Selective hydrolysis of borage oil with Candida rugosa lipase: Two factors affecting the reaction

A 46% γ-linolenic acid (GLA)-containing oil was produced by selective hydrolysis of borage oil (GLA content, 22%) at 35°C for 15 h in a mixture containing 50% water and 20 units (U)/g reaction mixture of Candida rugosa lipase. The GLA content was not raised over 46%, even though the hydrolysis extent was increased by extending the reaction time and by using a larger amount of the lipase. However, 49% GLA-containing oil was produced by hydrolysis in a reaction mixture with 90% water. This result suggested that free fatty acids (FFA) that accumulated in the mixture affected the apparent fatty acid specificity of the lipase in the selective hydrolysis and interfered with the increase of the GLA content. To investigate the kinetics of the selective hydrolysis in a mixture without FFA, glycerides containing 22, 35, and 46% GLA were hydrolyzed with Candida lipase. The result showed that the hydrolysis rate decreased with increasing GLA content of glycerides, but that the release rate of GLA did not change. Thus, it was found that the apparent fatty acid specificity of the lipase in the selective hydrolysis was also affected by glyceride structure. When 46% GLA-containing oil was hydrolyzed at 35°C for 15 h in a mixture containing 50% water and 20 U/g of the lipase, GLA content in glycerides was raised to 54% at 20% hydrolysis. Furthermore, GLA content in glycerides was raised to 59% when the hydrolysis extent reached 60% using 200 U/g of the lipase. These results showed that repeated hydrolysis was effective to produce the higher concentration of GLA oil. Because film distillation was found to be extremely effective for separating FFA and glycerides, large-scale hydrolysis of borage oil was attempted. As a result, 1.5 kg of 56% GLA-containing oil was obtained from 7 kg borage oil by repeated reaction.     

Study on the composition of rice bran oil and its higher free fatty acids value

The compositions of rice bran oils (RBO) and three commercial vegetable oils were investigated. For refined groundnut oil, refined sunflower oil, and refined safflower oil, color values were 1.5–2.0 Lovibond units, unsaponifiable matter contents were 0.15–1.40%, tocopherol contents were 30–60 mg%, and FFA levels were 0.05–0.10%, whereas refined RBO samples showed higher values of 7.6–15.5 Lovibond units for color, 2.5–3.2% for unsaponifiable matter, 48–70 mg% for tocopherols content, and 0.14–0.55% for FFA levels. Of the four oils, only RBO contained oryzanol, ranging from 0.14 to 1.39%. Highoryzanol RBO also showed higher FFA values compared with the other vegetable oils studied. The analyses of FA and glyceride compositions showed higher palmitic, oleic, and linoleic acid contents than reported values in some cases and higher partial glycerides content in RBO than the commonly used vegetable oils. Consequently, the TG level was 79.9–92% in RBO whereas it was >95% in the other oils studied. Thus, refined RBO showed higher FFA values, variable oryzanol contents, and higher partial acylglycerol contents than commercial vegetable oils having lower FFA values and higher TG levels. The higher oryzanol levels in RBO may contribute to the higher FFA values in this oil.     

Lipase‐catalyzed alcoholysis of vegetable oils

Fatty acid alkyl esters were produced from various vegetable oils by transesterification with different alcohols using immobilized lipases. Using n‐hexane as organic solvent, all immobilized lipases tested were found to be active during methanolysis. Highest conversion (97%) was observed with Thermomyces lanuginosa lipase after 24 h. In contrast, this lipase was almost inactive in a solvent‐free reaction medium using methanol or 2‐propanol as alcohol substrates. This could be overcome by a three‐step addition of methanol, which works efficiently for a range of vegetable oils (e.g. cottonseed, peanut, sunflower, palm olein, coconut and palm kernel) using immobilized lipases from Pseudomonas fluorescens (AK lipase) and Rhizomucor miehei (RM lipase). Repeated batch reactions showed that Rhizomucor miehei lipase was very stable over 120 h. AK and RM lipases also showed acceptable conversion levels for cottonseed oil with ethanol, 1‐propanol, 1‐butanol and isobutanol (50‐65% conversion after 24 h) in solvent‐free conditions. Methyl and isopropyl fatty acid esters obtained by enzymatic alcoholysis of natural vegetable oils can find application in biodiesel fuels and cosmetics industry, respectively.     

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.     

Production of thermostable alkaline lipase on vegetable oils from a thermophilic Bacillus sp. DH4, characterization and its potential applications as detergent additive

BACKGROUND: The alkaline lipase production on vegetable oils as sole carbon source, its characterization and effect of different commercial detergents and surfactants on enzymatic activity from thermophilic Bacillus sp. DH4 was investigated. RESULTS: The organism grew on mannose, but the amount of lipase secreted was significantly less than on vegetable oils. This study identified a simple substrate for lipase production and established the utility of groundnut oil for increasing the lipase yield. The enzyme was compatible with various ionic and non‐ionic surfactants as well as commercial detergents. Lipase activity was strongly inhibited by sodium dodecyl sulfate (SDS), but not by Triton X‐100 or Triton X‐114. The best assay conditions observed for this lipase was found to be pH 9.0 and 50 °C. The enzyme was stable at alkaline pH and considerable activity was observed at pH 10 and it retained 93% of the residual activity at 60 °C. The lipase showed a novel property of marked activation at alkaline pH. Wash performance analysis of commercial detergent for removal of fatty stains improved upon addition of lipase. CONCLUSION: The production on cheap vegetable oils, novel properties and resistance towards various surfactants and tolerance to commercial detergents make this lipase a potential additive for detergent formulations. Significance and impact of the study: Bacillus sp. produces alkaline and thermostable lipase on cheap vegetable oils and its compatibility can find use in the detergent industry. Copyright © 2008 Society of Chemical Industry     

In vitro Action of Pancreatic Lipase on Complex Glycerides from Thermally Oxidized Oils

The action of pancreatic lipase on complex glyceridic molecules originating during thermal oxidation of fats is defined. Four samples of increasing level of alteration compounds – 4.5, 37.1, 67.2 and 100% respectively – have been analyzed by means of a combination of chromatographic techniques, which allows quantification of the most representative groups of degradation compounds – oxidized triacylglycerol monomers, triacylglycerol dimers and polymers, diacylglycerols and fatty acids. The samples have been subjected to in vitro hydrolysis by pancreatic lipase at different reaction times and modifications originated have been defined by means of high performance size exclusion chromatography (HPSEC) as well as by titration of fatty acids released. The results clearly demonstrate that enzymatic hydrolysis depends on the molecular weight of the glycerides being slower as alteration compound weight increases. Moreover, by direct quantitation of complex glycerides remaining after the action of the enzyme, the influence of different variables (reaction time, alteration level, etc) can be deduced.     

Enzymatic Production of Fatty Acid Methyl Esters by Hydrolysis of Acid Oil Followed by Esterification

Acid oil, a by-product of vegetable oil refining, was enzymatically converted to fatty acid methyl esters (FAME). Acid oil contained free fatty acids (FFA), acylglycerols, and lipophilic compounds. First, acylglycerols (11 wt%) were hydrolyzed at 30 °C by 20 units Candida rugosa lipase/g-mixture with 40 wt% water. The resulting oil layer containing 92 wt% FFA was used for the next reaction, methyl esterification of FFA to FAME by immobilized Candida antarctica lipase. A mixture of 66 wt% oil layer and 34 wt% methanol (5 mol for FFA) were shaken at 30 °C with 1.0 wt% lipase. The degree of esterification reached 96% after 24 h. The resulting reaction mixture was then dehydrated and subjected to the second esterification that was conducted with 2.2 wt% methanol (5 mol for residual FFA) and 1.0 wt% immobilized lipase. The degree of esterification of residual FFA reached 44%. The degree increased successfully to 72% (total degree of esterification 99%) by conducting the reaction in the presence of 10 wt% glycerol, because water in the oil layer was attracted to the glycerol layer. Over 98% of total esterification was maintained, even though the first and the second esterification reactions were repeated every 24 h for 40 days. The enzymatic process comprising hydrolysis and methyl esterification produced an oil containing 91 wt% FAME, 1 wt% FFA, 1 wt% acylglycerols, and 7 wt% lipophilic compounds.     

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.     

Enrichment of polyunsaturated fatty acids withGeotrichum candidum lipase

Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.     

Strategies for the enzymatic enrichment of PUFA from fish oil

PUFA from oil extracted from Nile perch viscera were enriched by selective enzymatic esterification of the free fatty acids (FFA) or by hydrolysis of ethyl esters of the fatty acids from the oil (FA‐EE). Quantitative analysis was performed using RP‐HPLC coupled to an evaporative light scattering detector (RP‐HPLC‐ELSD). The lipase from Thermomyces lanuginosus discriminated against docosahexaenoic acid (DHA) most, resulting in the highest DHA/DHA‐EE enrichment while lipase from Pseudomonas cepacia discriminated against eicosapentaenoic acid (EPA) most, resulting in the highest EPA/EPA‐EE enrichment. The lipases discriminated between DHA and EPA with a higher selectivity when present as ethyl esters (EE) than when in FFA form. Thus when DHA/EPA were enriched to the same level during esterification and hydrolysis reactions, the DHA‐EE/EPA‐EE recoveries were higher than those of DHA/EPA‐FFA. In reactions catalysed by lipase from T. lanuginosus, at 26 mol% DHA/DHA‐EE, DHA recovery was 76% while that of DHA‐EE was 84%. In reactions catalysed by lipase from P. cepacia, at 11 mol% EPA/EPA‐EE, EPA recovery was 79% while that of EPA‐EE was 92%. Both esterification of FFA and hydrolysis of FA‐EE were more effective for enriching PUFA compared to hydrolysis of the natural oil and are thus attractive process alternatives for the production of products highly enriched in DHA and/or EPA. When there is only one fatty acid residue in each substrate molecule, the full fatty acid selectivity of the lipase can be expressed, which is not the case with triglycerides as substrates.     

Structure of unsaturated vegetable oil glycerides: Direct calculation from fatty acid composition

Composition and structure of unsaturated glycerides of vegetable oils can be calculated directly from the fatty acid composition of the oil. Fatty acid distribution on the 2 position as normally determined by lipase hydrolysis is calculated from the composition of the whole oil by applying the following three rules in their respective order: Saturated fatty acids and those with chain length greater than 18 carbons are first distributed equally and randomly on the 1 and 3 position of the glycerol moiety; oleic and linolenic acids are treated equally, or as a unit, and distributed equally and randomly on all three glyceride positions with any excess from the 1 and 3 position being added to the 2 position; and all remaining positions are filled by linoleic acid. Remarkably good agreement between the calculated and experimentally determined fatty acid distributions is shown for soybean, linseed, safflower and many other vegetable oils whose compositions are reported in the literature. An association between oleic and linolenic acid within the glyceride structure of some vegetable oils is evident. Presented in part at the T. P. Hilditch Symposium Annual Meeting AOCS, Houston, Texas, 1965. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Uptake of blood triglyceride by various tissues

Triglycerides are transported in the blood in chylomicrons and very low density lipoproteins. Electron microscopic studies indicate that these particles, which range in diameter from 0.03–0.6 μ, cannot cross the capillary endothelium in most tissues. There is now considerable evidence that the triglycerides are hydrolyzed to free fatty acids (FFA) during uptake and that this process is catalyzed by lipoprotein lipase. The enzyme is found in nearly all tissues that utilize circulating triglyceride, and the level of activity, in individual tissues, varies with nutritional and physiological states that affect triglyceride uptake, such as fasting, diabetes and pregnancy. Studies in perfused adipose tissue with doubly labeled chylomicrons showed that hydrolysis occurs outside of the blood stream. Two-thirds of the fatty acids are incorporated into tissue triglyceride and the rest are release as FFA, with glycerol, to the blood. Infusion of heparin causes immediate release of lipoprotein lipase activity to the blood and decreases the amount of chylomicron-triglyceride hydrolyzed by the tissue. Electron microscopic cytochemical studies showed that hydrolysis of blood glycerides by lipoprotein lipase in adipose tissue occurs within the capillary endothelial cells and in the subendothelial space near the pericytes, but not in the capillary lumen or near the fat cells. The results indicate that the fatty acids of chylomicrons cross the capillary endothelium as glycerides and FFA, within a membrane-bounded system, and cross the extravascular space to the fat cells as FFA. Presented at the AOCS Meeting, Atlantic City, October 1971.     

Enzymatic glycerolysis and transesterification of vegetable oil for enhanced production of feruloylated glycerols

Novel functional groups can be introduced into vegetable oils using enzymes, resulting in value-added products. The transesterification kinetics of ethyl ferulate with MAG, DAG, and TAG were examined. Transesterification was catalyzed by immobilized Candida antarctica lipase B in solventless batch and packed-bed reactors. Initial reaction rates with TAG were slightly sensitive to water activity, whereas rates with MAG and DAG were water activity independent. Transesterification was also three-to sixfold faster with MAG and DAG. These observations indicate that the reaction is rate limited by the acyl acceptor, and that oils with free hydroxyl groups are preferred acyl acceptors in comparison with TAG, which must undergo partial hydrolysis before becoming reactive.     

Prospects for application of enzymatic interesterification of oils in the production of modified fats

This review discusses a recent state of research in the field of enzymatic biocatalysis in application for the production of modified food fats. Properties of biocatalysts for enzymatic interesterification both been currently under development and already applied in industry of vegetable oils are discussed. The main directions of research on development of new biocatalysts, including those based on the novel recombinant lipase enzymes, as well as the potential of targeted modifications in the composition of oils by optimization of the catalytic process are covered. The relevant analysis of the enzymatic interesterification of oils shows its potential for development of energy efficient and environmentally friendly processes for production of the high quality food products with the specified characteristics.     

Organogel-Based Emulsion Systems,Micro-Structural Features and Impact on In Vitro Digestion

Organogels based on edible oils and specific mixtures of phytosterols can serve as structured systems with a low saturated fat content. These low-SAFA organogels can be used also to create o/w emulsions. Little is known about the structures formed in these specific organogels and at the emulsion interface. We studied o/w organogels on different length scales to describe and understand their micro-structural features. Very basic processing conditions such as composition, temperature and storage time were taken into account. Two different types of structure were observed; at the smallest scale, long thin crystals are formed out of the oil phase into the continuous water phase. We propose that these are needle-like crystals. Next, tube-like structures are identified and can be visualized as tubular micelles. A model is proposed which fits the dimension (~7 nm) with the length scale of the molecular building blocks (TAGs and sterols). As edible fats from food products are enzymatically hydrolyzed in the gut prior to absorption, we also looked into the impact on the lipase reaction speed. Simple in vitro enzymatic hydrolysis experiments showed a slower enzymatic digestion. Organogel systems and emulsion made thereof have interesting food structuring properties with possible advantages in composition (low SAFA) and digestion speed. All authors were full time employees or trainee (MC) of Unilever during actual execution of this study.     

Application of a specificity ofMucor miehei lipase to concentrate docosahexaenoic acid (DHA)

Lipases catalyze the reesterification or hydrolysis of fats and oils and may show specificity towards certain fatty acids. An immobilized lipase from the fungusMucor miehei shows specificity towards docosahexaenoic acid (DHA) (DHA being a poor substrate), whether the DHA is used as a free fatty acid (FFA) as substrate in esterification with methanol, or fatty acid methyl ester (FAME) is used as a substrate for hydrolysis. The specificity of the lipase fromM. miehei may be applied to concentrate DHA originating from a marine oil in either the FAME or the FFA fraction, which can be separated.     

Free fatty acid fractions from some vegetable oils exhibit reduced survival time-shortening activity in stroke-prone spontaneously hypertensive rats

Previously, we demonstrated that several vegetable oils that included low-erucic rapeseed oil markedly shortened the survival time (by ∼40%) of stroke-prone spontaneously hypertensive (SHRSP) rats as compared with perilla oil, soybean oil, and fish oil. We considered that a factor other than fatty acids is toxic to SHRSP rats, because the survival time-shortening activity could not be accounted for by the fatty acid compositions of these oils. In fact, a free fatty acid (FFA) fraction derived from lipase-treated rapeseed oil was found to be essentially devoid of such activity. A high-oleate safflower oil/safflower oil/perilla oil mixture exhibited a survival time-shortening activity comparable to that of rapeseed oil, but the activity of this mixed oil was also reduced by lipase treatment. A partially hydrogenated soybean oil shortened the survival time by ∼40%, but a FFA fraction derived from lipase-treated partially hydrogenated soybean oil shortened it by 13% compared with soybean oil. Fatty acid compositions of the rapeseed oil and a FFA fraction derived from lipase-treated rapeseed oil were similar, but those of hepatic phospholipids of rats fed the oil and FFA were slightly but significantly different. These results support the interpretation that the survival time-shortening activity exhibited by some vegetable oils is due to minor components other than fatty acids, and that an active component(s) were produced in or contaminated soybean oil during the partial hydrogenation processes.     

Isolation of tocopherol and sterol concentrate from sunflower oil deodorizer distillate

The isolation of tocopherols and sterols together as a concentrate from sunflower oil deodorizer distillate was investigated. The sunflower oil deodorizer distillate was composed of 24.9% unsaponifiable matter with 4.8% tocopherols and 9.7% sterols, 28.8% free fatty acid (FFA) and 46.3% neutral glycerides. The isolation technology included process steps such as biohydrolysis, bioesterification and fractional distillation. The neutral glycerides of the deodorizer distillates were hydrolyzed byCandida cylindracea lipase. The total fatty acids (initial FFA plus FFA from neutral glycerides) were converted into butyl esters withMucor miehei lipase. The esterified product was then fractionally distilled in a Claisen-vigreux flask. The first fraction, which was collected at 180–230°C at 1.00 mm of Hg for 45 min, contained mainly butyl esters, hydrocarbons, oxidized products and some amount of free fatty acids. The fraction collected at 230–260°C at 1.00 mm Hg for 15 min was rich in tocopherols (about 30%) and sterols (about 36%). The overall recovery of tocopherols and sterols after hydrolysis, esterification and distillation were around 70% and 42%, respectively, of the original content in sunflower oil deodorizer distillate.     

Lipase-mediated plant oil hydrolysis—Toward a quantitative glycerol recovery for the synthesis of pure allyl alcohol and acrylonitrile

Plant based triacylglycerides are a sustainable feedstock for the chemical, bioenergy, food and cosmetic sector. While fatty acids conversion has been intensively studied, processes for glycerol valorization have been scarce. In contrast to chemical hydrolysis of plant triacylglycerides enzymatic hydrolysis methods provide a cleaner glycerol stream. This study focuses on the selection of a commercial lipase capable of quantitative hydrolysis of rapeseed- and high oleic sunflower oils. Enzymatic process using only water as the reaction medium allows simplified recovery of pure glycerol. Among the six commercial lipase preparations tested, Candida rugosa lipase was identified as the most effective biocatalyst. Catalytic behavior in buffer and pure water was equivalent. Glycerol generated using a washed lipase was recovered by just three purification steps. The FTIR spectrum of the recovered glycerol was equivalent to pure glycerol standard. Over the entire recovery process, 82%–88% of the theoretical glycerol yield could be obtained. Purified glycerol was further didehydroxylated to allyl alcohol by a formic acid mediated open distillation process. The enriched allyl alcohol had a quality, which allows to use it for the synthesis of bio-based acrylonitrile.     

Enzymatic conversion of steryl esters to free sterols

Conversion of oleic acid phytosteryl esters (OASE) to free phytosterols (referred to as sterols) by an enzymatic process was attempted. Enzymatic hydrolysis of OASE reached a steady state at 55–60% hydrolysis, but addition of methanol (MeOH) significantly accelerated the conversion of OASE to sterols. Screening of commercially available enzymes indicated that Pseudomonas aeruginosa lipase was most effective for the conversion. Based on the study of several factors affecting the reaction, the optimal conditions were determined as follows: ratio of OASE to MeOH, 1∶2 (mol/mol); water content, 10 wt%; lipase amount, 20 U/g by weight of reaction mixture; temperature, 30°C. When the reaction was conducted for 48 h with stirring, the conversion reached 98%. FAME accumulated in the reaction mixture, but FFA did not, indicating that the FAME was poorly recognized as a substrate in the reverse conversion of sterols to OASE but the FFA was easily recognized as a substrate. The high conversion of OASE to sterols was therefore due to elimination of FFA from the reaction system. After the enzymatic reaction, the oil layer was fractionated at −20°C with 5 vol parts of n-hexane. Sterols were efficiently purified in the resulting precipitate (92% recovery, 99% purity).