Protein-phenolic interactions and reactions: Discrepancies, challenges, and opportunities.

Although noncovalent interactions and covalent reactions between phenolic compounds and proteins have been investigated across diverse scientific disciplines, a comprehensive understanding and identification of their products remain elusive. This review will initially outline the chemical framework and, subsequently, delve into unresolved or debated chemical and functional food-related implications, as well as forthcoming challenges in this topic. The primary objective is to elucidate the multiple aspects of protein-phenolic interactions and reactions, along with the underlying overwhelming dynamics and possibilities of follow-up reactions and potential crosslinking between proteins and phenolic compounds. The resulting products are challenging to identify and characterize analytically, as interactions and reactions occur concurrently, mutually influencing each other. Moreover, they are being modulated by various conditions such as the reaction parameters and, obviously, the chemical structure. Additionally, this review delineates the resulting discrepancies and challenges of properties and attributes such as color, taste, foaming, emulsion and gel formation, as well as effects on protein digestibility and allergenicity. Ultimately, this review is an opinion paper of a group of experts, dealing with these challenges for quite a while and aiming at equipping researchers with a critical and systematic approach to address current research gaps concerning protein-phenolic interactions and reactions.

Formation of Cysteine Adducts with Chlorogenic Acid in Coffee Beans.

The post-harvest processing of coffee beans leads to a wide range of reactions involving proteins. The formation of crosslinks between proteins and phenolic compounds present in high concentrations of coffee beans represents one of the most challenging and still not fully characterized reactions. The aim of this work was to assess the presence of products from such reactions in coffee samples, focusing on the adducts between cysteine and chlorogenic acids (CQAs). For this purpose, 19 green and 15 roasted coffee samples of the Coffea arabica, Coffea canephora, and Coffea liberica varieties were selected for this study and basically characterized. Then, targeted liquid chromatography mass spectrometry (LC-MS/MS) methods were developed to assess the formation of adducts between CQA and cysteine, glutathione, and N-acetylcysteine as the amino acid and peptide models, and quantified such adducts in coffee samples. The results of the characterization showed a heterogeneous distribution of the protein content (8.7-14.6%), caffeine (0.57-2.62 g/100 g), and antioxidant capacity (2-4.5 g ascorbic acid/100 g) in Arabica, Canephora, and Liberica samples. Glutamic acid, arginine, and proline were found to be the major amino acids, while 5-CQA (38-76%), 3-CQA (4-13%), and 4-CQA (4-13%) were the most abundant CQA derivatives of all coffee varieties. The model experiments for adduct formation demonstrated that cysteine binds to CQA via thiol groups and 5-CQA initially isomerizes to 3- and 4-CQA, depending on the conditions, allowing cysteine to bind to two different sites on 3-, 4- or 5-CQA molecules, thus, forming six different Cys-CQA adducts with m/z 476. The reaction was more favored at pH 9, and the adducts proved to be stable up to 90 °C for 10 min and up to 28 days at room temperature. The relative quantification of adducts showed peak area values ranging from 1100 to 3000 in green coffee bean samples, while no adducts were detected in roasted coffee beans. Overall, this work was the first attempt to demonstrate the presence of Cys-CQA adducts in coffee beans and paves the way for further investigations of such adduct formation at the protein level.

Cellulose-based adsorbents for solid phase extraction and recovery of pharmaceutical residues from water.

Cellulose has attracted interest from researchers both in academic and industrial sectors due to its unique structural and physicochemical properties. The ease of surface modification of cellulose by the integration of nanomaterials, magnetic components, metal organic frameworks and polymers has made them a promising adsorbent for solid phase extraction of emerging contaminants, including pharmaceutical residues. This review summarizes, compares, and contrasts different types of cellulose-based adsorbents along with their applications in adsorption, extraction and pre-concentration of pharmaceutical residues in water for subsequent analysis. In addition, a comparison in efficiency of cellulose-based adsorbents and other types of adsorbents that have been used for the extraction of pharmaceuticals in water is presented. From our observation, cellulose-based materials have principally been investigated for the adsorption of pharmaceuticals in water. However, this review aims to shift the focus of researchers to the application of these adsorbents in the effective pre-concentration of pharmaceutical pollutants from water at trace concentrations, for quantification. At the end of the review, the challenges and future perspectives regarding cellulose-based adsorbents are discussed, thus providing an in-depth overview of the current state of the art in cellulose hybrid adsorbents for extraction of pharmaceuticals from water. This is expected to inspire the development of solid phase exraction materials that are efficient, relatively cheap, and prepared in a sustainable way.

Limited enzymatic hydrolysis of green coffee protein as a technique for preparing new functional food components.

Valorisation of side-streams in food production has become an important booster for increased sustainability in food production. The objective of this work was to study and improve the functional properties of green coffee (GC) protein. Extraction of defatted GC meal by using PVPP slightly increased protein yield and significantly decreased the amount of covalently and non-covalently bound CQA, therefore decreasing the antioxidant activity of the meal. Peptic hydrolysis at pH 1.5 led to a significantly higher degree of hydrolysis (DH) than at pH 3. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weights of peptides of GC protein hydrolysates were in the range of 11-60 kDa, while peptides were in the range of 500-5000 Da using matrix-assisted laser desorption/ionization-time of flight mass spectroscopy (MALDI-TOF MS). Additionally, the enzymatic hydrolysis significantly improved the antioxidant activity of the GC protein. Finally, the results suggest that enzymatic hydrolysis with pepsin is an effective technique to provide bioactive compounds. The works presented in our manuscript may help in further exploiting the potential use of green coffee beans for food, cosmetic or pharmaceutical industry. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05646-3.

Targeted Bottom-Up Mass Spectrometry Approach for the Relative Quantification of Post-Translational Modification of Bovine κ-Casein during Milk Fermentation.

κ-casein (κ-CN) is one of the key components in bovine milk, playing a unique role in the structuration of casein micelles. It contains in its chemical structure up to sixteen amino acid residues (mainly serine and threonine) susceptible to modifications, including glycosylation and phosphorylation, which may further be formed during milk processing. In this study, changes in post-translational modification (PTM) of κ-CN during bovine milk fermentation were investigated. One-to-five-day fermented milk samples were produced. A traditional bottom−up proteomics approach was used to establish a multiple-reaction monitoring (MRM) method for relative quantification of κ-CN PTM. Endoproteinase Glu-C was found to efficiently digest the κ-CN molecule. The developed LC-MS method was validated by performing assessments of linearity, precision, repeatability, reproducibility, limit of detection (LOD), and limit of quantification (LOQ). Among the yielded peptides, four of them containing serine and threonine residues were identified and the unmodified as well as the modified variants of each of them were relatively quantified. These peptides were (1) IPTINTIASGEPTSTTE [140, 158], (2) STVATLE [162, 168], (3) DSPE [169, 172], and (4) INTVQVTSTAV [180, 190]. Distribution analysis between unmodified and modified peptides revealed that over 50% of κ-CN was found in one of its modified forms in milk. The fermentation process further significantly altered the composition between unmodified/modified κ-CN, with glycoslaytion being predominant compared to phosphorylation (p < 0.01). Further method development towards α and ß-CN fractions and their PTM behavior would be an asset to better understand the changes undergone by milk proteins and the micellar structure during fermentation.

Effect of the Post-Harvest Processing on Protein Modification in Green Coffee Beans by Phenolic Compounds.

The protein fraction, important for coffee cup quality, is modified during post-harvest treatment prior to roasting. Proteins may interact with phenolic compounds, which constitute the major metabolites of coffee, where the processing affects these interactions. This allows the hypothesis that the proteins are denatured and modified via enzymatic and/or redox activation steps. The present study was initiated to encompass changes in the protein fraction. The investigations were limited to major storage protein of green coffee beans. Fourteen Coffea arabica samples from various processing methods and countries were used. Different extraction protocols were compared to maintain the status quo of the protein modification. The extracts contained about 4-8 µg of chlorogenic acid derivatives per mg of extracted protein. High-resolution chromatography with multiple reaction monitoring was used to detect lysine modifications in the coffee protein. Marker peptides were allocated for the storage protein of the coffee beans. Among these, the modified peptides K.FFLANGPQQGGK.E and R.LGGK.T of the α-chain and R.ITTVNSQK.I and K.VFDDEVK.Q of ß-chain were detected. Results showed a significant increase (p < 0.05) of modified peptides from wet processed green beans as compared to the dry ones. The present study contributes to a better understanding of the influence of the different processing methods on protein quality and its role in the scope of coffee cup quality and aroma.

Development of Non-Targeted Mass Spectrometry Method for Distinguishing Spelt and Wheat.

Food fraud, even when not in the news, is ubiquitous and demands the development of innovative strategies to combat it. A new non-targeted method (NTM) for distinguishing spelt and wheat is described, which aids in food fraud detection and authenticity testing. A highly resolved fingerprint in the form of spectra is obtained for several cultivars of spelt and wheat using liquid chromatography coupled high-resolution mass spectrometry (LC-HRMS). Convolutional neural network (CNN) models are built using a nested cross validation (NCV) approach by appropriately training them using a calibration set comprising duplicate measurements of eleven cultivars of wheat and spelt, each. The results reveal that the CNNs automatically learn patterns and representations to best discriminate tested samples into spelt or wheat. This is further investigated using an external validation set comprising artificially mixed spectra, samples for processed goods (spelt bread and flour), eleven untypical spelt, and six old wheat cultivars. These cultivars were not part of model building. We introduce a metric called the D score to quantitatively evaluate and compare the classification decisions. Our results demonstrate that NTMs based on NCV and CNNs trained using appropriately chosen spectral data can be reliable enough to be used on a wider range of cultivars and their mixes.

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS.

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0-15 kDa, 15-35 kDa, 35-70 kDa and 70-250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.

Effect of Cereal α-Amylase/Trypsin Inhibitors on Developmental Characteristics and Abundance of Digestive Enzymes of Mealworm Larvae (Tenebrio molitor L.).

The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21% to 42% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8% and 14% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant.

Epigenetic DNA Methylation of EBI3 Modulates Human Interleukin-35 Formation via NFkB Signaling: A Promising Therapeutic Option in Ulcerative Colitis.

Ulcerative colitis (UC), a severe chronic disease with unclear etiology that is associated with increased risk for colorectal cancer, is accompanied by dysregulation of cytokines. Epstein-Barr virus-induced gene 3 (EBI3) encodes a subunit in the unique heterodimeric IL-12 cytokine family of either pro- or anti-inflammatory function. After having recently demonstrated that upregulation of EBI3 by histone acetylation alleviates disease symptoms in a dextran sulfate sodium (DSS)-treated mouse model of chronic colitis, we now aimed to examine a possible further epigenetic regulation of EBI3 by DNA methylation under inflammatory conditions. Treatment with the DNA methyltransferase inhibitor (DNMTi) decitabine (DAC) and TNFα led to synergistic upregulation of EBI3 in human colon epithelial cells (HCEC). Use of different signaling pathway inhibitors indicated NFκB signaling was necessary and proportional to the synergistic EBI3 induction. MALDI-TOF/MS and HPLC-ESI-MS/MS analysis of DAC/TNFα-treated HCEC identified IL-12p35 as the most probable binding partner to form a functional protein. EBI3/IL-12p35 heterodimers (IL-35) induce their own gene upregulation, something that was indeed observed in HCEC cultured with media from previously DAC/TNFα-treated HCEC. These results suggest that under inflammatory and demethylating conditions the upregulation of EBI3 results in the formation of anti-inflammatory IL-35, which might be considered as a therapeutic target in colitis.

Comparative quantification and differentiation of bracatinga (Mimosa scabrella Bentham) honeydew honey proteins using targeted peptide markers identified by high-resolution mass spectrometry.

Honey traceability is an important topic, especially for honeydew honeys, due to the increased incidence of adulteration. This study aimed to establish specific markers to quantify proteins in honey. A proteomics strategy to identify marker peptides from bracatinga honeydew honey was therefore developed. The proteomics approach was based on initial untargeted identification of honey proteins and peptides by LC-ESI-Triple-TOF-MS/MS, which identified the major royal jelly proteins (MRJP) presence. Afterwards, the peptides were selected by the in silico digestion. The marker peptides were quantified by the developed targeted LC-QqQ-MS/MS method, which provided good linearity and specificity, besides recoveries between 92 and 100% to quantify peptides from bracatinga honeydew honey. The uniqueness and high response in mass spectrometry were backed by further complementary protein analysis (SDS-PAGE). The selected marker peptides EALPHVPIFDR (MRJP 1), ILGANVK (MRJP 2), TFVTIER (MRJP 3), QNIDVVAR (MRJP 4), FINNDYNFNEVNFR (MRJP 5) and LLQPYPDWSWTK (MRJP 7), quantified by LC-QqQ-MS/MS, highlighted that the content of QNIDVVAR from MRJP 4 could be used to differentiate bracatinga honeydew honey from floral honeys (p < 0.05) as a potential marker for its authentication. Finally, principal components analysis highlighted the QNIDVVAR content as a good descriptor of the analyzed bracatinga honeydew honey samples.

Coffee By-Products as Sustainable Novel Foods: Report of the 2nd International Electronic Conference on Foods-"Future Foods and Food Technologies for a Sustainable World".

The coffee plant Coffea spp. offers much more than the well-known drink made from the roasted coffee bean. During its cultivation and production, a wide variety of by-products are accrued, most of which are currently unused, thermally recycled, or used as fertilizer or animal feed. Modern, ecologically oriented society attaches great importance to sustainability and waste reduction, so it makes sense to not dispose of the by-products of coffee production but to bring them into the value chain, most prominently as foods for human nutrition. There is certainly huge potential for all of these products, especially on markets not currently accessible due to restrictions, such as the novel food regulation in the European Union. The by-products could help mitigate the socioeconomic burden of coffee farmers caused by globally low coffee prices and increasing challenges due to climate change. The purpose of the conference session summarized in this article was to bring together international experts on coffee by-products and share the current scientific knowledge on all plant parts, including leaf, cherry, parchment and silverskin, covering aspects from food chemistry and technology, nutrition, but also food safety and toxicology. The topic raised a huge interest from the audience and this article also contains a Q&A section with more than 20 answered questions.

Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars.

Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, P81367 and P81368; both reviewed and a targeted LC-MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR (P81367) and TYMVR (P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.

Comprehensive Characterization and Relative Quantification of α-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS.

The α-amylase/trypsin inhibitors (ATIs) are discussed as being responsible for non-celiac wheat sensitivity (NCWS), besides being known as allergenic components for baker's asthma. Different approaches for characterization and quantification including proteomics-based methods for wheat ATIs have been documented. In these studies generally the major ATIs have been addressed. The challenge of current study was then to develop a more comprehensive workflow encompassing all reviewed wheat-ATI entries in UniProt database. To substantially test proof of concept, 46 German and Turkish wheat samples were used. Two extractions systems based on chloroform/methanol mixture (CM) and under buffered denaturing conditions were evaluated. Three aspects were optimized, tryptic digestion, chromatographic separation, and targeted tandem mass spectrometric analysis (HPLC-MS/MS). Preliminary characterization with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) documented the purity of the extracted ATIs with CM mixture and the amylase (60-80%)/trypsin (10-20%) inhibition demonstrated the bifunctional activity of ATIs. Thirteen (individual/common) biomarkers were established. Major ATIs (7-34%) were differently represented in samples. Finally, to our knowledge, the proposed HPLC-MS/MS method allowed for the first time so far the analysis of all 14 reviewed wheat ATI entries reported.

Comparison of Batch and Continuous Wet-Processing of Coffee: Changes in the Main Compounds in Beans, By-Products and Wastewater.

Many technical challenges still need to be overcome to improve the quality of the green coffee beans. In this work, the wet Arabica coffee processing in batch and continuous modus were investigated. Coffee beans samples as well as by-products and wastewaters collected at different production steps were analyzed in terms of their content in total phenols, antioxidant capacity, caffeine content, organic acids, reducing sugars, free amino group and protein content. The results showed that 40% of caffeine was removed with pulp. Green coffee beans showed highest concentration of organic acids and sucrose (4.96 ± 0.25 and 5.07 ± 0.39 g/100 g DW for the batch and continuous processing). Batch green coffee beans contained higher amount of phenols. 5-caffeoylquinic Acid (5-CQA) was the main constituent (67.1 and 66.0% for the batch and continuous processing, respectively). Protein content was 15 and 13% in the green coffee bean in batch and continuous processing, respectively. A decrease of 50 to 64% for free amino groups during processing was observed resulting in final amounts of 0.8 to 1.4% in the processed beans. Finally, the batch processing still revealed by-products and wastewater with high nutrient content encouraging a better concept for valorization.

Effect of Blanching Plus Fermentation on Selected Functional Properties of Mealworm (Tenebrio molitor) Powders.

The aim of this study was to determine the effect of blanching followed by fermentation of mealworms (Tenebrio molitor) with commercial meat starter cultures on the functional properties of powders produced from the larvae. Full fat and defatted powder samples were prepared from non-fermented and fermented mealworm pastes. Then the crude protein, crude fat, and dry matter contents, pH, bulk density, colour, water and oil binding capacity, foaming capacity and stability, emulsion capacity and stability, protein solubility, quantity of free amino groups, and protein composition of the powders were evaluated. Regardless of the starter culture used, the blanching plus fermentation process reduced the crude and soluble protein contents of the full fat powders and in general impaired their water and oil binding, foaming, and emulsifying properties. Defatting of the powders improved most functional properties studied. The o-phthaldialdehyde assay revealed that the amount of free amino groups was higher in the fermented powders while sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the soluble proteins of the fermented powders were composed of molecules of lower molecular mass compared to non-fermented powders. As molecular sizes of the soluble proteins decreased, it was clear that the protein structure was also modified by the fermentation process, which in turn led to changes in functional properties. In general, it was concluded that fermentation of mealworms with blanching as a pre-treatment does not contribute to the functional properties studied in this work. Nevertheless, the results confirmed that the properties of non-fermented powders are comparable to other food protein sources.

A New Approach of Extraction of α-Amylase/trypsin Inhibitors from Wheat (Triticum aestivum L.), Based on Optimization Using Plackett-Burman and Box-Behnken Designs.

Wheat is one of the most consumed foods in the world and unfortunately causes allergic reactions which have important health effects. The α-amylase/trypsin inhibitors (ATIs) have been identified as potentially allergen components of wheat. Due to a lack of data on optimization of ATI extraction, a new wheat ATIs extraction approach combining solvent extraction and selective precipitation is proposed in this work. Two types of wheat cultivars (Triticum aestivum L.), Julius and Ponticus were used and parameters such as solvent type, extraction time, temperature, stirring speed, salt type, salt concentration, buffer pH and centrifugation speed were analyzed using the Plackett-Burman design. Salt concentration, extraction time and pH appeared to have significant effects on the recovery of ATIs (p < 0.01). In both wheat cultivars, Julius and Ponticus, ammonium sulfate substantially reduced protein concentration and inhibition of amylase activity (IAA) compared to sodium chloride. The optimal conditions with desirability levels of 0.94 and 0.91 according to the Doehlert design were: salt concentrations of 1.67 and 1.22 M, extraction times of 53 and 118 min, and pHs of 7.1 and 7.9 for Julius and Ponticus, respectively. The corresponding responses were: protein concentrations of 0.31 and 0.35 mg and IAAs of 91.6 and 83.3%. Electrophoresis and MALDI-TOF/MS analysis showed that the extracted ATIs masses were between 10 and 20 kDa. Based on the initial LC-MS/MS analysis, up to 10 individual ATIs were identified in the extracted proteins under the optimal conditions. The positive implication of the present study lies in the quick assessment of their content in different varieties especially while considering their allergenic potential.

Cocoa Bean Proteins-Characterization, Changes and Modifications due to Ripening and Post-Harvest Processing.

The protein fractions of cocoa have been implicated influencing both the bioactive potential and sensory properties of cocoa and cocoa products. The objective of the present review is to show the impact of different stages of cultivation and processing with regard to the changes induced in the protein fractions. Special focus has been laid on the major seed storage proteins throughout the different stages of processing. The study starts with classical introduction of the extraction and the characterization methods used, while addressing classification approaches of cocoa proteins evolved during the timeline. The changes in protein composition during ripening and maturation of cocoa seeds, together with the possible modifications during the post-harvest processing (fermentation, drying, and roasting), have been documented. Finally, the bioactive potential arising directly or indirectly from cocoa proteins has been elucidated. The "state of the art" suggests that exploration of other potentially bioactive components in cocoa needs to be undertaken, while considering the complexity of reaction products occurring during the roasting phase of the post-harvest processing. Finally, the utilization of partially processed cocoa beans (e.g., fermented, conciliatory thermal treatment) can be recommended, providing a large reservoir of bioactive potentials arising from the protein components that could be instrumented in functionalizing foods.

Investigation of the post mortem zinc protoporphyrin IX fluorescence with respect to its protein-bound and unbound occurrence in aqueous meat extracts.

Zinc protoporphyrin IX (ZnPP) is known to accumulate in most meat products during storage. However, the pathway of its formation is not yet completely clarified. To gain more insights into the specificity of ZnPP occurrence, a SEC-HPLC-UV-fluorescence setup was established to screen the proteins in aqueous meat extracts for their ZnPP fluorescence during incubation. In accordance with previous studies it was identified by SDS-PAGE and MALDI-TOF-MS that ZnPP formation takes place in myoglobin. In this study, valuable new insights into the ZnPP forming pathway were gained, as our results indicated that a significant part of ZnPP - after being formed within the protein - is transitioned into free ZnPP during incubation. Additionally, the obtained results implied that ZnPP may also occur in proteins of higher molecular weight (>100 kDa).

Selected Plant Metabolites Involved in Oxidation-Reduction Processes during Bud Dormancy and Ontogenetic Development in Sweet Cherry Buds (Prunus avium L.).

Many biochemical processes are involved in regulating the consecutive transition of different phases of dormancy in sweet cherry buds. An evaluation based on a metabolic approach has, as yet, only been partly addressed. The aim of this work, therefore, was to determine which plant metabolites could serve as biomarkers for the different transitions in sweet cherry buds. The focus here was on those metabolites involved in oxidation-reduction processes during bud dormancy, as determined by targeted and untargeted mass spectrometry-based methods. The metabolites addressed included phenolic compounds, ascorbate/dehydroascorbate, reducing sugars, carotenoids and chlorophylls. The results demonstrate that the content of phenolic compounds decrease until the end of endodormancy. After a long period of constancy until the end of ecodormancy, a final phase of further decrease followed up to the phenophase open cluster. The main phenolic compounds were caffeoylquinic acids, coumaroylquinic acids and catechins, as well as quercetin and kaempferol derivatives. The data also support the protective role of ascorbate and glutathione in the para- and endodormancy phases. Consistent trends in the content of reducing sugars can be elucidated for the different phenophases of dormancy, too. The untargeted approach with principle component analysis (PCA) clearly differentiates the different timings of dormancy giving further valuable information.