Note: Descriptions are shown in the official language in which they were submitted.
~.33389~,
Background of the Invention and Prior Art
This invention relates to the proce~s of converting
high melting fats, such as high grade beef tallow, into a hi8h
yield of fatty acids and glycerol, comprising the hydrolysis of
beef tallow in the presence of a minor amount of coconut oil by
means of the castor bean lipase enzyme, at low temperatures, of
about 25-50C, and preferably about 37C, for use in the produc-
i tion of soap. The high yield of fatty acids (98% conversion to
fatty acids and glycerol), and the utilization of low temperatures
in this process is accomplished by the addition of a minor amount
of coconut oil or other vegetable oil, which permits the emulsi-
fication of the beef tallow at lower temperatures, (37C). This
process eliminates undesirable thermal products and provides a
savings in energy without resorting to undesirable additives.
The fatty acid and glycerol mixture is free of undesirable
extraneous materials, because the coconut oil additive also
hydrolyzes into fatty acids and glycerol.
Soap manufacturing is usually accompliahed by saponifi-
cation of high grade beef tallow with lye. This is a high
temperature reaction which has become expensive in recent year
due to the sharp rise in fuel costs. Hence, a low temperature
reaction was sought, and enzymatic hydrolysis with lipases was
investigated, as a substitute for the lye process.
The optimum conditions of lipase reactions is usually
as an emulsion at 37C. Unfortunately, the problem is that fats
such a~ high grade beef tallow, do not start melting until at
least 41C to 50C. Therefore, they do not form emulsions at
37C in water without additives. It is apparently for this
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~_ !
1 33389~
reason that in systems containing only beef tallow, water, and a
lipase preparation, the yields of fatty acids are always low or
not-reported. Moskowitz et al, J. Agric. Food Chem., 25 1146
(1977). Constantin et al, Biochim. et Biophys. Acta, 43, 103
(1960). Ralston, Fatty Acids and Their Derivatives, pp. 274-279,
Wiley (1948). Haley et al, J. Am. Chem. Soc., 43, 2664 (1921).
Raising the temperature of the lipase retction did
not solve this problem, because of the rapid loss of stability
of lipases at elevated temperatures. For example, pancreatic
lipase loses 36% of its activity after 10 minutes at 50C
because enzymes are denatured at raised temperatures.
In an attempt to overcome this problem, the reaction
system was modified by the addition of metallic additives such
as calcium and magnesium salts, which did give higher yields,
as disclosed in Constantin et al (supra); Altschul et al,
Federation Proc., 18, 180 (1959); Kokusho et al,
Jpn Kokai Tokkyo Koho 79, 95, 607 (1979); Benzonana et al,
Biochim. Biophys. Acta, 164, 47 (1968). However, the metal
soips formed in this reaction are hard soaps which provide
unsatisfactory foaming and cleansing action. Haley et al
(tupra) added petroleum ether as a fat solvent in order to get
better physical contact between eneyme and substrate (beef
tallow) to increase or accelerate hydrolysis. This process how-
ever is one of considerable danger due to the high explosion
potential of the solvent.
Since solid fats are very difficult to emulsify, a
study on the selection of emulsifiers of natural (solid)
fats was made by Lobreva, et al,
1333~3
Micro -biologiya, 48, 53 (1979) in an attempt to increase lipoly-
tic activity on fats. The emulsifying agents disclosed herein
for the lipolysis of animal fats such a~ lard, beef and lamb fats
; ! #~
are-Triton X-100, Triton X-305, egg albumen , gelatin, gum
arabic, lecithin , and Tween-60. Not all of these agents
were successful in emulsifying beef tallow. This lipolytic
reaction did not give a high yield of fatty acids and glycerol.
Furthermore, said emulsifying agents have the disadvantage of
providing undesirable materials to the hydrolysis mixture.
The use of the castor bean lipase in the hydrolysis
of fats, and its preparation, are well known in the art, as dis-
closed in U.S. Patent No. 2,485,779, wherein a solvent extracted,
ground castor bean meal, prepared at A temperature not exceeding
120F is used in the partial hydrolysis of fish oil. Diethyl
ether extracted, ground ca~tor seed kernels have been used in
the hydrolysis of low quality industrial fats, as disclosed in
Trosko et al, Maslo-Zhir, Prom-st, 1977, 27; and in the hydrolysis
of sunflower oil, as disclosed in Meerow et al, Prikl. Biokhem.
Mikrobiol., 12 934 (1976). Castor bean lipase prepared by cen-
trifuging a homogenate of the kernels into a fatty layer which
is extracted by ether in the pre~ence of a saturated salt
solution has been used in the hydrolysis of cottonseed oil,
mono- and diolein, castor oil and a cottonseed oil emulsion,
as disclosed in Altschul et al, (supra)l
Haley et al, (supra) discloses a method
of preparing a castor bean lipase by extracting the
hull-free kernels with petroleum ether
hydrolysis of fats and oil. Ralston, Fatty Acids and Their
Derivative, Wiley, p. 276 (1948) disclose~ variations in the
Jf~c~ 4-
1333893
method of preparing an active lipase from castor beans for use
in the hydrolysis of fat containing 40-50% water, and in the
! presence of a small amount of acetic acid or an activating salt
such as manganese sulfate. The general method includes grinding
the dehulled seeds in water, filtering the solids, and centrifuging
to form an emulsion. One variation thereof is grinding
the dry seeds in cottonseed oil and centrifuging the ~ixture.
Another method includes extracting the macerated beans with
petroleum ether, drying, pulverizing and sifting the product,
which retains its original activity, over a period of ten years.
However, there is no disclosure of the low temperature
hydrolysis of high melting point fats such as beef tallow with
a non-stereospecific animal or vegetable lipase, particularly the
castor bean lipase, in the presence of a vegetable oil emulsifying
agent such as coconut oil at an acidic pH and a low temperature
under 50C.
Summary of the Invention
It has now been found that the low temperature
enzymatic hydrolysis of high melting fats such as beef tallow,
utilizing a non-stereospecific animal or vegetable lipase enzyme,
such as castor bean lipase, and a vegetable oil emulsifying agent
such as coconut oil provides the almost quantative hydrolysis
of the mixture of said beef tallow and coconut oil into fatty
acids and glycerol free of extraneous undesirable materials.
Neutralization with sodium hydroxide forms a high grade soap
(i.e. free of contaminants).
13~8~3 62301-1448
Accordingly, it is an aim of the present invention to
provide a process of hydrolyzing high melting fats, such as
high grade beef tallow, into a high yield of fatty acids and
glycerol, at a low temperature, with a non-stereospecific
lipase such as castor bean lipase, in the presence of a
vegetable oil emulsifying agent.
Another aim of thi~ invention is to provide a process
of converting high melting beef tallow into fatty acids and
glycerol, ln high yield, at low temperatures by hydrolyzing a
mixture of beef tallow and a minor amount of coconut oil with
the castor bean lipase enzyme.
Still another aim of this invention is to provlde an
enzymatic hydrolysis process of converting a mixture of a high
melting fat and a low melting vegetable oil into a reaction
mixture of fatty acids and glycerol free of undesirable
minerals, i.e. thermal products and addltlves.
Another aim of this invention is to provide an enzyme
system to hydrolyze beef tallow and coconut oil into fatty
acids ~and glycerol) and neutrallzing said fatty acids wlth
NaOH, Na2C03 or NaHCO3 to form soap.
Additional advantages will be apparent from a
consideration of the following description and ln part wlll
become apparent to those skilled in the art upon examination of
the following or may be learned by practice of the lnventlon.
The objects and advantages of the invention may be realized and
attained by mean~ of the instrumentalities and combinations
particularly pointed out in the appended claims.
To achieve the foregoing in accordance with the
present invention, as embodied and broadly described herein,
the process of thls invention of converting high melting fats
.~ 6
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13~38~3
62301-1448
into fatty acids and glycerol comprises hydrolyzlng an
emulsified mixture of about 90-75% by weight of a high meltlng
beef tallow and about 10-25% by weight of the mixture of a
vegetable oil, preferably coconut oll, in an aqueous medium,
with a non-stereospecific animal or vegetable lipase enzyme, at
a temperature of about 25-50C, preferably 37C, and at a pH of
about 4-5.5, and recovering a final reaction mixture consisting
of fatty acids, glycerol and lipase. The mixture is agitated
for a sufficient period of time, about 2 to 48 hours, to obtain
substantially complete hydrolysis into fatty aclds and
glycerol. The final reaction mixture is free of undesirable
materials, and consists of three layers, a fatty acid top
layer, a lipase mixture middle layer, and an aqueous glycerin
(sweet water) bottom layer. The layers may be separated. The
fatty acid layer is skimmed off, neutralized with NaOH, Na2C03,
or NaHCO3, and the resulting soap purified in the usual manner
of soap manufacturers skilled in the art. The bottom layer is
separated, and the glycerine removed. The middle layer,
containing more than 50% of the original lipase still being
active, is reused after adding a lesser quantity of fresh
lipase.
The present lnvention also relates to a process of
producing a high grade soap, free of undesirable additlves,
which comprises hydrolyzing an emulsified mixture of a high
melting fat and about 10-25~ by weight of a vegetable oil in an
aqueous medium, with a non-stereospecific animal or vegetable
lipase at a temperature of about 25-50C and at a pH of about
4-5.5, agitating the mixture for a period of time to obtain
substantially complete hydrolysis into fatty acids and glycerol
separating the fatty acids from the glycerol, and neutralizing
~ 333~3
said fatty acids with an alkaline material to form a soap, free
of undesirable additives. More specifically, the final reac-
tion mixture consi~ts of three layers, a fatty ncid top layer,
a lipase mixture middle layer and an aqueous glycerin bottom
layer,andtheprocessincludesseparatingthetoplayeroffattyacidfromthefi~
reaction mixture, and neutralizing said fatty acids with an
alkaline material selected from the group consisting of
sodium hydroxide, sodium carbonate and sodium bicarbonate to
form a sodium soap substantially free of contaminants. The
resulting soap and glycerine is much lighter in color than the
corresponding colors after sulfuric acid hydrolysis of fats
or the high temperature sodium hydroxide saponification of fats.
More specifically, present invention relates to a
process of hydrolyzing a high melting fat into a high yield of
fatty acids for use in the production of soap free of under-
sirable additives, which comprises reacting a mixture of about
90-75% of a high melting beef tallow and about 10-25% coconut
oil, with castor bean lipase, in about 20-50% water acidified to
~ r~c a~
a pH of about 4-5.5 with a weak acid such as acetic acid, ~
phosphoric acid, phosphorous acid and carbonic acid, and at
a temperature of about 37C, agitating the reaction mixture
for about 3-48 hours, to obtain a final reaction mixture con-
sisting of three layers, a fatty acid top layer, a lipase mixture
in the middle layer and an aqueous glycerin bottom layer, separ-
ating the layers and neutralizing said fatty acids to form soap.
The middle layer which contains active castor bean lipase is
reused in~,the hydrolyzation process.
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1 333893 62301-1448
The llpase enzyme used as a catalyst in the present
hydrolysis process may be any animal or vegetable llpase which
is non-stereospecific, i.e. the lipase must split the beta
(middle carboxyl linkage) glycerlde linkage at about the same
rate as splittlng the alpha (outer carboxyl) llnkages.
Suitable examples of non-stereospecific lipase enzymes are
derived from castor bean, Candida cylindracea,
Propionibacterium acnes, Rhizopus arrhizus, Staphylococcus
aureus, Aspergillus flavus and Geotrichum candidum. Most
lipases are stereospecific and therefore are ineffective, e.g.,
porcine pancreatic lipase. Normally the extent of hydrolysis
with these enzymes does not exceed 70%, whereas the non-
stereospecific enzymes affect substantially complete
hydrolysis.
The preferred lipase enzyme utillzed herein is the
castor bean lipase (ricinus communis). It is lnsoluble in
water, and its activity is materially reduced by contact with
water. The enzyme is stabilized by the presence of fats. It
is rapidly inactivated by alkalls and functions only in a
neutral or slightly acidic medium. This enzyme is activated by
the presence of acids, preferably weak acids, such as acetic
acid, phosphoric acid, phosphorous acld, carbonlc acid, etc.,
which exerts the greatest accelerating effect. Accordingly,
the optimum temperature for ricinus lipase action is about
37C, and is inactivated at temperatures above about 50C. The
amount of lipase used in the hydrolysis process is about 3-15%
of the substrate (fat and oil) by weight, if the lipase
activity (LA) is unknown. If the LA is known, approximately
one LA unit is used for every 10 microequivalents of potential
acid. (See N. Pelch and M. C. Kranz, Anal Biochem, 112, 219-
222 ~1981) for a procedure for determination of llpase
activity).
~3338~3
An additional advantage in the use of the castor bean lipase in
the hydrolysi~ reaction is the ability to recover (~eparate)
said lipase from the final reaction mixture and recycle it for
use with a fresh substrate (fat and oil). This capability
results from the natural immobilization property of ca~.tor bean
1 ipas e .
The castor bean lipase cannot be obtained commercially,
but can be prepared as disclosed in the prior art previously
discussed. The castor bean lipase utilized herein is prepared by
dehulling castor beans, extracting the ~ oils by grinding the
dehulled beans in the presence of lowboiling petroleum ether,
f ilt ering the ground bean pcnace and discarding the filtrate, i.e.
ether layer containing endogenous oils, repeating the eXtraction
and filtration ~teps two more times, air drying the filtered
pomace and recovering a lipase preparation in the form of the
pomace. Another method of preparing the castor bean lipase
enzyme without allergen may also be used. This method of pre-
paration should be considered because of the pre~enc e of a potent
allergen in the bean. The dehulled bean is macerated
in water, rather than petroleum ether and then centrifuged.
The fat layer is separated from the aqueous layer, and the aqueous
layer is discarded. Since the lipase is in the spherosome~ along
with the endogenous oil, it will remain in the fat layer. The
fat layer is extracted with petroleum ether and saturated NAC1
solution. The petroleum ether contain~ the endogenous oil, and
is discarded. The saturated NaCl solution contains the lipase
in particulate matter.
--10--
~ 333893
The optimum condition~ for activity of castor bean
lipase i5 about 25-50C and preferably about 37 and a pH of
4 to 5.5. A dilute acid such as O.lN acetlc acid, or other
weak acids may be added to bring the pH to approximately 5Ø
The amount of the castor bean lipase enzyme used in the present
hydrolysis process should be sufficient to effect a substantial
degree of conversion of the beef tallow/coconut oil mixture
into fatty acids and glycerol. Amounts of about 3 to 15% and preferably about
10% by weight of thetriglyceride substrate mixture is used.
It has unexpectedly been found that the addition of
a vegetable oil such as coconut, corn, soybean, lin~eed, olive
and palm oil, to the high melting point beef tallow
enables the beef tallow to
emulsify at a lower temperature, about 37C. The lipase enzyme
hydrolyzes the emulsified tallow/vegetsble oil mixture almost
quantitatively at the lower temperature with a resultant
savings in energy. The vegetable oil such as coconut oil also
hydrolyze~ into fatty acids and glycerol. ThuA, no undesirable
extraneous ma~erial~ are present in the reaction mixture,
j thereby yielding a substantially pure soap upon neutralization
of the fatty acids with sodium hydroxide, sodium carbonate or
sodium bicarbonate. The coconut oil constitutes a lesser
amount by weight than the beef tallow in a mixture thereof.
The weight ratio of beef tallow to vegetable oil is about
75-90% to about 25-10% vegetable oil.
--1 1--
133389 ~
The data in Table I show that a mixture of high
grade edible beef tallow and coconut oil, at the same ratio as
may be used in soaps, was hydrolyzed approximately 98~ into fatty
acids at the described conditions. This is in clear contrast
to results reported in the literature (~eyet ~, ~), ~ ~ch
~fta~ ~ reacted ~th ~tor ~3~ ~e ~t ~ o~yhy~olyz~ d~OUt
3% (Table II). In the latter case, coconut oil was absent.
The combined results clearly show that the addition of coconut
oil unexpectedly yields a high conversion of the high melting
beef tallow into fatty acids. It is believed that the coconut
oil lowered the melting point of the beef tallow enough so that
it would form an emulsion at 37C-
Table I
:;
Extent of Castor Bean Lipase Hydrolysis of
Fats and Oils
Observed
I Lit, S.V.* Enzyme Hydrolysis Value
; Olive oil (control 186-196 185
83:17 Tallow:coco 203-213 203
j *Saponification Value is the weight (mg) of KOH required to
I saponify lg of fat, and is indicative of the extent of hydrolysis
of the fats and oils,
Table II
Hydrolysis of Beef Tallow by Castor Bean Lipase
Duration of Experiment (hrs.) Extent of Hydrolysis (%)
24 2.8
48 2.8
72 2.8
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1333893
The saponification value is determined
by conventional methods described in the literature,
M. Applewhite, Kirk-Othmr. Encycl. Chem. Tech., 3rd Ed., 9,
795 (1980). The saponification procedure utilized herein
comprises adding 30 ml of absolute ethanol to a 2 to 3g sample
in a covered flask, warming on a ~team bath (50-60C), adding
50 ml standardized 0.5N alcoholic KOH solution and boiling for
one hour, adding phenolphthalein indicator solution a~d titrating
with standardized 0.5N HCl to the disappearance of the pink
color, and the ~aponification value is determined.
Saponification of fats and oils in the absence of
lipase, to determine the maximum yield of soap availab~e from
fats and oils, wa~ conducted on four substrates. Triolein and
olive oil are standards used in literature. Beef tallow and
coconut oil are components of the soaps prepared herein. The
data in Table III shows that the observed results are in fairly
good agreement with the literature values for the two standards
and coconut oil, but low for beef tallow.
Table III
Saponification Values (SV)
Lit Obs.
Triolein 189 (calc) 190
Olive oil 186-196 182
Beef tallow 193-202 173
Coconut oil 250-264 253
.
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13338~3
:1,
i'
The advantages of the enzymatic hydrolysis process of
present invention are multifaceted. Higher yields of fatty
acids and glycerol are obtained, about 98% conversion. The
use of lower temperatures resulted in considerable cost savings.
The use of vegetable oil such as coconut oil which also hydro-
lyzes into fatty acids a~d glycerol yields fatty acids and
glycerol free of undesirable additives. Ssid fatty acids yield
a pure soap upon nuetralization with sodium hydroxide, carbonate, '
or bicarbonate. This low temperature hydrolysis reaction
requires less utilization of energy, and therefore, less atmos-
pheric pollution i~ produced. The use of a low reaction tem-
perature in this process yields fewer undesirable thermal pro-
ducts, and a lighter color.
Detailed Descriptioniofithe Invention
The following examples are merely illustrative
of the invention and are not to be construed as limiting
thereof.
1333893
Example 1
Preparation of Enzyme
Dehull 60g of castor beans. Grind the dehulled beans
in a Waring blender in the presence of 600ml of low boiling
j (30-60C) petroleum ether. The ether extracts endogenous oils
from the beans. Filter and discard filtrate. Repeat extraction
and filtration two more timesi. Air dry the crude lipase prepara-
tion.
Enzymatic Hydrolysis of Tallow/Coconut Mixture
Mix 1.67g of the lipase preparation snd 16.7g of
an 83:17 mixture of high grade beef tallow and coconut oil, and
10 cc of O.lN acetic acid. Stir approximately 24 hoursi at
37C. The final mixture consists of 3 layers, fatty acid on
the top, lipase mixture in the middle, and sweet water (aqueous
glycerine) on the bottom. The fatty acid layer is skimmed off,
neutralized with NaOH, Na2CO3, or NaHCO3, and the resulting soap
purified in the usual manner of soap manufacturers skilled in the
art. The bottom layer is separated, and th~ glycerine removed.
The middle layer, containing more than 50% of the original
lipa~e still being active, ls reused after adding a lesser
quantity of fresh lipaise.
Analysis of the extent of hydrolysis is determined
by adding 3.4 g of reaction mixture to 100 ml absolute alcohol,
and tritrating to pH 9.5 with O.lN alcoholic potassiium hydroxide.
Results: 98% conversion to fatty acids and glycerol.
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~3~3~93
Example 2
~reparation of Castor Bean Lipase
60g of dehulled castor beans is macerated in water and
centrifuged. The fat layer is separated from the aqueous layer,
which is discarded. The fat layer is extracted with 600 ml
low boiling petroleum ether (30-60C) and saturated NaCl
; solution. The petroleum ether is discarded, and the NaCl
r solution contains the lipase in particulate form. 1.67 g of this
particulate lipase is used in the hydrolysis of the tallow/
coconut mixture in accordance with the proces~ of Example 1.
Substan~ially complete hydrolysis into fatty acids and glycerol
is obtained.
Example 3
The high melting beef tallow is melted at a temperature
of about 42C until it is liquified. The liquified tallow is mixed
with the coconut oil in the weight ratio of 80:20 tallow:oil.
1.67g of the lipase preparation of Example 1 is mixed with
16.7 g of the liquified fat and oil mixture, and 10 cc of
O.lN acetic acid for about 24 hours at 37C. Substantially
complete hydroly~is into fatty acids and glycerol is obtained.
.
i
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i
~ 3~389~-
Other weak acids may be substituted for the
acetic acid in tne examples, such as phosphoric, phosphorous or
carbonic acid.
Lower melting point fats such as sheep tallow,
industrial quality beef tallow, lard and butter may be used in
the present lipase hydrolysis process.
Also, other vegetable oils such as corn, soybean,
linseed, olive and palm oil may be substituted for the coconut
oil in the example
It is understood that the foregoing detailed
description is given merely by way of illustration and that
variations may be made therein without departing from the spirit
of the invention. The "Abstract" given above is merely for the
convenience of technical searchers and is not to be given
any weight with respect to the scope of the inventlon.
