Hydrolysis Of Milk Fat Research Articles

An Improved Liquid Chromatographic Method for the Quantitative Determination of Free Fatty Acids in Milk Products

An existing HPLC method for the quantitative determination of the major FFA in milk fat was improved. Complete resolution of the p-bromophenacyl (PBP) ester derivatives of saturated and unsaturated FFA of acyl-chain length C4 to C18 was accomplished by gradient elution (60 to 100% acetonitrile in water) when the temperature of the reverse-phase column was maintained at 10°C. This allowed the determination of all the major FFA in milk fat within a single chromatographic analysis. The improved method should save time in analysis of FFA in milk fat and also can be used to quantify the low concentrations (78 to 270 μM±17%) of the major FFA (myristic, palmitic, stearic, and oleic acids) in fresh pasteurized milk and follow the time course of milk fat hydrolysis where low amounts of FFA are liberated.

Lipases in human milk: effect of gestational age and length of lactation on enzyme activity.

Human milk contains two lipases, bile salt-stimulated lipase (BSSL) and lipoprotein lipase (LPL). In the mammary gland, LPL provides long-chain fatty acid for milk fat synthesis. LPL has no known function in milk, but has been implicated in milk fat hydrolysis during cold storage. BSSL may have an important role in infant fat digestion. The aims of the present studies were to assess (1) the methodological validity of using whole milk to analyze BSSL activity, (2) the longitudinal variation of BSSL and LPL activity in the milk of mothers delivering premature and full-term infants, and (3) the stability of BSSL and LPL activity during cold storage. Diluted whole milk and purified BSSL were shown to have similar characteristics. LPL activity was equally stable at -20 and -70 degrees C, whereas BSSL activity was higher in milks stored at -70 than at -20 degrees C (38.8 +/- 0.88 vs 33.3 +/- 0.87 U/ml milk, respectively; 1U = 1 mumol free fatty acid release/min). Levels of BSSL activity in preterm and term milk were similar. LPL activity tended to be higher in term milk. Overall, BSSL activity showed significant longitudinal variation, being highest at 1 and 3 weeks of lactation (43.2 +/- 0.04 and 42.6 +/- 1.03 U/ml milk, respectively). For LPL, the longitudinal pattern of activity depended upon the length of pregnancy. Implications for infant nutrition and mammary gland biology are discussed.

Effect of Pregnancy, Milk Yield, and Somatic Cell Count on Bovine Milk Fat Hydrolysis

During 12 mo, 1818 milk samples were collected from Holsteins and Jerseys (n = 261) to evaluate effects of advancing lactation and pregnancy on milk fat hydrolysis. Aliquots, cooled immediately and stored 48h at 4°C, were analyzed for free fatty acid content. Holsteins had higher acid degree values than Jerseys (.90 vs. .62). No difference in values was detected between alternate a.m. (.74) and p.m. (.76) sampling times. Repeatability of acid degree values from lactation to lactation was low (.22). Days in milk, days pregnant, and milk yield had curvilinear effects on acid degree values, whereas SCC effects were linear. Estimated acid degree value at 335 d in milk (average dry-off) was lowered from .80 to .63 when adjusted for days pregnant and to .48 when adjusted also for milk yield. These responses agree with the increased acid degree values associated with two late lactation events: increasing day pregnant and decreasing milk yield. Estrogen secreted by the developing fetal-placental unit could mediate changes in milk composition that promote milk fat hydrolysis.

Lipases in Preterm Human Milk

SummaryTerm human milk contains two lipases: Bile salt stimulated lipase (BSSL—this lipase also has bile salt stimulated esterolytic activity, BSSE) and lipoprotein lipase (LPL). We have measured the activity of these lipases in preterm (25–36 weeks' gestation) and term (37–40 weeks' gestation) human milk collected from the time of parturition until 3 months of lactation. BSSL levels are similar in preterm and term milk and do not vary significantly as a function of length of lactation. In the three groups of women who delivered at 26–30, 31–36, and 37–40 weeks of gestation, BSSL levels were 22.5 ± 6.6, 21.0 ± 3.6. and 32.3 ± 7.6 U/ml in colostrum and 26.3 ± 1 1.4, 31.1 ± 3.4, and 33.3 ± 10.7 U/ml in milk collected at 3 months of lactation, respectively. These observations suggest that preterm milk has the same fat digesting potential as term milk. BSSE activity decreased as a function of length of gestation and lactation, 61.2 ± 8.78, 44.4 ± 4.6, and 44.9 ± 13.3 U/ml in colostrum and 40.8 ± 5.0, 35.6 ± 9.5, and 23.3 ± 3.4 U/ml in milk collected after 3 months of lactation in the three groups studied. In all milks examined, the esterase lost its bile salt dependency upon storage at ‐ 10°C for periods of 2 weeks, suggesting that spontaneous hydrolysis of milk fat and accumulation of free fatty acids can occur in milk stored at ‐ 10°C. LPL activity increased as a function of length of gestation and lactation. LPL activity increased from 12.2 ± 3.3 U/ml in colostrum to 101.6 ± 12.6 U/ml in milk collected at 6 weeks of lactation from the preterm mothers and from 17.0 ± 9.7 U/ml in colostrum to 270 ± 15.5 U/ml in milk collected at 3 weeks of lactation from mothers of term infants. Our data suggest: (a) Milk secreted by women who deliver premature infants (26–36 weeks' gestation) has the same fat digesting potential as milk secreted by mothers of term infants, (b) The esterolytic component of the BSSL develops earlier than the lipolytic activity, (c) “Drip ” milk has higher LPL activity than milk pumped concomitantly from the other breast, suggesting that there is no relationship between mammary cell damage and LPL release, (d) Since BSSL and BSSE activities are 200− to 3,000‐fold higher in human milk than LPL activity, and since BSSE loses its bile salt dependency during storage at −10°C, the rise in free fatty acid levels in banked milks could be related to BSSE rather than to LPL.

Inhibition of Lipolysis by Milk Fat Globule Membrane Materials in Model Milk Fat Emulsion

The milk fat globule membrane (MFGM) enclosing fat droplets in bovine milk was isolated, and its effects on hydrolysis of milk fat by lipases were investigated by using a gum arabic-stabilized milk fat emulsion as substrate. The addition of isolated MFGM to the reaction mixture markedly inhibited hydrolysis by pancreatic and microbial (Rhizopus delemer) lipases. The inhibition was completely lost on tryptic digestion of MFGM, suggesting that the protein moiety of MFGM played a role in the inhibition. Soluble glycoprotein (SGP) which was isolated from delipidated MFGM produced marked inhibitory activity. The inhibition by SGP was dependent on substrate concentration, suggesting that the inhibition was at least partly due to coverage and blockage of the substrate surface by SGP.

Inhibition of lipolysis by milk fat globule membrane materials in model milk fat emulsion.

The milk fat globule membrane (MFGM) enclosing fat droplets in bovine milk was isolated, and its effects on hydrolysis of milk fat by lipases were investigated by using a gum arabic-stabilized milk fat emulsion as substrate. The addition of isolated MFGM to the reaction mixture markedly inhibited hydrolysis by pancreatic and microbial (Rhizopus delemer) lipases. The inhibition was completely lost on tryptic digestion of MFGM, suggesting that the protein moiety of MFGM played a role in the inhibition. Soluble glycoprotein (SGP) which was isolated from delipidated MFGM produced marked inhibitory activity. The inhibition by SGP was dependent on substrate concentration, suggesting that the inhibition was at least partly due to coverage and blockage of the substrate surface by SGP.

Application of Lipolytic Enzymes to Flavor Development in Dairy Products

Hydrolysis of milk fat catalyzed by enzymes, though a major dairy industry problem because of the production of rancid flavor defects in milk, cream, and manufactured dairy products, commonly is applied as a controlled process for the development of desirable flavors in certain products. These applications include flavor development in various cheeses, manufacture of lipolyzed milk fat products for enhancement of butter-like flavors, and flavor development in milk chocolate. Our paper reviews research on the relationship between lipolysis and flavor, which involves a variety of tissue and microbial lipase or esterase enzymes. Comparative data on enzyme specificity and enzyme activity indicate a wide variety of flavor effects from various enzyme preparations. The technology of application of lipolytic enzymes to flavor development in dairy products is described. Commercial applications, including patents, involving lipolytic enzymes or products therefrom in various cheeses, lipolyzed milk fat products, bakery formulations and flavor preparations, are reviewed. Potential future applications also are considered.

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.

Determination of the Major Free Fatty Acids of Cheddar Cheese1,2

Summary The major individual free fatty acids (FFA), acetic through linolenic, were determined in 14 samples of Cheddar cheese. A technique utilizing two methods of column chromatography plus gas-liquid chromatography was necessary for resolution of the complete series of FFA. Formic and propionic acids were not observed in any of the cheeses. Acetic acid showed the greatest variability in concentration and was usually the most abundant. Among the FFA which can arise through the hydrolysis of milk fat, free butyric acid was always found in about twice the percentage reported for esterified butyric acid. The individual FFA from caproic through linolenic, however, were present in nearly the same ratio as the same esterified acids in milk fat. Excluding two rancid samples, the average concentrations (expressed as mg of FFA per kg of cheese) were as follows: 2:0, 865; 4:0, 115: 6:0, 38; 8:0, 41: 10:0, 49; 12:0, 81; 14:0, 218; 16:0, 503; 18:0, 172; 18:1, 467; 18:2, 69; 18:3, 40. In the two rancid samples the concentrations of acetic acid fell within the range found in the 12 other samples. Each of the individual FFA from butyric through linolenic, however, was present in the rancid samples in about ten times the average concentration found in the 12 other samples.

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.

The Specificity of Milk Lipase. I. Determination of the Lipolytic Activity in Milk toward Milk Fat and Simpler Esters1,2

Summary A study of the optimum conditions for the activity of milk toward different esters was made. This will provide the basis for the assay methods used in further specificity studies. The reactions were carried out with constant shaking. Under those conditions, appreciable hydrolysis of milk fat occurred with nonhomogenized cream. The emulsion was more stable, however, with homogenized creams. Cream homogenized at 2,500 p.s.i. was therefore used for the milk fat hydrolysis. The pH optima for the milk fat, Tween-20, and methyl butyrate hydrolyses were 8.8–9.1, 8.8, and 8.0, respectively. The pH optimum of 8.7–8.9 for the tributyrinase activity of milk was confirmed.