey protein product, whereby whey protein is denatured
by acidification and heating. The protein aggregates thus obtained
are used in food application.
[0008] GB 1079604 describes improvements in the manufacture of
cheese, whereby whey proteins undergo heat treatment at an optimum
pH value, in order to obtain insoluble whey proteins which are then
added to raw milk.
[0009] WO 93/07761 is concerned with the provision of a dry
microparticulated protein product which can be used as a fat
substitute.
[0010] U.S. Pat. No. 5,750,183 discloses a process for producing
proteinaceous microparticles which are useful as fat substitute
containing no fat.
[0011] A proteinaceous fat substitute is also disclosed in WO
91/17665 whereby the proteins are in the form of a
water-dispersible microparticulated denatured whey protein.
[0012] Apart from the food applications, proteins are also present
in many pharmaceutical and cosmetic compositions.
[0013] One of the problems encountered with the production of
products containing globular proteins in general, and whey protein
in particular, however is their limited processability in
industrial food production. Indeed, protein molecules when heated,
or when subjected to acidic or alkaline environment or in the
presence of salts tend to lose their native structure and
reassemble in various random structures such as gels, for
example.
[0014] The preparation of gelled aqueous compositions of whey
proteins is the subject of EP 1281322.
[0015] Elofsson et al. in International Dairy Journal, 1997, p.
601-608 describe cold gelling of whey protein concentrates.
[0016] Similarly, Kilara et al. in Journal of Agriculture and Food
20 Chemistry, 1998, p. 1830-1835 describes the effect of pH on the
aggregation of whey proteins and their gelation.
[0017] This gel effect presents limitation in terms of not only
processability (e.g. clogging of machines used in the manufacture
of protein-containing products) but also in terms of the texture
thus obtained, which may not be desirable for the wide range of
protein applications.
[0018] Controlled denaturation of proteins is thus desirable in
order to widen the use of proteins.
[0019] In the Proceedings of the Second International Whey
Conference, Chicago, October 1997, reported in International Dairy
Federation, 1998, 189-196, Britten M. discusses heat treatments to
improve functional properties of whey proteins. A process for
producing whey protein micro-particle dispersion at 95.degree. C.
is described.
[0020] Erdman in Journal of American College of Nutrition, 1990, p.
398-409 describes that the quality of microparticulated protein is
not affected despite using high shear and heat. EP 0603981 also
describes a heat stable oil-in-water emulsion containing
proteins.
[0021] Sato et al. in U.S. Pat. No. 5,882,705 obtained micellar
whey protein by heat treating a hydrolysed whey protein solution.
The micellar whey protein are characterised by an irregular
shape.
[0022] Thus, an object of the invention is to improve the usability
of proteins in industrial production processes.
SUMMARY OF THE INVENTION
[0023] Accordingly, this object is achieved by means of the
features of the independent claims. The dependent claims develop
further the central idea of the present invention.
[0024] To achieve this object, a method for the production of whey
proteins micelles concentrates is proposed, in a first aspect,
which comprises the steps of subjecting a solution containing
native whey proteins to a specific temperature at a specific pH and
concentrating the solution thus obtained to result in the
production of a whey protein micelle concentrate comprising whey
protein micelles having a diameter of less than 1 .mu.m.
[0025] In particular, the present invention relates to a process
for the production of whey protein micelles concentrate comprising
the steps of:
[0026] a. Adjusting the pH of a whey protein aqueous solution to a
value between 3.0 and 8.0,
[0027] b. Subjecting the aqueous solution to a temperature between
70 and below 95.degree. C. and
[0028] c. Concentrating the dispersion obtained in step b.
[0029] In a second aspect, the invention relates to the whey
protein micelles concentrate thus obtainable and to whey protein
micelles having a protein concentration greater than 12%. In a
further aspect, the present invention relates to the use of said
concentrate in nutritional and/or cosmetic and/or pharmaceutical
applications. A composition containing the whey protein concentrate
also falls under an aspect of the present invention.
[0030] Furthermore, the whey protein micelles concentrate may be
dried, in particular by freeze-drying, roller drying or
spray-drying, yielding a whey protein micelles powder.
[0031] Thus, according to another aspect, the invention provides a
whey protein micelles powder comprising at least 20% micelles.
[0032] The whey protein micelles concentrate may be spray-dried
with additional ingredients thus resulting in a mixed whey protein
powder comprising whey protein micelles and additional ingredients
in a weight ratio of 30:1 to 1:1000 according to a further aspect
of the invention.
[0033] The use of the whey protein powder or the mixed whey protein
powder for instance in the production of protein enriched
consumables and compositions comprising these powders and, for
instance, are all features of the present invention.
[0034] Whey protein micelles and consumable products comprising
said micelles are also features of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0035] The present invention will be further described hereinafter
with reference to some preferred embodiments shown in the
accompanying figures in which:
[0036] FIG. 1 shows the result of an experiment demonstrating the
effect of pH and heat treatment on the micellisation of
.beta.-lactoglobulin.
[0037] FIG. 2 is showing a mean to determine the pH of
micellisation for a commercial preparation (Bipro.RTM., Batch
JE032-1-420) using turbidity measurements at 500 nm.
[0038] FIG. 3 is a TEM (Transmission Electron Microscopy)
micrograph from whey protein micelles (2 wt. %, WPI 95, Lactalis)
at pH 7.4. Scale bar is 200 nm.
[0039] FIG. 4 shows the result of an experiment evaluating the
impact of the ionic strength (Arginine HCl) on the formation of
protein micelles at constant pH of 7.0.
[0040] FIG. 5 shows the volume stability (FVS) of foam stabilized
by 1 wt. % .beta.-lactoglobulin micelles (Davisco) at pH 7.0 in
presence of 60 mM Arginine HCl compared to non-micellised
.beta.-lactoglobulin.
[0041] FIG. 6 shows the intensity-based equivalent hydrodynamic
diameter of whey protein obtained by heat-treatment of a 1 wt %
.beta.-lactoglobulin dispersion for 15 min at 85.degree. C. at pH
ranging from 2 to 8. Whey protein micelles are obtained at pH 4.25
(positively charged with a zeta potential around +25 mV) and at pH
6.0 (negatively charged with a zeta potential around -30 mV).
Z-averaged hydrodynamic diameter of the micelles was 229.3 nm at pH
4.25 and 227.2 nm at pH 6.0. The corresponding micrographs of the
micelles obtained by TEM after negative staining are shown. Scale
bars are 1 .mu.m.
[0042] FIG. 7 shows a highly schematic structure of a whey protein
micelle.
[0043] FIG. 8 shows a SEM (Scanning electron microscopy) micrograph
of a whey protein micelle powder obtained after spray drying of a
20% protein content dispersion after microfiltration.
[0044] FIG. 9 is a negative staining TEM micrograph of a whey
protein micelles dispersion obtained at 4% protein content.
[0045] FIG. 10 is a negative staining TEM micrograph of a whey
protein micelle dispersion obtained at 20% protein content after
microfiltration.
[0046] FIG. 11 shows the heat stability of a whey protein micelle
dispersion obtained at 10% protein content after microfiltration at
pH 7.0 in presence of NaCl after heating at 85.degree. C. for 15
min.
[0047] FIG. 12 shows the heat stability of a whey protein
dispersion obtained at 4% protein content at pH 7.0 in presence of
NaCl after heating at 85.degree. C. for 15 min.
[0048] FIG. 13 is a negative staining TEM micrograph from a 4% whey
protein micelles dispersion based on a pure whey protein micelle
spray dried powder after dispersion at 50.degree. C. in deionised
water.
[0049] FIG. 14 is a graph showing the size distribution of micelles
obtained by the process of the invention using a 4% Prolacta 90
whey protein isolate treated at pH 5.9.
[0050] FIG. 15 is a SEM micrograph showing the internal structure
after cutting of a spray-dried powder granule that is presented on
FIG. 8.
[0051] FIG. 16 is a negative staining TEM micrograph of a 4% whey
protein micelles dispersion based on a pure freeze dried whey
protein micelle powder after at room temperature in deionised
water. Scale bar is 0.5 micrometre.
[0052] FIG. 17 is a schematic view of the WPM coating by SBO
(sulphated butyl oleate) upon increasing the mixing ratio at pH
3.0. Grey circle: WPM with positive surface charges. Black
head+tail: negatively charged head and hydrophobic tail from
SBO.
[0053] FIG. 18 is a photograph of a whey protein micelle
concentrate at 20% obtained after evaporation in which 4% NaCl is
added.
[0054] FIG. 19 is a bright field light microscopy micrograph of
whey protein micelle powder semi-thin section after toluidine blue
staining Scale bar is 50 microns.
[0055] FIG. 20 is a SEM micrograph of the hollow whey protein
micelle powder particle after cutting. Left: internal structure.
Right: Detail of the whey protein micelle composing the powder
particle matrix. Scale bars are 10 and 1 micron respectively.
DETAILED DESCRIPTION
[0056] FIG. 7 is a schematic representation of the micelles of the
present invention, wherein the whey proteins are arranged in such a
way that the hydrophilic parts of the proteins are oriented towards
the outer part of the agglomerate and the hydrophobic parts of the
proteins are oriented towards the inner "core" of the micelle. This
energetically favourable configuration offers good stability to
these structures in a hydrophilic environment.
[0057] The specific micelle structure can be seen from the figures,
in particular FIGS. 3, 9, 10, 13 and 15, wherein the micelles of
the present invention consist essentially of spherical agglomerates
of denatured whey protein. The micelles of the present invention
are particularly characterised by their regular, spherical
shape.
[0058] Due to their dual character (hydrophilic and hydrophobic),
this denatured state of the protein seems to allow interaction with
a hydrophobic phase, e.g. a fat droplet or air, and a hydrophilic
phase. The whey protein micelles therefore have perfect emulsifying
and foaming properties.
[0059] Furthermore, the micelles produced by the method of the
present invention have an extremely sharp size distribution (see
FIG. 14), such that more than 80% of the micelles produced will
have a size smaller than 1 micron, preferably between 100 nm and
900 nm, more preferably between 100-770 nm, most preferably between
200 and 400 nm.
[0060] The mean diameter of the micelles can be determined using
Transmission Electron Microscopy (TEM). In order to do so, the
liquid micelle samples are encapsulated in agar gel tubes. Fixation
is achieved by immersion in a solution of 2.5% glutaraldehyde in
0.1M, pH 7.4 cacodylate buffer and post-fixation with 2% Osmium
tetroxide in the same buffer, both solutions containing 0.04%
Ruthenium red. After dehydration in a graded ethanol series (70,
80, 90, 96, 100% ethanol), the samples are embedded in Spun resin
(Spurr/ethanol 1:1, 2:1, 100%). After polymerization of the resin
(70.degree. C., 48 hours), semi-thin and ultra-thin sections are
cut with a Leica ultracut UCT ultra-microtome. Ultra-thin sections,
stained with aqueous uranyl-acetate and lead citrate, are then
examined by transmission electron microscopy (Philips CM12, 80
kV).
[0061] Without wishing to be bound by theory, it is thought that
during micelle formation according to the process of the invention,
the micelle reach a "maximum" size, due to the overall
electrostatic charge of the micelle repelling any additional
protein molecule, such that the micelle cannot grow in size any
longer. This accounts for the narrow size distribution observed
(cf. FIG. 14).
[0062] The micelles described above are produced by a process
according to the present invention, said process being described in
detail in the following.
[0063] As the whey protein to be used in the present method, any
commercially available whey protein isolates or concentrates may be
used, i.e. whey protein obtained by any process for the preparation
of whey protein known in the art, as well as whey protein fractions
prepared therefrom or proteins such as .beta.-lactoglobulin (BLG),
.alpha.-lactalbumin and serum albumin. In particular, sweet whey
obtained as a by-product in cheese manufacture, acid whey obtained
as a by-product in acid casein manufacture, native whey obtained by
milk microfiltration or rennet whey obtained as a by-product in
rennet casein manufacture may be used as the whey protein. The whey
protein may be from a single source or from mixtures of any
sources. It is preferable that the whey protein does not undergo
any hydrolysis step prior to micelle formation. Thus, the whey
protein is not subjected to any enzymatic treatment prior to
micellisation. According to the invention, it is important that the
whey protein be used in the micelle formation process and not
hydrolysates thereof.
[0064] The present invention is not restricted to whey isolates
from bovine origin, but pertains to whey isolates from all
mammalian animal species, such as from sheep, goats, horses, and
camels. Also, the process according to the present invention
applies to mineralised, demineralised or slightly mineralised whey
preparations. By "slightly mineralised" is meant any whey
preparation after elimination of free minerals which are dialyzable
or diafiltrable, but which maintains minerals associated to it by
natural mineralisation after preparation of the whey protein
concentrate or isolate, for example. These "slightly mineralised"
whey preparations have had no specific mineral enrichment.
[0065] Whey proteins are an excellent source of essential amino
acids (AA) (45%). Compared to casein (containing 0.3 g cysteine/100
g protein), sweet whey proteins contain 7 times more cysteine, and
acid whey 10 times more cysteine. Cysteine is the rate limiting
amino acid for glutathione (GSH) synthesis, a tripeptide made of
glutamate cysteine and glycine which has primary important
functions in the defence of the body in case of stress.
Requirements in these amino acids may be increased in case of
stress and in elderly people. Also, glutathione oral
supplementation with whey protein has been shown to increase plasma
GSH levels of HIV-infected patients (Eur. J. Clin. Invest. 2001;
31, 171-178).
[0066] Other health benefits provided by whey proteins include
enhancement of muscle development and building, as well as muscle
maintenance in children, adults or elderly people, enhancement of
the immune function, improvement of cognitive function, control of
blood glucose such that they are suitable for diabetics, weight
management and satiety, anti-inflammatory effects, wound healing
and skin repair, lowering of the blood pressure, etc.
[0067] Whey proteins have a better protein efficiency ratio
(PER=118) compared for example to casein (PER=100). PER is a
measure of a protein quality assessed by determining how well such
protein supports weight gain. It can be calculated by the following
formula:
PER=body weight growth (g)/protein weight intake (g).
TABLE-US-00001 PER % Casein casein 3.2 100 Egg 3.8 118 Whey 3.8 118
Whole Soya 2.5 78 Wheat gluten 0.3 9
[0068] Examples:
[0069] For the process of the invention, whey proteins may be
present in an aqueous solution in an amount of 0.1 wt. % to 12 wt.
%, preferably in an amount of 0.1 wt. % to 8 wt. %, more preferably
in an amount of 0.2 wt. % to 7 wt. %, even more preferably in an
amount of 0.5 wt. % to 6 wt. %, most preferably in an amount of 1
wt. % to 4 wt. % on the basis of the total weight of the
solution.
[0070] The aqueous solution of the whey protein preparation as
present before the micellisation step may also comprise additional
compounds, such as by-products of the respective whey production
processes, other proteins, gums or carbohydrates. The solution may
also contain other food ingredients (fat, carbohydrates, plant
extracts, etc). The amount of such additional compounds preferably
does not exceed 50 wt. %, preferably 20 wt. %, and more preferably
does not exceed 10 wt. % of the total weight of the solution.
[0071] The whey protein may be used in purified form or likewise in
form of a crude product. According to a preferred embodiment, the
content of divalent cations in the whey protein for the preparation
of the whey protein micelles concentrate may be less than 2.5%,
more preferably less than 2%, even more preferably less than 0.2%.
Most preferably the whey proteins are completely demineralised.
[0072] According to the present finding, the pH and the ionic
strength are important factors in the present method. Thus, for
extensively dialyzed samples which are virtually devoid or depleted
of free cations such as Ca, K, Na, Mg, it has been found that when
performing the heat treatment during a time period of 10 s to 2
hours at a pH below 5.4, curd is obtained, while at a pH exceeding
6.8, soluble whey protein results (see FIG. 1). Thus, only in this
rather narrow pH window will whey proteins micelles having a
diameter of less than 1 .mu.m be obtained. These micelles will have
an overall negative charge. The same micelle form can also be
obtained symmetrically below the isoelectrical pH, i.e from 3.5 to
5.0, more preferably 3.8 to 4.5, resulting in micelles being
positively charged (see FIG. 6).
[0073] Thus, according to an embodiment, in order to obtain
positively charged micelles, micellisation of whey proteins may be
done in a salt free solution at a pH value adjusted between 3.8 and
4.5 depending on the mineral content of the protein source.
[0074] Preferably, the micelles obtained will have an overall
negative charge. Thus, in a preferred embodiment, the pH is
adjusted to a range of from 6.3 to 9.0, for a content in divalent
cations comprised between 0.2% and 2.5% in whey protein powder.
[0075] More specifically, to obtain negatively charged micelles,
the pH is adjusted to a range of from 5.6 to 6.4, more preferably
from 5.8 to 6.0 for a low divalent cation content (e.g. less than
0.2% of the initial whey protein powder). The pH may be increased
up to 8.4 depending on the mineral content of whey protein source
(concentrate or isolate). In particular, the pH may be between 7.5
to 8.4, preferably 7.6 to 8.0 to obtain negatively charged micelles
in the presence of large amounts of free minerals and the pH may be
between 6.4 to 7.4, preferably 6.6 to 7.2 to obtain negatively
charged micelles in the presence of moderate amounts of free
minerals. As a general rule, the higher the calcium and/or
magnesium content of the initial whey protein powder, the higher
the pH of micellisation.
[0076] In order to standardize the conditions of formation of the
whey protein micelles, it is most preferable to demineralise by any
of the known demineralisation techniques (dialysis,
ultrafiltration, reverse osmosis, ion exchange chromatography . . .
), any source of liquid native whey proteins with a protein
concentration ranging from that of sweet whey, microfiltration
permeate of milk or acid whey (0.6% protein content) to that of a
concentrate at 30% protein content. The dialysis can be done
against water (distilled, deionised or soft), but as this will only
allow removal of the ions weakly bound to the whey proteins, it is
more preferable to dialyse against an acid at pH below 4.0 (organic
or inorganic) to better control the ionic composition of the whey
proteins. By doing so, the pH of whey protein micelle formation
will be below pH 7.0, more preferably comprised between 5.8 to
6.6.
[0077] Prior to heating the whey protein aqueous solution, the pH
is generally adjusted by the addition of acid, which is preferably
food grade, such as e.g. hydrochloric acid, phosphoric acid, acetic
acid, citric acid, gluconic acid or lactic acid. When the mineral
content is high, the pH is generally adjusted by the addition of
alkaline solution, which is preferably food grade, such as sodium
hydroxide, potassium hydroxide or ammonium hydroxide.
[0078] Alternatively, if no pH adjustment step is desired, it is
possible to adjust the ionic strength of the whey protein
preparation while keeping the pH constant. Then, ionic strength may
be adjusted by organic or inorganic ions in such a way that allows
micellisation at a constant pH value of 7. FIG. 4 represents an
embodiment of the present invention, whereby micelles may be formed
at a constant pH value of 7.0 while the ionic strength is varied by
the addition of 70-80 mM of arginine HCl.
[0079] A buffer may be further added to the aqueous solution of
whey protein so as to avoid a substantial change of the pH value
during heat treatment of the whey protein. In principle, the buffer
may be selected from any food-grade buffer system, i.e. acetic acid
and its salts, such as e.g. sodium acetate or potassium acetate,
phosphoric acid and salts thereof, e.g. NaH.sub.2PO.sub.4,
Na.sub.2HPO.sub.4, KH.sub.2PO.sub.4, K.sub.2HPO.sub.4, or citric
acid and salts thereof etc.
[0080] Adjusting the pH and/or the ionic strength of the aqueous
solution, according to the present invention, results in a
controlled process yielding micelles having a size between 100
nm-900 nm, preferably between 100-700 nm, most preferably between
200-400 nm. Preferably, the proportion of micelles with an average
size comprised between 100-700 nm is greater than 80% when carrying
out the process of the invention (see FIG. 14).
[0081] In order to obtain regular shape micelles, it is also
important, according to the invention, that the whey protein does
not undergo any hydrolysation step prior to micelle formation.
[0082] In a second step of the process of the present invention,
the starting whey protein aqueous solution is then subjected to the
heat treatment. In this respect it has been found that for
obtaining whey protein micelles, it is important to have the
temperature in the range of from about 70 to below 95.degree. C.,
preferably from 80 to about 90.degree. C., more preferably of from
about 82 to about 89.degree. C., even more preferably of from about
84 to about 87.degree. C., most preferred at about 85.degree. C. It
has also been found that, on an industrial scale, it is important
that the temperature be preferably less than 95.degree. C., more
preferably between 80.degree. C. and 90.degree. C., most preferably
about 85.degree. C.
[0083] Once the desired temperature has been reached, it is kept at
this temperature for a minimum of 10 seconds and a maximum of 2
hours. Preferably, the time period during which the aqueous whey
protein solution is kept at the desired temperature ranges from 12
to 25 minutes, more preferably from 12 to 20 minutes, or most
preferably about 15 minutes.
[0084] The heat treatment may also be achieved in a microwave oven
or any similar equipment allowing heating by microwaves with a
time/quantity ratio of 10 s/10 mL for a 4 wt % protein solution
heated in a 1500 W apparatus up to boiling temperature (98.degree.
C. at an altitude of 833 m). A continuous process may also be used
by addition of 8 or more magnetrons around a glass tube potentially
prolonged by a holding tube to increase the time of incubation.
[0085] As shown in FIG. 2, turbidity measurements are an indication
of micelle formation. According to the present invention, the
turbidity measured by absorbance at 500 nm is at least 3 absorbance
units for 1% protein solution but can reach 16 absorbance units
when the yield of micellisation is above 80% (see FIG. 2).
[0086] To further illustrate the effect of micelle formation from a
physicochemical point of view, a 1 wt % dispersion of Bipro.RTM.
has been heated for 15 minutes at 85.degree. C. at pH 6.0 and 6.8
in MilliQ water. The hydrodynamic diameter of the aggregates
obtained after heat treatment was measured by dynamic light
scattering. The apparent molecular weight of the aggregates was
determined by static light scattering using the so-called Debye
plot. The surface hydrophobicity was probed using the hydrophobic
ANS probe and the free accessible thiol groups by the DTNB method
using cystein as the standard amino acid. Finally, the morphology
of the aggregates was studied by negative staining TEM. The results
are presented in table 1.
TABLE-US-00002 TABLE 1 Physicochemical properties of soluble whey
protein aggregates obtained by heat treatment (85.degree. C., 15
min) of a 1 wt % protein dispersion in presence or absence of NaC.
accessible hydrodynamic molecular .zeta.- protein surface SH groups
diameter weight M.sub.w potential hydrophobicity (nmol SH pH (nm)
(.times.10.sup.6 g mol.sup.-1) morphology (mV) (.mu.g mmol.sup.-1
ANS) mg.sup.-1 prot.) 6.0 120.3 .+-. 9.1 27.02 .+-. 8.09 Spherical
micelles -31.8 .+-. 0.8 105.4 3.5 .+-. 0.4 6.8 56.2 .+-. 4.6 0.64
.+-. 0.01 linear aggregates -27.9 .+-. 1.2 200.8 6.8 .+-. 0.5
[0087] From table 1, it is clear that the whey protein micelles
that were formed at pH 6.0 allow protein to decrease its specific
ANS surface hydrophobicity by a factor of 2 compared to
non-micellised whey protein heated in the same condition, but at pH
6.8. The micelle formation can be also seen on the very high
molecular weight of 27.times.10.sup.6 g.mol.sup.-1 compared to
0.64.times.10.sup.6 g.mol.sup.-1 for non-micellised protein,
indicating a very condensed state of the matter within the micelle
(low amount of water). Interestingly enough, the -potential of the
micelles is even more negative than the non-micellised proteins
even if the latter have been formed at a more basic pH than the
micelles. This is the result of a more hydrophilic surface of the
micelles being exposed to the solvent. Finally, one should note
that the thiol reactivity of the micelles is much lower than that
of the non-micellised protein because of the different pH of heat
treatment.
[0088] It has been found that the conversion yield of native whey
protein to micelles decreases when the initial protein
concentration is increased before pH adjustment and heat treatment.
For example, when starting with a whey protein isolate Prolacta 90
(lot 673 from Lactalis), the yield of formation of whey protein
micelles drops from 85% (when starting with 4% proteins) to 50%
(when starting with 12% of proteins). In order to maximize the
formation of whey protein micelles (>85% of the initial protein
content), it is better to start with an aqueous whey protein
solution having a protein concentration below 12%, preferably below
4%. Depending on the intended final applications, the protein
concentration is adjusted before heat treatment to manage the
optimal whey protein micelles yield.
[0089] The whey proteins micelles obtained according to the present
method shall have an average size of less than 1 .mu.m, preferably
of from 100 to 900 nm, more preferably from 100 to 700 nm, most
preferably from 200-400 nm.
[0090] Depending on the desired application, the yield of micelles
before concentration is at least 35%, preferably at least 50%, more
preferably at least 80% and the residual soluble aggregates or
soluble protein content is preferably below 20%. The average
micelle size is characterised by a polydispersity index below
0.200. It has been observed that whey protein micelles could form
aggregates around pH 4.5, with however no sign of macroscopic phase
separation after at least 12 hours at 4.degree. C.
[0091] The purity of whey protein micelles produced according to
the method of the present invention can be obtained by determining
the amount of residual soluble proteins. Micelles are eliminated by
centrifugation at 20.degree. C. and 26900 g for 15 min. The
supernatant is used to determine the protein amount in quartz
cuvettes at 280 nm (1 cm light pathlength). Values are expressed as
a percentage of the initial value before heat treatment.
Proportion of micelles=(Amount of initial proteins-amount of
soluble proteins)/Amount of initial proteins
[0092] An advantage of the method of the present invention is that
the whey protein micelles prepared accordingly have not been
submitted to any mechanical stress leading to reduction of the
particle size during formation, contrary to conventional processes.
This method induces spontaneous micellisation of whey proteins
during heat treatment in the absence of shearing.
[0093] The whey protein micelles may be used as such in any
composition, such as nutritional compositions, cosmetic
compositions, pharmaceutical compositions etc. Furthermore, the
whey protein micelles may be filled with an active component. Said
component may be selected from coffee, caffeine, green tea
extracts, plant extracts, vitamins, minerals, bioactive agents,
salt, sugar, sweeteners, aroma, fatty acids, oils, protein
hydrolysates, peptides etc. and mixtures thereof.
[0094] Furthermore, the whey protein micelles (pure or filled with
active components) of the present invention may be coated with an
emulsifier such as phospholipids, for example, or other coating
agents such as a protein, a peptide, a protein hydrolysate or a gum
such as acacia gum in order to modulate the functionality and the
taste of the whey protein micelles. When a protein is used as a
coating agent, it may be selected from any proteins having an
isoelectric point significantly higher or lower than whey protein.
These are, for example, protamine, lactoferrin and some rice
proteins. When a protein hydrolysate is used as coating agent, it
is preferably a hydrolysate from proteins such as protamine,
lactoferrin, rice, casein, whey, wheat, soy protein or mixtures
thereof. Preferably, the coating is an emulsifier selected from
sulphated butyl oleate, diacetyltartaric acid esters of mono- and
diglycerides, citric acid esters of monoglycerides, stearoyl
lactylates and mixtures thereof FIG. 17 is a schematic
representation of such coating with sulphated butyl oleate. Coating
may be carried out by any methods known in the art. Furthermore,
co-spraydrying, as described further herein, may also result in a
coating of the whey protein micelles.
[0095] The whey protein micelles have shown to be ideally suited
for use as an emulsifier, fat substitute, substitute for micellar
casein or foaming agent, since they are able to stabilize fat
and/or air in an aqueous system for prolonged period.
[0096] The foam stability is shown in FIG. 5 which compares the use
of non-micellised whey protein versus the micellised whey protein
of the present invention.
[0097] Thus, whey protein micelles may be used as an emulsifying
agent, for which the material is ideally suited, since it has a
neutral taste and no off-flavour is created by the use of such
material. They may also be used as micellar casein substitute.
[0098] In addition, the present whey protein micelles are still in
a condition to serve as whitening agent, so that with one compound
several tasks may be fulfilled. Since whey is a material abundantly
available, the use thereof reduces the cost of a product requiring
an emulsifying, filling, whitening or foaming agent, while at the
same time adding to its nutritional value.
[0099] Accordingly, the whey protein micelles obtained according to
the method of the present invention can be used for the preparation
of any kind of consumable product requiring stabilisation of an
emulsion or a foam, such as e.g. present in mousse or ice cream, in
coffee creamers, or also in low fat or essentially fat free dairy
products, or also where it finds application as a micellar casein
substitute. By "consumable" is meant any food product in any form,
including beverages, soups, semi-solid foods etc. which can be
consumed by a human or an animal. Examples of products, where the
present whey protein micelles may find application are for example,
dairy products, mayonnaise, salad dressing, pasteurized UHT milk,
sweet condensed milk, yoghurt, fermented milks, sauces, reduced fat
sauces such as bechamel sauce for instance, milk-based fermented
products, milk chocolate, white chocolate, dark chocolate, mousses,
foams, emulsions, ice creams, fermented cereal based products, milk
based powders, infant formula, diet fortifications, pet food,
tablets, liquid bacterial suspensions, dried oral supplement, wet
oral supplement, performance nutrition bars, spreads, fruit drinks,
coffee mixes.
[0100] Furthermore, the present whey protein micelles may be used
either alone or together with other active materials, such as
polysaccharides (e.g. acacia gum or carrageenans) to stabilise
matrices and for example milky foam matrices. Due to their neutral
taste, their whitening power and their stability after heat
treatment, the present whey proteins micelles may be used to
increase skimmed milk whiteness and mouth feel.
[0101] As well as increasing the whitening power of dairy systems
for the same total protein content, the fat content in a food
matrix may be reduced. This feature represents a particular
advantage of the present whey protein micelles, since it allows
producing low-fat products, for example adding a milk creamer
without adding additional fat derived from the milk as such.
[0102] In the method of the present invention, the whey protein
micelle dispersion obtained after heat treatment is concentrated to
yield a whey protein micelle concentrate.
[0103] Accordingly the concentration step may be carried out by
evaporation, centrifugation, sedimentation, ultrafiltration and/or
by microfiltration.
[0104] Evaporation may be carried out on the micelles dispersion by
feeding it to an evaporator under vacuum, having a temperature
between 50.degree. C. and 85.degree. C.
[0105] Centrifugation may be carried out with high acceleration
rate (more than 2000 g) or low acceleration rate (less than 500 g)
after acidification of the whey protein micelle dispersion at a pH
lower than 5, preferably 4.5.
[0106] Spontaneous sedimentation may also be carried out on the
whey protein micelle dispersion by acidification. Preferably, the
pH will be 4.5 and the sedimentation time is more than 12
hours.
[0107] Preferably, concentration of the whey protein micelles
according to the present invention may be achieved by
microfiltration of the micelles dispersion. This enriching
technique not only enables to concentrate whey protein micelles by
removing the solvent but also enables the removal of non-micellised
protein (such as native proteins or soluble aggregates). Thus, the
final product only consists of micelles (as checked by Transmission
Electron Microscopy--cf. FIGS. 9 and 10). In this case, the
concentration factor that is possible to achieve is obtained after
the initial flow rate of permeate through the membrane has dropped
to 20% of its initial value.
[0108] The whey protein concentrate obtained by the method of the
present invention will have a protein concentration of at least
12%. Furthermore, the concentrate will contain at least 50% of the
protein in the form of micelles.
[0109] It is interesting to note that the concentrate, if adjusted
to a protein content of 10% has the ability to withstand a
subsequent heat treatment at 85.degree. C. for 15 min at pH 7.0 in
presence for example of up to 0.15 M of sodium chloride, as shown
in FIG. 11. As a matter of comparison, a native whey protein
dispersion (Prolacta90, lot 500658 from Lactalis) forms a gel in
the presence of 0.1 M of sodium chloride at a protein concentration
of only 4% (cf. FIG. 12).
[0110] The present invention also presents the benefit that the
high stability of the micelle structure is preserved during the
concentration step. Furthermore, the micelles according to the
present invention have a Protein Efficiency Ratio equivalent to the
starting whey protein of at least 100, preferably at least 110,
which makes them important nutritional ingredients.
[0111] The enrichment of the whey protein micelles offers the
exceptional advantages that protein-enriched products may be
obtained at concentration previously not attainable. Furthermore,
since the concentrate may act as a fat substitute while maintaining
desirable structural, textural and organoleptic properties, a wider
variety of low-fat product may be obtained.
[0112] Additionally, it presents the cost advantage that a smaller
amount of concentrate is needed to obtain the desired effects.
[0113] The whey protein micelle concentrate (from evaporation or
microfiltration) can be used in liquid form as a dispersion or in
semi-solid form, or in a dried form. It may be used in a great
variety of applications such as those described above with respect
to the whey protein micelles applications.
[0114] For instance, the 20% protein concentrate obtained by
evaporation has a creamy, semi-solid texture (see FIG. 18) and can
be texturised in a spreadable texture by acidification using lactic
acid. This liquid, creamy, pasty texture can be used to prepare
acid, sweet, salty, aromatic, protein-rich consumables.
[0115] The whey protein micelles concentrate in any form may be
mixed with 5% of an acidic fruit base and 5% of sucrose in order to
obtain a stable whey protein enriched acidic fruit drink. It may
also be used in the manufacture of milk products, ice cream, or
used as coffee whitener amongst others.
[0116] Further applications include skin care and mouth care, such
as toothpaste, chewing gum, or gum-cleaning agent for instance.
[0117] The whitening power of the concentrate in any form is
tremendously increased in comparison to the non-concentrated
micelles or to the native protein powders. For example, the
whitening power of 4 mL of a 15% whey protein micelle concentrate
is equivalent to 0.3% of titanium oxide in 100 mL of a 2% soluble
coffee cup. Interestingly, it is possible to disperse soluble
coffee and sucrose into a whey protein micelle concentrate so that
a 3-in-1 concentrate having a total solids concentration of 60%
without fat is obtained.
[0118] The concentrate may be used as such or diluted depending on
the application.
[0119] For instance, the whey protein micelle concentrate in liquid
or dried form may be diluted to a protein content of 9% like in
sweet and condensed milk. The milk minerals, lactose and sucrose
can be added so that the final product will have similar
nutritional profile compared to milk, but only whey protein as the
protein source. This whey protein based blend is more stable than
sweet condensed milk against Maillard reaction (based on the speed
of development of a brown colour) when incubated 2 hours at
98.degree. C. (temperature of boiling water at an altitude of 833
m).
[0120] The dried form of the whey protein concentrate obtained by
the method of the present invention may be obtained by any known
techniques, such as spray-drying, freeze-drying, roller drying etc.
Thus, the whey protein concentrate of the present invention may be
spray-dried with or without addition of further ingredients and may
be used as a delivery system or a building block to be used in a
wide range of processes, e.g. consumables production, cosmetic
applications etc.
[0121] FIG. 8 shows a powder obtained by spray-drying without
addition of any further ingredients, having an average particle
diameter size greater than 1 micron due to the micelle aggregation
occurring during spray-drying. A typical average volume median
diameter (D.sub.43) of the powders of the invention is between 45
and 55 microns, preferably 51 microns. The surface median diameter
(D.sub.32) of the powders of the present invention is preferably
between 3 and 4 microns, more preferably it is 3.8 microns.
[0122] The moisture content of the powders obtained after
spray-drying is preferably less than 10%, more preferably less than
4%.
[0123] Such a whey protein micelle powder may comprise at least 85%
whey protein, from which at least 20%, preferably more than 50%,
most preferably more than 80% are in the micellar form.
[0124] Furthermore, the whey protein micelles powder of the present
invention have a high binding capacity for solvents such as water,
glycerol, ethanol, oil, organic solvents etc. The binding capacity
of the powders to water is at least 50%, preferably at least 90%,
most preferably at least 100%. For solvents such as glycerol and
ethanol, the binding capacity is of at least 50%. For oil, the
binding capacity is at least 30%. This property of the whey protein
micelle powders of the present invention allows these to be sprayed
or filled with further functional ingredients such as coffee,
caffeine, green tea extracts, plant extracts, vitamins, minerals,
bioactive agents, salt, sugar, sweeteners, aroma, fatty acids,
oils, protein hydrolysates, peptides etc. and mixtures thereof.
[0125] The functional ingredients may be included in the powder in
an amount of 0.1-50%. Thus, the powder may act as a carrier for
those functional ingredients. This presents the advantage that, for
instance, caffeine bitterness perception is reduced when filled
into the powders of the present invention and used in caffeinated
nutrition bars for instance.
[0126] Additional ingredients may be mixed to the whey protein
micelle concentrate prior to spray-drying. These comprise soluble
or non-soluble salts, peptides, protein hydrolysates e.g. cultured
wheat gluten hydrolysate for example, probiotic bacteria, stains,
sugars, maltodextrins, fats, emulsifiers, sweeteners, aroma, plant
extracts, ligands, bioactive agents, caffeine, vitamins, minerals,
drugs, milk, milk proteins, skimmed milk powder, micellar casein,
caseinate, vegetal protein, amino acids, polyphenols, pigment etc.
and any possible mixtures thereof. The resulting mixed whey protein
micelle powders comprise whey protein micelles and at least one
additional ingredient in a weight ratio ranging from 30:1 to
1:1000.
[0127] This co-spraydrying results in powders consisting of whey
protein micelles agglomerated or coated with an additional
ingredient. Preferably, the weight ratio of whey protein micelles
to additional ingredient is 1:1. This may further facilitate
solubilisation of these powders and may be of particular interest
in the manufacture of dehydrated food products such as soups,
sauces etc. comprising whey protein micelles.
[0128] The whey protein micelle powders obtained by the present
invention are characterised by an internal structure composed
mainly of hollow spheres but also of collapsed spheres (cf. FIG.
19). The hollow spheres structure can be easily explained by the
formation of the vapour droplet within the WPM concentrate droplet
during the spray drying. As the vapour droplet left the WPM droplet
due to a temperature above 100.degree. C., a hollow sphere
remained. The "bone-shape" is due to a combination of the water
evaporation from droplet and the external pressure within the
droplet.
[0129] The internal structure of the spherical hollow spheres was
investigated by SEM after sectioning the particle close to its
diameter (FIG. 20, left). The wall thickness of the particle was
around 5 .mu.m and seemed very smooth, whereas the inner structure
had a more grainy appearance. Increased magnification showed that
this graininess was in fact due to the presence of the initial WPM
that were fused to form the inner matrix of the powder particle.
Interestingly, the spherical shape of the micelles was kept during
spray drying as well the homogeneous particle size distribution
(FIG. 20, right).
[0130] Thus, on a microscopic basis, whey protein micelle powders
are characterised by a unique granule morphology of hollow or
collapsed spheres containing intact and individualised whey protein
micelles.
[0131] Whey protein micelle powders are characterised by a very
high flowability, which offers advantages not previously
obtainable. For instance, these powders behave almost as liquids
and present the advantages of easy usability and transferability.
The angle of repose of these powders is preferably below
35.degree., more preferably below 30.degree.. Such a low angle of
repose allows the powders of the present invention to be used as
flowing agents in food applications, for instance.
[0132] A very important feature of these powders, mixed or "pure"
is that the basic micelle structure of the whey proteins is
conserved. FIG. 15 shows a whey protein powder grain which has been
sectioned, and whereby the individual whey protein micelles are
observable. Furthermore, the micelle structure can be easily
reconstituted in solvents. It has been shown that the powders
obtained from whey protein micelle concentrate can be easily
redispersed in water at room temperature or at 50.degree. C. The
size and structure of the whey protein micelles are fully conserved
compared to the initial concentrate. For example, in FIG. 13, the
whey protein concentrate that was spray-dried at 20% protein
concentration has been redispersed in deionised water at 50.degree.
C. at a protein concentration of 4%. The structure of the micelles
has been probed by TEM and can be compared to FIG. 10. A similar
shape of micelles was obtained. The diameter of the micelles was
found to be 315 nm by dynamic light scattering with a
polydispersity index of 0.2. FIG. 16 also shows dispersion of a
freeze-dried whey protein micelle powder, wherein the micelles are
reconstituted.
[0133] The fact that the whey protein micelles and only a minor
aggregated fraction were observed in solution after reconstitution
of the spray-dried or freeze-dried powder confirms that whey
protein micelles are physically stable regarding spray-drying and
freeze-drying.
[0134] The powders of the present invention may be used in a wide
range of applications, such as all those described above in
relation to whey protein micelles and the concentrates thereof. For
instance, protein-enriched consumables, such as chocolate,
performance nutrition bars, dehydrated culinary products,
chewing-gum etc. can be easily produced by using the micelle
concentrate powders.
[0135] Due to their high stability to processing, the powders of
the present invention may also be further coated by emulsifiers or
gums, for instance. This may be advantageous to modulate the
functionality and the taste of these powders.
[0136] The following examples illustrate the present invention
without limiting it thereto.
EXAMPLES
[0137] The invention is further defined by reference to the
following examples describing in detail the preparation of the
micelles of the present invention. The invention described and
claimed herein is not to be limited in scope by the specific
embodiments herein disclosed, since these embodiments are intended
as illustrations of several aspects of the invention. Any
equivalent embodiments are intended to be within the scope of this
invention. Indeed, various modifications of the invention in
addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims.
Example 1
Micellisation of .beta.-Lactoglobulin by pH Adjustment
[0138] .beta.-Lactoglobulin (lot JE002-8-922, 13-12-2000) was
obtained from Davisco (Le Sueur, Minn., USA). The protein was
purified from sweet whey by ultra-filtration and ion exchange
chromatography. The composition of the powder is 89.7% protein,
8.85% moisture, 1.36% ash (0.079% Ca.sup.2+, 0.013% Mg.sup.2+,
0.097% K.sup.+, 0.576% Na.sup.+, 0.050% Cl.sup.-). All other
reagents used were of analytical grade (Merck Darmstadt,
Germany).
[0139] The protein solution was prepared at 0.2% concentration by
solvation of .beta.-lactoglobulin in MilliQ.RTM. water (Millipore),
and stirring at 20.degree. C. for 2 h. Then pH of aliquots was
adjusted to 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0
by HCl addition. The solutions were filled in 20 ml glass vials
(Agilent Technologies) and sealed with aluminum capsules containing
a silicon/PTFE sealing. The solutions were heated at 85.degree. C.
for 15 min (time to reach the temperature 2.30-3.00 min). After the
heat treatment, the samples were cooled in ice water to 20.degree.
C.
[0140] The visual aspect of products (FIG. 1) indicates that the
optimal pH of micellisation is 5.8.
Example 2
Micellisation of Whey Protein Isolate
[0141] Whey protein isolate (WPI) (Bipro.RTM., Batch JE032-1-420)
was obtained from Davisco (Le Sueur, Minn., USA). The composition
of the powder is reported in table 2.
[0142] The protein solution was prepared at 3.4% protein by
solvation of whey protein powder in MilliQ.RTM. water (Millipore),
and stirring at 20.degree. C. for 2 h. The initial pH was 7.2. Then
pH of aliquots was adjusted at 5.6, 5.8, 6.0, 6.2, 6.4 and 6.6 by
HCl 0.1N addition.
[0143] The solutions were filled in 20 ml glass vials (Agilent
Technologies) and sealed with aluminum capsules containing a
silicon/PTFE sealing. The solutions were heated at 85.degree. C.
for 15 min (time to reach the temperature 2.30-2.50 min). After the
heat treatment, samples were cooled in ice water to 20.degree.
C.
[0144] The turbidity of heated whey proteins has been determined at
500 nm and 25.degree. C., samples were diluted to allow the
measurement in the range of 0.1-3 Abs unit (Spectrophotometer
Uvikon 810, Kontron Instrument). Values were calculated for the
initial protein concentration 3.4%.
[0145] The pH of micellisation was considered to be reached upon
stability (less than 5% variation of the initial value) of the
absorbance measured at 500 nm within an interval of 10 minutes for
the same sample as illustrated by the FIG. 2. For this product the
optimal pH for micellisation was 6.0 to 6.2. For this pH adjusted
before heat treatment stable turbidity was 21 and residual soluble
protein evaluated by absorbance at 280 nm after centrifugation was
1.9%. We can conclude that 45% of initial proteins were transformed
in micelles at pH 6.0.
TABLE-US-00003 TABLE 2 Composition of WPI and sample
characteristics after micellisation Supplier Davisco Product name
Bipro Batch number JE 032-1-420 Composition (mg/100 g) Sodium 650
Potassium 44 Chloride* 10 if .ltoreq.40 10 Calcium 82 Phosphorus 49
Magnesium 6 Initial pH 7.2 pH micellisation 6.0 Turbidity (500
nm)for 21 3.4% protein in solution Residual Soluble protein (%) 1.9
by absorbance at 280 nm
Example 3
Microscopic Observation of Micelles
Production of Micelles:
[0146] Protein solution was prepared at 2% protein by solvation of
whey protein powder (WPI 90 batch 989/2, Lactalis, Retier, France)
in MilliQ.RTM. water (Millipore), and stirred at 20.degree. C. for
2 h. Then pHs of aliquots were adjusted using HC10.1N or NaOH
0.1N.
[0147] The solutions were filled in 20 ml glass vials (Agilent
Technologies) and sealed with aluminum capsules containing a
silicon/PTFE sealing. The solutions were heated at 85.degree. C.
for 15 min (time to reach the temperature 2.30-2.50 min). After the
heat treatment, the samples were cooled in ice water to 20.degree.
C. For this product the optimal pH for micellisation was 7.4.
Microscopic Observations:
[0148] Liquid micelle samples were encapsulated in agar gel tubes.
Fixation was achieved by immersion in a solution of 2.5%
glutaraldehyde in 0.1M, pH 7.4 cacodylate buffer and post-fixation
with 2% Osmium tetroxide in the same buffer, both solutions
containing 0.04% Ruthenium red. After dehydration in a graded
ethanol series (70, 80, 90, 96, 100% ethanol), the samples were
embedded in Spurr resin (Spurr/ethanol 1:1, 2:1, 100%). After
polymerization of the resin (70.degree. C., 48 hours), semi-thin
and ultra-thin sections were cut with a Leica ultracut UCT
ultra-microtome. Ultra-thin sections, stained with aqueous
uranyl-acetate and lead citrate, were examined in transmission
electron microscopy (Philips CM12, 80 kV).
[0149] TEM micrograph is presented in FIG. 3. Obtained micelles are
presenting a spherical shape with a diameter of 200 nm.
Particle Size Distribution
[0150] The intensity-based size distributions of micelles were
measured for those micelles obtained by heat-treatment of a 1 wt %
.beta.-lactoglobulin dispersion for 15 min at 85.degree. C. at pH
4.25 (positively charged with a zeta potential around +25 mV) and
at pH 6.0 (negatively charged with a zeta potential around -30 mV).
Z-averaged hydrodynamic diameter of the micelles was 229.3 mm at pH
4.25 an 227.2 at pH 6.0. .beta.-LG and whey protein aggregations
were followed using dynamic light scattering. A Nanosizer ZS
apparatus (Malvern Instruments, UK) equipped with a laser emitting
at 633 nm and with 4.0 mW power was used. The instrument was used
in the backscattering configuration, where detection is done at a
scattering angle of 173.degree.. This allows considerable reduction
of the multiple scattering signals found in turbid samples. Samples
were placed in a squared quartz cell (Hellma, pathlength 1 cm). The
path length of the light beam was automatically set by the
apparatus, depending on the sample turbidity (attenuation). The
fluctuation of the scattered intensity). The results are presented
in FIG. 6. It shows that the average particle is characterized by a
very narrow polydispersity index (<0.200).
Example 4
Micellisation of a .beta.-lactoglobulin at a Constant pH
[0151] The method described in example 1 was repeated using an
aqueous solution of 2% .beta.-lactoglobulin. The pH of this
solution has been adjusted to 7.0 after adding Arginine HCl
solutions to obtain a final salt concentration ranging from 5 to
200 mM and a final .beta.-lactoglobulin concentration of 1%.
Subsequent heat treatment (80.degree. C., 10 min, about 2 min
heating up) was carried out to produce micelles.
[0152] The results are shown in FIG. 4 and clearly indicate that
only in the ionic strength range of from about 50 to 70 mM, a
substantial turbidity can be observed, indicating the presence of
whey protein micelles.
Example 5
Preparing a Whitening Agent
[0153] Native whey proteins (WPI 95 batch 848, Lactalis; 8 wt-%
aqueous solution) were treated according to example 2. The
resulting product lightness (L) was measured in trans-reflectance
mode using a MacBeth CE-XTH D65 10.degree. SCE apparatus equipped
with a 2 mm measuring cell. The resulting lightness was L=74.8,
that could be compared to the value of L=74.5 for full-fat
milk.
Example 6
Preparing a Coffee Creamer
[0154] Native whey proteins (Bipro.RTM., lot JE 032-1-420, 0.5 wt-%
aqueous solution) were mixed at 50.degree. C. with 10 wt.-%
partially hydrogenated palm oil, 14 wt. % maltodextrin (DE 21) and
in presence of 50 mM phosphate-citrate buffer adjusted to the
micellisation pH of 6.0 for this Bipro.RTM.. The mixture was
homogenized under 400/50 bars using a Rannie homogeniser and
subsequently heat-treated for 15 minutes at 85.degree. C.
[0155] The emulsion obtained showed a high stability over a time
period of at least one month at the conditions of storage at
4.degree. C. and gave a whiteness of L=78 compared to a reference
liquid creamer (Creme a Cafe, Emmi, Switzerland) having a fat
content of 15% and a lightness of L=75.9.
Example 7
Preparing an Aqueous Foam
[0156] Native .beta.-lactoglobulin (Biopure, Davisco, lot JE
002-8-922, 2 wt-% aqueous solution) was mixed with 120 mM Arginine
HCl solution so that the final .beta.-lactoglobulin concentration
was 1 wt. % and Arginine HC160 mM. The pH was then adjusted to 7.0
by addition of 1N HCl. The mixture was then heat treated at
80.degree. C. for 10 minutes so that 90% of initial
.beta.-lactoglobulin was converted into micelles having a
z-averaged diameter of 130 nm. In this case, the diameter of the
micelles was determined using a Nanosizer ZS apparatus (Malvern
Instruments, UK). The sample was poured in a quartz cuvette and
variations of the scattered light were recorded automatically. The
obtained autocorrelation function was fitted using the cumulants
method so that the diffusion coefficient of the particles could be
calculated and thereafter the z-averaged hydrodynamic diameter
using the Stokes-Einstein law. For this measurement, the refractive
index of the solvent was taken as 1.33 and that of the micelles
1.45. A volume of 50 mL of the resulting dispersion of
.beta.-lactoglobulin micelles is then foamed by nitrogen sparging
through a glass frit generating bubbles of 12-16 .mu.m to produce a
foam volume of 180 cm.sup.3 using the standardised Foamscan.TM.
(ITConcept) apparatus. The volume stability of the foam was then
followed with time at 26.degree. C. using image analysis and
compared to the stability of the foam obtained with
.beta.-lactoglobulin treated in the same conditions, but without
Arginine HCl, where no micelles were formed. FIG. 5 shows that the
foam volume stability is greatly improved by the presence of
.beta.-lactoglobulin micelles.
Example 8
Whey Based Fermented Dairy Product--Fermentation Trials
Material
[0157] Whey protein isolate (WPI) (Bipro.RTM.) was obtained from
Davisco (Le Sueur, Minn., USA) (protein concentration 92.7%).
Spray dried whey permeate (Variolac 836): Lactose concentration:
83%-Minerals: 8%
Lactic Acid 50%
Edible Lactose (Lactalis)
[0158] De-ionized water
Method
[0159] The Bipro.RTM. powder was dissolved in de-ionized water in
order to have a protein concentration of 4.6%, i.e. for 3 litres of
solution 154.5 g of WPI powder and 2845.5 g of water. The hydration
time was 3 hours. After hydration, this solution has been divided
in samples of 200 ml to prepare the different trials:
TABLE-US-00004 TABLE 3 Whey permeate pH Heating 85.degree. C./15
Trial (%) Lactose (%) adjustment min 1 2.9 2.5 6.5 + 2 0 5 6 + 3 0
5 6.7 - 4 0 5 6.7 + 5 0 5 6.1 + 6 0 0 6 + 7 0 5 (added after pH 6 -
adjustment) 8 0 5 (added after pH 6 + adjustment)
[0160] For each solution, lactic acid at 50% has been added to
adjust the ph before heating.
[0161] Samples were heated with the double boiler up to 85.degree.
C. and maintain at this temperature during 15 minutes. After
heating, solutions were cooled at 40.degree. C. and inoculated with
Lactobacillus bulgaricus and Streptococcus thermophilus. Samples
were incubated 5 h30 in a steam room at 41.degree. C. before to be
placed in a cold room at 6.degree. C.
The results are presented in Table 4.
TABLE-US-00005 TABLE 4 Whey pH after Trial permeate Lactose pH
Heating 5 h 30 Aspect 1 + + 6.5 + 4.68 Very firm 2 - + 6 + 4.7 Firm
3 - + 6.7 - 5.78 Liquid 4 - + 6.7 + 4.81 Very firm 5 - + 6.1 + 4.59
Very firm 6 - - 6 + 4.99 Very firm 7 - added 6 - 4.87 Liquid with
after pH white speckles adjustment 8 - added 6 + 4.77 Firm after pH
adjustment
Example 9
Whey Protein Boosted Ice Cream with Reduced Fat Content
Material
[0162] Whey protein isolate (WPI, Prolacta90.RTM. from Lactalis,
Retiers, France) with a protein content of 90%
Skim milk powder with 35% protein content
Sucrose
Maltodextrins DE39
[0163] Anhydrous milk fat
Emulsifier
[0164] De-ionised water Edible hydrochloric acid 1M
Method
[0165] Using a double-jacketed 80 L tank, the Prolacta90.RTM.
powder was dispersed at 50.degree. C. in de-ionized water at a
protein concentration of 9.67 wt % under gentle stirring in order
to avoid foam formation, i.e. 3.3 kg of Prolacta90.RTM. were
dispersed in 31.05 kg of de-ionised water. After 1 hour of
dispersion, the pH of the dispersion was adjusted to the
micellisation pH by addition of HCl. The temperature of the
dispersion was raised to 85.degree. C. and maintained for 15
minutes in order to generate the whey protein micelles. After 15
minutes, the temperature was decreased to 50.degree. C. and the
additional ingredients were sequentially added to the micelles
dispersion (i.e. skim milk powder, maltodextrins DE39, sucrose,
emulsifier and anhydrous milk fat). The final amount of mix was 50
kg with total solids content of 39.5% and a fat content of 5 wt %.
After 30 minutes of hydration, the mix was two-step homogenised
(80/20 bars) and pasteurised (86.degree. C./30 s) before ageing
during overnight. The day after, the ice-cream mix was frozen at an
overrun of 100% using a Hoyer MF50 apparatus and hardened at
-40.degree. C. before storage at -20.degree. C. The final ice cream
contained 8 wt % proteins (20% caseins, 80% whey proteins) and 5 wt
% fat on the ice cream mix basis.
Example 10
Powdered Whey Protein Micelles Obtained by Spray-Drying
Material
[0166] Whey protein isolate (WPI, Prolacta90.RTM. from Lactalis,
Retiers, France) with a protein content of 90%
Edible lactose
Maltodextrins DE39
[0167] De-ionised water Edible hydrochloric acid 1M
Method
[0168] Using a double-jacketed 100 L tank, the Prolacta90.RTM.
powder was dispersed at 50.degree. C. in de-ionized water at a
protein concentration of 10 wt % under gentle stirring in order to
avoid foam formation, i.e. 11 kg of Prolacta90.RTM. were dispersed
in 89 kg of de-ionised water. After 1 hour of dispersion, the pH of
the dispersion was adjusted to the micellisation pH (around 6.3 in
that case) by addition of HCl. The temperature of the dispersion
was raised to 85.degree. C. and maintained for 15 minutes in order
to generate the whey protein micelles. After 15 minutes, the
temperature was decreased to 50.degree. C. and the 10 wt % whey
protein micelles dispersion was split in two batches of 50 kg. In a
first trial, 20 kg of lactose were dispersed in 50 kg of micelles
dispersion at 50.degree. C. and stirred for 30 min. Similarly, 20
kg of maltodextrins DE39 were added to the remaining 50 kg of whey
protein micelles dispersion.
[0169] The two mixtures were then spray dried into a NIRO SD6.3N
tower at a flow rate of 15 L/h. The air input temperature was
140.degree. C. and the air output temperature was 80.degree. C. The
water content of the obtained powders was lower than 5%.
[0170] The size of the whey protein micelles was determined in
presence of lactose and maltodextrin (DE39) in water using dynamic
light scattering before and after spray drying. The total protein
concentration was set to 0.4 wt % by dilution of the dispersion
before spray drying or reconstitution of the powder in order to be
in the dilute regime of viscosity for whey protein micelles. A
Nanosizer ZS apparatus (Malvern Instruments) was used and micelle
diameter was averaged from 20 measurements.
[0171] The particle diameter determined for whey protein micelles
in presence of lactose and maltodextrins (DE39) was 310.4 nm and
306.6, respectively. After reconstitution of the powders, the
respective diameters were found to be 265.3 nm and 268.5,
respectively. These measurements confirm than whey protein micelles
were physically stable regarding spray drying. The results were
corroborated by TEM microscopy observations of 0.1 wt % whey
protein micelles dispersions in water using negative staining in
presence of 1% phosphotungstic acid at pH 7. A Philips CM12
transmission electron microscope operating at 80 kV was used. Whey
protein micelles were observed in solution before spray drying and
after reconstitution of the spray-dried powder. No difference of
morphology and structure could be detected.
Example 11
Concentration by Evaporation
[0172] A whey protein isolate Prolacta 90 from Lactalis (lot
500648) has been reconstituted at 15.degree. C. in soft water at a
protein concentration of 4% to reach a final batch size of 2500 kg.
The pH was adjusted by addition of 1M hydrochloric acid so that the
final pH value was 5.90. The whey protein dispersion was pumped
through plate-plate APV-mix heat exchanger at a flow rate of 500
l/h. Pre-heating at 60.degree. C. was followed by heat treatment of
85.degree. C. for 15 minutes. Formation of whey protein micelles
was checked by measurement of particle size using dynamic light
scattering as well a turbidity measurement at 500 nm. The obtained
4% whey protein micelles dispersion was characterised by a
hydrodynamic radius of particles of 250 nm, a polydispersity index
of 0.13 and a turbidity of 80. The whey protein micelle dispersion
was then used to feed a Scheffers evaporator at a flow rate of 500
l/h. The temperature and vacuum in the evaporator were adapted so
that around 500 kg whey protein micelles concentrate having a
protein concentration 20% were produced and cooled down to
4.degree. C.
Example 12
Enrichment by Microfiltration
[0173] A whey protein isolate Prolacta 90 from Lactalis (lo 500648)
has been reconstituted at 15.degree. C. in soft water at a protein
concentration of 4% to reach a final batch size of 2500 kg. The pH
was adjusted by addition of 1M hydrochloric acid so that the final
pH value was 5.90. The whey protein dispersion was pumped through
plate-plate APV-mix heat exchanger at a flow rate of 500 L/h. A
pre-heating at 60.degree. C. was followed by heat treatment of
85.degree. C. for 15 minutes. Formation of whey protein micelles
was checked by measurement of particle size using dynamic light
scattering as well a turbidity measurement at 500 nm. The obtained
4% whey protein micelles dispersion was characterised by a
hydrodynamic radius of particles of 260 nm, a polydispersity index
of 0.07 and a turbidity of 80. The micelle form of the protein was
also checked by TEM, and micelle structures with an average
diameter of 150-200 nm were clearly visible (FIG. 9). The whey
protein micelle dispersion could be cooled at 4.degree. C. for
storage or directly used to feed a filtration unit equipped with a
6.8 m.sup.2 Carbosep M14 membrane at a flow rate of 180 L/h. In
that case, the concentration of the whey protein micelles was
performed at 10 to 70.degree. C. until the permeate flow rate
reached 70 L/h. In that case, the final whey protein concentrate
contained 20% of proteins. The structure of the micelles in the
concentrate was checked by TEM, and clearly no significant change
was visible compared to the 4% whey protein dispersion before
microfiltration (FIG. 10).
Example 13
Whey Protein Micelles Powder Comprising at Least 90% Whey
Protein
[0174] 200 kg of a whey protein micelle concentrate obtained by
microfiltration at 20% protein (see example above) were injected in
a Niro SD6.3N tower using an atomisation nozzle (O=0.5 mm, spraying
angle=65.degree., pressure=40 bars) at a product flow rate of 25
kg/h. The inlet temperature of product was 150.degree. C. and the
outlet temperature was 75.degree. C. The airflow in the tower was
150 m.sup.3/h. The moisture content in the powder was less than 4%
and the powder was characterized by a very high flowability.
Scanning electron microscopy of the powder exhibited very spherical
particles having an apparent diameter ranging from 10 to 100 .mu.m
(FIG. 8).
Example 14
Mixed Whey Protein Micelle Powder
[0175] 20 kg of a whey protein micelle concentrate were mixed with
1.7 kg of maltodextrins with a DE of 39 so that the final whey
protein micelle to maltodextrin ratio in powder is 70/30. This
mixture was injected in a Niro SD6.3N tower using an atomisation
nozzle (O=0.5 mm, spraying angle=65.degree., pressure=40 bars) at a
product flow rate of 25 kg/h. The inlet temperature of product was
150.degree. C. and the outlet temperature was 75.degree. C. The
airflow in the tower was 150 m.sup.3/h. The moisture content in the
powder was less than 4% and the powder was characterized by very
high flow ability.
[0176] The powders of examples 13 and 14, when reconstituted in
water, comprise essentially micelles having the same structure and
morphology as the whey protein micelle concentrate.
Example 15
Whey Protein Micelle Powder Obtained by Freeze-Drying
Material
[0177] Whey protein micelle concentrate at 20% protein produced by
microfiltration in example 12 with a protein content of 90%
Method
[0178] 100 g of whey protein micelles concentrate were introduced
in a plastic beaker and frozen at -25.degree. C. for one week. This
beaker was then placed in a lab-scale freeze drier Virtis equipped
with a vacuum pump. Sample was left for 7 days until the pressure
in the freeze drier remained constant at about 30 mbars. Around 20
g of freeze-dried whey protein micelles has been recovered.
Example 16
A Whey Protein Enriched Dark Chocolate without Sucrose
Material
TABLE-US-00006 [0179] Ingredients Percentage Whey protein micelle
powder from example 13 40-50% with a protein content of 90%
Sucralose 0.05-0.1% Anhydrous milk fat 3-5% Cocoa liquor 30-40%
Cocoa butter 5-15% Vanillin 0.005-0.015% Lecithin 0.1-1%
Method
[0180] Cocoa liquor is mixed with cocoa butter, butter fat, whey
protein micelle powder, sucralose, vanillin and lecithin. This
mixture is conched overnight at 65.degree. C. until a homogenous
paste is obtained. This chocolate mass is then moulded in chocolate
plates and cooled down. The dark chocolate is characterized by a
final whey protein content of 45-50%.
Example 17
A Whey Protein Enriched White Chocolate
Material
TABLE-US-00007 [0181] Ingredients Method 1 Method 2 Method 3 Whey
protein micelle 15-25% 25-35% 35-40% powder from example 13 with a
protein content of 90% Sucrose 40-45% 30-35% 30-35% Anhydrous milk
fat 1-10% 1-10% 1-10% Whey powder 2-10% 2-10% 0% Cocoa butter
20-30% 20-30% 20-30% Vanillin 0.01-0.1% 0.01-0.1% 0.01-0.1%
Lecithin 0.1-1% 0.1-1% 0.1-1%
Method 1
[0182] Whey protein micelles, whey powder, sucrose and vanillin are
mixed and ground until the desired particle size distribution is
obtained. This mixture is then conched overnight at 65.degree. C.
with cocoa butter, anhydrous milk fat and lecithin until a
homogenous paste is obtained. This chocolate mass is then moulded
in chocolate plates and cooled down. This white chocolate is
characterized by a final whey protein content of 20%.
Method 2
[0183] Whey protein micelles, whey powder, sucrose and vanillin are
mixed and ground until the desired particle size distribution is
obtained. This mixture is then conched overnight at 65.degree. C.
with cocoa butter, anhydrous milk fat and lecithin until a
homogenous paste is obtained. This chocolate mass is then moulded
in chocolate plates and cooled down. This white chocolate is
characterized by a final whey protein content of 30%.
Method 3
[0184] Whey protein micelles, sucrose and vanillin are mixed and
ground until the desired particle size distribution is obtained.
This mixture is then conched overnight at 65.degree. C. with cocoa
butter, anhydrous milk fat and lecithin until a homogenous paste is
obtained. This chocolate mass is then moulded in chocolate plates
and cooled down. This white chocolate is characterized by a final
whey protein content of 30-35%.
Example 18
Aqueous Dispersion of Whey Protein Micelles Coated with Sulfated
Butyl Oleate (SBO) or any Other Negatively Charged Emulsifier
Material
[0185] Whey protein micelle (WPM) powder from example 13 with a
protein content of 90%
SBO
[0186] Hydrochloric acid (1M)
Method
[0187] WPM powder described in example 13 is dispersed in MilliQ
water to achieve a final protein concentration of 0.1 wt %. This
dispersion is filtered on 0.45 .mu.m filters in order to remove
possible WPM aggregates. The pH of this WPM dispersion was brought
down to 3.0 by addition of hydrochloric acid 1M. A 1 wt %
dispersion of SBO is prepared at pH 3.0.
[0188] The hydrodynamic radius and zeta potential of these WPM was
determined using the Nanosizer ZS apparatus (Malvern Instruments
Ltd.). Diameter was 250 nm and electrophoretic mobility+2.5
.mu.m.cm.V.sup.-1.s.sup.-1. The hydrodynamic radius and
electrophoretic mobility of the SBO dispersion at pH 3.0 are 4 nm
and -1.5/-2.0 .mu.m.cm.V.sup.-1.s.sup.-1, respectively.
[0189] After having performed this preliminary characterization,
the SBO dispersion is used to titrate the WPM one, while following
evolution of hydrodynamic radius and electrophoretic mobility of
the mixture. It was found that the hydrodynamic radius was constant
around 250-300 nm until a WPM/SBO weight-mixing ratio of 5:1 was
reached. At this point, the hydrodynamic radius diverges
dramatically to 20000 nm and precipitation of complexes WPM SBO is
encountered. Upon further addition of SBO, higher than a mixing
ratio of 5:1, the hydrodynamic progressively decreased to 250 nm,
as found initially for WPM, levelling of from a ratio of 4:1 on.
Following the electrophoretic mobility of the mixture showed that
it decreased upon addition of SBO, reaching zero value for a mixing
ratio of 5:1. Then it continued to drop upon SBO addition, starting
levelling of at -3.0 .mu.m.cm.V.sup.-1.s.sup.-1 from ratio 4:1
on.
[0190] The explanation for these results is that the positively
charged WPM are, in a first step coated electrostatically with the
negative head of the SBO until full charge neutralisation is
achieved (mixing ratio 5:1). At this point, the hydrophobic tails
from the SBO are able to self-associate, leading to
over-aggregation with very large hydrodynamic diameter and
precipitation of complexes. Upon further addition of SBO, the
hydrophobic tails associate further to form a double coating,
exposing their negative head to the solvent. This lead to
negatively charged WPM with a double coating of SBO (see FIG. 17)
comparable to a full protein core liposome.
[0191] Similar results have been obtained with other acidic food
grade Emulsifiers such as DATEM, CITREM, SSL (from Danisco) in
aqueous solution at pH 4.2 where they are mainly ionized in their
anionic form (--COO.sup.- chemical functions).
Example 19
A Protein-Enriched Bechamel Sauce
Material
[0192] Mixed whey protein micelle powder from example 14 with a
protein content of 70%
Butter
Flour
[0193] Skim milk
Salt
Method
[0194] 30 g of mixed whey protein micelle powder are dispersed in 1
litre of skim milk under heating. 30 g of butter and 80 g of flour
are then added together with 2.85 g of salt. The mixture is then
boiled in order to produce a bechamel sauce having a whey protein
content of about 3 g/100 g.
Example 20
A Whey Protein-Enriched Base for Performance Bar
Material
TABLE-US-00008 [0195] Ingredients Percentage Mixed whey protein
micelle powder from 40-50% example 13 with a protein content of 90%
(moisture 3.5%) Brown rice syrup 35-45% Maltitol 5-10% Glycerol
10-15%
Method
[0196] Brown rice syrup is mixed with maltitol and glycerol at
25.degree. C. Whey protein micelle powder is then added and mixing
is performed for 10 minutes. A whey protein-enriched base for
performance bar is then obtained and can be mixed with other
ingredients (minerals, vitamins, flavours). This preparation
contains more proteins than milk (38%).
Example 21
Determination of Repose Angle for Spray Dried Whey Protein Micelle
Powder, Mixed Whey Protein Micelle Powder, Whey Protein Isolate
Powder and Low Heat Skim Milk Powder
Material
[0197] Whey protein micelle powder from example 12 with a protein
content of 90% (moisture 3.5%)
[0198] Mixed whey protein micelle powder from example 13 with a
protein content of 90% (moisture 3.5%)
[0199] Whey protein isolate powder Prolacta 90 (lot 500658 from
Lactalis, France; moisture 4%)
[0200] Low heat skim milk powder (lot 334314 from Emmi,
Switzerland; moisture 3.5%)
[0201] Measuring device described to measure repose angle for
powders according to ISO norm 4324
Method
[0202] The powder is placed in a funnel with a stem diameter of 99
mm and the powder is forced to flow using the agitator. The powder
falls on a transparent plastic vessel with diameter 100 mm and a
height of 25 mm. The angle of repose, .PHI., is measured from the
following equation:
Repose angle .PHI.=ARCTAN(2h/100)
[0203] Where h is the maximum height of the powder cone than can be
obtained, all surface of the plastic vessel being covered with
powder.
[0204] Results from the repose angle test (values are mean of 3
measurements and standard deviation is indicated).
TABLE-US-00009 Mixed whey Low heat Whey protein protein Whey
protein skim milk micelle powder micelle powder isolate powder
Repose 24.6 .+-. 1.1 27.3 .+-. 0.7 34.3 .+-. 0.5 43.8 .+-. 2.8
angle (.degree.)
[0205] Repose angle results clearly show that whey protein micelle
powder, pure or mixed with maltodextrins, exhibit a significantly
lower angle than the initial whey protein powder or even skim milk
powder. A repose angle lower than 35.degree. is characteristic of
very well flowing powders.
* * * * *