PH26178A - Enzymatic hydrolysis of beef tallow - Google Patents

Description

i - 1 26178

Background of the Invention and Prior Art

This invention relates to the process of converting high melting fats, such as high grade beef tallow, into a high yield of fatty acids and glycerol, comprising the hydrolysis of ! beef tallow in the presence of a minor amount of coconut oil by k means of the castor bean lipase enzyme, at low temperatures, of about 25-50°C, and preferably about 37°C, for use in the produc- tion of soap. The high yield of fatty acids (98% conversion to fatty acids and glycerol), and the utilization of low temperature 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, (37°C). This process eliminates undesirable thermal products and provides a savings in energy without resorting to undesirable additives. i 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 accomplished by saponifi- i cation of high grade beef tallow with lye. This is a high temperature reaction which has become expensive in recent years due to the sharp rise in fuel costs. Hence, a low temperature reaction was sought, and enzymatic hydrolysis with lipases was investigated, as a substiftute for the lye process,

The optimum conditions of lipase reactions is usually as an emulsion at 370C. Unfortunately, the problem is that fats such as high grade beef tallow, do not start melting until at least 41°C to 50°C. Therefore, they do not form emulsions at 379C in water without additives. It is apparently for this a

. i reason that in systems containing only beef tallow, water, and a lipase preparation, the yields of fatty acids are always low or

J 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 reaction 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 50°C ) because enzymes are denatured at raised temperatures. i 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,

Sn rr a reson + Lh 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 sodps formed in this reaction are hard soaps which provide unsatisfactory foaming and cleansing action. Haley et al (supra) added petroleum ether as a fat solvent in order to get better physical contact between enzyme 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,

} . : | 261 — . | * 7 8

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 as 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, ovis closed in U.S. Patent No. 2,485,779, wherein a solvent extracted, ground castor bean meal, prepared at a temperature not exceeding ’ 120°F is used in the partial hydrolysis of fish oil. Diethyl : ether extracted, ground castor seed kernels have been used in the hydrolysis of low quality industrial fats, as disclosed in {Fo et al, Maslo-Zhir, Prom-st, 1977, 27; and in the hydrolysis fi ; of sunflower oil, as disclosed in Meerow et al, Prikl. Biokhem. ' i 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 presence of a saturated salt i 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) {

Haley et al, (supra) discloses a method i of preparing a castor bean lipase by extracting the i hull-free kernels with petroleum ether hydrolysis of fats and oil. Ralston, Fatty Acids and Their

I Derivative, Wiley, p. 276 (1948) discloses variations in the

* ! , ! § } _ 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

Ehe dehulled seeds in water, filtering the solids, and centrifuging to form an emulsion. One variation thereof tof einaing the dry seeds in cottonseed oil and centrifuging the mixture. '

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 f hydrolysis of high melting point fats such as beef tallow with i a non-stereospecific animal or vegetable lipase, particularly the castor bean lipase, in the presence of a vegetable oil emulsifying i \ agent such as coconut oil at an acidic pH and a low temperature

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Wp 50°C. i 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). {

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Accordingly, it is an object of the present invention to provide a process of hydrolyzing high melting fats, such as high grade beef tallow, into a high yieldfof fatty acids and glycerol, at a low temperature, with a non-stereospecific lipase such as castor bean lipase, in the presence of a vege- table oil emulsifying agent.

Another object of this invention is to provide a pro- cess of converting high melting beef tallow into fatty acids and glycerol, in high yield, at low temperatures by hydrolyzing a mixture of beef tallow and a minor amount of coconut oil with ee eee ree the castor bean lipase enzyme.

Still another object of this invention is to provide 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 additives.

Another object of this invention is to provide an enzyme system to hydrolyze beef tallow and coconut oil into . fatty acids (and glycerol) and neutralizing said fatty acids with

NaOH, Na,CO3 or NaHCOq to form soap.

Additional objects and advantages will be apparent from a consideration of the following description and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention.

I! The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out/1n the appended claims.

To achieve the foregoing and other objects and in ac- cordance with the present invention, as embodied and broadly .

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I oo] 3 ¥ described herein, the process of this invention of converting high melting fats into fatty acids and glycerol comprises hydrolyzing an emulsified mixture of a high melting beef tallow and about 10-25% by weight of the mixture of a vegetable oil, preferably coconut oil, in an aqueous medium, with a non-

I stereospecific animal or vegetable lipase enzyme, at a tempera- ture of about 25-50°C, preferably 37°C, 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 acids anid glycerol. { 4 The final reaction mixture is free of undesirable materials, and consists of three layers, a fatty acid top layer, a lipase a mixture middle layer, and an aqueous glycerin (sweet water) bottom layer. The layers may be separated. The fatty acid a . | layer is skimmed off, neutralized with NaOH, NajCO3, or NaHCOg, fi aN | 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, 1s/ reused after adding a lesser quantity of fresh lipase.

The present invention also relates to a process of producing a high grade soap, free of undesirable additives, whic 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 25-500C 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

- ) 26178 . | .said fatty acids with an alkaline material to form a soap, free of undesirable additives, More specifically, the final reac- tion mixture consists of three layers, a fatty acid top layer, a lipase mixture middle layer and an aqueous glycerin bottom layer, and the process includes separating the top layer of fatty acid from the final 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 / 1 of 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- Cj sirable additives, which comprises reacting a mixture of about 90-75% of a high melting beef tallow and about 10-25% coconut , . 0il, with castor bean lipase, in about 20-50% water acidified 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 37°C, 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 mixtur 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 int the hydrolyzation process. )

The lipase enzyme used as a catalyst in the present hydrolysis process may be any animal or vegetable lipase which is non-stereospecific, i.e. the lipase must split the beta (middle carboxyl linkage) glyceride linkage at about the same

Type TZ ALA rate as splitting the alpha (outer carbons) linkages. 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 hydroylsis with these enzymes does not o £79 Ss alwlkL exceed 70%, whereas the non-stereospecific enzymes affect substantially complete hydrolysis.

The preferred lipase enzyme utilized herein is the castor bean lipase (ricinus communis). It is insoluble in water, and its activity is materially reduced by contact with water. The enzyme is stabilized by the presence of fats. . It is repidly inactivated by alkalis and functions only in a © neutral or slightly acidic medium. This enzyme ie activated by the presence of acids, preferafildy weak acids, such as acetic i acid, phosphoric acid, phosphorous acid, carbonic acid, etc, which exerts the greatest accelerating effect. Accordingly, the pprimun yemperature for ricinus lipase action is about

Lode a ed at temperatures above about 509cC. ’ p

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 lipase activity).

Co 9 6 : 178 : '

An additional advantage in the use of the castor bean lipase in the hydroflysis reaction is the ability to recover (separate) ! 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 castor bean lipase.

The castor bean lipase cannot be obtained commercially, but ‘can be prepared as disclosed in the prior art previously )

O discussed. The castor bean lipase utilized herein is prepared by dehulling castor beans, extracting the endogenous oils by grinding the dehulled beans in the presence of lowboiling petroleum ether, filtering the ground bean pomce and discarding the filtrate, i.e. ether layer containing endogenous oils, repeating the extraction and filtration steps 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 presence of a potent

E 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 aqueouy i layer is discarded. Since the lipase is in the spherosomes along oO | with the endogenous oil, it will remain in the fat layer. The ! fat layer is extracted with petroleum ether and satfated NaCl i solution. The petroleum ether contains the endogenous oil, and is discarded. The saturated NaCl solution contains the lipase } in particulate matter. i! -10-

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The optimum conditions for activity of castor bean lipase is about 25-500C and preferably about 370 and a pH of 4 to 5.5. A dilute acid such as 0.1IN acetic acid, or other ! : Lan eg Tes Be weak acids may be added to bring the pH to approximately 5.0.

The amount of the castor bean lipase enzyme used in the present hydrolysis process should be sufficient to effect a substantial ql degree of conversion of the beef tallow/coconut oil mixture into fatty acids and glycerol. Amounts of about 3 to 15% and preferably about

J 1072 by weight of thecriglyceride substrate mixture is used. :

It has unexpectedly been found that the addition of i a vegetable oil such as coconut, corn, soybean, linseed, olive } and palm oil, to the high melting point beef tallow enables the beef tallow to emulsify at a lower temperature, about 37°C. The lipase enzyme : hydrolyzés the emulsified tallow/vegetable oil mixture almost quantitatively at the lower temperature with a resultant savings in energy. The vegetable oil such as coconut oil also hydrolyzes into fatty acids and glycerol. Thus, no undesirable extraneous materials are present in the reaction mixture, i thereby yielding a substantially pure soap upon neutralization

I 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. : | -11- i ol !

The data im 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 il ‘| acids at the described conditions. This is in clear contrast ‘| to results reported in the literature (Haley et al, supra), in vhich beef tallow was reacted with castor bean lipase but was only hydrolyzed about ! i ) 3% (Table II). In the latter case, coconut oil was absent.

J The combined results clearly show that the addition of coconut 3 oil unexpectedly yields a high conversion of the high melting i beef tallow into fatty acids. It is believed that the coconut ot i oil lowered the melting point of the beef tallow enough so that 1 . ., 1t would form an emulsion at 37°C. ; Table I 1 bi Extent of Castor Bean Lipase Hydrolysis of

N Fats and Oils . Observed iy Lit, S.v.* Enzyme Hydrolysis Value .' Olive oil (control 186-196 185 iL 83:17 Tallow:coco 203-213 203 * —_t—— i *Saponification Value is the weight (mg) of KOH required to saponify lg of fat, and is indicative of the extent of hydrolysi of the fats and oils,

Table II li Hydrolysis of Beef Tallow by Castor Bean Lipase i i

Duration of Experiment (hrs.) Extent of Hydrolysis (7%) jpozZuratlion of bxperiment rs.’

N 24 2.8 i 48 2.8 i 72 2.8 12

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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 steam bath (50-60°C), adding 50 ml standardized 0.5N alcoholic KOH solution and boiling for one hour, adding phenolphthaléin indicator solution anid titrating

I with standardized 0.5N HCl to the disappearance of the pink color, and the saponification value is determined.

Saponification of fats and oils in the absence of lipase, to determine the maximum yield of soap available from fats and oils, was conducted on four substrates. Triolein and olive 0il are standards used in Lterature./ 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 II1I

Saponification Values (SV)

Lit Obs.

Triolein 189 (calc) 190

Olive oil 186-196 182

Beef tallow 193-202 173

Coconut oil 250-264 253 i oo 26178 . oo fH

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 0il such as coconut oil which also hydro- ! lyzes into fatty acids ard glycerol yields fatty acids and glycerol free of undesirable additives. Said 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 1s produced. The use of a low reaction tem- !| perature in this process yields fewer undesirable thermal pro- ducts, and a lighter/ color.

Detailed Dédscriptioniofi the Invention

The following examples are merely illustrative of the invention and are not to be construed as limiting thereof. i

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Example 1 ! Preparation of Enzyme . Dehull 60g of castor beans. Grind the dehulled beans i i in a Waring blender in the presence of 600ml of low boiling (30-60°C) petroleum ether. The ether extracts endogenous oils from the beans. Filter and discard filtrate. Repeat extraction and filtration two more times. Air dry the crude lipase prepara- tion.

Enzymatic Hydrolysis of Tallow/Coconut Mixture

Mix 1.67g of the lipase preparation and 16.7g of an 83:17 mixture of high grade beef tallow and coconut oil, and cc of 0.1N acetic acid. Stir approximately 24 hours at ¥ 379C. 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, Na,COj3, or NaHCO3, and the resulting soap k purified in the usual manner of soap manufacturers skilled in the art. The bottom layer is separated, and the glycerine removed, . | The midfile layer, containing more than 50% of the original lipase still being active, is reused after adding a lesser quantity of fresh lipase.

Analysis of the extent of hydrolysis is determined by adding 3.4 g of reaction mixture to 100 ml absolute alcohol,

Iq and tritrating to pH 9.5 with O.1N alcoholic potassium hydroxide. d Results: 98% conversion to fatty acids and glycerol.

I o oo 2613 *, {

Example 2 ' Preparation of Castor Bean Lipase 3 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 (30-60°C) and saturated NaCl 3 solution. The petroleum ether is discarded, and the NaCl y 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 process of Example 1.

Substantially complete hydrolysis into fatty acids and glycerol ! i is obtained. 1 Example 3 ; The high melting beef tallow is melted at a temperature of about 42°C until it is liquified. The liquified ftallow is mixed : with the coconut oil in the weight ratio of 80:20 tallow:oil. i 1.67g of the lipase preparation of Example 1 is mixed with

I 16.7 g of the liquified fat and oil mixture, and 10 cc of 0.IN acetic acid for about 24 hours at 37°C. Substantially ) complete hydrolysis into fatty acids and glycerol is obtained. !

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Other weak acids may be substituted for the i acetic acid in the examples, such as phosphoric, phosphorous or i| carbonic acid. i Lower melting point fats such as sheep tallow, industrial quality beef tallow, lard and butter may be used in the present lipase hydrolysis process. [ il Also, other vegetable oils such as corn, soybean, fH linseed, olive and palm oil may be substituted for the coconut 0il in the examples. i

It is understood that the foregoing detailed description is given merely by way of illustration and that if variations may be made therein without departing from the spirit f 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 invention. 1 i il t

Claims (1)

1. HI : ) : aE 26178 . ; WHAT IS CLAIMED IS:

   1. A process of converting high melting fats in- i $o a high yield of fatty ecids and glycerol which com- prises hydrolyzing an emulsified mixture of a high melt~ j 5 ing beef tallow and a vegetable oil in the weight ratio 1 of about 75-90% tallow to 25-10% oil, in en agueous me- } i diun, with an enimal or vegetable lipase which ie non- g ] stereospecific at a temperature of about 25-50°C, end at ] a pH of about 4-5.5 agitating the mixture for a suffi- § 10 cient period of time of about 3-48 hours to obtain 3 substantially complete hydrolysis into fatty acids and glycerol and recovering a final reaction mixture consist- 8 A ing of fatty acids, glycerol end lipase.

   1 2. The process according to Claim 1, wherein the 2 16 vegetable oil is coconut oil. 4 3, The process according to Claim 2, wherein the 2 f mixture of the beef tallow and coconut oil is in the weight 4 3 ratio of 83:17, ‘ 3 | 4. Te process according to Claim 1, wherein the EEL Jipase cbhstitutes about 3-15% of the weight of the tallow 4 and vegetable oil mixture,

   a. BAD ORIGINAL E 4 5+ The process according to Claim 1, wherein the 4 1 Lo lipase enzyme is derived from castor bean, Candida cylin-

   i 8 F dracea, Propionibacterium acnes, Rhizopus arrhizus, f Staphylococcus aureus, Asperigillus flavus and Geotri- , chun candidum,

   6. A process according to Claim 5, wherein the i 5 lipase enzyme is a castor bean lipase which is prepared by dehulling the castor beans, extracting the lipase by 4 grinding the dehulled beans in the presence of a low boil- , ing petroleum ether, Filtering the ground beans, extract- 3 ing and filtering the lipase again, air drying the fi1-~ tered lipase ang recovering a dried castor bean lipgse’ 1 Te A process according to Claim 5, wherein the 1 enzyme is a castor bean lipase which ig prepared by mgce~ rating the dehulled beans in water, centrifuging the mix- 3 ture to form fat layer and an aqueous lgyer, separating the fat layer from the aqueous layer, extracting the fat : layer with petroleum ether and saturated sodium chloride Solution, and Tecovering the lipase from the sodium chlo- ride solution,

   8. A process of producing high grade B0ap which ‘ 20 comprises hydrolyzing an emulsified mixture of a high melt- ing beef tallow and about 10-25% of a vegetable oil, in an aqueous medium, in the presence of non-stereospeci fic lipase enzyme, at a temperature of about 25-50% and at a pH of about 4-5.5, agitating the mixture for a period BAD ORIGINAL

   : — ’ of time of about 3-48 hours to obtain substantially com~ plete hydrolysis into fatty acids and glycerol, Separat- : ing the glycerol from the fatty acids, and neutralizing sald fatty acid with an alkaline material to form g 80ap free of undesirable additives, 9« A process according to Claim 8, wherein the alkaline neutralizing material ig selected from the group consisting of sodium hydroxidx, sodium carbonate and sodium bicarbonate;

   10. A process according to Claim 8, wherein the lipese enzyme is castor bean lipase;

   11. A process according to Claim 6, wherein the castor bean lipase ig separated from the fatty acids and glycerol and jg recycled for use in hydrolyzing o fresh mixture of fat ang oil, 12, A process according to Claim 8, wherein the vegetable oil is cocomt oil,’ 13, A process of hydrolyzing g high melting fat ; into a high yield of fatty acids for use in the production Of a soap free of undesirable additives, which comprises reacting a mixture of about 90-75% by weight of a high BAD ORIGINAL melting beef tallow ang about 10-25% coconut oil with castor bean lipase in about 20-50% water acidified to a pH of about iH 2 i] i 1 4-5.5 and at a temperature of about 37°¢,, agltating 1 } the reaction mixture for about 3-48 hours to obtain a final reaction mixture consisting of three layers, a g * ; fatty acid top layer, a lipase mixture middle layer and E 5 an sgueous glycerin bottom layer, separating the top 3 layer from the other layer and neutralizing acid fatty J acids with a sodium salt selected from the group consist- 3 ing of sodium hydroxide, sodium carbonate, and sodium bi-~ carbonate to form soap.

   14. A process according to Claim 13, wherein the middle leyer containing active castor bean lipase is re- used in the hydrolyzation process.

   15. A process according to Claim 13, wherein the water is acidified with a weak acid.

   16. A process according to Claim 15, wherein the 8 weak acid is citric acid.

   17. A process according to Claim 13, wherein the fatty acids are neutralized with sodium hydroxide, : 18. A process according to Claim 7, wherein the p 20 castor bean lipase is separated from the fatty acids and ] glycerol and is recycled for use in hydrolyzing a fresh [ mixture of fat and oil. ed ORIGINAL ee et

   1 26178 8 - 7 ; 4-5.5 end at a temperature of about 37°Cs, egltating the reaction mixture for about 3-48 hours to obtain a y final reaction mixture consisting of three lgyers, a } 1 fatty acid top layer, a lipase mixture middle layer and =n 5 an egueous glycerin bottom layer, separating the top - layer from the other layer and neutralizing acid fatty acids with a sodium salt selected from the group consist ing of sodium hydroxide, sodium carbonate, and sodium bi~ ; carbonate to form soap, 14, A process according to Claim 13, wherein the middle layer containing active castor bean lipase 1s re—- used in the hydrolyzation process,

   15. A process according to Claim 13, wherein the water is acidified with a weak acids

   16. A process according to Claim 15, wherein the weak acid is citric acid.

   17. A process according to Claim 13, wherein the fatty acids are neutralized with sodium hydroxides

   18. A process according to Claim 7, wherein the castor bean lipase is separated from the fatty acids end glycerol end is recycled for use in hydrolyzing a fresh mixture of fet and oil, .. — DRIGINAL

   ) i: i ¢ 1 «7 4 1 19. A jrocess according to Claim 10, wherein the } 3 castor bean lipase is Separated from the Tatty acids ang 4 8lycerol and ig recycled for use in hydrolyzing a fresh mixture of fat ang oil, ; EDVARD ALBERT pAvsg A EDVARD RIGEN ¥ Inventors % 3 § ! hy: a rE | x if FNGIIAL = /