Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Effect of milk serum proteins on aggregation, bacteriostatic activity and digestion of lactoferrin after heat treatment
    Xiong, Ling ; Boeren, Sjef ; Vervoort, Jacques ; Hettinga, Kasper - \ 2020
    Food Chemistry 337 (2020). - ISSN 0308-8146
    Bacteriostatic activity - In vitro digestion - Lactoferrin - Milk serum proteins - Thermal aggregation

    To establish the effect of the presence of milk serum proteins on heat-induced changes to lactoferrin, lactoferrin alone, and lactoferrin mixed with either milk serum or β-lactoglobulin was heated at 65 °C, 70 °C and 75 °C for 30 min. After heating, the effect of milk serum proteins on aggregation of lactoferrin was characterized, after which the effect of such aggregation on digestion and bacteriostatic capacity of lactoferrin were determined. The presence of milk serum proteins accelerated the aggregation of lactoferrin during heating through thiol/disulphide interchange. Lactoferrin also formed disulphide-linked aggregates when it was heated with β-lactoglobulin. Protein aggregates formed at 75 °C were much more resistant to infant digestion, causing decreased peptide release from lactoferrin. Heating lactoferrin and milk serum proteins together accelerated the loss of bacteriostatic activity upon heating. In conclusion, heat-induced aggregation of lactoferrin with milk serum proteins affected both its digestion and its bacteriostatic activity.

    Characterizing the changes of bovine milk serum proteins after simulated industrial processing
    Liu, Yaowei ; Zhang, Wenjin ; Zhang, Lina ; Hettinga, Kasper ; Zhou, Peng - \ 2020
    Food Science and Technology = Lebensmittel-Wissenschaft und Technologie 133 (2020). - ISSN 0023-6438
    ELISA - Lactoferrin - LC-MS/MS - Proteomics - Thermal treatments

    This study investigated the changes and lactose glycosylation of milk serum proteins under simulated industrial processing conditions, including raw milk (R), holder pasteurization (L), high temperature short time pasteurization (H), extended shelf life (E), ultra-high temperature sterilization (U) and spray-drying (S). Through label-free proteomics, 433 proteins were identified in the samples. Several immune-related proteins, such as lactoferrin, complement C3, lactadherin, cystain, and lactoperoxidase, decreased in abundance after severe thermal treatments, while the abundance of caseins increased. No immunoglobulins and xanthine oxidase could be detected in milk after E, U or S treatments while 30%–60% of immunoglobulins was retained after pasteurizations. In detail, lactoferrin showed a better retention in H treatment while IgG showed a better retention in L treatment. UPLC-MS results showed that a slight lactose glycosylation occurred to α-LA and β-LG after severe thermal treatments (E, U and S). In addition, the results of LC-MS/MS based proteomics were verified by determining the lactoferrin and IgG content using ELISA. The observations here would update current information on the changes of milk proteins during traditional thermal processing and help to optimize current dairy processing.

    Peptide release after simulated infant in vitro digestion of dry heated cow’s milk protein and transport of potentially immunoreactive peptides across the caco-2 cell monolayer
    Zenker, Hannah E. ; Wichers, Harry J. ; Tomassen, Monic M.M. ; Boeren, Sjef ; Jong, Nicolette W. De; Hettinga, Kasper A. - \ 2020
    Nutrients 12 (2020)8. - ISSN 2072-6643 - 24 p.
    Allergenicity - Caco-2 cell - Cow’s milk protein - Glycation - Immunogenicity - Peptides

    Dry heating of cow’s milk protein, as applied in the production of “baked milk”, facilitates the resolution of cow’s milk allergy symptoms upon digestion. The heating and glycation-induced changes of the protein structure can affect both digestibility and immunoreactivity. The immunological consequences may be due to changes in the peptide profile of the digested dry heated milk protein. Therefore, cow’s milk protein powder was heated at low temperature (60 °C) and high temperature (130 °C) and applied to simulated infant in vitro digestion. Digestion-derived peptides after 10 min and 60 min in the intestinal phase were measured using LC-MS/MS. Moreover, digests after 10 min intestinal digestion were applied to a Caco-2 cell monolayer. T-cell epitopes were analysed using prediction software, while specific immunoglobin E (sIgE) binding epitopes were identified based on the existing literature. The largest number of sIgE binding epitopes was found in unheated samples, while T-cell epitopes were equally represented in all samples. Transport of glycated peptide indicated a preference for glucosyl lysine and lactosyl-lysine-modified peptides, while transport of peptides containing epitope structures was limited. This showed that the release of immunoreactive peptides can be affected by the applied heating conditions; however, availability of peptides containing epitopes might be limited.

    Hydrophobicity drives receptor-mediated uptake of heat-processed proteins by THP-1 macrophages and dendritic cells, but not cytokine responses
    Deng, Ying ; Govers, Coen ; Teodorowicz, Malgorzata ; Liobyte, Ieva ; Simone, Ilaria de; Hettinga, Kasper ; Wichers, Harry J. - \ 2020
    PLoS ONE 15 (2020)8 August. - ISSN 1932-6203

    Although an impact of processing on immunogenicity of food proteins has clearly been demonstrated, the underlying mechanisms are still unclear. We applied 3 different processing methods: wet heating (60 C) and low- or high-temperature (50 C or 130 C, respectively) dry-heating in absence or presence of reducing sugars, to β-lactoglobulin (BLG), lysozyme and thyroglobulin, which represent dietary proteins with different pI or molecular weight. Uptake of the soluble fraction of the samples was tested in two types of, genetically homogeneous, antigen-presenting cells (macrophages and dendritic cells derived from THP-1 monocytes). This revealed a strong correlation between the uptake of the different protein samples by macrophages and dendritic cells, and confirmed the key role of hydrophobicity, over aggregation, in determining the uptake. Several uptake routes were shown to contribute to the uptake of BLG by macrophages. However, cytokine responses following exposure of macrophages to BLG samples were not related to the levels of uptake. Together, our results demonstrate that heat-treatment-induced increased hydrophobicity is the prime driving factor in uptake, but not in cytokine production, by THP-1 macrophages.

    Short communication: Short-time freezing does not alter the sensory properties or the physical stability of ultra-high-temperature hydrolyzed-lactose milk
    Bottiroli, R. ; Zhang, C. ; Aprea, E. ; Fogliano, V. ; Hettinga, K. ; Gasperi, F. - \ 2020
    Journal of Dairy Science (2020). - ISSN 0022-0302
    aroma compound - freezing - hydrolyzed-lactose milk - physical stability - sensory property

    In this study, the effect of milk freezing was studied, focusing on the changes in 1% and 3% fat UHT hydrolyzed-lactose milk after slow- (−20°C) and fast-rate freezing (−80°C) for 72 h. Changes on the sensory properties were first assessed by discriminant analysis (triangle test), and then by volatile organic compounds and color analysis. The milk emulsion stability was characterized by optical centrifugation, particle size analysis, and confocal microscopy. The sensory panel was not able to distinguish the milk subjected to freezing from the control (72 h at 20°C). The volatile organic compounds and color analysis demonstrated that both freezing rates did not cause any significant changes in the milk aroma or color characteristics. The results of physical properties confirmed that short-time freezing did not lead to a distinct destabilization, except for a slight increase in the mean particle diameter at −80°C. Taking all the results together, UHT hydrolyzed-lactose milk was not significantly altered during the operation of freezing and thawing and, therefore, short-time freezing at both −20°C and −80°C can be used for milk storage without altering the product.

    Opportunities for fraudsters : When would profitable milk adulterations go unnoticed by common, standardized FTIR measurements?
    Yang, Yuzheng ; Hettinga, Kasper A. ; Erasmus, Sara W. ; Pustjens, Annemieke M. ; Ruth, Saskia M. van - \ 2020
    Food Research International 136 (2020). - ISSN 0963-9969
    Ammonium chloride (PubChem CID: 25517) - Ammonium sulphate (PubChem CID: 6097028) - Dicyandiamide (PubChem CID: 10005) - Formaldehyde (PubChem CID: 712) - Fourier transform infrared - Fructose (PubChem CID: 5984) - Glucose (PubChem CID: 79025) - Hydrogen peroxide (PubChem CID: 784) - Lactose (PubChem CID: 104938) - Maltodextrin (PubChem CID: 68229136) - Melamine (PubChem CID: 7955) - Milk adulteration - Milk composition - Milkoscan measurements - One class classification - Profitability - Sodium bicarbonate (PubChem CID: 516892) - Sodium carbonate (PubChem CID: 10340) - Sodium citrate (PubChem CID: 23666341) - Sodium hydroxide (PubChem CID: 14798) - Starch (PubChem CID: 24836924) - Sucrose (PubChem CID: 5988) - Urea (PubChem CID: 1176)

    Milk is regarded as one of the top food products susceptible to adulteration where its valuable components are specifically identified as high-risk indicators for milk fraud. The current study explores the impact of common milk adulterants on the apparent compositional parameters of milk from the Dutch market as measured by standardized Fourier transform infrared (FTIR) spectroscopy. More precisely, it examines the detectability of these adulterants at various concentration levels using the compositional parameters individually, in a univariate manner, and together in a multivariate approach. In this study we used measured boundaries but also more practical variance-adjusted boundaries to set thresholds for detection of adulteration. The potential economic impact of these adulterations under a milk payment scheme is also evaluated. Twenty-four substances were used to produce various categories of milk adulterations, each at four concentration levels. These substances comprised five protein-rich adulterants, five nitrogen-based adulterants, seven carbohydrate-based adulterants, six preservatives and water, resulting in a set of 360 samples to be analysed. The results showed that the addition of protein-rich adulterants, as well as dicyandiamide and melamine, increased the apparent protein content, while the addition of carbohydrate-based adulterants, whey protein isolate, and skimmed milk powder, increased the apparent lactose content. When considering the compositional parameters univariately, especially protein- and nitrogen-based adulterants did not raise a flag of unusual apparent concentrations at lower concentration levels. Addition of preservatives also went unnoticed. The multivariate approach did not improve the level of detection. Regarding the potential profit of milk adulteration, whey protein and corn starch seem particularly interesting. Combining the artificial inflation of valuable components, the resulting potential profit, and the gaps in detection, it appears that the whey protein isolates deserve particular attention when thinking like a criminal.

    Implications of differences in safety and hygiene control practices for microbial safety and aflatoxin M1 in an emerging dairy chain : The case of Tanzania
    Ledo, James ; Hettinga, Kasper A. ; Kussaga, Jamal B. ; Luning, Pieternel A. - \ 2020
    Food Control 118 (2020). - ISSN 0956-7135
    Critical sampling location - Customised assessment tool - Fresh milk - Microbial contamination - Moulds

    The varying performance of safety and hygiene control practices by chain actors can influence the consistent production of milk of good quality and safety in dairy chains. Therefore, the study aimed to investigate if differences in safety and hygiene control practices translate into distinctions in milk quality and safety at the farm, and to analyse the implications for actors further in the Tanzanian dairy chain. A previously developed diagnostic tool, customised for emerging dairy chains, was applied to assess and differentiate the performance of safety and hygiene control practices of actors from the farm to local retail shops. Based on interviews and on-site visits, each safety and hygiene control practice were differentiated into a poor, basic, intermediate or standard level. Milk samples were collected with a 7-day interval over three-time points to determine total bacterial counts (TBC), coliforms and Staphylococcus aureus. Besides, aflatoxin M1 (AFM1) occurrence was determined in farm milk as an indication of feed storage and monitoring practices. Data showed that none of the chain actors attained the standard level on any of the safety and hygiene control practices. Cluster analysis of on-farm safety and hygiene control practices generated two clusters, which differed mainly on the scores for udder and teat care, and disease detection practices. Differences in safety and hygiene control practices observed among farmers did not translate into differences in milk quality and safety. The analysis for AFM1 showed that 22% exceeded the maximum limit of the United States Food and Drug Authority Standard. Also, the microbial data showed that the farm milk already exceeded maximum limits of the East Africa Community (EAC) standard to the extent that no continued growth was observed further in the chain. The study demonstrates that improvements in milk quality and safety would require multiple practices to be upgraded to the standard level. Research is needed to advance the performance of control practices towards compliance with international standard requirements.

    Binding of CML-Modified as Well as Heat-Glycated β-lactoglobulin to Receptors for AGEs Is Determined by Charge and Hydrophobicity
    Zenker, Hannah E. ; Teodorowicz, Malgorzata ; Ewaz, Arifa ; Neerven, R.J.J. van; Savelkoul, Huub F.J. ; Jong, Nicolette W. De; Wichers, Harry J. ; Hettinga, Kasper A. - \ 2020
    International Journal of Molecular Sciences 21 (2020)12. - ISSN 1661-6596
    CD36 - charge - Galectin-3 - glycation - milk protein - Nε-carboxymethyl lysine - sRAGE

    Intake of dietary advanced glycation end products (AGEs) is associated with inflammation-related health problems. Nε-carboxymethyl lysine (CML) is one of the best characterised AGEs in processed food. AGEs have been described as ligands for receptors present on antigen presenting cells. However, changes in protein secondary and tertiary structure also induce binding to AGE receptors. We aimed to discriminate the role of different protein modifications in binding to AGE receptors. Therefore, β-lactoglobulin was chemically modified with glyoxylic acid to produce CML and compared to β-lactoglobulin glycated with lactose. Secondary structure was monitored with circular dichroism, while hydrophobicity and formation of β-sheet structures was measured with ANS-assay and ThT-assay, respectively. Aggregation was monitored using native-PAGE. Binding to sRAGE, CD36, and galectin-3 was measured using inhibition ELISA. Even though no changes in secondary structure were observed in all tested samples, binding to AGE receptors increased with CML concentration of CML-modified β-lactoglobulin. The negative charge of CML was a crucial determinant for the binding of protein bound CML, while binding of glycated BLG was determined by increasing hydrophobicity. This shows that sRAGE, galectin-3, and CD36 bind to protein bound CML and points out the role of negatively charged AGEs in binding to AGE receptors.

    Loss of allergy-protective capacity of raw cow's milk after heat treatment coincides with loss of immunologically active whey proteins
    Abbring, Suzanne ; Xiong, Ling ; Diks, Mara A.P. ; Baars, Ton ; Garssen, Johan ; Hettinga, Kasper ; Esch, Betty C.A.M. van - \ 2020
    Food & Function 11 (2020)6. - ISSN 2042-6496 - p. 4982 - 4993.

    The allergy-protective capacity of raw cow's milk was demonstrated to be abolished after heat treatment. The heat-sensitive whey protein fraction of raw milk is often implied to be the source of this allergy-protective effect, but a direct link between these proteins and the protection against allergic diseases is missing. This study therefore aimed at investigating the mechanistic relation between heat damage to whey proteins and allergy development. Raw cow's milk was heated for 30 min at 50, 60, 65, 70, 75, or 80 °C and the native whey protein profile of these differentially heated milk samples was determined using LC-MS/MS-based proteomics. Changes in the native protein profile were subsequently related to the capacity of these milk samples to prevent the development of ovalbumin-induced food allergy in a murine animal model. A substantial loss of native whey proteins, as well as extensive protein aggregation, was observed from 75 °C. However, whey proteins with immune-related functionalities already started to denature from 65 °C, which coincided with the temperature at which a loss of allergy protection was observed in the murine model. Complement C7, monocyte differentiation antigen CD14, and polymeric immunoglobulin receptor concentrations decreased significantly at this temperature, although several other immunologically active whey proteins also showed a decrease around 65 °C. The current study demonstrates that immunologically active whey proteins that denature around 65 °C are of importance for the allergy-protective capacity of raw cow's milk and thereby provides key knowledge for the development of microbiologically safe alternatives to raw cow's milk.

    Serum Protein N-Glycans in Colostrum and Mature Milk of Chinese Mothers
    Elwakiel, Mohèb ; Bakx, Edwin J. ; Szeto, Ignatius M. ; Li, Yitong ; Hettinga, Kasper A. ; Schols, Henk A. - \ 2020
    Journal of Agricultural and Food Chemistry 68 (2020)25. - ISSN 0021-8561 - p. 6873 - 6883.
    glycoproteins - glycosylation - intestinal mucosal barrier - oligosaccharides

    To study the Chinese human milk N-glycome over lactation, N-glycans were released and separated from serum proteins, purified by solid-phase extraction, and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 66 different putative N-glycans were found in the colostrum (week 1) and mature milk (week 4) of seven Chinese mothers. A clear difference was observed between milk of five secretor and two nonsecretor mothers, based on the type and relative amounts of the individual N-glycans. The relative levels of the total neutral nonfucosylated and the fucosylated N-glycans in milk of five secretor mothers increased and decreased over lactation, respectively. This pattern could not be observed for the milk from the two nonsecretor mothers. Overall, this was the first study that provided detailed information on individual N-glycans in milk among mothers and over time as well as that fucosylation of N-glycans in milk was associated with the mother's secretor status.

    The effect of low vs. high temperature dry heating on solubility and digestibility of cow's milk protein
    Zenker, Hannah E. ; Raupbach, Jana ; Boeren, Sjef ; Wichers, Harry J. ; Hettinga, Kasper A. - \ 2020
    Food Hydrocolloids 109 (2020). - ISSN 0268-005X
    Crosslinking - Digestibility - Dry heating - Maillard reaction - Milk protein - Solubility

    Dry heating of cow's milk protein in the presence of the milk sugar lactose leads to a loss of solubility and digestibility. Most studies that investigated the loss of solubility in milk protein powders suggested that, besides structural changes, hydrophobic interaction, hydrogen bonds, disulphide bonds, and Maillard reaction-induced crosslinking are responsible for this. However, little is known about the direct contribution of these inter- and intramolecular interactions on loss of solubility and protein digestibility. Low temperature (60 °C) and high temperature (130 °C) dry heating of cow's milk protein in the presence of lactose was applied after which both the soluble and insoluble fractions were analysed with SDS-PAGE and LC-MS/MS. The Maillard reaction was monitored by quantification of Nε-carboxymethyllysine, Nε-carboxyethyllysine, and pentosidine with LC-MS/MS. Loss of solubility was analysed with solvent solubility tests. Protein hydrolysis after simulated infant in vitro digestion, and after hydrolysis with single enzymes, was monitored using SDS-PAGE and the o-phthaldialdehyde assay. The results indicated that caseins are the main proteins that become insoluble upon dry heating, independent of the heating temperature. The decreased solubility of low temperature dry heated cow's milk protein is induced by hydrogen bonds and hydrophobic interactions and did not impair protein hydrolysis. At the same time, covalent protein crosslinking is an important determinant in protein solubility and digestibility of high temperature dry heated cow's milk protein.

    Processing-induced physicochemical changes of dietary proteins : Implications for digestion, intestinal uptake and immunological responses
    Deng, Ying - \ 2020
    Wageningen University. Promotor(en): H.J. Wichers, co-promotor(en): K.A. Hettinga; C.C.F.M. Govers. - Wageningen : Wageningen University - ISBN 9789463953818 - 167

    Heat processing is a common procedure in producing cow’s milk products in industry, amongst others to extend their shelf life. Thermal processing is reported to influence a protein’s structure and potentially its immunogenicity. This thesis describes the impact of various types of processing on structural characteristics of ß-lactoglobulin (BLG), as the major allergen in cow’s milk allergy, and on the immune response of antigen presenting cells (APCs) in particular. These cells were chosen as they are the first immune cells to come in contact with the exogenous food protein and subsequently initiate further immune responses, including the sensitization step of the allergic reaction. Next to BLG, thyroglobulin and lysozyme were studied in the thesis to estimate whether generally applicable conclusions could be drawn on the relationship between structural modifications of proteins and the subsequent immune responses.

    This thesis studied a comprehensive set of parameters that were considered to be relevant for this question, from a protein’s accessibility to intestinal APCs after processing, its physicochemical structural modifications to its uptake by APCs and further immune responses. To generate a range of structural modifications, including unfolding, aggregation and glycation, three different heating methods were used. Reducing saccharides were also included in the heating process to induce glycation, which quite commonly occurs in milk processing and is expected to be important for structural modifications and immunogenicity of the protein. The methods were wet-heating (W; heating in solution at 60 °C for 3 days) which led to aggregation of protein, high-temperature dry-heating (H; heating at 130 °C for 10 minutes) for advanced glycation and low-temperature dry-heating (L; heating at 50 °C for 9 hours) for mild glycation.

    In Chapter 2, the changes of the accessibility of BLG to intestinal immune cells after different processing methods was studied. The intestine is the main surface where exogenous proteins would interact with the immune system, thus the digestion and transport of proteins across the intestinal epithelial cell layer may be important for its accessibility to intestinal immune cells. A modified version of the current consensus in vitro digestion INFOGEST protocol and a Caco-2 insert transwell system, which mimicked the intestinal epithelial monolayer, were used. The gastric digestibility increased significantly when the protein structure was severe modified (as observed for wet-heated BLG), being specifically enhanced by the exposure of hydrophobic regions, while the influence from the presence of lactose during processing was limited. Translocation studies of BLG across Caco-2 cell monolayers showed a lower translocation rate of wet-heated BLG compared to the otherwise processed BLG-samples. Our study indicates that structural modifications of BLG after heating (especially wet-heating) enhance its digestibility, in particular gastric digestibility, and reduce the translocation efficiency across intestinal epithelial cells. Of all the heat treatment methods, the wet-heating method decreased the accessibility of BLG to intestinal APCs the strongest.

    After the gastric digestion and being translocated through the epithelial layer, protein uptake by APCs would be the first step for its interaction with immune cells. In Chapter 3, macrophages derived from THP-1 monocytes were exposed to fluorescently labelled BLG samples and the uptake was calculated after measuring the intracellular fluorescent signal. Wet-heated BLG showed significantly higher uptake by macrophages compared to native and otherwise processed BLG samples. The presence of saccharides during processing, and hence the resulting glycation, was found to be not relevant for uptake efficiency. To link its strong uptake with the structural properties of processed BLG, a set of physicochemical parameters including molecular weight, hydrophobicity, amyloid-like structure, degree of glycation, surface charge, and secondary structure was measured. Statistical analysis demonstrated that uptake was correlated strongly to hydrophobicity, amyloid-like structures, and aggregation of the protein. Due to the fact that both amyloid-like structures were reported to be related to the increase of hydrophobicity, we postulate that the exposure of hydrophobic regions and aggregation are the leading physicochemical characteristics to explain the significantly higher uptake of wet heated BLG samples by THP-1 macrophages.

    In Chapter 4, the immune response of APCs upon exposure to differently processed BLG was measured by both gene transcription and expression levels. In addition to macrophages, whose role is mainly in antigen phagocytosis, dendritic cells as another type of APCs that play an important role in signaling between the innate and adaptive immune system were also included in our study. A biological response is determined by the actor (in the case of this thesis processed protein samples) and the recipient (in this thesis the innate immune cells). To be able to obtain a clearer picture of the importance of variation in the structure of the processed proteins, a read-out system with a maximally homogenous genetic background was developed. For these experiments, macrophages and dendritic cells were both derived from THP-1 monocytes. The differences between macrophages and dendritic cells were clearly shown by their different morphology, transcription (microarray), and protein expression levels, which is in line with their specific functions in inducing an inflammatory response and T cell signaling, respectively. After incubation with native and differently heat treated BLG, stronger transcriptional responses were found in macrophages than dendritic cells. For macrophages specifically, native and low-temperature dry-heated BLG showed stronger transcription-modulating effects, while there was not a clear impact from the otherwise treated BLG samples. This might be due to the strong structural modifications of BLG in these other samples. (Limited) glycation, as occurring in low-temperature dry-heated BLG, might be related to its T helper 2 (anti-inflammatory) inducing property. For immature dendritic cells, hardly any transcriptional response to processing-modified BLG was observed. Our results thus indicate that processing-induced structural modification influence the transcriptional, and in particular the immune response, of macrophages.

    Chapter 3 showed that the uptake of BLG by THP-1 macrophages differed after different processing methods and was positively correlated with hydrophobicity and aggregation. In Chapter 5, we applied the same three processing methods that were applied to BLG also to lysozyme and thyroglobulin, which have different pI or molecular weight compared to BLG, respectively. Uptake was tested in both THP-1 monocytes-derived macrophages and dendritic cells. A strong correlation between the uptake of the different protein samples by macrophages and dendritic cells was found and the former cells showed stronger uptake capability than the latter ones. The key role of hydrophobicity, over aggregation, in determining the uptake was confirmed by the results for lysozyme and thyroglobulin and the chemically crosslinked form of all three proteins. Several uptake routes were shown to contribute to the uptake of BLG by macrophages. However, cytokine responses following exposure of macrophages to BLG samples were not related to the levels of uptake. Together, our results demonstrate that heat-treatment-induced increased hydrophobicity is the prime driving factor in uptake, but also shows that the further immune response by APCs might be governed by other parameters than uptake capability.

    In conclusion, this thesis showed that physicochemical modifications of proteins, e.g. exposure of hydrophobic regions as a consequence of the wet-heating processing, influences its accessibility and uptake by APCs stronger than the other two heating methods. Moreover, the potential inherent pro-inflammatory effect of BLG on macrophages was reduced by severe structural changes. These outcomes may contribute to reduce the incidence and prevalence of CMA, and possible adverse immune effects from milk proteins, by better understanding of the underlying immunological mechanisms.

    Heat treatment of β-lactoglobulin affects its digestion and translocation in the upper digestive tract
    Deng, Ying ; Govers, Coen ; Tomassen, Monic ; Hettinga, Kasper ; Wichers, Harry J. - \ 2020
    Food Chemistry 330 (2020). - ISSN 0308-8146
    Caco-2 monolayer - Heat treatment - Hydrophobicity - Immune system - In vitro digestion - Translocation - β-Lactoglobulin

    Heat treatment is a commonly applied unit operation in the processing of β-lactoglobulin containing products. This does, however, influence its structure and thereby impacts its activity and digestibility. We describe how various heat-treatments of β-lactoglobulin change the digestibility using a modified version of the current consensus INFOGEST protocol. Additionally, protein was investigated for its translocation over the intestinal epithelial barrier, which would bring them in contact with immune cells. The extent of gastric digestibility was higher when the protein structure was more modified, while the influence of glycation with lactose was limited. Translocation studies of protein across Caco-2 cell monolayers showed a lower translocation rate of protein heated in solution compared to the others. Our study indicates that structural modifications after different heat-treatments of β-lactoglobulin increase in particular gastric digestibility and the translocation efficiency across intestinal epithelial cells.

    Short communication: Volatile profile of matured Tronchón cheese affected by oxytetracycline in raw goat milk
    Quintanilla, P. ; Hettinga, K.A. ; Beltrán, M.C. ; Escriche, I. ; Molina, M.P. - \ 2020
    Journal of Dairy Science 103 (2020)7. - ISSN 0022-0302 - p. 6015 - 6021.
    antibiotic residue - goat cheese - oxytetracycline - volatile profile

    The presence of antibiotics in milk destined for cheese production may affect the biological processes responsible for the formation of volatile compounds, leading to alterations in the characteristic cheese flavor expected by consumers. The aim of this study was to evaluate the effect of the presence of oxytetracycline in goat milk on the volatile profile of ripened cheeses. Traditional mature Tronchón cheeses were manufactured from raw goat milk spiked with different concentrations of oxytetracycline (50, 100, and 200 µg/kg). Cheese made from antibiotic-free goat milk was used as control. We analyzed the residual amounts of the antibiotic and the volatile profile of the experimental cheeses on a fortnightly basis during maturation using liquid chromatography tandem mass spectrometry and then solid-phase microextraction followed by gas chromatography–mass spectrometry. Our results suggested that oxytetracycline was widely transferred from milk to cheese: drug concentrations in the cheeses were 3.5 to 4.3 times higher than those in raw milk. Although the residual amounts of oxytetracycline significantly decreased during maturation (88.8 to 96.5%), variable amounts of residues remained in cheese matured for 60 d (<10 to 79 µg/kg). In general, the presence of oxytetracycline in goat milk did not affect the volatile profile of Tronchón cheeses; volatile profile was significantly modified by ripening time. Still, the presence of oxytetracycline residues in cheeses ripened for 60 d could be of great concern for public health.

    Organic milk: understanding intrinsic characteristics and their origin, and development of novel means of detection
    Liu, N. - \ 2020
    Wageningen University. Promotor(en): S.M. van Ruth, co-promotor(en): K.A. Hettinga; A.M. Pustjens. - Wageningen : Wageningen University - ISBN 9789463953986 - 150

    Organic milk has been receiving more and more attention in recent years. However, it is also susceptible to fraud considering its high retail price and the strict requirements of organic production. Since the quality of milk is affected by many factors, such as seasons, environment, lactation stage, feed, cow breed, etc., it is challenging to distinguish organic milk from conventional milk. To ensure fair competition and consumer confidence, there is a strong need to confirm the identity of organic products including milk. Therefore, this study was carried out to elucidate the differences in characteristics, in terms of fatty acid (FA) and volatile organic compound (VOC) profiles, of organic milk and other milks, and to study their underlying causes.

    In Chapter 2 and Chapter 4, the FA profiles of different farm (raw) milks and retail milks were investigated by gas chromatography (GC). Both the types of silages and grazing management strategies in different dairy production systems impacted on the FA profiles of milks, where the impact of the latter one was more significant. Corresponding to this result for farm (raw) milks, the differences of the FA profiles of different milks have also been observed among retail milks. Hence, industrial processing and milk pooling did not erase all the characteristics of organic milks. However, due to the relatively flexible regulations of pasture milk, the differences between pasture retail milk and conventional retail milk were less significant than the differences between pasture raw milk and conventional raw milk.

    Similarly, the VOC profiles of raw milks and retail milks were analysed in Chapter 3 and Chapter 6. By using proton transfer reaction quadrupole mass spectrometry (PTR-(Quad)MS) and proton transfer reaction time of flight mass spectrometry (PTR-(ToF)MS), the characteristics of different milks in winter and summer were investigated. Similar to the results of FA analysis, the grazing management had a greater impact on VOC profiles than the type of silages. This is not only due to the VOCs resulting from the feedstuff consumed by the cows, but relates also to the FA composition of the milks. Moreover, the characteristics of the VOC profiles of retail milks were investigated as well, which revealed that the differences between milks from organic and other production systems in summer and winter could still be observed.

    In general, pasture milk was closer to conventional milk in winter and closer to organic milk in summer, in terms of its FA profile and VOC profile, whereas the differences between organic milk and conventional milk were significant both in summer and winter.

    Subsequently, different analysis approaches were compared for various stakeholders. PTR-(ToF)/(Quad)MS and GC told the differences between organic milk and milks from other production system (including conventional and pasture systems). They provide options for the authorities to confirm the authenticity of organic milk. Fourier transform near-infrared spectroscopy (FT-NIR) and micro near infrared spectroscopy (Micro-NIR) could distinguish organic milk from conventional retail milk but not from pasture milk. They can provide a first, on site check of the identity of organic milk. The novel handheld near-infrared spectroscopy, SCIO, can be easily operated by consumers and generate some relevant information of the products.

    The results of this study could contribute to the authenticity of organic milk and help protect the rights of stakeholders.

    Prevalence of milk fraud in the Chinese market and its relationship with fraud vulnerabilities in the chain
    Yang, Yuzheng ; Zhang, Liebing ; Hettinga, Kasper A. ; Erasmus, Sara W. ; Ruth, Saskia M. Van - \ 2020
    Foods 9 (2020)6. - ISSN 2304-8158
    China - Fourier transform-infrared spectroscopy - Fraud vulnerability - Milk adulteration - Milk composition - One-class classifications

    This study aimed to assess the prevalence of ultra-high-temperature (UHT) processed milk samples suspected of being adulterated on the Chinese market and, subsequently, relate their geographical origin to the earlier determined fraud vulnerability. A total of 52 UHT milk samples purchased from the Chinese market were measured to detect possible anomalies. The milk compositional features were determined by standardized Fourier transform-infrared spectroscopy, and the detection limits for common milk adulterations were investigated. The results showed that twelve of the analysed milk samples (23%) were suspected of having quality or fraud-related issues, while one sample of these was highly suspected of being adulterated (diluted with water). Proportionally, more suspected samples were determined among milks produced in the Central- Northern and Eastern areas of China than in those from the North-Western and North-Eastern areas, while those from the South were in between. Combining the earlier collected results on fraud vulnerability in the Chinese milk chains, it appears that increased fraud prevalence relates to poorer business relationships and lack of adequate managerial controls. Since very few opportunities and motivations differ consistently across high and low-prevalence areas, primarily the improvement of control measures can help to mitigate food fraud in the Chinese milk supply chains.

    Variability in immune-active human milk components
    Elwakiel, Mohèb - \ 2020
    Wageningen University. Promotor(en): H.A. Schols; K.A. Hettinga. - Wageningen : Wageningen University - ISBN 9789463953443 - 166

    Human milk contains, among others, a range of biofunctional components, like serum proteins and human milk oligosaccharides (HMOs). This PhD thesis provides information on the variability in serum proteins, serum protein N-glycans and HMOs in milk of individual mothers during lactation, and offers insights on the degradation of proteins using a newly developed infant (0–3 months) in vitro digestion model.

    The 15 most abundant HMOs cover >95% of the total HMO content. The mother’s secretor (Se) and Lewis (Le) histo-blood groups are related to specific fucosylated HMOs. Based on the total neutral fucosylated HMO concentrations in both Chinese and Dutch human milk, for the first time, Se+Le+ subgroups were identified. Variation among mothers was found in the composition of serum proteins in both colostrum and mature milk, although the group of immune-active proteins, enzymes, and transport proteins were the most abundant for all mothers. These three protein groups encompass many of the 15 most abundant proteins, covering >95% of the total protein concentrations, in both the Chinese and Dutch milk serum proteome. The serum protein N-glycan composition in human milk from 2 different lactational periods was investigated. Analysis showed that neutral fucosylated and nonfucosylated N-glycans dominated the human milk serum glycoproteome. Based on the levels of the individual N-glycans, for the first time, a clear difference can be observed between the milk of secretor and nonsecretor mothers. It also indicated that specific fucosylated N-glycans can be synthesized in the gland of nonsecretor mothers and even in much higher concentrations compared to secretor mothers. In an in vitro infant digestion model, the total milk protein content decreased from the start to the end of infant in vitro digestion with large variation between mothers, especially in the gastric phase (remaining between 25–80%). After intestinal digestion, still  some undigested proteins could be found, ranging from 0.5% to 4.2% of the initial protein content, although no differences could be observed between colostrum and mature milk. More than 40 serum proteins could be detected after intestinal digestion. Overall, caseins are digested more than most serum proteins during digestion. Especially immune-active serum proteins, which are digested to a lower extent, might protect infants from pathogens.

    Maternal allergy and the presence of nonhuman proteinaceous molecules in human milk
    Dekker, Pieter M. ; Boeren, Sjef ; Wijga, Alet H. ; Koppelman, Gerard H. ; Vervoort, Jacques J.M. ; Hettinga, Kasper A. - \ 2020
    Nutrients 12 (2020)4. - ISSN 2072-6643
    Allergen transfer - Human milk - Maternal allergy - Nonhuman proteins - β-lactoglobulin

    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.

    The Chinese milk supply chain: A fraud perspective
    Yang, Yuzheng ; Huisman, Wim ; Hettinga, Kasper A. ; Zhang, Liebing ; Ruth, Saskia M. van - \ 2020
    Food Control 113 (2020). - ISSN 0956-7135
    China - Dairy farmer - Economically motivated adulteration - Fraud vulnerability assessment - Milk processor - Milk supply chain

    Food fraud has become a serious concern all over the world and especially in China. The melamine contaminated infant formula in 2008 has brought food fraud in the spotlights. This incident had grave consequences for the Chinese citizens as well as the Chinese milk industry. Fraud vulnerability assessments are the first step towards food fraud prevention and mitigation. To combat food fraud, one has to think like a criminal. In the current study, we determined the most vulnerable points in the Chinese milk supply chain, and examined the underlying causes. The fraud vulnerability perceived by 90 Chinese dairy farmers and 14 milk processors was evaluated with the SSAFE food fraud vulnerability assessment tool. Overall, actors perceived the milk supply chain as low to medium vulnerable to food fraud. Farmers appeared significantly more vulnerable than processors due to enhanced opportunities and motivations, and less adequate controls. Both geographical location of the farms and their size affected their perceived fraud vulnerability significantly.

    Dataset on proteomic changes of whey protein after different heat treatment
    Xiong, Ling ; Boeren, Sjef ; Vervoort, Jacques ; Hettinga, Kasper - \ 2020
    Data in Brief 29 (2020). - ISSN 2352-3409
    Heat treatment - Milk - Pasteurization - Proteomics - Whey protein

    Hereby we provide data from a shot-gun proteomics experiment, using filtered-aided sample preparation (FASP), and liquid chromatography with tandem mass spectrometry (LC-MS/MS), to relatively quantify the changes in the protein profile of whey proteins after heating milk at either 65 °C, 70 °C, 75 °C, 80 °C, or 85 °C for 30 min. The data supplied in this article supports the accompanying publication [1]. The raw mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier “PXD016436”.

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