Using an ultrafiltration membrane (molecular cut-off, 3,000), low molecular weight compounds in bovine milk were collected (YM-3 filtrate). A hydrogen peroxide (H2O2)-like substance was generated in the YM-3 filtrate. This substance was undetected at 0 h, but increased in a time-dependent manner, peaking after 2 h of incubation at 38 degrees C. After incubating the YM-3 filtrate with catalase and lactoperoxidase, the signal showing the presence of this substance disappeared. The substance was quantified using one chemiluminescence and three colorimetric H2O2 detection systems. In all systems, their estimates were within the same range. The amount of substance, as estimated by the chemiluminescence H2O2 detection system, was correlated with that estimated by the other three colorimetric systems (r=0.98, 0.95 and 0.87). The substance was eluted at the same position as H2O2 by gel filtration on Superdex 30. Thus, the substance had the same characteristics as H2O2. An H2O2-generating substance in either the YM-3 filtrate or whey had a molecular mass of about 600. In this study, we clarify that bovine milk is capable of generating H2O2 by utilizing a low molecular weight compound. Thus, we present a new type of H2O2-supplying system in bovine milk.

Mastitis is the most frequent and prevalent production disease in dairy herds in developed countries. Based on a milk somatic cell count (SCC) of either >300,000 or <200,000 cells/ml in this study, we defined the quarter as either inflamed or uninflamed, respectively. The electrical conductivity (EC) of milk was used as an indicator of udder epithelial cell damage. We determined the amount of H2O2 produced by utilizing a small molecular weight compound in milk, and examined the characteristics of H2O2 production and EC in milk from inflamed and uninflamed quarters. In cows with milk of delivery grade (control population), H2O2 production and EC were 3.6+/-1.3 nmol/ml and 5.4+/-0.4 mS/cm (mean+/-sd), respectively. In 37 inflamed quarter milk samples, the production of H2O2 was 1.9+/-1.0 nmol/ml and was significantly smaller than that in the control population (P<0.01). Production of H2O2 was moderately but significantly correlated with EC (r<-0.71). In 20 cows with inflamed quarters, the production of H2O2 in milk from inflamed quarters was significantly smaller than that in milk from uninflamed quarters (P<0.01). In 18 out of 20 cows, milk from inflamed quarters showed the smallest H2O2 production among all tested quarters in each cow. We conclude that inflammation caused a decrease in H2O2 production in milk. In this study, we present parameters for evaluating the lactoperoxidase/H2O2/thiocyanate antibacterial defence system in bovine milk.

In dairy cows, hydrogen peroxide (H₂O₂) produced from a low-molecular-weight compound in milk from inflamed quarters was lower than that in milk from un-inflamed quarters. In milk of delivery grade, characteristics of H₂O₂ production in milk with high electrical conductivity (EC) were examined in this study. Milk samples were collected from a total of 230 cows at 1-month intervals, and the EC of skimmed milk was determined. Based on the highest and the lowest EC of a cow's quarter milk, the inter-quarter difference of ≥0.6 mS/cm (mean + t₀.₀₁ SE) was taken as a high EC. Milk with high EC was found in 52 quarters. In cows with milk of high EC, H₂O₂ production in milk with normal EC was higher than that in milk with high EC in the same animal but was lower than that in the control population. In milk with high EC, the decrease of H₂O₂ production correlated with the increase in EC. The production of H₂O₂ decreased in particular when the inter-quarter difference exceeded 0.8 mS/cm. In milk collected from the same quarter 1 month before, EC changed from normal to high, and H₂O₂ production decreased. In milk from the other three quarters, EC remained normal and H₂O₂ production remained unchanged. We concluded that milk with high EC appeared in low H₂O₂ -producing cows. The results suggest that the degree of decrease in H₂O₂ production reflects the extent of quarter abnormality.