Preparation of 2-Monoglycerides

Chapter 5 Preparation of 2-Monoglycerides Adam W. Mazur, George D. Hiler, II, and Magda El-Nokaly

Downloaded by NORTH CAROLINA STATE UNIV on April 14, 2013 | http://pubs.acs.org Publication Date: December 26, 1991 | doi: 10.1021/bk-1991-0448.ch005

Food and Beverage Technology Division, Miami Valley Laboratories, The Procter and Gamble Company, P.O. Box 398707, Cincinnati, OH 45239-8707

We present a method for the selective hydrolysis of triglycerides resulting in an efficient preparation of 2-monoacyl glycerols or specific mixtures of the latter compounds with 1,2-diacyl glycerols. These derivatives are important substrates for the synthesis of tailored triglycerides. The hydrolysis reactions are carried out in a simple biphasic system consisting of hexane, alkyl alcohol, and enzyme-containing buffer, without added emulsifiers. The analysis of reaction kinetics shows that selectivity of this hydrolysis depends upon the specific composition of solvents and the key factor in the selectivity may be modification of the interface properties by solvents. The work on selective hydrolysis of triglycerides to 2-acyl glycerols, described in this chapter, has been part of our research on the preparation of tailored triglycerides. These are important materials for the investigations of functional and biological properties of pure fat components. Unfortunately, there are no published procedures suitable for preparation on a large scale of specific triglycerides having different three acyl groups. We found that the reactions between 2-monoacyl glycerols and fatty acid anhydrides catalyzed by lipase in organic solvents can produce diglycerides selectively with very little formation of triglycerides (Figure 1). Acylation of diglycerides with another acyl anhydride in the presence of an enzyme or a chemical catalyst, such as 4-N,N-dimethylaminopyridine, produces the required specific triglycerides. These products are also expected to have an excess of one enantiomer since the NMR spectra of diglycerides measured in the presence of a chiral shift reagent indicate that the reaction in Figure 1 is stereoselective (Mazur, A. W.; Hiler, G. D., The Procter & Gamble Co., unpublished data).

0O97-6156/91/0448-O051$O6.00/0 © 1991 American Chemical Society In Microemulsions and Emulsions in Foods; El-Nokaly, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

MICROEMULSIONS AND EMULSIONS IN FOODS


52

However, the key substrates, 2-acyl glycerols, were not readily available i n imiltigram quantities. Typical chemical methods (1) used for their preparation require several steps and yields are frequently affected by iscmerization of the acyl group between 1(3)- and 2- positions of glycerol. Another possible source of 2-monoacyl glycerols i s hydrolysis of triglycerides with 1,3-specific lipases. The reaction produces mono- and diglycerides as intermediates (2) before finally giving glycerol (Figure 2). Unfortunately, selectivity i n a simple aqueous hydrolysis i s not easy to control and preparation of 2-monoglycerides i s not practical by this route. Recently, i t was reported that the formation of miarcemulsion phase, by addition

(R, C 0 ) 0

-OH

-OCOR-

2

Lipozyme Organic Solvent

OCOR

-OCOR OH

Figure 1.

Regioselective Esterification of 2-Acyl Glycerols

..OCOR

ÔCOR t)COR

-OCOR

k_

-OCOR

1

OH

rjG)

k OH

(DG)

1

1

2

2

-OCOR

*0H

(MG)

OH

2

[MG][A] - k [DG] 2

[MG][A] - k^ [MG]

d[G]/dt = k^ [MG]

Figure 2.

(G)

+ RCOOH (A)

[DG][A]

d[DG]/dt = k [TG] - k_T [DG][A] + k_ d[MG]/dt = k [DG] - k_

ÔH

+ RCOOH (A)

+ RCOOH (A) d[TG]/dt = -k-,[TG] + k_

ÔH

Hydrolysis of Triglycerides

In Microemulsions and Emulsions in Foods; El-Nokaly, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


5. MAZUR ET AL.

Preparation of 2-Monoglycerides

of emulsifiers, can induce selectivity i n selectivity i n lipolysis and good yields of 2-monoacyl glycerols were obtained (3-4). However, this method i s not convenient as the product has to be purified from the mixtures containing substantial quantities of emulsifiers. Due to these limitations, our objective was to find an efficient method for the preparation of 2-monoacyl glycerols i n large quantities. It i s known that enzymatic lipolysis takes place at the oil/water interface (5) for typical biphasic mixtures and micrOemulsions. The latter systems, of fine dispersion of micelles are optically transparent. Physically, they are s t i l l biphasic with the enzymes located i n the micellar water pools (6), the micelle palisade, or i n the interfacial film. Mcroemulsions can promote selectivity because association structures created by emulsifiers and solvents modify properties of the interface thus influencing enzyme activity (6-11) as well as kinetics and equilibria (7b, 12-14) of the reactions. The reactants present in the lipolysis mixtures including triglycerides, diglycerides, monoglycerides and fatty acids, can create various association structures such as liquid crystals or microemulsions depending icon a specific composition of the mixture (15-18). The presence of organic solvents i s one of the important factors affecting self-association of amphophilic molecules (19-20). We were interested i n influencing the process of micaxDstructure formation and the properties of interface by proper selection of solvent and, ultimately, controlling the selectivity of the biphasic hydrolysis without emulsifiers. MATERIALS AND METHODS

Hexane and butanols were purchased from J.T. Baker Go., phosphate buffer from Fisher and Lipolase from Novo. The quantitative GC analysis of the reaction mixtures including triglycerides, diglycerides, monoglycerides, glycerol, and acid was done losing HP 588QA instrument equipped with a capillary column (DB^l 15 m χ 0.247 mm, film thickness 0.25 micron). Samples were analyzed under the following conditions: injection port temperature 340o, s p l i t injection (60:1), flame ionization detector at 325°C. Oven temperature profile: 70°C to 300°C at 15°C/min hold 300°C for 10 min. A l l samples were silylated with Ν,Ο-bis- (trimethylsilyl) - trifluoroacetamide (Pierce) prior to the injection into column i n order to convert glycerol and i t s partial esters into volatile derivatives. Changes i n concentrations of these species with time were recorded and used for plotting the reaction progress curves as well as calculating the individual rates for each hydrolysis step. Quantitative analysis of the kinetic data was performed with the program GEAR Iterator (21) on the VAX 785 computer. The program was purchased from the Quantum Chemistry Exchange Programs at Indiana University. The points on the graphs are the experimental data while the continuous lines represent the computer generated f i t (vide supra).



54

MICROEMULSIONS AND EMULSIONS IN FOODS

Hvdrolvsis of Trioctanoin i n Bichasic Mixtures. Quantities of reagents and solvents used i n the reactions are shown i n Table 1. In the reactions with primary alcohol, 20 ml of 1-butanol were used. The mixtures were stirred vigorously at 25°C. We did not follow the pH during the reactions but GC analysis showed that at least 85% of free acids formed i n each hydrolysis partitioned into the organic phase. After stirring was stopped, a l l the reaction mixtures gave good phase separation upon standing and only two phases, upper organic and lower aqueous, were seen. For isolation of 2-octanoyl glycerol, organic phase was evaporated at the temperature below 3CrC to a viscous residue which was dissolved i n hexane. Ihe product precipitating from the solution at -78°C was filtered at this temperature and dried under vacuum. The yield was 70%. Hydrolysis of Other Triglycerides. Fully saturated triglycerides, up to trimyristin ( C ) , can be hydrolyzed analogously to trioctanoin. Beginning*with tripalmitin ( C ), preparation of 2-monoglycerides according to our procedure i s not practical since hydrolysis occurs very slowly. However, unsaturated triglycerides regardless of the chain length undergo hydrolysis similarly to trioctanoate. 1 4 : 0

1 6 ; 0

ANALYSIS OF ΊΗΕ REACTION KINETICS To investigate the phenomena responsible for the selectivity of lipolysis, we have analyzed the reaction progress curves (Figures 3 and 4 are examples) and variations i n the rate constants (Table I) as calculated from the kinetic model (Figure 2). Effect of a Primary Alcohol. Figure 3 shows the reaction progress curve for the hydrolysis of triglyceride i n buffer without addition of organic solvents. Tricaprylin i s not soluble in the buffer and the reaction mixture i s biphasic. The reaction reaches an equilibrium or a steady state characterized by a relatively low degree of triglyceride hydrolysis and low monoglyceride concentration. This confirms our earlier assumption that l i t t l e selectivity i n formation of 2-monoglycerides can be expected i n the unmodified hydrolysis system. In contrast, hydrolysis i n the presence of hexane and 1-butanol (Figure 4) produces excellent yields of 2-monoglycerides. Caprylic acid i s esterified i n the latter process to butyl ester thus moving the reaction equilibrium towards products. One could certainly argue at this point whether the properties of interface are really important for the displayed selectivity, or the good yield of 2-monoglyceride i s solely the result of the equilibrium shift caused by acid esterification with butanol. That the interface modification i s indeed important for the selectivity, can be concluded from the results discussed below. Effects of a Secondary and longer Chain Alcohols. Upon changing the alcohol to 2-butanol, which i s not esterified i n the reaction, the selectivity of 2-monoglyceride formation was s t i l l


5. MAZUR ET

55

Preparation of 2-Monoglycerides

Table 1. Biphasic Hydrolysis of Trioctanoin Effects of Solvent Composition, and Triglyceride Concentration

Entry

TG

Solvent

k


None Hexane 2-Butanol 2-Butanol 2-Butanol 2-Butanol 2-Butanol 2-Butanol

Hexane Hexane Hexane Hexane Hexane

2.03 0.70 1.40 0.72 0.60 0.41 0.31 0.21

3

3

10"

[M] 1 2 3 4 5 6 7 8

k

*2

l

332.0 225.0 346.0 217.0 214.0 256.0 204.0 202.0

4.69 20.30 2.20 5.59 5.95 7.22 7.41 8.61

72.9 108.0 106.0 78.7 89.6 78.0 94.1 70.3

0.77 7.6 44.5 13.70 4.7

Preparation of 2-Monoglycerides

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