Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CA 02286094 1999-10-14 r3!~':Z.~
1
Use of at least one protein fraction extracted from Hibiscus
esculentus seeds and cosmetic composition containing such a
fraction
The present invention concerns the field of cosmetology, in
particular cutaneous and capillary applications, and relates
to the use of at least one protein fraction extracted from
Hibiscus esculentus and to a composition containing at least
one such extract.
The Hibiscus esculentus (Abelmoshus esculentus or okra from
the Malvaceae family) is a plant of African origin introduced
into the United States and East Indies under the Spanish name
of gumbo. It is one of the botanical species which has been
cultivated for its pods for more than 2,000 years.
okra grows in numerous regions of the world such as India,
Malaysia, the Philippines, America (Mid-West), Mediterranean
regions, Africa and, more generally, in tropical regions.
The fruits (pods) which are eaten young as vegetables are
long, green and tapering; they have a delicate flavour and a
mucilaginous internal texture.
Apart from the pods, which are of interest on account of the
gum which they contain, research has also been carried out on
the seeds of Hibiscus esculentus in order to study their
potential as a new source of proteins.
To this end, the chemical composition of the whole seed of
different varieties of okra (outer skin plus endosperm) has
been determined.
Similarly, the properties (protein solubility, amino acid
composition, emulsification capacity, foaming capacity,
CA 02286094 1999-10-14
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nutritional value) of various products obtained from the seeds
(whole flour prepared from skinned seeds, delipidated flour,
concentrate and protein isolate) have been studied for food
purposes (see, for example, Bryant LA, Montecalvo J, Morey KS,
Loy B: "Processing, functional and nutritional properties of
okra seed products", Journal of food science, vol. 53, No. 3,
818-816).
okra seed mainly contains the following substances in o by
weight, relative to the dry material:
17.7 to 21.80 of proteins
- 14.7 to 20.060 of lipids
- 4.33 to 4.620 of ash
- 6.84 to 7.920 of water
- 0.0032a of gossypol,
and, in particular, traces of the following substances:
- Calcium: 282.26
mg/100
g
- Iron: 10.26 mg/100
g
- Thiamine: 0.69 mg/100
g
- Riboflavin: 0.14 mg/100
g
- Niacin: 4.01 mg/100
g
- a-tocopherol: 30.4 mg/100
g.
(See Karakoltsidis PA, Constantidines SM: "okra seed: a new
protein source", J. Agric Food Chem., 1975, 23 No. 6, 1204-
1207/Wandawi AL: "Chemical composition of seeds of two okra
cultivars Abelmoschus esculentus", Journal of agricultural and
food chemistry, 1983, 31 No. 6, 1355-1358).
The amino acid composition of concentrated Hibiscus esculentus
seed proteins or protein isolates resembles that of Soya
proteins and is close to that of casein (see table below).
CA 02286094 1999-10-14
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Amino Hibiscus HibiscusProtein Protein Soya Casein
acid (seeds) (seeds) concentrateisolate (seed) Karakot-
g/16 KarakoltsidisBryant Bryant Bryant Karakot-sidis
g et al et al LA LA et sidis et
of Mandawi AL et al al et al
nitrogen al
Asp 11.82 to 15.4711.57 10.89 12.12 17.00 7.11
Thr 3.02 to 4.38 2.86 3.37 2.90 5.47 4.65
Ser 5.25 to 6.71 5.07 5.23 4.97 7.42 6.02
Glu 20.48 to 22.0815.91 19.15 17.35 21.05 21.19
Pro 3.83 to 6.06 3.79 4.88 4.98 7.71 11.54
Gly 5.79 to 6.66 4.78 7.77 4.16 4.32 1.97
Ala 5.89 to 6.66 4.83 4.53 4.59 6.13 3.07
Val 4.0 to 6.4 4.24 4.42 4.33 5.26 6.72
Cys 1.54 to 2.53 3.63 1.89 1.90 1.61 0.36
Met 1.29 to 1.85 1.83 2.18 2.21 1.25 2.78
Ile 3.15 to 4.65 2.96 3.06 3.13 4.46 5.40
Leu 6.68 to 8.47 6.21 6.96 6.97 9.35 9.49
Tyr 3.6 to 3.83 3.46 5.15 4.03 3.72 5.81
Phe 3.93 to 4.7 4.41 4.80 4.85 5.26 5.23
Lys 7.24 to 8.9 6.22 6.19 6.47 8.00 8.80
His 1.78 to 2.99 2.34 3.61 3.83 2.67 2.91
Arg 11.04 to 12.4611.17 10.02 9.98 10.07 3.74
Trp 0.85 to 0.96 2.02 2.57 2.03 nd nd
With regard to the casein, close contents of threonine,
serine, glutamic acid, valine, isoleucine, leucine,
phenylalanine, lysine and histidine are noted in particular.
The various aforementioned studies therefore demonstrate the
value of Hibiscus esculentus seeds as potential sources of
proteins from a food point of view.
Furthermore, the use for dermatological purposes of a viscous
liquid product obtained by hot extraction and/or extraction
under pressure from the fruits of Hibiscus esculentus (FR-A-2
679 443) as well as the use of a powdered material composed of
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polysaccharide extracted from immature Hibiscus esculentus
seeds in a cosmetic product (JP-A-57/199969) are also known.
Now the inventors have found that it was possible to use
extracts of Hibiscus esculentus seeds directly in cosmetics
and that the use of at least one preferably soluble protein
fraction extracted from Hibiscus esculentus or okra seeds, in
particular as a substitute for casein, in a cosmetic
composition or product yielded a composition or product having
surprising and advantageous specific properties.
A strong cellular nutritive power, a smoothing and biofilm-
forming effect, conditioning, restructuring and repairing
effects as well as anti-irritant, light-protecting, soothing
and cutaneous anti-ageing effects have thus been found.
The aforementioned extracts can be used not only for skin care
and hygiene applications (products for the face or for the
body, day or night products, solar products, anti-wrinkle
hygiene products, slimming products), but also in the field of
hair care and hygiene (lotions or shampoos; creams; mousses;
protective products, repairing products, softeners, film-
forming agents and light protectors; perming and colouring
products).
Proteins can be prepared by conventional methods of extraction
of vegetable proteins and preparation of protein concentrates
or isolates known to a person skilled in the art and
described, in particular, in the aforementioned article by
Bryant LA, Montecalvo J, Morey KS and Loy B.
The raw material consists of Hibiscus esculentus seeds or seed
flour (protein content - 21.6o relative to the dry material).
The flour thus obtained may be delipidated by extraction in
hexane at 45°C (advantageously 3 successive extractions).
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The oil yield is 16.2 to 23.90 by weight, depending on whether
the starting material is a whole seed flour or a flour from
seeds partially freed of the outer skins or shells by sifting.
The delipidated flours partially freed of the seed shells
contain between 37 and 44% by weight of proteins (N x 6.25).
Various processes for obtaining and preparing extracts of
Hibiscus esculentus or okra seeds will be described
hereinafter by way of illustrative, non-limiting examples.
Example 1
100 g of non-delipidated enriched flour (partially freed of
the shell waste) in one litre of the following media:
- distilled water,
- distilled water containing 1 g/1 of NaCl,
- distilled water containing 5 g/1 of NaCl,
- distilled water containing 10 g/1 of NaCl
are added.
After stirring for 15 minutes, the pH of the solution is
adjusted to 9 with NaOH 4 N.
Extraction is carried out for one and a half hours at ambient
temperature while keeping the pH at 9.
After centrifugation, the upper lipidic layer is removed and
the aqueous supernatant liquid is collected.
The golden yellow supernatant liquid is adjusted to pH - 7.5
then. filtered to 0.22 ~.m; it is noted that the higher the salt
content of the solvent, the easier filtration is.
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The protein content of the filtrate is determined by the
biuret method. The supernatant liquids remain opalescent.
The following results are obtained:
Extraction solvent Proteins in the biuret
filtered extract (g/1)
distilled water 16.9
NaCl 1 g/1 13.3
NaCl 5 g/1 9,5
NaCl 10 g/1 10.2
Example 2
25 g of enriched delipidated flour (freed from shell debris)
are added in 250 ml of distilled water containing 5 g/1 of
sodium chloride.
After stirring for 15 minutes, the pH of the solution is
adjusted to the following pH according to the test: 6-6.5-7-
7.5.
Extraction is carried out for one hour at ambient temperature.
After centrifugation, the supernatant liquid is recovered,
then filtered over 5 Vim.
The following results are obtained:
pH Biuret proteins Proteins (N x 6.25)
(g/1) (g/1)
6 12.5 13.8
6.5 13.3 14.8
7 14.6 15.1
7.5 15.5 . 16.1
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Example 3
25 g of enriched delipidated flour are added in 250 ml of
distilled water containing 5 g/1 of sodium chloride.
The pH of the solution is adjusted to 8 after stirring for 15
minutes.
Extraction is carried out for 6 hours at ambient temperature.
After centrifugation, the supernatant liquid is recovered and
the pH adjusted to 7.5, then the solution is filtered over 5
~.m .
The following results are obtained:
Duration (hours) Biuret proteins (g/1)
2 18.45
4 20.3
19.7
Example 4
25 g of enriched delipidated flour are added in 250 ml of
distilled water containing 5 g/1 of sodium chloride.
After stirring for 15 minutes, the pH of the solution is
adjusted to pH 7.5.
Extraction is carried out for 6 hours at 45°C.
After centrifugation, the supernatant liquid is recovered, its
pH is adjusted to 7.5 then the solution is filtered over 5 Vim.
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The following results are obtained:
Duration (hours) Biuret proteins (g/1)
18.0
4 18.1
13 8
Example 5
300 g of enriched delipidated flour are added in 3 1 of
distilled water containing 5 g/1 of sodium chloride.
After stirring for 15 minutes, the pH of the solution is
adjusted to pH 7.5.
Extraction is carried out for 2 hours at 50°C.
After centrifugation, the supernatant is recovered then
filtered over 5 ~.m.
The following results are obtained:
Biuret proteins: 13.7 g/1
Kjeldahl proteins: 14.6 g/1
One litre of supernatant liquid is removed and its pH is
adjusted to 4.5 using 4N sulphuric acid .
After 30 minutes of stirring, the solution is centrifuged and
the precipitate is collected, then washed with water at pH
4.5. It is then freeze-dried: a powder of which the protein
content is 85.30 by weight is obtained.
Example 6
600 g of enriched delipidated flour in 6 litres of distilled
water containing 5 g/1 of NaCl are added while stirring in a
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thermostatically controlled reactor. The pH measured is
adjusted to 7.5.
The solution is pumped into an ultrasonic tube with an
integral passage by means of a peristaltic pump (ultrasonic
power 600 4~1 - frequency 20,000 Hz), the flow rate being 60
1/h.
Two discontinuous passages by successive charges are produced
(the extract is collected after passage in the ultrasonic tube
in a second reactor).
After centrifugation, then filtration over 5 ~.m, the following
results are obtained:
- proteins from the extract after the first passage: 14.2 g/1
- proteins from the extract after the second passage: 15.2 g/1
Example 7
300 g of enriched delipidated flour in 6 litres of distilled
water containing 5 g/1 of NaCl are added in a thermostatically
controlled reactor while stirring. The measured pH is adjusted
continuously to 7.5.
The solution is introduced continuously for 1 hour in a closed
circuit in the integral passage ultrasonic tube from example 6
(ultrasonic power: 700 W - frequency: 20,000 Hz), the flow
rate being 60 1/h.
A circulation of cold water allows the temperature of the
solution in the vessel to be kept at about 33°C.
Samples are taken after 15 min, 30 min, and 45 min of
extraction.
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After centrifugation, then filtration of the extracts over 5
~,m, the following results are obtained:
Duration of extraction (min) Proteins in the biuret
filtered extract (g/1)
8.23
30 8.56
45 8.89
60 9.89
Example 8
25.0 kg of distilled water are introduced into a reactor and
the following operations are carried out in succession:
- dispersing with stirring 2.5 kg of whole flour obtained by
crushing whole hibiscus seeds,
- adjusting the pH to 9 with NaOH 4N after 15 minutes of
dispersion,
- extracting with stirring for 2 hours at ambient temperature
while keeping the pH at 9 by addition of NaOH 4N,
- centrifuging for 10 minutes at 5,000 g,
- recovering the cloudy beige supernatant liquid,
- adjusting the pH to 7.5 by addition of H2S04 4N,
- centrifuging,
- clarifying by renewed centrifugation,
- recovering the opalescent supernatant liquid,
- filtering to 0.5 ~tm,
- recovering the supernatant liquid,
- spraying.
A clear beige powder can therefore be recovered with a spray
yield of 62.70 by weight.
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Example 9
In a first variation of example 8, the following operations
can also be carried out:
- adjusting the pH of a proportion of the extract prepared
according to example 8 to 4.1 by addition of H2S04 4N,
- leaving to rest at +4°C (formation of a precipitate - about
500 of the total volume),
- centrifuging to 5,000 g for 15 min,
- washing the caps with distilled water at pH = 4.1,
- centrifuging to 5,000 g for 15 min,
- collecting the precipitate,
- reconstituting the precipitate in distilled water (l00 of
the volume prior to precipitation),
- adjusting the pH to 7.5 by adding NaOH 4N while stirring,
- homogenising to assist solubilisation and homogenisation,
- centrifuging for 10 min at 5,000 g to eliminate the
insoluble material,
- spraying the protein concentrate (spray yield based on the
dry extract: 82.90a by weight).
Example 10
In a second variation of example 8, the following operations
can be carried out:
- adjusting the pH of a proportion of the extract prepared in
example 8 to pH 5 by addition of H2S04 4N,
- leaving to rest for one hour at ambient temperature,
- centrifuging,
- collecting the precipitate,
- reconstituting the precipitate in l00 of the volume prior to
precipitation without carrying out the washing stage,
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- adjusting the pH to 7.5 by adding NaOH 4N while stirring,
- homogenising to assist solubilisation and homogenisation,
- centrifuging for 10 min to 5,000 g to eliminate the
insoluble material,
- atomising the protein concentrate.
Gel permeation analysis over a column of the superose 12 HR
type of the fraction extracted by the various methods
described hereinbefore allows at least 6 different fractions
to be characterised as shown in the diagrams in Fig. 1 to 6 of
the accompanying drawings, in which:
Fig. 1 shows the molecular weight distribution of the proteins
of an extract of whole seed flour of Hibiscus esculentus
(extraction: 2 hours at ambient temperature in water, pH = 9),
Fig. 2 shows the molecular weight distribution of the proteins
of an extract of dehusked seed flour of Hibiscus esculentus
(extraction: 1.5 hour at ambient temperature in water, pH -
9) ,
Fig. 3 shows the molecular weight distribution of the proteins
of an extract of dehusked seed flour of Hibiscus esculentus
(extraction: 1.5 hour at ambient temperature in NaCl 10 g/l,
pH = 9),
Fig. 4 shows the molecular weight distribution of the proteins
of an extract of dehusked seed flour of Hibiscus esculentus
(extraction in an ultrasonic tube, 1 hour in NaCl 5 g/l, pH -
7.5),
Fig. 5 shows the molecular weight distribution of the proteins
of a protein concentrate obtained by precipitation at pH = 4.1
of an extract of whole seed flour of Hibiscus esculentus, and
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Fig. 6 shows the molecular weight distribution of the proteins
of a protein concentrate obtained by precipitation at pH - S
of an extract of whole seed flour of Hibiscus esculentus.
The following summary table (in two parts) shows the
distribution of the various protein fractions extracted.
Extraction pH = pH = 9 pH = pH = pH = pH =
9 9 9 6.5 8
conditions water NaCl 1 NaCl NaCl NaCl NaCl
5 10 5 5
g/1 g/1 g/1 g/1 g/1
4 h
PM > 500,000 13.7 9.8 4.4 3.7 2.9 2,7
Da
PM between 100,000
and 500,000 Da 12.2 11.2 10.7 14.4 9.7 17.3
PM between 30,000
and 100,000 Da 4 3.3 7.6 7.7 14.7 7.9
PM between 5,000
and 30,000 Da 41.2 44.7 46.3 51.2 49.6 45.9
PM lower than
or
equal to 5,000 28.9 31 31 23 23.1 26.2
Da
Extraction pH = pH = pH = pH Protein Protein
7.5 7.5 7.5 =
9
conditions NaCl NaCl NaCl Hz0 concentrateconcentrate
5 5 5
g/1 g/1 g/1
whola
6 h 15 min 60 min flour (pH 4.1) (pH 5)
US US
PM > 500,000 4 13 20.9 30.5 35.5 46.6
Da
PM between 100,000
and 500,000 Da 13.1 14.2 3.8 15 12.2 17.5
PM between 30,000
and 100,000 Da 4.3 6.1 15.1 5.9 8.1 7,3
PM between 5,000
and 30,000 Da 50.2 46 35.3 26.4 35.4 22.6
PM lower than
or
equal to 5,000 28.4 20.7 24.9 22.2 8.8 6
Da
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The aforementioned 6 protein fractions consist of:
- a fraction (designated F1) having a very high molecular
weight (between 1,000,000 and 1,500,000 Da according to
column calibration),
- a fraction (designated F2) having a molecular weight between
250,000 and 350,000 Da visible mainly on the extracts of
whole seed flour,
- a fraction (designated F3) having a molecular weight between
130,000 and 180,000 Da,
- a fraction (designated F4) corresponding to a molecular
weight between 17,000 and 22,000 Da,
- two molecular weight fractions of about 3,300 Da (F5) and
about 2,300 to 2,600 Da (F6) respectively.
According to the extraction conditions, the fraction F1
constitutes between 2.7 and 50.OOo of the proteins and the
fraction F4 constitutes between 16 and 520 (average 400) of
the total proteins.
It is noted that, the higher the salt content of the
extraction solvent at the outset, the smaller the fraction F1.
The extracts obtained by means of the examples of the
described processes can be used directly in liquid form or
after drying by conventional dehydration techniques (spraying,
freeze-drying, etc.).
The protein fractions extracted according to the
aforementioned examples have the advantage, relative to the
natural state in which they occur in the seeds, of having an
identical native chemical structure, of being able to be
partially or completely purified (that is freed of other seed
components such as lipids, fibres, saccharoses or the like)
and of having a composition which can be varied as a function
CA 02286094 1999-10-14
of the extraction and/or purification process employed (total
proteins of the crude extract, protein concentrate or purified
protein fraction (s) .
In relation to lactic casein, improved solubility is noted in
addition to the desired renewable vegetable origin in the
cosmetics field, while maintaining an amino acid composition
close to casein, of which the nutritional, moisturising and
film-forming properties for skin and the superficial body
growths are known.
The protein fractions can be used either in their native form
without modification of the initial structures or in the form
of natural associations of two or of all the extracted
fractions corresponding to the various peaks of the
chromatograms shown in the accompanying drawings or again in
isolated form, that is the use of one or more fractions
corresponding to one or more peaks of the aforementioned
chromatograms.
The protein fractions can also be used in the form modified or
functionalised by any one of the following treatments:
- polymerisation,
- chemical hydrolysis of the hibiscus proteins
- enzymatic hydrolysis of the hibiscus proteins. To this end,
proteases originating from extracts of animal, vegetable,
microbial or fungal origin can be used to modify the hibiscus
proteins: pepsin, trypsin, chymotrypsin/papain, pronase,
bromelain/endoproteinase, thermitase, proteases of Bacillus
subtilis, Aspergillus Niger and Aspergillus
Oryzae/subtilisine, alcalase, neutrase.
- microbiological transformation with use of hibiscus proteins
as substrate for fermentation by various microorganisms such
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as yeasts (Saccharomyces), mould fungus (of the Aspergillus
type), bacteria such as Bacillus or the like.
- chemical or enzymatic functionalisation by processes such as
desamidation, succinylation or phosphorylation.
- quaternisation.
- grafting of saccharidic or lipidic molecules.
The invention also relates to a cosmetic composition, in
particular for topical application, for the skin and/or the
superficial body growths, characterised in that it contains at
least one preferably soluble protein fraction extracted from
Hibiscus esculentus seeds as substitute for casein.
According to a characteristic of the invention, the protein
fractions) is (are) extracted from non-delipidated or
delipidated flour of whole or dehusked Hibiscus esculentus
seeds by water or saline solutions at various pH.
According to a preferred embodiment of the invention, the
protein fracbon(s) is (are) extracted in aqueous solution
under the influence of ultrasound and this fracb on(s) is
(are) purified by a purification process selected from the
group formed by precipitation, absorption, ion or affinity
exchange chromatography and ultrafiltration.
The total or native protein fraction is selected from the
group of total and native protein fractions extracted from
Hibiscus esculentus seeds and having apparent molecular
weights when filtered over gel of 1,000,000 to 1,500,000 Da,
250,000 to 350,000 Da, 130,000 to 180,000 Da, 17,000 to 22,000
Da, 3,300 Da and 2,300 to 2,600 Da.
The aforementioned protein fracbon(s) can consist of~ a
chemical or enzymatic hydrolysate prepared from native
proteins, can be obtained by polymerisation or
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depolymerisation of native proteins or can be chemically
modified by grafting.
According to a first embodiment of the invention, the cosmetic
composition contains at least two protein fractions of
different apparent molecular weights.
According to a second embodiment of the invention, the
cosmetic composition contains an extract of Hibiscus
esculentus seeds consisting of all the soluble protein
fractions naturally present in these seeds.
Said cosmetic composition according to one of claims 1 to 11,
characterised in that it preferably contains between O.Olo and
50.OOo by weight, preferably between 1 and 25a by weight, of
protein fractions) extracted from Hibiscus esculentus seeds
as obtained in any one of the examples of processes described
hereinbefore.
As non-limiting embodiments of the invention, various cosmetic
products or compositions containing at least one soluble
protein fraction extracted from Hibiscus esculentus or okra
seeds will be described hereinafter.
Example 1
A cosmetic product according to the invention, in the form of
a moisturising smoothing day cream for the face and body can,
for example, have a composition by weight made up of the
following fractions A, B, C and D, as indicated hereinafter.
Fraction A:
- Cutina MD 14.OOo
- Eutanol G 6.OOo
- Cetiol B 6.OOo
- Eumulgin B1 1.500
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- Eumulgin B2 1.500
Fraction B:
- Extracts of total native proteins from hibiscus
according to the aforementioned example 1 8.OOo
Fraction C:
- Allantoin 0.200
- Methylparaben 0.200
- Germall 115 0.300
- Distilled water 62.OOo
Fraction D:
- Fragrance 0.300
The process for the preparation and production of the
aforementioned day cream essentially involves heating fraction
A to 75°C, preparing fraction C at 75°C, pouring fraction A
into fraction B with turbine stirring, adding fraction C at
about 50°C then continuing planetary stirring to ambient
temperature and finally adding fraction D.
Example 2
A cosmetic product according to the invention in the form of a
restructuring, anti-wrinkle nourishing repairing night cream
could, for example, have a composition by weight consisting of
the following fractions A, B, C and D, as mentioned
hereinafter.
Fraction A:
- Miglyol 810 6.OOo
- Myrj 51 3.OOo
- Arlatone 983 S 2.OOo
- Stearic acid TP 4.00%
- Cetyl alcohol 3.00%
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Fraction B:
- Propylene glycol 3.OOo
- Elestab LS 388
(Laboratoires Serobiologiques) 2.500
- Distilled water 61.200
Fraction C:
- Sprayed extracts of native hibiscus proteins
according to the aforementioned example 9 2.OOo
- Distilled water 13.OOo
Fraction D:
- Fragrance 0.30%
The process for preparing and producing the aforementioned
night cream essentially involves preparing and heating
fractions A and B to 75°C, pouring fraction A into fraction B
with turbine stirring, preparing (separately and
extemporaneously) the solute of fraction C by stirring of the
atomisate in distilled water, adding fraction C to emulsion A
+ B while cooling to about 50°C, then continuing planetary
stirring from 45°C and to ambient temperature while adding the
fragrance at about 40°C.
Example 3
A cosmetic product according to the invention in the form of a
protective conditioning covering light-protective
microemulsion for hair could, for example, have a composition
by weight made up of the following fractions A, B, C and D, as
mentioned hereinafter.
Fraction A:
- Brij 96 11.800
- Arlatone G 10.000
- Paraffin oil 13.OOo
CA 02286094 1999-10-14
Fraction B:
- Distilled water 53.700
- Methylparaben 0.20a
- Elestab 4112
(Laboratoires Serobiologiques) 0.300
Fraction C:
- Native hibiscus proteins in the form of a protein
concentrate according to the aforementioned example 6
5.OOo
- Distilled water S.OOo
Fraction D:
- Fragrance 0.20%
- Tween 20 0.80%
The process for preparing and producing the aforementioned
microemulsion essentially involves preparing and heating
fraction A and B to 75°C, pouring fraction A into fraction B
with turbine stirring, carrying out progressive cooling and,
at about 50°C, adding the fraction C then the fraction D and
continuing stirring until cooling to ambient temperature and
perfect homogenisation.
The invention is obviously not limited to the embodiments
described and illustrated in the accompanying drawings.
Modifications are possible, in particular with regard to the
composition of the various elements or by substitution of
technical equivalents, without departing from the scope of
protection of the invention.
