Off-flavor Compounds Research Articles

Occurrence of Brettanomyces/Dekkera in Brazilian red wines and its correlation with ethylphenols

The yeast Brettanomyces/Dekkera can cause important spoilage in wines, with the production of ethylphenols and other off-flavor compounds. This study aimed at determining the presence of this yeast and the ethylphenols produced by them in Brazilian red wines, establishing their relationship with other chemical characteristics. Isolates of Brettanomyces/Dekkera were quantified by plating 126 samples of dry red wine in selective culture medium, while ethylphenols were analyzed by solid phase extraction and GC/FID. Free and total SO2, alcohol, total dry extract, residual sugar, total and volatile acidity, and pH were also determined. Brettanomyces/Dekkera was present in 27% of samples. Ethylphenols were detected in most samples, with amounts higher than the threshold limit of 426 μg/L found in 46.03% of samples. The majority of wine samples showed inadequate levels of SO2 and residual sugars, facts that might facilitate microbial spoilage. The passage in barrels and the grape varieties (Cabernet Sauvignon and Merlot), did not show any influence on the levels of contamination or ethylphenols contents. The prevalence of Brettanomyces/Dekkera and the concentrations of ethylphenols were high considering the sensory impact they can cause. The growth of Brettanomyces/Dekkera was dependent on the levels of SO2 and alcohol of wines. Knowledge of the contamination, the presence of ethylphenols, and their relationship with the chemical characteristics of wines can entice effective measures to prevent Brettanomyces/Dekkera and contribute to improve the general quality of Brazilian red wines.

Factors Influencing the Formation of Medicinal Off-Flavor from Ascorbic Acid and α,β-Unsaturated Aldehydes

In a previous study, the formation and formation pathway of 6-propylbenzofuran-7-ol as an off-flavor compound formed from ascorbic acid and (E)-hex-2-enal in a test apple beverage were investigated. In the present study, elucidating the pH, temperature, and (E)-hex-2-enal and ascorbic acid concentrations that lead to the generation of 6-propylbenzofuran-7-ol in various model solutions was performed. The quantities of 6-propylbenzofuran-7-ol generated in model solutions increased when the initial concentrations of (E)-hex-2-enal and ascorbic acid increased, and linear correlations between the quantities and concentrations were observed. The quantity of 6-propylbenzofuran-7-ol steadily rose in step with pH from pH 2.24, peaked at pH 3.73, and steadily decreased in step with pH to pH 4.24. The quantity of 6-propylbenzofuran-7-ol increased sharply as the storage temperature rose. Meanwhile, no 6-propylbenzofuran-7-ol was produced when short-duration heating processes such as heat sterilization were applied. These results will allow to us prevent the occurrence of off-flavor by regulating the initial pH, storage temperature, and (E)-hex-2-enal and ascorbic acid concentrations. Reactions with ascorbic acid and other α,β-unsaturated aldehydes were also investigated, and it was confirmed that corresponding 6-alkylbenzofuran-7-ols were formed.

Elucidating the Formation Pathway of the Off-Flavor Compound 6-Propylbenzofuran-7-ol

In a previous work, we identified 6-propylbenzofuran-7-ol as an off-flavor compound formed from ascorbic acid and (E)-hex-2-enal in a test apple beverage. In this study, we elucidate the pathway by which 6-propylbenzofuran-7-ol formed. Isotope labeling studies revealed that the propyl group of 6-propylbenzofuran-7-ol derives from (E)-hex-2-enal and that 6-propylbenzofuran-7-ol contains carbons 2-6 of ascorbic acid. Two compounds, namely, 2,3-dihydro-6-propylbenzofuran-3,7-diol and 3-(2-furoyl)hexanal, were identified as byproducts of a model reaction of ascorbic acid and (E)-hex-2-enal. Each of these compounds was dissolved in an aqueous solution of citric acid and stored at 60 °C for 1 week. After storage, 6-propylbenzofuran-7-ol was detected from a solution of 2,3-dihydro-6-propylbenzofuran-3,7-diol, but not from 3-(2-furoyl)hexanal. 6-Propylbenzofuran-7-ol was formed by isolating tricyclic hemiacetal lactone derived from the Michael addition of ascorbic acid to (E)-hex-2-enal, mixing the tricyclic hemiacetal lactone with the aqueous solution of citric acid, and applying heat. This confirmed that 6-propylbenzofuran-7-ol was formed via the Michael adduct.

Aroma characteristics of fermented Korean soybean paste (Doenjang) produced by Bacillus amyloliquefaciens

Four strains of Bacillus amyloliquefaciens with high proteolytic, fibrinolytic, and angiotensin converting enzyme (ACE) inhibitory activities were used to make Bacillus-koji suitable for the production of functional doenjang. Volatile compounds in Bacillus-koji doenjang were analyzed by headspace-solid phase microextraction-GC-MS (HS-SPME-GC-MS) to compare the aroma profile with those of commercially produced doenjang made with traditional meju and with modified koji. Extraction temperatures for SPME were adjusted to 30 and 90°C. Commercial samples contained significantly more esters than Bacillus-koji doenjang and demonstrated a more complex volatile compound profile. Pentanoic acid, butanoic acid, or 2/3-methyl butanoic acid, known as off-flavor compounds in doenjang, were not detected in one of the Bacillus-koji doenjang when extracted at 30°C. From the sensory test results, the cheese character was less intense in Bacillus-koji doenjang than commercial samples, although not all sensory properties projected a significant difference.

The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties

The yeast Dekkera/Brettanomyces bruxellensis can cause enormous economic losses in wine industry due to production of phenolic off-flavor compounds. D. bruxellensis is a distant relative of baker's yeast Saccharomyces cerevisiae. Nevertheless, these two yeasts are often found in the same habitats and share several food-related traits, such as production of high ethanol levels and ability to grow without oxygen. In some food products, like lambic beer, D. bruxellensis can importantly contribute to flavor development. We determined the 13.4Mb genome sequence of the D. bruxellensis strain Y879 (CBS2499) and deduced the genetic background of several “food-relevant” properties and evolutionary history of this yeast. Surprisingly, we find that this yeast is phylogenetically distant to other food-related yeasts and most related to Pichia (Komagataella) pastoris, which is an aerobic poor ethanol producer. We further show that the D. bruxellensis genome does not contain an excess of lineage specific duplicated genes nor a horizontally transferred URA1 gene, two crucial events that promoted the evolution of the food relevant traits in the S. cerevisiae lineage. However, D. bruxellensis has several independently duplicated ADH and ADH-like genes, which are likely responsible for metabolism of alcohols, including ethanol, and also a range of aromatic compounds.

Impact of depuration of earthy-musty off-flavors on fillet quality of Atlantic salmon, Salmo salar, cultured in a recirculating aquaculture system

Over the past decade in the United States, there has been increased interest in the establishment and use of land-based, closed-containment systems [e.g., recirculating aquaculture system (RAS)] for salmonid culture. These culture systems have unique challenges compared to net pen culture of salmonids, including maintenance of pumps and filters as well as the potential growth of certain bacteria within the systems that can render fish off-flavored. The purpose of this study was to determine the impact of implementing a depuration process to purge the “earthy” and “musty” off-flavor compounds geosmin and 2-methylisoborneol (MIB) from Atlantic salmon fillets on fillet quality characteristics (e.g., lipid content, color). During two depuration trials, salmon were depurated without feed in a flow-through tank, a recently “cleaned” RAS system or the originally stocked grow-out tank for up to 20 days. Results from both trials determined that the salmon required depuration in odor-free water for 10–15 days in either a flow-through system or a recently cleaned RAS to obtain the lowest residual levels of geosmin and MIB in the fish flesh. In trial 1, after 20 days, fish had lost significantly more weight (5.8%) compared to day 5 (3.8%). In the second trial, lipid content of the fillet also significantly dropped from 8.2% to 5.1% and moisture content increased from 69.3% to 71.1%. Fillet color quality was not compromised during the 20-day depuration periods. In trial 1, MIB was the main off-flavor compound present in salmon fillets while geosmin was at higher levels than MIB in fish flesh in trial 2. During the second depuration study, three geosmin-producing species of actinomycetes were isolated from the recirculating system and were attributed as the likely sources of geosmin in the salmon fillets. Because fillet color quality was not compromised during the depuration periods used in these studies, the main fillet quality concerns for producers of RAS-cultured salmon are flavor, texture and lipid levels during the pre-harvest purging process.

Stability of Citral in Emulsions Coated with Cationic Biopolymer Layers

Multilayer emulsions containing citral were prepared by the layer-by-layer deposition technique based on the electrostatic interaction between negatively charged emulsion droplets and two positively charged biopolymer coatings, chitosan (CS) and ε-polylysine (EPL). The optimum concentrations of both CS and EPL were determined through the ζ-potential and particle size measurements and were found to be 1.5 mg/mL for CS and 6 mg/mL for EPL. Quartz crystal microbalance with dissipation monitoring (QCM-D) was conducted to monitor the binding between emulsion droplets and cationic polymers, and our results proved the existence of strong interactions between emulsions and the cationic polymer coatings. The stability of citral and the production of the off-flavor compounds were analyzed by solid-phase microextraction gas chromatography (SPME-GC). The results suggested that the addition of the cationic CS interfacial layer was effective in improving the stability of citral during storage.

Trace level analysis of corky off-flavor compounds: Development of a new analytical method based on solid phase extraction and analysis by multidimensional gas chromatography with mass spectrometric detection

This work describes the development of a trace level (<1ngL−1) analysis of haloanisoles in complex wine matrix. The suggested method involves sample preparation based on solid phase extraction, a clean-up to remove acidic compounds, concentration of the haloanisole fraction and large volume on-column injection into a multidimensional GC–MS system. Mass spectrometric detection in the selected ion mode allowed reliable quantification of 2,4,6-trichloroanisole (TCA) or 2,4,6-tribromoanisole (TBA), via their highly deuterated ([2H5]) isotopologues as internal standards (stable isotope dilution analysis; SIDA), which had prior been synthesized in house. The development of this new method had become necessary, as a one-dimensional HS-SPME–GC-ECD method, routinely applied for analysis of TCA in cork soaks, had to be extended for TeCA and TBA determination, but failed due to co-elutions within wine matrices. The newly developed SPE//MDGC–MS method provided detection limits well below olfactory thresholds of the analytes with 0.05ngL−1 (LOD), 0.19ngL−1 (LOQ) for TCA, 0.06ngL−1 (LOD), 0.21ngL−1 (LOQ) for TeCA, and 0.09ngL−1 (LOD), 0.34ngL−1 (LOQ) for TBA.

The management of undesirable cyanobacteria blooms in channel catfish ponds using a constructed wetland: Contribution to the control of off-flavor occurrences

An exploratory study on the management of undesirable cyanobacteria blooms with respect to off-flavor problems using an integrated vertical-flow constructed wetland (CW) was performed at a small commercial-scale channel catfish farm from 2004 to 2007. The results of the three-year experiment indicated that water treatment by the CW could reduce the possibility of dominance by undesirable cyanobacteria species that often cause off-flavor problems. A detailed investigation in 2007, showed that the concentrations of geosmin, MIB (2-methylisoborneol), and β-cyclocitral in the water of the recirculating pond (4.3 ng L −1, U.D. (undetected) and 0.2 ng L −1, respectively) treated by the CW were significantly lower than those in the control pond (152.6 ng L −1, 63.3 ng L −1 and 254.8 ng L −1, respectively). In addition, the relationships among the cyanobacteria species, the off-flavor compounds and ten environmental variables were explored by canonical correspondence analysis (CCA). The results showed that Oscillatoria sp., Oscillatoria kawamurae and Microcystis aeruginosa were the main sources of off-flavor compounds in the catfish ponds. The successful manipulation of undesirable cyanobacteria species potentially resulted in lower concentrations of odorous compounds in the water of the recirculating pond. An investigation of the concentrations of geosmin and MIB in catfish fillets showed that the levels of odorous compounds were below the OTC (odor threshold concentration) values in the recirculating pond but were above the OTC values from July to October in the control pond. Water recycling by the CW could potentially be one of the best management practices to control off-flavor occurrences in aquaculture.

Changes in oxidation-derived off-flavor compounds of roasted sesame oil during accelerated storage in the dark

Oil oxidation and off-flavor compounds were evaluated in roasted sesame oil during accelerated storage at 70 °C in the dark for 4 weeks. Oil oxidation was monitored by measuring contents of conjugated dienoic acid (CDA) and polar compounds as well as by analyzing fatty acid composition by gas chromatography (GC). Off-flavor compounds were evaluated with the headspace gas analysis using a solid phase microextraction (SPME) and GC. The roasted sesame oil showed little change in fatty acid composition during storage, and contents of CDA and polar compounds increased slowly. Among off-flavor compounds including pentane, hexane, hexanal, heptanal, 1-pentanol, acetic acid, and furfuryl alcohol, hexanal, heptanal, or 1-pentanol content increased with storage time and showed a high correlation with CDA values of the oil. Acetic acid and furfuryl alcohol did not show consistent trends throughout the 4 week storage. The results confirmed high oxidative stability of roasted sesame oil and suggested use of hexanal, heptanal, or 1-pentanol as an indicator off-flavor compound to monitor the autoxidation of roasted sesame oil.

Development of phytoplankton communities and common off-flavors in a biofloc technology system used for the culture of channel catfish (Ictalurus punctatus)

The use of biofloc technology production systems continues to increase in the aquaculture industry worldwide. Recent research demonstrated that outdoor biofloc systems can be used to produce high yields of channel catfish (Ictalurus punctatus). However, studies have not yet been performed to determine the development and composition of phytoplankton communities and related off-flavor problems in these biofloc production systems. In this study, water samples were collected biweekly from May to November and channel catfish samples were collected during harvest in November from nine 18.6m2 biofloc culture tanks. Water and fillet samples were analyzed for levels of the common off-flavor compounds geosmin and 2-methylisoborneol (MIB). The development and composition of phytoplankton communities in each culture tank was also monitored. In addition, water and biofloc samples were evaluated to assess the microbial sources of geosmin and MIB within the culture tanks. Phytoplankton (including algae and cyanobacteria attached to bioflocs) biomass, as determined by concentrations of chlorophyll a in the water, gradually increased in all tanks over time. Phytoplankton communities that developed in the culture tanks were dominated by fast-growing, unicellular and small colonial types of green algae (chlorophytes) and diatoms (bacillariophytes) and slower growing, small colonial types of cyanobacteria (cyanophytes). A positive correlation (p<0.05) between cumulative feed addition and chlorophyll a concentration was found. Although geosmin and MIB were present in the culture water of each tank during most of the study, levels were typically low and only one tank yielded catfish with geosmin and MIB in their flesh at levels high enough to be designated as off-flavor. A positive correlation (p<0.05) between cumulative feed addition and MIB concentrations in the water of culture tanks indicates a greater potential for MIB-related off-flavor problems at high feed application rates. The microbial sources responsible for production of geosmin and MIB in the culture tanks remain unknown.

Integrated expression of the α-amylase, dextranase and glutathione gene in an industrial brewer’s yeast strain

Genetic engineering is widely used to meliorate biological characteristics of industrial brewing yeast. But how to solve multiple problems at one time has become the bottle neck in the genetic modifications of industrial yeast strains. In a newly constructed strain TYRL21, dextranase gene was expressed in addition of α-amylase to make up α-amylase's shortcoming which can only hydrolyze α-1,4-glycosidic bond. Meanwhile, 18s rDNA repeated sequence was used as the homologous sequence for an effective and stable expression of LSD1 gene. As a result, TYRL21 consumed about twice much starch than the host strain. Moreover TYRL21 speeded up the fermentation which achieved the maximum cell number only within 3days during EBC tube fermentation. Besides, flavor evaluation comparing TYRL21 and wild type brewing strain Y31 also confirmed TYRL21's better performances regarding its better saccharides utilization (83% less in residual saccharides), less off-flavor compounds (57% less in diacetyl, 39% less in acetaldehyde, 67% less in pentanedione), and improved stability index (increased by 49%) which correlated with sensory evaluation of final beer product.

Identification of the Medicinal Off-Flavor Compound Formed from Ascorbic Acid and (E)-Hex-2-enal

A test apple beverage made up of apple juice (20%), high-fructose corn syrup (11.5%), citric acid (0.43%), trisodium citrate (0.02%), apple-odor flavor (0.1%), and ascorbic acid (0.02%) was stored at 40 °C and then analyzed for the change of odor in the beverage. Although no thermoacidophilic bacteria (TAB) were detected, a medicinal off-flavor was perceived after the 8 weeks of storage. Model experiments on the ingredients of the test apple beverage revealed that the off-flavor compound had been formed by ascorbic acid and (E)-hex-2-enal. Synthesis and NMR (¹H, ¹³C, HMQC, and HMBC) analyses identified the compound as 6-propylbenzofuran-7-ol. The odor quality, retention index (RI), and mass spectrum of synthetic 6-propylbenzofuran-7-ol were identical with those of the medicinal odor compound from the test apple beverage. Sensory evaluation revealed the recognition thresholds for medicinal odor were 31.4 ppb in water and 24.0 ppb in apple beverage, and the detection thresholds were 19.6 ppb in water and 8.6 ppb in apple beverage, respectively. The quantified concentration of 6-propylbenzofuran-7-ol formed in test apple beverage was 90 ppb, approximately. This concentration was well above the odor threshold, so it was concluded that the compound was the source of the medicinal off-flavor.

Inhibition of Citral Degradation by Oil-in-Water Nanoemulsions Combined with Antioxidants

The aim of the present study was to investigate the effects of oil-in-water (O/W) nanoemulsions combined with six different natural antioxidants on the stability of citral. Acidic emulsions (lecithin-stabilized palm kernel lipid in pH 3 buffer) containing 1000 ppm citral and 1000 ppm antioxidants (black tea extract, ascorbic acid, naringenin, tangeretin, β-carotene, and tanshinone) were stored at 25 and 50 °C, respectively. The emulsions with and without antioxidants were analyzed by solid phase microextraction gas chromatography (SPME-GC) to monitor the degradation process of citral and the formation of different off-flavor compounds, such as α,p-dimethylstyrene and p-methylacetophenone. The results suggested that encapsulation of citral in emulsions and the addition of the appropriate antioxidants (β-carotene, tanshinone, and black tea extract) could greatly enhance citral's chemical stability during storage.

Determination of Geosmin and 2-Methylisoborneol in Water by Headspace Liquid-Phase Microextraction Coupled with Gas Chromatography-Mass Spectrometry

Geosmin (GSM) and 2-methylisoborneol (MIB) were extracted from water samples, adsorbed in organic solvent microdrop by headspace liquid-phase microextraction (HS-LPME), and were analyzed by gas chromatography-mass spectrometry (GC-MS). Influence factors such as the extraction solvent types, headspace and microdrop volumes, stirring rate, equilibrium and extraction time, and ionic strength for HS-LPME efficiency were thoroughly evaluated. Under optimized extraction and detection conditions, the calibration curves of GSM and MIB were linear in the range of 5–1000 ng/L. The detection limits of GSM and MIB were 1.1 and 1.0 ng/L, respectively. Average recoveries of 95.45–113.7% (n = 5) were obtained and method precisions were also satisfactory. Trace levels of the off-flavor compounds at ng/L in tap water and raw water were successfully quantified.

Construction of Amylolytic Industrial Brewing Yeast Strain with High Glutathione Content for Manufacturing Beer with Improved Anti-Staling Capability and Flavor

Glutathione in beer works as the main antioxidant compounds which correlates with beer flavor stability. High residual sugars in beer contribute to major non-volatile components which correlate to high caloric content. In this work, Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and Scharomycopsis fibuligera ALP1 gene encoding alpha-amylase were co-expressed in industrial brewing yeast strain Y31 targeting at alpha-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), and new recombinant strain TY3 was constructed. The glutathione content from the fermentation broth of TY3 increased to 43.83 mg/l compared to 33.34 mg/l from Y31. The recombinant strain showed high alpha-amylase activity and utilized more than 46% of starch after 5 days growing on starch as sole carbon source. European Brewery Convention tube fermentation tests comparing the fermentation broth of TY3 and Y31 showed that the flavor stability index increased to 1.3 fold and residual sugar concentration were reduced by 76.8%, respectively. Due to the interruption of ILV2 gene and ADH2 gene, the amounts of off-flavor compounds diacetyl and acetaldehyde were reduced by 56.93% and 31.25%, comparing with the amounts of these from Y31 fermentation broth. In addition, as no drug-resistance genes were introduced to new recombinant strain, consequently, it should be more suitable for use in beer industry because of its better flavor stability and other beneficial characteristics.

Selectable one-dimensional or two-dimensional gas chromatography–olfactometry/mass spectrometry with preparative fraction collection for analysis of ultra-trace amounts of odor compounds

A novel selectable one-dimensional ( 1D) or two-dimensional ( 2D) gas chromatography–olfactometry/mass spectrometry with preparative fraction collection (selectable 1D/ 2D GC–O/MS with PFC) system was developed. The main advantages of this system are the simple and fast selection of 1D GC–O/MS or 2D GC–O/MS or 1D GC-PFC or 2D GC-PFC operation with a mouse click (without any instrumental set-up change), and total transfer of enriched compounds with thermal desorption (TD) on the same system for identification with 2D GC–O/MS analysis. Recovery of PFC enrichment with 20 injection cycles of 15 model compounds at 500 pg each (e.g. alcohol, aldehyde, ester, lactone, and phenol) was very good with recoveries in the range of 98–116%. The feasibility and benefit of the proposed system was demonstrated with an identification of off-flavor compounds (e.g. 2,4,6-trichloroanisole (TCA), 2-isobutyl-3-methoxypyrazine (IBMP), and geosmin) in spiked wine at odor perception threshold level (5–50 ng L −1). After parallel stir bar sorptive extraction (SBSE) for 20 aliquots of a sample and subsequent PFC enrichment for the odor-active fractions from the 20 stir bars, three off-flavor compounds were clearly resolved and detected with TD– 2D GC–O/MS in scan mode. The good efficiency of SBSE-PFC enrichment in the range of 71–78% shows that all analytical steps, e.g. SBSE, TD, 1D/ 2D GC–O/MS, and PFC, are quantitative and identification of off-flavor compounds at ng L −1 level in wine is possible.

Evaluation of ozonation on levels of the off-flavor compounds geosmin and 2-methylisoborneol in water and rainbow trout Oncorhynchus mykiss from recirculating aquaculture systems

Common “off-flavors” in fish cultured in recirculating aquaculture systems (RAS) are “earthy” and “musty” due to the presence of the off-flavor metabolites geosmin and 2-methylisoborneol (MIB), respectively. Previously, ozone addition has been applied to RAS at relatively low doses to break refractory organic molecules (i.e., reducing color), microflocculate fine particulate matter (i.e., increasing solids removal), and oxidize nitrite to nitrate, but the effect of ozone addition at these dosing rates on levels of off-flavor compounds was unknown. Ozonation has been used in municipal drinking water facilities to reduce the levels of these compounds, to improve water taste, and to subsequently reduce consumer complaints. In this study, the effects of ozone addition to the inlet water of the RAS culture tanks on levels of geosmin and MIB in the culture water and fish flesh were evaluated. Water and rainbow trout (Oncorhynchus mykiss) samples were obtained twice after maximum feed rates were reached during a 6-month grow-out period. Results indicated that ozone addition to maintain an oxidation reduction potential of 248mV (<1μg/L of ozone residual) did not significantly reduce levels of these off-flavor compounds in the water and fish flesh, though it did significantly improve culture tank water quality. Higher dosages of ozone might be beneficial in removing geosmin and MIB in the RAS water, but the inclusion of ultraviolet (UV) irradiation would be required to prevent mortality associated with ozone toxicity.

On-tap passive enrichment, a new way to investigate off-flavor episodes in drinking water

Because taste and odor events in drinking water are often fleeting and unpredictable phenomena, an innovative enrichment sampler has been developed to trap off-flavor compounds directly at the consumer's tap. The ARISTOT (Advanced Relevant Investigation Sampler for Taste & Odor at Tap) consists of a tap adapter in which seven polydimethylsiloxane (PDMS) coated stir bars are placed, allowing the stir bar sorptive extraction (SBSE) of organic compounds during each tap opening. In order to study the efficiency of ARISTOT, a private network pilot unit has been constructed in our laboratory, equipped with four faucets in parallel, solenoid valves for an automation of the system and a mixing chamber to spike drinking water with odorous compounds in order to have homogenously smelling water at each tap. After enrichment, the stir bars are taken out, in-line thermo-desorbed and analyzed by gas chromatography coupled with a mass spectrometer. The results showed the high sensitivity of ARISTOT, which was able to quickly monitor odorous compounds at the sub ng/L level. A “multishot” method was developed to analyze chemicals concentrated on the seven stir bars in only one chromatographic run, thereby increasing the sensitivity of the system. Higher enrichment factors were obtained under low water flow rates or by using longer stir bars and/or stir bars with a higher PDMS film thickness. No significant loss of extracted compounds was reported for flow rates between 2 and 4 L/min. This allowed us to spike the stir bars with an internal standard prior to sampling in order to monitor the analytical variations. It was also observed that hot water increases the loss of enriched solutes but the quantification can be corrected by internal standard addition.

Loss of Ripening Capacity of Pawpaw Fruit with Extended Cold Storage

The fruit ripening traits of pawpaw [ Asimina triloba (L.) Dunal] were examined after harvest and after cold storage at -2, 2, 4, and 6 degrees C for up to 12 weeks. Generally, fruits stored at 2-4 degrees C for 4 weeks ripened normally, but those stored at -2 degrees C did not ripen normally, those stored at 6 degrees C were overripe, and by 6-8 weeks those stored at 2-4 degrees C had a lower respiration rate and ethylene production, lower firmness, and lower pH than fruit cold-stored for 4 weeks or less. These changes, and the occasional development of brown discoloration in the pulp once the fruits were moved back to room temperature, were evidence of chilling injury by 6 weeks. After harvest and through 4 weeks of cold storage, the main volatile compounds produced by fruit were methyl and ethyl octanoates and hexanoates. Volatile production significantly increased >5-fold in fruit ripened for 72 h after harvest or after removal from up to 4 weeks of cold storage. Fruit cold-stored for 6 weeks or more produced fewer total volatiles and esters but increased levels of such off-flavor compounds as ethyl acetate, ethyl propionate, and hexanoic and decanoic acids. Alcohol acyltransferase (AAT) activity declined in cold-stored fruit but was not correlated with either total volatile production or total ester production. Alcohol dehydrogenase activity did not change during ripening after harvest or cold storage. Lipoxygenase activity was highest just after harvest or after 2 weeks of cold storage, but was low by 4 weeks. Thus, ripening of pawpaw fruit seems to be limited to 4 weeks at 2-4 degrees C with loss of ability to continue ripening and chilling injury symptoms evident at colder temperatures and after longer periods of cold storage.