Production of microbial lipases for the study of triglyceride structure

Lipases from different microorganisms are known to differ in their site of attack on triglycerides, and recent evidence has indicated that they may be useful in studying triglyceride structure. This paper is concerned with the most recent developments in the production, recovery, and stability of lipases from three of these microorganisms. The lipase fromStaphylococcus aureus, which attacks both the 1-, 2-, and 3-positions of triglycerides, is produced in an aerated, tryptic digest of casein at 30C in 1–2 days. The lipase fromGeotrichum candidum, which attacks primarily unsaturated fatty acid linkages and shows some stereospecificity, is produced in a static culture grown on a mineral salts-glucoseprotein hydrolysate medium incubated at 20C for 4–5 days. An improved method is described for preparing lyophilized preparations of these, and thePseudomonas fragi lipase, which are quite stable when stored in a refrigerator.     

Low-calorie triglyceride synthesis by lipase-catalyzed esterification of monoglycerides

Monoglycerides of erucic acid (C_22:1, Δ13), prepared by conventional methods, were reacted with caprylic acid (octanoic acid, C_8.0) by using lipases as catalysts with the intention of synthesizing a triglyceride that contains two molecules of caprylic acid and one molecule of erucic acid (caprucin). The reaction was carried out by mixing lipase powder, a small quantity of water, and the reactants in a temperature-controlled stirred batch reactor. Organic solvents or emulsifying agents were not required. When the nonspecific lipase fromPseudomonas cepacia was used, a yield of approximately 37% caprucin was obtained, together with a complex mixture of di- and triglycerides that resulted from the random transesterification of the erucic acid. The fatty acid-specific lipase fromGeotrichum candidum promoted minimal transesterification of erucic acid and resulted in a yield of 75% caprucin and approximately 10% interesterification products. Lipase fromCandida rugosa exhibited a similar, although less pronounced, specificity to that fromG. candidum and promoted more transesterification of erucic acid. Optimum conditions forG. candidum lipase were at 50°C and an initial water content of 5.5%. After the reaction, erucic acid was converted to behenic acid by hydrogenation, thereby converting caprucin into caprenin, a commercially available low-calorie triglyceride.     

Characterizing cocoa butter seed crystals by the oil-in-water emulsion crystallization method

Unambiguous quantitative evidence for the catalytic action of seed crystals in cocoa butter is presented. We used an ultrasound velocity technique to determine the isothermal growth of solid fat content in cocoa butter oil-in-water emulsions, in which the probability of finding a seed crystal in any one droplet was around 0.37 at 14.2°C. The upper limit for the size of seed crystals in West African cocoa butter was around 0.09 μm, the Gibbs free energy for nucleation was 0.11 mj m⁻², and the concentration of seed crystals was in the range of 10¹⁶ to 10¹⁷ m⁻³. X-ray diffraction measurements showed that emulsified cocoa butter crystallizes in the α polymorph and does not appear to transform to the β′ form within the first 25 min of crystallization. Primary nucleation events in cocoa butter emulsions are accounted for by seed crystals. Collision-mediated nucleation, a secondary nucleation mechanism, in which solid droplets (containing seed crystals) catalyze nucleation in liquid droplets, is shown to account for subsequent crystallization. This secondary nucleation mechanism is enhanced by stirring.     

Specificity of a lipase fromGeotrichum candidum forcis-octadecenoic acid

A lipase fromGeotrichum candidum released mostly oleic acid from glyceryl 1-elaidate-2,3-dioleate and very littletrans fatty acid from margarine. When cod liver, Macadamia nut, peanut and safflower oils were substrates, the oleic acid content of the free acids was always in excess of the amount of the acid in the intact triglycerides. Congo palm oil was digested by bothG. candidum and pancreatic lipases and the fatty acid compositions of the products of hydrolysis compared. The results obtained with the aid ofG. candidum lipase tend to substantiate existence of some of the triglyceride isomers predicted from pancreatic lipase data. Scientific contribution No. 134, Agricultural Experiment Station, University of Connecticut, Storrs.     

Phase behavior and extended phase scheme of static cocoa butter investigated with real-time X-ray powder diffraction

A complete isothermal phase-transition scheme of cocoa butter under static conditions is presented, based on time-resolved X-ray powder diffraction experiments. In contrast to what is known from literature, not only β V, but also β VI can be obtained directly through transformation from β′. Another remarkable result is that β′ exists as a phase range rather than as two separate phases. Within this β′ phase range no isothermal phase transitions have been observed. More detailed information concerning the observed cocoa butter polymorphs was obtained by determination of melting ranges, using time-resolved X-ray powder diffraction. Also standard X-ray powder diffraction patterns of the γ, the α, and the two β phases and parts of the β′ phase range have been recorded. The observed phase behavior of cocoa butter has been explained based on the concept of individual crystallite phase behavior of cocoa butter     

Selective hydrolysis of polyunsaturated fatty acid-containing oil withGeotrichum candidum lipase

Polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can be concentrated in glycerides by hydrolyzing tuna oil withGeotrichum candidum lipase, the main components in the resulting oil being triglycerides. The reaction mechanism of this selective hydrolysis was investigated. Although the lipase acted well on the esters of oleic, linoleic, and α-linolenic acids, it did not affect the esters of γ-linolenic acid, arachidonic acid, EPA, and DHA as much. The action of PUFA-glycerides was mono-> di- > triglycerides. Furthermore, the condensation of PUFA-partial glycerides and PUFA occurred even in the presence of a large amount of water, and the partial glycerides converted to the triglycerides by transacylation. These results suggested that the PUFA-rich triglycerides were accumulated in the glyceride fraction by the following mechanism: The PUFA-partial glycerides generated by the hydrolysis were converted to PUFA-triglycerides by condensation and transacylation reactions. As the PUFA-triglycerides formed were the poor substrates of lipase, they were accumulated in the reaction mixture.     

Adipose hormone-sensitive lipase preferentially releases polyunsaturated fatty acids from triglycerides

Rat adipose hormone-sensitive lipase-mediated release of fatty acids from triglycerides was studied in three model systems: i) cultured preadipocytes containing polyunsaturated fatty acid-enriched triglyceride; ii) perfused epididymal fat pads; and iii)in vitro incubations of crude preparations of hormone-sensitive lipase with synthetic triglyceride-analogues as substrates. We found that cultured preadipocytes challenged with 10μM norepinephrine tended to release more ω6 and ω3 polyunsaturated fatty acids than saturated fatty acids. Fat pads perfused with 10 μM norepinephrine preferentially released arachidonate and α-linolenate but tended to retain oleate and linoleate. Finally, crude preparations of hormonesensitive lipase released from the triglyceride-analogue substrates α-linolenate twice as fast as oleate. We conclude that rat adipose hormone-sensitive lipase preferentially releases polyunsaturated fatty acids from triglycerides. We suggest that this may be a mechanism by which these fatty acids are kept from being trapped in fat depots and maintained in the circulation.     

Modification of selected indian vegetable fats into cocoa butter substitutes by lipase-catalyzed ester interchange

A few solid and semi-solid fats of tree origin in India, namely sal (Shorea robusta), kokum(Garcinia indica), mahua (Madhuca latifolia), dhupa (Vateria indica) and mango (Mangifera indica), were chosen for modification into cocoa butter substitutes by lipase-catalyzed ester interchange with methyl palmitate and/or stearate. Hexane solutions of mixtures of fat and methyl ester(s) in various molar proportions were passed through a column of Lipozyme™, a lipase fromMucor miehei immobilized on a macroparticulate ion-exchange resin. The interesterified fats were purified by extraction with 95% ethanol followed by silica column chromatography. Interesterified dhupa, kokum and sal fats compared well with cocoa butter in the total fatty acid composition and the 2-position of triacylglycerols, as well as glyceride composition. In particular, interesterified kokum fat resembled cocoa butter well in solid fat content and peak melting temperature as determined by differential scanning calorimetry. IICT Communication No. 2743.     

Synthesis of cocoa butter equivalent by lipase-catalyzed interesterification in supercritical carbon dioxide

With supercritical carbon dixoide as a reaction medium, the syntheses of cocoa butter equivalent by interesterification with various lipases were investigated. The study showed that among those five lipases tested, lipase IM-20 from Mucor miehei was the most effective and specific in synthesizing this cocoa butter equivalent product by interesterification. The yields of cocoa butter equivalent are affected by pressure, substrate oil composition, solubility and co-solvent. The best reaction conditions were: reaction pressure at 1500 psi, triglyceride with high content of POP (P, palmitate; O, oleate) and POO, reaction medium with 5.0% water, and reaction temperature at 50°C. The major component of cocoa butter, POS (S, stearate), can be increased by 6.0% by adding a small amount of carbon dioxide. The yield and melting point of the purified cocoa butter equivalent are 53.0% and 34.3°C, respectively.     

Determination of the fully saturated triglyceride composition of fats

A method is described for the quantitative determination of the fully saturated triglyceride composition of fats. The method involves a quantitative oxidation of the unsaturated triglycerides in the fat to a mixture of α ketol and dihydroxy compounds, using aqueous alkaline potassium permanganate and a phase transfer catalyst in a two phase reaction. The fully saturated triglycerides are fractionated from the oxidation products by column chromatography on silicic acid and their composition is quantified by gas liquid chromatography (GLC) using a flame ionization detector. Analyses of samples of lard, cocoa butter, olive oil, soybean oil, crude palm oil (ex Malaysia) and the stearin fraction from palm oil are given as examples of the method. Presented as a poster presentation at the 14th World Congress for Fat Research in Brighton, England (1978).     

The interpretation of GLC triglyceride data for the determination of cocoa butter equivalents in chocolate: A new approach

A new approach to interpret triglyceride data obtained by gas liquid chromatography (GLC) in order to determine cocoa butter equivalents (CBE) in chocolate is described. The approach is based on the known straight line relationship which exists between the C₅₀ and C₅₄ triglycerides of cocoa butter of different origins and the realization that, for currently available CBE conforming to CAOBISCO’s criteria, a similar band relationship exists. The technique described enables the quantity of unspecified CBE in a chocolate containing an unknown cocoa butter to be determined to an accuracy of ±1.5% when present in chocolate at the 5% level. Nut oils (almond, walnut or hazelnut) are sometimes present in mainland European chocolates and, should CBE also be present, it is possible to calculate the combined percentages of nut oil and CBE in the chocolate. The method of interpretation described is not dependent on a particular GLC technique for determining triglycerides. Interpretation of other laboratories’ results obtained using different GLC instruments and procedures has shown that the method enables any CBE present in the fat under examination to be determined accurately. The method compensates for variations in the composition of CBE and for the differences between cocoa butters of different origin. A detailed knowledge of CBE compositions is not required and only a few cocoa butter/CBE standards are necessary. The method described is graphical, enabling small laboratories not equipped with microcomputers to utilize the method. The calculation can, however, be programmed for a computer.     

Contribution of triglycerides from cocoa butter to the physical properties of milkfat fractions

Milkfat was separated into major chainlength fractions by solid-phase extraction. The effect on thermal behavior and texture of replacing both saturated and monounsaturated long-chain triglycerides from milkfat by long-chain monounsaturated triglycerides with an unsaturated fatty acid in thesn-2 position is reported. Increasing proportions of cocoa butter were added to fractions of short-to medium-chain triglycerides (C₂₂−C₄₄) and medium- to long-chain triglycerides (C₃₆−C₄₈) isolated from milkfat. Thermal behavior and texture of the mixtures were measured. Results indicated that long-chain monounsaturated triglycerides from cocoa butter enhanced co-crystallization and co-operative melting and did not induce polymorphic transitions upon crystallization and melting of the fractions. At 4°C, they acted as texture builder if present in proportions of more than 30%, whereas below this level, they acted as texture softeners. The effect of the long-chain monounsaturated triglycerides on the texture of fractions that melt at low temperature could not be predicted from the proportion of solid fat at that temperature. Presented at the 1995 AOCS Annual Meeting & Expo, San Antonio, Texas, May 1995.     

Polymorphism of cocoa butter

Largely by x-ray diffraction six crystalline states, I–VI, in order of increasing melting point, have been identified for cocoa butter. Of these states II, IV, V and VI are pure and identifiable with previously (or presently) identified polymorphs of 2-oleoylpalmitoyl stearin (POS), namelyα-2,β′-2,β-3 (“V”) andβ-3 (“VI”); V and VI representing distinct but very closely related crystalline structures. State I is a definite but fleeting and not readily characterized subα state and may be a phase mixture, as state III may be also. Melting points, heats of fusion and dilatometric data are reported for all states to the extent that their stability permits. The normal state of cocoa butter in chocolate is apparently V, certainlyβ-3. While it is true that “bloom” has not been observed for pure V nor observed to exist in the absence of VI, it is premature to say that VI is specifically the phase of chocolate “bloom”.     

Lipase-catalyzed enrichment of long-chain polyunsaturated fatty acids

Lipase hydrolysis was evaluated as a means of selectively enriching long-chain ω3 fatty acids in fish oil. Several lipases were screened for their ability to enrich total ω-3 acids or selectively enrich either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA). The effect of enzyme concentration, degree of hydrolysis, and fatty acid composition of the feed oil was studied. Because the materials that were enriched in long-chain ω3 acids were either partial glycerides or free fatty acids, enzymatic reesterification of these materials to triglycerides by lipase catalysis was also investigated. Hydrolysis of fish oil by eitherCandida rugosa orGeotrichum candidum lipases resulted in an increase in the content of total ω3 acids from about 30% in the feed oil to 45% in the partial glycerides. The lipase fromC. rugosa was effective in selectively enriching either DHA or EPA, resulting in a change of either the DHA/EPA ratio or the EPA/DHA ratio from approximately 1:1 to 5:1. Nonselective reesterification of free fatty acids or partial glycerides that contained ω3 fatty acids could be achieved at high efficiency (approximately 95% triglycerides in the product) by using immobilizedRhizomucor miehei lipase with continuous removal of water.     

A new concept for determining triglyceride composition of fats and oils by liquid chromatography

The matrix concept was used to characterize the chromatographic rules in the elution of molecular species of triglyceride. To prove the hypothesis experimentally, cacao butter, palm oil, linseed oil, olive oil, rapeseed oil and triglyceride of “Ogonori” (Gracilaria verrucosa) were examined. A matrix model was suggested to help determine the individual molecular species of naturally occurring triglycerides.     

The determination of cocoa butter equivalents in chocolate

A method of determining cocoa butter equivalents in chocolate and cocoa butter is described. The method relies on a new approach for interpreting data obtained by triglyceride gas liquid chromatography (GLC). This technique provides information on the composition of a fat according to the carbon number of the triglycerides (C_n). Examination of the data for a wide range of cocoa butters shows that a straight line relationship between the C₅₀ and C₅₄ contents exists. This relationship has been used as the basis for a quantitative method determining the amount and type of cocoa butter equivalent added to chocolate. The application of the method to both plain and milk chocolate is described. The method is also used to determine the amount of milk fat in chocolate.     

Triglyceride characteristics of cocoa butter from cacao fruit matured in a microclimate of elevated temperature1

Generally, the melting characteristic of cocoa butter is relatively constant. However, softer than normal butter is sometimes encountered. In Brazil the occurrence of soft butter has been correlated with mean daily temperature during the cropping season. The temperature effect was, therefore, studied more fully by positioning heat lamps near fruit of cacao to create a microclimate of elevated temperature during the most active period of lipid biosynthesis. As determined by differential scanning calorimetry, cocoa butter from these fruits, contained more solid fat at 16 C, and 20 C and 24 C than butter from control fruit matured in a normally lower temperature climate. The temperature effect on softness was in accordance with differences found in triglyceride types. S₂U and SU₂ triglycerides from heat-exposed samples were 88.5% and 8.9%, respectively, compared to 79.3% and 18.3% for cocoa butter from control fruit. Additional evidence of differences due to growth temperature was obtained by analysis of triglyceride fractions separated by high-performance liquid chromatography. Presented in part at 1979 American Oil Chemists' Society Meeting, San Francisco.     

Rapid MS method for analysis of cocoa butter TAG

Ammonia negative ion CI-MS was applied to analyze the M.W. distribution and regioisomeric structure of TAG in cocoa butter and in cocoa butter equivalents. The M.W. distribution results obtained for a reference cocoa butter were consistent with corresponding results obtained in an intercomparison study by chromatographic methods. Minor but statistically significant differences were observed when proportions of the three major M.W. species (52∶1, 54∶1, and 50∶1; acyl carbon number/number of double bonds) in a mixture of nine cocoa butters and in mixtures containing 10 or 20% (w/w) of specific cocoa butter equivalents were compared. Tandem MS was used to determine the regioisomeric structure of the three major TAG M.W. species in cocoa butter and in cocoa butter equivalents. The regioisomeric structure in cocoa butter and in all the equivalents analyzed were nearly identical, oleic acid being located primarily in the sn-2 position. These results cannot be exploited in detecting added foreign fats in this case. However, the present study shows that useful TAG composition data, which may be used to detect foreign fats in cocoa butter by applying chemometric data evaluation, can be obtained by MS in a significantly shorter time compared to chromatographic methods.     

Production of a very low saturate oil based on the specificity of Geotrichum candidum lipase

Lipase B (GCB) produced by the fungus Geotrichum candidum CMICC 335426 is known for its high specificity towards cis-Δ9 unsaturated fatty acids. The wild-type lipase (not genetically modified) as well as the lipase obtained by heterologous expression of the corresponding gene in Pichia pastoris (genetically modified) were studied in a process aiming to produce an oil containing very little saturated fatty acids (SAFA). The approach described in this paper is based on the selective hydrolysis of sunflower oil (12% SAFA) using the G. candidum type B (GCB) lipases. Depending on the lipase input, up to 60% w/w degree of hydrolysis was obtained within 6–8 h. Because of the high specificity of the GCB lipases (specificity factor ∼30), the level of unsaturates in the free fatty acid fraction was >99% w/w. In contrast with literature data, no loss of specificity was observed, even at the highest degree of hydrolysis obtained. Though both GCB lipases are stable at 30°C, the rate of hydrolysis decreased considerably during the process. Product inhibition as well as time-dependent deactivation (half-life ≈2 h) were shown to be involved. After separation of the oil phase, the unsaturated free fatty acids were recovered from the mixture by evaporation and reconverted to triglycerides by enzymatic esterification with glycerol. Because the GCB lipases have a very low efficiency for esterification, this reaction was carried out with immobilized Rhizomucor miehei lipase. Under continuous removal of the water generated during the process, >95% triglycerides were obtained in less than 24 h. Standard deodorization resulted in an odorless, colorless, and tasteless oil with less than 1% SAFA.     

Seeding effects on solidification behavior of cocoa butter and dark chocolate. I. Kinetics of solidification

Effects of seeding of fat crystals on the crystallization kinetics of cocoa butter and dark chocolate were examined with a rotational viscometer. The seed crystals employed were cocoa butter, 1,3-distearoyl-2-oleoylglycerol (SOS), 1,3-dibehenoyl-2-oleoylglycerol (BOB) and 1,2,3-tristearoylglycerol (SSS). The seed powders were prepared by pulverization below —50°C, the dimensions being in a range from 20–70 μm. Particular attention was paid to the influence of polymorphism of the seed crystal. We found that all of the above seed materials accelerated the crystallization, the degree of acceleration being in a following order; SOS (β ₁) > cocoa butter (Form V) > SOS (a mixture ofβ’ andβ ₂) > BOB (β ₂) > BOB (pseudo-β’) > SSS (β). Precise measurements of the crystallization kinetics showed that the most influential factors in the seeding effects are the physical properties of the seed materials—above all, thermodynamic stability, and similarity in the crystal structure to cocoa butter are the most determinative.