Antibacterial Activity of Oregano (Origanum vulgare L.) Essential Oil Vapors against Microbial Contaminants of Food-Contact Surfaces.

The antimicrobial effect of eight essential oils' vapors against pathogens and spoilage bacteria was assayed. Oreganum vulgare L. essential oil (OVO) showed a broad antibacterial effect, with Minimum Inhibitory Concentration (MIC) values ranging from 94 to 754 µg cm-3 air, depending on the bacterial species. Then, gaseous OVO was used for the treatment of stainless steel, polypropylene, and glass surfaces contaminated with four bacterial pathogens at 6-7 log cfu coupon-1. No viable cells were found after OVO treatment on all food-contact surfaces contaminated with all pathogens, with the exception of Sta. aureus DSM 799 on the glass surface. The antimicrobial activity of OVO after the addition of beef extract as a soiling agent reduced the Sta. aureus DSM 799 viable cell count by more than 5 log cfu coupon-1 on polypropylene and glass, while no viable cells were found in the case of stainless steel. HS-GC-MS analysis of the headspace of the boxes used for the antibacterial assay revealed 14 different volatile compounds with α-Pinene (62-63%), and p-Cymene (21%) as the main terpenes. In conclusion, gaseous OVO could be used for the microbial decontamination of food-contact surfaces, although its efficacy needs to be evaluated since it depends on several parameters such as target microorganisms, food-contact material, temperature, time of contact, and relative humidity.

Liquid submerged fermentation by selected microbial strains for onion skins valorization and its effects on polyphenols.

Onion skins, actually recycled as organic fertilizers, could be used as a substrate in environmental-friendly bioprocesses to recover high-value bioactive compounds and food ingredients.In this work, a bioprospecting method was carried out including 94 bacterial and 45 yeast strains from several agri-food and environmental niches to verify their ability to grow on onion skins as unique nutrients source.Red and yellow onion skins were assessed by newly selected starter-driven liquid submerged fermentation assays mainly aimed at the release and modification of polyphenols through microbial activities. Fermented onion skins were also investigated as a inexpensive favourable source of microbial enzymes (amylases, proteases, lipases, esterases, cellulases, xylanases).In red onion skins, the treatment with Lactiplantibacillus plantarum TB 11-32 produced a slight increase of bioactive compounds in terms of total phenolics, whereas with the yeast strain Zygosaccharomyces mrakii CL 30 - 29 the quercetin aglycone content increased of about 25% of the initial raw material.In yellow onion skins inoculated, the highest content of phenolic compounds was detected with the yeast strain Saccharomyces cerevisiae En SC, while quercetin aglycone increased of about 60% of the initial raw material in presence of the bacterial strain L. plantarum C 180 - 34.In conclusion, the proposed microbial pre-treatment method can be a potential strategy to re-use onion skins as a fermentation substrate, and as a first step in the development of a biorefinery process to produce value-added products from onion by-products.

Plant Antimicrobials for Food Quality and Safety: Recent Views and Future Challenges.

The increasing demand for natural, safe, and sustainable food preservation methods drove research towards the use of plant antimicrobials as an alternative to synthetic preservatives. This review article comprehensively discussed the potential applications of plant extracts, essential oils, and their compounds as antimicrobial agents in the food industry. The antimicrobial properties of several plant-derived substances against foodborne pathogens and spoilage microorganisms, along with their modes of action, factors affecting their efficacy, and potential negative sensory impacts, were presented. The review highlighted the synergistic or additive effects displayed by combinations of plant antimicrobials, as well as the successful integration of plant extracts with food technologies ensuring an improved hurdle effect, which can enhance food safety and shelf life. The review likewise emphasized the need for further research in fields such as mode of action, optimized formulations, sensory properties, safety assessment, regulatory aspects, eco-friendly production methods, and consumer education. By addressing these gaps, plant antimicrobials can pave the way for more effective, safe, and sustainable food preservation strategies in the future.

Quality and Safety of Ready-to-Eat Golden Thistle (Scolymus hispanicus L.): A New Product for Traditional Italian Dishes.

Golden thistle (Scolymus hispanicus L.) is a wild edible plant belonging to Asteraceae family, with a great potential for food applications. The aim of this study was to identify the best cooking procedure able to provide a high-quality, ready-to-use product. For this purpose, leaf midribs (the most used edible part of the plant) were cooked by boiling, steaming, and 'sous vide', and the cooked products were compared for their phenolic content and composition, antioxidant activity, sugar and inorganic ion content, organoleptic characteristics, and microbial safety, this latter also during storage. In general, boiling caused a decrease in the value of these parameters, despite being the best product for taste and overall acceptability. On the contrary, steaming and 'sous vide' resulted in the best treatments to preserve antioxidant activity, total phenols, and chlorogenic acid. In particular, in 'sous vide' cooked samples, a significant increase in the value of these parameters and a remarkable decrease in nitrate content were found. Moreover, 'sous vide' resulted in the best treatment also regarding microbial safety during shelf life; actually, after 15 days of storage at 8 °C, Enterobacteriaceae and mesophilic aerobic bacteria were not detectable in 'sous vide' samples. These results contributed to increase the knowledge of a wild edible plant with high nutritional properties and promoting its consumption by obtaining a ready-to-use product with good organoleptic characteristics and endowed with a long period of shelf life.

Use of Selected Lactic Acid Bacteria for the Fermentation of Legume-Based Water Extracts.

In this study, the effect of selected Lactobacillus acidophilus ATCC 4356, Limosilactobacillus fermentum DSM 20052, and Lacticaseibacillus paracasei subsp. paracasei DSM 20312 strains on the sensory characteristics, and protein and amino acid content of fermented water extracts derived from lupin, pea, and bean grains is reported. Even though all strains were able to grow over 7 log cfu mL-1 and to decrease pH in the range of -0.52 to -1.25 within 24 h, the release of an unpleasant ferric-sulfurous off-odor from the fermented bean water extract prohibited further characterization. Lupin and pea grain-based beverages underwent an in-depth sensory evaluation using a simplified check-all-that-apply (CATA) method, finding new and appreciable sensory notes such as cooked ham, almonds, and sandalwood. Fermented lupin water extract showed higher total protein content (on average, 0.93 mg mL-1) in comparison to that of pea grains (on average, 0.08 mg mL-1), and a free amino acid content (on average, 3.9 mg mL-1) close to that of cow milk. The concentrations of these nutrients decreased during refrigerated storage, when the lactic acid bacteria load was always higher than 7 log cfu mL-1. The results of this study indicated that lactic fermentation improves the sensory characteristics of these innovative legume-based beverages, which sustained high loads of viable lactobacilli up to the end of cold storage.

A green approach to develop zeolite-thymol antimicrobial composites: analytical characterization and antimicrobial activity evaluation.

In this work, the development, analytical characterization and bioactivity of zeolite-thymol composites, obtained using wet, semi-dry and dry processes, were carried out in order to obtain sustainable and powerful antimicrobial additives. FT-IR, XRD, DSC, TGA, SEM and B.E.T. analyses were carried out to gain comprehensive information on the chemical-physical, thermal, and morphological features of the composites. GC-MS analyses allowed quantifying the active molecule loaded in the zeolite, released by the functionalized composites and its stability over time. Among the three procedures, the dry approach allowed to reach the highest thymol loading content and efficiency (49.8 ± 1.6% and 99.6 ± 1.2%, respectively), as well as the highest composite specific surface area value, feature which promises the best interaction between the surface of the composite and the bacterial population. Therefore, the bioactive surface of composites obtained by this solvent-free method was assayed for its antimicrobial activity against four microbial strains belonging to Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans species. The higher antimicrobial activity produced by the solvent-free composite in comparison with that of pure thymol, at the same thymol concentration, was ascribed to the large interfacial contact between the composite and the bacterial target. This feature, together with its enhanced storage stability, suggested that this composite could be employed as effective additives for the development of antimicrobial biointerfaces for food, home and personal care applications.

Comparison of Antibacterial and Antioxidant Properties of Red (cv. Negramaro) and White (cv. Fiano) Skin Pomace Extracts.

Wine pomace has attracted the attention of the food industry, due to its high content in bioactive compounds, and its multiple healthy activities. In this work, whole and separated skin pomaces from fermented (red) and un-fermented (white) grape by-products were characterized for their antioxidant and antimicrobial activities in order to exploit them as functional food ingredient. Antioxidant activity, measured by both ORAC and TEAC assays, was higher in whole than in skin pomace extracts. The characterization of phenolic composition in whole and skin pomace extracts confirmed the peculiarity of some compounds such as anthocyanins (107.84 + 10.3 mg/g TP) in red skin pomace and a great amount of flavanols (80.73 + 4.04 mg/g TP) in white skin pomace. Whole and skin pomace extracts displayed the same antibacterial activity at 250 µg gallic acid equivalents (GAE)/mL. Red and white skin pomace extracts showed a Minimum Inhibitory Concentration (MIC) of 31.25-62.5 GAE/mL against Staphylococcus aureus and Enterococcus faecalis. Pseudomonas spp. were more sensitive to red skin pomace extracts rather than white skin pomace extracts. Given these results, both red and white pomace extracts could be exploited for future application in food, pharmaceutical and cosmetic industry.

Innovative Eco-Friendly Hydrogel Film for Berberine Delivery in Skin Applications.

Hydrogel formulations (masks or patches, without tissue support) represent the new frontier for customizable skin beauty and health. The employment of these materials is becoming popular in wound dressing, to speed up the healing process while protecting the affected area, as well as to provide a moisturizing reservoir, control the inflammatory process and the onset of bacterial development. Most of these hydrogels are acrylic-based at present, not biodegradable and potentially toxic, due to acrylic monomers residues. In this work, we selected a new class of cellulose-derived and biodegradable hydrogel films to incorporate and convey an active compound for dermatological issues. Films were obtained from a combination of different polysaccharides and clays, and berberine hydrochloride, a polyphenolic molecule showing anti-inflammatory, immunomodulatory, antibacterial and antioxidant properties, was chosen and then embedded in the hydrogel films. These innovative hydrogel-based systems were characterized in terms of water uptake profile, in vitro cytocompatibility and skin permeation kinetics by Franz diffusion cell. Berberine permeation fitted well to Korsmeyer-Peppas kinetic model and achieved a release higher than 100 µg/cm2 within 24 h. The latter study, exploiting a reliable skin model membrane, together with the biological assessment, gained insights into the most promising formulation for future investigations.

Unravelling the Antifungal Effect of Red Thyme Oil (Thymus vulgaris L.) Compounds in Vapor Phase.

The aim of this work was to evaluate the antifungal activity in vapor phase of thymol, p-cymene, and γ-terpinene, the red thyme essential oil compounds (RTOCs). The Minimum Inhibitory Concentration (MIC) of RTOCs was determined against postharvest spoilage fungi of the genera Botrytis, Penicillium, Alternaria, and Monilinia, by measuring the reduction of the fungal biomass after exposure for 72 h at 25 °C. Thymol showed the lowest MIC (7.0 µg/L), followed by γ-terpinene (28.4 µg/L) and p-cymene (40.0 µg/L). In the case of P. digitatum ITEM 9569, resistant to commercial RTO, a better evaluation of interactions among RTOCs was performed using the checkerboard assay and the calculation of the Fractional Inhibitory Concentration Index (FICI). During incubation, changes in the RTOCs concentration were measured by GC-MS analysis. A synergistic effect between thymol (0.013 ± 0.003 L/L) and γ-terpinene (0.990 ± 0.030 L/L) (FICI = 0.50) in binary combinations, and between p-cymene (0.700 ± 0.010 L/L) and γ-terpinene (0.290 ± 0.010 L/L) in presence of thymol (0.008 ± 0.001 L/L) (FICI = 0.19), in ternary combinations was found. The synergistic effect against the strain P. digitatum ITEM 9569 suggests that different combinations among RTOCs could be defined to control fungal strains causing different food spoilage phenomena.

Design and Characterization of a Novel Fermented Beverage from Lentil Grains.

The experimental activities carried out in this study aimed at designing a lentil-based beverage rich in soluble and digestible proteins. In order to extract soluble proteins, lentil grains were soaked in water overnight, blended, treated with proteolytic enzymes and fermented with Lactobacillus strains. Protein enzymatic hydrolysis, carried out with four commercial food grade enzyme preparations, showed that bromelin, at the enzyme to substrate ratio of 10%, was the best solution to produce this novel beverage. Even though the seven Lactobacillus strains were all able to ferment aqueous extract within 24 h, L. acidophilus ATCC 4356, L. fermentum DSM 20052 and L. paracasei subsp. paracasei DSM 20312 showed the highest growth rate and the lowest pH values. In fermented lentil-based beverages, the antinutritional factor phytic acid decreased up to 30%, similarly, the highest reduction in raffinose oligosaccharides content reached about 12% the initial concentration. It is worthy of note that the viable density of all strains remained higher than 7 log cfu/mL after 28 days of cold storage. The results here reported show for the first time the possibility to obtain a probiotic lentil-based beverage rich in soluble proteins, peptides and amino acids with low content in main antinutritional factors.

Phenotype and genomic background of Arcobacter butzleri strains and taxogenomic assessment of the species.

In this study the phenotypic and genomic characterization of two Arcobacter butzleri (Ab) strains (Ab 34_O and Ab 39_O) isolated from pre-cut ready-to-eat vegetables were performed. Results provided useful data about their taxonomy and their overall virulence potential with particular reference to the antibiotic and heavy metal susceptibility. These features were moreover compared with those of two Ab strains isolated from shellfish and a genotaxonomic assessment of the Ab species was performed. The two Ab isolated from vegetables were confirmed to belong to the Aliarcobacter butzleri species by 16S rRNA gene sequence analysis, MLST and genomic analyses. The genome-based taxonomic assessment of the Ab species brought to the light the possibility to define different subspecies reflecting the source of isolation, even though further genomes from different sources should be available to support this hypothesis. The strains isolated from vegetables in the same geographic area shared the same distribution of COGs with a prevalence of the cluster "inorganic ion transport and metabolism", consistent with the lithotrophic nature of Arcobacter spp. None of the Ab strains (from shellfish and from vegetables) metabolized carbohydrates but utilized organic acids and amino acids as carbon sources. The metabolic fingerprinting of Ab resulted less discriminatory than the genome-based approach. The Ab strains isolated from vegetables and those isolated from shellfish endowed multiple resistance to several antibiotics and heavy metals.

Initiated Chemical Vapor Deposition of Crosslinked Organic Coatings for Controlling Gentamicin Delivery.

A coating consisting of a copolymer of methacrylic acid and ethylene glycol dimethacrylate was deposited over a gentamicin film by initiated chemical vapor deposition with the aim of controlling the drug release. Gentamicin release in water was monitored by means of conductance measurements and of UV-vis Fluorescence Spectroscopy. The influence of the polymer chemical composition, specifically of its crosslinking density, has been investigated as a tool to control the swelling behavior of the initiated chemical vapor deposition (iCVD) coating in water, and therefore its ability to release the drug. Agar diffusion test and microbroth dilution assays against Staphylococcus aureus and Pseudomonas aeruginosa on cellulose coated substrates confirmed that the antibacterial activity of the drug released by the coating was retained, though the release of gentamicin was not complete.

Autochthonous and Probiotic Lactic Acid Bacteria Employed for Production of "Advanced Traditional Cheeses".

Microbial characterization of two Italian traditional cheeses, Giuncata and Caciotta Leccese, was carried out, with the aim to isolate autochthonous bacterial strains to be used as starters to improve and standardize the quality of these cheeses. More than 400 bacterial isolates were found, using PCR-based identification, to belong to 12 species of the Streptococcus, Lactococcus, Lactobacillus, and Leuconostoc genera. The dominant strains were screened for antagonistic activity against pathogenic and spoilage bacteria and exopolysaccharide production, acidification, and proteolytic activity. Since Streptococcus macedonicus was found to be the most prevalent lactic acid bacteria species present in milk and in both types of cheese, the best performing strain of this species was successfully used, alone or in combination with a selected autochthonous Lactococcus lactis strain, in pilot-scale productions of Giuncata and Caciotta Leccese cheeses, respectively. The combined inoculums of selected autochthonous strains positively influenced the sensory characteristics of both Giuncata and Caciotta cheeses. Finally, the selected autochthonous cultures were enriched with a potentially probiotic Lactobacillus rhamnosus strain and successfully used in pilot-scale productions of these traditional cheeses. To the best of our knowledge, this is the first study reporting the use of an autochthonous S. macedonicus strain as a starter for the production of cheeses with added probiotics. In addition, the identification of the probiotic strain in the feces of healthy volunteers fed with the advanced traditional cheese proved its effectiveness as a carrier for the delivery of probiotics to the human body.

Exploitation of Prunus mahaleb fruit by fermentation with selected strains of Lactobacillus plantarum and Saccharomyces cerevisiae.

The organoleptic attributes of Prunus mahaleb, a fruit representing a new source of bioactive compounds, are so pronounced that it can be consider non-edible. This study was designed to evaluate the acceptance of P. mahaleb fruits after fermentation with different Saccharomyces cerevisiae and Lactobacillus plantarum protechnological strains. Four different bacterial and one yeast strains, as single or mixed starter formulation, were used to inoculate an aqueous suspension of P. mahaleb fruits. The fermented fruits and fermentation broths were subjected to physico-chemical characterization and the organoleptic properties of both samples were also assessed by a hedonic panel. The obtained results indicated that all the employed strains were able to grow and to ferment the matrix. However, the mixed starter FG69 + Li180-7 (L. plantarum/S. cerevisiae) had the best impact on sensory characteristics of P. mahaleb fruit and fermented medium. The adopted protocol allowed us to attain edible fruits and a new fermented non-dairy drink with valuable probiotic health-promoting properties. In our knowledge, this is the first study concerning the exploitation of P. mahaleb fruits. This investigation confirmed the potential of yeasts and lactic acid bacteria co-inoculation in the design of starter tailored for this kind of food applications.

Genomic Characterization of Arcobacter butzleri Isolated From Shellfish: Novel Insight Into Antibiotic Resistance and Virulence Determinants.

Arcobacter (A.) butzleri is an emerging pathogenic microorganism, whose taxonomy has been recently suggested to be emended to the Aliarcobacter (Al.) butzleri comb. nov. Despite extensive taxonomic analysis, only few fragmented studies have investigated the occurrence and the prevalence of virulence and antibiotic resistance determinants of this species in strains isolated from shellfish. Herein we report for the first time the whole genome sequencing and genomic characterization of two A. butzleri strains isolated from shellfish, with particular reference to the antibiotic, heavy metals and virulence determinants. This study supported the taxonomic assignment of these strains to the Al. butzleri species, and allowed us to identify antibiotic and metal resistance along with virulence determinants, also additional to those previously reported for the only two A. butzleri strains from different environments genomically characterized. Moreover, both strains showed resistance to ß-lactams, vanocomycin, tetracycline and erythromycin and susceptibility to aminoglycosides and ciprofloxacin. Beside enlarging the availability of genomic data to perform comparative studies aimed at correlating phenotypic differences associated with ecological niche and geographic distribution with the genetic diversity of A. butzleri spp., this study reports the endowment of antibiotic and heavy metal resistance and virulence determinants of these shellfish-isolated strains. This leads to hypothesize a relatively high virulence of A. butzleri isolated from shellfish and prompt the need for a wider genomic analysis and for in vitro and in vivo studies of more strains isolated from this and other ecological niches, to unravel the mechanism of pathogenicity of this species, and the potential risk associated to their consumption.

Plasma-Deposited Nanocapsules Containing Coatings for Drug Delivery Applications.

Coatings consisting in gentamicin-containing nanocapsules have been synthetized by means of an aerosol-assisted atmospheric pressure plasma deposition process. The influence of different parameters affecting the process has been extensively investigated by means of a morphological and chemical characterization of the coatings. Scanning electron microscopy highlighted the presence of nanocapsules whose size and abundance depend on power input and deposition time. A detailed analysis carried out with matrix-assisted laser desorption ionization coupled to high-resolution mass spectrometry allowed to detect and identify the presence of gentamicin embedded in the coatings and its rearrangement, as a result of the interaction with the plasma. The release of gentamicin in water has been monitored by means of UV-vis fluorescence spectroscopy, and its biological activity has been evaluated as well by the disk diffusion assay against Staphylococcus aureus and Pseudomonas aeruginosa. It is confirmed that the antibacterial activity of gentamicin is preserved in the plasma-deposited coatings. Preliminary cytocompatibility investigations indicated that eukaryotic cells well tolerate the release of gentamicin from the coatings.

Lactobacillus reuteri suppresses E. coli O157:H7 in bovine ruminal fluid: Toward a pre-slaughter strategy to improve food safety?

The bovine gastrointestinal tract (GIT) is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Therefore, it is crucial to develop strategies, such as EHEC suppression by antagonistic microorganisms, to reduce EHEC survival in the GIT of cattle and to limit shedding and food contamination. Most human-derived Lactobacillus reuteri strains produce hydroxypropionaldehyde (HPA), an antimicrobial compound, during anaerobic reduction of glycerol. The capacity of L. reuteri LB1-7, a strain isolated from raw bovine milk, to produce HPA and its antimicrobial activity against an O157:H7 EHEC strain (FCH6) were evaluated in bovine rumen fluid (RF) under strict anaerobiosis. EHEC was totally suppressed when incubated in RF inoculated with L. reuteri LB1-7 and supplemented with 80 mM glycerol (RF-Glyc80). The addition of LB1-7 or glycerol alone did not modify EHEC survival in RF. Glycerol was converted to HPA (up to 14 mM) by LB1-7 during incubation in RF-Glyc80, and HPA production appeared to be responsible for EHEC suppression. The bactericidal activity of L. reuteri LB1-7, the concentration of glycerol required and the level of HPA produced depended on physiological and ecological environments. In vitro experiments also showed that EHEC inoculated in rumen fluid and exposed to L. reuteri and glycerol had a very limited growth in rectal contents. However, L. reuteri exerted an antimicrobial activity against the rumen endogenous microbiota and perturbed feedstuff degradation in the presence of glycerol. The potential administration of L. reuteri and glycerol in view of application to finishing beef cattle at the time of slaughter is discussed. Further in vivo studies will be important to confirm the efficiency of L. reuteri and glycerol supplementation against EHEC shedding in ruminants.

Silver-loaded chitosan coating as an integrated approach to face titanium implant-associated infections: analytical characterization and biological activity.

The present work focuses on the idea to prevent and/or inhibit the colonization of implant surfaces by microbial pathogens responsible for post-operative infections, adjusting antimicrobial properties of the implant surface prior to its insertion. An antibacterial coating based on chitosan and silver was developed by electrodeposition techniques on poly(acrylic acid)-coated titanium substrates. When a silver salt was added during the chitosan deposition step, a stable and scalable silver incorporation was achieved. The physico-chemical composition of the coating was studied by X-ray photoelectron spectroscopy (XPS), while atomic force microscopy in intermittent contact mode (ICAFM) was used to explore the coating morphology. The amount of silver released from the coating up to 21 days was evaluated by inductively coupled plasma mass spectrometry (ICP-MS). The capability of the proposed coating to interact in vitro with the biological environment in terms of compatibility and antibacterial properties was assessed using MG-63 osteoblast-like cell line and S. aureus and P. aeruginosa strains, respectively. These studies revealed that a coating showing a silver surface atomic percentage equal to 0.3% can be effectively used as antibacterial system, while providing good viability of osteoblast-like cells after 7 days. The antibacterial effectiveness of the prepared coating is mainly driven by a contact killing mechanism, although the low concentration of silver released (below 0.1 ppm up to 21 days) is enough to inhibit bacterial growth, advantaging MG-63 cells in the race for the surface.

Draft Genome Sequence of Pseudomonas fluorescens Strain ITEM 17298, Associated with Cheese Spoilage.

Pseudomonas fluorescens is a genetically and phenotypically heterogeneous species that is often reported as a spoiler of fresh foods, but it has recently been implicated in clinical infection. In this study, we sequenced the genome of P. fluorescens strain ITEM 17298, isolated from mozzarella cheese and able to cause several alterations under cold storage.

Reduction of whey protein concentrate antigenicity by using a combined enzymatic digestion and ultrafiltration approach.

The global interest in saving food resources is leading to recycle wasted-food materials to extract useful nutrients. In dairy industry, the recycling of whey proteins determines their utilization in the healthy-addressed foods, which, however, can cause immunological responses in allergic subjects. In this work, a whey protein concentrate (WPC) was alternatively hydrolyzed with pepsin, papain, trypsin and rennin in order to attenuate or abolish the ß-lactoglobulin (BLG) antigenicity. The electrophoretic profiles of both pepsin and papain WPC hydrolysates proved the disappearance of the BLG band, even though a slight antigenicity was still found by ELISA. Pepsin hydrolysates, filtered through a 10-kDa cut-off membrane, did not produce immunological response. A deeper investigation carried out on pepsin digested and ultrafiltered samples by LC-MS/MS showed the disappearance of the immunoreactive BLG-fragment IVTQMKGLDIQKVAGTW. The remaining peptides, partially overlapped to major IgE binding epitopes, were not able to give immunoreactivity response. The combined WPC pepsin digestion with ultrafiltration confirmed to be a user-friendly strategy to reduce markedly the WPC antigenicity. The improvement of this two-steps process could be used to produce novel hypoallergenic infant food formulas.