Note: Descriptions are shown in the official language in which they were submitted.
1335054
EXTRACTION OF FRESH LIQUID EGG YOLK
This invention relates to the isolation of lecithin from
fresh liquid egg yolk of hens eggs and more particularly it
relates to the isolation of lecithin (phospholipids), neutral
egg yolk oil ( neutral lipids which are mainly triglycerides)
and egg yolk protein from fresh liquid egg yolk of hens eggs.
Lecithin is useful in the food and pharmaceutical industry
while egg oil is useful in the preparation of infant formula
food because of its fatty acid composition. Egg yolk protein
is useful as a source of protein.
We have now found, and herein lies our invention, that
fresh liquid egg yolk from hens eggs can be subjected to a
unique novel process using a precise combination of solvent and
temperature to provide a high yield of egg yolk protein, leci-
thin (phospholipids) and neutral egg yolk oil (triglycerides).
Thus according to our invention we provide a processfor the separation of fresh liquid egg yolk into a yolk protein
fraction, a neutral egg oil fraction and an egg lecithin frac-
tion which comprises treating fresh liquid egg yolk with
aqueous ethanol at an elevated temperature to provide a slurry
thereof, filtering the slurry to provide solid yolk protein and
an aqueous ethanolic filtrate, and thereafter subjecting the
filtrate to low temperature crystallization to provide a crys-
talline neutral egg oil fraction and removing this crystalline
fraction to provide a residual aqueous ethanolic solution con-
taining egg lecithin.
Fresh liquid egg yolk from hens eggs contains roughly
about 50% water and about 50% solid matter and the latter is
essentially made up of yolk protein and lipids. These lipids
are a complex mixture of triglycerides (also called neutral
lipids or neutral egg oil) and phospholipids which are polar
lipids containing lecithin, together with cholesterol. It is
possible to use the process of the present invention to isolate
the yolk protein essentially free from lipids and thereafter to
separate the residual lipids into a neutral lipid fraction
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known as neutral egg oil (triglycerides) and egg lecithin which
is essentially phospholipids, i.e. polar lipids containing
lecithin. These products thus obtained by the process of this
invention are essentially free from, or contain only very minor
amounts of, cholesterol.
Fig. 1 is a schematic flow sheet of the process showing
how fresh liquid egg yolk may be separated into solid yolk
residue (protein) along with neutral egg oil (triglycerides)
and egg lecithin (phospholipids). This flow sheet shows a
preferred embodiment of the process wherein 1 part by weight of
fresh liquid egg yolk is stirred with 4 volumes of 95% aqueous
ethanol (95% ethanol and 5% water) at a temperature of about
60 C for about 15 minutes. The slurry thus obtained is
filtered and the solid yolk residue is essentially yolk
protein free from lipids. The residual aqueous ethanolic
filtrate is subjected to low temperature crystallization, e.g.
a temperature of about 2 C to 5 C. The crystalline product
which separates is essentially neutral triglycerides which is
commonly known as neutral egg oil since it is crystalline at
the temperature of 2 C to 5 C but liquefies at ambient temper-
ature. Once the crystalline product has been removed by
filtration at that low temperature, the residual aqueous
ethanolic filtrate may be dried in a vacuum evaporator to
remove the aqueous ethanol and provide a final solid residue
which is essentially egg lecithin.
Thus, according to a further feature of the invention,
we provide a process for the separation of fresh liquid egg
yolk into a yolk protein fraction, a neutral egg oil fraction
and an egg lecithin fraction which comprises treating about 100
parts by weight of fresh liquid egg yolk with about 400 volumes
(4:1 v/w) of aqueous ethanol (5% water and 95% ethanol) at a
temperature of about 60 C to provide a slurry thereof, filter-
ing said slurry to provide solid yolk protein and an aqueous
ethanolic filtrate, and thereafter subjecting said filtrate to
low temperature crystallization to provide a crystalline neu-
tral egg oil fraction and removing said crystalline fraction to
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provide a residual aqueous ethanolic solution containing egg
lecithin.
According to yet an additional feature of the invention
we provide a process for the separation of fresh liquid egg
yolk into a yolk protein fraction, a neutral egg oil fraction
and an egg lecithin fraction which comprises treating about 100
parts by weight of fresh liquid egg yolk with about 400 volumes
(4:1 v/w) of aqueous ethanol (5% water and 95% ethanol) at a
temperature of about 60 C to provide a slurry thereof, filter-
ing said slurry to provide solid yolk protein and an aqueous
ethanolic filtrate, and thereafter subjecting said filtrate
crystallization at a temperature of from about 2 C to about 5 C
to provide a crystalline neutral egg oil fraction and removing
said crystalline fraction to provide a residual aqueous etha-
nolic solution containing egg lecithin.
By the use of the novel process of this invention it is
possible to separate fresh liquid egg yolk from hens eggs into
three products. The first product is yolk protein, the second
product is neutral egg yolk oil so-called because it is an oil
at ambient temperature and a crystalline product at a lower
temperature of about 2 C to 5 C while the third product is egg
leci-thin. These products contain little or no undesirable by-
products and they are therefore very desirable products for use
in the food and pharmaceutical industries.
The expression aqueous ethanol is intended to mean
ethanol containing from about 2% v/v of water, i.e. about 98%
ethanol, to about 10% v/v of water, i.e. about 90% ethanol. A
preferred aqueous ethanol is one containing from about 4% v/v
to about 6% v/v of water, i.e. about 96% to about 94% ethanol,
and, more especially, one containing about 5% v/v of water,
i.e. 95% ethanol. The volume (v) of aqueous ethanol used in
the process in relation to the weight (w) of fresh liquid egg
yolk used as starting material may vary from about 2 volumes,
i.e. 2 v/w, to about 5 volumes, i.e. S v/w, and preferably from
about 3 volumes to about 5 volumes (3 v/w to 5 v/w) and more
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particularly about 4 volumes, i.e. 4 v/w. In the latter case,
it is preferred to use, for example, about 400 volumes of
aqueous ethanol, desirably 95% ethanol, for about 100 parts by
weight of fresh liquid egg yolk used as starting material thus
giving a volume to weight ratio of about 4:1 v/w.
The expression elevated temperature is intended to mean
a temperature of from about 45 C to about 75 C, preferably from
about 55 C to about 65 C and more particularly a temperature of
from about 58 C to about 62 C. A most preferred temperature is
about 60 C.
The time taken for treatment of the fresh liquid egg
yolk with aqueous ethanol to prepare a slurry thereof may vary
from about 5 minutes to about 60 minutes but it has generally
been found that the separation of the solid yolk protein
fraction occurs fairly quickly. A treatment time of about 10
to 20 minutes, more particularly about 12 to 18 minutes, pre-
ferably about 15 minutes, provides a satisfactory separation.
As a preferred method of separation, it has been found
that when starting with about 100 parts by weight of fresh
liquid egg yolk and using about 400 volumes of about 95%
ethanol, a preferred operating temperature is about 60 C and
the time for treatment is generally about 15 minutes before the
slurry thus obtained is ready for separation into the desired
products.
Treatment of the fresh liquid egg yolk with aqueous
ethanol according to the process of this invention is effective
in precipitating the solid yolk protein fraction while the
residual aqueous ethanolic liquid contains essentially the
neutral egg yolk oil and the lecithin. After removal of the
solid yolk protein fraction, the residual aqueous ethanolic
solution can be cooled to a sufficiently low temperature to
permit the neutral egg yolk oil to crystallize out from the
solution. A suitable low temperature below ambient temperature
may be, for example, a temperature of from about 0 C to-about
10 C, preferably from about 2 C to about 8 C, and especially to
a temperature of from about 2 C to about 5 C. After the crys-
~ 5 ~ 13~505~
talline product has been removed, for example by filtration at
low temperature, the residual aqueous ethanolic filtrate may be
dried, for example by use of a rotary vacuum evaporator, to
remove the water and ethanol under relatively mild conditions.
The solid residue thus obtained is essentially lecithin which
may be further purified, as required, by known means.
The crystalline product which is neutral egg oil in its
crystalline state may be washed with a small amount of cold
aqueous ethanol to remove adhering traces of the filtrate from
which it has been separated. The crystalline product may then
be allowed to warm up to ambient temperature when it liquefies
and the neutral egg yolk oil thus obtained is essentially a
mixture of triglycerides. The oil may be further purified by a
recrystallization procedure.
The originally isolated egg yolk protein may be re-
treated with aqueous ethanol to remove residual traces of unde-
sired products and the slurry thus obtained may be filtered to
obtain purified egg yolk protein.
The invention is illustrated by, but not limited by,
the following Examples.
EXAMPLE 1
100 g Of fresh liquid egg yolk which contains approxi-
mately 50%, i.e. 50 g of solid matter and 50 g of water, is
diluted with 400 ml (4:1 v/w) of 95% aqueous ethanol (95%
ethanol and 5% v/v water) in an extraction chamber and the
mixture is stirred for 15 minutes at 60 C. There is thus
obtained an aqueous ethanol egg yolk slurry, and the slurry is
filtered to provide a solid residue which is essentially lipid
free egg yolk protein. The aqueous ethanolic filtrate contains
egg yolk lipids (fat). The aqueous ethanolic filtrate is then
cooled to a temperature of from about 2 C to about 5 C for a
period of about 12 hours (roughly overnight). The neutral
lipids (triglycerides) in the aqueous ethanolic filtrate crys-
tallize and are precipitated at the bottom. The bottom crys-
talline product (mainly triglycerides) is separated from the
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aqueous ethanol solution by filtering through a *Whatman #2
ashless filter paper. The crystalline product thus collected
is transferred into a round bottomed flask with several rins-
ings of hot ethanol and is then dried (remove aqueous ethanol)
by a rotary vacuum evaporator. The product is designated as
"Neutral Egg Oil" (97.03% triglycerides).
The remaining aqueous ethanolic filtrate, after the
removal of neutral egg oil (triglycerides), is dried in a
rotary vacuum evaporator at 40 C. The solid fraction thus
obtained is designated as "Egg Lecithin" (89.16% phospholip-
ids).
The initially obtained solid residue which is essent-
ially lipid free egg yolk protein may be subjected to a further
treatment with 100 ml of 95% aqueous ethanol in order to remove
residual traces of lipids. The slurry thus produced is fil-
tered and the solid residue of egg yolk protein thus obtained
is essentially free from lipids.
The neutral egg oil (97.03% triglycerides) contains only
trace amounts (0.04% to 0.12%) of cholesterol. This cholest-
erol may be reduced or removed completely by subjecting the egg
oil to a recrystallization procedure using aqueous ethanol or
by subjecting the egg oil to a supercritical fluid extraction
procedure.
EXAMPLE 2
In order to evaluate the variation in the rate of ex-
traction over different time periods (15, 30 and 60 minutes)
and at different temperatures (40 C and 60 C), the process
described in Example 1 was further evaluated as follows:-
To 100 g of fresh liquid egg yolk (48.1% water, 35.0%
lipids, 15.1% protein and 1.8% ash) were added 400 ml of 95%
aqueous ethanol (4:1 v/w). The slurry was homogenized in a
blender and charged into the chamber of an extraction/filtra-
tion (EF) unit in the extraction mode. Extraction was con-
tinued by stirring for 15, 30 or 60 minutes. In a filtration
mode of the EF unit, the slurry was isothermally filtered at
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40 C or 60 C with a gradually increasing pressure in order to
prevent clogging of the filter. A triple layer of FYNTEX 300
mesh (pore size 79 ~m) nylon filter was found to be suitable
for this purpose.
The aqueous ethanolic filtrate was collected and stored
overnight (about 12 to 16 hours) at 2 C to 5 C. Two phases
formed, one a crystalline precipitate at the bottom of the
flask, which is essentially triglycerides, and an isotropic
liquid phase at the top, which contains polar lipids, mainly
lecithin. The two phases were separated by filtering using a
*Whatman #2 ashless filter paper in a cold room temperature (2
C to 5 C). The crystalline phase containing neutral egg oil
(triglycerides) and the liquid upper phase containing mainly
polar lipids (lecithin) were each subjected to a drying process
by evaporation at 40 C in a rotary vacuum evaporator.
It will be seen in the following tables that extraction
at 60 C gives better results on the basis of the amount of
products isolated. However, neither extraction rate nor purity
of crude oil appeared to be influenced by the length of extrac-
tion time. A time of 15 minutes extraction under the above
described conditions appeared to be satisfactory.
Effect of varying extraction time and temperature
on fresh liquid egg yolk extraction with 95%
aqueous ethanol.
At 40 C
Time (min)
Yield (g)11.16~1.1010.92+0.4412.15+1.37
LecithinPL10.17+0.97 9.88+0.4810.85+1.44
FC0.71+0.07 0.77+0.03 0.75+0.11
TG0.25+0.10 0.23+0.02 0.40+0.31
Yield (g)18.20+1.1017.57+0.9117.31+1.08
Neutral PL1.02+0.49 0.92+0.15 0.66+0.18
Egg Oil FC0.06+0.02 0.06+0.01 0.04+0.04
TG17.13+0.9016.58+0.7716.60+1.28
* Trade Mark
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Total Lipid
Extracted (g)29.35+1.1928.49+0.8229.45+2.44
Extractability (%)83.90+3.4181.44+2.3484.18+6.99
At 60 C
Time (min)
Yield (g)11.04+0.3611.76+0.23 11.39+0.63
LecithinPL9.79+0.24 10.42+0.2910.16+0.52
FC0.88+0.09 0.82+0.25 0.71+0.19
TG0.33+0.05 0.36+0.08 0.44+0.20
Yield (g)22.68+0.3222.72+1.1121.77+0.41
Neutral PL1.34+0.10 1.19+0.08 0.90+0.18
Egg Oil FC0.11+0.01 0.12+0.09 0.04+0.03
TG21.22+0.4321.40+1.1820.85+0.31
Total Lipid
Extraction (g)33.72+0.6834.48+1.1933.16+0.43
Extractability (%)96.36+1.9398.54+3.3994.77+1.24
PL = phospholipids (polar lipids containing lecithin).
FC = free cholesterol.
TG = triglycerides (neutral lipids).
EXAMPLE 3
In an attempt to try to determine the most suitable
volume of aqueous ethanol to be used for the v/w ratio, an
experiment was carried out using 200, 300, 400 or 500 ml of 95%
ethanol as follows:-
100 g Of fresh liquid egg yolk was extracted with 200,
300, 400 or 500 ml of 95% aqueous ethanol at 60 C for 15
minutes. Extraction, filtration and cold temperature frac-
tionation were performed as described in Example 2. As seen in
the following table, 4:1 v/w ratio gives 96.36% extractability,
producing 11 g of crude lecithin and 22 g of neutral egg oil
per 100 g fresh liquid egg yolk with a high purity. A ratio
133505~
higher than 4:1 (v/w) of 95% ethanol appears to increase the
total extractability but the lecithin fraction becomes less
pure mainly due to the shift of triglycerides to the lecithin
fraction in the cold temperature fractionation step. There-
fore, 400 ml per 100 g (4:1 v/w ratio) was adopted as the best
procedure.
95% aqueous ethanol volume (ml)
200 300 400 500
Crude (g)6.50+0.069.84+0.31 11.04+0.36 12.73+0.07
Lecithin
Crude (g)24.45+0.8621.95+0.76 22.68+0.32 21.49+0.19
Oil
Total Lip-30.95+0.8431.74+1.04 33.72+0.68 34.22+0.23
ids (g)
Extractab-88.45+2.4090.71+2.98 96.36+1.93 97.78+0.64
ility (%)
The process of this invention, as exemplified in the
foregoing Examples 2 and 3, may be conveniently operated in a
bench top model extractor as illustrated in Figs. 2 and 3.
Referring to Fig. 2, diagram A of a bench top model extractor
shows a metal stand (8) supporting a temperature controlled
extraction chamber (1) surrounded by a water jacket (7) fitted
with a water inlet (3) and a water outlet (4). The extraction
chamber is fitted with an upper removable screw cap (2) and a
lower interchangeable filter plate (6) with a filter set
adjacent to a removable bottom screw cap (5). In diagram B,
the extractor is shown assembled for the extracting mode fitted
with a plastic insert (1) and an electrical stirrer (2). In
diagram C, the extractor is shown in the extruding and filter-
ing mode wherein the extraction chamber is fitted with an
extruding piston with shaft (1) and the interchangeable plate
with filter set is shown with a supporting Teflon O-ring (2),
A * Trade-mark
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triple-layered circular polyethylene 300 mesh filters (*Fyntex)
and a perforated stainless filter supporting plate (4). In
diagram D, the perforated stainless filter supporting plate is
shown in the circular plate (1) and perforated circular plate
(2).
Fig 3 is a photograph of the bench top model in a disas-
sembled mode showing the temperature-controlled extraction
chamber (1), the removable bottom screw cap (2), the triple-
layered circular polyethylene 300 mesh *Fyntex filters (3), the
perforated stainless filter supporting plate (4), a metal
bushing (5) and steel arm (6), the stirrer (7), the lower
removable screw cap (8), the upper removable screw cap (9), the
extruding piston and the water jacket (11) in the form of a
circulating water bath having a heating element.
* Trade Mark
