Effect of in vitro gastrointestinal digestion on the antibacterial activity of bioactive dairy formulas supplemented with lactoferrin against Cronobacter sakazakii

Milk is a source of proteins with high nutritional value and relevant biological activities. Bioactive milk proteins, like lactoferrin, are important for newborn development and can also be used as ingredients in functional products to improve health. Lactoferrin is essential in infant’s diet, since protects against infections and promotes immune system maturation. Bovine lactoferrin is used to supplement formula milk in order to strengthen baby’s defences against some pathogenic bacteria. Thus, lactoferrin supplemented formula can be a barrier against emergent pathogens, such as Cronobacter sakazakii, which has caused great concern in the last few years. Milk proteins generate bioactive peptides in the digestion process, and it is known that industrial processing can modify their susceptibility to digestion. Treatments such as heating have been shown to denature whey proteins and make them more easily digestible. Therefore, the aim of this study was to analyze the effect of technological treatments and gastrointestinal digestion on the antibacterial activity against C. sakazakii of proteins present in dairy formulas supplemented with lactoferrin. Commercial bovine lactoferrin has been shown to have antibacterial activity against C. sakazakii, both in the native state and after static in vitro gastrointestinal digestion. In addition, the digests obtained from dairy formulas subjected to technological treatments, either homogenization or pasteurization, have higher antibacterial activity than non-treated formulas. The release of low molecular weight peptides during the in vitro gastric digestion is probably the cause that would explain the enhanced antibacterial activity of the digested dairy formulas

Fracciones bioactivas del lactosuero: aislamiento y evaluación de la actividad antirrotavirus in vitro.

El rotavirus pertenece a la familia Reoviridae y causa diarreas severas y moderadas en niños menores de 5 años, produciendo muchas muertes al año en todas partes del mundo. El lactosuero obtenido durante la elaboración del queso es un subproducto de la industria alimentaria que contiene proteínas con interés nutritivo y biológico. De su separación por métodos de ultrafiltración se pueden obtener fracciones con actividad biológica. Algunas proteínas y péptidos presentes en el lactosuero bovino, como el glicomacropéptido (GMP) y el componente 3 de la proteosa peptona (PP3), exhiben actividad frente a bacterias patógenas, pero existe poca información sobre su actividad frente a virus. El objetivo de este Trabajo de Fin de Máster ha sido el aislamiento y caracterización de proteínas y péptidos derivados del lactosuero bovino por medio de métodos cromatográficos y el fraccionamiento del lactosuero por métodos de ultrafiltración. Se ha evaluado la capacidad del GMP, sus hidrolizados, un producto comercial enriquecido en GMP, la PP3 y fracciones del lactosuero para neutralizar la infección causada por la cepa de rotavirus bovino WC3 utilizando la línea celular MA104. El GMP, la PP3, la fracción del lactosuero con un peso molecular superior a 10 kDa y la fracción con un peso molecular inferior a 3 kDa exhiben actividad neutralizante frente a los rotavirus. El grado de neutralización se incrementa cuando se aumenta la concentración de proteína. No se observa neutralización para la fracción de 3-10 kDa, ni para el producto comercial enriquecido en GMP ni para sus hidrolizados.<br /

Antirotaviral activity of dairy byproducts enriched in fractions from hyperimmune bovine colostrum: the effect of thermal and high hydrostatic pressure treatments

Nowadays, rotaviruses remain a major health burden, especially in developing countries, and strategies complementary to vaccination are needed. In this view, dairy fractions have attracted great scientific interest, due to their high content of bioactive compounds. The objective of this study was to evaluate the antiviral activity of whey and buttermilk enriched in proteins from hyperimmune bovine colostrum (HBC) against rotavirus. The enriched fractions were spray-dried and subsequently tested for their neutralizing activity against the bovine rotavirus WC3 strain in vitro, using differentiated Caco-2/TC7 cells. The highest antirotaviral activity was observed when whey and buttermilk were enriched in purified immunoglobulin G (IgG), showing complete rotavirus neutralization at concentrations of 3 and 6 mg mL−1 for whey and buttermilk, respectively. Additionally, the use of a crude immunoglobulin fraction also gave satisfactory results. The inhibitory activities of all samples significantly decreased after the application of heat, except for the IgG-enriched buttermilk which showed a slight increase of activity following the application of short-time treatments (75 or 85 °C for 20 s). This sample also showed a significant increase of activity (13%) after the application of low-intensity high hydrostatic pressure treatment (400 MPa for 5 min). The maximum loss of bioactivity was observed at 600 MPa for 10 min (31 and 20% for whey- and buttermilk-based formulas, respectively). This study provides relevant information on the potential of whey, buttermilk, and HBC to be part of functional products as complementary strategies to combat rotavirus infections

Dairy by-products and Lactoferrin exert antioxidant and antigenotoxic activity on intestinal and hepatic cells

The dairy industry generates a large volume of by-products containing bioactive compounds that may have added value. The aim of this study was to evaluate the antioxidant and antigenotoxic effects of milk-derived products, such as whey, buttermilk, and lactoferrin, in two human cell lines: Caco-2 as an intestinal barrier model and HepG2 as a hepatic cell line. First, the protective effect of dairy samples against the oxidative stress caused by menadione was analyzed. All these dairy fractions significantly reversed the oxidative stress, with the non-washed buttermilk fraction presenting the greatest antioxidant effect for Caco-2 cells and lactoferrin as the best antioxidant for HepG2 cells. At concentrations that did not impact cell viability, we found that the dairy sample with the highest antigenotoxic power against menadione, in both cell lines, was lactoferrin at the lowest concentration. Additionally, dairy by-products maintained their activity in a coculture of Caco-2 and HepG2, mimicking the intestinal-liver axis. This result suggests that the compounds responsible for the antioxidant activity could cross the Caco-2 barrier and reach HepG2 cells on the basal side, exerting their function on them. In conclusion, our results show that dairy by-products have antioxidant and antigenotoxic activities, which would allow revaluing their use in food specialties

The transmittable through stinging microbiota differs between honeybees and wasps: a potentially greater microbial risk of the wasp sting for humans

The present research investigated whether accidental contact through stinging with honeybees, wasps, and hornets could represent a microbial hazard for humans. It has been previously suggested that such contact may transmit pathogens causing infections that could even be fatal for some susceptible individuals. Stinging simulation experiments were performed in the lab with live insects collected from the environment in Lemnos Island (north-eastern Greece), while different selective agar media targeting some clinically important bacteria (i.e., Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis/faecium, and Pseudomonas aeruginosa) were used as substrates for microbial recovery and identification. Results revealed none of the target pathogenic bacterial species in the honeybee samples, with bacilli, staphylococci, and micrococci dominating their surveyed microbiota. However, most of the suspect colonies isolated from wasps and hornets belonged to important hygienic indicators (i.e., enterococci, Proteus mirabilis, and coliforms), implying possible contact of these insects with fecal origin materials. To sum up, the microbiota that may be transmitted to humans through stinging appears to differ between honeybees and wasps/hornets, while the isolation from the latter samples of some other important opportunistic pathogens, such as Enterobacter spp. and Klebsiella spp., also known for multidrug resistance, could be an additional reason of concern

Comparison of qualitative characteristics of propolis extracts using different purification methods

Purification of extracts is an essential procedure in order to remove wax and other undesirable ingredients while retaining the polyphenolic fraction which contains most of bioactive components. This study investigates how different purification methods affect the qualitative characteristics of propolis extracts. Five different purification methods were conducted both for ethanolic and methanolic extracts: (a) centrifugation for 30 min at 370 g; (b) centrifugation at −5 °C for 10 min at 1850 g twice with a 15 min interval; (c) centrifugation at 3340 g for 2.5 min twice with a 15 min interval; (d) filtration through a 0.22 μm polyethersulfone membrane; (e) filtration through a 0.45 μm pore size nylon membrane filter using Buchner vacuum system. Methods were evaluated with reference to the post-treatment antioxidant activity (radical scavenging), total phenolic content and total flavonoid content of each extract. Two different methods of centrifugation were found to be the most effective and stable: centrifugation at 3340 g for 2.5 min or for 10 min at 1850 g, at −5 °C

Evaluation of in vitro antirotaviral activity of lactoferrin from different species using a human intestinal model

Rotaviruses are the leading agents of severe diarrheal disease in infants and young children and complementary strategies to vaccination are needed. Therefore, we evaluated the activity of lactoferrin (LF) from different species against rotavirus in differentiated Caco-2/TC7 cells. The mechanism of rotavirus inhibition and the influence of iron saturation and sialic acid content were also investigated. Furthermore, the effect of bovine LF on Toll-like receptor expression (TLRs) during rotavirus infection was studied. LF presented high inhibitory activity against rotavirus above 8 mg mL−1, the bovine protein being the most potent. The antirotaviral mechanism of LF was mainly related to the virus neutralisation at an early step of infection. Interestingly, a direct relationship between rotavirus neutralisation activity and content of sialic acid in LFs was observed. Finally, the neutralisation of rotavirus by LF leaded to a decreased expression of TLRs in Caco-2/TC7 cells, with a more evident effect on TLR7

Bioactivity of fucoidan-rich extracts from fucus vesiculosus against rotavirus and foodborne pathogens

Marine algae are sources of bioactive components with defensive properties of great value against microbial infections. This study investigated the bioactivity of extracts from brown algae Fucus vesiculosus against rotavirus, the worldwide leading cause of acute gastroenteritis in infants and young children. Moreover, one of the extracts was tested against four foodborne bacteria: Campylobacter jejuni, Escherichia coli, Salmonella Typhimurium, and Listeria monocytogenes, and the non-pathogenic: E. coli K12. In vitro tests using MA104 cells revealed that both whole algae extracts and crude fucoidan precipitates neutralized rotavirus in a dose-responsive manner. The maximum neutralization activity was observed when the rotavirus was incubated with 100 μg mL−1 of the hydrochloric acid-obtained crude fucoidan (91.8%), although crude fucoidan extracted using citric acid also demonstrated high values (89.5%) at the same concentration. Furthermore, molecular weight fractionation of extracts decreased their antirotaviral activity and high molecular weight fractions exhibited higher activity compared to those of lower molecular weight. A seaweed extract with high antirotaviral activity was also found to inhibit the growth of C. jejuni, S. Typhimurium, and L. monocytogenes at a concentration of 0.2 mg mL−1. Overall, this study expands the current knowledge regarding the antimicrobial mechanisms of action of extracts from F. vesiculosus