Pregastric Esterase Research Articles

Purification and characterization of pregastric esterase from calf

Calf pregastric esterase (PGE) was purified from calf gullet tissues. The tissue was excised and lyophilized, and lipid materials were extracted with acetone and n-butanol at −20 °C. Proteins were extracted from the delipidated tissue with a buffer containing a chaotropic salt (NaSCN) to solubilize hydrophobically bound protein aggregates. Calf PGE precipitated from the crude extract at pH 5.0. The precipitated, solubilized proteins were subjected to anion-exchange chromatography on DEAE-Sephacel, and the enzymatic activity was eluted using a linear gradient from 0.10 to 0.50 m NaCl at pH 8.0. Fractions with high specific activities were then chromatographed twice using gel filtration on Sephadex G-100. The resultant enzyme was shown to be pure upon discontinuous electrophoresis in 12% polyacrylamide containing 0.1% sodium dodecyl sulfate (SDS-PAGE). From SDS-PAGE gel patterns, a molecular weight of 49,000 was determined. The amino acid composition of the enzyme allowed calculation of an “average hydrophobicity” (Bigelow index) of 1150 cal/residue. This indicates that calf PGE is relatively hydrophobic, being similar to proteins such as α-lactalbumin and bovine serum albumin in average hydrophobicity.

Characterization of Pregastric Esterases

Sephadex gel chromatography and polyacrylamide gel electrophoresis were used to estimate molecular weights and demonstrate heterogeneity of pregastric esterases obtained from commercial extracts of sublingual tissue of calves, kids, and lambs. The molecular weights of calf, kid, and lamb esterases were approximately 172,000, 168,000, and 150,000. Each esterase preparation appeared to contain several enzymes that had similar molecular weights and electrophoretic mobilities.

Effects of Pregastric Esterase on Utilization of Whole Milk by Preruminant Calves

The role of pregastric esterase was investigated in four abomasally-cannulated calves during a sequence of ten 3-day periods beginning when calves were 2 to 5 days of age. Two calves were fed whole milk from nipple bottles to maximize exposure to pregastric esterases, and two other calves were given milk by infusion directly into the abomasum to minimize exposure. The initial system of feeding each calf was continued for eight periods after which treatments were reversed. Postfeeding lipolytic activity of samples of abomasal contents and of intragastric lipolysis of milk fat were markedly greater for orally-fed calves than for abomasally-fed. Systems of feeding effected no significant differences in a) digestibility of dry matter, fat, crude protein, and nitrogen-free extract, b) retention of nitrogen, and c) percentage weight gains. Digestibility of dry matter, crude protein, and nitrogen-free extract of milk and retention of nitrogen were greater in later periods than in initial. Although age of calves was a positive factor associated with utilization of milk, decreased exposure of milk to pregastric esterase and the Resulting reduction of lipolysis in the abomasum did not effect a detectable lowering of milk utilization by calves in age groups studied.

Diffusion of Salt, Fatty Acids, and Esterases in Mozzarella Cheese

Diffusion rates of sodium chloride, butanoic acid, octanoic acid, and pregastric esterase in Mozzarella cheese were estimated. Solutions of sodium chloride, butanoic acid, octanoic acid, and carbon-14 labeled pregastric esterase were absorbed into chromatography paper, these “paper wicks” were placed between two slabs of cheese, and rates of migration of the solutes in cheese were measured. The approximate equilibration times of sodium chloride, butanoic acid, and octanoic acid over a 1.5 cm-thick section of Mozzarella cheese were 7, 28, 160 days. Pregastric calf esterase did not migrate during 11 wk of diffusion, probably because of its high molecular weight. Diffusion of sodium chloride, the smallest molecule evaluated, was equivalent across or parallel to the fibrous structure of Mozzarella cheese. Butanoic acid applied to the paper wick as a water or octanol solution migrated at equal rates, suggesting that butanoic acid should migrate freely through either water or fat phases of cheese.

IN VITRO OBSERVATIONS ON FACTORS AFFECTING CALF PREGASTRIC ESTERASE ACTIVITY

Three in vitro experiments were conducted to determine the efficacy of calf pregastric esterase (PGE) for hydrolyzing various fats and the influence of various factors on its lipolytic activity. Calf PGE was effective in hydrolyzing butterfat and coconut oil, hydrolyzed smaller amounts of the other plant oils tested and had a very limited capacity for lard and the six grades of tallow studied. The esterase retained good lipolytic activity for butterfat over the pH range normally encountered in the calf abomasum after feeding liquid diets. Rennet clotting of reconstituted skim-milk at pH 6.1 reduced enzyme hydrolysis of butterfat by 30%, presumably due to fat occlusion in the clot. Lecithin, skim-milk powder, casein, and lactalbumin markedly increased PGE activity; Ca++ had no effect. The bile salts taurodeoxycholate, glycochenodeoxycholate and taurochenodeoxycholate markedly inhibited PGE lipolysis, whereas others (taurocholate, deoxycholate, cholate, glycocholate) had little or no effect.

Properties of the Esterase Produced by Mucor miehei to Develop Flavor in Dairy Products

The development of Italian cheese flavor is attributed in large part to the action of animal pregastric esterases added to the milk prior to coagulation. Comparable flavor in Fontina and Romano cheese can be developed with an esterase preparation derived from Mucor miehei. The unique ability of this esterase to replace the animal-derived enzymes prompted a study of its properties. Esterase and microbial rennet are produced by Mucor miehei in the same fermentation. The enzymes are separated by filtration at pH 5.0. The insoluble esterase then is recovered by extraction of the filter cake at pH 10.5. The enzyme has a pH optimum of 7.0 on tributyrin, has a temperature optimum of 50C, and is stable over the pH range of 4.0 to 10.0 and up to 55C at pH 5.3. The enzyme will hydrolyze a number of synthetic triglycerides, with optimum activity on tributyrin and trioctanoin. Higher molecular weight fatty acid containing triglycerides are hydrolyzed less readily. Sorbitol esters of fatty acids also are attacked. The esterase will hydrolyze a number of animal fats and vegetable oils and is relatively unaffected by high concentrations of various salts, ethylene diamine tetraacetate, or sulfhydryl inhibitors.

Plasma lipids, ketone bodies, and glucose concentrations in calves fed high- and low-fat milk replacers.

Twenty-four 5-day-old male calves were fed twice daily milk replacers containing either 5% (low-fat) or 25% (high-fat) lard. Plasma lipids, blood glucose, and ketone bodies were determined in jugular blood before feeding and every hour during 8 h after feeding. The high-fat diet caused in the 1st h after feeding a sharp increase of triglycerides and phospholipids followed by a sharp decrease; these two increased slowly during the following 5 h. Within the first 2 h after feeding, there was an increase of cholesterol esters, free cholesterol, and nonesterified fatty acids. With the low-fat diet, triglycerides and cholesterol esters showed a small increase during the 4 h following meal whereas phospholipids, free cholesterol, and nonesterified fatty acids were not affected significantly. With both diets, blood glucose reached a maximum of 110 mg/100ml 1 h after feeding; ketone bodies were not altered significantly. With the high-fat diet, lipid digestion would occur in two phases; firstly, part of the fat would be lipolyzed quickly by pregastric esterase before clot formation in the abomasum; secondly, the rest of the lipids, slowly released by progressive lysis of the coagulum would be digested under the action of gastric and pancreatic lipases. The first phase did not occur with the low-fat diet.

Enhancement of cheese flavors with microbial esterases

AbstractThe characteristic flavor of hard Italian cheeses is associated with the presence of fatty acids, particularly butyric acid, liberated from milk fat during the ripening process. To ensure proper development and control of flavor, animal pregastric esterases or lipases are routinely added to the milk before coagulation of the curd. Such esterases are also used to generate flavor in enzyme modified cheese and other dairy products.Esterases from microbial sources have been investigated as agents to enhance flavor in cheese. We have found that an esterase from Mucor miehei exhibits the type of lipolytic activity needed for this application. Romano and fontina cheeses of excellent quality have been prepared by the use of this esterase. It has also been used successfully in the preparation of enzyme modified cheese, and, in turn, processed American cheese.

Flavor Development in Fontina and Romano Cheese by Fungal Esterase

Abstract Flavor character and intensity in pilot-size lots of Fontina cheese and Romano cheese made with Mucor miehei esterase were compared with flavor properties developed in control cheeses which received pregastric esterase preparations. Changes in flavor were assessed organoleptically and chemically as water-soluble acids and trichloroacetic acid soluble nitrogen during normal and extended ripening periods. In the Fontina cheese, the desired flavor developed parallel to that in the control cheese when fungal esterase and pregastric calf esterase were incorporated at equivalent lipolytic activity. Attainment of desired flavor sharpness in Romano required a fivefold greater addition of fungal esterase, based on lipolytic activity, than pregastric kid esterase. No objectionable flavors or aromas appeared in cheese ripened far beyond its usual market age.

Pregastric Esterase in Milk Sham Fed to Adult Jersey Steers1,2

Pregastric esterase activity was detected in reconstituted nonfat milk sham fed from a nipple pail to two 4-yr-old rumen-fistulated steers. Lipolytic activity, determined in a medium containing 5% tri-n-butyrin, averaged 8.6±.4 lipase units. Further assays, in which activity was measured by free fatty acids released from a condensed milk substrate, averaged 166.9±9.2 μmol. These values are higher than those noted for young calves, indicating that secretion of pregastric esterase may persist in cattle beyond calfhood. Esterase activity in one of the steers fed whole milk until he was 2 yr of age showed no marked residual effect of earlier intake of milk fat.

Marine Fat Fed to Young Calves

In two calves fed milk replacers containing hydrogenated marine fat, collection of the duodenal chyme was carried out over a 24 hr. period, when the calves were about one and a half weeks old. The fat was partly broken down to free fatty acids (F.F.A.) before entering the duodenum. Hydrolysis was probably due to pregastric esterase. No hydrogenation of the unsaturated fatty acids was observed.

The influence of chemical modification on the hydrolysis of fats by pancreatic lipase and pregastric esterase with reference to fat digestion in the pre-ruminant calf.

AbstractThe hydrolysis of various dietary fats and pure triglycerides by pregastric esterase and pancreatic lipase was studied in vitro in conjunction with a series of digestibility trials to examine the effect of various chemical modifications to beef tallow on its digestibility in milk replacers for pre‐ruminant calves. Using pure triglycerides, it was shown that pregastric esterase preferentially hydrolyses triglycerides containing short chain fatty acids; the rate was highest with glyceryl tributyrate and decreased markedly with increase in chain length. Using butterfat, tallow and various modified forms of tallow, it was shown that the inclusion of butyric acid in tallow by interesterification, which markedly improved its digestibility, substantially increased its rate of hydrolysis by pregastric esterase. The initial rate of hydrolysis of tallow by pancreatic lipase was substantially less than that of butterfat. Little improvement in the rate of hydrolysis and solubilisation was obtained when the tallow sample was interesterified or when it was supplemented with the long chain fatty acids, myristic and oleic, or with the short chain fatty acid, butyric. A considerable increase in the initial rate of hydrolysis and solubilisation did occur with tallow samples with a reduced content of stearic acid.The results suggest that hydrolysis by pregastric esterase is a major site of discrimination between different fats in the pre‐ruminant calf and an important factor in determining overall digestibility.

Relative importance of pancreatic lipase and pregastric esterase on lipid absorption in calves 1-2 weeks of age.

An attempt has been made to determine the relative importance of pancreatic lipase and pregastric esterase on lipid absorption in calves 1-2 weeks of age. This was carried out by measuring the quantity of esterified lipid in lymph and the products of lipid hydrolysis in jejunal contents of 10 calves fed either milk or fatty whey in the presence or absence of pancreatic juice.

The importance of lipolytic enzymes in milk-fed and ruminating calves.

An attempt has been made to compare the activity and specificity of pregastric esterase and pancreatic lipase from calves 1-2 weeks of age in an in vitro system using washed milk-fat globules as substrate. In addition, the changes in activity of pancreatic lipase and pancreatic phospholipase have been investigated as milk-fed calves change from a monogastric to a ruminant type of digestion.

Gastric Lipase Characterization and Utilization in Cheese Manufacture

Gastric lipase has been demonstrated in some rennet paste preparations but not in commercial rennet extracts. Lamb gastric lipase had a pH optimum from 7.0 to 7.6 and was most stable at pH 7.0. It hydrolyzed tributyrin most rapidly at 45C and β-naphthyl laurate very slowly. Lamb gastric lipase hydrolyzed mono- and dibutyrins more rapidly than lamb pregastric esterase. It hydrolyzed milk fat very slowly but may have a role in cheese flavor development. Cheddar and Provolone cheeses were organoleptically preferred when gastric lipase preparations were included in their manufacture.

Lamb Gastric Lipase and Proteases in Cheese Manufacture

The value of lipase and proteases from lamb gastric tissues in cheese flavor development was demonstrated independently of lamb pregastric esterase and calf rennet activities. Parmesan, Romano, Cheddar cheese, and directly acidified Pizza curd were improved in body breakdown and flavor development when treated with lamb gastric extract. The best cheese flavor resulted when both gastric and pregastric extracts were incorporated. Protease system from lamb gastric tissue was active at pH 9.0 and had casein-agar gel diffusion and caseolytic characteristics that were different from calf rennet or swine pepsin.

Effect of Fat Source on Relative Activity of Pregastric Esterase and on Gleceride Composition of Intestinal Contents1,2

Sham-fed nonfat milk was incubated with filled milks containing 8% of various fats and oils. When related to evaporated milk (7.9% fat, relative activity 100), activities for butteroil, colostrum fat, coconut fat, choice white pork grease, refined lard, tallow, soybean oil, and corn oil were 87.3, 87.2, 70.8, 28.5, 14.1, 14.2, 22.9 and 18.1.In a second study, residual glycerides in various sections of the gastrointestinal tract of seven-day old calves were examined after milk replacers containing lard or milk fat were fed. Significantly greater proportions of triglycerides were in contents of the abomasum, descending and ascending duodenum, and first 2m of jejunum of calves fed lard-filled milks than of calves fed milk fat-filled milks. Abomasal diglyceride and monoglyceride fractions were lower (P<0.01) for calves fed lard-filled milk than for calves receiving milk fat-filled milk; however, by the time intestinal contents reached the jejunum, only monoglycerides were significantly lower (P<0.05).

Action of Pregastric Esterase on Synthetic Triglycerides Containing Butyric Acid

Pregastric esterase hydrolyzed synthetic triglycerides containing butyric acid at greatly different rates. The triglycerides of lower molecular weight were attacked most rapidly. All of the diglycerides from the digestions contained butyric acid; therefore, the diglycerides were not representative of the long-chain portion and were not suitable for stereospecifie analysis. Some of the monoglycerides also contained butyric acid.

Effects of in Vivo and in Vitro Acid Environments on Activity of Pregastric Esterase

Abomasal samples were collected at 0, 30, 60, 120, and 180 minutes post-feeding from a rumen-fistulated calf fed nonfat milk. Abomasal pH decreased progressively with time after feeding. Lipolytic activity, presumably due to pregastric esterase, was apparent in all samples; however, activity was considerably less in samples taken 180 minutes post-feeding.In another trial, nonfat sham-fed milk, collected by way of an esophageal fistula, was subjected in vitro to acid conditions of pH 4.0, 3.5, 3.0, 2.5, and 2.0 for intervals of 15, 30, 60, 120, and 180 minutes. The pH of each preparation was then adjusted to 6.1 and incubated at 37C with evaporated milk. Lipolytic activity of samples subjected to pH 4.0, 3.5, and 3.0 was about 65% of the control, irrespective of time held at these levels of acidity. Samples subjected to pH 2.5 and 2.0 lost nearly all of their lipolytic activity irrespective of time interval.

Lipolysis of Milk Fat by Pregastric Esterase in Vivo1,2

Three male Holstein calves, surgically fitted with rumen fistulas, were fed milk by nipple pail and by direct infusion into the abomasum. Samples withdrawn from the abomasum at .5, 1 and 2 hr postfeeding were analyzed for glyceride and fatty acid composition. The molar percentages of tri-, di- and monoglycerides for calves fed by nipple pail were 57.5, 30.8 and 11.5; 53.3, 32.9 and 13.8; and 49.4, 35.0 and 15.6 at .5, one and two hours, respectively. For calves given milk directly into the abomasum, the molar percentages were 88.7, 8.9 and 2.4; 84.9, 11.2 and 3.9; and 75.0, 17.5 and 7.5, respectively. Fatty acid composition analysis of abomasal glycerides from orally fed calves revealed no butyric or caproic acids in monoglycerides. Smaller quantities of caprylic, capric, lauric and oleic acids were found in monoglycerides than in di- or triglycerides. Palmitic acid was present in greatest amounts in monoglycerides. Butyric, caproic, caprylic, capric and lauric acids were present in the free fatty acid fraction of samples from orally fed calves.