Optimization of oil retention in sesame based halva using emulsifiers and fibers: an industrial assay.

Oil bleeding during storage oleaginous seeds based confectionery products is a major problem affecting acceptance by consumers. Halva is a popular sweet food prepared from a sesame paste and a sugar mixture. The objective of this work was to improve the oil retention in this product by incorporating commercial fibers and emulsifiers: soya lecithin and monoglycerides (MG1 or MG2) during manufacturing. Oil retention yield was optimized on small batches, by response surface methodology using a central composite design applied with two factors, emulsifier concentration (0.25-2.25 %) and fibers concentration (0-2 %) at three levels. A centrifugation test was optimized to assess oil retention in halva samples. The experimental response (oil retention) was fitted with quadratic equations for each emulsifier, using multiple regression analysis. The emulsion stability increased with increasing the emulsifier concentration, particularly to 2.25 %. The oil bleeding assessed at 45 °C was slow but yielded similar results to those estimated by centrifugation test. The latter seems an attractive rapid method to quantify oil retention in oleaginous seeds and crops based food matrices. At an industrial scale, the increase of MG1 concentration to 2.25 % in halva enhances the oil retention of the product but does not affect its color or textural characteristics. Microscopic observations allowed us to explain high oil retention in this product by a homogeneous dispersion of oil droplets in the aqueous phase.

Culture of Staphylococcus xylosus in fish processing by-product-based media for lipase production.

AIMS: The objective of this study was to demonstrate that fish-processing by-products could be used as sole raw material to sustain the growth of Staphylococcus xylosus for lipase production. METHODS AND RESULTS: Bacterial growth was tested on supernatants generated by boiling (100 degrees C for 20 min) of tuna, sardine, cuttlefish and shrimp by-products from fish processing industries. Among all samples tested, only supernatants generated from shrimp and cuttlefish by-products sustained the growth of S. xylosus. Shrimp-based medium gave the highest growth (A(600) = 22) after 22 h of culture and exhibited the maximum lipase activity (28 U ml(-1)). This effect may be explained by better availability of nutrients, especially, in shrimp by-products. Standard medium (SM) amendments to sardine and tuna by-product-based media stimulated the growth of S. xylosus and the highest A(600) values were obtained with 75% SM. Lipase activity, however, remained below 4 U ml(-1) for both sardine and tuna by-product-based media. CONCLUSIONS: Fish by-products could be used for the production of highly valuable enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of fish by-products in producing S. xylosus-growth media can reduce environmental problems associated with waste disposal and, simultaneously, lower the cost of biomass and enzyme production.

Cloning and molecular modelling of turkey pancreatic lipase: structural explanation of the increased interaction power with lipidic interface.

Starting from total pancreatic mRNAs, turkey pancreatic lipase (TPL) cDNA was synthesized by RT-PCR and cloned into the PGEM-T vector. Amino acid sequence of the TPL is compared to that of human pancreatic lipase (HPL). A 3-D structure model of TPL was built using the 3-D structure of HPL as template, given the high amino acid sequence homology between the two lipases. Based on this model, the enhanced interaction power of TPL, as compared to that of HPL, into a phosphatidylcholine monolayer film, could be explained. We concluded that an increase in the exposed hydrophobic residues on the surface of TPL would be responsible for an enhanced interaction with a lipidic interface.

[Enzymatic synthesis of homogenous triacylglycerol in media without solvent]. / Synthèse enzymatique de triacylglycérols homogènes dans un milieu sans solvant.

The study of triacylglycerols synthesis using 1,3-regiospecific immobilized Rhizomucor miehei lipase (lipozyme) or non-regiospecific immobilized Candida antarctica lipase (novozyme) as biocatalyst was carried out in pure substrates conditions. Our results show that long-chain triacylglycerols were synthesized from glycerol and free fatty acids at a higher rate than medium-chain triacylglycerols which were themselves synthesized at a higher rate than short-chain ones. Furthermore, it is clearly shown that linoleic acid is more slowly esterifled than oleic acid which is itself more slowly esterified than octadecanoic acid. The higher the number of unsaturation, the lower the rate of synthesis and the final yield. On the other hand, the final yield of synthesis is comparable when using specific or non-specific lipase, as biocatalyst.

Dromedary pancreatic lipase: purification and structural properties.

Dromedary pancreatic lipase was purified from delipidated pancreases. Pure dromedary pancreatic lipase (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephadex G-100 gel filtration, anion-exchange (Mono Q Sepharose) and size exclusion column using high performance liquid chromatography (HPLC). The pure lipase is a monomer and has a molecular mass of about 45 kD and a pI of around 4.8. A specific activity of 5900 U/mg was measured on tributyrin as substrate at 37 degrees C in the presence of colipase and 2 mM NaTDC. The first 11 N-terminal amino acid residues and 10 peptides obtained by endoproteinase Glu-C digestion were sequenced. Dromedary pancreatic lipase is very similar to other pancreatic lipases as compared with their N-terminal and some peptides sequences. DrPL is activated by interfaces. The interfacial activation could be related to the presence of a lid and in fact one fragment of this lid domain (P9) was sequenced here: its' role will be discussed below.

Kinetic behaviour of pancreatic lipase in five species using emulsions and monomolecular films of synthetic glycerides.

In the absence of colipase and bile salts, using tributyrin emulsions or monomolecular films of dicaprin at low surface pressure, we observed that no significant lipase activity can be measured with Human Pancreatic Lipase (HuPL), Horse Pancreatic Lipase (HoPL) or Dog Pancreatic Lipase (DPL). Only Porcine Pancreatic Lipase (PPL) and recombinant Guinea Pig Pancreatic Lipase Related Protein of type 2 (r-GPL) hydrolyse pure tributyrin in the absence of any additive, as well as dicaprin films at low surface pressures. The former lipases may lack enzyme activity because of irreversible interfacial denaturation due to the high energy existing at the tributyrin/water interface and at the dicaprin film surface at low surface pressures. The enzyme denaturation cannot be reflected in the number of disulfide bridges, since all the pancreatic lipases tested here contain six disulfide bridges, but behaved very differently at interfaces. We propose to use the surface pressure threshold, as determined using the monomolecular technique, as a criterion for classifying lipases in terms of their sensitivity to interfacial denaturation.

Inactivation of pancreatic and gastric lipases by THL and C12:0-TNB: a kinetic study with emulsified tributyrin.

THL is a potent inhibitor of pancreatic (PPL) and gastric (HGL, RGL) lipases. Inactivation occurs preferentially at the oil/water interface (method B, C). In the aqueous phase (method A), the inhibition of HGL was accelerated by the presence of bile salts. C12:0-TNB, a disulfide reagent, specifically inactivates gastric lipases and had no effect on the pancreatic lipase (in the presence of bile salts) whatever the method used. The capacity of THL and C12:0-TNB to inactivate lipases using Methods B and C was found to depend directly upon the interfacial area of the system used. Consequently, inactivation can be reduced or prevented by further addition of a water-insoluble substrate which reduces the surface density of inactivator molecules. With a heterogeneous system of this kind, typical of lipolysis, the use of a classical Michaelis-Menten model is irrelevant and hence the traditional kinetic parameters (Km, KI, Vmax) are only apparent values.

Screening of preduodenal lipases in several mammals.

The tissular localization of preduodenal lipases was studied from the tongue to the pyloric portion of the stomach in 11 mammals. Lipolytic activities were clearly differentiated from those of pancreas. All lipase activities show an acidic pH optimum, except the gastric enzyme from hog. For every mammal tested, preduodenal lipase activity was associated mainly with only a single tissue located either in tongue, or in the pharyngeal area, or in the stomach. Resistance to acidic pH medium allows the classification of lipase activities into three groups. These results are related to the dietary habits and zoologic classification of the different animal species.

Purification, characterization and kinetic properties of the rabbit gastric lipase.

Rabbit gastric lipase was purified from an acetonic powder of rabbit stomach fundus. 25 mg of pure rabbit gastric lipase (glycerol ester hydrolase, EC 3.1.1.3) was obtained from 30 rabbit stomachs after ammonium sulfate fractionation, Sephadex G-100 gel filtration and cation exchange (mono S column) using a fast protein liquid chromatography (FPLC) system. The pure enzyme obtained was resistant to acidic pH conditions, and had specific activities of 1200, 850 and 280 U/mg, using, respectively, short- (tributyroylglycerol (TC4)), medium- (trioctanoyl- to tridecanoylglycerol (TC8-TC10)) and long-chain (soybean oil) triacylglycerols. The amino-acid composition was determined, and the first 30 N-terminal amino-acid residues were sequenced. Interfacial denaturation and catalytic properties on triacylglycerol emulsions were studied. Rabbit gastric lipase turned out to be structurally and kinetically very similar to human gastric lipase.

Inhibition of pancreatic and microbial lipases by proteins.

We have compared the effect of several proteins, including melittin, beta-lactoglobulin A, serum albumin, ovalbumin and myoglobin, on the hydrolysis of tributyrin and triolein by lipases from various origins. All proteins tested inactivate pancreatic lipase in absence of colipase and bile salt. Inhibition is not significantly reversed by colipase in absence of bile salt except in systems containing tributyrin and melittin or triolein and beta-lactoglobulin A. In all other cases, activation of pancreatic lipase by colipase in presence of inhibitory protein requires the presence of bile salt. Lipase from Rhizopus delemar is also inhibited by the proteins that inactivate pancreatic lipase. In contrast, the activity of lipase from Rhizopus arrhizus is not affected by the proteins in the same concentration range. Inhibition of lipase activity by amphiphiles such as proteins or detergents appears to be a general phenomenon not directly related to a decrease in tension at the triacylglycerol-water interface. Inhibition could be the result of desorption of lipase from its substrate due to a change in interfacial quality.

Importance of human gastric lipase for intestinal lipolysis: an in vitro study.

Using soybean triacylglycerols emulsified with egg lecithin we have studied, in vitro, the influence of substrate prehydrolysis by human gastric lipase upon subsequent degradation by the pancreatic lipase-co-lipase system. Fatty acids liberated by pure human gastric lipase or juice trigger immediate activity of human pancreatic lipase. Gastric lipolysis appears to be of prime importance for dietary lipid digestion in human.

Ajoene prevents fat digestion by human gastric lipase in vitro.

Human gastric lipase (HGL) is a sulfhydryl enzyme which has been shown by Gargouri et al. (Gargouri, Y., Moreau, H., Piéroni, G. and Verger, R. (1988) J. Biol. Chem. 263, 2159-2162) to be inhibited by hydrophobic disulfides. Since HGL is involved in the digestion and absorption of dietary fats we have investigated in vitro the ability of ajoene, a natural disulfide to inactivate HGL. Ajoene is derived from ethanolic garlic extracts. The finding that ajoene inactivates HGL is consistent with the fact that it is reactive towards sulfhydryl compounds and also corroborates previous reports on the ability of garlic to lower triacylglycerol blood levels. These data may explain the age-old Mediterranean and Oriental belief in the 'blood-thinning' effects of garlic on a molecular and physiological basis.

Crystallization of pancreatic procolipase and of its complex with pancreatic lipase.

The human pancreatic lipase-porcine procolipase complex has been crystallized in space group P3(2)21 (a = b = 80.3 A and c = 251 A) from a solution containing polyethylene glycol, NaCl and beta-octyl glucoside. The crystals diffract to 2.6 A on a synchrotron beam. The complex in the presence of bile salts and phospholipids crystallizes in a tetragonal space group P4(2)2(1)2 (a = b = 133.4 A, c = 92.6 A). Crystals of procolipase alone were obtained under slightly different experimental conditions (space group I432, a = b = c = 164.3 A).

Immunocytochemical localization of rabbit gastric lipase and pepsinogen.

Lipase and pepsin activities were determined in rabbit gastric biopsy specimens. Lipase activity was found to be restricted to a small part of the fundic mucosa, near the cardia, whereas pepsin activity spread over about two thirds of the total fundic area, overlapping that of lipase. The cells producing these two enzymes were labeled by immunofluorescence using polyclonal antibodies against rabbit gastric lipase (RGL) or antibodies against rabbit pepsinogen. The immunocytochemical localization showed unequivocally that RGL and pepsinogen, which were both present in the cardial area, were in fact located in different gastric cells. The cells producing pepsinogen were in the lower base of the gastric fundic glands, whereas the cells producing RGL were in the upper base of the same glands. The cells producing pepsinogen and RGL showed no significant morphological differences. In the part of the fundic area, where only pepsin activity was detected, cells producing pepsinogen covered both the lower and the upper base of the gastric glands. No chief cells were observed in the antral mucosa. RGL and pepsinogen could represent useful gastric enzyme markers for cellular differentiation studies.

Importance of sulfhydryl group for rabbit gastric lipase activity.

We have shown recently that rabbit gastric lipase (RGL) purified from gastric tissue presents catalytic properties comparable with those of human gastric lipase (HGL). We report here that only one sulfhydryl group was modified per molecule of native RGL after incubation at pH 8.0 with 5,5'-dithiobis(2-nitrobenzoic acid) (NbS2) for 4 h or 4,4'-dithiopyridine (4-PDS) for 60 min. With both reagents, a direct correlation was found between the modification of one sulfhydryl group per enzyme molecule and loss of RGL activity. Incubation of RGL with the new hydrophobic sulfhydryl reagent, dodecyldithio-5-(2-nitrobenzoic acid) (C12-NbS), at 30-fold molar excess, at pH 3.0, 5.0 and 8.0, induced immediate and complete inactivation of RGL. Unlike NbS2 and 4-PDS, C12-NbS almost instantaneously stopped the course of tributyrin hydrolysis by RGL, in contrast to porcine pancreatic lipase (PPL). RGL can be included with HGL in the group of sulfhydryl enzymes.

Characterization of turkey pancreatic lipase.

Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAE-Sepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37 degrees C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37 degrees C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65 degrees C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (beta-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N-terminal amino acid residues were sequenced. This sequence was similar to those of mammal's pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals.

Biochemical and molecular characterization of Staphylococcus simulans lipase.

Staphylococcus simulans strain secretes a non-induced lipase in the culture medium. Staphylococcus simulans lipase (SSL), purified to homogeneity, is a tetrameric protein (160 kDa) corresponding to the association of four lipase molecules. The 30 N-terminal amino acid residues were sequenced. This sequence is identical to the one of Staphylococcus aureus PS54 lipase (SAL PS54) and exhibits a high degree of homology with Staphylococcus aureus NCTC8530 lipase (SAL NCTC8530), Staphylococcus hyicus lipase (SHL) and Staphylococcus epidermis RP62A lipase (SEL RP62A) sequences. But the cloning and sequencing of the part of the gene encoding the mature lipase show some differences from SAL PS54 sequence, which suggest that it is a new sequence. The lipase activity was maximal at pH 8.5 and 37 degrees C. SSL is able to hydrolyze triacylglycerols without chain length specificity. A specific activity of about 1000 U/mg was measured on tributyrin or triolein as substrate at 37 degrees C and at pH 8.5 in the presence of 3 mM CaCl(2). In contrast to other staphylococcal lipases previously characterized, Ca(2+) is not required to express the activity of SSL. SSL was found to be stable between pH 4 and pH 9. The enzyme is inactivated after a few minutes when incubated at 60 degrees C. Using tripropionin as substrate, SSL does not present the interfacial activation phenomenon. In contrast to many lipases, SSL is able to hydrolyze its substrate in the presence of bile salts or amphiphilic proteins.

Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique.

Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).

Horse pancreatic lipase. Interaction with colipase from various species.

Horse pancreatic lipase has been purified from tissue homogenates. Molecular and catalytic properties of horse lipase are comparable to those of the pancreatic lipases previously isolated. Kinetic studies of the inhibition of horse lipase activity by bile salts and of reactivation by pure colipase from three species (horse, ox and pig) allowed to calculate the apparent dissociation constant (Kd) of the lipase-colipase complex in the presence of the substrate (triolein). Identical values of Kd were found in all three cases (Kd = 1.1 10(-9) M). These values are lower by several orders of magnitude than that published for the binding between lipase and colipase in the absence of substrate. Qualitative experiments show that the activation of horse lipase can be accomplished by rat, dog and chicken colipase as well. The interaction between lipase and colipase is enhanced when the complex is adsorbed at the lipid-water interface. This specific protein-protein interaction is preserved in heterologous mixtures using colipases from other animal species.

Interactions of lipases with lipid monolayers. Facts and questions.

Among the proteins, lipolytic enzymes provide a valuable model for studying protein-lipid interactions. Lipases having a catalytic action which is strictly dependent upon the presence of a lipid interface were used in the present study in order to gain better insight into protein-lipid interactions. Most of the data presented here were obtained using the monolayer technique, by recording (either independently or simultaneously) the lipolytic activity, the amount of protein adsorbed to the lipid monolayer, and the surface pressure variations following protein adsorption. Several non-enzymatic proteins were used as controls in order to determine how lipase behaviour differs from that of other proteins. At all initial surface pressures tested, with zwitterionic monolayers, a good correlation was observed between the amount of lipase bound to the monolayer and the surface pressure increase, in agreement with previous studies. Conversely, with neutral lipid monolayers the amount of lipase bound to the monolayer was not found to be surface pressure dependent. This latter behaviour observed with lipases on neutral films is not specific to lipases, since it was also observed with bovine serum albumin and beta-lactoglobulin A. Lipase activity in the presence of various proteins was investigated with monomolecular films of glycerol didecanoate, either at constant surface area or at constant surface pressure. Depending upon the nature of the lipase and the protein, inhibition of lipase activity was either observed or not. Inhibition was correlated with a decrease in lipase surface concentration. The ability of the various proteins to inhibit lipolysis is: (i) a function of their excess versus lipase in the bulk phase, and: (ii) correlated with their penetration capacity (i.e., the initial rate of surface pressure increase of a glycerol didecanoate monolayer having an initial surface pressure of 20 dyn/cm, after the injection-of the protein). Since lipase inhibition was observed with low surface densities of inhibitory proteins, a long-range effect is probably involved in the mechanism of interfacial lipase inhibition. The nature of the ionic charge added to the monolayer by the protein is not critical for determining lipase adsorption or desorption. It is hypothesized that the lack of lipase adsorption to, or desorption from, the lipid monolayer results from a change in the organization of the hydrocarbon moiety of the lipid.