Detection and Quantification of Milk Ingredients as Hidden Allergens in Meat Products by a Novel Specific Real-Time PCR Method

Abstract

Milk ingredients are often included in a wide range of meat products, such as cooked hams and sausages, to improve technological characteristics. However, milk proteins are also important food allergens. The aim of this study was the development of a highly sensitive and specific real-time PCR system targeting the 12S rRNA gene of Bos domesticus for the detection and quantification of milk as an allergenic ingredient in processed meat products. The method was able to achieve an absolute limit of detection (LOD) of 6 fg of milk DNA. Using a normalized approach (∆Ct method) for the detection of milk protein concentrate (MPC), it was possible to obtain sensitivities down to 0.01% (w/w) of MPC in model hams (raw and cooked) and autoclaved sausages, and 0.005% in raw sausage mixtures. The developed systems generally presented acceptable PCR performance parameters, being successfully validated with blind samples, applied to commercial samples, and further compared with an immunochemical assay. Trace amounts of milk material were quantified in two out of 13 samples, but the results mostly infer the excessive practice of the precautionary labeling.

Development and verification of a quantitative real‐time PCR method to identify and quantify gelatin derived from animal hide

“…This sensitivity level is much higher than other reported values, such as 0.05 ng for bovine DNA (Xiao, Qin, Wenju, & Qin, 2016). For practical sensitivity, we observed a sensitivity level of 5% to 0.1% w/w, which is lower than the reported levels for other food products, such as 0.01% w/w for hams and sausages (Villa, Costa, & Mafra, 2019). The intensive processing conditions explained the compromised practical sensitivity as far as gelatin products were concerned.…”

“…Gelatin is processed under more intense conditions than meat products, rendering quantification more difficult. Compared to the reported relative error for meat products, which stands at ± 25% for a 4% mixture (Villa et al., 2019), the relative error of 26% for 5% gelatin mixture was slightly high, but for the purpose of semiquantitation and discrimination between intentional and unintentional mislabeling, such a relative error is acceptable.…”

Selected Instrumental Techniques Applied in Food and Feed: Quality, Safety and Adulteration Analysis

“…Zhang and coworkers [ 141 ] developed a qPCR method to test simultaneously for peanut, soybean, and sesame seed in processed foods. Villa and coworkers [ 140 ] used RT-PCR to assess milk proteins in meat-based products detecting as low as 100 and 50 mg·kg −1 milk protein concentrate in hams/autoclaved sausages and raw sausage mixtures, respectively. Miyazaki and coworkers [ 144 ] detected food allergens, including wheat, buckwheat, and peanuts.…”

Production of Liquid Milk Protein Concentrate with Antioxidant Capacity, Angiotensin Converting Enzyme Inhibitory Activity, Antibacterial Activity, and Hypoallergenic Property by Membrane Filtration and Enzymatic Modification of Proteins

“…Furthermore, due to the absence of lactase, a hydrolytic enzyme in brush border of epithelial cells in the small intestine, the milk sensitive community frequently suffers with symptoms of lactose maldigestion [20,21]. However, concentrations of lactose and fat in milk protein concentrate are significantly low; in some cases, food formulas fortified with milk protein concentrate offer immunoglobulin-mediated allergies among people of all ages [30,31]. A plethora of literature about thermal and non-thermal processing technologies have been adopted to combat milk protein allergens [32,33].…”