Rotaviral gastroenteritis is associated with significant morbidity in developed countries and a high rate of infant mortality in developing countries. Diverse studies have demonstrated that a wide range of milk-derived fractions exhibit antirotaviral activity. The present study shows the antirotaviral activity of some bovine and ovine dairy byproducts, buttermilk, butter serum, and milk fat globule membrane (MFGM), and evaluates the effect of cream washing and heat treatment on that activity. Furthermore, the rotavirus-neutralizing activity was evaluated for some MFGM proteins, such as xanthine oxidase and lactophorin. Ovine and bovine buttermilk reached rotavirus-neutralizing values of 51.3 and 32.2%, at 1 mg/mL, respectively. The cream washing process led to a significant decrease in the antirotaviral activity of fractions. This activity was also influenced by heat treatment. Treatment at 75 °C for 20 s caused 24.6 and 36.1% decreases of activity in bovine and ovine buttermilk, respectively, and 85 °C for 10 min caused decreases of 80.9 and 79.0% in both fractions, respectively.
Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value.
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