JPH0730352B2 - Enzymatic purification of fats and oils - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for purifying fats and oils by an enzyme, and more specifically, a fat and oil to which monoglyceride lipase and / or diglyceride lipase (hereinafter collectively referred to as partial glyceride lipase) is applied. , A monoglyceride and / or diglyceride mixed in triglyceride in fats and oils (hereinafter collectively referred to as partial glycerides) are hydrolyzed, and a method for refining fats and oils.

That is, an object of the present invention is to decompose and remove partial glyceride, which is an impurity contained in fats and oils, to provide fats and oils having a high triglyceride content.

BACKGROUND ART Oils and fats contain an ester of fatty acid and glycerol, that is, triglyceride as a main component, and a small amount of partial glyceride, free fatty acid and unsaponifiables as components other than triglyceride.

Partial glycerides mixed in fats and oils are those synthesized in vivo in the plants and animals and microorganisms from which they originate, and triglycerides have been partially hydrolyzed by the action of lipase or non-enzymatic action contained in the fats and oils during storage. Or a product produced as a by-product in the process of processing fats and oils such as transesterification and synthesis of fats and oils from fatty acid and glycerol. Among natural oils and fats, especially palm oil collected from the flesh of oil palm (Elaeis guineensis) and olive oil collected from the fruit of olives (Olea europaea) have a high water content, so that they are hydrolyzed by enzymes during the oil collection process and storage. Since rice bran, which is a source of rice oil, contains a strong lipase activity, each fat has a high partial glyceride content.

Conventionally, as a method of removing impurities in fats and oils, first, alkali purification for the purpose of removing fatty acids, vacuum steam distillation for the purpose of removing odorous components and other volatile components and fatty acids, and gums, carbohydrates, Degumming is performed for the purpose of removing proteins and the like, and removal of these impurities is relatively easy. However, the separation of tri, di and monoglycerides from each other is carried out by means of such analysis, such as column chromatography using silicic acid, thin layer chromatography and gel permeation chromatography utilizing the molecular sieve effect. The method of is not used too much. Further, for the purpose of removing fatty acids and fractionating the constituent triglycerides of fats and oils, fractional crystallization utilizing the difference in melting point of triglyceride, liquid-liquid extraction utilizing the difference in solubility in a solvent, and fractional distillation utilizing the difference in boiling point and molecules. Distillation has been carried out, but diglyceride was difficult to remove by these methods although some monoglyceride was removed.

Problems to be Solved by the Invention The removal of partial glycerides from fats and oils by the above-mentioned chromatographic method has not been adopted at all on an industrial scale even though it is possible in the laboratory. Further, in the above-mentioned means for the purpose of separating the constituent triglycerides in the oil and fat, diglyceride and triglyceride form a eutectic mixture, so that these cannot be separated. That is, it can be said that there has been substantially no method suitable for the purpose, although it has been conventionally desired to industrially remove partial glyceride from fats and oils.

The inclusion of partial glycerides has various undesirable effects on fats and oils. First, partial glyceride has an action of preventing the formation of triglyceride crystal nuclei. For example, the presence of about 2% or more of monoglyceride in palm oil inhibits the growth of triglyceride crystal nuclei, and about 13% of triglyceride crystal nuclei exist.
% Diglyceride significantly increases the lifetime of α-type crystals, and generally the presence of diglyceride in fats or oil products suppresses the crystal transition of β ′ to β-type of triglyceride (Olaginiaux, 2
9 , 421,1974; Oil Palm News, 22 , 10-18,1977; J. Sci. Food
Agric., 32 , 1197,1981 and Fette Seifen Anstrichm, 8
5 , 64, 1983).

Next, diglyceride makes a eutectic mixture with triglyceride as described above, not only makes it difficult to separate them, but also has the action of reducing the solid fat index of fats and oils, so that the high melting point triglyceride involved in crystals is Since the apparent content is lower than the actual content, the yield of the solid part is reduced,
At the same time, a part of the solid part remains in the liquid part, so that triglyceride is not completely separated. Further, the partial glyceride itself has an emulsifying action, which causes a decrease in the efficiency in the fractional crystallization of triglyceride using a polar organic solvent.

In addition to the effect on the fractionation of the triglyceride, as an example where the presence of partial glyceride in fats and oils becomes a problem,
In the case of fats and oils products that require a sharp melting point, such as cocoa substitute fat, partial glyceride is a causative substance that makes the melting point of fats and oils unclear and wide.

Means and Actions for Solving Problems According to the present invention, by causing a partial glyceride lipase to act on fats and oils, only partial glycerides mixed in the fats and oils are hydrolyzed, and as a result, separation from triglycerides becomes easy.

The partial glyceride lipase used in the present invention is a so-called monoglyceride in which any one of the three hydroxyl groups of glycerol is esterified with a fatty acid and / or glycerol at 1,2 (or 2,3) to 1,3 positions. The enzyme has a property of hydrolyzing a so-called diglyceride whose hydroxyl group is esterified with a fatty acid, but has no or little specificity for triglyceride esterified with a fatty acid at all three positions. So-called triglyceride lipase, which has high specificity not only for partial glycerides but also for triglycerides, cannot be used for the purpose of the present invention because it hydrolyzes triglycerides first rapidly.

Examples of the partial glyceride lipase used in the present invention, rat small intestine, monoglyceride lipase or diglyceride lipase derived from animal organs such as porcine adipose tissue, having specificity for monoglyceride and diglyceride produced by filamentous fungi of the genus Penicillium. Examples include lipase. A lipase derived from a Penicillium spp., Particularly preferably Penicillium cyclopium (Peni) deposited at the American Type Culture Collection under the accession number ATCC 34613
The partial glyceride lipase produced by Cillium cyclopium) is used. The strain is listed in the catalog of the depositary institution and can be obtained by any person. It is known that penicillium cyclopium produces a partial glyceride lipase simultaneously with triglyceride lipase (J. Biochem., 87 , 205-211, 1980). However, we are not aware of any prior literature suggesting that the partial glyceride lipase can be used for the purposes of the present invention.

In order to obtain the partial glyceride lipase used in the present invention from a strain of the genus Penicillium, the penicillium strain having the ability to produce a partial glyceride lipase is grown in a medium used for culturing a normal microorganism, and then in a culture. Partial glyceride lipase is accumulated and can be collected from this. The partial glyceride lipase collected from the culture can be purified by a known purification means, but it is not necessary to purify it to a pure state for the purpose of the present invention. However, if the crude enzyme contains triglyceride lipase, it must be removed. The culture of Penicillium cyclopium ATCC 34613 strain which is particularly preferably used in the present invention contains triglyceride lipase, but the crude enzyme solution is chromatographed, particularly preferably chromatograph using DEAE-Sepharose CL-6B. The triglyceride lipase can be separated and removed by applying E. coli.

The fats and oils to be refined in the present invention are natural and synthetic fats and oils, processed fats and oils thereof, and fats and oils products containing them. Natural fats and oils include crude crude oil extracted from animal tissues, plant fruits and seeds, cultures of microorganisms, and the like, and alkali refining and degumming of these crude crude oils,
Refined oils that have been subjected to deodorization and the like are included, synthetic fats and oils include fats and oils synthesized from fatty acids and glycerol in the presence of a catalyst, and processed fats and oils are commonly used in the art for fats and oils. Those that have undergone processing techniques such as fractionation, hydrogenation and transesterification are included. More specifically, soybean oil, cottonseed oil, palm oil, coconut oil, olive oil,
Crude crude oils such as rice oil, sesame oil, corn oil, safflower oil, beef tallow, pork oil, fish oil and refined oils thereof, as well as hardened oils obtained by processing them, transesterified oils, tempura oils, frying oils, salad oils, hard butters. , Margarine, short toning, refined lard, drying oil and the like.

The conditions for the reaction between the fat and oil and the partial glyceride in the present invention may be any conditions as long as the hydrolysis activity of the partial glyceride lipase used can be discovered. For example, the reaction temperature can be selected at an appropriate value depending on the optimum temperature of the partial glyceride lipase used and the melting point of the fat or oil. Usually, 20 to 55 ° C is preferable. The reaction time may be selected such that the partial glyceride in the oil or fat falls to the required value. The lipase reaction is generally reversible, and catalyzes a hydrolysis reaction when the water content of the reaction system is high and an ester synthesis reaction when the water content is low. Therefore, in the present invention, the presence of water is indispensable, and it can be said that the higher the water content, the more preferable, but the addition of too much water only causes troublesome removal of water during the purification of fats and oils after the enzymatic reaction. The suitable water content is 0.5 to 10 times (v / w) with respect to fats and oils. Water or a buffer solution having a pH suitable for the partial glyceride lipase used is used for the adjustment of water content. The amount of partial glyceride lipase used can be changed depending on the amount of partial glyceride present in fats and oils, reaction temperature, time, etc.
0.1 to 1,000 units, particularly preferably 1 to 100 units.
In the present invention, the partial glyceride lipase can be bound to an insoluble carrier and used as an immobilized enzyme. The immobilized enzyme is preferable because the enzyme can be used again or continuously.

Furthermore, in the present invention, it is preferable to add an organic solvent to the reaction system. The addition of an organic solvent has the effects of reducing the water content of the reaction system, dissolving high-melting oils and fats, and promoting the reaction rate. The organic solvent used dissolves fats and oils, but may be any one as long as it does not inhibit the hydrolysis activity of the partial glyceride lipase. For example,
Preferred organic solvents include n-hexane, cyclohexane, decane, undecane, hexadecane, tetradecane, pentadecane, petroleum ether, diethyl ether,
Acetone and methyl ethyl ketone are mentioned. The amount of the organic solvent added is preferably 0.1 to 10 times (v / w) of the oil and fat.

Regarding the operation of the reaction, since the fat and the water-soluble enzyme inherently lack affinity, it is desired to adjust the physical conditions so as to improve the contact with each other. For example, beads can be placed in a reaction vessel equipped with a stirrer and reacted to increase the contact surface between the fat and oil and the partial glyceride lipase, thereby allowing the reaction to proceed rapidly.

According to the present invention, partial glycerides in fats and oils hydrolyzed by partial glyceride lipase produce glycerol and free fatty acids, which are automatically separated from triglycerides because glycerol immediately migrates to the aqueous layer, while free fatty acids Although it remains in the oil tank as it is, it can be easily separated from triglyceride by the conventional means for refining fats and oils, such as alkali refining, fractional crystallization, vacuum distillation, and vacuum distillation of ester with lower alcohol.

The present invention will be described in detail below with reference to test examples and examples. However, the analysis of the components of fats and oils and the definition of the unit of partial glyceride lipase are as follows.

Analysis of components of oils and fats Raw oils and fats or reaction mixtures of oils and fats and partial glyceride lipase are extracted into petroleum ether, and the oil layer components are applied to a preparative silica gel thin layer plate (PLK5, Whatman), and petroleum ether: diethyl ether: Acetic acid (80:30:
(1, volume ratio), and spots were detected with iodine vapor. The spots corresponding to each glyceride and fatty acid were collected, and glyceride was measured with an enzymatic glyceride analysis reagent composition using Triglyceride lipase (trade name: Triglyceride G-test wako, manufactured by Wako Pure Chemical Industries, Ltd.). Reagent composition for enzymatic free fatty acid analysis using acyl CoA synthetic oxygen (trade name: NEFA C-test wak
o, manufactured by the same company). The ratio of each molecular species is shown by the molar ratio (%).

Definition of units of partial glyceride lipase Mix 0.95 ml of 50 mM acetate buffer (pH 5.6) containing 2.5 mM p-nitrophenyllauric acid and 2.0% Triton X-100 with 0.05 ml of enzyme solution, and mix at 37 ° C for 15 minutes. After the incubation, the reaction is stopped by adding 2.0 ml of acetone.
Then, the amount of p-nitrophenol released in the reaction was adjusted to 41
It is determined from the intensity of absorbance at 0 nm. The activity of partial glyceride lipase was defined as 1 unit of the amount of the enzyme that liberates 1 micromol of p-nitrophenol in 1 minute in the above reaction.

Test Example Preparation of partial glyceride lipase Penicillium cyclopium was added to a liquid medium (pH 6.0) consisting of 4% rice bran and 3% corn staple liquor.
Inoculated with ATCC 34613, aerated at 26 ° C with stirring,
Cultured for 2 days. After filtering the bacterial cells from the obtained culture solution, the filtrate was concentrated by ultrafiltration. DEAB this concentrate
-Cepharose CL-6B (manufactured by Pharmacia) was subjected to chromatography to separate and remove coexisting triglyceride lipase to obtain a purified partial glyceride lipase preparation. It was confirmed that this preparation hydrolyzes monoglyceride and diglyceride, but has little effect on triglyceride.

This partial glyceride lipase was used in all of the following explanations.

Example 1 1.0 g each of two kinds of unrefined palm oils having different production lots
And partial glyceride lipase aqueous solution 1.0 ml (20 units)
And n-hexane 3.0 ml and diameter 5 mm glass beads 30
Put the pieces in an Erlenmeyer flask with a capacity of 100 ml,
It was shaken for time (amplitude 3 cm, cycle 140 times / min). Original 2
The types of palm oil contained diglyceride and monoglyceride in total of 6.48% and 13.31%, respectively, but none were detected after the reaction (shown in Table 1). In the table, TG, FA, DG and MG represent triglyceride, fatty acid, diglyceride and monoglyceride, respectively (the same applies hereinafter).

Example 2 1.0 g of a purified palm oil product from which fatty acids were removed, 0.5 ml (20 units) of an aqueous solution of partial glyceride lipase, and four types of reaction mixtures prepared by mixing the amounts of water and n-hexane shown in Table 2 below were prepared. The reaction was carried out according to Example 1. The original palm oil contains a total of diglyceride and monoglyceride 2.
Although it was contained in an amount of 06%, it was not detected at all after the reaction was completed (shown in Table 2).

Example 3 The same operation as in Example 2 was performed using a refined rice oil product from which fatty acids were removed instead of palm oil. As shown in Table 3, the content of partial glyceride could be significantly reduced by adding water and n-hexane in appropriate amounts to the reaction mixture.

Example 4 1.0 g of processed comb oil, aqueous solution of partial glyceride lipase 1.
0 ml (10 units) and 3.0 ml of various organic solvents shown in Table 4 below
Alternatively, 2.0 ml of 50 mM acetate buffer (pH 5.6) was mixed and reacted according to Example 1. By adding these organic solvents, partial glyceride could be remarkably decomposed even with a low water content (shown in Table 4).

Example 5 Each of the following processed fats and oils, that is, palm oil, olive oil, rice oil, rapeseed oil, sesame oil and cottonseed oil, 1.0 g, 5.0 ml of 50 mM acetate buffer (pH 4.5) and the activity shown in Table 5 below are included. 1.0 ml of an aqueous solution of partial glyceride lipase was mixed, and the temperature and reaction time shown in the same table and operation were carried out according to Example 1. Table 5 shows the composition of fats and oils before and after the reaction.

Example 6 1,000 g of crude palm oil having a composition of 76.4% triglyceride, 10.3% fatty acid, 8.9% 1,3-diglyceride, 2.8% 1,2-diglyceride and 1.6% monoglyceride and 1,000 ml of an aqueous solution of partial glyceride lipase (20,000 Unit) and 3,000 ml of n-hexane are mixed and stirred under stirring to 40
The reaction was carried out at 0 ° C for 1 hour. The reaction mixture was allowed to stand and the oil layer and the water layer were separated, then 151 ml of 20 ° B sodium hydroxide was added to the obtained oil layer, and the mixture was stirred at 60 ° C., and the formed precipitate of Soken was filtered off. The oily substance obtained was subjected to vacuum distillation to remove the solvent, and 833 g of purified palm oil was obtained. The composition of this oil is 99.98% triglyceride, 0.0
In 2%, no partial glyceride was detected.

Effects of the Invention According to the present invention, partial glycerides mixed as impurities in fats and oils are enzymatically hydrolyzed into fatty acids and glycerol without decomposition of triglycerides, and as a result, separation from triglycerides is facilitated. The fats and oils refined according to the present invention have a high triglyceride content and their physical properties such as crystallinity and melting point are improved, and can be high-quality and high-value-added fats and oils products.

Claims (2)

[Claims] 1. A lipase having a property of acting on monoglycerides and / or diglycerides derived from the penicillium genus of fats and oils but not substantially on triglycerides is added in an amount of 0.1 to 1,000 units per 1 g of fats and oils, and the water content is relative to the fats and oils. 0.1 to 10 times (v / w), and if necessary, one or more organic solvents selected from n-hexane, cyclohexane, decane, undecane, hexadecane, tetradecane, pentadecane, petroleum ether, diethyl ether, acetone and methyl ethyl ketone. 0.1 to 10 times (v
/ w) in the presence of the enzyme to hydrolyze monoglyceride and / or diglyceride present in fats and oils. 2. The lipase is penicillium cyclopium.
The method for purifying fats and oils with an enzyme according to claim 1, which is a lipase produced by the ATCC34613 strain.