Purification of a lipase from the hepatopancreas of oil sardine (Sardinella longiceps linnaeus) and its characteristics and properties

Lipase has been purified from the hepatopancreas of oil sardine (Sardinella longiceps) by defatting, water extraction, ammonium sulphate fractionation and chromatography on DEAE Sephadex and Sephadex G-100. The preparation was homogeneous on polyacrylamide disc gel electrophoresis and on gel filtration through Sephacryl S-200. The enzyme showed a molecular weight of 54000±57000 with 6.1% of carbohydrate. The pH and temperature optima of purified sardine lipase were 8 and 37°C respectively. Sardine lipase remained stable up to 45°C (15 min) and in the pH range 5 to 9.5. The K_m values obtained for the substrates tributyrin and triacetin were 4 × 10^?2 and 30 × 10^?2, respectively. The effect of halogens and various metal ions on sardine lipase activity, substrate specificity, amino acid and carbohydrate composition are also reported.     

Gel Matrix Influence on Hydrolysis of Triglycerides by Immobilized Lipases

The hydrolytic activities and specificities of gel-entrapped C. cylindracae lipase (CCL) and R. arrhizus lipase (RAL) toward olive oil and tributyrin were investigated. Lipases in hydrophobic gels with the longest chain lengths generally displayed highest activity. The optimal temperature was 30–35° for free and 37–40° for gel-entrapped lipases. The ratio of the activity on tributyrin to that of olive oil (expressed as T/O ratio), an indicator of substrate specificity, increased from 0.3 for free lipases to 12.3 ± 2.3 for CCL lipase in ENTP-2000-formed gel and 16.2 ± 0.3 for RAL lipase in ENTP-4000-formed gel.     

Factors Influencing the Activity of a Heat-Resistant Lipase of Pseudomonas

The heat resistant lipase of Pseudomonas spp. MC50 hydrolyzed coconut oil, corn oil, butter oil and olive oil. Lipase activity was maximum at 1% corn oil. The effects of pH and temperature on lipase activity varied with the substrate. The pH optima were in the range pH 8–9; the temperature optima were in the range 35–40°C. Of several emulsifiers tested, four supported significant lipase actiivity, and three supported no lipase activity and may have inhibited the lipase. Emulsifier concentration influenced lipase activity. All stabilizers tested supported lipase activity; above 0.36% stabilizer concentration had no effect.     

Lipase Mediated Production of Flavor and Fragrance Esters from Fusel Oil

Mixtures of C₃, C₄ and C₅ chain length aliphatic alcohols found in fusel oil were converted to their acetic acid and butyric acid esters using a lipase (from Candida cylindracea) mediated process in a water immiscible fluid (hexane). Incubation temperature was 30°C with shaking (150 rpm). Maximum production of the mixed butyric acid ester product (0.62 mol/L; yield = 65.8%) and acetic acid ester product (0.052 mol/L; yield = 46.4%) were obtained in 48 hr. Enzyme could be reused three times before loss of activity for acetic acid ester synthesis but enzyme activity decreased after one use for butyric acid ester synthesis.     

Migration of silver from nanosilver–polyethylene composite packaging into food simulants

An analytical method for the determination of silver in food simulants (3% (w/v) aqueous acetic acid or 95% (v/v) aqueous ethanol) was developed based on inductively coupled plasma mass spectrometry (ICP-MS). Recoveries ranged from 87% to 109% and RSDs ranged from 0.7% to 7.8%. The procedure was applied to determine the migration of silver from nanosilver–polyethylene food packaging film into food simulants at different temperatures and migration times. Results indicated that the maximum migration ratios were 1.70%, 3.00% and 5.60% for 3% (w/v) aqueous acetic acid at 20, 40 and 70°C, respectively, while for 95% (v/v) aqueous ethanol the maximum migration ratios were 0.24%, 0.23% and 0.22% at 20, 40 and 70°C.     

Kinetic Properties and Enantioselectivity of The Lipases Produced by Four Aspergillus Species

Four high lipase-producing Aspergillus species, selected in our laboratory, were compared in terms of their stability and reactivity for enantioselective esterification between (R, S)-2-octanol with octanoic acid in n-hexane. We determined the pH and temperature reactions dependences of lipases activities, and we found that these enzymes exhibited various pH sensitivities. The optimum pH observed for Aspergillus terreus lipase was 5.5, for A. niger and A. oryzae lipases in the range of 6.0 to 6.5 and pH 7.0 for A. flavus lipase. Good stability was observed at pH ranging from 5.0 to 8.5 after 24 hours at 40° C, and the optimum activity was observed at 35–40° C for all lipases tested. The lipases from A. terreus and A. niger were highly thermostable, retaining 60% and 50% activity at 60° C after 1 hour, respectively. The lipases from A. niger and A. terreus lipases provided the best results in terms of enantioselectivity (E) in the esterification of (R, S)-2-octanol with octanoic acid in n-hexane (E = 4.9 and E = 4.5, respectively). These properties make these lipases good candidates for biocatalysis in organic media.     

Characterization of a multilayer film activated with Lactobacillus curvatus CRL705 bacteriocins

BACKGROUND: Bacteriocins produced by lactic acid bacteria offer enormous promise for food safety preservation. In this study an active multilayer film obtained by the incorporation of lactocin 705 and lactocin AL705, two bacteriocins produced by Lactobacillus curvatus CRL705 with antimicrobial activity against Lactobacillus plantarum CRL691 and Listeria innocua 7, respectively, was characterized for its potential application in active packaging technology. Film activity performance at different storage conditions, bacteriocins transfer into water and sunflower oil, and film surface properties were evaluated. RESULTS: Film activity against L. innocua 7 was maintained during 2, 4 and 6 weeks at 30, 10 and 5 °C respectively. At 30 and 10 °C, activity loss against L. plantarum CRL691 was observed on the second week of storage and after the fourth week at 5 °C. Results showed no significant difference for active multilayer film contact angle and seal properties compared to the control (without bacteriocins). A decrease in lactocin 705 inhibitory activity after sunflower oil contact was observed, while lactocin AL705 remained unaffected. After water contact, film activity was retained for both bacteriocins. CONCLUSIONS: As demonstrated by antimicrobial activity and physico‐mechanical properties retention, lactocin 705 and AL705 active multilayer film present potential for application in active packaging technology. Copyright © 2011 Society of Chemical Industry     

Laundry detergent-stable lipase from Pacific white shrimp (Litopenaeus vannamei) hepatopancreas: Effect of extraction media and biochemical characterization

Extraction and biochemical properties of a new lipase from the hepatopancreas of Pacific white shrimp were studied. Recovery of the hepatopancreas powder with 50 mM Tris-HCl, pH 7.0 containing 0.2% (v/v) Brij35 gave a higher recovery of lipase activity than other extractants tested (p < 0.05). The optimal pH and temperature for lipase activity were 8.5 and 60°C, respectively, when p-nitrophenyl palmitate was used as a substrate. The enzyme was stable to heat treatment up to 40°C and over a pH range of 7.0–10.0 for 30–120 min. Lipase activities continuously decreased as the sodium deoxycholate (NaDC) concentration increased, but activities increased as NaCl concentration increased up to 3.0 M. Hydrolytic activity was enhanced by NaN₃, but strongly inhibited by Hg²⁺, Cu²⁺, Al³⁺, and phenylmethanesulfonyl fluoride. The lipase was evaluated as highly stable against surfactants (Tween 20, Tween 80, Triton X-100, and gum arabic). However, the enzyme was unstable against sodium dodecyl sulphate. Stability of the lipase with commercial liquid and solid detergents (Attack^®, Bres^®, Omo^®, and Pao^®) was also investigated. The lipase exhibited substantial stability and compatibility with tested commercial liquid and solid laundry detergents for 30–60 min. The overall properties of the lipase from Pacific white shrimp hepatopancreas, thus leading us to propose that it is an excellent candidate for use as biocatalysts for better detergent formulation.     

Lipase-catalysed acidolysis of lard with caprylic acid to produce structured lipid

Enzymatic acidolysis of lard with caprylic acid was investigated. Of the five lipases that were tested in the initial screening, immobilised lipase TL IM from Thermomyces lanuginosus resulted in the highest incorporation of caprylic acid into lard. This enzyme was further studied for the effect of enzyme load, organic solvent, substrate ratio, reaction time and temperature. HPLC was used to analyse the products from the acidolysis reaction. The highest incorporation was attained at 15% enzyme load. Among the solvents tested, n‐hexane was the best reaction medium for the acidolysis of lard with caprylic acid. Time course studied suggests that the incorporation of caprylic acid into lard was increased up to 37.7 mol% after 24 h. Desirable mole ratio of lard to caprylic acid was 1:2, caprylic acid incorporation up to 34.2 mol%. Temperature had no significant effect on enzyme activity in the range of 40–80 °C.     

Modification of chitosan using hydrogen peroxide and ascorbic acid and its physicochemical properties including water solubility,oil entrapment and in vitro lipase activity

Chitosan was modified using H₂O₂ and ascorbic acid with different incubation temperatures (4–40 °C). The properties of modified chitosan, including its oil entrapment ability, water solubility and the lipase-resistant activities, using in vitro intestine model system were determined. Nuclear magnetic resonance analysis showed that ascorbic acid was bound to modified chitosan. All of the modified chitosan from 4 to 40 °C demonstrated improved water solubility (even in pH 7) compared to non-modified chitosan, which was only soluble at pH 4. Modified chitosan from 4 °C exhibited 27.40% of oil entrapment ability which was approximately four times higher than 6.87% of non-modified chitosan. Modified chitosan from 4 and 40 °C had increased resistance against lipase activity compared to other biopolymers, including beeswax, carnauba wax and non-modified chitosan. Modified chitosan could be used as a new food ingredient due to their high water solubility, oil-entrapping ability and resisting lipase activity.     

Investigations into and an Enzymic Assay Method of Transglucosidase Activity of Commercial Glucoamylase Preparations

An enzymatic assay method was developed for the quantitative determination of transglucosidase activity in commercial glucoamylase preparations. The method is based on the determination of residual glucose after incubation of the glucoamylase preparation with this substrate under welldefined conditions (pH 4,2 and 30°C). Glucose determination was carried out by the glucose oxidase-peroxidase method and transglucosidase activity expressed in international enzyme activity units. The over-all time requirement of the analysis is 3 h with liquid and 26 h with powdered preparations. The variation coefficient was less than 3% in the former and 4% in the latter case. Optimum substrate concentration was found to be 8,3% w/v, higher glucose concentrations markedly inhibited the reaction at 30°C. Temperature optima were observed at 40 and 50°C, respectively, more elevated temperatures up to 60°C caused a strong decrease in activity.     

Variation in short chain fatty acid and ethanol concentration resulting from the natural fermentation of wheat and barley for inclusion in liquid diets for pigs

Fifty‐six samples of wheat and 44 samples of barley were taken, at harvest, from locations across the UK. Lactic acid bacteria (LAB) and yeasts were enumerated before the samples were ground. Following grinding, triplicate 30‐g samples of each cereal were mixed with sterile distilled water and incubated at 30, 35 or 40 °C. Samples were taken immediately after mixing and at 24‐h intervals for analysis of short‐chain fatty acids (SCFA) and ethanol by isocratic ion‐exclusion liquid chromatography. The number of LAB and yeasts present in samples ranged from 0 to 5.0 (mean 2.25 ± 1.31) and 3.30 to 6.25 (mean 4.96 ± 0.74) log₁₀ colony‐forming units (cfu) ml^?1 respectively. At 30 °C the mean concentrations (mmol l^?1) of SCFAs and ethanol were, lactic acid 59.6 ± 40.0 (range 0.14–134.9), acetic acid 23.2 ± 11.1 (range 2.9–51.4), butyric acid 17.2 ± 16.8 (range 0.0–62.2) and ethanol 15.0 ± 9.0 (range 4.6–53.7) respectively. After fermentation for 24 h only 9 of 300 fermentations produced more than 75 mmol l^?1 lactic acid, which has previously been demonstrated to prevent the growth of Salmonella in liquid pig feed. Fermenting at 35 or 40 °C had no effect on lactic acid concentration but significantly (p < 0.001) increased the concentrations of acetic and butyric acids and ethanol. These results indicate that natural fermentation cannot be relied upon to produce levels of SCFAs that will prevent the proliferation of enteropathogens. Copyright © 2004 Society of Chemical Industry     

Lipase‐catalysed biosynthesis of hexyl butyrate by direct esterification: optimisation by response surface methodology

The ability of immobilised lipase from Rhizomucor miehei (Lipozyme IM‐77) to catalyse the direct esterification of hexanol and butyric acid was investigated. Response surface methodology (RSM) and a four‐factor/five‐level central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters such as reaction time (2–10 h), temperature (25–65 °C), substrate molar ratio of hexanol to butyric acid (1:1–3:1) and enzyme amount (10–50%; 0.24–1.18 BAUN) on the percentage molar conversion of hexyl butyrate by direct esterification. All the parameters had significant effects on the percentage molar conversion. Based on ridge maximum analysis, the optimal conditions for synthesis were: reaction time 8.0 h, temperature 46.9 °C, substrate molar ratio 1:1.2 and enzyme amount 36.4% (0.87 BAUN). The predicted value was 100% and the actual experimental value 98.2% molar conversion. Copyright © 2003 Society of Chemical Industry     

Properties of Trypsin from the Pyloric Ceca of Atlantic Cod (Gadus morhua)

Trypsin (EC 3.4.21.4) was isolated from the pyloric ceca of Atlantic cod and purified to homogeneity by affinity chromatography. The enzyme catalyzed the hydrolysis of benzoyl arginine p-nitroanilide (BAPA, pH 8.2 and 25°C) such that V_max was 250 BAPA units per micromole trypsin and K_m was 1.48 mM. For the hydrolysis of tosyl arginine methyl ester (TAME, pH 8.1 and 25°C), V_max was 18.2 × 10³ TAME units/micromole trypsin, and K_m 0.22 mM. The pH and temperature optima with BAPA substrate were 7.5 and 40°C, respectively. Atlantic cod trypsin was most active and stable at alkaline pH. The enzyme was heat labile, losing more than 50% of its activity after incubation at 50°C for 30 min. Amino acid analysis of Atlantic cod trypsin revealed that the enzyme was rich in residues such as serine, glycine, glutamate and aspartate, but poor in basic amino acid residues compared to trypsins from warm blooded animals.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

Improvement of palm oil and sunflower oil blends by enzymatic interestrification

Palm oil (PO) and sunflower oil (SFO) blends with varying proportions were subjected to enzymatic interesterification (EIE) using a 1,3‐specific immobilised lipase. The interesterified blends were evaluated for their slip melting point (SMP), solid fat content (SFC) at 10–40 °C, p‐anisidine value, peroxide value, free fatty acids (FFA), induction period of oxidation at 110 °C (IP110) and composition of fatty acids by gas chromatography. Under EIE treatment, the blends of PO and SFO in different proportions (20:80, 40:60, 50:50, 60:40 and 80:20) had saturated and unsaturated fatty acid content in the range of 37.6–52.0% and 48.0–62.4%, respectively. The blends showed a considerable reduction in their SFC, SMP, peroxide value and oxidative stability at 110 °C, but presented increase in FFA and p‐anisidine value. The optimum condition for minimising the fatty acid in oil was obtained, at 64 °C, using 8.9% enzyme and 3 h reaction time.     

Partial Characterization of the Action of An A. niger Lipase on Butteroil Emulsions

A commercially available lipase (A. niger, APF-12) was employed to hydrolyze an emulsified butteroil substrate. HPLC analyses of the reaction products indicated that short chain fatty acid residues (e.g., butyric acid) were preferably hydrolyzed at pH 5 whereas the overall rate of hydrolysis for all major fatty acids showed a pH optimum between 6 and 7. This indicated the possibility of directing the selectivity of lipolysis in butteroil to enhance production of short-chain fatty acids associated with flavor development. The optimal temperature was ca. 35°C. Different cations also affected rate of hydrolysis.     

Polyphenols Extracted from Black Tea (Camellia sinensis) Residue by Hot‐Compressed Water and Their Inhibitory Effect on Pancreatic Lipase in vitro

Abstract: Polyphenols, retained in black tea wastes following the commercial production of tea beverages, represent an underutilized resource. The purpose of this study was to investigate the potential use of hot‐compressed water (HCW) for the extraction of pancreatic lipase‐inhibiting polyphenols from black tea residues. Black tea residues were treated with HCW at 10 °C intervals, from 100 to 200 °C. The resulting extracts were analyzed using high‐performance liquid chromatography‐mass spectrometry and assayed to determine their inhibitory effect on pancreatic lipase activity in vitro. Four theaflavins (TF), 5 catechins, 2 quercetin glycosides, quinic acid, gallic acid, and caffeine were identified. The total polyphenol content of extracts increased with increasing temperature but lipase inhibitors (TF, theaflavin 3‐O‐gallate, theaflavin 3′‐O‐gallate, theaflavin 3,3′‐O‐gallate, epigallocatechin gallate, and epicatechin gallate) decreased over 150 °C. All extracts inhibited pancreatic lipase but extracts obtained at 100 to 140 °C showed the greatest lipase inhibition (IC₅₀s of 0.9 to 1.3 μg/mL), consistent with the optimal extraction of TFs and catechins except catechin by HCW between 130 and 150 °C. HCW can be used to extract pancreatic lipase‐inhibiting polyphenols from black tea waste. These extracts have potential uses, as dietary supplements and medications, for the prevention and treatment of obesity. Practical Application: Active forms of lipase inhibitors can be recovered from black tea residues. They could be used as dietary supplements or medications.     

Monoacylglycerols derived from butter oil by Penicillium roquefortii in suspension cultures

Structural isomers of monoacylglycerols (monoglycerides, MAGs) were identified and compared after degradation of butter oil by two strains of Penicillium roquefortii and a commercial lipase from P roquefortii (EC 3.1.1.3) at pH 7.0 and 10 °C. The conditions were selected as they were comparable with those used in the manufacture of blue mould‐ripened cheese. The commercial lipase was selected to compare with the fungal strains in terms of acyl migration. Results showed that the main isomers formed by lipolysis with the commercial lipase were sn‐2 MAGs (64 mol%), whilst spores and emerging mycelia of P roquefortii produced mainly sn‐1(3) MAGs (83–90 mol%). The work reported here may lead to further assessment of different MAG structural isomers as natural preservatives in foods and dairy products. © 2002 Society of Chemical Industry     

Phytate dephosphorylation by Lactobacillus pentosus CFR3

Studies on phytate‐degrading enzymes from lactobacilli are scarce, despite its potential in improving the nutritional quality of plant‐based foods. Therefore, the current investigation deals with the phytate‐degrading enzyme produced by a native Lactobacillus pentosus strain. Phytase activity was highest towards the end of the exponential phase. Activity increased in the presence of maltose (381.1%) compared with glucose. The presence of phytate in the media stimulated the enzyme production. The enzyme of interest was a 70 kDa protein with a pH and temperature optima of 5.0 and 55–60 °C, respectively. It retained 46% of activity after exposure to 70 °C for 20 min and also showed broad substrate specificity. It was completely inhibited by Hg²⁺, Fe²⁺ and PMSF while being activated by Co²⁺. This report is the first to show dephytinisation of autoclaved finger millet flour either by fermentation with L. pentosus or by treatment with the corresponding cell‐free extract.