Enzyme Activity In Milk Research Articles

Relationship Among NAGase Enzyme Activity, CMT Score and Somatic Cell Count in Milk of Crossbred Dairy Cows with Sub-Clinical Mastitis in North-Western Province of Sri Lanka

The assay procedure for N-acetyl-β-D-glucosaminidase (NAGase) was considered to be the most reliable, simple, and rapid enzymatic method for estimating the severity of mammary tissue damage. This study was carried out to assess the relationship of NAGase enzyme activity, California mastitis test score (CMTS) and somatic cell counts (SCC) in milk and the influence of breed, parity and age of the cow on NAGase activity. The results showed that prevalence of sub-clinical mastitis (SCM) was 74.5% (41/55) in the population of cows tested. The isolated pathogens were Staphylococcus spp. (34%, 14/41), Escherichia coli (34%, 14/41) and Streptococcus spp. (32%, 13/41). The SCC showed a significant (p<0.05) positive correlation with the activity of NAGase (R2= 0.796) and CMTS (R2= 0.709). A significant correlation was observed between the NAGase activity and SCC in SCM positive milk, where the correlation was stronger (R2= 0.748) in the former than in the latter. The parity (R2= 0.843) and age (R2= 0.758) of the cow also showed a significant (p<0.05) positive correlation with enzyme activity. The mean SCC and enzyme activity of different crossbreds of the study population did not show a significant correlation, but mean values of SCC and enzyme activity were higher when an animal was phenotypically more related to European breeds (779– 1848 x103 SCC/ml;0.64 -0.89 μmole/min/ml) than tropical breeds (427–534x103 SCC/ml; 0.26-0.43 μmole/min/ml). It could be concluded that, the prevalence of SCM in crossbred cows in North-Western Province of Sri Lanka is considerably high. The determination of NAGase enzyme activity in milk could be used as a method for early detection of SCM more accurately than CMTS and SCC.

Short communication: Diets supplemented with starch and corn oil, marine algae, or hydrogenated palm oil differently affect selected metabolite concentrations in cow and goat milk

The objective was to investigate the effects of species (cow vs. goat) and of various dietary lipid supplements, known to modulate milk fat content, on selected metabolites and enzymes in milk and to explore their correlations with performance traits. Twelve Holstein cows and 12 Alpine goats, all multiparous and nonpregnant, and at 86 ± 24.9 and 61 ± 1.8 DIM, respectively, were fed a basal diet (45% forage + 55% concentrate) not supplemented (CTL) or supplemented with corn oil plus wheat starch [COS, 5% of diet dry matter (DM)], marine algae powder (MAP, 1.5% of diet DM), or hydrogenated palm oil (HPO, 3% of diet DM) in a replicated 4 × 4 Latin square design with 28-d experimental periods. Intake, milk production and composition, milk fatty acid profile, and plasma metabolite concentrations were previously reported. Concentrations of 9 milk metabolites [β-hydroxybutyrate (BHB), glucose, glucose-6-phosphate, isocitrate, choline, glutamate, urea, cholesterol, and free amino groups] and 2 milk enzyme activities (alkaline phosphatase and lactate dehydrogenase) were measured on d 24 of each experimental period. Dairy performance data showed marked species and diet effects on milk fat content. Irrespective of diet, cow milk was richer in alkaline phosphatase and glucose compared with goat milk (16 and 3 times more, respectively), whereas goat milk had greater urea and glucose-6-phosphate concentrations compared with cow milk (1.9 and 5.3 times more, respectively). In cows, COS decreased milk BHB and choline (-25 and -43%, respectively) compared with CTL, whereas no effects were observed in goats. The COS and MAP diets increased milk isocitrate compared with CTL in cows, but COS decreased isocitrate concentrations in goat milk. Milk choline was correlated with milk fat content in cows (Spearman r, rS = +0.73) and goats (rs = +0.58), and lactate dehydrogenase activity was correlated with milk somatic cell count (rs = +0.66) in cows but not in goats. We provide evidence of different milk metabolite responses according to species and diets. Metabolites and enzymes secreted in milk may be indicators of specificities of lipid metabolism among ruminant species and may contribute to a better understanding of mechanisms regulating milk fat secretion. Changes in the concentrations of some metabolites considered minor components of milk may be valuable diagnostic tools of mammary gland and animal metabolism as well as of milk processing characteristics.

Serum enzyme activities in blood and milk in the different stage of lactation in holstein dairy cows

The objective of this study was to determine correlation between serum blood and milk enzyme activities of aspartate-aminotransferase (AST), alanine- aminotransferase (ALT), alkaline-phosphatase (ALP in the 36 dairy Holstein cows divided into three groups according to production period. Group 1 consisted cows in the start of lactation (n = 12); Group 2 -consisted of early lactation cows (n=12) and Group 3 included mid lactation cows (n=15). Statistically significant higher (P&lt;0.01) activity of AST in blood serum was established in early lactation groups of cows as compared to mid lactation group of cows. ALT activity showed a lower (P&lt;0.01) serum activities in early lactation groups of cows than in the mid lactation cows. Higher values ALP in blood and milk are determined in early lactation groups of cows as compared to mid laltation cows, but without statistical significance (P&gt;0.05). Research results showed possibility of mild degree of hepatic lesions, probably due to fat infiltration in early lactation cows. No significant difference (P&gt;0.05) was observed in milk serum value for AST, ALT and ALP between the three groups of cows. No significant correlations among AST, ALT and ALP activities in blood and milk serum were determined (P&gt;0.05) and shows that activity of these enzyme in the milk are not used as markers for early diagnosis of subclinical metabolic disease.

Assessment of Lactate Dehydrogenase Enzyme Activity in Milk as a Marker for Detection of Subclinical Mastitis

Sub clinical mastitis is responsible for heavy economic losses throughout the world in dairy sector. The objective of present study was to investigate lactate dehydrogenase (LDH) enzyme activity in milk for detection of subclinical mastitis in buffaloes Milk samples from 60 buffaloes, including 40 subclinical infected and 20 healthy were subjected to the estimation of LDH using a LDH activity assay kit. LDH concentration in milk of buffaloes having subclinical mastitis was reported to be high (1.88±0.91 μmole/ml/min) as compared to healthy animals (0.26±0.10 μmole/ml/min). The receiver operating characteristics analysis at cut off value of 0.44 μmole/ml/min for LDH activity exhibited sensitivity and specificity of 95% each and area under curve was 0.959. Results of the correlation matrix revealed significantly strong positive interdependence of somatic cell count with enzyme LDH (r = 0.722**; P <0.01). Measuring LDH activity in milk was found both easy and cost effective with high sensitivity and specificity indicating that LDH activity has great potential as a diagnostic tool for detection of sub clinical mastitis in buffaloes.

Effect of oxytocin on enzyme activities in bovine milk

The objective of this study was to determine the effect of administration of oxytocin prior to milking on enzyme activities and thiocyanate concentration in bovine milk. Intramuscular administration of oxytocin (20 IU) was made to a group of cattle comprising 8 animals, whilst a second group was kept as control. Significant (p < 0.01) differences were measured in enzymatic levels of oxytocin-treated and control bovine milk. Alkaline phosphatase and lipase activity were reduced in milk of oxytocin-treated cows. However, the trend was the opposite for acid phosphatase, where high enzyme activity was observed in milk after oxytocin treatment (0.010 U mL−1) compared with the control (0.004 U mL−1). Treating cattle with oxytocin did not significantly (p < 0.01) affect lactoperoxidase concentration but increased thiocyanate levels of milk by ∼6.5%. In conclusion, oxytocin administration to cows cause variations in milk enzyme activities and might consequently affect milk stability against certain deteriorative changes.

Comparison of Milk Enzyme Activity in Different Lactation Periods

The study mainly measured enzyme activity of alkaline phosphatase and amylase in the different periods of milk, and seeking the best conditions for the enzyme activity of the respective in different pH, different temperature. Results showed that the activity of amylase in bovine colostrum was higher than in mature milk and terminal milk. The highest amylase activity, 103.28 U/dL, was detected at the temperature 40oC and pH 6. The best condition for Bovine colostrum of alkaline phosphatase activity is higher than mature milk and terminal milk. Alkaline phosphatase activity is the highest temperature at 40oC and pH 10, about 96.38 U/mL.

Influence of udder infection status on milk enzyme activities and somatic cell count throughout early lactation in goats

At present the analysis of somatic cell count (SCC) used for the detection of intramammary infections (IMI) in bovine milk is also recommended for goat milk, but due to the various factors influencing SCC it allows only limited conclusions on the udder health of goats. The research on enzyme activity in milk appears to show promise in finding an approach with more suitable indicators of the early detection of IMI in goats. Therefore, the present study aimed to investigate the influence of goat udder infection status on different milk enzyme activities and SCC throughout early lactation. A total of 60 dairy goats were sampled at weekly intervals over a period of 6 weeks after kidding and the bacteriological status, milk SCC and the activity of N-acetyl-β-d-glucosaminidase (NAGase), β-glucuronidase and lactate dehydrogenase (LDH) of udder halves were analysed. Infections with minor or major pathogens were identified in 47% of all animals over the sampling period. Coagulase-negative staphylococci (CNS) represented the main group of pathogens in bacteria isolates (16.4%). Corynebacteria and major pathogens were detected in 7.2% and 5.7% of udder half samples. Excluding the first week after parturition, the study revealed a highly significant influence of lactation week on log10 SCC (F4,255=11.63, p<0.001) and log10 LDH (F4,285=5.02, p<0.01) and must be acknowledged as the most dominating predictor on NAGase activity (F4,250=29.62, p<0.001) in early lactation. Levels in β-glucuronidase activity were not influenced by the stage of lactation. The infection status of udder halves had a highly significant effect on log10 SCC (F3,528=18.88, p<0.001), log10 β-glucuronidase (F3,407=11.02, p<0.001) and log10 LDH (F3,534=12.39, p<0.001). We revealed no effect of different pathogenic groups on NAGase activity, thus this parameter proved not to be suitable as an indicator for udder infections in early lactation. The proposed milk enzymes β-glucuronidase and LDH might be indicative of inflammatory processes, but the influence of parity and lactation stage on the overall results should be considered in the assessment of udder health in dairy goats.

Evaluation of Milk Trace Elements, Lactate Dehydrogenase, Alkaline Phosphatase and Aspartate Aminotransferase Activity of Subclinical Mastitis as and Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis).

Mastitis is a highly morbid disease that requires detection at the subclinical stage. Tropical countries like India mainly depend on milch buffaloes for milk. The present study was conducted to investigate whether the trace minerals viz. copper (Cu), iron (Fe), zinc (Zn), cobalt (Co) and manganese (Mn) and enzyme activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in riverine buffalo milk can be used as an indicator of subclinical mastitis (SCM) with the aim of developing suitable diagnostic kit for SCM. Trace elements and enzyme activity in milk were estimated with Atomic absorption Spectrophotometer, GBC 932 plus and biochemical methods, respectively. Somatic cell count (SCC) was done microscopically. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. A statistically significant (p<0.01) increase in SCC, Fe, Zn, Co and LDH occurred in SCM milk containing gram positive bacterial agents only. ALP was found to be elevated in milk infected by both gram positive and negative bacteria. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×105 cells/ml of milk as the benchmark. Only ALP and Zn, the former being superior, were found to be suitable for diagnosis of SCM irrespective of etiological agents. LDH, Co and Fe can be introduced in the screening programs where Gram positive bacteria are omnipresent. It is recommended that both ALP and Zn be measured together in milk to diagnose buffalo SCM, irrespective of etiology.

SOLVENT EXTRACTION OF β-GALACTOSIDASE FROM KLUYVEROMYCES LACTIS YIELDS A STABLE AND HIGHLY ACTIVE ENZYME PREPARATION

ABSTRACT The yeast Kluyveromyces lactis was cultured in cheese whey and β-galactosidase was extracted from cells by an organic solvent procedure. Under optimum conditions (pH 8.5, 25C), approximately 90% of the intracellular β-galactosidase activity was released into 0.1 M potassium phosphate solution with 2% chloroform after 9 h. The cell-free extract containing β-galactosidase activity was concentrated and the enzyme partially characterized. This enzyme has exhibited an optimum activity at a pH range of 6.2–6.6 and temperature of 40–45C on O-nitrophenyl-β-D-galctopyranoside. The maximum enzyme activity in milk was found at 45C and the concentrated enzymatic extract was found to have 19 U/mg protein. A feasible preparation for commercial application containing 20% glycerol maintained 89% of activity after 2 months at 4C, demonstrating stability. This preparation analyzed on 5% nondenaturing polyacrylamide gel electrophoresis exhibited two bands suggesting two aggregation forms with β-galactosidase activity. PRACTICAL APPLICATIONS Kluyveromyces lactis yeast cells are a safe source of β−galactosidase for food and pharmaceutical industries. Their application to prior hydrolysis of lactose in milk products has become commonplace in alleviating the symptoms of lactose intolerance. It is also of great interest to prevent lactose crystallization in concentrated or frozen dairy products such as condensedmilk, ice cream and “dulce de leche,” thereby increasing consumers' acceptability. In addition, treatment of cheese whey with β-galactosidase increases its fermentability to convert cheese whey lactose into ethanol helping to reduce the pollution caused by large volumes of this by-product released in the environment. Solvent extraction of β-galactosidase from K. lactis is a low-cost process and yields a stable and highly active enzyme preparation.

Milk Enzyme Activities and Subclinical Mastitis Among Women in Guinea-Bissau

Subclinical mastititis (SCM) is a condition with raised milk concentration of sodium and milk immune factors. The milk enzymes N-acetyl-beta-D-glucosaminidase (NAGase), lactate dehydrogenase (LDH), acid phosphatase (AcP), and alkaline phosphatase (AP) have attracted attention in dairy research as indicators of SCM, udder health, and milk quality. To investigate if milk enzyme activities and the inflammatory interleukin 8 (IL-8) level are increased in women with SCM, we measured sodium, potassium, NAGase, LDH, AcP, AP, and IL-8 in breastmilk samples collected at 2 months postpartum from 797 women in Guinea-Bissau, West Africa. SCM, as indicated by a molar milk sodium/potassium ratio (Na/K ratio) greater than 0.6, was present in 26% of the women, and 9% had Na/K ratios larger than 1. The Na/K ratio was positively associated with NAGase, LDH, AcP, AP, and IL-8 in univariate linear regression (p < 0.001 for linear trend for all milk enzymes and IL-8). A positive association between the Na/K ratio and the breastmilk enzymes NAGase, LDH, AcP, and AP was found. Breastmilk enzymes have not previously been investigated in relation to SCM in women, and further research is warranted to highlight their specific role in the etiology of SCM.

Chemiluminescent determination of xanthine oxidase activity in milk.

A chemiluminescent method for determining xanthine oxidase (XOD) activity was developed and applied to the assay of milk enzyme activity using a photomultiplier luminometer. Various kinds of milk and cream samples were analysed for XOD content. In pasteurized milk, XOD activity depended on the fat content and in UHT milk it disappeared owing to the heat treatment. Milk sample preparation was very simple, requiring only homogenization at 40 degrees C followed by a 1:10 dilution with UHT ('XOD-free') milk. The assay was carried out at 25 degrees C. The response obtained from XOD standard solutions in milk was linear from 0.1 to 500 enzyme units (U) l-1, but for the actual milk samples values ranged only from 1 to 135 U l-1. The detection limit at 2 SD was 0.1 U l-1 in milk, while in buffer it was 100 times lower. The intra-assay and interassay CV for XOD activity in milk were 6-12%.

LIPOSOMAL ENCAPSULATION OF ?-GALACTOSIDASE: COMPARISON OF TWO METHODS OF ENCAPSULATION AND IN VITRO LACTOSE DIGESTIBILITY

β-Galactosidase from Aspergillus flavus was encapsulated in liposomes by dehydration-rehydration vesicle (DRV) and reverse-phase evaporation vesicle (REV) preparative methods involving different membrane compositions. Method of preparation influenced both encapsulation efficiency and rate of enzyme inactivation significantly (p < 0.05), apparently due to membrane composition differences in the resulting liposomes. Mean encapsulation efficiencies in DRV and REV liposomes were 18.3 and 27.6%, respectively. Corresponding values for enzyme inactivation were 13.5 and 28.0%. When suspended in milk at refrigeration temperature for 20 days, DRV liposomes hydrolyzed 65% of the laciose while REV liposomes hydrolyzed only 20% of the lactose, indicating better protection of lactose from β-galactosidase by REV liposomes in milk. Total enzyme activity in milk, however, was not influenced by the method used. Both DRV and REV liposomes digested up to 50% of lactose in milk under simulated gastrointestinal conditions. Results indicated that the stability of lactose in milk is better with REV liposomes than that with DRV liposomes.

Paracellular Leakage of Lipoprotein Lipase Across the Mammary Epithelium of the Goat

The ability of lipoprotein lipase to move across the mammary epithelium by a paracellular route was investigated. Five goats were milked hourly to activate the paracellular pathway. Three goats responded to hourly milking with a fivefold increase in milk lipoprotein lipase activity as compared with nonresponding goats. Massage of the mammary gland was necessary in the two nonresponding goats too cause increased lipoprotein lipase activity in milk. Oxytocin treatment during hourly milking also increased enzyme activity in milk from a nonresponding goat. Activation of the paracellular pathway by hourly milking increased milk sodium and protein and decreased potassium and lactose concentrations. After a 12-h milking interval, lipoprotein lipase activity was distributed primarily in the serum (48%) and cream (40%) fractions and, to a lesser extent, in the casein (12%) fraction. Hourly milking increased enzyme activity distributed in the serum fraction (62%), whereas enzyme activity associated with the cream (32%) and casein (6%) fractions decreased. Possible mechanisms for the origin of lipoprotein lipase in milk are discussed.

Thermal Denaturation of Whey Proteins in Skim Milk

High-pressure processing (HPP), microwaves (MW) and ultrasound (US) are used for pasteurization with minimum heat input. They also alter physico-chemical properties of milk proteins and enzymes. This article aims at identifying the important changes in milk proteins imparted by these three processing technologies. HPP dissociates casein micelles at low pH (<6.7) and concentrations (<4% w/w), while β-LG is the most pressure sensitive whey protein due to the presence of free thiol groups. Milk enzyme activity is inhibited at higher pressures (>400 MPa). MW treatment denatures whey proteins rapidly, even below their thermal denaturation temperatures. High-power MW treatment (e.g. 60 kW) deactivates enzymes by denaturing them. However, low-power controlled MW irradiation (e.g. 30 W) improves enzyme activity. Ultrasound can homogenize protein aggregates in dairy systems and cause whey protein denaturation. Sonication under applied pressure and heat (e.g. 3.5 kg/cm2, 126.5 °C) causes enzyme inhibition while mild sonication conditions can improve enzyme activity.HPP, MW and US are gaining popularity in the dairy industry due to their ability to pasteurize and functionalize dairy streams with minimal heat input. This review offers insights into how these technologies can be used in isolation or in combination to alter milk proteins and enzyme activity for different academic and industrial applications. However, to fully understand the potential of HPP, MW and US treatment on dairy systems, further research is required in several areas including health related nutritional changes in milk and milk products caused by these technologies.

Relationship of Human Milk pH During Course of Lactation to Concentrations of Citrate and Fatty Acids

Human milk pH was measured in 309 samples obtained from 52 women who had delivered at term and lactated for as long as 10 months thereafter. The mean pH decreased from 7.45 for colostrum to a nadir of 7.04 during the second week of lactation. Thereafter, the pH of milk remained between 7.0 and 7.1 until 3 months postpartum and then increased gradually to 7.4 by 10 months. The change in hydrogen ion concentration in milk was associated with corresponding changes throughout lactation in the concentration of citrate but not with the concentration of lactose. Lactose concentration increased gradually for 3 weeks; the concentration of saturated medium-chain fatty acids increased more rapidly. One interpretation of these findings is that the hydrogen ions and citrate generated by mammary secretory cell metabolism are used after the second week of lactation for de novo synthesis of fatty acids more rapidly than they are synthesized. Milk samples from ruminants were found to have concentrations of hydrogen ions and citrate that are greater than and pH that is less than the respective measurements in human milk. The significance for the recipient infant of the predictable changes in human milk pH during lactation and of the higher pH of human milk throughout lactation relative to bovine milk is unknown. However, drug excretion into milk, milk enzyme activity, milk leukocyte function, and neonatal gastrointestinal function are affected by ambient pH and may be influenced by the pH of milk.

N-Acetyl-B-D-Glucosaminidase Activity and Somatic Cells in Goat Milk

N-Acetyl-B-D-glucosaminidase activity, somatic cell count, and udder infection status were determined in milk of nine Saanen goats. Plasma enzyme activity was also measured. Individual half udder milk samples were taken for 12 d over a 3-wk period and animals were bled weekly.Three of the 18 udder halves were infected with coagulase-negative staphylococci over all 12 sampling d. The N-acetyl-B-D-glucosaminidase and somatic cells in milk were significantly elevated in samples where minor pathogens were isolated. Plasma enzyme was variable among goats but not within goats or across weeks. Greater daily variation was seen in somatic cell count as compared to milk enzyme activity. Correlation between milk N-acetyl-B-D-glucosaminidase and somatic cell count was .54.

Persistence of Bovine Milk Xanthine Oxidase Activity after Gastric Digestion In Vivo and In Vitro

Seventy rats were fasted for 20h and used in two experiments. In the first experiment fasted rats were intubated with 5ml marketed milk of known xanthine oxidase activity. After dosing, food was withheld, and rats were sacrificed at intervals. The stomach of each rat was excised and its contents assayed polarographically for xanthine oxidase activity. Activity decreased with time. At .5 and 1.5h the recovered activities were 62 and 45%, and in some rats activity persisted up to 8h. In the second experiment, fasted rats received 2ml marketed milk. Enzyme activity dropped faster than in the first experiment. Typical gastric transit times for ingested milk were 30 to 40min. Enzyme activity in milk began to decrease at pH 6.70; below pH 3.90 the enzyme was inactivated completely. Inactivation was partly reversible at pH 2.50 and above by adjusting the pH to 7.3. When simulated gastric juice and milk were combined in ratios of 3:1, 2:1, 1:1, 1:2, and 1:3, pH's were 1.85, 2.08, 3.57, 5.16, and 5.56, and respective activities were 0, 0, 0, 14.2, and 23.8%. In one in vitro experiment, when juice and milk (1:2) were intubated at 37C for 0, .5, 8, and 24h, their pH's were 5.20, 5.26, 5.13, and 5.15 while recovered activities were 19.2, 18.7, 14.9, and 9.9%. When juice and milk (1:2) were incubated for .5h and followed by pancreatin solution for 0, .5, 3.5, and 7.5h, the recovered activities were 13.5, 11.9, 7.2, and 6.1%. Considerable xanthine oxidase in milk is not inactivated in the gastrointestinal tract but is available for absorption.

Assaying Sterile Concentrated Milk for Native Proteolytic Enzymes

Summary Deproteinized filtrates of hemoglobin that have been acted upon by a protease have an optical density peak at 400 mμ due to hemin-containing split products. This technique was used to assay milk in borate buffer, pH 8.6, for native proteolytic enzymes. Proteolysis in raw milk was evident by increases in the optical density of filtrates after adding heated enzyme-free hemoglobin and incubating. Preheating at 94C for 4sec greatly retarded enzyme activity in milk and in its concentrate. Concentrated milk sterilized at 150C, its autoclaved control, and autoclaved hemoglobin revealed the same linear optical density curves at 280 and 400 mμ, indicating no measurable proteolytic activity during storage for 30 days. Optical densities of filtrates from three concentrated milks were essentially the same at 400 mμ after storage for 14 days; whereas, at 280 mμ they were significantly different due to nonenzymatic proteolysis. Incubating sterile concentrated milk at 65 and 80C for 63 hr increased nonprotein N which was heat-induced, but the milk was unaffected at 20 to 65C for 37 hr. Native milk proteases were of no significance in the gelation of concentrated milk sterilized by the high-temperature short-time method.