Pregastric Lipase Research Articles

The effect of probiotic crops and lipolytic enzymes on the properties of dairy products in the cheese biotechnology under development

This article aims to evaluate the influence of probiotic microorganisms and pregastric lipase on organoleptic and biochemical characteristics of soft cheese made from a mixture of milk and buttermilk. The study was designed to determine how the temperature and exposure at this temperature affect the growth and development of lactose and bifidobacteria, as well as their quantity in the finished product. In order to increase the biological and nutritional value of the developed soft cheese, protein-carbohydrate raw materials rich in vitamins A, B12, D, E, phosphate and minerals were introduced into the production mixture. The addition of buttermilk increased the biological value of the product and improved its consistency. The taste and consistency of cheese was improved by a pregastric lipase preparation, widely used in the cheesemaking industry for production of various cheeses with high and low temperatures of the second heating. As a result of directed lipolysis, the finished product accumulates free fatty acids and diacylglycerols, acting as plasticizers and affecting the texture and organoleptic characteristics of the cheese. The results show that combining buttermilk and milk in an optimal ratio and addition of lipase produce a product with the best physicochemical parameters and a maximum evaluation of organoleptic characteristics. In the presence of a probiotic starter culture (provided there is a ready-made substrate), an active synthesis of enzymes necessary for the complete hydrolysis of milk fat begins. This all creates a functional product.

RETRACTED: Enhancing the lipolysis of feta-type cheese made from ultrafiltered cow's milk

Abstract This study was conducted to investigate the effects of adding different levels of commercial pregastric lipase enzyme (calf lipase) on the free fatty acid profile of ultrafiltered Feta type cheese during ripening period. With an increase in added lipase level and ripening period, the main components of cheese including total solids, fat, fat in total solids, salt and salt in total solids did not change significantly but water soluble nitrogen increased. Increasing the added pregastric lipase level resulted in a significant decrease in the percentages of C 4:0 -C 8:0 free fatty acids while that of C 12:0 -C 18:0 and C 18:1 free fatty acids increased. According to sensory analysis of cheese samples, the best samples had the combination of 20, 40 and 60 days of ripening with 6 g/100 kg, 4 g/100 kg and 2 g/100 kg lipase in retentate, respectively. The addition of lipase to cheese milk could be recommended for the acceleration of flavor development and ripening of UF-Feta cheese over a short ripening period.

Factors influencing the dynamics of emulsion structure during neonatal gastric digestion in an in vitro model

We investigated the chemical, electrochemical and physical changes in the composition of a formula milk following digestion in an in vitro system that replicated the shear rates that would normally be encountered in the infant stomach. We examined these changes over a range of shear rates and pH levels that would be encountered in stomach lumen postprandially, and in the presence and absence of proteolytic and lipolytic enzymes. Digestion was conducted either with a fungal lipase that is commonly used in ex vivo systems (Rhizopus oryzae) or a mammalian gastric lipase (calf pregastric lipase).Prior to digestion the fat droplets in the formula milk were considerably smaller than those in human breast milk, and were stabilised by an adsorbed layer of milk protein. Droplet size increased progressively by coalescence during in vitro digestion at pHs between 3.5 and 4.5 when both lipase and protease were present, but not when either enzyme was omitted or when pH levels were outside this range. Coalescence was augmented by shear, notably at rates above the normal physiological range.Hence the stability and structural properties of formula emulsions appear to be influenced by disruption of the proteinaceous oil/water interface during digestion, being most susceptible to the concerted activity of pepsin and gastric lipase over a limited range of pH. Given that the onset of secretion of pepsin, lipase and hydrochloric acid does not occur synchronously in the developing infant stomach, inappropriately formulated milks may lower digestive efficiency. The fidelity of in vitro systems does not appear to be compromised by the use of fungal lipases but may be compromised by the use of inappropriately high stirring rates.

Assessment of proteolysis and lipolysis in cheeses made using artisanal lamb rennet paste

AbstractRennet paste contains chymosin and pepsin as major enzymatic coagulant, and lipolytic enzymes such as pre-gastric lipase (PGL) and gastric lipase (GL), able to release free fatty acids during cheese ripening. Main factors influencing rennet paste features are diet (milk feeding, weaning) and slaughter age. Little is known about the effects of probiotic addition to milk substitute on the microbial and enzyme feature of rennet, and thus on the characteristics of cheese. In the present study the effects of different rennet pastes on the Pecorino foggiano cheese in terms of proteolytic and lipolytic characteristics were investigated. Twenty-four lambs were divided into six groups of four according to a 3x2 factorial design with lambs being subjected to three different feeding systems: mother suckling (MS), artificial rearing (AR), and artificial rearing with Lactobacillus acidophilus (Strain DSM 20079; DSMZ, Braunshweig-Germany; cells were suspended in milk replacer at the concentration of 7log 10 cfu...

Lipolysis, proteolysis and sensory properties of ewe’s raw milk cheese (Idiazabal) made with lipase addition

In this paper, we describe the effect of the addition of pregastric lipase on the composition and sensory properties of Idiazabal cheese. Free fatty acids (FFA), partial glycerides, free amino acids (FAA), gross composition and sensory characteristics were determined at different ripening times in cheeses manufactured with three different amounts of commercial animal lipase or with lipase-containing artisanal lamb rennet paste. The addition of lipase increased the content of total FFA, particularly of short-chain FFA, and that of total partial glycerides in cheeses. Unexpectedly, lipase utilization significantly affected total FAA concentration, which decreased in cheeses elaborated with high lipase amount. In general, Val, Glu and Leu were the major FAA, and their concentrations depended, mainly, on ripening time. Lipase addition had significant influence on the sensory characteristics of the cheeses, increasing scores for most of the flavour and odour attributes of the cheese. Principal component analysis (PCA) was done including dry matter, FFA, FAA, partial glycerides and odour and flavour attributes of the cheeses. It indicated that aroma and flavour parameters of Idiazabal cheese and the content of short-chain FFA and diglycerides were highly correlated to first principal component (PC1), while texture parameters, compositional variables and FAA were correlated to the second principal component (PC2).

Microstructural properties of fat during the accelerated ripening of ultrafiltered-Feta cheese

A commercial pregastric lipase was used to accelerate lipolysis of Iranian ultrafiltered-Feta (UF-Feta) cheese. Scanning electron microscopy images showed that with an increase in lipase levels from 2.0 to 6.0 g 100 kg −1 of retentate, disruption of fat globules increased significantly. On day 3 of ripening and enzyme level of 2.0 g 100 kg −1 of retentate, individual fat globules and fat aggregates were clearly observed on the cheese samples. An increase in the lipase level or ripening period resulted in an increase in the rate of disappearance of fat globules. After 20 days of ripening, no apparent fat globules were observed and fewer fingerprints and voids of free fat were detected, compared to when no enzyme was used.

Assessment of proteolysis and lipolysis in cheeses made using artisanal lamb rennet paste

Rennet paste contains chymosin and pepsin as major enzymatic coagulant, and lipolytic enzymes such as pre-gastric lipase (PGL) and gastric lipase (GL), able to release free fatty acids during cheese ripening. Main factors influencing rennet paste features are diet (milk feeding, weaning) and slaughter age. Little is known about the effects of probiotic addition to milk substitute on the microbial and enzyme feature of rennet, and thus on the characteristics of cheese. In the present study the effects of different rennet pastes on the Pecorino foggiano cheese in terms of proteolytic and lipolytic characteristics were investigated. Twenty-four lambs were divided into six groups of four according to a 3x2 factorial design with lambs being subjected to three different feeding systems: mother suckling (MS), artificial rearing (AR), and artificial rearing with Lactobacillus acidophilus (Strain DSM 20079; DSMZ, Braunshweig-Germany; cells were suspended in milk replacer at the concentration of 7log 10 cfu ml-1) supplementation (ARLb) and slaughtered at 20d and 40d for each feeding treatment. Lambs’ abomasa were processed according to a traditional protocol to obtain rennet paste used for ovine cheese production. The extent of proteolysis and lipolysis in the cheese was monitored by determining the levels of free amino acids (FAAs) and free fatty acids (FFAs) in cheeses at 60 days of ripening. Total FAAs detected in MS cheeses was higher (P<0.05) than in other experimental cheeses, at both age of slaughtering of lambs. FAAs can be substrate for successive flavor forming reactions which convert FAA to various alcohols, aldehydes, acids, esters. Lowest levels of total FAA in ARLb cheeses could be related to the highest lactic microflora loads in rennet paste. In fact, in a previous note the supplementation of milk replacer with Lb. acidophilus was found to influence the growth dynamics of lactic acid bacteria in the rennet paste, with greater lactobacilli cell loads in cheese in the first stages of ripening. A reduction of individual FFA in cheese was observed in all treatments passing from 20 to 40 days of slaughtering of lambs. The most abundant free fatty acid were: butyric acid (C4:0), caproic acid (C6:0), palmitic acid (C16:0), oleic acid (C18:1) and linoleic acid (C18:2) according to milk fatty acid content and other work on ovine cheese. ARLb cheeses displayed higher levels of short chain FFA (C4:0-C10:0) and medium chain FFA (C12:0-C16:0) than MS cheeses, at 20 and 40 days, probably due to the impact of the diet on lipolytic enzymes in the rennet paste. When lambs are feed milk supplemented with Lb. acidophilus rennet paste is enriched in pregastric lipases able to release preferentially short-chain FFA. In conclusion, diet and age of slaughtering of lambs were found to influence the enzyme profile in rennet paste and thus the proteolytic and lipolytic patterns of cheese at 60 days of ripening.

The influence of the enzymatic composition of lamb rennet paste on some properties of experimentally produced PDO Fiore Sardo cheese

An experiment on PDO (Protected Designation of Origin) Fiore Sardo cheese making was carried out to assess the influence of rennet paste on cheese quality. Four different preparations of lamb rennet paste (A, B, C and D) were used. Rennet A came exclusively from suckling lambs slaughtered immediately after suckling, rennet B from suckling lambs not fed for 12 h, and rennets C and D from suckling lambs allowed to graze and slaughtered in the same way as A and B, respectively. In vitro lipolysis and multiple cheese-making trials were carried out for each rennet. The enzymatic composition of the prepared rennets was different. Lipolytic activity was assessed in vitro by measuring the quantity of free fatty acids released by hydrolysis of a sheep milk cream substrate. Rennet A had the greatest lipolytic activity, which had a strong preference for esters of short-chain fatty acids. The cheeses made using the different rennets did not differ significantly in their moisture, protein and fat content or in the nitrogen fractions which resulted from proteolysis. The cheese produced with rennet A had the highest lipolysis. Short-chain fatty acids, in particular butyric acid, were prevalent, as they were in the in vitro trials. The diet and the presence of milk in the abomasa was responsible for the enzymatic peculiarities of rennet A. Suckling stimulated the secretion of pre-gastric lipase from the pharingeal tissue. Thus, if one wishes to produce a rennet with the particular enzymatic composition necessary for a particular cheese, the diet and the slaughtering conditions of the lambs should be controlled.

Assessment of industrial lipases for flavour development in commercial Idiazabal (ewe's raw milk) cheese

Three commercial lipases (lipases A1 and A2 were pregastric lipases and lipase A3 was a fungal lipase) from three different manufacturers were studied to develop the characteristic pungent flavour of Idiazabal (sheep's raw milk) cheese in experimental productions (50 L vats), both in summer and in winter. In the experimental productions, all lipases significantly increased the concentration of total free fatty acids (FFA), both after 90 and 180 days of ripening. Lipases A1 and A2 increased primarily the concentration of short-chain FFA (C4, C6 and C10), whereas C16:0 and C18:1 were the main FFA obtained with lipase A3. Cheeses made with no lipase had the lowest concentrations of total FFA, with C16:0 and C18:1 as the major FFA. The percent of short-chain FFA was 70–80% of the total for lipases A1 and A2, but 30–40% in cheeses made with lipase A3 or with no lipase. Sensory analysis was performed after 90 and 180 days of ripening. Cheeses made with lipases A1 or A2 or with lamb rennet paste (which contains pregastric lipase and was used as control for sensory analysis) had significantly higher scores in 6 of the 22 attributes analyzed (odour and flavour intensity, sharp odour, pungent flavour and rennet odour and flavour) than cheeses made with lipase A3 or with no lipase added. Lipase A1 was selected to conduct commercial cheese productions (200 L vats) made by artisan cheese makers adding 94 lipase units (LU)/50 L (high amount) or 8 LU/50 L (low amount). Control cheeses were made with lamb rennet paste having comparable amounts of lipolytic activity. After 90 and 180 days of ripening, cheeses made with the same amount of lipase (either as lipase A1 or present in lamb rennet paste) were indistinguishable from each other, both from the sensory and analytical points of view (comparable amounts of total FFA, and percent FFA composition). Those with low amount of lipase were rated as ‘mild’, whereas those with high amount of lipase were rated as ‘strong’. An industrial production (5000 L vat) with 100 LU/50 L yielded cheeses of ‘intermediate strength’ and intermediate levels of lipolysis. A linear correlation was observed between percent of short-chain FFA and the score for pungent flavour, for all amounts of lipase used in this study. We conclude that lipase A1 is an adequate commercial lipase to develop the characteristic flavour of Idiazabal cheese, both in artisan and industrial fabrications.

Changes in lipid fractions and sensory properties of Idiazabal cheese induced by lipase addition.

This work studied the addition of an adequate lipase to enhance lipolysis reactions and the development of piquant flavour and sharp odour in Idiazabal cheese, as an alternative to the use of lamb rennet paste. Cheeses were manufactured from bulk raw ewes' milk in 50 l vats with commercial bovine rennet and 80 lipase units of pregastric or 180 lipase units of fungal lipase and ripened for 180 days. A higher lipolytic activity was induced by lipase addition promoting strong changes in odour and flavour attributes. Both fungal and pregastric lipases increased the content of total free fatty acids (FFA), but the fungal lipase released mainly medium- and long-chain FFA. In contrast, the pregastric lipase preferably released short-chain FFA. Diglyceride (DG) content was considerably higher in cheeses made with added pregastric lipase compared with those made with fungal lipase or with no lipase. Monoglycerides (MG) were detected only in cheeses made with either lipase added, reaching comparable concentrations after ripening for 180 days. The cheeses made with pregastric lipase had the highest scores for odour and flavour intensity, and sharp and rennet odours, desirable attributes for the Idiazabal cheese made with lamb rennet paste. None of the texture attributes were significantly influenced by the concentrations of MG and DG in the cheeses made with either lipase. Thus, the pregastric lipase was more appropriate than the fungal lipase to develop a more traditionally-flavoured Idiazabal cheese.

Determination of esterolytic and lipolytic activities of lactic acid bacteria.

Lactic acid bacteria (LAB) are considered weakly lipolytic compared with many other groups of bacteria (e.g., Pseudomonas, Bacillus, and Achromobacter). The esterolytic and lipolytic systems of dairy LAB remain poorly characterized. Esterases from lactic acid bacteria, yeasts, and Pseudomonas organisms may be involved in the development of fruity flavors in foods, and pregastric lipase and esterases are essential for the development of typical flavor in Italian cheese. Microbial lipases and esterases may improve quality or accelerate the maturation of cheeses, cured bacon, and fermented sausages. Lipases are defined as glycerol ester hydrolases (EC 3.1.1.3) that hydrolyze tri-, di-, and monoglycerides present at an oil-water interface. Esterases (EC 3.1.1.6) hydrolyze esters in solution and may also hydrolyze tri- and especially di- and monoglycerides containing short-chain fatty acids. Some probiotic strains of LAB can hydrolyze the triglycerides, releasing most short and medium chain, and essential fatty acids, which are valuable to today's health-conscious consumer. Medium chain fatty acids (C6-C14), in particular, have become accepted treatment for patients with malabsorption symptoms, a variety of metabolic disorders, cholesterol problems, and infant malnutrition. These probiotic bacteria could alleviate lipase deficiency in the digestive tract during digestion (steatorrhea). In this chapter, we describe different methods routinely used in our laboratory to determine the esterolytic and lipolytic activity of LAB. These techniques include the use of alpha- and beta-naphthyl derivatives of fatty acids (chromogenic method), the p-nitrophenyl (pNP) derivative of fatty acids (chromogenic method), and triglycerides (agar-well assay technique and titrimetric test) as substrates.

The Inhibitory Effects of Brans and Their Aqueous Extracts on the Lipolysis of Tributyrin Catalyzed by Calf Pregastric Lipase

ABSTRACTThe ability of red and white wheat brans and oat bran to inhibit calf pregastric lipase (CPGL)‐catalyzed hydrolysis of tributyrin has been studied in vitro at pH 6.5, 37 °C. Brans that had been presoaked in Bis‐Tris buffer at pH 6.5, 4 °C reduced CPGL activity by 13% to 20% after being soaked for 0.25 h and by 17% to 30% after being soaked for 24 h. Maximum reduction of CPGL activity was seen within 2 to 5 min incubation between coarse red wheat bran and CPGL. The results are discussed in terms of likely inhibitory components within the brans and their water‐soluble components and their roles in inhibiting pregastric and other lipase‐catalyzed hydrolysis of dietary lipids.

Effect of Soluble Dietary Fibers on Lipase‐catalyzed Hydrolysis of Tributyrin

ABSTRACT:The ability of soluble fibers to inhibit calf pregastric lipase (CPGL)‐catalyzed hydrolysis of tributyrin (TBG) has been measured at pH 6.5, 37 °C (CPGL 0.02 mg/mL). Fibers tested were pectin, carboxymethylcellulose (CMC), carrageenan, and gum arabic. The fibers (5 g/L), solubilized in Bis‐Tris buffer (50 mM, pH 6.5) at 4 °C for either 15 min or 24 h, were added to an emulsion containing TBG, before initiating the reaction by addition of CPGL. An immediate inhibitory effect of 8% to 19% was observed. The value of the CPGL–pectin dissociation constant, Ki, was 0.76 to 2.34 mM. Dialysis of pectin solution reduced the inhibitory effect by 14% and the concentration of galacturonic acid by 15%.

Changes of free fatty acid contents and sensory properties of white pickled cheese during ripening

In this study, the effects of commercial pregastric lipase enzyme on the accelerated ripening of white pickled cheese were investigated. Sensory evaluation of cheese samples was also performed. Commercial pregastric lipase was added to cheese milk before rennet addition at the level of 2, 4, 6 g/100 l milk and white pickled cheese was made from this milk. The main components (total solids, fat, total nitrogen, salt and titratable acidity), pH and free fatty acids were analysed in cheese samples after 1, 10, 20 and 30 days of ripening. The main components and pH of cheese samples were not significantly affected by the addition of pregastric lipase levels. Generally, when the pregastiric lipase levels increased, volatile free fatty acids (VFFAs) and free fatty acids (FFAs) also significantly increased ( P<0.05). Generally, sensory scores (appearance, flouvour, body and texture and odour) were significantly higher in both 4 g/100 l treated milk added and after 20 days ripening ( P<0.05).

The antimicrobial properties of milkfat after partial hydrolysis by calf pregastric lipase

Studies on the kinetic characteristics of calf pregastric lipase (EC 3.1.1.3) have shown that it preferentially releases short chain fatty acids (SCFAs) from bovine milkfat. The released fatty acids form mixed micelle structures. The aim of this investigation has been to test whether hydrolysed milkfat is antimicrobial, and how the state of the emulsion alters the bactericidal or bacteriostatic effects. Partial hydrolysis of milkfat by pregastric lipase was carried out in two types of emulsion systems, containing either Triton X-100 or casein/lecithin, plus milkfat in citrate/phosphate buffer (pH 5.0–6.0). The concentrations and compositions of fatty acids were determined by gas chromatography. The minimum percentages of hydrolysed milkfat which affected growth and survival of selected Gram-positive and Gram-negative bacteria were measured. The bacterial experiments were repeated using pure fatty acids at similar concentrations. Lauric acid (C12:0) was found to be the most potent bactericidal fatty acid against Enterococcae (Gram-positive), and caprylic acid (C8:0) was the most potent against coliforms (Gram-negative). Use of Triton X-100 for milkfat emulsification provided a more compatible medium for studying bacterial growth in the hydrolysed milkfat than did use of casein/lecithin. The results also show that the antimicrobial effects of individual fatty acids released from hydrolysed milkfat were at least additive and suggest that hydrolysis of milkfat may be a significant factor in controlling growth of organisms imbibed with food in pre-weaned animals. The amount of pregastric catalyzed triglyceride hydrolysis in the digestive tract is sufficient to produce an antibacterial concentration of fatty acids and monoglycerides.

Early weaning of calves using feedstuffs. A rationalization based on inhibition of lipolysis.

The ability of broll (a combination of the wheat-milling byproducts bran and pollard, i.e., a mixture of wheat bran, husk, and flour) and blackstrap molasses (an ingredient of calf feed) to inhibit calf pregastric lipase (CPGL)-catalyzed hydrolysis of tributyrylglycerol (TBG) has been studied in vitro. Lipolysis was measured at pH 6.5 and 37 degrees C (CPGL at 0.02 mg/mL) with stirring at 300 rpm. The broll soaked in Bis-Tris buffer (50 mM, pH 6.5) at 4 degrees C for either 24 h or 15 min, and then added to an emulsion containing TBG, before initiation of the reaction by addition of CPGL, exhibited 22% inhibitory effect. A solution of blackstrap molasses (50%, v/v) exhibited inhibitory effects of 50% in the absence and 45% in the presence of Bis-Tris buffer. The initial rate of lipolysis in the presence of the dialyzed molasses retentate (10%, v/v) increased a little, compared with the same amount of crude molasses, from a mean value of 69% to a mean value of 74%. The results have been discussed in terms of the chemical nature and composition of broll and molasses and their roles as components of feedstuffs used in development of the rumen in early weaning of calves.

Determining the regio- and typo-selectivity of calf pregastric lipase

A selection of natural lipids, milk fat, cocoa butter and soybean oil, and three synthetic triglycerides 1,3-dipalmitoyl-2-oleoyl- rac-glycerol (POP), 1(3),2-palmitoyl-3(1)-oleoyl- rac-glycerol (PPO), and 1,3-dipalmitoyl-2-butyryl- rac-glycerol (PBP) were used as substrates for calf pregastric lipase (CPGL) catalysed hydrolysis at pH 6.5, 37.5 °C. CPGL preferentially releases short-chain fatty acids at the sn-3 position and unsaturated fatty acids during the hydrolysis of lipids containing long-chain fatty acids, and its activity increases with increasing unsaturation in the carbon chain of the long-chain fatty acids. The relative rate constants of hydrolysis were 1.56, 0.99, 0.73 h −1 for C4:0, C6:0, C8:0 esters, respectively, at sn-3; 0.0097, 0.0047 h −1 for the C16:0, C18:0 esters at sn-1,3, respectively; 0.0094 h −1 for the C18:1 ester at sn-2; and 0.0073, 0.0085, 0.012 h −1 for C18:1, C18:2, C18:3 esters, respectively, at sn-1,2,3. These last results confirmed that CPGL enzyme does not exhibit positional selectivity towards lipid substrates. The rate constant of hydrolysis was 1.14 h −1 for the C4:0 ester at sn-2. PBP was synthesised in four steps from commercially available glycerol as starting material. Glycerol was first selectively protected as a bis-silylether at the primary alcohol ( sn-1,3) positions. The free alcohol at the sn-2 position was then esterified with butyric anhydride. The protecting groups at C1 and C3 were then removed, and the resultant hydroxyl groups esterified with palmitic acid.

EFFECT OF LIPASE ENZYME ON THE RIPENING OF WHITE PICKLED CHEESE

ABSTRACTThe effect of addition of pregastric lipase enzyme on the accelerated ripening of white pickled cheese was investigated. Commercial pregastric lipase was added to milk before rennet addition at a level of 0,5, 8, 11 g per 100 L of milk and cheeses were made from this milk. Total solids, fat, total nitrogen, salt, titratable acidity, pH and free fatty acids (C2‐C18:1) were analysed in the samples during 1–90 days of ripening period at 15 days intervals. Total solids, fat, total nitrogen, salt, titratable acidity, and pH of cheeses slightly increased during the ripening period. Free fatty acids and volatile free fatty acid contents in cheeses made from pregastric lipase added milk were affected by pregastric lipase and their contents were increased significantly (P&lt;0.01) during the ripening period. Particularly, when cheese had a high level (11 g per 100 L milk) pregastric lipase, the amounts of butyric, caproic and caprylic acids in white pickled cheese were quite high. The relative amounts of volatile free fatty acids varied with storage time and pregastric lipase levels.

Meal-stimulated gastric acid secretion in healthy subjects correlates with postprandial but not fasting gastric acidity

A partially purified pregastric lipase from lambs was used as a catalyst for the hydrolysis of tributyroylglycerol. The reaction was followed by pH-stat autotitration of the released butyric acid. Integral values for pH and temperature were selected for determination of full Michaelis-Menten plots, and a kinetic surface was derived to characterize the activity of the enzyme preparation over a wide combination of these conditions. This surface enclosed a region of maximum activity centered on optimal pH (pH 6.4) and temperature (43°C), although optima were not sharply delineated. The activity in the surrounding region was generally high, and the value of the Michaelis-Menten constant was < 0.2 mM, indicating that tributyroylglycerol was generally a highly favored substrate.

Design, synthesis and evaluation of two diastereoisomeric phosphonate inhibitors of lamb pregastric lipase

A mixture of the two diastereoisomers of (2 R)-1,2-di- O-butyl- sn-glycerol-3- O- p-nitrophenyl- n-propylphosphonate, varying only in their configuration at phosphorus, was synthesised from ( R)-3- O-benzyl- sn-glycerol. The diastereoisomers were separated by normal phase HPLC and fully characterised. Both diastereoisomers were irreversible inhibitors of the activity of lamb pregastric lipase (LPGL), although the isomer which eluted first in the HPLC separation was significantly more effective.