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.

Discovery of Polyhydroxyalkyl Pyrazine Generation upon Coffee Roasting by In-Bean Labeling Experiments.

The major non-volatile reaction products formed from free amino acids during the early stage of coffee roasting were investigated using biomimetic in-bean experiments with labeled and unlabeled free amino acids. Comprehensive untargeted screening by ultra-high performance liquid chromatography-electrospray-ionization-quadrupole time-of-flight-tandem mass spectrometry (UHPLC-ESI-QToF-MS) in data-independent acquisition (DIA) mode was carried out and modeling by orthogonal partial least-squares discriminant analysis (OPLS-DA) helped in revealing 11 pyrazine structures identified in coffee for the first time. 2-(2',3',4'-Trihydroxybutyl)-(5/6)-methyl-pyrazine (1) and 2,(5/6)-bis(2',3',4'-trihydroxybutyl)-pyrazine (2) were the most prominent compounds, while 2-(3',4'-dihydroxybutyl)-(5/6)-methyl-pyrazine (5) and 2-(2',3',4'-trihydroxybutyl)-(5/6)-(2'-hydroxyethyl)-pyrazine (10) were not even previously identified in other food matrices. The structures could be verified by means of additional biomimetic in-bean experiments with labeled sucrose leveraging the carbon module labeling (CAMOLA) approach. Based on these results, plausible formation pathways could be formulated fitting into the known Maillard reaction mechanisms. Sucrose was highlighted as the predominant precursor of the carbon backbone of all identified pyrazines butonly 33-55% of the nitrogen atoms originated from free amino acids.

Quantitative Validation of the In-Bean Approach in Coffee Roasting.

The representativeness of the so-called biomimetic "in-bean" technique was studied by following the formation of target aroma compounds during the roasting course (10 points from 0 to 400 s) in recombined coffee beans and non-treated green coffee reference beans. For this purpose, the water-soluble fraction was replaced by a biomimetic recombinate in reconstituted beans prior to roasting. The targeted analysis of key aroma compounds was performed by means of the stable isotope dilution assay and solid-phase microextraction-gas chromatography-mass spectrometry of roasted and ground coffee samples. The results were compared to the quantitative data on a green coffee reference roasted under the same conditions. The results showed similar formation kinetics for most of the evaluated volatiles, such as Strecker aldehydes, alkylpyrazines, or α-diketones. In addition, the final quantities of key odorants in both types of samples were quite comparable. Hence, the refined biomimetic approach was validated as a valuable tool in studying different aspects of flavor formation upon coffee roasting.

Generation of α-Diketones and 4-Hydroxy-2,5-dimethyl-3(2H)-furanone upon Coffee Roasting-Impact of Roast Degree on Reaction Pathways.

The formation pathways of α-diketones (2,3-butanedione and 2,3-pentanedione) and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) upon coffee roasting were investigated in a kinetic study applying labeled and unlabeled sucrose (CAMOLA approach) in biomimetic in-bean experiments. The results highlighted that not only did the contribution of sucrose to the level of α-diketones in roasted coffee change with the roasting degree but also the portion of the individual reaction pathways. At early roasting stages, 2,3-butanedione was formed from sucrose mainly via the intact sugar skeleton, whereas from the middle of the roasting course, the formation foremost occurred from sugar fragments, primarily by C1/C3 and C2/C2 recombinations. In contrast, 2,3-pentanedione was generated from sucrose mainly via an intact sugar skeleton during the whole roasting cycle; nevertheless, the share of 2,3-pentanedione formed by recombination of fragments (mainly C2/C3) progressively increased with roasting time. HDMF was generated from sucrose almost exclusively via cyclization of an intact skeleton, irrespective of the roast time.

New Insight into the Role of Sucrose in the Generation of α-Diketones upon Coffee Roasting.

The origin and formation pathways of the buttery-smelling α-diketones 2,3-butanedione and 2,3-pentanedione upon coffee roasting were studied by means of biomimetic in-bean experiments combined with labeling experiments. For this purpose natural sucrose in the coffee bean was replaced by fully or partially 13C-labeled sucrose or by a mixture of unlabeled and fully 13C-labeled sucrose (CAMOLA approach). The obtained data point out that sucrose contributes to both α-diketones; however, its importance and reaction pathways clearly differ. Whereas the major part of 2,3-pentanedione originates from sucrose (about 76%), its contribution to 2,3-butanedione is much lower (about 35%). Formation from intact sugar skeleton is the major pathway generating 2,3-pentanedione from sucrose, whereas 2,3-butanedione is mainly generated by recombination of sucrose fragments. The contribution of glucose and fructose moieties of sucrose to both α-diketones is comparable. Finally, kinetic experiments with fully labeled sucrose showed that the contribution of sucrose changes during roasting.

Identification of modified lysozyme peptides upon photo-oxidation by LC-TOF-MS.

Protein oxidation can have major implications on the quality and safety of foods, but the majority of methods to evaluate oxidative damage lack specificity. Therefore, this study aimed to identify specific markers for protein oxidation. A well-characterized protein, lysozyme, was modified by photo-oxidation and subsequently hydrolyzed prior to peptide analysis by LC-TOF-MS. A semiquantitative analysis of the peptides indicated that from the seven peptides containing sensitive amino acids, two peptides (HGLDNYR and WWCNDGR) were highly affected upon photo-oxidation and have the potential to serve as markers for protein oxidation. Site-specific modifications enabled the description of the degradation pathway of several lysozyme peptides but also indicated that the surrounding amino acids and the 3D structure of the protein have an impact on the induced modifications. It is therefore advisable to evaluate protein oxidation on the intact protein.

Mycotoxin co-occurrence in rice, oat flakes and wheat noodles used as staple foods in Ecuador.

The co-occurrence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2), ochratoxin A (OTA), deoxynivalenol (DON), fumonisin B1 (FB1), zearalenone (ZEN), and HT-2 and T-2 toxins in the main Ecuadorian staple cereals (rice, oat flakes, and yellow and white wheat noodles) was evaluated. A ultra high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) method was developed and validated to screen for the presence of these mycotoxins in those cereal matrices. Matrix-matched calibration curves were used to compensate for ion suppression and extraction losses and the recovery values were in agreement with the minimum requirements of Regulation 401/2006/EC (70-110%). For most mycotoxins, the LODs obtained allowed detection in compliance with the maximum permitted levels set in Regulation EC/2006/1881, with the exception of OTA in all cereals and AFB1 in yellow noodles. Extra target analysis of OTA in oat flakes and wheat noodles was performed by HPLC with fluorescence detection. High rates of contamination were observed in paddy rice (23% DON, 23% FB1, 7% AFB1, 2% AFG1 and 2% AFG2), white wheat noodles (33% DON and 5% OTA) and oat flakes (17% DON, 2% OTA and 2% AFB1), whereas the rates of contamination were lower in polished rice (2% AFG1 and 4% HT-2 toxin) and yellow noodles (5% DON). Low rates of co-occurrence of several mycotoxins were observed only for white wheat noodles (5%) and paddy rice (7%). White noodles were contaminated with DON and/or OTA, while combinations of AFG1, AFB1, DON and FB1 were found in paddy rice. Yellow noodles were contaminated with DON only; oat flakes contained DON, OTA or AFB1, and polished rice was contaminated with AFG1 and HT-2 toxin.

The impact of photo-induced molecular changes of dairy proteins on their ACE-inhibitory peptides and activity.

Among all dietary proteins, dairy proteins are the most important source of bio-active peptides which can, however, be affected by modifications upon processing and storage. Since it is still unknown to which extent the biological activity of dairy proteins is altered by chemical reactions, this study focuses on the effect of photo-induced molecular changes on the angiotensin I converting enzyme (ACE) inhibitory activity. Milk proteins were dissolved in phosphate buffer containing riboflavin and stored under light at 4 °C for one month during which the molecular changes and the ACE-inhibitory activity were analysed. An increase in the total protein carbonyls and the N-formylkynurenine content was observed, besides a decrease in the free thiol, tryptophan, tyrosine and histidine content. These changes were more severe in caseins compared with whey proteins and resulted moreover in the aggregation of caseins. Due to these photo-induced molecular changes, a significant loss of the ACE-inhibitory activity was observed for casein peptides. A peptide analysis moreover illustrated that the decreased activity was not attributed to a reduced digestibility but to losses of specific ACE-inhibitory peptides. The observed molecular changes, more specifically the degradation of specific amino acids and the casein aggregation, could be assigned as the cause of the altered peptide pattern and as such of the loss in ACE-inhibitory activity.

A two-year investigation towards an effective quality control of incoming potatoes as an acrylamide mitigation strategy in french fries.

The current entrance quality control used in the french fries industry is done based on colour evaluation with a United States Department of Agriculture/Munsell colour chart (after a short frying test, typically 180°C for 3 min). On the basis of a study carried out during two consecutive potato storage seasons, the possibility of a more effective entrance control of the raw potato tubers in order to identify batches of potatoes prone to acrylamide formation was evaluated. The current entrance control was compared to two other colour evaluation methods (CIE L*a*b* colour parameters and a process-specific Agtron analyser) and to reducing sugar content determination. Seasonal variability did not affect the slopes of the linear correlation models, for most of the parameters studied. The determination of colour formation measured by the Agtron methodology and reducing sugar content allowed a better identification of batches of potatoes prone to acrylamide formation compared with the current entrance control. Different scenarios represented by decision trees for quality control measures for incoming potatoes were evaluated while considering the investigation value of 600 µg kg⁻¹ for french fries recently prescribed by the European Commission. Samples were categorised based on predictions of threshold values and acrylamide levels in the final product.

Implementation of acrylamide mitigation strategies on industrial production of French fries: challenges and pitfalls.

This study evaluated various additives or process aids on the industrial production of French fries, based on their acrylamide mitigation potential and other quality parameters. The application of acetic and citric acid, calcium lactate and asparaginase was investigated on the production of frozen par-fried French fries at the beginning and end of the 2008 and 2009 potato storage season. Despite the fact that some of these treatments significantly reduced acrylamide content of the final product in preliminary laboratory experiments, their application on the industrial production of French fries did not result in additional acrylamide reductions compared to the standard product. Asparaginase was additionally tested in a production line of chilled French fries (not par-fried). Since for this product a longer enzyme-substrate contact time is allowed, a total asparagine depletion was observed for the enzyme treated fries after four days of cold storage. French fries upon final frying presented acrylamide contents below the limit of detection (12.5 µg kg⁻¹) with no effects on the sensorial properties of the final product.

Hypochlorous and peracetic acid induced oxidation of dairy proteins.

Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

Effective quality control of incoming potatoes as an acrylamide mitigation strategy for the French fries industry.

The correlation between sugar levels in raw material (potatoes), brown colouring and formation of acrylamide in French fries was investigated. The objective was to identify incoming potatoes (raw material) with a high potential for acrylamide formation. Ten different potato varieties commonly used in the Western European French fries industry were stored at 8 degrees C and samples were taken throughout the storage time. The current quality control used in the French fries industry for incoming potatoes is poorly correlated with acrylamide in the final product (r = 0.74). Changing the quality control parameter from colour to reducing sugars in raw material did not improve the correlation (r = 0.72). The best correlation was obtained with the Agtron colour measurement after blanching and a two-stage frying (r = -0.88). It was concluded that alternative entrance control measurements could provide better mitigation of the acrylamide issue in French fries from the start of production. These alternatives, however, are less cost-effective and more difficult to implement.

Importance of oil degradation components in the formation of acrylamide in fried foodstuffs.

This study investigates the importance of selected oil degradation components and some analogues in the formation of acrylamide. For this, a model system containing silica gel, PBS buffer, and oil was heated in a closed tubular reactor, under practically relevant heating conditions. Several probable acrylamide precursors were mixed together with free asparagine in the model system, such as partial glycerides, glycerol, acrolein, acrylic acid, and several aldehydes. Only the heated model system containing acrolein and asparagine showed a significantly higher acrylamide content compared to the control to which only asparagine was added. It was postulated that a nucleophilic 1,2-addition of the alpha-amino group of free asparagine to the carbonyl function of acrolein would lead to the formation of acrylamide. This hypothesis could partially be confirmed, replacing acrolein with other alpha,beta-unsaturated aldehydes. However, the contribution of acrolein to the overall formation of acrylamide appeared to be negligible in the presence of a reducing sugar, indicating that in foodstuffs the importance of acrolein and other oil degradation products is probably small.

Importance of a canteen lunch on the dietary intake of acrylamide.

A food and drink intake survey was carried out among university students and staff members. Consumption data were collected on days when the participants took hot lunch in a university canteen. The dietary acrylamide exposure was calculated through a probabilistic approach and revealed a median intake of 0.40 microg/kg bw/day [90% confidence interval: 0.36-0.44], which is in accordance with previous exposure calculations. Biscuits (35.4%), French fries (29.9%), bread (23.5%), and chocolate (11.2%) were identified to be the main sources of dietary acrylamide. Foodstuffs consumed in between the three main meals of the day (so called snack type foods) contributed the most to the intake (42.2%). The exposure was lower in an intervention group which received free portions of fruit and vegetables, indicating that a nutritionally balanced diet may contribute to a decreased acrylamide intake. French fries had a significant impact on the acrylamide intake, due to the frequent consumption in the canteen. This demonstrates the important responsibility of caterers and canteen kitchens in the mitigation of acrylamide exposure through reduction of acrylamide in their prepared products, in particular in French fries.

Role of water upon the formation of acrylamide in a potato model system.

The moisture sorption isotherms of a commercial potato powder were investigated at 20 degrees C for water activities ranging from 0.11 to 0.97. The sorption isotherms were typical type-II sigmoidal curves, with a steep increase in moisture content for water activities above 0.9 and exhibiting hysteresis over the whole water activity range. On the basis of the isotherms, the influence of the initial water activity and moisture content on both Maillard browning and acrylamide formation was determined by heating oil containing potato powder mixtures in a closed stainless-steel tubular reactor. The Maillard browning, as determined spectrophotometrically, showed an optimum at intermediate water activities. The yields of acrylamide, expressed relatively to the molar amount of asparagine, remained constant below 0.8 aw and below moisture contents of about 20% (on a dry basis). For the more intense heat treatments, an increased acrylamide yield was however observed at higher moisture contents, with an optimum at water contents of about 100% (on a dry basis). However, this increase and optimum was not observed at less intense heat treatments. At moisture contents above 100%, a significant decrease in acrylamide yields was assessed, although the water activity increased only marginally in this area of the sorption isotherms. It was thus observed that the acrylamide content was rather dependent upon the moisture content than upon the water activity in the high-moisture potato powder model system.

Selection criteria for potato tubers to minimize acrylamide formation during frying.

A number of parameters linked to the selection of potato tubers were evaluated with regard to their potential to influence acrylamide formation in French fries. The formation of acrylamide, which is a potential human carcinogen, can be minimized for a big extent by the selection of an appropriate tuber. This study focused on the following selection criteria: variety as influenced by storage time and soil type, underwater weight, and tuber size. A total of 16 varieties were compared, concerning their potential for acrylamide formation. From that survey, certain varieties, such as Tebina and Quincy, could be appointed as unsuitable for frying. The differences in the potential of acrylamide formation between the varieties could mainly be explained by the reducing sugar content of the potato (R2 = 0.82, n = 96). The investigated type of soil and storage time at 8 degrees C appeared to have a minor influence on the acrylamide formation during frying. On the other hand, the tuber size of the potato did contribute in a significant manner to the acrylamide formation. Smaller tubers were more susceptible to acrylamide formation and should be avoided in the frying process. The last selection parameter, the underwater weight, appeared to be of minor importance in the acrylamide formation. On the basis of these simple selection criteria, it is possible to make a first screening of potatoes to reduce the acrylamide formation during frying.

Influence of fertilization on acrylamide formation during frying of potatoes harvested in 2003.

The quality of the potato has been found to vary, when grown under different agricultural and environmental conditions, such as the level of fertilization. Consequently these factors may influence the acrylamide formation during the preparation of French fries. These assumptions were studied on three varieties: Bintje, Ramos, and Saturna from the harvest of 2003. Decreasing N fertilization caused increases in the reducing sugar concentration from 60% up to 100% on DM for all varieties studied. Due to a high correlation between the reducing sugar content and the generation of acrylamide during frying, this resulted in a parallel increase in the acrylamide concentration of the French fries. Thus by lowering the amount of N fertilizer, an increase of 30-65% of the acrylamide generation during frying could be observed. It seems of extreme importance to find an appropriate balance between the level of N fertilizer in order to diminish acrylamide formation but on the other hand to obtain an acceptable tuber and to consider the environmental impact. All results reported should be seen in the perspective of the warm growing season of 2003.

Influence of storage practices on acrylamide formation during potato frying.

A number of parameters linked to storage of potatoes were evaluated with regard to their potential to influence the acrylamide formation in French fries. Acrylamide, which is a potential human carcinogen, is reported to be formed during the frying of potatoes as a result of the reactions between asparagine and reducing sugars. This study was conducted using three potato varieties (Bintje, Ramos, and Saturna) typically used in Belgium, The Netherlands, and the northern part of France for French fry and crisp production. Saturna, mainly used in crisp production, appeared to be the least susceptible for acrylamide formation during frying. Especially storage at low temperatures (4 degrees C) compared to storage at 8 degrees C seemed to enhance acrylamide formation due to a strong increase in reducing sugars caused by low-temperature storage. Because of the reversible nature of this physiological reaction, it was possible to achieve a significant reduction of the reducing sugars after a reconditioning of the cold-stored potatoes for 3 weeks at 15 degrees C. All changes in acrylamide concentrations could mainly be explained by the reducing sugar content of the potato (R2 = 0.84, n = 160). This means that, by ensuring a low reducing sugar content of the potato tuber, the risk for acrylamide formation will largely be reduced. Finally the use of a sprout inhibitor did not influence the composition of the potato, and thus acrylamide formation was not susceptible to this treatment.

Influence of oil type on the amounts of acrylamide generated in a model system and in French fries.

Acrylamide formation was studied by use of a new heating methodology, based on a closed stainless steel tubular reactor. Different artificial potato powder mixtures were homogenized and subsequently heated in the reactor. This procedure was first tested for its repeatability. By use of this experimental setup, it was possible to study the acrylamide formation mechanism in the different mixtures, eliminating some variable physical and chemical factors during the frying process, such as heat flux and water evaporation from and oil ingress into the food. As a first application of this optimized heating concept, the influence on acrylamide formation of the type of deep-frying oil was investigated. The results obtained from the experiments with the tubular reactor were compared with standardized French fry preparation tests. In both cases, no significant difference in acrylamide formation could be found between the various heating oils applied. Consequently, the origin of the deep-frying vegetable oils did not seem to affect the acrylamide formation in potatoes during frying. Surprisingly however, when artificial mixtures did not contain vegetable oil, significantly lower concentrations of acrylamide were detected, compared to oil-containing mixtures.