Integrating fruit by-products and whey for the design of folate-bioenriched innovative fermented beverages safe for human consumption.

Global concerns over folate deficiency, the risks of excessive synthetic folic acid consumption, and food loss implications for environmental sustainability and food security drive needs of innovative approaches that align food by-product valorisation with folate bio-enrichment. This study explored the use of three fruit by-products extracts (grape, passion fruit, and pitaya) and whey to develop a folate bio-enriched fermented whey-based beverage. Three strains (Lacticaseibacillus rhamnosus LGG, Bifidobacterium infantis BB-02, and Streptococcus thermophilus TH-4) were tested for folate production in different fermentation conditions in modified MRS medium and in a whey-based matrix prepared with water extracts of these fruit by-products. B. infantis BB-02 and S. thermophilus TH-4, alone and in co-culture, were the best folate producers. The selection of cultivation conditions, including the presence of different substrates and pH, with grape by-product water extract demonstrating the most substantial effect on folate production among the tested extracts, was crucial for successfully producing a biofortified fermented whey-based beverage (FWBB). The resulting FWBB provided 40.7 µg of folate per 100 mL after 24 h of fermentation at 37 °C, effectively leveraging food by-products. Moreover, the beverage showed no cytotoxicity in mouse fibroblast cells tests. This study highlights the potential for valorising fruit by-products and whey for the design of novel bioenriched foods, promoting health benefits and contributing to reduced environmental impact from improper disposal.

Husk Separation (Kubessa Method) Impacts the Aging Chemistry of Beer.

The removal of husks before the mashing process, also known as the Kubessa method, is an established brewing practice often positively associated with smoothness and better flavor-stability of beer. Empirical evidence on the effect of the Kubessa method on beer, however, has been lacking. Similarly, our study's comprehensive analysis of established brewing attributes revealed that traditional methods do not fully capture the impact of husk separation in beer brewing. Conclusive evidence of the Kubessa method's impact on beer aging chemistry was obtained through ultrahigh resolution mass spectrometry (FT-ICR-MS), revealing intricate molecular details inaccessible to conventional analytical techniques. The compositional information on thousands of molecules in Kubessa beer was resolved and compared to whole malt mashing. Machine learning algorithms applied to aging experiments identified over 500 aging-related compounds inhibited by husk separation. Complementary Time of flight mass spectrometry (ToF-MS) coupled with chromatography further confirmed that the mashing of husks introduces sulfur-containing lipid compounds. These significant differences in the beer composition provide valuable insights for further investigation into the staling protective effect of husk-separation (Kubessa process) during beer production, as empirically demonstrated in this work.

Power-to-vitamins: producing folate (vitamin B9) from renewable electric power and CO2 with a microbial protein system.

We recently proposed a two-stage Power-to-Protein technology to produce microbial protein from renewable electric power and CO2. Two stages were operated in series: Clostridium ljungdahlii in Stage A to reduce CO2 with H2 into acetate, and Saccharomyces cerevisiae in Stage B to utilize O2 and produce microbial protein from acetate. Renewable energy can be used to power water electrolysis to produce H2 and O2. A drawback of Stage A was the need for continuous vitamin supplementation. In this study, by using the more robust thermophilic acetogen Thermoanaerobacter kivui instead of C. ljungdahlii, vitamin supplementation was no longer needed. Additionally, S. cerevisiae produced folate when grown with acetate as a sole carbon source, achieving a total folate concentration of 6.7 mg per 100 g biomass with an average biomass concentration of 3 g l-1. The developed Power-to-Vitamin system enables folate production from renewable power and CO2 with zero or negative net-carbon emissions.

Development of a high-throughput UHPLC-MS/MS method for the analysis of Fusarium and Alternaria toxins in cereals and cereal-based food.

A QuEChERS (quick, easy, cheap, effective, rugged, and safe)-based multi-mycotoxin method was developed, analyzing 24 (17 free and 7 modified) Alternaria and Fusarium toxins in cereals via ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A modified QuEChERS approach was optimized for sample preparation. Quantification was conducted using a combination of stable isotope dilution analysis (SIDA) for nine toxins and matrix-matched calibration for ten toxins. Quantification via a structurally similar internal standard was conducted for four analytes. Alternariol-9-sulfate (AOH-9-S) was measured qualitatively. Limits of detection (LODs) were between 0.004 µg/kg for enniatin A1 (ENN A1) and 3.16 µg/kg for nivalenol (NIV), while the limits of quantification were between 0.013 and 11.8 µg/kg, respectively. The method was successfully applied to analyze 136 cereals and cereal-based foods, including 28 cereal-based infant food products. The analyzed samples were frequently contaminated with Alternaria toxins, proving their ubiquitous occurrence. Interestingly, in many of those samples, some modified Alternaria toxins occurred, mainly alternariol-3-sulfate (AOH-3-S) and alternariol monomethyl ether-3-sulfate (AME-3-S), thus highlighting the importance of including modified mycotoxins in the routine analysis as they may significantly add to the total exposure of their parent toxins. Over 95% of the analyzed samples were contaminated with at least one toxin. Despite the general contamination, no maximum or indicative levels were exceeded.

Assessing a Fermented Whey Beverage Biofortified with Folate as a Potential Folate Source for Humans.

Folate, a vital water-soluble vitamin (B9), requires specific attention as its recommended daily intake frequently is not reached in countries without mandatory fortification. In this regard, biofortification with microorganisms like Bifidobacterium and Streptococcus offers a compelling approach for enhancing food with natural folates. A randomized, nonblinded, and monocentric human pilot study is conducted to assess the bioavailability of a folate-biofortified fermented whey beverage, comprising 3 intervention days and a controlled replenishment phase before and during the assay. Folate plasma concentration (5-CH3-H4folate) is determined using a stable isotope dilution assay and LC-MS/MS detection. Biokinetic parameters (cmax and tmax) are determined, and areas under the curve (AUC) normalized to the basal folate plasma concentration are calculated. An average bioavailability of 17.1% in relation to the 5-CH3-H4folate supplement, ranging from 0% to 39.8%, is obtained. These results reiterate the significance of additional research into folate bioavailability in general and dairy products. Further investigations are warranted into folate-binding proteins (FBP) and other potential limiting factors within the food and individual factors. In summary, biofortification via fermentation emerges as a promising avenue for enhancing the natural folate content in dairy and other food products.

Pyrrolizidine alkaloids and tropane alkaloids in milk samples from individual dairy farms of the German federal states of Bavaria and Schleswig-Holstein.

1,2-Dehydro-pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO) and tropane alkaloids (TA), are toxic plant metabolites. If plant material, containing these toxins, is present in the feed of dairy cows these toxins can be transferred into milk. Here, milk was sampled directly from dairy farms in the German federal states of Bavaria and Schleswig-Holstein in 2020-2022 in order to investigate a possible contamination of milk at the production stage. In total, 228 milk samples were analysed for 54 PA/PANO and two TA by a sensitive LC-ESI-MS/MS method. In addition, a subset of milk samples (n = 85) was independently analysed for TA by a cooperating laboratory for verification. PA/PANO were found in 26 samples (11%) with a low median sum content of the contaminated samples of 0.024 µg/L. The highest level of contamination was 5.6 µg/L. Senecionine-, lycopsamine- and heliotrine-type PA/PANO were detected. In four samples (1.8%), atropine was determined up to 0.066 µg/L. The toxin levels in the milk samples hardly contributed to the total daily exposure. These data are first-time results on contamination rates and levels occurring in milk from individual dairy farms, based on a large sample number.

European food safety research: An explorative study with funding experts' consultation.

The European research area exhibits considerable opacity and fragmentation in food safety research funding and organizational structures, impeding the exploitation of existing research potential across European countries. Given that food safety is inherently linked to the societal challenges of our time, identifying and removing existing barriers to research funding in this area is crucial. Towards investigating this matter, interviews were conducted with funding bodies from six European countries to assess key issues related to research funding in general and food safety in particular. Funding experts were then invited to a workshop to jointly discuss the challenges identified and explore strategies to address them. Evaluation of the food safety research funding situation in selected European countries revealed both convergences and significant differences among national funding bodies. Engaging with funding experts provided invaluable insights into the issues encountered with research funding, such as inadequate call management staff or insufficient research funds, culminating in a set of recommendations for action to remedy the situation.

Molecular Characterization of Cooking Processes: A Metabolomics Decoding of Vaporous Emissions for Food Markers and Thermal Reaction Indicators.

Thermal processing of food plays a fundamental role in everyday life. Whereas most researchers study thermal processes directly in the matrix, molecular information in the form of non- and semivolatile compounds conveyed by vaporous emissions is often neglected. We performed a metabolomics study of processing emissions from 96 different food items to define the interaction between the processed matrix and released metabolites. Untargeted profiling of vapor samples revealed matrix-dependent molecular spaces that were characterized by Fourier-transform ion cyclotron resonance-mass spectrometry and ultra-performance liquid chromatography-mass spectrometry. Thermal degradation products of peptides and amino acids can be used for the differentiation of animal-based food from plant-based food, which generally is characterized by secondary plant metabolites or carbohydrates. Further, heat-sensitive processing indicators were characterized and discussed in the background of the Maillard reaction. These reveal that processing emissions contain a dense layer of information suitable for deep insights into food composition and control of cooking processes based on processing emissions.

FT-ICR-MS reveals the molecular imprints of the brewing process.

The study of fermentation and brewing has a long history of pioneering discoveries that continue to influence modern industrial food production. Since then, numerous research endeavors have yielded conventional criteria that guide contemporary brewing practices. However, the intricate open challenges faced today necessitate a more exhaustive understanding of the process at the molecular scale. We have developed an ultra-high-resolution mass spectrometric analysis (FT-ICR-MS) of the brewing process that can rapidly and comprehensively resolve thousands of molecules. This approach allows us to track molecular fluctuation during brewing at the level of chemical compositions. Employing biological triplicates, our investigation of two brewing lines that are otherwise identical except for the malt used revealed over 8,000 molecular descriptors of the brewing process. Metabolite imprints of both the similarities and differences arising from deviating malting temperatures were visualized. Additionally, we translated traditional brewing attributes such as the EBC-value, free amino nitrogen, pH-value, and concentration curves of specific molecules, into highly correlative molecular patterns consisting of hundreds of metabolites. These in-depth molecular imprints provide a better understanding of the molecular circumstances leading to various changes throughout the brewing process. Such chemical maps go beyond the observation of traditional brewing attributes and are of great significance in the investigation strategies of current open challenges in brewing research. The molecular base of knowledge, along with advancements in technological and data integration schemes, can facilitate the efficient monitoring of brewing and other productions processes.

The Pharmacokinetics of Individual Conjugated Xanthohumol Metabolites Show Efficient Glucuronidation and Higher Bioavailability of Micellar than Native Xanthohumol in a Randomized, Double-Blind, Crossover Trial in Healthy Humans.

SCOPE: Prenylated chalcones and flavonoids are found in many plants and are believed to have beneficial effects on health when consumed. Xanthohumol is present in beer and likely the most consumed prenylated chalcone, but poorly absorbed and rapidly metabolized and excreted, thus limiting its bioavailability. Micellar formulations of phytochemicals have been shown to improve bioavailability. METHODS AND RESULTS: In a randomized, double-blind, crossover trial with five healthy (three males and two females) volunteers, a single dose of 43 mg was orally administered as a native or micellar formulation. The major human xanthohumol metabolites are quantified in plasma. Unmetabolized free xanthohumol makes 1% or less of total plasma xanthohumol. The area under the plasma concentration-time curve of xanthohumol-7-O-glucuronide following the ingestion of the micellular formulation is 5-fold higher and its maximum plasma concentration is more than 20-fold higher compared to native xanthohumol. CONCLUSION: Metabolism of orally ingested xanthohumol is complex and efficiently converts the parent compound to predominantly glucuronic acid and to a lesser extent sulfate conjugates. The oral bioavailability of micellar xanthohumol is superior to native xanthohumol, making it a useful delivery form for future human trials.

An improved folate stable isotope dilution assay of unexploited food sources from Brazil.

Brazil has a diverse plant community, including underutilized non-conventional food crops (PANCs), which have the potential to be a rich source of food and contribute to food security. For assessing the folate content in a range of Brazilian PANCs, we extended the validation of an existing stable isotope dilution assay (SIDA) for the stably 13C-labelled 10-formyl-Pte[13C5]Glu (10-CHO-Pte[13C5]Glu). The SIDA method with an enzymatic treatment, purification step, and an LC-MS/MS measurement was validated regarding linearity, precision, LoD/LoQ, and recovery for 10-CHO-PteGlu. After successful validation, the study of some underutilized Brazilian non-conventional fruits and leaves from the São Paulo State University campus revealed them as an important source of folates. It provided the first insights into the folate content of unexploited food sources from Brazil. Pequi had the highest folate content among the fruits studied, with mean values of 333 µg/100 g based on fresh weight (FW). The analysis also shows that different cultivars of fruit or fruits from different growing locations have a high variability in folate content or other nutritional factors. In most fruits, the main vitamer was 5-CH3-H4folate, but jenipapo and taioba showed the highest content of 10-CHO-PteGlu with 28.22 µg/100 g (FW) in jenipapo peel and 75.64 µg/100 g (FW) in the taioba leaves. Thus, this study also provides results on the importance of the folate vitamer 10-CHO-PteGlu contributing to the total folate content.

Challenges in the determination of total vitamin B12 by cyanidation conversion: insights from stable isotope dilution assays.

Previous methods for vitamin B12 (B12) analysis have extensively used cyanidation conversion with the intention of converting all cobalamins to cyanocobalamin (CNCbl) for total B12 determination. This approach has been favored for its advantages in reducing the number of analytes, increasing analyte concentration, and improving analyte stability. However, the present study revealed underlying limitations associated with this approach. First, a stable isotope dilution assay (SIDA) determining total B12 as CNCbl after cyanidation conversion (conversion SIDA method) was developed. Method validation demonstrated good sensitivity, recovery, accuracy, and reproducibility for the target analyte CNCbl. However, subsequent application of the conversion method to real meat samples showed incomplete conversions of cobalamins. These inconsistencies revealed day-to-day variability and reliability challenges associated with the cyanidation process. It was not possible to identify this issue during method validation as CNCbl was spiked as the sole analyte and it requires no further cyanidation conversion. The application of LC-MS/MS enabled the detection of trace amounts of unconverted cobalamins. Nevertheless, this approach remains restricted by instrument sensitivity and stability as well as the performance of immunoaffinity purification for different vitamers. Further development of a reliable monitoring method is a prerequisite for further optimization of the cyanidation process. However, significant improvements of analytical instrumentation in terms of sensitivity and stability are required to overcome the current limitations.

Building a novel strategic research agenda for METROFOOD-RI: design process and multi-stakeholder engagement towards thematic prioritization.

Introduction: The European research landscape suffers widely from fragmentation and little cross-border research collaboration. Efforts are underway to bring the European Research Area to a higher level of performance and capacity in cutting-edge science, with high anticipations for the promotion of multidisciplinary research infrastructures of transnational engagement. A European distributed research infrastructure active in this framework is METROFOOD-RI, committed to promoting metrology in food and nutrition with particular focus on measurement research related to agrifood systems. Methods: For research infrastructures, streamlining resources among partner organizations and establishing priorities around specific topics is critical for ensuring smooth operation. Similarly, METROFOOD-RI faced the challenge of exploring its strategic direction and research priorities as revealed in its first Strategic Research and Innovation Agenda (SRIA). This report details how the internal process of topic identification and prioritization progressed within the METROFOOD-RI SRIA and what obstacles were encountered along the way. A dual-track strategy was taken for locating future SRIA topics, applying a top-down and bottom-up approach, followed by internal consultation with METROFOOD-RI experts. The topic prioritization drew on a vote among the METROFOOD-RI Management Committee employing a custom-designed numerical rating scale questionnaire. Based on the maximum scores obtained for each topic, appropriate thresholds were introduced for classifying individual topics into high, medium, low, and very low priority ones. Results: A total of 80 topics categorized into eight major clusters of challenges were located as potential SRIA candidates. Upon prioritization, 9 topics of very high priority and 16 topics of medium priority were identified as key research thematic areas of the newly developed SRIA. Discussion: As a strategic framework, the SRIA occupies a central position and sets not only the scientific focus of the research infrastructure in the coming years, but also contributes to realizing the full potential and excellence of METROFOOD-RI, selectively expanding the existing portfolio and thus contributing to maximum efficiency and sustainability. It is anticipated that the lessons learned by METROFOOD-RI and its experiences shared are a valuable stimulus and guide for those who are taking on the challenge of setting-up a SRIA and are looking for edifying and constructive information on how to do so.

Root uptake and metabolization of Alternaria toxins by winter wheat plants using a hydroponic system.

Fungi of the genus Alternaria are ubiquitous in the environment. Their mycotoxins can leach out of contaminated plants or crop debris into the soil entering the plant via the roots. We aim to evaluate the importance of this entry pathway and its contribution to the overall content of Alternaria toxins (ATs) in wheat plants to better understand the soil-plant-phytopathogen system. A hydroponic cultivation system was established and wheat plants were cultivated for up to two weeks under optimal climate conditions. One half of the plants was treated with a nutrient solution spiked with alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA), whereas the other half of the plants was cultivated without mycotoxins. Plants were harvested after 1 and 2 weeks and analyzed using a QuEChERS-based extraction and an in-house validated LC-MS/MS method for quantification of the ATs in roots, crowns, and leaves separately. ATs were taken up by the roots and transported throughout the plant up to the leaves after 1 as well as 2 weeks of cultivation with the roots showing the highest ATs levels followed by the crowns and the leaves. In addition, numerous AOH and AME conjugates like glucosides, malonyl glucosides, sulfates, and di/trihexosides were detected in different plant compartments and identified by high-resolution mass spectrometry. This is the first study demonstrating the uptake of ATs in vivo using a hydroponic system and whole wheat plants examining both the distribution of ATs within the plant compartments and the modification of ATs by the wheat plants.

DBDIpy: a Python library for processing of untargeted datasets from real-time plasma ionization mass spectrometry.

MOTIVATION: Plasma ionization is rapidly gaining popularity for mass spectrometry (MS)-based studies of volatiles and aerosols. However, data from plasma ionization are delicate to interpret as competing ionization pathways in the plasma create numerous ion species. There is no tool for detection of adducts and in-source fragments from plasma ionization data yet, which makes data evaluation ambiguous. SUMMARY: We developed DBDIpy, a Python library for processing and formal analysis of untargeted, time-sensitive plasma ionization MS datasets. Its core functionality lies in the identification of in-source fragments and identification of rivaling ionization pathways of the same analytes in time-sensitive datasets. It further contains elementary functions for processing of untargeted metabolomics data and interfaces to an established ecosystem for analysis of MS data in Python. AVAILABILITY AND IMPLEMENTATION: DBDIpy is implemented in Python (Version ≥ 3.7) and can be downloaded from PyPI the Python package repository (https://pypi.org/project/DBDIpy) or from GitHub (https://github.com/leopold-weidner/DBDIpy). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Therapeutic drug monitoring in breast cancer therapy - LC-MS/MS method for quantification of the CDK4/6 inhibitors abemaciclib, palbociclib, ribociclib, and major metabolites abemaciclib M20 and M2 in human serum.

The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors palbociclib, ribociclib, and abemaciclib were approved by the U.S. Food and Drug Administration (FDA) and European Medicine Agency for the treatment of breast cancer between 2015 and 2018. Oral tumor therapeutics extend the options for cancer therapy, but also challenge physicians and patients. The aim of the present work was to establish a semi-automated liquid-chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of abemaciclib, its active metabolites abemaciclib M20 and M2, palbociclib, and ribociclib in human serum. Detuning of ribociclib enabled the development of a simultaneous quantification method for abemaciclib, M20, M2, palbociclib, and ribociclib in the respective relevant concentration ranges based on semi-automated sample preparation with isotope dilution LC-MS/MS. The method was validated according to the guidance of the FDA. The LC-MS/MS method was successfully validated according to FDA and showed inaccuracies ≤ 10.7% and imprecisions ≤ 8.51%. Linearity was given from 20 to 800 ng/mL for abemaciclib, 15-600 ng/mL for M20, 10-400 ng/mL for M2 and palbociclib, and 100-4000 ng/mL for ribociclib. Normalized matrix factors and process efficiency showed no significant matrix effects regardless of the analytes. To demonstrate the applicability of the method, authentic samples were also analyzed. This novel semi-automated LC-MS/MS method covering all previously approved CDK4/6 inhibitors as well as the similarly pharmacologically active metabolites in human serum simultaneously was developed for potential future use in routine analysis in order to improve personalized therapy, patient safety, and treatment success.

Real-Time Monitoring of Miniaturized Thermal Food Processing by Advanced Mass Spectrometric Techniques.

Mass spectrometry is a popular and powerful analytical tool to study the effects of food processing. Industrial sampling, real-life sampling, or challenging academic research on process-related volatile and aerosol research often demand flexible, time-sensitive data acquisition by state-of-the-art mass analyzers. Here, we show a laboratory-scaled, miniaturized, and highly controllable setup for the online monitoring of aerosols and volatiles from thermal food processing based on dielectric barrier discharge ionization (DBDI) mass spectrometry (MS). We demonstrate the opportunities offered by the setup from a foodomics perspective to study emissions from the thermal processing of wheat bread rolls at 210 °C by Fourier transformation ion cyclotron resonance MS. As DBDI is an emerging technology, we compared its ionization selectivity to established atmospheric pressure ionization tools: we found DBDI preferably ionizes saturated, nitrogenous compounds. We likewise identified a sustainable overlap in the selectivity of detected analytes with APCI and electrospray ionization (ESI). Further, we dynamically recorded chemical fingerprints throughout the thermal process. Unsupervised classification of temporal response patterns was used to describe the dynamic nature of the reaction system. Compared to established tools for real-time MS, our setup permits one to monitor chemical changes during thermal food processing at ultrahigh resolution, establishing an advanced perspective for real-time mass spectrometric analysis of food processing.

Metabolic folate profiling as a function of time during cultivation suggests potential C2-metabolism in Saccharomyces cerevisiae.

Yeasts are reported to be rich in folates, a group of vitamers known to be involved in several biosynthetic reactions such as methylation reactions, oxidation and reduction processes, and nucleotide synthesis. Not being able to synthesize folates, humans rely on external folate supply. Here, we show the application of LC/MS-MS methods using SIDA (stable isotope dilution analysis) assays for the quantitative analysis of different folate mono- and polyglutamates during growth of Saccharomyces cerevisiae. Molecular networking (MN) was applied for detailed analysis of further folate metabolites. Highest folate contents of 13,120 µg/100 g were observed after 20 h of cultivation. The main vitamers 5-CH3-H4folate and H4folate decreased during cultivation, while 5-CHO-H4folate increased during cultivation. The hexa- and heptaglutamate of 5-CH3-H4folate accounted for >96% of the total 5-CH3-H4folate content. A shift of the major polyglutamate from hexa- to heptaglutamate was observed after 29 h. MN unraveled two groups of novel folates which could be assigned to a potentially existing C2-metabolism in yeast. In detail, 5,10-ethenyl-tetrahydrofolate and a further CO-substituted 5-CH3-H4folate were identified as hexa- and heptaglutamates. The latter was neither identified as 5-acetyl-tetrahydrofolate nor as EthylFox, the oxidation product of 5-ethyl-tetrahydrofolate. The structure needs to be elucidated in future studies.