Elena Molina

Egg White Ovalbumin Digestion Mimicking Physiological Conditions

Gastrointestinal digestion of ovalbumin (OVA) was simulated using an in vitro system in two steps, which mimicked digestion in the stomach and duodenum, to assess the effect of different gastric pHs, different concentrations of proteases, and the presence of surfactants, such as phosphatidylcholine (PC) and bile salts (BS). OVA was very resistant to pepsin action at an enzyme/substrate ratio that would resemble a physiological situation (1:20 w/w, 172 units/mg) at pH values equal or above 2. The presence of PC did not change the susceptibility of OVA to proteolysis with pepsin. Fluorescence experiments showed that OVA interacted with PC vesicles, particularly at acidic pH, but it is likely that the protein maintained a high degree of conformational stability, resisting pepsin action. The presence of BS at physiological concentrations considerably increased the proteolysis of OVA by a mixture of pancreatic enzymes. The addition of PC made OVA even more sensitive to proteolytic degradation, suggesting that OVA could associate with the surfactants under duodenal conditions, increasing its exposure to pancreatic proteinases. Immunoreactivity against IgE from sera of allergic patients was retained after in vitro gastric digestion, depending on the reactivity of the sera, but it decreased considerably after in vitro duodenal digestion.

Human immunoglobulin e (IgE) binding to heated and glycated ovalbumin and ovomucoid before and after in vitro digestion

This study focuses on the effect of heating and Maillard reaction (MR) on the in vitro digestibility and rabbit IgG- and human IgE-binding properties of ovalbumin (OVA) and ovomucoid (OM) to estimate the impact of processing on their allergenicity. With the human sera studied, heat treatment significantly reduced IgE binding to both OVA and OM, whereas MR reduced the IgE binding to OVA but increased IgE binding to OM. In contrast, heat treatment significantly favored OVA digestibility but glycation impaired it, and these treatments did not affect the digestibility of OM. The changes observed in the digestibility affected the immunogenicity of the digests accordingly, so that the higher the digestibility, the lower the antibody binding. Heat treatment and glycation by MR showed an influence on the potential allergenicity of the main egg white proteins that could be related to their resistance to denaturation and digestive enzymes.

Isolation of Mycobacterium avium subsp. paratuberculosis from muscle tissue of naturally infected cattle

Johnes disease or paratuberculosis is a chronic granulomatous inflammation of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Recent studies suggest an association between MAP and Crohns disease in humans. MAP can become widely distributed within the tissues of infected animals, and meat may be a possible route of exposure of MAP to humans. In this study, 47 dairy and beef cattle were examined for the occurrence of viable MAP in diaphragm muscle. At the slaughterhouse, gut tissues, diaphragm muscle, blood, and feces of the 47 animals were collected for bacteriological culture, as well as gut samples for histopathological analysis. MAP was detected by bacteriological culture and conventional and real-time IS900 polymerase chain reaction in the diaphragm muscle of six infected cattle at slaughter (13%). The six animals showing evidence of MAP in diaphragm muscle had diffuse lesions and severe granulomatous inflammation in ileocecal lymph nodes, jejunal lymph nodes, ileocecal valve, and ileum. All six had heavy bacterial load in mesenteric lymph nodes, ileocecal valve, ileum, and jejunum, and four showed clinical signs of paratuberculosis. Two animals did not show clinical signs but had viable MAP in intestinal tissues and in diaphragm muscle as well. MAP was found in blood of only one of the six animals showing evidence of MAP in diaphragm muscle and in feces of three of them. In general, there was a positive association between enteric lesion severity, clinical signs of paratuberculosis, heavy bacterial load in intestinal tissues, fecal shedding of MAP, and the presence of disseminated MAP infection in diaphragm muscle. The results of this study demonstrated that MAP can be detected and cultured from muscle of MAP-infected cattle destined for human consumption and suggest a possible risk of exposure of humans to MAP via contaminated meat.

Capillary electrophoresis characterization of the casein fraction of cheeses made from cows', ewes' and goats' milks.

Casein fractions and their breakdown products in Iberico-type cheeses made from the milk of cows, ewes or goats were analysed by capillary electrophoresis in order to characterize them. The actions of plasmin and chymosin on caseins were evaluated by comparing the electropherograms of caseins from milk and from cheese, both with and without treatment with plasmin. Characteristic capillary electrophoresis patterns were obtained for cheeses made from the milk of each of the three species, and the main components were identified. Caprine para-kappa-casein and bovine beta-caseins, eluting at the first and at the last part of the electropherogram respectively, were found to be indicative of the presence of the milks of these species.

Effect of processing technologies on the allergenicity of food products

Heat treatment has been used since ancient times for food processing, first to ensure the safety of food and its storage, but also to transform its characteristics (in its raw form) and obtain new textures, flavors, or novel foods. However, the transformation experienced by food components when heated, or processed, can dramatically affect the allergenicity of food, either reducing or increasing it. To date, most of the articles published dealing with the changes in the potential allergenicity of food are focused on heat treatment and the Maillard reaction. However, it is also important to give prominence to other group of new technologies developed nowadays, such as high-pressure processing, microwaves and food irradiation. These techniques are not likely to replace traditional processing methods, but they are becoming attractive for the food industry due to different reasons, and it is expected in the near future to have different products on the market processed with these new technologies at an affordable cost. Moreover, other biochemical modifications, particularly enzymatic cross-linking of proteins, have attracted wide-spread attention and will be considered as well in this review, because of its great opportunities to induce protein modification and thus affect food allergenicity. Together with the effect of processing of food allergens, this review will place special attention on gastroduodenal digestion of processed allergens, which directly affects their allergenicity.

Significant reduction in bacterial shedding and improvement in milk production in dairy farms after the use of a new inactivated paratuberculosis vaccine in a field trial

BackgroundParatuberculosis vaccination has been in use in some regions for many decades, but results have not been widely spread. A new Mycobacterium avium subsp. paratuberculosis (MAP) killed vaccine was studied in relationship with its effects on fecal shedding and milk production in four farms while other two were kept as controls submitted to a test and cull scheme.FindingsFecal detection (n = 1829) and milking records (n = 2413) have been analyzed after two (5 herds) and four (1 herd) years of the beginning of the intervention. Shedder prevalence was reduced by 100% in three of the four vaccinated farms, 68% in the total of vaccinated animals and 46% in the two control farms. Total amount of MAP shed was reduced 77% in the vaccinated farms and 94% in the control farms. Overall milk production increased up to 3.9% after vaccination, while there was no significant difference in production after intervention in the non-vaccinated farms.ConclusionMAP shedding reduction can be quickly accomplished both by vaccination and by testing and culling. However, vaccination appears to be a less expensive and more sustainable strategy since it required one single intervention and was also associated with an increase in milk production.

In vitro digestibility of bovine β-casein with simulated and human oral and gastrointestinal fluids. Identification and IgE-reactivity of the resultant peptides

Stability during digestion is considered an important feature in determining the allergenicity of food proteins. This study aimed to provide an immunological characterisation of the digestion products of the major cows milk allergen β-casein (β-CN) produced by in vitro orogastrointestinal hydrolysis with simulated and human digestive fluids. β-CN was unaffected by oral digestion, but quickly broke down during the early stages of gastric digestion. The degradation with human fluids was faster than that with commercial enzymes. There were similarities in the peptide patterns of the hydrolysates produced in both models, showing 20 peptides in common after gastric digestion. After gastroduodenal digestion, the human fluids gave less numerous and shorter peptides. The IgE binding of most of the individual sera used to the hydrolysates produced with simulated and human fluids increased at the end of the gastric phase and decreased when the duodenal digestion was completed. Two IgE-binding synthetic peptides: β-CN (57-68) and β-CN (82-93), which matched fragments released by β-CN following in vitro digestion with simulated and human fluids, consisted of the most immunoreactive areas of the protein. The similarities found between the in vitro simulated digestion system and that using human digestive fluids suggest that the former would provide a reasonably good estimation of the potential allergenicity of protein digests.

Immunoreactivity of hen egg allergens: Influence on in vitro gastrointestinal digestion of the presence of other egg white proteins and of egg yolk

Hen egg white comprises of a complex mixture of proteins, which greatly differ in their physicochemical characteristics and relative abundance. We aimed to identify potential undiscovered egg allergens within the egg white proteome and investigated the existence of matrix effects on the proteolytic stability and resultant IgE-binding of the allergenic proteins. In addition to the main egg allergens: ovalbumin (OVA), ovomucoid (OM) and lysozyme (LYS), two minor egg white proteins, tentatively identified as ovoinhibitor and clusterin, were found to react with serum IgE from egg-allergic patients. Egg white exhibited residual immunoreactivity after gastrointestinal digestion due to the presence of intact OVA and LYS, as well as of several IgE-binding peptides derived from OVA. The presence of egg yolk slightly increased the susceptibility to hydrolysis of egg white proteins and abrogated bile salt-induced precipitation of LYS in the duodenal medium. However, the resultant immunoreactivity against IgE of egg white proteins after in vitro digestion was not significantly modified by the presence of yolk components.

Development and Evaluation of a Novel Multicopy-Element-Targeting Triplex PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Feces

ABSTRACT The enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused by Mycobacterium avium subsp. paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detect M. avium subsp. paratuberculosis in feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02 (an insertion sequence of M. avium subsp. paratuberculosis present in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10 M. avium subsp. paratuberculosis cells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900 and ISMap02 PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection of M. avium subsp. paratuberculosis in fecal samples from cattle and a very valuable tool to be used in PTB control programs.

Immunization of adult dairy cattle with a new heat-killed vaccine is associated with longer productive life prior to cows being sent to slaughter with suspected paratuberculosis

The objective of this study was to evaluate the efficacy of a new heat-killed Mycobacterium avium ssp. paratuberculosis (MAP) vaccine for control of premature culling in tuberculosis-free dairy cattle. Feces and gastrointestinal tissues were collected from 50 vaccinated cows and 38 nonvaccinated cows at slaughter and analyzed by bacteriological culture and histopathology. Vaccination was associated with a significant reduction of the frequency of vaccinated animals with MAP in feces and gut tissues compared with the nonvaccinated animals. In addition, the frequency of vaccinated animals with heavy bacterial load in gut tissues was 40% lower than the frequency of the nonvaccinated animals with the same MAP load. The peak age of paratuberculosis-associated culling was from 4.5 to 5 yr old (21%) in the vaccinated animals and from 3 to 4.5 yr old (60%) in the nonvaccinated animals. The vaccinated and nonvaccinated animals with suspected paratuberculosis were culled at an average age of 4.7 and 3.7 yr old, respectively. Therefore, a significant increase in the productive life of the vaccinated animals sent to slaughter with suspected paratuberculosis was observed. In addition, our analysis revealed a positive effect of the vaccine on the carcass weights of the animals with severe histopathological lesions at slaughter compared with the nonvaccinated animals. In summary, our findings suggest a therapeutic effect of the vaccine and a significant attenuation of pre-existing infection in cows naturally infected with paratuberculosis that were adults at the time of vaccination.