A Novel Double-Blind, Placebo-Controlled Food Challenge Matrix for Milk and Raw Egg.

BACKGROUND: The double-blind, placebo-controlled food challenge (DBPCFC) is still considered to be the gold standard in food allergy diagnosis. This test is however not common practice in routine due to several practical limitations, especially for non-IgE-mediated food allergy with its typical delayed food allergic reactions. OBJECTIVE: The aim of this study was to develop and evaluate DBPCFC matrices for the diagnosis of milk and egg allergies which can be applied at home for the diagnosis of delayed food allergic reactions. The main focus was the blinding of milk and raw egg and the development of matrices which can be prepared and consumed conveniently at home with a sufficiently long shelf life (+/- 6 months or longer). METHODS: A sensory test evaluated the blinding of the egg and milk in the matrices. The microbiological analysis confirmed the safety and stability of the developed matrices. To assess the applicability of the matrices, a pilot DBPCFC study for milk including 7 patients was conducted. RESULTS: Sensory tests confirmed that the masking of the allergenic ingredients was sufficient. Microbial safety and stability of the matrices were confirmed up to 6 months of storage at ambient temperatures in the dark. The DBPCFC for milk showed different outcomes and proved its applicability for use at home. CONCLUSION: A novel stable DBPCFC matrix for milk and raw egg has been developed that allows convenient use at the patients' home.

Selecting Processing Robust Markers Using High-Resolution Mass Spectrometry for the Detection of Milk in Food Products.

BACKGROUND: Cow's milk allergy is one of the most reported food allergies in Europe. To help patients suffering from food allergies it is important to be able to detect milk in different foods. An analytical method that is gaining interest in the field of allergen detection is ultrahigh performance liquid chromatography-tandem mass spectrometry, where the analyte is a target peptide. When these peptide biomarkers are selected, the effect of food processing should be taken into account to allow a robust detection method. OBJECTIVE: This work aims at identifying such processing stable peptide markers for milk for the ultrahigh performance liquid chromatography-tandem mass spectrometry based detection of food allergens in different food products. METHOD: Milk-incurred food materials that underwent several processing techniques were produced. This was followed by establishing tryptic peptide profiles from each matrix using ultrahigh performance liquid chromatography-high resolution mass spectrometry. RESULTS: A careful comparison of peptide profiles/intensities and the use of specific exclusion criteria resulted in the selection of eight peptide biomarkers suitable for application in ultrahigh performance liquid chromatography-tandem mass spectrometry based milk detection methods. One of these markers is an α-lactalbumin specific peptide, which has been determined to be stable in different incurred materials for the first time. CONCLUSIONS: To our knowledge, this is the first systematic and experimentally based approach for the selection of suitable milk peptide biomarkers robust toward multiple, often applied food processing techniques for milk. Ensuring the exact knowledge of the food processing circumstances by starting from well-defined raw material and using fully controlled settings to produce incurred test material allowed the construction of a peptide database with robust markers. These robust markers can be used for the development of a robust detection method for milk in different food matrixes. HIGHLIGHTS: To facilitate food allergen detection in processed food, processing stable peptide markers for the detection of milk in food products were determined using Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry on well-defined raw materials which were processed in accordance with often used processing techniques.

Selection of universal peptide biomarkers for the detection of the allergen hazelnut in food trough a comprehensive, high resolution mass spectrometric (HRMS) based approach.

The interest of using LC-MS/MS as a method for detection of allergens in food is growing. In such methods, peptides are used as biomarkers for the detection and quantification of the allergens. The selection of good biomarker peptides is of high importance to develop a specific, universal and sensitive method. Biomarkers should, for example, be robust to food processing. To evaluate robustness, test material incurred with hazelnut having undergone different food processing techniques was produced. Proteins of these materials were extracted, digested and further analyzed using HRMS. After peptide identification, selection was carried out using several criteria such as hazelnut specificity and amino acid composition. Further selection was done by comparing peptide MS intensities in the different food matrices. Only peptides showing processing robustness were retained. Eventually, eight peptides coming from three major hazelnut proteins were selected as the best biomarkers for hazelnut detection in processed foods.

Seasonal influence on heat-resistant proteolytic capacity of Pseudomonas lundensis and Pseudomonas fragi, predominant milk spoilers isolated from Belgian raw milk samples.

Psychrotolerant bacteria and their heat-resistant proteases play a major role in the spoilage of UHT-processed dairy products. Summer and winter raw milk samples were screened for the presence of such bacteria. One hundred and three proteolytic psychrotolerant bacteria were isolated, characterized by API tests, rep-PCR fingerprint analysis and evaluated for heat-resistant protease production. Twenty-nine strains (representing 79% of the complete collection) were further identified by 16S rRNA gene sequencing, rpoB gene sequencing and DNA-DNA hybridizations. A seasonal inter- and intra-species influence on milk spoilage capacity (e.g. growth rate and/or protease production) was demonstrated. Moreover, this polyphasic approach led to the identification of Pseudomonas fragi and Pseudomonas lundensis (representing 53% of all isolates) as predominant producers of heat-resistant proteases in raw milk. The role of Pseudomonas fluorescens, historically reported as important milk spoiler, could not unequivocally be established. The use of more reliable identification techniques and further revision of the taxonomy of P. fluorescens will probably result in a different perspective on its role in the milk spoilage issue.

Development and validation of a standardized double-blind, placebo-controlled food challenge matrix for raw hazelnuts.

BACKGROUND: Double-blind, placebo-controlled food challenge (DBPCFC) is considered the gold standard for food allergy diagnosis. However, this test is rarely performed routinely in clinical practice because of various practical issues, e.g. the lack of a standardized matrix preparation. The aim of this study was to develop and validate a convenient DBPCFC matrix, that can easily be implemented in daily clinical practice. The focus of this study was the blinding of hazelnuts, whereby the hazelnuts retained as much as possible their allergenicity and could be mixed homogenously in low-doses to the matrices. METHODS: A basophil-activation test (BAT), microbial tests and an LC-MS/MS test were performed to assess respectively the allergenicity of the used hazelnuts, the microbial stability of the novel developed matrices and the homogeneity of the hazelnuts in the matrices. A sensory test was conducted to validate the blinding of the hazelnuts in the matrices. A pilot DBPCFC study included eight patients as proof of concept. RESULTS: The BAT-test gave the first insights concerning the retained allergenicity of the hazelnuts. The microbial safety could be assured after 12 months of storage. Sufficient masking was assessed by several sensory tests. Homogeneous hazelnut distribution could be achieved for the different hazelnut concentrations. The DBPCFC's results showed diverse allergic responders (from no reactions to distinct objective symptoms). CONCLUSION: A novel stable and validated DBPCFC matrix using raw hazelnuts has been developed that allows easy preparation in a standardized way for convenient use in daily clinical practice.Trial registration EC Project number: EC/2015/0852; Date of registration: 13 Oct 2015; End date: 01 Feb 2017.

Pilot-scale production of cloudy juice from low-quality pear fruit under low-oxygen conditions.

In this study, a process for the production of premium quality yellowish, cloudy pear juice from low-quality fruit under low-oxygen conditions was developed. The production process consisted of (1) shredding, (2) pressing with spiral-filter technology including a vacuumised extraction cell, (3) holding in an inert gas buffer tank, (4) pasteurisation, (5) and refrigerated storage. First, the system parameters of a spiral-filter press were optimised with the aim of producing a yellowish, cloudy pear juice with the highest possible juice yield. A maximum juice yield of 78% could be obtained. Enzymatic browning during juice extraction could be suppressed as a result of the fast processing and the low air (oxygen) levels in the extraction chamber of the spiral-filter press. Furthermore, we observed that instantaneous pasteurisation at 107 °C for 6s, subsequent aluminium laminate packaging and cold storage had only a minimum effect on the phenolic composition.

A comparative study between spiral-filter press and belt press implemented in a cloudy apple juice production process.

In this study, advantages and disadvantages of the innovative, low-oxygen spiral-filter press system were studied in comparison with the belt press, commonly applied in small and medium size enterprises for the production of cloudy apple juice. On the basis of equivalent throughput, a higher juice yield could be achieved with spiral-filter press. Also a more turbid juice with a higher content of suspended solids could be produced. The avoidance of enzymatic browning during juice extraction led to an attractive yellowish juice with an elevated phenolic content. Moreover, it was found that juice produced with spiral-filter press demonstrates a higher retention of phenolic compounds during the downstream processing steps and storage. The results demonstrates the advantage of the use of a spiral-filter press in comparison with belt press in the production of a high quality cloudy apple juice rich in phenolic compounds, without the use of oxidation inhibiting additives.

Heterogeneity of heat-resistant proteases from milk Pseudomonas species.

Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.