Pregastric Esterase Research Articles

Changes in Milk Products Sham Fed to Calves. III. Effects of Concentration of Fat

Liberation of volatile soluble fatty acids from sham-fed milk products (separated milk, 0.03% fat; whole milk, 4.4%; low-fat cream, 23%; and high-fat cream, 49%) and the accompanying changes in pH and bacterial counts were used as indicators of the lipolytic capacity of pregastric esterase in calves. Samples were collected for analysis immediately after sham feeding and serially during a subsequent ten-hour period of incubation at 38C.The magnitude of lipolysis in the sham-fed products generally corresponded to the concentrations of fat therein. After two hours of incubation, the rate of change in the higher fat products declined markedly. During, terminal stages of incubation, acidity in sham-fed separated milk increased, apparently as a result of bacterial metabolism.Bacterial counts of the milk products increased as a result of contamination in sham feeding. During subsequent incubation, the numbers in whole milk and creams decreased, but subsequently increased. The decline probably was the result of toxicity of certain fatty acids.Pregastric esterase normally secreted by calves consuming whole milk seems to have the capacity to hydrolyze milk lipids in excess of quantities normally in the diet.

Assay and Characterization of Pregastric Esterase

A recording pH stat has been adapted to the assay of pregastric esterase preparations. Assay substrate containing 5ml n-tributyrin, 0.5ml 10% soy lecithin in mineral oil, 0.6g acid casein, and 95ml water is emulsified in a blender. Enzyme inoculum is prepared via rehydration of 0.2 to 1.0g powder in 75–80ml 0.5 m NaCl solution for 20min at ambient temperatures. The suspension is made up to 100ml and 1ml is inoculated into 5ml of substrate on a pH stat, set to control at pH 6.20 and 40C. The resultant slope is a function of enzyme activity. This assay is more precise than currently used colorimetric tests. Calf pregastric esterase was found most stable at pH 5.5, using the pH stat. The enzyme was most active at 45C. Kid and lamb enzymes also had maximum activity at 42–45C. pH optima occurred at 5.3 to 5.5 on both tributyrin and milk fat substrates. Minor peaks occurred at pH 8.5 and 8.7 for calf and kid, respectively. Pregastric esterase has esterase characteristics when evaluated on solutions and emulsions of triacetin. The enzyme is not as active upon 1-mono- and dibutyrin as pancreatic, milk, or Aspergillus niger lipases.

Lipolysis of Milk Fat by Pregastric Esterase in the Abomasum of the Calf1,2,3

The role of pregastric esterase in the abomasal digestion of milk fat was studied in calves having fistulae both in the rumen and the duodenum. Two systems of feeding were employed. Whole milk either was nipple-fed (oral feeding) or was infused directly into the abomasum via the rumen fistula (abomasal feeding). The latter method bypasses the site of pregastric esterase secretion. After feeding, serial collections of the digesta passing from the abomasum into the duodenum were taken for 10 hr. Each hourly sample was analyzed for butyric acid, both total and free, and for higher fatty acids, both total and free. When the milk was fed orally, a high proportion of the total butyric was present as the free acid. When pregastric esterase was excluded by abomasal feeding, however, only a small percentage of the total butyric was present as the free acid. A similar contrast between the two feeding systems was noted also for the higher fatty acids, though the difference was not so great as that observed for butyric acid. These observations suggest that pregastric esterase may be of greater importance than gastric lipase in the abomasal digestion of milk fat.

Source of Lipolytic Enzymes in the Abomasum of the Calf , ,

An investigation was conducted to ascertain whether gastric lipase has a primary role in the hydrolysis of milk fat in the abomasum of the calf. The treatments of rumen-fistulated calves were: (a) fresh whole milk given orally from a nipple-feeder; (b) fresh whole milk passed through a tube directly into the abomasum; (c) same as (b) except that the calf was sham-fed immediately before abomasal feeding; and (d) same as (b) except that sham-fed milk from another calf was given in lieu of fresh milk. Abomasal contents withdrawn at various intervals after feeding were analyzed for total free fatty acids. The level of free fatty acids was high only in those treatments involving the passage of milk through the oral cavity, the site of pregastric esterase secretion. This observation indicated that gastric lipase, if present at all, contributed little to the abomasal hydrolysis of fat. Electrophoretic analyses of both abomasal and intestinal digesta collected after the oral feeding of milk revealed a pattern of esterases similar to that reported previously for pregastric esterase. These observations substantiate the belief that pregastric esterase is primarily responsible for the digestion of milk fat in the abomasum.

Development of Domestic Feta Cheese

Summary A manufacturing method for Feta cheese was developed using pasteurized milk, Streptococcus lactis and Lactobacillus casei cultures, a pregastric esterase (Capalase-KL), and liquid rennet. Cheese had a pleasant acetic acid, salty and mild characteristic rancid flavor, and a smooth, creamy, soluble, and sliceable body. A mixed culture of S. lactis with either L. casei or Lactobacillus acidophilus was necessary for the development of Feta cheese characteristics. The product made without lactobacilli lacked flavor and smoothness of body, whereas cheese without streptococcus was extremely soluble and gassy. Higher levels of soluble protein in the lactobacillus cheese supported the organoleptic evaluation that curd creaminess and curd solubility might be synonymous. The flavor of Feta cheese of high quality differed from the bitter, atypical rancid and unclean flavors produced by the lipases of milk, Capalase-K, or Italase. Homogenization accentuated the defects caused by these lipases. Desirable rancid flavor developed in cheese made with Capalase-KL and Capalase-L. Chemical analyses indicated that the rancid flavor in Feta cheese was associated with the free fatty acids from C 2 through C 10 . Objectionable rancid flavor developed in cheese with high levels of C 12 and higher fatty acids.

Electrophoretic Characterization of Pregastric Esterase from the Calf1,2

Summary This investigation was conducted primarily to determine whether pregastric esterase from the calf is a single enzyme or a group of enzymes. Accordingly, sham-fed water and extracts of various alimentary and related tissues were subjected to electrophoresis in starch gel, which was stained histochemically to locate individual esterases. The results indicate that pregastric esterase is composed of at least six different enzymes. Resolution of these esterases was obtained in gel containing 8g of starch per 100ml of buffer, but not in that containing 15g per 100ml. Thus, the components of pregastric esterase are believed to be of high molecular weight. Moreover, these esterases precipitate at a slightly acid pH, thereby providing a means of separating pregastric esterase from nonsecretory esterases that are present also in tissue extracts.

Role of Pregastric Esterase in the Abomasal Hydrolysis of Milk Fat in the Young Calf1,2

Summary The hydrolysis of milk fat in the abomasum of the young calf was studied. Whole milk either was nipple-fed (oral feeding) or was placed directly into the abomasum (abomasal feeding). The purpose of the latter system was to minimize the entrance of pregastric esterase. At 1, 2, and 3 hr. after feeding, the contents of the abomasum were sampled and analyzed for total free fatty acids (FFA). The FFA from the oral-fed samples were also separated into three fractions: butyric, valeric, and higher fatty acids. The mean concentrations of total FFA (μ M /5g. contents) were as follows for the oral and abomasal systems of feeding, respectively: 1 hr., 86, 19; 2 hr., 101, 20; and 3 hr., 120, 30. For the oral-fed samples, the mean concentrations of butyric, valeric, and higher fatty acids were as follows, respectively: 1 hr., 34, 7, 53; 2 hr., 19, 4, 77; and 3 hr., 11, 3, 119.

Substrate Specificity of Pregastric Esterase from the Calf

Substrate Specificity of Pregastric Esterase from the Calf

Effects of Age and Diet on the Secretion of Pregastric Esterase in Calves1,2

Summary The effects of age and diet on the quantity of pregastric esterase secreted during the nursing of milk were studied in six Holstein calves. During the first 180 days of life, there was no appreciable change in enzyme secretion. Limited observations on two calves suggest that this trend continues through at least 1 yr. of age. Calves sustained on a diet of hay and grain secreted, when nursing milk, approximately the same quantity of pregastric esterase as did those animals restricted to a diet of milk. The output of enzyme during the actual consumption of hay and grain, however, appeared to be considerably less than that stimulated by the nursing process. There was no correlation between the total volume of saliva secreted and the output of pregastric esterase.

Effects of Continuous Nursing, Length of the Nursing Period, and Rate of Milk Consumption on the Secretion of Pregastric Esterase by Calves1,2

Summary The effects of continuous nursing, length of the nursing period, and rate of milk consumption on the secretion of pregastric esterase by calves were investigated. When 50 lb. of milk was fed continuously, as ten successive 5-lb. increments, the enzyme content of each increment decreased progressively. The quantity of enzyme secreted into a single 5-lb. unit of milk, however, was increased markedly by increasing the length of the nursing period. Increasing the rate of milk consumption appeared to stimulate the output of pregastric esterase slightly. The samples of sham-fed milk collected in the morning contained more enzyme than did those collected in the afternoon.