Assessment of Protein Quality and Digestibility in Plant-Based Meat Analogues.

In this first work, commercial steak-like (n = 3) and cured meat (n = 3) analogues with different legume and cereal formulations were studied and compared to their animal-based (n = 3) counterparts. Plant-based products showed lower protein content than meat controls but a good amino acidic profile even though the sum of essential amino acids of plant-cured meats does not fulfill the requirements set by the Food and Agriculture Organization for children. A comparable release of soluble proteins and peptides in the digestates after in vitro digestion was observed in meat analogues as meat products, whereas the digestibility of proteins was lower in plant-based steaks and higher in plant-based cured meats than their counterparts. The overall protein quality and digestibility of products are related to both the use of good blending of protein sources and processes applied to produce them. An adequate substitution of meat with its analogues depends mostly on the quality of raw materials used, which should be communicated to consumers.

Assessment of Enzymatic Improvers in Flours Using LC-MS/MS Detection of Marker Tryptic Peptides.

Enzymatic improvers are enzymes obtained from microbial or fungal cultures, added as technical adjuvants to flour, with the aim of improving the dough characteristics in bakery products. They are used in a low ppm range and, being technical adjuvants, can go undeclared on the label. Many types of enzymatic improvers are present on the market, such as amylases, lipases, proteases, xylanases, glucose oxidases, and others, each with a different function. Analytical methods capable of detecting these enzymes are needed, particularly for bakery companies, in order to monitor the quality of raw materials and to detect any undeclared presence. In the present work, specific peptide markers, obtained by enzymatic digestion, have been used to detect the presence of enzymatic improvers by LC-MS/MS techniques. Promising results were obtained for some enzymes acting on the carbohydrate fraction (glucoamylase, glucose oxidase, xylanase) in which amounts as low as 20 ppm could be identified in blind flour samples. For lipases and proteases the method proved to be very effective in terms of specific identification, even if less sensitive.

Shotgun proteomics, in-silico evaluation and immunoblotting assays for allergenicity assessment of lesser mealworm, black soldier fly and their protein hydrolysates.

Since 2018, insects have belonged the category of Novel Foods and the presence of allergens represents one of the main hazards connected to their consumption, also due to the potential cross-reactivity with Arthropoda pan-allergens. In the present work, the allergenicity assessment of black soldier fly and lesser mealworm was performed with a shotgun bottom-up proteomic approach combined with in-silico assessment, followed by IgG- and IgE-immunoblotting experiments. The peptides identified, filtered for their abundance and robustness, belonged mainly to muscle proteins, which represented the most abundant protein group. The relevant potential allergens were in-silico identified by sequence similarity to known allergens, and among them tropomyosin resulted the most abundant insect allergen. IgG-immunoblotting analysis with anti-Tropomyosin I antibodies and IgE-immunoblotting assay with serum from patient allergic to crustacean tropomyosin were performed in order to assess the immunoreactivity in both insects. The immunoassays were carried out also on protein hydrolysates extracted by treating insects with Protease from Bacillus licheniformis (1%, 60 °C, pH 7.5). While IgG-immunoblotting demonstrated the loss of immunoreactivity for both hydrolysates, IgE-immunoblotting showed a partial immunoreactivity preservation, also after hydrolysis, in the case of black soldier fly hydrolysate, and a total loss of immunoreactivity for lesser mealworm hydrolysate.

Regular monitoring of breast-feeding rates: feasible and sustainable. The Emilia-Romagna experience.

OBJECTIVE: An efficient breast-feeding monitoring system should be in place in every country to assist policy makers and health professionals plan activities to reach optimal breast-feeding rates. Design/Setting/Subjects From March to June 2015, breast-feeding rates at 3 and 5 months of age were monitored in Emilia-Romagna, an Italian region, using four questions added to a newly developed paediatric immunization database with single records for each individual. Data were collected at primary-care centres. Breast-feeding definitions and 24 h recall as recommended by the WHO were used. Direct age standardization was applied to breast-feeding rates. Record linkage with the medical birth database was attempted to identify maternal, pregnancy and delivery factors associated with full breast-feeding rates at 3 and 5 months of age. RESULTS: Data on breast-feeding were collected for 14044 infants. The mean regional full breast-feeding rate at 3 months was 52 %; differences between local health authorities ranged from 42 to 62 %. At 5 months of age, the mean regional full breast-feeding rate dropped to 33 % (range between local health authorities: 26 to 46 %). Record linkage with the birth certificate database was successful for 93 % of records. Total observations more than doubled with respect to the previous regional survey. CONCLUSIONS: The new monitoring system implemented in 2015 in Emilia-Romagna region, totally integrated with the immunization database, has proved to be feasible, sustainable and more efficient than the previous one. This system can be a model for other regions and countries where the vast majority of mothers obtain vaccinations from public health facilities and that already have an immunization database in place.

Simulated gastrointestinal digestion of Pru ar 3 apricot allergen: assessment of allergen resistance and characterization of the peptides by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry.

RATIONALE: Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceae family. The severity of allergic reactions often relates to resistance of the allergen to digestion. Thus, it is important to evaluate the digestibility of these proteins and characterise the peptides generated in the gastrointestinal tract. METHODS: Simulated gastrointestinal digestion of purified allergen Pru ar 3 was performed using pepsin for the gastric phase in aqueous HCl at pH = 2 and chymotrypsin and trypsin for the intestinal phase in aqueous NH(4)HCO(3) at pH = 7.8. The peptide mixture obtained was analysed by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry (UPLC/ESI-MS). Peptide sequences were identified by comparing their molecular mass to that obtained by in silico digestion, and were confirmed by the ions obtained by in-source fragmentation. Semi-quantification was performed for the intact protein by comparison with internal standards. RESULTS: The resistance to gastrointestinal digestion of Pru ar 3 allergen was evaluated to be 9%. This value is consistent with that found for grape LTP, but much lower than the resistance found for peach LTP (35%). All the peptides generated were identified by ESI-MS on the basis of their molecular mass and from the ions generated from in-source fragmentation. Apart from low molecular mass peptides, five high molecular mass peptides (4500-7000 Da) containing disulphide bridges were identified. ESI-MS of the intact protein indicated a less compact folded structure when compared to that of the homologous peach LTP. CONCLUSIONS: An extensive characterisation of the peptides generated from the gastrointestinal digestion of Pru ar 3 allergen was performed here for the first time via UPLC/ESI-MS analysis. The digestibility of the allergen was evaluated and compared with that of other LTPs, demonstrating that only a small amount of undigested protein remains, and that specific proteolytic action involves immunodominant epitopes. These data might explain the lower allergenicity of apricot LTP compared to peach LTP, despite their high sequence homology.

In vitro gastrointestinal digestion of the major peach allergen Pru p 3, a lipid transfer protein: molecular characterization of the products and assessment of their IgE binding abilities.

A simulated gastrointestinal digestion has been carried out on purified peach lipid transfer protein, one of the main allergens among the population of the Mediterranean area and the major allergen of peach allergic patients. The percentage of intact protein, after extensive digestion, measured by comparison with a non-digestible peptide analogue used as internal standard, was found to be about one-third of the original protein content. The peptides formed in digested fraction were characterized by means of LC/MS. The products of the digestion essentially derived from trypsin action, whereas the protein appeared to be resistant to pepsin and chymotrypsin. The identified peptides could be classified as low molecular weight and high molecular weight peptides. The latter consisted of the full protein, with the disulfide bridges still intact, deprived of the smaller peptides. The different digestion products, including the high and low molecular weight peptides, were purified by LC and assessed, together with the intact protein, by dot-blot analysis with sera of allergic patients, allowing to estimate their potential allergenicity. The intact protein and the high molecular weight peptides were found to be recognized by patients' sera, whereas the small peptides were found to be not reactive.