Microbial Phospholipase Research Articles

Evaluation of Microbial Phospholipase and Lipase Activity Through the Chromatographic Analysis of Crude, Degummed, and Transesterified Soybean Oil for Biodiesel Production.

The present study aimed at synthesizing fatty acid methyl esters in a combined enzymatic method by applying degumming and transesterification of soybean oil. A soluble lipase from Serratia sp. W3 and a recombinant phosphatidylcholine-preferring phospholipase C (PC-PLC) from Bacillus thuringiensis were used in a consecutive manner for phosphorus removal and conversion into methyl esters. By applying 1% of recombinant PC-PLC almost 83% of phosphorus was removed (final content of 21.01mg/kg). Moreover, a sensitive and selective high-performance liquid chromatography method coupled to tandem mass spectrometry was applied to obtain a comprehensive lipid profile for the simultaneous evaluation of phospholipids removal and diacylglycerol (DAG) increase. A significant increase for all the monitored DAG species, up to 138.42%, was observed by using the enzymatic degumming, in comparison to the crude sample, resulting in an increased oil yield. Serratia sp. W3 lipase was identified as a suitable biocatalyst for biodiesel production, converting efficiently the acylglycerols. The results regarding the physical-chemical characteristics show that the cetane level, density and pour point of the obtained biodiesel are close to current regulation requirements. These findings highlight the potential of a two-step process implementation, based on the combination of lipase and phospholipase, as a suitable alternative for biodiesel production.

Case study of chemical and enzymatic degumming processes in soybean oil production at an industrial plant

The vegetable oil degumming process plays a critical role in refining edible oil. Phospholipids (PL) removal from crude extracted soybean oil (SBO) by the enzymatic degumming process has been investigated in this work. Enzymatic degumming of extracted SBO with microbial phospholipase A1 PLA-1 Quara LowP and Lecitase Ultra enzymes have also been studied comparatively. The main novelty of our work is the use of the enzymatic degumming process on an industrial scale (600 tons a day). Many parameters have been discussed to understand in detail the factors affecting oil losses during the degumming process. The factors such as chemical conditioning (CC) by phosphoric acid 85%, the enzyme dosage mg/kg (feedstock dependent), the enzymatic degumming reaction time, and the characteristics of the plant-processed SBO have been discussed in detail. As a main point, the degummed oil with a phosphorus content of < 10 mg/kg increases yield. Quara LowP and Lecitase Ultra enzymes are not specific for certain phospholipids PL; however, the conversion rate depends on the SBO phospholipid composition. After 4 h, over 99% of Phospholipids were degraded to their lysophospholipid LPL (lysolecithin). The results showed a significant effect of operating parameters and characteristics of different origins of SBO, fatty acids FFA content, Phosphorus content and total divalent metals (Calcium Ca, Magnesium Mg and Iron Fe mg/kg) content on the oil loss. The benefit of using enzymatic degumming of vegetable oils rather than traditional chemical refining is that the enzymatic degumming process reduces total oil loss. This decrease is known as enzymatic yield. The enzymatic degumming also decreases wastewater and used chemicals and running costs; moreover, it enables physical refining by lowering the residue phosphorus to < 10 mg/kg.

Persistent organic pollutants in model fungal membranes. Effects on the activity of phospholipases

Soils are the final sink for multiple organic pollutants emitted to the environment. Some of these chemicals which are toxic, recalcitrant and can bioaccumulate in living organism and biomagnify in trophic chains are classified persistent organic pollutants (POP). Vast areas of arable land have been polluted by POPs and the only economically possible means of decontamination is bioremediation, that is the utilization of POP-degrading microbes. Especially useful can be non-ligninolytic fungi, as their fast-growing mycelia can reach POP molecules strongly bond to soil minerals or humus fraction inaccessible to bacteria. The mobilized POP molecules are incorporated into the fungal plasma membrane where their degradation begins. The presence of POP molecules in the membranes can change their physical properties and trigger toxic effects to the cell. To avoid these phenomena fungi can quickly remodel the phospholipid composition of their membrane with employing different phospholipases and acyltransferases. However, if the presence of POP downregulates the phospholipases, toxic effects and the final death of microbial cells are highly probable. In our studies we applied multicomponent Langmuir monolayers with their composition mimicking fungal plasma membranes and studied their interactions with two different microbial phospholipases: phospholipase C (α-toxin) and phospholipase A1 (Lecitase ultra). The model membranes were doped with selected POPs that are frequently found in contaminated soils. It turned out that most of the employed POPs do not downregulate considerably the activity of phospholipases, which is a good prognostics for the application of non-ligninolytic fungi in bioremediation.

Host phospholipid peroxidation fuels ExoU-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology.

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.

Current Developments on the Cloning and Expression of Microbial Phospholipase D

Phospholipase D (PLD, EC 3.1.4.4) is a suite of enzymes catalyzing the hydrolysis of the phosphodiester bond of phospholipids, and the transfer of phosphatidic acid to primary alcohols to generate different phospholipids. The transphosphatidylation reaction makes PLD have wide applications in food, pharmaceutical and cell biology fields. However, large-scale industrial production of PLD has been restricted by many factors. Genetic engineering technology has therefore been adopted to clone and express PLD genes to obtain PLD with high quality, high purity and high transphosphatidylation activity. In this review, current progresses on the cloning and high-efficiency expression of microbial phospholipase D are analyzed, with a hope to guide the large-scale industrial production of PLD and lay a solid foundation for the development and utilization of related products.

Spatiotemporal control of phosphatidic acid signaling with optogenetic, engineered phospholipase Ds.

Phosphatidic acid (PA) is both a central phospholipid biosynthetic intermediate and a multifunctional lipid second messenger produced at several discrete subcellular locations. Organelle-specific PA pools are believed to play distinct physiological roles, but tools with high spatiotemporal control are lacking for unraveling these pleiotropic functions. Here, we present an approach to precisely generate PA on demand on specific organelle membranes. We exploited a microbial phospholipase D (PLD), which produces PA by phosphatidylcholine hydrolysis, and the CRY2-CIBN light-mediated heterodimerization system to create an optogenetic PLD (optoPLD). Directed evolution of PLD using yeast membrane display and IMPACT, a chemoenzymatic method for visualizing cellular PLD activity, yielded a panel of optoPLDs whose range of catalytic activities enables mimicry of endogenous, physiological PLD signaling. Finally, we applied optoPLD to elucidate that plasma membrane, but not intracellular, pools of PA can attenuate the oncogenic Hippo signaling pathway. OptoPLD represents a powerful and precise approach for revealing spatiotemporally defined physiological functions of PA.

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology.

Emulsifying properties of hydrolysed and low HLB sunflower lecithin mixtures

Most commonly processes for lecithin modification are deoling, fractionation with absolute ethanol to produce phosphatidylcholine or phosphatidylinositol‐enriched fractions, or enzymatic hydrolysis of these compounds. These modifications are important for achieving an optimal balance between hydrophilic and lipophilic compounds (HLB) and ensure a good food processing ability. The aim of this work was to improve the O/W emulsifying properties of modified lecithins with low HLB mixing them with hydrolysed sunflower lecithin obtained using a microbial phospholipase A2 (PLA2). Thereby, three modified sunflower lecithins (hydrolysed (HL), deoiled (DSL), and PI fraction (PIf)) were obtained. These modified lecithins and their mixtures were applied as emulsifying agent of coarse and fine O/W emulsions (Φm = 0.3). Stability of different emulsions was evaluated through the evolution of backscattering profiles, particle size distribution, and mean particle diameters. Mixtures with a hydrolysed lecitihin increased the HLB (addition of lysophospholipids) and improved the O/W emulsifying properties of DSL and PIf in each case. Mixtures 0.5:0.5 of DSL/HL or PIf/HL produced a similar behavior than 1% HL emulsions, constituting a good alternative for production of new bioactive agents.Practical applications: This study deals about the increase of the O/W emulsifying properties and thereby the commercial value of modified sunflower lecithins with low HLB values. The main target of this research work is to contribute with useful information to the food industry.Flow diagram of the process used for producing different modified lecithins to analyze their emulsifier properties in mixtures.

Enzymatic synthesis and modification of structured phospholipids: recent advances in enzyme preparation and biocatalytic processes.

Phospholipids (PLs) containing specific polar head groups and fatty acids, artificially synthesized from a complex mixture of natural PLs, have considerable industrial applications. The biocatalytic approaches to synthesizing structured PLs are of great interest because the enzymes used show high selectivity and performance under mild conditions, leading to the generation of products that cannot easily be obtained by chemical catalysis. Although the limited supply of phospholipases (e.g., phospholipase D) has thus far been an obstacle to the widespread use of enzymatic processing, recent advances in enzyme preparation have opened up various applications for PL modification. In this review, attempts to increase the productivity and utility of microbial phospholipases and lipases are presented. We also summarize recent developments in enzyme-catalyzed modification of PLs, focusing particularly on the relevant reactions, bioreactor design, and novel proof-of-concept experiments.

Enzymatic degumming: Degumming efficiency versus yield increase

Enzymatic degumming trials were performed on crude extracted soybean (SBO) and rapeseed oils (RSO) with a microbial phospholipase A1 (Lecitase Ultra®). We obtained a degummed oil with a phosphorus content &lt; 10 mg/kg when applying an enzyme dosage of 30 mg/kg and (feedstock dependent) a contact time of 10–120 min. While a good degumming efficiency can already be obtained after a relative short reaction time, it was observed that a longer reaction time (1–2 h) is required for complete degradation of the phospholipids (PL), which results in a yield increase. We demonstrated that Lecitase Ultra® has no specificity for a given PL, but that the rate of conversion depends on the PL composition of the crude oil. After 60 min, 80% of phosphatidyl ethanolamine (PE) was degraded to its lyso‐form while only 40% of phosphatidyl inositol (PI) was converted in the same time interval. The formation of glycerophospholipids (GPL) was only observed after 60 min reaction.Practical applications: Practical advantages and disadvantages of the use of Lecithase Ultra for enzymatic degumming of oils is described, focusing on soybean oil and rapeseed oil. The information will be valuable and important for the oil refining industry.Degumming of soybean and rapeseed oils with phospholipase A1 (Lecithase Ultra) was performed and it was demonstrated that there is no specificity for a given phospholipid. However, the conversion rate depends on the phospholipid composition of the crude oils.

Characterization of novel phospholipase C from Bacillus licheniformis MTCC 7445 and its application in degumming of vegetable oils

A novel microbial phospholipase C (PLC) from Bacillus licheniformis MTCC 7445 was purified to homogeneity by ammonium sulphate fractionation, dialysis, anion exchange chromatography and gel exclusion chromatography. The bacteria growing on vegetable oils secreted significantly high amount of PLC. The enzyme was purified to 23.4-fold with 46% recovery and specific activity 398 U/mg. It exhibited optimum activity at 70°C and pH 10.0. Using diphosphatidylglycerol as substrate the PLC of B. licheniformis MTCC 7445 had a Vmax and Km of 0.68 mM/min and 32 mM, respectively. It hydrolyzed phosphatidylinositol and phosphatidylserine as well as phosphatidylcholine but not other glycerophospholipids. Its activity was enhanced by 113% with Mn2+ and 110% with Mg2+. During degumming of vegetable oils with this enzyme preparation, the phosphorus content of the oil became lower than 4 mg/kg after 5 h of enzyme treatment at 40°C. The novel PLC from B. licheniformis MTCC 7445 is potentially useful for the refining of high quality oils with 95% removal of phospholipids with attractive yield.

Improved purification and some properties of a novel phospholipase C from Bacillus mycoides strain 970

A novel microbial phospholipase C from Bacillus mycoides strain 970 was purified to apparent homogeneity by an improved purification process, in which DEAE-Cellulose adsorption was first used to remove bulk of dark brown impurities and then a combination of DEAE-Cellulose ion-exchange, Phenyl Sepharose hydrophobic interaction and Sephacryl S-100 gel filtration chromatography was used to separate the enzyme from remained contaminants. The enzyme appeared to be a single peptide of 75.1 kDa on SDS-PAGE. The optimum temperature and pH of the enzyme were 60°C and 7.0 - 7.5, respectively. The enzyme was stable at temperatures lower than 50°C and pH 5 - 9.5. This purified phospholipase C was characterized as a metallophospholipase C and Ca2+, Mg2+, Zn2+ , Ba2+ were essential for its PLC activity. The enzyme showed no lecithinase activity on egg yolk agar and also no hemolytic activity on blood agar. Key words: Phospholipase C, purification, metalloenzyme, nonhemolytic activity,Bacillus mycoides.

Insight into the Enzymatic Degumming Process of Soybean Oil

AbstractAn enzymatic degumming trial of soybean oil was carried out at a capacity of 400 tons/day by applying microbial phospholipase A1 from Thermomyces lanuginosus/Fusarium oxysporum. When the pH was kept in the range of 4.8–5.1, less than 10 mg/kg of phosphorous content of The oil was obtained. The gum and oil were easily separated after centrifugation and the oil loss was minimal under the process conditions. Through analysis of phospholipids compounds in the gum by Electrospray Ionization‐Mass Spectrometer and phosphorous content, it could be seen that both glycerophospholipids and lysophospholipids existed with contents of 45.7 and 54.3%, respectively. The performance of enzymatic degumming was found to be related to the production of glycerophospholipids.

Insights into the structure of plant α‐type phospholipase D

Phospholipases D play an important role in the regulation of cellular processes in plants and mammals. Moreover, they are an essential tool in the synthesis of phospholipids and phospholipid analogs. Knowledge of phospholipase D structures, however, is widely restricted to sequence data. The only known tertiary structure of a microbial phospholipase D cannot be generalized to eukaryotic phospholipases D. In this study, the isoenzyme form of phospholipase D from white cabbage (PLDalpha2), which is the most widely used plant phospholipase D in biocatalytic applications, has been characterized by small-angle X-ray scattering, UV-absorption, CD and fluorescence spectroscopy to yield the first insights into its secondary and tertiary structure. The structural model derived from small-angle X-ray scattering measurements reveals a barrel-shaped monomer with loosely structured tops. The far-UV CD-spectroscopic data indicate the presence of alpha-helical as well as beta-structural elements, with the latter being dominant. The fluorescence and near-UV CD spectra point to tight packing of the aromatic residues in the core of the protein. From the near-UV CD signals and activity data as a function of the calcium ion concentration, two binding events characterized by dissociation constants in the ranges of 0.1 mm and 10-20 mm can be confirmed. The stability of PLDalpha2 proved to be substantially reduced in the presence of calcium ions, with salt-induced aggregation being the main reason for irreversible inactivation.

Phospholipases: Occurrence and production in microorganisms, assay for high‐throughput screening, and gene discovery from natural and man‐made diversity

AbstractVarious kinds of phospholipids have wide industrial applications such as in food and nutraceuticals, cosmetics, agricultural products, and pharmaceuticals. The demand for reliable biocatalysts for the production of phospholipid products, such as phospholipases A1, A2, C, and D, has steadily increased over the past several decades. A large number of microbial phospholipases have been isolated and characterized, and the increasing availability of these enzymes could eventually lead to the sustained development of phospholipid‐related biotechnology. Although a number of reactions have been performed using phospholipases, a reliable and efficient supply of superior phospholipases in quantity is still a challenge for their practical application. High‐throughput functional assay methods for phospholipases should be devised to develop superior new species from the huge diversity of phospholipases. Recent biotechnological advances in the discovery of new phospholipase genes from natural sources, such as extremophiles of phospholipases by protein engineering, such as directed evolution, can provide valuable means of rapidly developing practical uses of phospholipases for various applications.

Enzymatic oil-degumming by a novel microbial phospholipase

Phospholipase A-mediated oil-degumming is a well-established process step (Enzy-Max®) in physical refining of vegetable oils (rape seed, soy bean, sunflower seed). A screening programme for microbial phospholipases of type A has been carried out. The target has been to develop a stable and robust phospholipase with optimal oil-degumming performance in the pH-range 4—5 and in the temperature range 30— 70 °C. One phospholipase of type A1 from Fusarium oxysporum, given the trade name Lecitase® Novo, has been studied in detail. Some of the characteristics of this novel microbial phospholipase in the oil-degumming application are: pH optimum ∼5, temperature optimum 40—45 °C. In laboratory tests the new phospholipase Lecitase® Novo has proven to be superior to porcine pancreatic Lecitase® 10L and other phospholipases with respect to oil-degumming performance, and it has proven to be suited for degumming of different oil qualities ranging from water-degummed to crude oil. A further advantage is that the new phospholipase acts at very low water content, which will make the problematic sludge recycling in the EnzyMax® process superfluous. As demonstrated by an HPLC study, phospholipase-mediated degumming is a unique process quite distinct from the well-known acid degumming variations, since the phospholipids (both hydratable and non-hydratable) present in the oil are hydrolysed to the corresponding lyso-phospholipids, which migrate to the aqueous phase under the conditions employed. Lecitase® Novo was introduced successfully for degumming of rapeseed oil at Cereol (Mannheim, Germany) mid 2000.

Enzymatic oil-degumming by a novel microbial phospholipase

Phospholipase A-mediated oil-degumming is a well-established process step (EnzyMax®) in physical refining of vegetable oils (rape seed, soy bean, sunflower seed). A screening programme for microbial phospholipases of type A has been carried out. The target has been to develop a stable and robust phospholipase with optimal oil-degumming performance in the pH-range 4-5 and in the temperature range 30-70 °C. One phospholipase of type A1 from Fusarium oxysporum, given the trade name Lecitase® Novo, has been studied in detail. Some of the characteristics of this novel microbial phospholipase in the oil-degumming application are: pH optimum ∼5, temperature optimum 40-45°C. In laboratory tests the new phospholipase Lecitase® Novo has proven to be superior to porcine pancreatic Lecitase® 10L and other phospholipases with respect to oil-degumming performance, and it has proven to be suited for degumming of different oil qualities ranging from water-degummed to crude oil. A further advantage is that the new phospholipase acts at very low water content, which will make the problematic sludge recycling in the EnzyMax® process superfluous. As demonstrated by an HPLC study, phospholipase-mediated degumming is a unique process quite distinct from the well-known acid degumming variations, since the phospholipids (both hydratable and non-hydratable) present in the oil are hydrolysed to the corresponding lyso-phospholipids, which migrate to the aqueous phase under the conditions employed. Lecitase® Novo was introduced successfully for degumming of rapeseed oil at Cereol (Mannheim, Germany) mid 2000. Une nouvelle phospholipase A1 capable de catalyser la reaction de demucilagination dans le procede de raffinage de l'huile vegetale a ete isolee de Fusarium oxysporum (son nom commercial etant Lecitase Novo). Ses caracteristiques techniques ont ete evaluees. Elle apparait superieure aux enzymes existantes utilisees dans ce procede. Elle est deja utiliseees pour la demucilagination de l'huile de colza a l'huilerie Cereol (Mannhein, Allemagne) depuis mi-2000.

Inhibition of potassium transport and growth of mycobacteria exposed to clofazimine and B669 is associated with a calcium-independent increase in microbial phospholipase A2 activity.

Altered phospholipase A2 (PLA2) activity and its relationship to cation (K+, Ca2+) uptake and growth were investigated in mycobacteria exposed to the riminophenazine antimicrobial agents, clofazimine and B669 (0.15-2.5 mg/L). Microbial PLA2 activity was measured using a radiometric thin-layer chromatography procedure, whereas K+ and Ca2+ transport were measured using 86Rb+ or 42K+ and 45Ca2+, respectively. Short-term exposure (15-30 min) of Mycobacterium aurum A+ or the virulent and avirulent isolates of Mycobacterium tuberculosis H37R to the riminophenazines resulted in dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+ influx and growth. Uptake of Ca2+ by mycobacteria was unaffected, or minimally affected, by the riminophenazines at concentrations of < or = 0.6 mg/L, whereas higher concentrations resulted in increased uptake of the cation in the setting of decreased microbial ATP concentrations. The results of kinetic studies using a fixed concentration (2.5 mg/L) of B669 demonstrated that riminophenazine-mediated enhancement of PLA2 activity and inhibition of K+ uptake in mycobacteria are rapid and probably related events that precede, by several minutes, any detectable effects on microbial ATP concentrations and uptake of Ca2+. Inclusion of the extracellular and intracellular Ca2+-chelating agents EGTA (0.2-7.2 g/L) and BAPTA/FURA-2 (0.2-9.5 mg/L), individually or in combination, did not prevent the effects of B669 on mycobacterial PLA2 activity or K+ transport, whereas alpha-tocopherol, which neutralizes PLA2 primary hydrolysis products, antagonized the inhibitory effects of the riminophenazines on microbial K+ uptake and growth. These results demonstrate that the antimycobacterial activities of clofazimine and B669 are related to a Ca2+-independent increase in mycobacterial PLA2, leading to interference with microbial K+ transport.

Effects of lecithin and corn oil on site of digestion, ruminal fermentation and microbial protein synthesis in sheep.

Six Hampshire wethers with ruminal and duodenal cannulas were fed three diets in a replicated 3 X 3 latin square to compare phospholipids with triglycerides for their effects on ruminal digestion. The diets (56% concentrate, 44% bermuda-grass hay, air-dried basis) contained either no added fat (control), 5.2% soybean lecithin or 2.4% corn oil on a DM basis. All diets were isonitrogenous and both fat-supplemented diets had similar fatty acid and energy contents. Fat added to the diet, regardless of source, reduced digestibilities of DM, energy, ADF and fatty acids in the rumen but had no effect on total tract digestibility coefficients. Lecithin slightly increased (P = .06) fatty acid digestion in the hindgut compared to corn oil (91.0 and 87.0%, respectively). Both fat sources decreased (P less than .01) ruminal ammonia concentration and increased (P less than .10) N flow to the duodenum. Added fat also reduced ruminal (P less than .01) and total tract (P less than .05) N digestibilities. Microbial N flow to the hindgut was not affected by diet, but adding fat increased (P less than .06) true efficiency of microbial protein synthesis. Overall, phospholipids from soybean lecithin inhibited ruminal fermentation similarly to triglycerides from corn oil. Despite ruminal degradation of lecithin by microbial phospholipases as shown in other studies, feeding lecithin tended to increase fatty acid digestion in the hindgut.

Phospholipase Activation and Arachidonic Acid Release in Intestinal Epithelial Cells from Patients with Crohn's Disease

A method for studying the mobilization of free arachidonic acid (AA) in viable isolated human intestinal epithelial cells has been developed and applied to the study of patients with Crohn's disease. Cells were isolated from morphologically unaffected parts of the distal ileum and incubated with 14C-AA; most of the incorporated 14C-AA was then found in phospholipids (mainly phosphatidylcholine) and in a pool of neutral lipids (mainly triacylglycerols). Cells from patients with Crohn's disease incorporated more 14C-AA into their neutral lipids than did cells from control patients. When the labeled cells were stimulated with phospholipase C from Clostridium perfringens or with the calcium ionophore A23187, they released significant amounts of AA, mainly from phosphatidylcholine. There was no difference between cells from Crohn patients and controls in the 14C-AA amounts released, but unstimulated and phospholipase C-stimulated cells from prednisolone-treated Crohn patients released less AA than cells from control patients. The A23187-stimulated AA release was completely inhibited by the phospholipase A2 inhibitor 4-bromophenacyl bromide, whereas the phospholipase C-stimulated release was not. These findings suggest that AA release in human small-intestinal epithelial cells may be caused by calcium-mediated phospholipase A2 activation or by products of microbial phospholipase C activity and that prednisolone reduces the mobilization of free AA in intestinal epithelial cells. They also illustrate the potential use of isolated epithelial cells for revealing mechanisms underlying AA release in the intestinal mucosa in different disease states.