Improvement of Robusta coffee aroma by modulating flavor precursors in the green coffee bean with enzymatically treated spent coffee grounds: A circular approach.

Spent coffee grounds (SCG) are by-products obtained from the industrial process of instant coffee production or alternatively after brewing of coffee at the point of consumption. This solid residue represents one of the largest waste materials worldwide, making this fraction a rational target for valorization. The composition of SCG varies significantly depending on the brewing and extraction methods. However, this by-product is mainly composed of cellulose, hemicellulose polysaccharides and lipids. Here, we report on the enzymatic hydrolysis of industrial SCG by the use of a combination of specific carbohydrate active enzymes, enabling sugar extraction yield of 74.3 %. The generated sugar-rich extract, primarily composed of glucose (8.41 ± 1.00 % of total SCG mass) and mannose (2.88 ± 0.25 % of total SCG mass), is separated from hydrolyzed grounds and soaked with green coffee. After drying and roasting, the coffee soaked with SCG enzymatic extract displayed lower earthy, burnt and rubbery notes as well as smoother and more acidic notes in the flavor profile as compared to untreated reference. Aroma profiling performed by SPME-GC-MS corroborated the sensorial effect, with a 2-fold increase in the generation of sugar-derived molecules such as Strecker aldehydes and diketones after soaking and roasting and a 45 % and respectively 37 % reduction in phenolic compounds and pyrazines. This novel technology could represent an innovative in situ valorization stream for the coffee industry, coupled with sensory improvement of the final cup.

Unlocking the Aromatic Potential of Native Coffee Yeasts: From Isolation to a Biovolatile Platform.

Postharvest processing of coffee has been shown to impact cup quality. Yeasts are known to modulate the sensory traits of the final cup of coffee after controlled fermentation at the farm. Here, we enumerated native coffee yeasts in a Nicaraguan farm during dry and semidry postharvest processing of Arabica and Robusta beans. Subsequently, 90 endogenous yeast strains were selected from the collected endogenous isolates, identified, and subjected to high-throughput fermentation and biovolatile generation in a model system mimicking postharvesting conditions. Untargeted volatile analysis by SPME-GC-MS enabled the identification of key aroma compounds generated by the yeast pool and demonstrated differences among strains. Several genera, including Pichia, Candida, and Hanseniaspora, showed both strain- and species-level variability in volatile generation and profiles. This fermentation platform and biovolatile database could represent a versatile opportunity to accelerate the development of yeast starter cultures for generating specific and desired sensory attributes in the final cup of coffee.

Cereal bran protects vitamin A from degradation during simmering and storage.

Food supplementation with vitamin A is an efficient strategy to combat vitamin A deficiency. The stability of vitamin A during cooking and storage is, however, low. We here show that cereal bran protects retinyl palmitate (RP) during simmering and storage. Native wheat bran stabilized RP the most during simmering. About 75% RP was recovered after 120 min of cooking, while all RP was lost after 80 min in the absence of bran. Heat-treated rice bran protected RP the best during forced storage, with a 35% recovery after 8 weeks. RP was degraded entirely in the absence of bran in less than one week. Results suggested that the physical entrapment of oil within the large wheat bran particles protects RP from the action of water and pro-oxidants during simmering. During storage, the high amount and diversity of lipid components present in rice bran are presumably responsible for its protective effect.

Influence of Various Processing Parameters on the Microbial Community Dynamics, Metabolomic Profiles, and Cup Quality During Wet Coffee Processing.

Post-harvest wet coffee processing is a commonly applied method to transform coffee cherries into green coffee beans through depulping or demucilaging, fermentation, washing, soaking, drying, and dehulling. Multiple processing parameters can be modified and thus influence the coffee quality (green coffee beans and cup quality). The present study aimed to explore the impacts of these parameters, including processing type (depulping or demucilaging), fermentation duration, and application of soaking, on the microbial community dynamics, metabolite compositions of processing waters (fermentation and soaking) and coffee beans, and resulting cup quality through a multiphasic approach. A large-scale wet coffee processing experiment was conducted with Coffea arabica var. Catimor in Yunnan (China) in duplicate. The fermentation steps presented a dynamic interaction between constant nutrient release (mainly from the cherry mucilage) into the surrounding water and active microbial activities led by lactic acid bacteria, especially Leuconostoc and Lactococcus. The microbial communities were affected by both the processing type and fermentation duration. At the same time, the endogenous coffee bean metabolism remained active at different stages along the processing, as could be seen through changes in the concentrations of carbohydrates, organic acids, and free amino acids. Among all the processing variants tested, the fermentation duration had the greatest impact on the green coffee bean compositions and the cup quality. A long fermentation duration resulted in a fruitier and more acidic cup. As an ecological alternative for the depulped processing, the demucilaged processing produced a beverage quality comparable to the depulped one. The application of soaking, however, tempered the positive fermentation effects and standardized the green coffee bean quality, regardless of the preceding processing practices applied. Lastly, the impact strength of each processing parameter would also depend on the coffee variety used and the local geographical conditions. All these findings provide a considerable margin of opportunities for future coffee research.

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome.

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

Enrichment of anaerobic heterotrophic thermophiles from four Azorean hot springs revealed different community composition and genera abundances using recalcitrant substrates.

DGGE analysis combined with a metagenomic approach was used to get insights into heterotrophic anoxic enrichment cultures of four hot springs of Vale das Furnas, Portugal, using the recalcitrant substrate spent coffee ground (SCG). Parallel enrichment cultures were performed using the major components of spent coffee ground, namely arabinogalactan, galactomannan, cellulose, and proteins. DGGE revealed that heterotrophic thermophilic bacteria are highly abundant in the hydrothermal springs and significant differences in community composition depending on the substrate were observed. DNA, isolated from enrichment cultures of different locations that were grown on the same substrate were pooled, and the respective metagenomes were analyzed. Results indicated that cultures grown on recalcitrant substrate SCG consists of a totally different thermophilic community, dominated by Dictyoglomus. Enrichments with galactomannan and arabinogalactan were dominated by Thermodesulfovibrio, while cultures with casein and cellulose were dominated by Thermus. This study indicates the high potential of thermophilic bacteria degrading recalcitrant substrate such as SCG and furthermore how the accessibility to complex polymers shapes the bacterial community.

Following Coffee Production from Cherries to Cup: Microbiological and Metabolomic Analysis of Wet Processing of Coffea arabica.

A cup of coffee is the final product of a complex chain of operations. Wet postharvest processing of coffee is one of these operations, which involves a fermentation that inevitably has to be performed on-farm. During wet coffee processing, the interplay between microbial activities and endogenous bean metabolism results in a specific flavor precursor profile of the green coffee beans. Yet, how specific microbial communities and the changing chemical compositions of the beans determine the flavor of a cup of coffee remains underappreciated. Through a multiphasic approach, the establishment of the microbial communities, as well as their prevalence during wet processing of Coffea arabica, was followed at an experimental farm in Ecuador. Also, the metabolites produced by the microorganisms and those of the coffee bean metabolism were monitored to determine their influence on the green coffee bean metabolite profile over time. The results indicated that lactic acid bacteria were prevalent well before the onset of fermentation and that the fermentation duration entailed shifts in their communities. The fermentation duration also affected the compositions of the beans, so that longer-fermented coffee had more notes that are preferred by consumers. As a consequence, researchers and coffee growers should be aware that the flavor of a cup of coffee is determined before as well as during on-farm processing and that under the right conditions, longer fermentation times can be favorable, although the opposite is often believed.IMPORTANCE Coffee needs to undergo a long chain of events to transform from coffee cherries to a beverage. The coffee postharvest processing is one of the key phases that convert the freshly harvested cherries into green coffee beans before roasting and brewing. Among multiple existing processing methods, the wet processing has been usually applied for Arabica coffee and produces decent quality of both green coffee beans and the cup of coffee. In the present case study, wet processing was followed by a multiphasic approach through both microbiological and metabolomic analyses. The impacts of each processing step, especially the fermentation duration, were studied in detail. Distinct changes in microbial ecosystems, processing waters, coffee beans, and sensory quality of the brews were found. Thus, through fine-tuning of the parameters in each step, the microbial diversity and endogenous bean metabolism can be altered during coffee postharvest processing and hence provide potential to improve coffee quality.

Vitamin A degradation in triglycerides varying by their saturation levels.

Vitamin A deficiency has a widespread occurrence globally and is considered as one of the world's most serious health risk factors. Potential solutions to address this deficiency include dietary diversification or supplementation, but food fortification is generally accepted as the most cost-effective solution. The main issue with food fortification of this vitamin is related to its high instability in food matrices. Dilution of vitamin A in triglycerides is a natural and appropriate way to stabilize this compound. We show here that vitamin A palmitate stability increases with increasing concentration of triglycerides. Moreover, we found that vitamin A palmitate displays improved stability in more saturated oils. Using various temperatures, and Arrhenius plots of experiments performed at storage temperatures between 30°C and 60°C for oils varying by their saturation and crystallinity, we demonstrate that crystallization is not responsible for this phenomenon. Additionally, we show by centrifugation that vitamin A is preferably solubilized in the liquid phase compared to the crystalline phase, explaining that triglyceride crystallization does not stabilize vitamin A palmitate. It is proposed that unsaturated fats generate more oxidation products such as radicals and peroxides, leading to a quicker degradation of vitamin A.

The pseudoenzyme PDX1.2 boosts vitamin B6 biosynthesis under heat and oxidative stress in Arabidopsis.

Vitamin B6 is an indispensable compound for survival, well known as a cofactor for numerous central metabolic enzymes and more recently for playing a role in several stress responses, particularly in association with oxidative stress. Regulatory aspects for the use of the vitamin in these roles are not known. Here we show that certain plants carry a pseudoenzyme (PDX1.2), which is involved in regulating vitamin B6 biosynthesis de novo under stress conditions. Specifically, we demonstrate that Arabidopsis PDX1.2 enhances the activity of its catalytic paralogs by forming a heterododecameric complex. PDX1.2 is strongly induced by heat as well as singlet oxygen stress, concomitant with an enhancement of vitamin B6 production. Analysis of pdx1.2 knockdown lines demonstrates that boosting vitamin B6 content is dependent on PDX1.2, revealing that this pseudoenzyme acts as a positive regulator of vitamin B6 biosynthesis during such stress conditions in plants.

It takes two to tango: defining an essential second active site in pyridoxal 5'-phosphate synthase.

The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2) that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5'-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5'-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.

Lipophilicity determination of highly lipophilic compounds by liquid chromatography.

Different experimental strategies using short columns in both conventional liquid chromatography (HPLC) and ultra-high pressure liquid chromatography (UHPLC) were evaluated to allow, for the first time with these techniques, the lipophilicity determination of compounds with log P>5. Various organic modifiers, stationary phases, and elution modes were tested on 14 rigid compounds with a CLogP between 5 and 8, and 38 compounds with log P(oct) from 0 to 5. The best results in HPLC were obtained with the 20-mm Discovery RP Amide C16 stationary phase in isocratic mode using MeOH as organic modifier. To improve analysis time, the UHPLC approach was then evaluated. Consequently, a generic method was developed with a 30-mm Acquity BEH Shield RP18 column in gradient mode using MeOH as organic modifier, allowing a fourfold gain of time compared to the HPLC method, for the highly lipophilic compounds tested. Finally, the most rapid and accurate results were obtained with a 10-mm Hypersil GOLD Javelin HTS stationary phase in UHPLC, enabling an eightfold gain of time compared to the HPLC method.

Intersubunit cross-talk in pyridoxal 5'-phosphate synthase, coordinated by the C terminus of the synthase subunit.

Vitamin B(6) is essential in all organisms, due to its requirement as a cofactor in the form of pyridoxal 5'-phosphate (PLP) for key metabolic enzymes. It can be synthesized de novo by either of two pathways known as deoxyxylulose 5-phosphate (DXP)-dependent and DXP-independent. The DXP-independent pathway is the predominant pathway and is found in most microorganisms and plants. A glutamine amidotransferase consisting of the synthase Pdx1 and its glutaminase partner, Pdx2, form a complex that directly synthesizes PLP from ribose 5-phosphate, glyceraldehyde 3-phosphate, and glutamine. The protein complex displays an ornate architecture consisting of 24 subunits, two hexameric rings of 12 Pdx1 subunits to which 12 Pdx2 subunits attach, with the glutaminase and synthase active sites remote from each other. The multiple catalytic ability of Pdx1, the remote glutaminase and synthase active sites, and the elaborate structure suggest regulation of activity on several levels. A missing piece in deciphering this intricate puzzle has been information on the Pdx1 C-terminal region that has thus far eluded structural characterization. Here we use fluorescence spectrophotometry and protein chemistry to demonstrate that the Pdx1 C terminus is indispensable for PLP synthase activity and mediates intersubunit cross-talk within the enzyme complex. We provide evidence that the C terminus can act as a flexible lid, bridging as well as shielding the active site of an adjacent protomer in Pdx1. We show that ribose 5-phosphate binding triggers strong cooperativity in Pdx1, and the affinity for this substrate is substantially enhanced upon interaction with the Michaelis complex of Pdx2 and glutamine.