Distinct Organotypic Platforms Modulate Rainbow Trout (Oncorhynchus mykiss) Intestinal Cell Differentiation In Vitro.

In vitro organotypic cell-based intestinal platforms, able to faithfully recapitulate the complex functions of the organ in vivo, would be a great support to search for more sustainable feed ingredients in aquaculture. We previously demonstrated that proliferation or differentiation of rainbow trout intestinal cell lines is dictated by the culture environment. The aim of the present work was to develop a culture platform that can efficiently promote cell differentiation into mature enterocytes. We compared four options, seeding the RTpiMI cell line derived from the proximal intestine on (1) polyethylene terephthalate (PET) culture inserts ThinCert™ (TC), (2) TC coated with the solubilized basement membrane matrix Matrigel® (MM), (3) TC with the rainbow trout fibroblast cell line RTskin01 embedded within the Matrigel® matrix (MMfb), or (4) the highly porous polystyrene scaffold Alvetex® populated with the abovementioned fibroblast cell line (AV). We evaluated the presence of columnar cells with a clear polarization of brush border enzymes, the formation of an efficient barrier with a significant increase in transepithelial electrical resistance (TEER), and its ability to prevent the paracellular flux of large molecules but allow the transit of small compounds (proline and glucose) from the apical to the basolateral compartment. All parameters improved moving from the simplest (TC) through the more complex platforms. The presence of fibroblasts was particularly effective in enhancing epithelial cell differentiation within the AV platform recreating more closely the complexity of the intestinal mucosa, including the presence of extracellular vesicles between fibroblasts and epithelial cells.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Current insights into non-thermal preservation technologies alternative to conventional high-temperature short-time pasteurization of drinking milk.

Milk is an important nutritional food source characterized by a perishable nature and conventionally thermally treated to guarantee its safety. In recent years, an increasing focus on competing non-thermal food processing technologies has been driven mainly by consumers' expectations for minimally processed products. Due to the heat sensitivity of milk, much research interest has been addressed to mild non-thermal pasteurization processing to keep safety, 'fresh-like' taste and to maintain the organoleptic qualities of raw milk. This review provides an overview of the current literature on non-thermal treatments as standalone alternative technologies to high-temperature short-time (HTST) pasteurization of drinking milk. Results of lab-scale experimentations suggest the feasibility of most emerging non-thermal processing technologies, including high hydrostatic pressure, pulsed electric field, cold plasma, cavitation and light-based technologies, as alternative to thermal treatment of drinking milk with premium in shelf life duration. Nevertheless, a series of regulatory, technological and economical hurdles hinder the industrial scaling-up for most of these substitutes. To date, only high hydrostatic pressure treatments are applied as alone alternative to HTSH pasteurization for processing of "cold pasteurized" drinking milk. Milk submitted to HTST treatment combined to ultraviolet light is currently accepted in EU countries as novel food.

Impact of in vitro static digestion method on the release of ß-casomorphin-7 from bovine milk and cheeses with A1 or A2 ß-casein phenotypes.

Beta-casomorphin-7 (BCM7) represents the fragment Val60-Ile66 of bovine ß-casein (ß-CN), and there is evidence that it is more easily released during gastrointestinal digestion (GID) of A1 ß-CN variant, in comparison to the A2 variant. This study aimed at investigating the effect of type of enzymes and the protease/protein (P/S) ratio on BCM7 release during the intestinal step of in vitro static GID of bovine milk and cheeses with A1 or A2 ß-CN phenotypes. BCM7 occurred in digests of both A1 and A2 samples, being the release more marked for A1 counterparts. Nonetheless, the BCM7 release depended on both the type of GID enzymes and the P/S ratio. These findings highlight the importance of GID conditions which may affect the outcomes for possible differences between A1 and A2 milk based on BCM7 release during in vitro GID.

Evaluation of the Characteristics and Infectivity of the Secondary Inoculum Produced by Plasmopara viticola on Grapevine Leaves by Means of Flow Cytometry and Fluorescence-Activated Cell Sorting.

Plasmopara viticola, the oomycete causing grapevine downy mildew, is one of the most important pathogens in viticulture. P. viticola is a polycyclic pathogen, able to carry out numerous secondary cycles of infection during a single vegetative grapevine season, by producing asexual spores (zoospores) within sporangia. The extent of these infections is strongly influenced by both the quantity (density) and quality (infectivity) of the inoculum produced by the pathogen. To date, the protocols for evaluating all these characteristics are quite limited and time-consuming and do not allow all the information to be obtained in a single run. In this study, a protocol combining flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) was developed to investigate the composition, the infection efficiency and the dynamics of the inoculum produced by P. viticola for secondary infection cycles. In our analyses, we identified different structures within the inoculum, including degenerated and intact sporangia. The latter have been sorted, and single sporangia were directly inoculated on grapevine leaf discs, thus allowing a thorough investigation of the infection dynamics and efficiency. In detail, we determined that, in our conditions, 8% of sporangia were able to infect the leaves and that on a susceptible variety, the time required by the pathogen to reach 50% of total infection is about 10 days. The analytical approach developed in this study could open a new perspective to shed light on the biology and epidemiology of this important pathogen. IMPORTANCE P. viticola secondary infections contribute significantly to the epidemiology of this important plant pathogen. However, the infection dynamics of asexual spores produced by this organism are still poorly investigated. The main challenges in dissecting the grapevine-P. viticola interaction in vitro are attributable to the biotrophic adaptation of the pathogen. This work provides new insights into the infection efficiency and dynamics imputable to P. viticola sporangia, contributing useful information on grapevine downy mildew epidemiology. Moreover, future applications of the sorting protocol developed in this work could yield a significant and positive impact in the study of P. viticola, providing unmatched resolution, precision, and accuracy compared with the traditional techniques.

Transmission routes, preventive measures and control strategies of SARS-CoV-2 in the food factory.

SARS-CoV-2 virus represents a health threat in food factories. This infectious virus is transmitted by direct contact and indirectly via airborne route, whereas contamination through inanimate objects/surfaces/equipment is uncertain. To limit the potential spread of the pathogen in the food industry, close working between individuals should be avoided and both personal and respiratory hygiene activities should be enforced. Despite the high infectivity, SARS-CoV-2, being an enveloped virus with a fragile lipid envelop, is sensitive to biocidal products and sanitizers commonly used in the food factory. In the context of the building design, interventions that promote healthy air quality should be adopted, especially in food areas with high-occupancy rates for prolonged times, to help minimize the potential exposure to airborne SARS-CoV-2. Air ventilation and filtration provided by heating, ventilation and air conditioning systems, are effective and easy-to-organize tools to reduce the risk of transmission through the air. In addition to conventional sanitation protocols, aerosolization of hydrogen peroxide, UV-C irradiation or in-situ ozone generation are complementary techniques for an effective virucidal treatment of the air.

Model infant biscuits release the opioid-acting peptides milk ß-casomorphins and gluten exorphins after in vitro gastrointestinal digestion.

Infant biscuits (IBs) are commonly used during the complementary feeding of infants from the 6th month of life. They contain wheat flour and dairy ingredients, which can release the opioid-acting peptides ß-casomorphins (BCMs) and gluten exorphins (GEs) after gastrointestinal digestion. In the present study, five model IBs were prepared with or without gluten and different powdered milk derivatives in the formulations. IBs were digested simulating an in vitro static gastrointestinal digestion for infants aged 6-12 months. BCMs and GEs were identified and quantified by UPLC/HR-MS. The amounts of BCM7 and the GE A5 were related to the ß-CN and gluten content of the formulations. To date, levels of BCMs and GEs in digests of IBs have not been reported in literature. This work represents an in vitro investigation regarding the release of opioid-acting peptides in IBs. It could add additional knowledge on complementary foods for infant health.

Gastrointestinal In Vitro Digests of Infant Biscuits Formulated with Bovine Milk Proteins Positively Affect In Vitro Differentiation of Human Osteoblast-Like Cells.

Infant biscuits (IBs) are part of complementary feeding from weaning up to the age of five years. They normally contain bovine milk proteins, which can influence bone development. This potential effect was investigated using experimental baked IBs, which were prepared from doughs containing different type of dairy proteins: milk protein concentrate (IB1), whey protein isolate (IB2), and skimmed milk powder (IB3). Dairy protein-free (IB0) and gluten-free (IB4) biscuits were also formulated. The in vitro gastrointestinal digests of IBs (IBDs) were tested on a co-culture of Caco-2/HT-29 70/30 cells as an in vitro model of human small intestine. None of the IBDs influenced cell viability and monolayer integrity, while IBD0 and IBD4 increased Peptide-YY production. The basolateral contents of Transwell plates seeded with Caco-2/HT-29 70/30 co-culture, mimicking metabolized IBDs (MIBDs), were tested on Saos-2 cells, an in vitro model of human osteoblast-like cells. After incubation, MIBD0, lacking dairy proteins, decreased the cell viability, while MIBD2, containing whey protein isolate, increased both the viability and the number of cells. MIBD2 and MIBD4, the latter containing both casein and whey proteins, increased alkaline phosphatase activity, a bone differentiation marker. These results highlight that IBs containing dairy proteins positively affect bone development.

Effect of protein fortification on heat damage and occurrence of ß-casomorphins in (un)digested donor human milk intended for nutrition of preterm infants.

Pasteurized donor human milk (PDHM) for preterm infant nutrition is fortified with hydrolyzates of cow's milk proteins, which have been poorly investigated in relation to heat-damage and occurrence of the bioactive peptides ß-casomorphins (BCMs). Therefore, thermal protein modifications of three commercial fortifiers were assessed by measuring well-recognized indexes of heat load. The fortifiers did not contain pyrraline, whereas furosine and lysinoalanine levels roughly overlapped the lowest values reported for liquid formulas addressed to term infant nutrition. Bovine BCMs 3 to 7 and human BCMs 3 to 9 were searched. Bovine BCMs 3, 4, 6 and 7 were found in the undigested fortifiers. Following in vitro digestion simulating the digestive conditions of premature infant, bovine BCMs still occurred in fortified PDHM; the human BCMs 3, 7, 8 and 9 formed. Overall, these results better address the nutritional features of protein fortifiers and fortified PDHM intended for nutrition of preterm infants.

Bovine whey peptides transit the intestinal barrier to reduce oxidative stress in muscle cells.

Health benefits are routinely attributed to whey proteins, their hydrolysates and peptides based on in vitro chemical and cellular assays. The objective of this study was to track the fate of whey proteins through the upper gastrointestinal tract, their uptake across the intestinal barrier and then assess the physiological impact to downstream target cells. Simulated gastrointestinal digestion (SGID) released a selection of whey peptides some of which were transported across a Caco-2/HT-29 intestinal barrier, inhibited free radical formation in muscle and liver cells. In addition, SGID of ß-lactoglobulin resulted in the highest concentration of free amino acids (176 nM) arriving on the basolateral side of the co-culture with notable levels of branched chain and sulphur-containing amino acids. In vitro results indicate that consumption of whey proteins will deliver bioactive peptides to target cells.

Functional characterization of Lactobacillus plantarum ITEM 17215: A potential biocontrol agent of fungi with plant growth promoting traits, able to enhance the nutritional value of cereal products.

In this work, we explored the potential of 25 Lactobacillus plantarum strains isolated from cereals and milk-based products, testing characteristics related to antifungal activity and to nutritional quality. The tested strains demonstrated interesting beneficial traits, such as the ability to utilize fructo-oligosaccharides, prebiotic substances that help probiotic microorganisms to grow in the human gut, and to reduce phytate, an antinutrient present in cereal sector. Regarding mould inhibition, we highlighted the ability of the strains to inhibit Penicillium roqueforti, Mucor circinelloides and mycotoxinogenic moulds associated with cereal grains as Aspergillus flavus, A. niger, Fusarium verticillioides. Moreover, a moderate reduction of the bioavailability of aflatoxin AFB1 was detected. The selected L. plantarum strain ITEM 17215, showed a strong inhibitory ability towards fungal growth and was able to produce 1,2-dihydroxybenzene, benzoic acid, p-hydroxyphenyllactic acid and 3-phenyllactic acid. The latter compound, already described as efficient antifungal inhibitor, was the most abundant and its concentration was further increased by adding phenylalanine and phenylpyruvic acid in the growth medium. The metabolites produced by strain ITEM 17215 could also be related to the ability of the strain to induce cereal germination and promote plant growth. This aspect, not yet investigated in L. plantarum, could have interesting applications in the agro-food sector.

Streptococcus thermophilus urease activity boosts Lactobacillus delbrueckii subsp. bulgaricus homolactic fermentation.

The proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in the yogurt consortium enhances the growth rate and size of each population. In contrast, the independent growth of the two species in milk leads to a slower growth rate and a smaller population size. In this study, we report the first evidence that the urease activity of S. thermophilus increases the intracellular pH of L. delbrueckii in the absence of carbon source. However, in milk, in the presence of lactose the alkalizing effect of urea-derived ammonia was not detectable. Nevertheless, based on glucose consumption and lactic acid production at different pHin, L. delbrueckii showed an optimum of glycolysis and homolactic fermentation at alkaline pH values. In milk, we observed that ammonia provided by urea hydrolysis boosted lactic acid production in S. thermophilus and in L. delbrueckii when the species were grown alone or in combination. Therefore, we propose that urease activity acts as an altruistic cooperative trait, which is costly for urease-positive individuals but provides a local benefit because other individuals can take advantage of urease-dependent ammonia release.

Protein breakdown and release of ß-casomorphins during in vitro gastro-intestinal digestion of sterilised model systems of liquid infant formula.

Protein modifications occurring during sterilisation of infant formulas can affect protein digestibility and release of bioactive peptides. The effect of glycation and cross-linking on protein breakdown and release of ß-casomorphins was evaluated during in vitro gastro-intestinal digestion (GID) of six sterilised model systems of infant formula. Protein degradation during in vitro GID was evaluated by SDS-PAGE and by measuring the nitrogen content of ultrafiltration (3kDa) permeates before and after in vitro GID of model IFs. Glycation strongly hindered protein breakdown, whereas cross-linking resulting from ß-elimination reactions had a negligible effect. Only ß-casomorphin 7 (ß-CM7) was detected (0.187-0.858mgL(-1)) at the end of the intestinal digestion in all untreated IF model systems. The level of ß-CM7 in the sterilised model systems prepared without addition of sugars ranged from 0.256 to 0.655mgL(-1). The release of this peptide during GID was hindered by protein glycation.

Gastrointestinal digestates of Grana Padano and Trentingrana cheeses promote intestinal calcium uptake and extracellular bone matrix formation in vitro.

In the present work, Grana Padano (GP) and Trentingrana (TN) cheeses at different ripening time were in vitro digested. To study calcium uptake and utilization, the intact digestates (selected doses that do not alter cell viability and Transepithelial Electrical Resistance) were administered to Caco2/HT-29 70/30 cells, cultured on a semipermeable membrane in transwells, as a model of human intestinal epithelium. Intact digestates as well as the whole basolateral solutions (mimicking the passage of digestates through intestinal cells before reaching the blood flow and bone) in parallel were further administered to human osteoblast-like cells SaOS-2 to study the extracellular bone matrix formation. In vitro digestates deriving from GP and TN promoted calcium uptake and extracellular bone matrix formation independently of both the cheese type and its ripening period (13, 19 or 26months). The present study reports the ability of whole digestates of GP and TN cheeses to improve intestinal calcium absorption and bone matrix formation in vitro. Once fully explored at bone level, this finding could better support the role of cheese in ameliorating calcium deficiencies and associated diseases in vivo.

Heat damage and in vitro starch digestibility of puffed wheat kernels.

The effect of processing conditions on heat damage, starch digestibility, release of advanced glycation end products (AGEs) and antioxidant capacity of puffed cereals was studied. The determination of several markers arising from Maillard reaction proved pyrraline (PYR) and hydroxymethylfurfural (HMF) as the most reliable indices of heat load applied during puffing. The considerable heat load was evidenced by the high levels of both PYR (57.6-153.4 mg kg(-1) dry matter) and HMF (13-51.2 mg kg(-1) dry matter). For cost and simplicity, HMF looked like the most appropriate index in puffed cereals. Puffing influenced starch in vitro digestibility, being most of the starch (81-93%) hydrolyzed to maltotriose, maltose and glucose whereas only limited amounts of AGEs were released. The relevant antioxidant capacity revealed by digested puffed kernels can be ascribed to both the new formed Maillard reaction products and the conditions adopted during in vitro digestion.

Occurrence and fate of ACE-inhibitor peptides in cheeses and in their digestates following in vitro static gastrointestinal digestion.

The occurrence of the casein-derived angiotensin converting enzyme-inhibitor (ACE-I) peptides VPP, IPP, RYLGY, RYLG, AYFYPEL, AYFYPE, LHLPLP and HLPLP were investigated in 12 different cheese samples by Ultra Performance Liquid Chromatography/High-Resolution Mass Spectrometry. The total amount of ACE-I peptides was in the range 0.87-331mgkg(-1). VPP and IPP largely prevailed in almost all cheeses. Following in vitro static gastrointestinal digestion of Cheddar, Gorgonzola, Maasdam and Grana Padano cheeses, type and amount of ACE-I peptides changed, and only VPP, IPP, HLPLP and LHLPLP were detected in the intestinal digestates. The results evidenced that the degree of proteolysis itself cannot be regarded as a promoting or hindering factor for ACE-I peptide release during cheese digestion. Moreover, the data indicated that the ACE-I potential of cheeses cannot be inferred based on the type and amount of ACE-I peptides present in undigested samples.

Modulation of fecal Clostridiales bacteria and butyrate by probiotic intervention with Lactobacillus paracasei DG varies among healthy adults.

BACKGROUND: The modulation of gut microbiota is considered to be the first target to establish probiotic efficacy in a healthy population. OBJECTIVE: This study was conducted to determine the impact of a probiotic on the intestinal microbial ecology of healthy volunteers. METHODS: High-throughput 16S ribosomal RNA gene sequencing was used to characterize the fecal microbiota in healthy adults (23-55 y old) of both sexes, before and after 4 wk of daily consumption of a capsule containing at least 24 billion viable Lactobacillus paracasei DG cells, according to a randomized, double-blind, crossover placebo-controlled design. RESULTS: Probiotic intake induced an increase in Proteobacteria (P = 0.006) and in the Clostridiales genus Coprococcus (P = 0.009), whereas the Clostridiales genus Blautia (P = 0.036) was decreased; a trend of reduction was also observed for Anaerostipes (P = 0.05) and Clostridium (P = 0.06). We also found that the probiotic effect depended on the initial butyrate concentration. In fact, participants with butyrate >100 mmol/kg of wet feces had a mean butyrate reduction of 49 ± 21% and a concomitant decrease in the sum of 6 Clostridiales genera, namely Faecalibacterium, Blautia, Anaerostipes, Pseudobutyrivibrio, Clostridium, and Butyrivibrio (P = 0.021), after the probiotic intervention. In contrast, in participants with initial butyrate concentrations <25 mmol/kg of wet feces, the probiotic contributed to a 329 ± 255% (mean ± SD) increment in butyrate concomitantly with an ∼55% decrease in Ruminococcus (P = 0.016) and a 150% increase in an abundantly represented unclassified Bacteroidales genus (P = 0.05). CONCLUSIONS: The intake of L. paracasei DG increased the Blautia:Coprococcus ratio, which, according to the literature, can potentially confer a health benefit on the host. The probiotic impact on the microbiota and on short-chain fatty acids, however, seems to strictly depend on the initial characteristics of the intestinal microbial ecosystem. In particular, fecal butyrate concentrations could represent an important biomarker for identifying subjects who may benefit from probiotic treatment. This trial was registered at www.controlled-trials.com/isrctn as ISRCTN56945491.

'Melatonin isomer' in wine is not an isomer of the melatonin but tryptophan-ethylester.

Melatonin is a neurohormone, chronobiotic, and antioxidant compound found in wine and deriving directly from grapes and/or synthesized by yeast during alcoholic fermentation. In addition, a melatonin isomer has been detected in different foods, wine among them. The special interest for melatonin isomer related to the fact that it was found in greater quantities than melatonin and probably shares some of its biological properties. Despite this, its chemical structure has not yet been defined; although some researchers hypothesize, it could be melatonin with the ethylacetamide group shifted into position N1. Thus, the aim of our study was to identify the structures of the melatonin isomer. For this purpose, melatonin and melatonin isomer in Syrah wine were separated chromatographically by a sub-2 µm particle column and detected by tandem mass spectrometry. The sample was then purified and concentrated by solid-phase extraction, hydrolyzed with alkali or esterase, and substrates and products quantified by UPLC-MS/MS. Moreover, melatonin, melatonin isomer, and their product ions were evaluated by high-resolution mass spectrometry. The amount of melatonin isomer and melatonin in the wine was 84 ± 4 and 3 ± 0 ng/mL, respectively. In the solutions, containing diluted alkali or esterase, melatonin isomer was hydrolyzed in about 8 min. Correspondingly, tryptophan was detected, and its amount increased and reached the maximum concentration in about 8 min. Melatonin concentration was not affected by diluted alkali or esterase. The fragmentation pattern of melatonin isomer was different from that of melatonin but comparable to that of tryptophan-ethylester. Finally, the so-called melatonin isomer identity was verified by cochromatography with authentic standard of tryptophan-ethylester.

Murein lytic enzyme TgaA of Bifidobacterium bifidum MIMBb75 modulates dendritic cell maturation through its cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) amidase domain.

Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system.

Targeted peptides for the quantitative evaluation of casein plasminolysis in drinking milk.

In addition to proteose peptones (PP), the extent of plasminolysis in different classes of drinking milk during storage has been evaluated by the quantification of the peptides αs2-CN (f1-25) 4P and αs2-CN (f1-21) 4P by UPLC/HR-MS. The rate of increase in the levels of all the studied peptides during storage depended on the heating process. The samples of drinking milk showed different levels of plasminolysis at their expiration dates, as revealed by αs2-CN (f1-25) 4P and αs2-CN (f1-21) 4P amounts. The different treatments applied during the manufacturing of extended shelf life (ESL) milk samples resulted in different levels of plasminolysis, confirming the heterogeneity of this class of drinking milk. The peptides from αs2-CN accumulated faster than PP in all the samples with the exception of UHT milk. Therefore, these peptides can be considered as sensitive indices of early plasminolysis in pasteurised and ESL milk.