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Selecting an adequate model to represent the mass transfer mechanisms occurring in a chromatographic process is generally complicated, which is one of the reasons why monolithic chromatography is scarcely simulated. In this study, the chromatographic separation of model proteins bovine serum albumin (BSA), β-lactoglobulin-A, and β-lactoglobulin-B on an anion exchange monolith was simulated based on experimental parameter determination, simultaneous model testing, and validation under three statistical criteria: retention time, dispersion accuracies, and Pearson correlation coefficient. Experimental characterization of morphologic, physicochemical, and kinetic parameters was performed through volume balances, pressure drop analysis, breakthrough curve analysis, and batch adsorptions. Free Gibbs energy indicated a spontaneous adsorption process for proteins and counterions. Dimensionless numbers were estimated based on height equivalent to a theoretical plate analysis, finding that pore diffusion controlled β-lactoglobulin separation, whereas adsorption/desorption kinetics was the dominant mechanism for BSA. The elution profiles were modeled using the transport dispersive model and the reactive dispersive model coupled with steric mass action (SMA) isotherms because these models allowed to consider most of the mass transport mechanisms that have been described. RDM-SMA presented the most accurate simulations at pH 6.0 and at low (250 mM) and high (400 mM) NaCl concentrations. This simulation will be used as reference to forecast the purification of these proteins from bovine whey waste and to extrapolate this methodology to other monolith-based separations using these three statistical criteria that have not been used previously for this purpose., (© 2024 Wiley‐VCH GmbH.)

Quantification of trace amounts of proteins is technically challenging because proteins cannot be directly amplified like nucleic acids. To improve the analytical sensitivity and to complement conventional protein analysis methods, we developed a highly sensitive and homogeneous detection strategy called Protein-Induced DNA Dumbbell Amplification (PINDA). PINDA combines protein recognition with exponential nucleic acid amplification by using protein binding probes made of DNA strands conjugated to protein affinity ligands. When a pair of probes bind to the same target protein, complementary nucleic acid sequences that are conjugated to each probe are brought into close proximity. The increased local concentration of the probes results in the formation of a stable dumbbell structure of the nucleic acids. The DNA dumbbell is readily amplifiable exponentially using techniques such as loop-mediated isothermal amplification. The PINDA assay eliminates the need for washing or separation steps, and is suitable for on-site applications. Detection of the model protein, thrombin, has a linear range of 10 fM-100 pM and detection limit of 10 fM. The PINDA technique is successfully applied to the analysis of dairy samples for the detection of β-lactoglobulin, a common food allergen, and Salmonella enteritidis, a foodborne pathogenic bacterium. The PINDA assay can be easily modified to detect other targets by changing the affinity ligands used to bind to the specific targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

β-Lactoglobulin (β-Lg) is a prevalent allergenic protein found in most dairy products, which poses great food safety risks for individuals with allergies, especially for infants. Sensitive and effective detection methods for such allergens are essential to reduce the risk of allergies in daily life. Herein, a fluorescent aptamer bioassay based on a dual and cyclic bidirectional strand displacement means is developed for the sensitive detection of β-Lg in infant rice porridge and milk. The aptamer in the duplex DNA probe binds β-Lg to release the assistance strand to further hybridize with two hairpins, which triggers the initiation of two cyclic amplification cycles through the polymerization, displacement, and nicking of the strands under the action of DNA polymerase and endonuclease restriction enzymes. The amplification cycles lead to the unfolding of many fluorescently quenched signal probes to exhibit substantially enhanced fluorescence recovery for detecting β-Lg. The assay can realize detection of β-Lg in concentrations as low as 4.41 pM within the range of 0.01 to 10 nM. Furthermore, our sensing method has the capability to discriminate β-Lg from other proteins with high selectivity, resulting in low levels of β-Lg detection in rice porridge and milk samples, demonstrating promising potentials of the developed sensing method for monitoring various food allergens., Competing Interests: Declarations. Ethics approval: This research did not involve human or animal samples. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

This study prepared a novel, portable and cost-effective microfluidic paper-based electrochemical analysis device (μ-PAD) using black phosphorus nanosheets@carboxylated multi-walled carbon nanotubes (BPNSs@MWCNTs-COOH) nanocomposites for β-lactoglobulin (β-LG) detection. At the appreciate ratio, the synthesized BPNSs@MWCNTs-COOH was demonstrated to not only serve as a high-quality substrate for the specific aptamer immobilization, but also improve the electron transfer capability of the sensing interface. The μ-PADs, utilizing BPNSs@MWCNTs-COOH and aptamer recognition, exhibited a wider detection range (10-1000 ng mL -1 ) and lower detection limit (LOD: 0.12 ng mL -1 ) for β-LG, and demonstrated enhanced specificity, satisfactory anti-interference ability and stability. When applied to the β-LG determination in dairy samples, the μ-PAD yielded β-LG concentrations highly correlated with those obtained using the HPLC method (R 2 : 0.9982). These results emphasized the reliable performance of the developed μ-PADs in β-LG allergen quantification, highlighting their potential as an efficient platform for the rapid screening of β-LG allergens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study is focused on fractionation of insulin-like growth factor I (IGF-I) and transforming growth factor-β2 (TGF-β2) using a new electro-based membrane process calledelectrodialysis with filtration membranes (EDFM). Before EDFM, different pretreatments were tested, and four pH conditions (4.25, 3.85, 3.45, and 3.05) were used during EDFM. It was demonstrated that a 1:1 dilution of defatted colostrum with deionized water to decrease mineral content followed by the preconcentration of GFs by UF is necessary and allow for these compounds to migrate to the recovery compartment during EDFM. MS analyses confirmed the migration, in low quantity, of only α-lactalbumin (α-la) and β-lactoglobulin (β-lg) from serocolostrum to the recovery compartment during EDFM. Consequently, the ratio of GFs to total protein in recovery compartment compared to that of feed serocolostrum solution was 60× higher at pH value 3.05, the optimal pH favoring the migration of IGF-I and TGF-β2. Finally, these optimal conditions were tested on acid whey to also demonstrate the feasibility of the proposed process on one of the main by-products of the cheese industry; the ratio of GFs to total protein was 2.7× higher in recovery compartment than in feed acid whey solution, and only α-la migrated. The technology of GF enrichment for different dairy solutions by combining ultrafiltration and electrodialysis technologies was proposed for the first time.

Beta-lactoglobulin (β-Lg), a prominent milk protein, is a major contributor to milk allergies. The quantitative assessment of β-Lg is a valuable method for assessing the allergenic potential of dairy products. In this study, a specific aptamer, β-Lg-01, with an affinity constant ( K D ) of 28.6 nM for β-Lg was screened through seven rounds of magnetic bead SELEX (MB-SELEX). A novel bio-layer interferometry (BLI)-based aptasensor was developed, which had a limit of detection (LOD) of 0.3 ng mL -1 , a linear range of 1.5 ng mL -1 -15 μg mL -1 , and a recovery rate of 102-116% among the milk samples. This aptasensor provides a potential tool for the detection and risk assessment of β-Lg within 10 min.

Bovine β-lactoglobulin (BLG) is a common allergen found in milk, and the immunoglobulin E (IgE) epitope plays a crucial role in cow milk allergy. Therefore, targeting the IgE epitope could be useful in accurately detecting BLG and assessing its allergenicity. However, producing an IgE epitope-specific antibody (IgE-EsAb) through traditional methods requires complex and time-consuming procedures. Here, IgE-EsAb was purified from rabbit anti-BLG sera by immunomagnetic beads in one step. Then, a sandwich ELISA (sELISA) based on the IgE-EsAb was developed to detect BLG and predict the potential milk allergenicity in foods. The obtained IgE-EsAb could specifically recognize the target IgE epitope of BLG and exhibited high affinity and specificity. The developed IgE-EsAb-based sELISA demonstrated an ultra-wide linear range of 3.9-1.28 × 10 5 ng/mL, with a limit of detection of 0.49 ng/mL for BLG. Additionally, the proposed immunoassay showed high specificity and recoveries (91.24-109.61%). The ability of the IgE-EsAb-based sELISA to evaluate the potential milk allergenicity in foods was validated using sera from cow milk allergy patients. These results suggest that immunomagnetic beads are an effective tool for rapidly obtaining the IgE-EsAb, and our proposed sELISA could be a reliable and user-friendly method for monitoring trace amounts of BLG and predicting the potential milk allergenicity of food samples.

Thermal processing (e.g., pasteurization and sterilization) is a critical step ensuring the microbial safety of our foods. Previous work from our laboratory has examined the covalent reactions occurring between proteins and a broad selection of flavor compounds under ambient storage temperatures (25-45 °C). However, similar research on reactions of flavor compounds with a protein under thermal processing conditions has not been investigated. In the current study, covalent adduct formation between β-lactoglobulin (BLG) and 46 flavor compounds encompassing 13 different classes of functional groups was investigated under pasteurization and sterilization conditions by UPLC-ESI-QTOF-MS. BLG was chosen as a representative protein for this study because it is structurally well characterized, its molecular weight is well suited for ESI-MS analysis (18.2 kDa), and it is broadly used in the food industry. Schiff base, aza-Michael addition, and disulfide linkages were the main types of covalent interactions occurring across the reactive samples. Among them, isothiocyanates, aldehydes, and thiol-containing compounds were generally very reactive. Increasing the severity of the thermal treatment [high-temperature-short-time (HTST) pasteurization, in-container pasteurization (IC), and ultra-high-temperature (UHT) sterilization conditions] accelerated the reactions of BLG with flavor compounds, which revealed reactivity of three flavor compounds not previously observed to react at room temperature (eugenol, 4-vinyl phenol, and 3-nonen-2-one). Ketones [other than 2-hydroxy-3-methyl-2-cyclopenten-1-one (cyclotene), diketones, and unsaturated ketones], alcohols, acids, alkenes (terpenes), esters, lactones, 3-acetylpyridine, methyl anthranilate, vanillin, 2-methylthiophene, and dimethyl sulfone did not show measurable reactivity with BLG under the thermal processing conditions examined. An overall view of the data shows that the HTST heat treatment (72 °C for 15 s) had the least effect on the extent of reaction while in-container pasteurization conditions (63 °C for 30 min) produced a similar extent of reaction as the UHT (130 °C 30 s) heat treatment. These varying extents of adductation are in reasonable accord with what one might expect, given that the rates of most classes of chemical reactions occurring near ambient temperature increase by a factor of 2-4 for each increase of 10 K in temperature. Unfortunately, our methodology did not permit us to obtain meaningful data using the most aggressive standard sterilization thermal conditions (110 °C for 30 min) because extensive aggregation/coagulation removed essentially all of the BLG protein from the reaction mixtures prior to MS analysis.

Dairy processing can alter the digestion stability and bioavailability of cow milk proteins in the gastrointestinal tract. However, analysis of stable linear epitopes on cow milk allergens that could enter into intestinal mucosal is limited. Thus, this study aimed to investigate the digestion and transportation properties and residual allergen epitopes entering into gastrointestinal mucosa of 3 commercial dairy products, including pasteurized milk (PM), ultra-heat-treated milk (UHTM), and dried skim milk (DSM). In this work, the digestive stability of the 3 kinds of dairy products has been performed in a standard multistep static digestion model in vitro and characterized by Tricine-SDS-polyacrylamide gel electrophoresis and reversed-phase HPLC. With respect to gastrointestinal digestion in vitro, the main allergens including β-lactoglobulin (β-LG), α-lactalbumin (α-LA), and caseins were degraded gradually, and the resistance peptides remained in the PM with a molecular weight of range from 3.4 to 5.0 kDa. Simultaneously, the potential allergenicity of the cow milk proteins was diminished gradually and is basically consistent after 60 min of gastrointestinal digestion. After gastrointestinal digestion, the remaining peptides were transported via an Ussing chamber and identified by liquid chromatography-MS/MS. By alignment, 10 epitopes peptides were identified from 16 stable peptides, including 5 peptides (AA 92-100, 125-135, 125-138, and 149-162) in β-LG, 2 peptides in α-LA (AA 80-93 and 63-79), 2 peptides in α S1 -casein (AA 84-90 and 125-132), and 1 peptide (AA 25-32) in α S2 -casein were identified by dot-blotting mainly exist in UHTM and PM. This study demonstrates dairy processing can affect the digestion and transport characteristics of milk proteins and in turn alter epitope peptides release., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

β-Lactoglobulin (BLG) is one of the prevalent whey protein in cattle. To date, several variants of bovine BLG have been found, but the most common are A and B, which differ from each other by SNPs rs109625649 and rs110066229. Numerous studies showed effects of A and B variants of BLG on milk yield, fat and protein content and cheese-making properties. To date, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), allele-specific polymerase chain reaction (ASPCR), PCR single-strand conformation polymorphism (PCR-SSCP) and high resolution melting (HRM) methods have been proposed for detection of A and B variants of bovine BLG. These methods involve multistep sample processing, which is an essential disadvantage in conducting large-scale cattle genotyping projects. This article describes a development of TaqMan PCR assay for detection of A and B variants (rs109625649) of bovine BLG. In this method a primer pair, initiating amplification of 101-bp fragment of BLG gene, and two allele-specific TaqMan probes are used. Identification of B and A variants of BLG is based on comparison of final fluorescence intensity of FAM and VIC dyes, respectively. The developed one-step method requires less time and is more suitable for large-scale genotyping of cattle compared to the commonly used PCR-RFLP.

The possibility to apply hyperbaric storage (HS) at room temperature (20 °C) as a sustainable approach for preservation of raw skim milk was studied. Samples were stored at 200 and 150 MPa for up to 6 days. Optimal pressure for milk HS was found to be 150 MPa, since no clotting was detected for up to 6 days. 150 MPa-HS caused the irreversible inactivation of inoculated Escherichia coli (5.13 ± 0.33 logCFU mL -1 ) and Staphylococcus aureus (5.66 ± 0.93 logCFU mL -1 ) within 2 and 6 days, respectively. Inactivation of total and faecal coliforms (3.0 log reductions) below the detection limit was achieved after just 2 days, whereas lactic acid bacteria and coagulase-positive Staphylococci were inactivated after 6 days. Pressurized storage also caused an increase in proteose peptones and the release of submicelles from casein micelles. Micelles progressively aggregated with pressure-unfolded β-Lactoglobulin. These phenomena led to milk presenting up to 4-fold better foaming capacity, probably due to β-Lactoglobulin unfolding or higher proteose peptones content. This work demonstrated the capability of HS to guarantee milk preservation during storage, and brought attention on the opportunity to consider the technology for milk pasteurization and functionality improvement., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Although bovine milk is regarded as healthy and nutritious, its high content of saturated fatty acids (FA) may be harmful to cardiovascular health. Palmitic acid (C16:0) is the predominant saturated FA in milk with adverse health effects that could be countered by substituting it with higher levels of unsaturated FA, such as oleic acid (C18:1cis-9). In this work, we performed genome-wide association analyses for milk fatty acids predicted from FTIR spectroscopy data using 1811 Norwegian Red cattle genotyped and imputed to a high-density 777k single nucleotide polymorphism (SNP)-array. In a follow-up analysis, we used imputed whole-genome sequence data to detect genetic variants that are involved in FTIR-predicted levels of C16:0 and C18:1cis-9 and explore the transcript profile and protein level of candidate genes., Results: Genome-wise significant associations were detected for C16:0 on Bos taurus (BTA) autosomes 11, 16 and 27, and for C18:1cis-9 on BTA5, 13 and 19. Closer examination of a significant locus on BTA11 identified the PAEP gene, which encodes the milk protein β-lactoglobulin, as a particularly attractive positional candidate gene. At this locus, we discovered a tightly linked cluster of genetic variants in coding and regulatory sequences that have opposing effects on the levels of C16:0 and C18:1cis-9. The favourable haplotype, linked to reduced levels of C16:0 and increased levels of C18:1cis-9 was also associated with a marked reduction in PAEP expression and β-lactoglobulin protein levels. β-lactoglobulin is the most abundant whey protein in milk and lower levels are associated with important dairy production parameters such as improved cheese yield., Conclusions: The genetic variants detected in this study may be used in breeding to produce milk with an improved FA health-profile and enhanced cheese-making properties., (© 2022. The Author(s).)

A fluorescence detection method based on quantum dot-aptamer-graphene oxide probes (QD-Apt-GO) was developed to detect β-lactoglobulin (β-LG) in foods. When β-LG was present in the samples, it specifically bound to the aptamer, inhibiting the binding of probes to graphene oxide (GO), and the fluorescence of the probes could be detected. When β-LG was not present, the probes could bind to GO through π-π stacking, and the fluorescence was consequently quenched. The detection range of the optimized assay for β-LG detection was 0.36-500 mg L -1 . The limit of detection (LOD) for β-LG was 96.91 μg L -1 . The method was also validated for food sample detection. In the spike and recovery experiments of Neocate amino acid infant formula, infant millet cookies, and infant rice porridge, the recoveries were in the range of 83.33-114.53%, which met the required range of the addition recoveries. At the same time, the results were consistent with those of commercial ELISA kits. Three types of random food products purchased from a local market were analyzed for β-LG via the developed assay and using a commercial ELISA kit. The results showed good accuracy and consistency between the proposed method and the commercial ELISA kit.

Background: Milk samples from 10,641 dairy cattle were screened by a mass spectrometry method for extreme concentrations of the A or B isoforms of the whey protein, β-lactoglobulin (BLG), to identify causative genetic variation driving changes in BLG concentration., Results: A cohort of cows, from a single sire family, was identified that produced milk containing a low concentration of the BLG B protein isoform. A genome-wide association study (GWAS) of BLG B protein isoform concentration in milk from AB heterozygous cows, detected a group of highly significant single nucleotide polymorphisms (SNPs) within or close to the BLG gene. Among these was a synonymous G/A variation at position + 78 bp in exon 1 of the BLG gene (chr11:103256256G > A). The effect of the A allele of this SNP (which we named B') on BLG expression was evaluated in a luciferase reporter assay in transfected CHO-K1 and MCF-7 cells. In both cell types, the presence of the B' allele in a plasmid containing the bovine BLG gene from -922 to + 898 bp (relative to the transcription initiation site) resulted in a 60% relative reduction in mRNA expression, compared to the plasmid containing the wild-type B sequence allele. Examination of a mammary RNAseq dataset (n = 391) identified 14 heterozygous carriers of the B' allele which were homozygous for the BLG B protein isoform (BB'). The level of expression of the BLG B' allele was 41.9 ± 1.0% of that of the wild-type BLG B allele. Milk samples from three cows, homozygous for the A allele at chr11:103,256,256 (B'B'), were analysed (HPLC) and showed BLG concentrations of 1.04, 1.26 and 1.83 g/L relative to a mean of 4.84 g/L in milk from 16 herd contemporaries of mixed (A and B) BLG genotypes. The mechanism by which B' downregulates milk BLG concentration remains to be determined., Conclusions: High-throughput screening and identification of outliers, enabled the discovery of a synonymous G > A mutation in exon 1 of the B allele of the BLG gene (B'), which reduced the milk concentration of β-lactoglobulin B protein isoform, by more than 50%. Milk from cows carrying the B' allele is expected to have improved processing characteristics, particularly for cheese-making., (© 2022. The Author(s).)

The nanopore is emerging as a means of single-molecule protein sensing. However, proteins demonstrate different charge properties, which complicates the design of a sensor that can achieve simultaneous sensing of differently charged proteins. In this work, we introduce an asymmetric electrolyte buffer combined with the Mycobacterium smegmatis porin A (MspA) nanopore to form an electroosmotic flow (EOF) trap. Apo- and holo-myoglobin, which differ in only a single heme, can be fully distinguished by this method. Direct discrimination of lysozyme, apo/holo-myoglobin, and the ACTR/NCBD protein complex, which are basic, neutral, and acidic proteins, respectively, was simultaneously achieved by the MspA EOF trap. To automate event classification, multiple event features were extracted to build a machine learning model, with which a 99.9% accuracy is achieved. The demonstrated method was also applied to identify single molecules of α-lactalbumin and β-lactoglobulin directly from whey protein powder. This protein-sensing strategy is useful in direct recognition of a protein from a mixture, suggesting its prospective use in rapid and sensitive detection of biomarkers or real-time protein structural analysis.

Infant formulas, designed to provide similar nutritional composition and performance to human milk, are recommended when breastfeeding is not enough to provide for the nutritional needs of children under 12 months of age. In this context, the present study aimed to assess the protein quality and essential amino acid content of both starting (phase 1) and follow-up (phase 2) formulas from different manufacturers. The chemical amino acid score and protein digestibility corrected by the amino acid score were calculated. The determined protein contents in most formulas were above the maximum limit recommended by FAO and WHO guidelines and at odds with the protein contents declared in the label. All infant formulas contained lactoferrin (0.06 to 0.44 g·100 g -1 ) and α-lactalbumin (0.02 to 1.34 g·100 g -1 ) below recommended concentrations, whereas ĸ-casein (8.28 to 12.91 g·100 g -1 ), α-casein (0.70 to 2.28 g·100 g -1 ) and β-lactoglobulin (1.32 to 4.19 g·100 g -1 ) were detected above recommended concentrations. Essential amino acid quantification indicated that threonine, leucine and phenylalanine were the most abundant amino acids found in the investigated infant formulas. In conclusion, infant formulas are still unconforming to nutritional breast milk quality and must be improved in order to follow current global health authority guidelines.

The bovine milk allergenic protein, 'β-lactoglobulin' is one of the leading causes of milk allergic reaction. In this research, a novel label-free non-faradaic capacitive aptasensor was designed to detect β-lactoglobulin using a Laser Scribed Graphene (LSG) electrode. The graphene was directly engraved into a microgapped (~ 95 µm) capacitor-electrode pattern on a flexible polyimide (PI) film via a simple one-step CO 2 laser irradiation. The novel hybrid nanoflower (NF) was synthesized using 1,1'-carbonyldiimidazole (CDI) as the organic molecule and copper (Cu) as the inorganic molecule via one-pot biomineralization by tuning the reaction time and concentration. NF was fixed on the pre-modified PI film at the triangular junction of the LSG microgap specifically for bio-capturing β-lactoglobulin. The fine-tuned CDI-Cu NF revealed the flower-like structures was viewed through field emission scanning electron microscopy. Fourier-transform infrared spectroscopy showed the interactions with PI film, CDI-Cu NF, oligoaptamer and β-lactoglobulin. The non-faradaic sensing of milk allergen β-lactoglobulin corresponds to a higher loading of oligoaptamer on 3D-structured CDI-Cu NF, with a linear range detection from 1 ag/ml to 100 fg/ml and attomolar (1 ag/ml) detection limit (S/N = 3:1). This novel CDI-Cu NF/LSG microgap aptasensor has a great potential for the detection of milk allergen with high-specificity and sensitivity., (© 2021. The Author(s).)

Human clinical trials have shown that a specific partially hydrolyzed 100% whey-based infant formula (pHF-W) reduces AD risk in the first yeast of life. Meta-analyses with a specific pHF-W (pHF-W1) confirm a protective effect while other meta-analyses pooling different pHF-W show conflicting results. Here we investigated the molecular composition and functional properties of the specific pHF-W1 as well as the stability of its manufacturing process over time. This specific pHF-W1 was compared with other pHF-Ws. We used size exclusion chromatography to characterize the peptide molecular weight (MW), a rat basophil degranulation assay to assess the relative level of beta-lactoglobulin (BLG) allergenicity and a preclinical model of oral tolerance induction to test prevention of allergic sensitization. To analyze the exact peptide sequences before and after an HLA binding assay, a mass cytometry approach was used. Peptide size allergenicity and oral tolerance induction were conserved across pHF-W1 batches of production and time. The median MW of the 37 samples of pHF-W1 tested was 800 ± 400 Da. Further oral tolerance induction was observed using 10 different batches of the pHF-W1 with a mean reduction of BLG-specific IgE levels of 0.76 log (95% CI = -0.95; -0.57). When comparing pHF-W1 with three other formulas (pHF-W2 3 and 4), peptide size was not necessarily associated with allergenicity reduction in vitro nor oral tolerance induction in vivo as measured by specific IgE level ( p < 0.05 for pHF-W1 and 2 and p = 0.271 and p = 0.189 for pHF-W3 and 4 respectively). Peptide composition showed a limited overlap between the formulas tested ranging from 11.7% to 24.2%. Furthermore nine regions in the BLG sequence were identified as binding HLA-DR. In conclusion, not all pHF-Ws tested have the same peptide size distribution decreased allergenicity and ability to induce oral tolerance. Specific peptides are released during the different processes used by different infant formula producers.

Background: beta-lactoglobulin (BLG) is one of the major cow's milk proteins and the most abundant allergen in whey. Heating is a common technologic treatment applied during milk transformational processes. Maillardation of BLG in the presence of reducing sugars and elevated temperatures may influence its antigenicity and allergenicity., Primary Objective: to analyze and identify lactosylation sites by capillary electrophoresis mass spectrometry (CE-MS)., Secondary Objective: to assess the effect of lactosylated BLG on antigenicity and degranulation of mast cells., Methods: BLG was lactosylated at pH 7, a water activity (aw) of 0.43, and a temperature of 65 °C using a molar ratio BLG:lactose of 1:1 by incubating for 0, 3, 8, 16 or 24 h. For the determination of the effect on antibody-binding capacity of lactosylated BLG, an ELISA was performed. For the assessment of degranulation of the cell-line RBL-hεIa-2B12 transfected with the human α-chain, Fcε receptor type 1 (FcεRI) was used., Results: BLG showed saturated lactosylation between 8 and 16 incubation hours in our experimental setup. Initial stage lactosylation sites L1 (N-terminus)-K47, K60, K75, K77, K91, K138 and K141-have been identified using CE-MS. Lactosylated BLG showed a significant reduction of both the IgG binding ( p = 0.0001) as well as degranulation of anti-BLG IgE-sensitized RBL-hεIa-2B12 cells ( p < 0.0001)., Conclusions and Clinical Relevance: this study shows that lactosylation of BLG decreases both the antigenicity and degranulation of mast cells and can therefore be a promising approach for reducing allergenicity of cow's milk allergens provided that the process is well-controlled.

Bovine whey protein was hydrolysed using cardosins A and B purified from dried flowers of Cynara cardunculus by combining diafiltration, anion-exchange chromatography and ultrafiltration. The proteolysis experiments were performed using different whey protein concentrations and enzyme/substrate (E/S) ratios. Complete hydrolysis of the main whey proteins, β-Lactoglobulin (β-Lg) and α-lactalbumin (α-La), was achieved after 4 h, at E/S ratios of 1/150 U/mg, regardless the initial protein concentration. In previous reports, the authors suggested that cardosins could not hydrolyse β-lactoblogulin. However, our promising results open up new possibilities to further explore the action of cardosins on whey proteins for the production of bioactive peptides., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Whey represents a valuable protein source for human nutrition. Whey composition varies with respect to process characteristics during milk processing. For efficient exploitation of this dairy side stream, reliable analytical methods are essential. The aim of this study was to develop and validate an RP-HPLC-DAD method for the simultaneous quantification of the minor (lactoferrin, lactoperoxidase, bovine serum albumin) and major (α-lactalbumin, β-lactoglobulin) whey proteins. Seven RP-columns were compared and the composition of the mobile phase was optimized to achieve baseline separation. In validation experiments the limits of detection (LOD < 8 mg/L) and quantification (LOQ < 24 mg/L) were determined. Validity was proofed by precision (>96%), accuracy (95% - 103%) and recovery (96% - 102%) measurements. Peak homogeneity was confirmed by SDS-PAGE. The individual working ranges were adjusted to the estimated protein concentrations in whey, allowing direct analysis without sample preparation at a method runtime of 23 min., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Identification of glycomacropeptide (GMP) and β-lactoglobulin (β-lg) present in cheese whey is difficult on SDS-PAGE due to their close proximity during electrophoresis and poor sensitivity of commonly used staining dye 'coomassie brilliant blue' (CBB) towards GMP. A simple method has been developed for the detection of GMP and β-lg by staining acrylamide gel after tricine SDS-PAGE using cationic 'stains all' dye. After staining and destaining major whey proteins, viz. ɑ-lactalbumin (ɑ-la) and β-lg appear red while GMP stains blue. The method can be used for the identification of these macromolecules in cheese whey and the detection of adulteration of milk with rennet whey., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In this study, three skimmed and one whole-fat spray-dried camel milk powders were produced and their characteristics were compared to those of bovine milk powders. The physicochemical analysis of the produced powders indicated that camel milk powders (whether skimmed or not) presented higher ash and whey protein contents as compared to those of bovine milk powders. Our results indicated that the investigated camel and bovine milk powders exhibited a high solubility index (>99%) with poor dispersibility and wettability indexes due to their small particles size (d 50 ≤ 12 µm) and their narrow size distribution (span ≤ 2). In addition, although camel and bovine milk powders presented the same total fat content, lower free fat content was measured for camel milk powders. Besides, the whey protein nitrogen index and the SDS-PAGE electrophoresis underlined that camel and bovine milk proteins remained intact after drying with low denaturation extent. It is worth noticed that camel milk proteins were less denaturized due to the absence of the heat-sensitive β-lactoglobulin in camel milk. Moreover, the low denaturation extent participated in the enhancing of the foaming capacity and stability of camel and bovine milk powders. Finally, the calorimetric analysis showed that higher fat melting temperatures were recorded in whole-fat camel milk powder and in their anhydrous form as compared to those of bovine milk. PRACTICAL APPLICATION: Camel milk powder is an emerging non-bovine dairy product. Understanding its rehydration ability and evaluating the impact of spray drying on its protein quality are promising approaches to obtain high-quality camel milk powder with high reconstitution ability. Findings of this study indicated that spray drying is a suitable technique to produce highly soluble camel milk powders with low denaturation extent. These results will benefit the research and development department of food industry (especially those producing camel milk powder) as well as the direct consumers., (© 2020 Institute of Food Technologists®.)

β-lactoglobulin is one of the nutrition allergens present in the milk of many mammals, with the exception of human. This protein belongs to the family of lipocalins, consisting of nine antiparallel β-strands (β-A to β-I) and one α-helix. This structure allows it to serve as a nanotransporter of various nature ligands in a pH dependent manner, which allows us to confidently consider it as a reliable carrier of drugs directly into the intestine, bypassing the destructive acidic environment of the stomach. Based on the latest data, this review describes the currently known methods of reducing the allergenicity of beta-lactoglobulin, as well as the mechanisms and methods of forming complexes of this protein with ligands, which emphasizes its importance and versatility and explains the growing interest in studying its properties in recent decades, and also opens up prospects for its practical application in medicine and pharmaceuticals., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Surface activity is an intrinsic protein feature, leading to the capability of aqueous protein solutions to form foam. This feature provides opportunities for downstream processing, such as usage of foam fractionation for purification. In order to investigate the impact of the surface activity on the performance of the foam fractionation process, protein solutions with different surface activity were produced by different thermal denaturation of aqueous β-lactoglobulin solutions. The effectiveness of the denaturation procedure was verified with circular dichroic spectroscopy, and the impact on surface activity was determined via dynamic surface tension measurement. The increased surface activity resulted in higher foamate flow rates. Furthermore, the effects could be correlated with secondary structure changes and with the dynamic surface pressure. The new result of this study is that the effect of the denaturation of a protein on foam fractionation depends on the protein concentration. At the lower feed concentration, effects became visible, which could not be observed at the higher concentration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A simple and low-cost electrochemical sensor based on multimodified screen-printed electrodes (SPEs) was successfully synthesized for the sensitive detection of β-lactoglobulin (β-Lg). The surface treatment of SPEs was accomplished by a simple drip coating method using polyethyleneimine (PEI), reduced graphene oxide (rGO), and gold nanoclusters (AuNCs), and the treated SPEs showed excellent electrical conductivity. The modified SPEs were then characterized with UV-Vis, SEM, TEM, and FTIR to analyze the morphology and composition of the AuNCs and the rGO. An anti-β-Lg antibody was then immobilized on the composite material obtained by modifying rGO with PEI and AuNCs (PEI-rGO-AuNCs), leading to the remarkable reduction in conductivity of the SPEs due to the reaction between antigen and antibody. The sensor obtained using this novel approach enabled a limit of detection (LOD) of 0.08 ng/mL and a detection range from 0.01 to 100 ng/mL for β-Lg. Furthermore, pure milk samples from four milk brands were measured using electrochemical sensors, and the results were in excellent agreement with those from commercial enzyme-linked immunosorbent assay (ELISA) methods.

Beta-lactoglobulin is a natural milk protein and the main cause of infant milk allergy. In this work, a sensitive, selective and inexpensive electrochemical biosensor for the detection of β-lactoglobulin was developed. In this sensor, a DNA aptamer was used instead of an expensive antibody as the recognition group highly selective for β-lactoglobulin. The flower-like BiVO 4 microspheres were firstly found to have peroxidase mimic catalytic activity and used to amplify the electrochemical signal. The aptamer can bind β-lactoglobulin and fall off from the working electrode, after which the DNA2/Au/BiVO 4 probe can be fixed to the DNA1/AuNPs/ITO working electrode by the hybridization of DNA2 with DNA1. Therefore, a higher concentration of β-lactoglobulin leads to increased fabrication of the DNA2/Au/BiVO 4 probe on the surface of the working electrode, and thereby increases the electrochemical signal. This electrochemical biosensor exhibited a wide detection range from 0.01 to 1000 ng mL -1 , with a limit of detection (LOD) of 0.007 ng mL -1 , which indicates a good potential application in the field of food analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We investigated the effect of seasonal variations on the acid gelation properties of bovine milk in a seasonal-calving New Zealand herd for 2 full milking seasons. We tested the formation of acid gels in 2 milk systems: unstandardized skim milk and standardized whole milk (4.6% protein, 4.0% fat). For unstandardized skim milk, late-season milk acid gels had a longer gelation time and a lower gelation pH than early- and mid-season milk acid gels, but we found no consistent seasonal variation in the final storage modulus. For standardized milk, late-season milk had the most inferior acid gelation properties during the year, including the lowest final storage modulus, the lowest gelation pH, and the longest gelation time. Standardization alleviated but did not eliminate the prolonged gelation time of late-season milk. These results indicated that the physicochemical properties of seasonal milk contributed greatly to its acid gelation, independent of differences in protein content. Standardization was not adequate to stabilize the acid gelation properties of late-season milk. Desirable acid gelation properties correlated with lower glycosylated κ-casein content, lower β-lactoglobulin:α-lactalbumin ratio, lower extent of whey protein-casein micelle association, and lower total calcium and ionic calcium content. We discuss the possible effects of the correlating variables on the acid gelation properties of seasonal milk. Natural variations in the glycosylation degree of κ-casein might play an important role in acid gel structural development by altering the electrostatic and hydrophobic interactions among the milk proteins., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The influence of ultrasonic pretreatment on the structure, antioxidant activity, and IgG/IgE binding activity of β-lactoglobulin (β-Lg) during digestion in vitro were studied by spectroscopy, chromatography and ELISA. After β-Lg was treated by ultrasonic prior to digestion in vitro, the treated β-Lg showed high intrinsic fluorescence emission, more of the hydrolytic products and high antioxidant activity, which depend on the proteolysis promoted by ultrasonic, and produced many small-molecule antioxidant peptides. Native β-Lg was resistant to gastric digestion and retained its allergenicity. However, the allergenicity of ultrasonicated β-Lg after gastric digestion was increased due to ultrasonic promotes the production of peptides with intact structure and immunogenicity. Subsequent gastrointestinal digestion, ultrasonicated β-Lg has a diametrically opposite results because the increase of small peptides with the decreasing of immunogenicity. Therefore, the structural changes of β-Lg by ultrasonic and gastrointestinal digestion were responsible for improving the antioxidant activity and reducing the IgG/IgE binding activity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Recently, we were consulted about a challenging case, where an infant died by poisoning and the drug-dependent mother insisted that she unintentionally gave the toxic drug through breast milk. Accordingly, we investigated the utility of immunoblotting and microRNA (miRNA) profiling of the infant's gastric content (GC) to differentiate between breast-feeding and formula-feeding. As a pilot study, we sampled the GC from breast-fed (GCB) and formula-fed (GCF) infants, as well as gastric juice (GJ) from fasted adults at autopsy. Breast milk (BM) samples were collected from volunteers within 1 year post-delivery. By immunoblotting, lactoferrin and gross cystic disease fluid protein (GCDEP) were clearly detected in BM, but could not be detected in GCB. Similarly, β-lactoglobulin was detected in formula milk, but could not be detected in GCF. Meanwhile, miRNA sequencing revealed that the miRNA expression profile of GCB was closer to BM than GCF and GJ. Especially, miR-151a and miR-186 were more abundant in BM and GCB than in GCF and GJ. Our study is the first to elucidate the human GJ miRNA profile and demonstrate the possibility that miR-151a and miR-186 in GC may be the biomarker of breast-feeding.

Human milk contains proteins and/or protein fragments that originate from nonhuman organisms. These proteinaceous molecules, of which the secretion might be related to the mother's allergy status, could be involved in the development of the immune system of the infant. This may lead, for example, to sensitization or the induction of allergen-specific tolerance. The aim of this study was to investigate the relation between maternal allergy and the levels of nonhuman proteinaceous molecules in their milk. In this study, we analysed trypsin-digested human milk serum proteins of 10 allergic mothers and 10 nonallergic mothers. A search was carried out to identify peptide sequences originating from bovine or other allergenic proteins. Several methods were applied to confirm the identification of these sequences, and the differences between both groups were investigated. Out of the 78 identified nonhuman peptide sequences, 62 sequences matched Bos taurus proteins. Eight peptide sequences of bovine β -lactoglobulin had significantly higher levels in milk from allergic mothers than in milk from nonallergic mothers. Dietary bovine β -lactoglobulin may be absorbed through the intestinal barrier and secreted into human milk. This seems to be significantly higher in allergic mothers and might have consequences for the development of the immune system of their breastfed infant.

An aptamer-amphiphile was designed that binds to β-lactoglobulin (β-LG), a major allergen from cow's milk. For this work, a 23-nucleotide ssDNA aptamer β-LG-23, capable of forming antiparallel G-quadruplexes was used, and its specificity and binding affinity of 22 ± 2 nM for β-LG were evaluated via enzyme-linked apta-sorbent assay (ELASA). The β-LG-23 aptamer was synthesized as an amphiphile by conjugating it to a C 16 double tail via different spacers, and the effect of the spacers on the binding affinity and secondary structure of the aptamer was investigated. From all amphiphiles tested, direct conjugation of the aptamer to the tail gave the lowest binding affinity to β-LG (37 ± 2 nM), while maintaining the antiparallel G-quadruplex secondary structure of the aptamer. As a proof of concept, the β-LG-23 aptamer-amphiphile was used to decorate the interface of a liquid crystal (LC) and effectively detected 10 nM or 0.18 ppm of β-LG with a 20 min equilibration time, thus demonstrating that it has the potential to be used for fast and label-free detection of β-LG.

The accurate determination of disulfide bonds for protein identification is in high demand. In this study, a simple electrochemical-mass spectrometry (EC-MS) method that possesses advantages of real-time information, simultaneous disulfide bond electroreduction and tagging was developed. In this EC-MS, an ITO glass corner functions as a counter electrode and spray system, and allows the direct sampling of the droplet-scale reacting solution in real-time. The application of this method was successfully demonstrated by electrochemical reduction of oxidized glutathione (GSSG) with one disulfide bond as well as insulin with multiple disulfide bonds. The preferred electroreduction of intermolecular-bonded disulfides for insulin has been observed and the intramolecular bond was not favored. Moreover, simultaneously tagging the formed thiol residues from electroreduction of GSSG using electrogenerated intermediates such as dopamine orthoquinone (DQ) and benzoquinone (Q) was performed. A proof-of-concept was also demonstrated with a large molecule, β-lactoglobulin A. The relationship between signal strength and operating parameters was also studied. This method successfully detected the reduction reaction of the disulfide bond in the polypeptide and protein. The detection limit (S/N ≥ 3) is 0.398 μg mL-1. These results suggest that this EC-MS platform can count cysteine moieties in proteins using a single drop of sample and in real-time and is promising for protein identification experiments.

Proteinaceous matter can leak into the permeate stream during ultrafiltration (UF) of milk and whey and lead to financial losses. Although manufacturers can measure protein content in the finished permeate powders, there is currently no rapid monitoring tool during UF to identify protein leak. This study applied front-face fluorescence spectroscopy (FFFS) and chemometrics to identify the fluorophore of interest associated with the protein leak, develop predictive models to quantify true protein content, and classify the types of protein leak in permeate streams. Crude protein (CP), nonprotein nitrogen (NPN), true protein (TP), tryptone-equivalent peptide (TEP), α-lactalbumin (α-LA), and β-lactoglobulin (β-LG) contents were measured for 37 lots of whey permeate and 29 lots of milk permeate from commercial manufacturers. Whey permeate contained more TEP than did milk permeate, whereas milk permeate contained more α-LA and β-LG than did whey permeate. The types of protein leak were thus identified for predictive model development. Based on excitation-emission matrix (EEM) of high- and low-TP permeates, tryptophan excitation spectra were collected for predictive model development, measuring TP content in permeate. With external validation, a useful model for quality control purposes was developed, with a root mean square error of prediction of 0.22% (dry basis) and a residual prediction deviation of 2.8. Moreover, classification models were developed using partial least square discriminant analysis. These classification methods can detect high TP level, high TEP level, and presence of α-LA or β-LG with 83.3%, 84.8%, and 98.5% cross-validated accuracy, respectively. This method showed that FFFS and chemometrics can rapidly detect protein leaks and identify the types of protein leak in UF permeate. Implementation of this method in UF processing plants can reduce financial loss from protein leaks and maintain high-quality permeate production., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Milk by-products such as whey and caseinate are widely used as ingredients or processing aids in food industry. However, since they could cause allergic reactions they are included in Allergen Control Plans. β-Lactoglobulin is the major whey protein and caseins are main proteins in milk. Selection of a unique target to analyze the presence of milk in foods could be insufficient when the source of milk proteins is unknown. A new test based on lateral flow immunocromatography that combines the simultaneous and independent detection of both proteins (β-lactoglobulin and casein) in one rapid test was developed. The assay was validated according to AOAC guidelines being able to detect β-lactoglobulin (0.5 ppm), casein (2 ppm), whey and powder milk (1-5 ppm). No cross-reactivity was found with a panel of 38 food commodities. The method is a rapid and suitable tool to identify milk proteins in processed food, ingredients, and rinsing water., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The exploration of nanomaterials with mimic enzyme activity (named nanozyme) has gained extensive attention in the fields of advanced analytical chemistry and materials science. Herein, the gold nanoparticles doped covalent organic frameworks (COFs) were prepared, which exhibited not only excellent mimic nitroreductase activity but also robust stability. By replacing the traditional natural enzyme tag in an enzyme-linked immunosorbent assay (ELISA), we employed the proposed nanozyme to label the detecting antibody. According to the catalytic properties of the nanozyme, 4-nitrothiophenol (4-NTP) was introduced as the substrate, which can be transformed to 4-aminothiophenol (4-ATP) in the presence of NaBH 4 . In a surface enhanced Raman scattering (SERS) assay, 4-ATP was capable of functioning as a powerful bridge to connect the gold nanostars (with excellent SERS performance) by both the Au-S bond and electrostatic force to further produce a Raman "hot spot". Meanwhile, the Raman signal of 4-nitrothiophenol at 1573 cm -1 was weakened, and a new signal at 1591 cm -1 generated by 4-ATP was turned on, leading to the generation of a ratiometric SERS signal. Based on this performance, a ratiometric nanozyme-linked immunosorbent assay (NELISA) strategy was developed delicately, which was applied to detect β-lactoglobulin (allergenic protein) by monitoring the ratiometric signal of I 1591 / I 1573 with a limit of detection (LOD) of 0.01 ng/mL. The linear range is 25.65-6.2 × 10 4 ng/mL, covering more than 3 orders of magnitude. The developed method showed many advantages such as low-cost, higher recovery, and lower cross-reactivity, providing new insight into the application of SERS technology for trace target analysis.

With the development of deep eutectic solvents (DESs), more DES-based functional materials have been explored and applied in various areas. In this work, a novel choline chloride-acrylic acid (ChCl-AA) DES polymer, on a 2D magnetic base, was prepared for the recognition of β-lactoglobulin (β-LG) biomacromolecules in milk and for the inhibition of common bacteria such as Escherichia coli (E. coli), Pseudomonas fluorescens (P. fluorescens), Staphylococcus aureus (S. aureus), and Bacillus subtilis (B. subtilis). The ChCl-AA DESs were polymerized on the surface of 2D MoS 2 sheets doped with nano Fe 3 O 4 particles, and the resulting polymer was abbreviated poly(ChCl-AA DES)@Fe 3 O 4 @MoS 2 . The free energy (ΔG=-92) of ChCl-AA DES was calculated using the Gaussian software, the composition and structure of poly(ChCl-AA DES)@Fe 3 O 4 @MoS 2 were characterized by field emission scanning electron microscopy, transmission electron microscopy, etc., the qualitative and quantitative analyses of β-LG were done by fluorescence spectra, sodium dodecyl sulfate polyacrylamide gel electrophoresis and high performance liquid chromatography, and the bioactivity of bacteria was analyzed by flat colony counting. Based on the present analysis, poly(ChCl-AA DES)@Fe 3 O 4 @MoS 2 specifically recognized β-LG in a good fitting Langmuir isotherm (R 2 = 0.9909) and second-order kinetic model (R 2 = 0.9989) by affinity, and evidently inhibited three bacteria, namely, E. coil (65%), S. aureus (50%), and B. subtilis (54%), effectively reducing the relative colony number. As the poly(ChCl-AA DES)@Fe 3 O 4 @MoS 2 material did not only exhibit specific recognition of biomacromolecules, but also had an antimicrobial effect against common bacteria, it could be an ideal separation media or carrier for biomacromolecules in real samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Quantitative detection of protein biomarkers is crucial to medical diagnosis. Fluorescent probes have been frequently used for protein detection, but they suffered from various weaknesses such as lack of versatility. In particular, most of the reported probes were not capable of simultaneous qualitative and quantitative detection for various proteins. In this paper, we developed novel nanoparticle array-based near-infrared (NIR) ratiometric probes for potent protein analysis, in which the specific protein was able to be distinguished and quantitated within a group of 11 common proteins. The activity of β-galactosidases (β-gal) was temporarily inhibited by the adsorption to magnetic nanoparticles and restored to certain content by replacement with detected proteins, leading to distinctive readout of the enzyme-activatable NIR probe (DCM-β-gal). The readout of the sensor array against 11 proteins, as verified by isothermal titration calorimetry, was processed and transformed into canonical factors with the help of linear discrimination analysis. Moreover, the ratiometric signals of DCM-β-gal were translated to quantitatively detect proteins within the concentration range of 0-100 μg/mL. Based on clear differentiation within both two-dimensional and three-dimensional plots, different proteins could be detected with 100% accuracy with their concentration simultaneously determined, which endowed the sensing system with great potential in clinical diagnosis.

Immunochemical detection of food allergens is usually based on the use of polyclonal or monoclonal immunoglobulins G (IgG) antibodies. However, due to differences in epitopes recognition between IgG and IgE, an epitope modification during food processing can potentially alter allergenicity and detection in such different way. For that reason, the use of traditional immunological methods to anticipate or study the protein allergenicity is not recommended. The objective of this work was to develop a standardized competitive ELISA, based on recombinant IgE antibodies to β-lactoglobulin (β-lg), to reveal and to measure changes in β-lg immunoreactivity provoked by technological processes in foods containing this milk protein. As a result, a sensitive immunochemical method (Limit of Detection, LOD < 0.2 μg mL -1 ) has been developed for β-lg recognition. Also, this technique has been confirmed to detect modifications in β-lg-IgE binding after food processing (thermal treatment) in a similar way to those results obtained with allergic patients in previous works. Consequently, data obtained with this assay could be used as a preliminary test to predict in vitro β-lg allergenic behavior in allergenicity studies in processed foods., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The question whether and which nonhuman peptides or proteins are present in human milk was raised many decades ago. However, due to cross-reactivity or nonspecific antibody recognition, the accuracy of detection by immunochemical methods has been a concern. Additionally, the relative low-abundance of nonhuman peptides/proteins in the complex milk sample makes them a challenging target to detect. Here, by deep proteome profiling, we detected several nonhuman peptides, which could be grouped as nonhuman proteins. We next estimated their concentration in human milk by combining data-dependent shotgun proteomics and parallel reaction monitoring. First, we fractionated human milk at the protein level and were able to detect 1577 human proteins. Additionally, we identified 109 nonhuman peptides, of which 71 were grouped into 36 nonhuman proteins. In the next step, we targeted 37 nonhuman peptides and nine of them could be repeatedly quantified in human milk samples. Peptides/proteins originating from bovine milk products were the dominant nonhuman proteins observed, notably bovine caseins (α-S1-, α-S2-, β-, κ-caseins) and β-lactoglobulin. The method we present here can be expanded to investigate more about nonhuman peptides and proteins in human milk and give a better understanding of how human milk plays a role in allergy prevention.

Cow's milk allergy is one of the most frequent and severe IgE-induced food allergies for children, demanding sensitive analytical methods, and β-lactoglobulin (BLG) can be as an important biomarker for detection of milk protein. In this study, a highly sensitive sandwich enzyme-linked immunosorbent assay (sELISA) based on a specific polyclonal antibody against human IgE linear epitopes of BLG and an anti-BLG polyclonal antibody-platinum nanoparticles probe was described for detection of BLG. This sELISA exhibited an ultrawide linear range of 0.49-1.6 × 10 4 ng/mL, covering more than four orders of magnitude. The limit of detection was 0.12 ng/mL, which was 16-fold lower than that using traditional sELISA with the same antibodies. Furthermore, the proposed approach showed high recoveries (93.53%-111.95%) and low coefficient of variation (1.49%-12.50%) after analysis of samples fortified with BLG. The presence of allergenic BLG residues also could be detected in partially hydrolyzed infant formulas. These results, in comparison with conventional and commercial BLG detection sELISAs, highlight that this proposed sELISA could be a reliable and user-friendly tool to monitor trace amounts of BLG and its potentially allergenic residues in foods.

The milk fat globule membrane (MFGM) plays an important role in stabilizing fat in the aqueous phase, and the components of this membrane are involved in multiple biological functions. Here, we investigated changes in the protein composition of the MFGM fraction between raw and heated whole milk using a label free proteomic approach. In total, 612 MFGM-related proteins were identified in all groups. Compared with raw milk, the number of proteins that were not identified in the MFGM fraction was increased from pasteurized milk to ultra-high-temperature milk, whereas the number of milk proteins incorporated into the MFGM fraction was similar among heated groups. The abundances of several milk proteins (β-lactoglobulin and β-casein) were increased in the heated milk groups in a temperature-dependent manner. From our functional analysis, proteins that were not identified in the MFGM fraction of heated milk were mainly associated with multiple biological functions. These findings provided novel insights into the effects of heat procedures on MFGM protein components and their potential physiological functions, thereby yielding data on the appropriate heating procedures to use for raw milk., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Recent data indicate that almost 60% of colostrum samples do not have enough antibodies to provide adequate protection for the calf. Thus, the aim of this study was to determine the influence of the level of production of cows on the content of immunostimulating components of protein fraction of colostrum. The observations were conducted on two farms that keep dairy cows. One of the farms was selected to represent a typical performance level for the mass population of dairy cows in Poland, about 5500 kg of milk per cow per year (LI) and the second one was characterized by a higher level of production, about 8000 kg (HI)., Results: Based on the data obtained, 100% of the samples collected from LI had an immunoglobulin G (IgG) content >50 gL -1 , in the first collection. In the case of HI, 63% of the samples had an IgG content >50 gL -1 , with a distribution in the range of 10.7 to 72.7 gL -1 . The colostrum of LI cows was characterized by a higher content of lactoferrin, lysozyme, α-lactalbumin, and β-lactoglobulin compared to the milk of HI cows., Conclusions: The immunostimulating components of colostrum declined rapidly in subsequent milkings or with the increased productivity of the cows. The concentration of IgG was negatively correlated with the performance level of cows, which means that high-producing cows had colostrum with a low concentration of IgG. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

In this work, we present a setup for mid-IR measurements of the protein amide I and amide II bands in aqueous solution. Employing a latest generation external cavity-quantum cascade laser (EC-QCL) at room temperature in pulsed operation mode allowed implementing a high optical path length of 31 μm that ensures robust sample handling. By application of a data processing routine, which removes occasionally deviating EC-QCL scans, the noise level could be lowered by a factor of 4. The thereby accomplished signal-to-noise ratio is better by a factor of approximately 2 compared to research-grade Fourier transform infrared (FT-IR) spectrometers at equal acquisition times. Employing this setup, characteristic spectral features of three representative proteins with different secondary structures could be measured at concentrations as low as 1 mg mL -1 . Mathematical evaluation of the spectral overlap confirms excellent agreement of the quantum cascade laser infrared spectroscropy (QCL-IR) transmission measurements with protein spectra acquired by FT-IR spectroscopy. The presented setup combines performance surpassing FT-IR spectroscopy with large applicable optical paths and coverage of the relevant spectral range for protein analysis. This holds high potential for future EC-QCL-based protein studies, including the investigation of dynamic secondary structure changes and chemometrics-based protein quantification in complex matrices.

Bovine milk is a recognized allergenic food source with β-lactoglobulin (BLG) as its major allergen. Reliable detection of BLG epitopes can, therefore, be a useful marker for the presence of milk in processed food products, and for potential allergenicity. At the present, enzyme-linked immunosorbent assays (ELISA) for the detection of BLG are time-consuming and generally not specific to BLG IgE epitopes. In this study, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-activated anti-BLG IgE epitope monoclonal antibody (mAb 1G9) was covalently bound onto the KOH-treated microtiter plate surface. Using this mAb-bound plate in sandwich combination with biotinylated anti-BLG polyclonal antibody-labeled gold nanoparticles, a linear dynamic range between 31.25 and 64 × 10 3  ng mL -1 with a limit of detection for BLG of 0.49 ng mL -1 was obtained, which is 32 times wider and 16 times more sensitive than conventional sandwich ELISA (sELISA). Total recovery of BLG in spiked food samples was found, without matrix effects. Also in partially hydrolyzed infant formulas, the allergenic BLG residues were detected quantitatively. Compared with conventional and commercial BLG detection sELISAs, our sELISA is reliable, highly BLG epitope-specific, user-friendly, and time-saving and allows accurate detection of potentially allergenic residues in different types of processed foods. This improved sELISA protocol can be easily extended to detect other well-identified and characterized food allergens. Graphical abstract IgE epitope mAb-bound plate in sandwich combination with gold probe for sensitive and rapid detection of bovine β-lactoglobulin and its potentially allergenic residues.

Many infants and young children are fed nutritional milk formulas. Although products are commonly based on cow milk, goat milk provides an alternative. We directly compared digestion of cow and goat milk proteins, varying pH, enzyme concentrations and incubation times to simulate infant and young child gastric conditions. Protein digestion and peptide formation were evaluated using electrophoresis and chromatography. Digestion of higher molecular weight whey proteins increased with decreased pH and higher enzyme concentrations of young child gastric digestion conditions compared to infant conditions. β-lactoglobulin was poorly digested under all gastric digestion conditions. Caseins reacted to pH changes differently compared to whey proteins, with less digestion of casein at pH 3.0 than at pH 5.0. Caseins from goat milk tended to be more efficiently digested compared to caseins from cow milk and peptide profiles from goat milk were distinct from cow milk., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The practice of adding adulterating substances in milk in order to raise profits is unfortunately worldwide. In addition, higher priced milk, coming from minor dairy species, is often illegally integrated with the lower priced cow milk. The presence of species-specific proteins, different from those declared in label, may be a serious problem for people with allergies. The development of proper analytical methods is therefore essential to protect consumer benefits and product authenticity. In this study, a proteomic approach for the detection of adulteration processes of specific milks in mixtures is proposed. Few microliters of milk samples have been digested with trypsin and chymotrypsin and analyzed by nanoLC-ESI-IT-MS/MS. A post-database processing was performed to obtain confident peptide sequence assignments, allowing the detection of milk adulteration at a level lower than 1%. Species-specific peptides from bovine β-lactoglobulin and αS1 casein were identified as suitable peptide markers of milk authenticity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A method for simultaneous analysis of egg and milk allergens using LC-QTOF-MS was developed. The proteins measured were α-casein, β-lactoglobulin, and ovalbumin, which are the main protein allergens in milk and eggs. The proteins were digested using trypsin, and the digests were analyzed by LC-QTOF-MS. Sixteen peaks were detected that confirmed the amino acid sequences of the digests, and a MRM method with high resolution (MRM-HR) using product ions of these peaks was applied for quantification. Next, validation studies were performed using beverage products to which milk and egg standard protein solutions had been added. Good linearity was achieved over the concentration range of 1.25 to 20 μg/g of milk and egg protein, and acceptable reproducibility and accuracy were obtained at 10 μg/g. Moreover, good agreement was also observed between LC-QTOF-MS and ELISA. These findings suggest that this LC-QTOF-MS method may be useful for determining the milk and egg protein contents of beverages.

Background: Bovine β-lactoglobulin (BLG) is the major allergen in cows' milk, and the specific epitope plays a key role in food allergy. Developing a method specifically bind to the IgE epitope is necessary for testing BLG and its allergenic residues., Results: The monoclonal antibody (1G9) specific to the IgE linear epitope for BLG was identified as high affinity and specificity. Based on 1G9, a sensitive fluorescent sandwich enzyme-linked immunosorbent assay (sELISA) was successfully developed using catalase-mediated fluorescence quenching of thiolated CdTe quantum dots in the presence of hydrogen peroxide as fluorescent signal output. The fluorescent sELISA showed high sensitivity and specificity, the limit of detection was 0.49 ng mL -1 , which was 16-fold lower than horseradish peroxidase (HRP)-based sELISA. The linear range for BLG detection were 125-4000 ng mL -1 (r = 0.9939) and 0.48-62.5 ng mL -1 (r = 0.9919). The recoveries and coefficients of variation were 94.25-109.83% and 4.38-20.29%, respectively. Allergenic residues were also detected in hydrolysed infant formulas. The results of fluorescent sELISA showed good performance as HRP-based sELISA and commercial sELISA kit., Conclusion: This proposed fluorescent sELISA could be employed to detect BLG and its allergenic residues in food with highly sensitivity, reliability, and recovery. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

The current study was designed to examine the role of galloyl moieties in the interaction of β-lactoglobulin (β-Lg) with (-)-epigallocatechin-3-gallate (EGCG) using fluorescence and CD spectroscopic methods. The interactions with β-Lg were investigated for EGCG, (-)-epigallocatechin (EGC), and the phenolic compound methyl gallate (MeG) at pH values 3.0, 5.0 and 7.4. The spectroscopic data indicated the binding constants for polyphenol complexes with β-Lg in phosphate buffer pH 7.4 to be in the order of MeG>EGCG>EGC. MeG exhibited the strongest quenching efficiency compared to EGCG and EGC, and EGC exhibited the weakest quenching efficiency. The quenching efficiency of EGCG on β-Lg fluorescence decreased with increasing pH. Polyphenols binding results in a slight secondary structural change of β-Lg, consistent with the fluorescence analysis. This study confirmed that the high binding affinity of EGCG to β-Lg is due to the galloyl functional group., (Copyright © 2017 Elsevier Ltd. All rights reserved.)