Olive Oil Substrate Research Articles

Characterisation of the lipolytic enzymatic activities of fungal rizoenzymes from Rhizopus oryzae in comparison to pancreatin from pigs

Abstract Aim In case of exocrine pancreatic insufficiency (EPI), the replacement of digestive enzymes with, for example porcine pancreatin or fungal rizoenzymes, is unavoidable under certain conditions. Current guidelines indicate that preparations from porcine pancreas have more advantageous physicochemical properties compared to those from fungi, especially at high bile salt concentrations, and that the latter can, therefore, only be used clinically to a limited extent. Since rizoenzymes are increasingly used in clinical practice, the present in vitro study investigated efficiency of enzymatic activity of rizoenzymes in comparison to pancreatin under various physiological and partly extreme environmental conditions. Materials and Methods The lipolytic properties of two typical preparations containing digestive enzymes from porcine pancreatin and rizoenzymes were compared (same dosage of activity units) at different pH values (pH 3–9), with the addition of different bile salt concentrations (0–15 mmol/L sodium taurocholate). Lipolytic activity was measured by quantifying the fatty acids released from olive oil substrate by liquid chromatography–tandem mass spectrometry after enzymatic digestion. Results For both enzyme preparations, the lipolytic activity maximum was reached at pH 7 with comparable fatty acid conversion rates (57% pancreatin, 58% rizoenzymes). However, in contrast to pancreatin, rizoenzymes were already active for certain fatty acids from pH 3 to 4. At a bile salt concentration up to 10 mmol/L taurocholate, there was an increase in activity of both enzyme preparations (rizoenzymes 69% vs. pancreatin 58% enzymatic conversion). Only at rather unphysiological concentration of 15 mmol/L during EPI, there was a slight decrease in activity (to 56%) for the rizoenzymes. Conclusions Rizoenzymes are an alternative therapeutic option for EPI. Relevant differences between rizoenzymes and pancreatin with regard to lipolytic activity under different physicochemical conditions could not be demonstrated in this study, whereas a potential advantage in favor of rizoenzymes activity at already low pH values was observed.

Lipase Enzyme Inhibitory Activity of Jombang Leaves Extract (Taraxacum officinale F.H. Wigg)

Background: Obesity is a condition that occurs due to an imbalance between energy intake and expenditure. Lipase enzymes play an important role in the process of fat metabolism, making it a target in obesity treatment strategies. Jombang plant has been used for generations as an alternative treatment for various diseases. The main content of secondary metabolite compounds in jombang plants are phenolics, flavonoids, terpenoids, and alkaloids. Objective: This study was conducted to determine the activity and potential inhibition of jombang plant (Taraxacum officinale F.H. Wigg.) against lipase enzyme in vitro. Materials and Methods: The phytochemical screening was determined by a color change reaction with certain reagents, while total phenolic content was carried out using Folin-Ciocalteau reagent spectrophotometrically. The inhibitory activity of 96% ethanol extract of jombang leaves as well as orlistat as positive control was measured by titration method and using olive oil substrate. The extract concentrations used were 125 ppm, 250 ppm, and 500 ppm. Results: Jombang leaf extract contains alkaloid, phenolic, flavonoid and terpenoid compounds qualitatively. Determination of total phenolic content in 96% ethanol extract of jombang leaves was obtained at 12.69 ± 0.91 mgGAE/g. The highest percentage of inhibition was found at a concentration of 500 ppm which amounted to 103.85%. The difference in the percentage of inhibition in all groups showed no significant difference (Sig>0.05). Conclusion: This study shows that jombang leaf extract is able to inhibit lipase enzyme activity with the highest percentage of inhibition was 103,85%.

Immobilization microbial cellulase and lipase as bio-catalyst agent using maltodextrin for biofuel synthesis

The alternative method of using enzyme technology and modification methods will reduce the cost of biofuel production. New solution for biofuel production by improving the advantages from potential microbial to produce biocatalyst such as cellulase and lipase as an alternative catalyst for replacing the chemical catalyst for transesterification process. This study was developing new method of enzyme immobilization for feasibility biofuel production which is more efficient and effective process in industries. The result obtained in this work allowed concluding that the highest value for the total cellulolytic activity was obtain using carboxymethyl cellulose as substrate reaching value 4.225 U/ml for 24 h with optimization of pH 7 and temperature 50 °C. Instead of cellulase, the potential Aspergillus niger InaCC F538 can produce extracellular lipase and the value found for lipase activity in olive oil substrate is 3.710 U/ml for 24 h incubation time with optimization of pH 8 and temperature 50 °C. The immobilization process of cellulase and lipase using maltodextrin was enhancing the activity to 19.615 U/ml and 24.123 U/ml. The biofuel production based on microbial will be widely used for continuously used for renewable raw material from waste agriculture biomasses for biofuel production. New enzymes combination for improving enzyme stability for industrial application based on fermentation through the economically feasible.

Isolation and Purification of Lipase from the Riboflavin Overproducing Fungus Eremothecium Ashbyii

An extracellular lipase was observed in the hemiascomycete riboflavin overproducing fungus Eremotheciumashbyii . It was isolated and purified by salting out, dialysis, ion exchange chromatography on DEAE Sephadex and FPLC to 75-fold purity. The molecular weight of the purified enzyme was estimated to be 66 KDa and it showed a preference for unsaturated fatty acids. Maximum lipase was induced on lipid substrates containing a large percentage of unsaturated fatty acids. Thus olive oil substrate induced maximum lipase activity. It had an optimum pH of 7.0, an optimum temperature of 30°C, was stimulated by Zn 2+ , Mg 2+ , Fe 3+ , Ca 2+ and inhibited byCu 2+ , Co 2+ . An inhibition of activity upon incubation with PMSF and guanidine chloride, suggested the involvement of Serine and Arginine respectively in catalysis, whereas absence of inhibition by mercapto ethanol ruled out involvement of thiol groups in catalysis. This is the first report on the isolation and characterization of the lipase from the riboflavin over producer E. ashbyii. The possibility of lipase providing precursors for riboflavin over production can be exploited further to improve riboflavin production.

Psychrotrophic Lipase Producers from Arctic Soil and Sediment Samples

Culturable microorganisms were successfully isolated from soil and sediment samples collected in 2011 on the northern coast of Hornsund, West Spitsbergen. A total of 63 single colony isolates from three sampling sites obtained were subjected to temperature dependence study to assess whether they are obligate psychrophilic or psychrotrophic strains. From initial temperature screening, only 53 psychrotrophic isolates were selected that are capable of growing between 4-28 degrees C. The rest that were capable of tolerating higher temperatures up to 37 degrees C were not included in this study. These isolates were chosen for lipase enzyme screening confirmation with the standard plate assay of olive oil and fluorescent dye Rhodamine B. Six lipase positive isolates were also subjected for subsequent lipase enzyme plate screening on tributyrin, triolein, olive oil and palm oil agar. Lipase production by these six isolates was further assayed by using colorimetric method with palm oil and olive oil as the substrate. These isolates with promising lipase activity ranging from 20 U/ml up to 160 U/ml on palm oil and olive oil substrate were successfully identified. Molecular identification by using 16S rRNA revealed that five out of six isolates were Gram-negative Proteobacteria and the other one was a Gram-positive Actinobacteria.

Activation and Characterization of Trypsin-Treated Lipase

Effect of trypsin hydrolysis on lipase in activation, characteristics and change in thermal stability were studied. Lipase was found to be increased in activity from 584U mL-1 to 759U mL-1 via trypsin treatment at the concentration of 1.5mg mL-1, 30oC and pH7.0 for 30min. The trypsin-treated lipase showed a lower Km value (79mg mL-1 olive oil substrate) than the native lipase (100mg mL-1), indicating an improved affinity for olive oil substrate. The optimum pH value of the trypsin-treated lipase maintained basically unchanged while the optimum temperature (45oC) showed lower than the native lipase (50oC). The half-inactivation time for the trypsin-treated lipase at 45oC, 50oC and 60oC was calculated as 131min, 35.5min and 4min respectively, while for the native lipase at 50oC and 60oC was calculated as 128min and 13min respectively, indicating that the thermal stability of lipase is lowered after trypsin treatment.

Activation and Characterization of Trypsin-Treated Lipase

Effect of trypsin hydrolysis on lipase in activation, characteristics and change in thermal stability were studied. Lipase was found to be increased in activity from 584U mL -1 to 759U mL -1 via trypsin treatment at the concentration of 1.5mg mL -1 , 30°C and p H 7.0 for 30min. The trypsin-treated lipase showed a lower K m value (79mg mL -1 olive oil substrate) than the native lipase (100mg mL -1 ), indicating an improved affinity for olive oil substrate. The optimum p H value of the trypsin-treated lipase maintained basically unchanged while the optimum temperature (45°C) showed lower than the native lipase (50°C). The half-inactivation time for the trypsin-treated lipase at 45°C, 50°C and 60°C was calculated as 131min, 35.5min and 4min respectively, while for the native lipase at 50°C and 60°C was calculated as 128min and 13min respectively, indicating that the thermal stability of lipase is lowered after trypsin treatment.

AKTIVITAS LIPOLITIK Rhizopus microsporus var. rhizopodiformis ISOLAT UICC No. 6

The lipolytic activity of Rhizopus microsporus var rhizopodiformis isolat UICC No. 6. A study was carried out to examine the effect of peptone concentration (2,5% or 5%), inoculum concentration (0,1%, 0,5% or 1%) and incubation period (0 – 96 hours, interval 12 hours) on the lipolytic activity of Rhizopus microsporus var rhizopodiformis isolat UICC No. 6. Fermentation was done using the basal medium from Samad and the lipolytic activity on olive oil substrate was measured employing titration method with 0,05M NaOH. Lipolytic activity is expressed as unit/ml and one unit is defined as 1 􀁭mol fatty acid liberated per minute. Results show that optimum lipolytic activity was obtained from 1 % inoculum, 5% peptone after 72 hours incubation period. Keywords: lipolytic, Rhizopus, inoculum, incubation

A comparative study of the lipase yield by solid state and submerged fermentations using fungal species from biopharmaceutical oil waste

A comparative study of lipase enzyme yields by solid state fermentation (SSF) and submerged fermentation (SmF) was performed here. Three fungal colonies were isolated from biopharmaceutical oil waste collected from “Oushadhi” (The Pharmaceutical Corporation (IM) Kerala Ltd). The pure colonies were then used as inoculums for solid state and submerged fermentation of lipase. Strains were identified as Aspergillus sp., Trichoderma sp. and Penicillium sp. The oil waste itself was used as a substrate for SSF and it was supplemented with ((NH4)2SO4 5.0 g/l; Na2HPO4 6.0 g/l; KH2PO4 2.0 g/l; MgSO4.7H2O 3.0 g/l and CaCl2 3.0 g/l) at pH 6. The composition of production media (pH 6) used for SmF was glucose-10, peptone-20, NaCl-5 and yeast extract-5 (g/l).The comparison of enzyme yields showed that the yield in SSF was higher than that of SmF in most cases. Out of the three fungal strains, lipase produced by Aspergillus sp. showed the highest enzyme activity on assay by olive oil substrate emulsion method.

A comparative study of the lipase yield by solid state and submerged fermentations using fungal species from biopharmaceutical oil waste

A comparative study of lipase enzyme yields by solid state fermentation (SSF) and submerged fermentation (SmF) was performed here. Three fungal colonies were isolated from biopharmaceutical oil waste collected from “Oushadhi” (The Pharmaceutical Corporation (IM) Kerala Ltd). The pure colonies were then used as inoculums for solid state and submerged fermentation of lipase. Strains were identified as Aspergillus sp., Trichoderma sp. and Penicillium sp. The oil waste itself was used as a substrate for SSF and it was supplemented with ((NH4)2SO4 5.0 g/l; Na2HPO4 6.0 g/l; KH2PO4 2.0 g/l; MgSO4.7H2O 3.0 g/l and CaCl2 3.0 g/l) at pH 6. The composition of production media (pH 6) used for SmF was glucose-10, peptone-20, NaCl-5 and yeast extract-5 (g/l).The comparison of enzyme yields showed that the yield in SSF was higher than that of SmF in most cases. Out of the three fungal strains, lipase produced by Aspergillus sp. showed the highest enzyme activity on assay by olive oil substrate emulsion method.

Total Phenolics, Antioxidant Activities, and Anthocyanins of Different Grape Seed Cultivars Grown in Jordan

The total phenolics, antioxidant activities, anthocyanins, vitamine E, and tert-butylhydroxytoluene (BHT) of different cultivars of grape seed extracts (GSE) grown in Jordan were evaluated. In addition, antioxidant activities of GSE were investigated using olive oil substrate by oxidative stability instrument (OSI). Results of chemical composition showed that Baladi black and Asbani black had the highest amount of fat content 14.52 and 14.22 g /100 g seed, respectively, followed by Baladi green (13.28 g /100 g seed), Ajloni green (12.24 g/100 g seed), and Khudari green (10.92 g/100 g seed), respectively. The total phenolics and anthocyanins of GSE ranged from 4.66 to 5.12 g/100 g extracts and 0.14 to 0.68 g/100 g extracts, respectively. Antioxidant activities of GSE ranged from 66.4 to 81.40%, while vitamin E and BHT were 90.34 and 94.70%, respectively. Antioxidant activity of the extracts using OSI ranged from 3.10 to 41.13 h induction time, while vitamin E and BHT had 16.33 and 17.20 h, respectively. GSE had high amounts of antioxidants and can be used to retard lipid oxidation in a variety of food products.

Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 °C

Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during cooled semen storage. In the first experiment, seminal plasma (1.0ml) was assayed for lipase activity based upon hydrolysis of triglycerides (olive oil substrate) into free fatty acids and subsequent titration of pH change (Sigma Diagnostic Lipase Kit). Lipase activity in stallion seminal plasma was 0.36±0.02 Sigma units/ml, (mean+S.E.M.; n=16 ejaculates from six stallions). In the second experiment, equine semen (three ejaculates from each of four stallions) was divided into five treatment aliquots. In Treatment 1, semen was extended 1:3 with nonfat dried skim milk extender (NFDSM). In treatment groups 2 through 5, spermatozoa were washed by centrifugation (300×g for 15min) and resuspended in NFDSM to a final concentration of 25×106spermatozoa/ml. Porcine pancreatic lipase (pPL) was added to Treatment 3 (10 pPL units/ml), Treatment 4 (100 pPL units/ml) and Treatment 5 (100 pPL units/ml, heat inactivated at 100°C for 5min), while Treatment 2 had no pancreatic lipase added and served as the control. Samples were cooled slowly to 5°C, and stored at 5°C until evaluation. Sperm motility was evaluated at time 0, 24, 48 and 72h by computerized semen analysis, and data were analyzed via repeated measures ANOVA. The addition of 100units/ml but not 10units/ml of pPL decreased (P<0.01) total and progressive motility of stored sperm. Heat-inactivated pPL (Treatment 5) did not significantly decrease motility of spermatozoa during storage. Because the lipase activity assayed (Sigma units) and the lipase activity added to cooled semen (pPL units) were not equivalent, pPL was assayed in the Sigma Diagnostic Lipase assay. The relationship between Sigma Units (Y) and pPL units (X) appeared to be a log-linear relationship with log(Y)=−0.912+0.007X; R2=0.90. Mean lipase activity assayed in stallion seminal plasma was equivalent to approximately 64 pPL units/ml. These data suggest that endogenous lipase activity in stallion seminal plasma may be a factor in the adverse effects of seminal plasma on cooled spermatozoa in some stallions.

Optimization of lipase activity from Rhizopus sp. in triglyceride hydrolysis using a modified simplex method

The simultaneous effect of reaction parameters and component concentrations on the microbial lipase from Rhizopus sp. was investigated. The modified simplex optimization method was applied to determine the best operating conditions. The activity measurement was carried out with the pH-stat method where the fatty acids liberated during the enzymic hydrolysis of the olive oil substrate were titrated with NaOH in an automatic tritrimeter. Two independent simplex optimizations resulted in a fully converged simplex with respect to all of the parameters. Moreover, the converged optimum parameter levels provided the maximum activity, indicating that the global optimum has been found. The maximum activity of 17·9 U/mg was obtained at 40°C, pH 8·3, 41·8 mg/ml gum arabic, and 0·6 mg/ml sodium taurocholate. This response was more than 100% better than a previous one obtained by the ‘one-factor-at-a-time’ method.

Continuous hydrolysis of olive oil by immobilized lipase in organic solvent

Lipase (EC 3.1.1.3) from Candida rugosa was immobilized with DEAE-Sephadex A50, Sephadex G50, Sephadex LH-20, Amberlite IRA94, and Amberlite XAD-7. The enzyme immobilized with DEAE-Sephadex A50 was found to be most effective for continuous hydrolysis of olive oil in isooctane. For the continuous reaction, 0.2 g of dry immobilized enzyme was swollen with predetermined amount of water, and packed in a glass column reactor. When the organic solvent (Isooctane) containing olive oil substrate was co-currently fed with aqueous buffer, the two phases were evenly distributed throughout the packed bed without surfactant supplement or prior mixing of the two phases. A small amount of the surfactant (AOT) was used only in packing procedure, and no additional surfactant was necessary thereafter. Effects of initial water content of the swollen gel, buffer types, and strength were examined in the continuous reaction. Our results suggest that the operational half-life was affected by desorption of the bound enzyme. Under the conditions of 20% olive oil in isooctane and 25 mM triethanolamine buffer (pH 7.0), operational half life was 220 h at 30 degrees C. The reactor was also operable with n-hexane, but the operational stability of the immobilized enzyme in n-hexane was only half of that in isooctane. Our results indicate that various enzyme carrier having hydrophilic or amphiphilic properties could be used for two-phase continuous reaction in packed-bed column, reactor without any surfactant supply or prior dispersion of the two immiscible phases.

Serum lipase determination in the dog: a comparison of a titrimetric method with an automated kinetic method.

An enzymatic, kinetic method for determining serum lipase activity was evaluated and compared to a standard manual method for use in dogs. The kinetic method was a commercial kit adapted for use on a tandem access clinical chemistry analyzer and utilized a series of coupled enzymatic reactions based on the hydrolysis of 1,2-diglyceride by lipase. The manual method was the Cherry-Crandall technique based on the titration of base against the acid formed by hydrolysis of an olive oil substrate by lipase. The correlation between the two methods was very good (r = 0.94). The reference range for 56 clinically healthy dogs assayed by the kinetic method was 90 to 527 U/L. Diseases associated with a greater than twofold elevation in serum lipase activity as determined by the kinetic method included pancreatitis, gastritis with liver disease, and oliguric renal failure with metabolic acidosis. In some cases, pancreatitis was seen with other clinical problems, such as gastroenteritis, diabetic ketoacidosis, duodenal mass, disseminated intravascular coagulation, and septic peritonitis. Diseases associated with serum lipase activity within the reference range or elevated less than twofold included gastritis, gastric ulcer, cholestasis, phenobarbital-induced hepatopathy, colitis, copper hepatopathy, abdominal hematoma, apocrine gland adenocarcinoma, and thrombocytopenia with pneumonia.

Application of Modified Super-Simplex Optimization to Food Processing

The simultaneous effect of reaction parameters and component concentrations on the microbial lipase from Rhizopus sp. was investigated. The modified simplex optimization method was applied to determine the best operating conditions. The activity measurement was carried out with the pH-stat method where the fatty acids liberated during the enzymic hydrolysis of the olive oil substrate were titrated with NaOH in an automatic tritrimeter. Two independent simplex optimizations resulted in a fully converged simplex with respect to all of the parameters. Moreover, the converged optimum parameter levels provided the maximum activity, indicating that the global optimum has been found. The maximum activity of 17·9 U/mg was obtained at 40°C, pH 8·3, 41·8 mg/ml gum arabic, and 0·6 mg/ml sodium taurocholate. This response was more than 100% better than a previous one obtained by the ‘one-factor-at-a-time’ method.

Simplified Photometric Copper-Soap Method for Rapid Assay of Serum Lipase Activity

We modified the method of Yang and Biggs [Clin. Chem. 17, 512 (1971)] for greater speed and simplicity. Serum is incubated with an emulsified olive oil substrate, the fatty acids extracted, the copper soap formed, and phases separated, all in a single tube. An aliquot of the organic phase is removed for color development. One analysis can be completed in 35 min, 20 in less than 2 h. The method is accurate and precise. Results correlate well with the unmodified colorimetric method, and with the classical titrimetric lipase procedure. The reference interval (normal range) is 20 to 180 U/liter.

Proposed Standard Method for Measuring Lipase Activity in Serum by a Continuous Sampling Technique

Abstract A method is presented for measuring lipase activity in serum by use of either triolein or olive oil as substrate. The method requires 3 to 8 min for each measurement and 5 to 8 min for initial set-up. The reaction mixture is maintained at the optimum pH of 8.8 by automatic addition of sodium hydroxide (10 mmol/liter) with a " pH-Stat." Amount of sodium hydroxide delivered per unit time reflects lipase activity. When reaction conditions—such as pH, substrate concentration, emulsifier concentration, and type and concentration of added bile salts—were made optimal, the reaction kinetics were zero order. Data presented indicate that adding sodium glycocholate prevents inactivation of lipase and does not lead to activation of this enzyme. Comparative studies, in which triolein and olive oil, respectively, are used as substrates show that triolein is the superior substrate for lipase, but olive oil can be substituted for triolein for routine clinical laboratory use. The suggested upper limit of normal is 200 and 160 U/liter for triolein and olive oil substrates, respectively.

Lipolytic enzyme content of rat tissues.

Summary1. Studies have been made of the concentration in the tissues of the rat of enzyme that catalyzes the hydrolysis of olive oil and tributyrin. 2. Serum liberated from an emulsified olive oil substrate an average of 6.6 micromoles of fatty acid per ml during the first hour of hydrolysis. 3. Liver, kidney and spleen were found to contain 3 to 6 times as much enzyme that splits olive oil as observed in serum. Pancreas showed 6000 times as much enzyme that splits olive oil as found in serum. Brain yielded low and questionable values. 4. In analyses with emulsified tributyrin as substrate, tissue extracts liberated 4 to 100 times as much fatty acid as was split from olive oil under the same conditions.

Serum Lipase Determination with an Olive Oil Substrate Using a Three-Hour Incubation Period

Abstract The effect of various emulsifying agents, pH, buffers, and accelerators on the Cherry-Crandall procedure for determining serum lipase was studied. A method is proposed which uses a 3-hour incubation period. Olive oil emulsified with 7% acacia is the substrate. This is buffered with tris buffer, pH 8.0; magnesium acetate and EDTA are added as accelerators. Other conditions which result in an increased rate of hydrolysis are discussed.