Use of microbial beta-lactamase to destroy penicillin added to milk.

A simple method is described for destruction of penicillin residues in bulk milk to an undetectable amount (less than .003 U/ml) with commercially available crude beta-lactamase enzyme. Milk containing .1 or .5 U/ml penicillin G was treated with .01 to 1.0 mU/ml of beta-lactamase (Bacillus cereus) for up to 96 h. The Bacillus stearothermophilus var. calidolactis assay was used to quantify penicillin in milk between .003 to 1.0 U/ml. The .5 U/ml of penicillin G was reduced to an undetectable amount within 18 h at 4 degrees C by 1.0 mU/ml of beta-lactamase. The development of titratable acidity over 5 to 6 h in contaminated milks treated with beta-lactamase and inoculated with Streptococcus thermophilus GH, Streptococcus cremoris, Streptococcus lactis, or a commercial starter culture was the same as for control milk samples containing no additives or only enzyme. Pilot-scale manufacture of Swiss and Cheddar cheeses from contaminated milks treated with beta-lactamase yielded cheeses of comparable quality, to control cheeses produced from penicillin-free milk. There were no delays in acid production as judged from pH measurements during production and ripening of the cheeses. About 50% of beta-lactamase activity added to milk remained after pasteurization at 63 degrees C for 30 min. The safety for human consumption of cheese containing small quantities of penicillin degradation products from milk treated with beta-lactamase remains to be established.

Photodegradation of DNA with fluorescent light in the presence of riboflavin, and photoprotection by flavin triplet-state quenchers.

Superoxide anion was photogenerated upon illumination of nucleic acids with fluorescent light in a solution containing phosphate buffer, pH 7.8 and riboflavin. DNA was a better reducing substrate for this reaction than was RNA. A similar riboflavin-sensitized photoreaction caused single- and double-strand scissions of supercoiled PM2 DNA as detected by electrophoresis in agarose gels. None of specific scavengers or quenchers for superoxide anion and other active oxygen species prevented the DNA strand breaks. However, among the flavin triplet-state quenchers, potassium iodide, butylated hydroxyanisole, and ferricytochrome c protected the supercoiled DNA from photodegradation; butylated hydroxytoluene, alpha-tocopherol, tyrosine and hemoglobin did not have any protective effect. These results indicate that triplet-state riboflavin or a derivative formed from it participate directly in the observed riboflavin-sensitized DNA photodegradation and that active oxygen species are not directly involved.

Initiation of oxidative changes in foods.

Initiation of lipid peroxidation in foods may be accomplished by a variety of mechanisms. Two principal initiation reactions involve homolytic scission of preformed peroxides as catalyzed by metal ions and heme proteins and the reaction of activated oxygen species with the lipid substrate to yield peroxides and free radicals. Copper and cytochromes in the milk fat globule membrane may serve as focal points for initiation of lipid peroxidation by catalyzing homolytic scission of peroxides. Activated oxygen species which may be important in initiating oxidative changes in foods include singlet oxygen, hydroxyl radical, ozone, superoxide anion (perhydroxyl radical at low pH), and hydrogen peroxide. Chemical and enzymic reactions in biological materials can generate singlet oxygen, hydroxyl radical, superoxide anion, and hydrogen peroxide. Ozone is primarily a product of photoreactions in polluted air. Reactions involving singlet oxygen, hydroxyl radical, and ozone with food constituents ultimately can yield peroxides which decompose to initiate oxidative chain reactions. Superoxide anion and hydrogen peroxide are relatively inert toward organic molecules but can decompose to produce the more reactive singlet oxygen and hydroxyl radical. Inhibition of reactions initiated by reactive oxygen species in foods should be very important in preserving the oxidative stability of foods. This paper presents a brief review of possible initiation reactions for lipid peroxidation and inhibition of reactions of activated oxygen species that are of importance in food systems.

Absence of fatty livers in rhesus monkeys fed orotic acid.

Pairs of rhesus monkeys were fed for 10 wk a basal diet containing 1% orotic acid or 10% nonfat milk powders. Amounts of total lipids in the liver and hepatic morphology were normal after 10 wk indicating that orotic acid in the diet did not induce fatty livers in rhesus monkeys.

Superoxide Dismutase Activity in Bovine Milk Serum.

Superoxide dismutase activity was shown to be present in bovine milk serum and was quantified by measuring the capacity of retentate from dialyzed milk serum to inhibit reduction of cytochrome c by xanthine-xanthine oxidase-generated superoxide anion. One unit of enzyme was defined as the quantity of superoxide dismutase which inhibits cytochrome c reduction by 20%. By this definition 19,500 units of enzyme were present per liter of retentate from dialyzed milk serum. This amount is equivalent to about 2.4 mg of purified bovine erythrocyte superoxide dismutase per liter. Polyacrylamide gel electrophoresis of a partially-purified superoxide dismutase from acid whey, followed by staining for enzymic activity, confirmed the presence of the enzyme in milk serum which was identical in electrophoretic properties to those of bovine erythrocyte copper-zinc superoxide dismutase. Pasteurization at 63 C for 30 min did not decrease superoxide dismutase activity in milk serum. Heating of purified bovine erythrocyte-superoxide dismutase at 100 C for 1 min resulted in almost complete loss of enzymic activity, whereas the partially-purified superoxide dismutase from acid whey still retained 40% of the original activity under these conditions. Bovine milk superoxide dismutase may be an important naturally-occurring antioxidant for increasing oxidative stability of milk and other dairy products.

Activated oxygen species and oxidation of food constituents.

Activated oxygen species which may be important in initiating oxidative changes in foods include singlet oxygen, hydroxyl radical, ozone, superoxide anion (perhydroxyl radical at low pH), and hydrogen peroxide. Chemical and enzymic reactions known to occur in biological materials can generate singlet oxygen, hydroxyl radical, superoxide anion, and hydrogen peroxide. Ozone is primarily a product of photoreactions in polluted air. Reactions involving singlet oxygen, hydroxyl radical, and ozone with food constituents can ultimately yield peroxides which decompose to initiate oxidative chain reactions. Superoxide anion and hydrogen peroxide are relatively inert toward organic molecules but can decompose to produce the more reactive singlet oxygen and hydroxyl radical. Inhibition of reactions initiated by reactive oxygen species in foods should be very important in preserving the oxidative stability of foods. The generation, detection, measurement, reaction, and inhibition of reactions of active oxygen species are surveyed in this review.