Materials and Methods
Before lactoperoxidase activity determination, each milk sample was skimmed by centrifugation at 3000g for 30 min at 15°C.
The milk samples analyzed were:
T0 Control (C)
T27 Microencapsulated (M)
T27 Control (C)
T29 Microencapsulated (M)
T29 Control (C)
T29 Microencapsulated (M)
T35 Control (C)
T35 Microencapsulated (M)
Lactoperoxidase activity was evaluated on each skimmed milk samples by a continuous spectrophotometric rate determination using as substrate 2,2’-Azinobis (3-Ethylbenzthiazoline-6-Sulfonic Acid) (ABTS, Sigma Chemical Co) according to Pruitt and Kamau .
The method is based on the reaction: H2O2 + reduced ABTS → 2H2O + oxidized ABTS. The final reaction mix contained: 100 mM potassium phosphate pH5.5; 23 mM ABST; 0.0008% (w/w) hydrogen peroxide; the reaction was started by the addition of 10μl of skimmed milk.
Absorbance measurements were made at 3 and 5 min, using a 436 nm wavelength. One unit of lactoperoxidase is defined as the amount of the enzyme that oxidizes 1.0 μmole of ABST per minute at pH 5.5 and 25°C. The calculation of the enzymatic activity was made as follows:
Units/ml enzyme = (ΔA436nm/min test- ΔA436nm/min blank) x (total mL of assay)/(29.3) x (mL of enzyme), were A436nm/min blank is the absorbance of the potassium phosphate solution with the substrate and the hydrogen peroxide but in absence of milk; 29.3 is the millimolar extinction coefficient of oxidized ABTS at 436nm.
Data were analyzed by the method of least squares using the general linear model procedures of SAS [SAS. Statistical analysis system (2001). SAS/STAT guide for personal computers. Version 8
Edition, Cary, NC.]. Significant differences between means were indicated when P < 0.05.
The lactoperoxidase enzymatic activity were calculated on the milk sample T0 (C and T) T27 (C and T), T29 (C and T) T35 (C and T), and the results are showed in table 1. In table 2 are the results on the statistical analysis.
Table 1. Lactoperoxidase enzymatic activity on milk samples, raw data
Table 2. Statistical analysis
|Group C (control)||Group M (Microencapsulated)|
Different letters on the same row show statistical differences (C: P<0.001)
From the results it is evident that after 35 days of treatment with the microencapsulated there is a significant increase on the lactoperoxidase activity (P<0.001). Lactoperoxidase member of the peroxidase-cyclooxygenase superfamily and one of the most abundant enzymes in bovine milk, constituting about 1% of whey proteins. Some structural similarities between bovine LPO, cytochrome c peroxidase, and horseradish peroxidase have been demonstrated, using nuclear magnetic resonance. Lactoperoxidase is an antioxidant enzyme, is a natural antimicrobial system that eliminates the harmful effects of microorganisms in milk. It has a wide range of applications and is also preferred in cosmetic and clinical applications, as well as used in foods. The use of antioxidants is well recognized in the food and feed industries to improve the shelf life of products.
Lactoperoxidase can increase the shelf life of raw milk. Inactivation of lactoperoxidase occurs at 80 °C in 15 sec whereas residual lactoperoxidase activity is detected following treatment at 72 °C. At this purpose, is used as an index of pasteurization efficiency in milk.
 Pruitt KM, Kamau DN (1994) Quantitative analysis of bovine lactoperoxidase
system components and of the effects of the activated system on bacterial
growth and survival. In: Indigenous Antimicrobial Agents of Milk. International
Dairy Federation Brussels, Belgium, 73-87.