Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis.

AIM: Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae. METHODS AND RESULTS: Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small-scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1:2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l⁻¹ (0.22 mol l⁻¹)]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1:5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1:10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0.42 ± 0.01 g ethanol (g glucose)⁻¹ were observed for both yeasts in 1:10 hydrolysate. In small-scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1:5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1:2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate. CONCLUSIONS: Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates.

Effect of starter culture inoculation on feed hygiene and microbial population development in fermented pig feed composed of a cereal grain mix with wet wheat distillers' grain.

AIMS: Investigating the influence of an added starter culture on the properties of fermented liquid pig feed. METHODS AND RESULTS: Diets of cereal grain blended with wet wheat distillers' grain that were either not inoculated (WWDG), inoculated with a silage starter culture at start (WWDGsc1) or at start and at each backslopping (replacement of 80% the content with fresh mixture, simulating feed outtake, WWDGsc5) were fermented for 5 days, followed by 5 days of daily backslopping. Numbers of undesirable micro-organisms (enterobacteria, moulds) were reduced in all fermentations; particularly enterobacteria in the starter culture inoculated diets. Lactobacillus plantarum present in the starter culture became dominant in diets WWDGsc1 and WWDGsc5. However, Lactobacillus panis that was dominating WWDG was also abundant in WWDGsc1 and WWDGsc5. Yeast populations were not influenced by the starter culture, with Pichia fermentans dominating all fermentations. All diets had similar chemical characteristics with the exception of a significant increase of all tested organic acids in WWDGsc5. CONCLUSIONS: The addition of a starter culture influences the bacterial population in fermented liquid feed, but there is also a strong impact of the flora already present in the feed ingredients. The yeast population is not influenced by adding a lactic acid bacteria (LAB) starter culture. A consortium of LAB and yeast strains adapted to the fermentation should be used as starter culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that it is possible to influence the current unpredictable and spontaneous process of feed fermentation when appropriate starter cultures are used. For this purpose, LAB and yeasts with desirable characteristics should be isolated.

Genetic characterization of the nonconventional yeast Hansenula anomala.

We describe genetic, molecular and taxonomic characteristics of the yeast Hansenula anomala. Pulsed-field gel electrophoresis of chromosomal DNAs from 19 H. anomala strains and related species indicated that H. anomala had a clearly different karyotype. Chromosome length polymorphism of the H. anomala strains was independent of their geographic origin and source of isolation. The strains were classified into four groups of similar karyotypes and one strain showed a unique profile. The sizes of chromosomes ranged from 850 to 3500 kb in different strains. The haploid chromosome number of H. anomala is at least nine. We have found RAPD primers discriminating at both the species and strain levels. All the primers tested except the M13 core sequence generated unique patterns with most strains. The results indicate the usefulness of PCR analysis with primer M13 for identification of the H. anomala species. Screening of the CBS (Utrecht) collection strains of H. anomala showed that they are rather difficult objects for genetic hybridization analysis. The strains have low fertility, viz. very poor sporulation, low mating type activities and, as a rule, nonviable ascospores. The majority of the hybrids obtained are polyploid, probably tetraploid, as judged by the segregation of control auxotrophic markers. Nevertheless, some monosporic cultures of the strains studied, including the biocontrol yeast J121, formed diploid hybrids with regular meiotic segregation of control auxotrophic markers. As a rule, H. anomala isolates are homothallic, showing delayed self-diploidization. Rare stable heterothallic strains of H. anomala also occur.

Barley straw decomposition with varied levels of microbial grazing by Folsomia fimetaria (L.) (Collembola, Isotomidae).

Folsomia fimetaria (L.) were added (0, 5, 10, 20 animals) to 0.100 g barley straw which had been inoculated 10 days (244 h) earlier with a natural soil microflora. Respiration (CO2 evolution) was monitored continuously. Mass loss, fungal standing crop (total and FDA-active), bacterial and protozoan biomass were estimated 42 days (1,000 h) after microbial inoculation. The degree of surface cover by hyphae was surveyed at regular intervals. No significant differences (P>0.05) were found in respiration, mass loss or microbial biomass, but the density of surface hyphae were reduced by addition of Collembola. Fungal production was low, less than 5% of the estimated microbial production, and could not account for all collembolan growth during incubation. F. fimetaria appeared to consume mainly bacteria and protozoa, and had little impact on carbon mineralization.

Volatiles for mycological quality grading of barley grains: determinations using gas chromatography-mass spectrometry and electronic nose.

The possibility of using an electronic nose or gas chromatography combined with mass spectrometry (GC-MS) to quantify ergosterol and colony forming units (CFU) of naturally contaminated barley samples was investigated. Each sample was split into three parts for (i) ergosterol and CFU analysis, (ii) measurements with the electronic nose and (iii) identification of volatiles collected on an adsorbent with a GC-MS system. Forty samples were selected after sensory analysis to obtain 10 samples with normal odour and 30 with some degree of off-odour. The data set of volatile compounds and the data collected from the electronic nose were evaluated by multivariate analyse techniques. SIMCA classification (soft independent modelling of class analogy) was used for objective evaluation of the usefulness of the data from the GC-MS or electronic nose measurements for classification of grain samples as normal or with off-odour. The main volatile compounds of grain with normal odour were 2-hexenal, benzaldehyde and nonanal, while 3-octanone, methylheptanone and trimethylbenzene were the main volatile compounds of grain with off-odours. Using data from the electronic nose three samples of 40 were misclassified, while data analysis of the volatile compounds detected with the GC-MS, led to six misclassified samples. Regression models (partial least-squares, PLS) were built to predict ergosterol- and CFU-levels with data from the GC-MS or electronic nose measurements. PLS models based on both GC-MS and electronic nose data could be used to predict the ergosterol levels with high accuracy and with low root mean square error of prediction (RMSEP). CFU values from naturally infected grain could not be predicted with the same degree of confidence.

Detection and quantification of ochratoxin A and deoxynivalenol in barley grains by GC-MS and electronic nose.

Mycotoxin contamination of cereal grains can be detected and quantified using complex extraction procedures and analytical techniques. Normally, the grain odour, i.e. the presence of non-grain volatile metabolites, is used for quality classification of grain. We have investigated the possibility of using fungal volatile metabolites as indicators of mycotoxins in grain. Ten barley samples with normal odour, and 30 with some kind of off-odour were selected from Swedish granaries. The samples were evaluated with regard to moisture content, fungal contamination, ergosterol content, and levels of ochratoxin A (OA) and deoxynivalenol (DON). Volatile compounds were also analysed using both an electronic nose and gas chromatography combined with mass spectrometry (GC-MS). Samples with normal odour had no detectable ochratoxin A and average DON contents of 16 microg kg(-1) (range 0-80), while samples with off-odour had average OA contents of 76 microg kg(-1) (range 0-934) and DON contents of 69 microg kg(-1) (range 0-857). Data were evaluated by multivariate data analysis using projection methods such as principal component analysis (PCA) and partial least squares (PLS). The results show that it was possible to classify the OA level as below or above the maximum limit of 5 microg kg(-1) cereal grain established by the Swedish National Food Administration, and that the DON level could be estimated using PLS. Samples with OA levels below 5 microg kg(-1) had higher concentration of aldehydes (nonanal, 2-hexenal) and alcohols (1-penten-3-ol, 1-octanol). Samples with OA levels above 5 microg kg(-1) had higher concentrations of ketones (2-hexanone, 3-octanone). The GC-MS system predicted OA concentrations with a higher accuracy than the electronic nose, since the GC-MS misclassified only 3 of 37 samples and the electronic nose 7 of 37 samples. No correlation was found between odour and OA level, as samples with pronounced or strong off-odours had OA levels both below and above 5 microg kg(-1). We were able to predict DON levels in the naturally contaminated barley samples using the volatile compounds detected and quantified by either GC-MS or the electronic nose. Pentane, methylpyrazine, 3-pentanone, 3-octene-2-ol and isooctylacetate showed a positive correlation with DON, while ethylhexanol, pentadecane, toluene, 1-octanol, 1-nonanol, and 1-heptanol showed a negative correlation with DON. The root mean square error of estimation values for prediction of DON based on GC-MS and electronic nose data were 16 and 25 microg kg(-1), respectively.

Electronic nose for microbial quality classification of grains.

The odour of grains is in many countries the primary criterion of fitness for consumption. However, smelling of grain for quality grading should be avoided since inhalation of mould spores or toxins may be hazardous to the health and determinations of the off-odours are subjective. An electronic nose, i.e. a gas sensor array combined with a pattern recognition routine might serve as an alternative. We have used an electronic nose consisting of a sensor array with different types of sensors. The signal pattern from the sensors is collected by a computer and further processed by an artificial neural network (ANN) providing the pattern recognition system. Samples of oats, rye and barley with different odours and wheat with different levels of ergosterol, fungal and bacterial colony forming units (cfu) were heated in a chamber and the gas in the chamber was led over the sensory array. The ANN could predict the odour classes of good, mouldy, weakly and strongly musty oats with a high degree of accuracy. The ANN also indicated the percentage of mouldy barley or rye grains in mixtures with fresh grains. In wheat a high degree of correlation between ANN predictions and measured ergosterol as well as with fungal and bacterial cfu was observed. The electronic nose can be developed to provide a simple and fast method for quality classification of grain and is likely to find applications also in other areas of food mycology.

Performance of mycological media in enumerating desiccated food spoilage yeasts: an interlaboratory study.

Dichloran 18% glycerol agar (DG18) was originally formulated to enumerate nonfastidious xerophilic moulds in foods containing rapidly growing Eurotium species. Some laboratories are now using DG18 as a general purpose medium for enumerating yeasts and moulds, although its performance in recovering yeasts from dry foods has not been evaluated. An interlaboratory study compared DG18 with dichloran rose bengal chloramphenicol agar (DRBC), plate count agar supplemented with chloramphenicol (PCAC), tryptone glucose yeast extract chloramphenicol agar (TGYC), acidified potato dextrose agar (APDA), and orange serum agar (OSA) for their suitability to enumerate 14 species of lyophilized yeasts. The coefficient of variation for among-laboratories repeatability within yeast was 1.39% and reproducibility of counts among laboratories was 7.1%. The order of performance of media for recovering yeasts was TGYC > PCAC = OSA > APDA > DRBC > DG 18. A second study was done to determine the combined effects of storage time and temperature on viability of yeasts and suitability of media for recovery. Higher viability was retained at -18 degrees C than at 5 degrees C or 25 degrees C for up to 42 weeks, although the difference in mean counts of yeasts stored at -18 degrees C and 25 degrees C was only 0.78 log10 cfu/ml of rehydrated suspension. TGYC was equal to PCAC and superior to the other four media in recovering yeasts stored at -18 degrees C, 5 degrees C, or 25 degrees C for up to 42 weeks. Results from both the interlaboratory study and the storage study support the use of TGYC for enumerating desiccated yeasts. DG18 is not recommended as a general purpose medium for recovering yeasts from a desiccated condition.

Image analysis for monitoring the barley tempeh fermentation process.

AIMS: To develop a fast, accurate, objective and nondestructive method for monitoring barley tempeh fermentation. METHODS AND RESULTS: Barley tempeh is a food made from pearled barley grains fermented with Rhizopus oligosporus. Rhizopus oligosporus growth is important for tempeh quality, but quantifying its growth is difficult and laborious. A system was developed for analysing digital images of fermentation stages using two image processing methods. The first employed statistical measures sensitive to image colour and surface structure, and these statistical measures were highly correlated (r=0.92, n=75, P<0.001) with ergosterol content of tempeh fermented with R. oligosporus and lactic acid bacteria (LAB). In the second method, an image-processing algorithm optimized to changes in images of final tempeh products was developed to measure number of visible barley grains. A threshold of 5 visible grains per Petri dish indicated complete tempeh fermentation. When images of tempeh cakes fermented with different inoculation levels of R. oligosporus were analysed the results from the two image processing methods were in good agreement. CONCLUSION: Image processing proved suitable for monitoring barley tempeh fermentation. The method avoids sampling, is nonintrusive, and only requires a digital camera with good resolution and image analysis software. SIGNIFICANCE AND IMPACT OF THE STUDY: The system provides a rapid visualization of tempeh product maturation and qualities during fermentation. Automated online monitoring of tempeh fermentation by coupling automated image acquisition with image processing software could be further developed for process control.

Survival of the biocontrol yeast Pichia anomala after long-term storage in liquid formulations at different temperatures, assessed by flow cytometry.

AIMS: Investigate the survival of liquid formulations of the biocontrol yeast Pichia anomala J121 at different temperatures, and develop a system for comparative studies of different storage conditions and formulations. METHODS AND RESULTS: The survival of P. anomala in liquid formulations with lactose, starch and trehalose amendments was measured during prolonged storage at temperatures ranging from -20 to +30 degrees C. The relative survival of the stored cells was rapidly estimated by flow cytometry. After 4 weeks incubation at 4 and 10 degrees C, 75-90% of the cells were viable, with no significant differences between the various formulations. Supplementing the storage buffer with lactose or trehalose increased the survival after longer incubations (8 and 12 weeks) at all temperatures (-20 to 30 degrees C). Trehalose was the most effective protectant at 20 and 30 degrees C (>20% viable cells after 12 weeks at 20 degrees C). The biocontrol activity was maintained after formulation and prolonged storage of P. anomala. CONCLUSIONS: The storage potential of liquid formulated P. anomala cells can be increased by supplementation with lactose or trehalose. The combination of a custom made incubation chamber and flow cytometry was suitable to evaluate stability of P. anomala formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: Liquid formulated P. anomala have a long shelf life. The developed test system can be used to study different formulations of other biocontrol agents.

Comparison of methods for estimating the biomass of three food-borne fungi with different growth patterns.

To evaluate the effectiveness of steps taken to reduce the growth of molds in food and feed, methods that can accurately quantify the degree of fungal contamination of solid substrates are needed. In this study, the ergosterol assay has been evaluated by comparing the results of this assay with spore counts and hyphal length measurements made with a microscope and with CFU counts. Three fungi with different growth patterns during cultivation on a synthetic agar substrate were used in these experiments. For the nonsporulating Fusarium culmorum, there was good agreement between changes in hyphal length, CFU, and ergosterol content. Penicillium rugulosum and Rhizopus stolonifer produced many spores, and the production of spores coincided with large increases in CFU but not with increases in hyphal length or ergosterol content. Spores constituted between 3 and 5% of the total fungal mass. Changes in ergosterol level were closely related to changes in hyphal length. It was concluded that ergosterol level is a suitable marker for use in quantitatively monitoring fungal growth in solid substrates.

Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.

Pichia anomala inhibits the growth of Penicillium roqueforti and Aspergillus candidus on agar. In this investigation, antagonistic activity on agar against 17 mold species was determined. The abilities of Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae to inhibit the growth of the mold Penicillium roqueforti in nonsterile high-moisture wheat were compared by adding 10(3) Penicillium roqueforti spores and different amounts of yeast cells per gram of wheat. Inoculated grain was packed in glass tubes, incubated at 25 degrees C with a restricted air supply, and the numbers of yeast and mold CFU were determined on selective media after 7 and 14 days. Pichia anomala reduced growth on agar plates for all of the mold species tested in a dose-dependent manner. Aspergillus fumigatus and Eurotium amstelodami were the most sensitive, while Penicillium italicum and Penicillium digitatum were the most resistant. Pichia anomala had the strongest antagonistic activity in wheat, with 10(5) and 10(6) CFU/g completely inhibiting the growth of Penicillium roqueforti. Inhibition was least pronounced at the optimum temperature (21 degrees C) and water activity (0.95) for the growth of Penicillium roqueforti. Pichia guilliermondii slightly reduced the growth of Penicillium roqueforti in wheat inoculated with 10(5) and 10(6) yeast CFU/g. S. cerevisiae inhibited mold growth only weakly at the highest inoculum level. Pichia anomala grew from 10(3) to 10(7) CFU/g of wheat in 1 week. To reach the same level, Pichia guilliermondii had to be inoculated at 10(4) CFU while S. cerevisiae required an inoculum of 10(5) CFU to reach 10(7) CFU/g of wheat.

Biocontrol of Mold Growth in High-Moisture Wheat Stored under Airtight Conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.

Volume 61, no. 3, p. 1029, Table 1, boxhead over columns 2 to 4: "(CFU/plate)" should read "(log CFU/plate)." FIG. 1 [This corrects the article on p. 1027 in vol. 61.].

Fluorescein diacetate hydrolysis as a measure of total microbial activity in soil and litter.

Spectrophotometric determination of the hydrolysis of fluorescein diacetate (FDA) was shown to be a simple, sensitive, and rapid method for determining microbial activity in soil and litter. FDA hydrolysis was studied in soil and straw incubated for up to 3 h. Hydrolysis was found to increase linearly with soil addition. FDA hydrolysis by pure cultures of Fusarium culmorum increased linearly with mycelium addition both in shake cultures and after inoculation into sterile soil. FDA hydrolysis by Pseudomonas denitrificans increased linearly with biomass addition. The FDA hydrolytic activities in soil samples from different layers of an agricultural soil were correlated with respiration. Acetone was found to be suitable for terminating the reaction.

Inhibitory effects of foscarnet on herpesvirus multiplication in cell culture.

The effect of phosphonoformate (INN; foscarnet sodium) on Herpes Simplex Virus type 1 replication in cell-culture has been studied. At 55 microM, foscarnet reduced the yield by 50 per cent which correlated well with a 50 per cent reduction of plaque formation at 60 microM. Foscarnet had to be added before 8 hours post infection to inhibit virus production. The inhibition of herpesvirus plaque formation by the presence of foscarnet for 24 hours was not reversed by the removal of the drug. The inhibition of virus replication by foscarnet could, in contrast to the inhibition by acycloguanosine, not be reversed by addition of deoxynucleosides.

Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound.

The antifungal activity spectrum of Lactobacillus coryniformis subsp. coryniformis strain Si3 was investigated. The strain had strong inhibitory activity in dual-culture agar plate assays against the molds Aspergillus fumigatus, A. nidulans, Penicillium roqueforti, Mucor hiemalis, Talaromyces flavus, Fusarium poae, F. graminearum, F. culmorum, and F. sporotrichoides. A weaker activity was observed against the yeasts Debaryomyces hansenii, Kluyveromyces marxianus, and Saccharomyces cerevisiae. The yeasts Rhodotorula glutinis, Sporobolomyces roseus, and Pichia anomala were not inhibited. In liquid culture the antifungal activity paralleled growth, with maximum mold inhibition early in the stationary growth phase, but with a rapid decline in antifungal activity after 48 h. The addition of ethanol to the growth medium prevented the decline and gave an increased antifungal activity. The activity was stable during heat treatment and was retained even after autoclaving at 121 degrees C for 15 min. Maximum activity was observed at pH values of between 3. 0 and 4.5, but it decreased rapidly when pH was adjusted to a level between 4.5 and 6.0 and was lost at higher pH values. The antifungal activity was fully regained after readjustment of the pH to the initial value (pH 3.6). The activity was irreversibly lost after treatment with proteolytic enzymes (proteinase K, trypsin, and pepsin). The antifungal activity was partially purified using ion-exchange chromatography and (NH(4))(2)SO(4) precipitation, followed by gel filtration chromatography. The active compound(s) was estimated to have a molecular mass of approximately 3 kDa. This is the first report of the production of a proteinaceous antifungal compound(s) from L. coryniformis subsp. coryniformis.

Chitinolytic properties of Bacillus pabuli K1.

The chitinolytic properties of Bacillus pabuli K1 isolated from mouldy grain was studied. Chitinase activity was measured as the release of p-nitrophenol from p-nitrophenyl-N,N'-diacetylchitobiose. Influences of substrate concentration and different environmental variables on growth and chitinase activity were determined. The optimum environmental conditions for chitinase production were: 30 degrees C, initial pH 8, initial oxygen 10% and aw > 0.99. Chitinase production was induced when B. pabuli K1 was grown on colloidal chitin. The smallest chito-oligosaccharide able to induce chitinase production was N,N'-diacetylchitobiose, (GlcNAc)2. Production was also induced by (GlcNAc)3 and (GlcNAc)4. When the bacterium was grown on glucose or N-acetylglucosamine, no chitinases were formed. The highest chitinase production observed was obtained with colloidal chitin as substrate. The production of chitinases by B. pabuli K1 growing on chitin was repressed by high levels (0.6%) of glucose. The production was also repressed by 0.6% starch, laminarin and beta-glucan from barley and by glycerol. The addition of pectin and carboxymethyl cellulose increased chitinase production.

Evaluation of a chromogenic chito-oligosaccharide analogue, p-nitrophenyl-beta-D-N,N'-diacetylchitobiose, for the measurement of the chitinolytic activity of bacteria.

Three methods of quantifying chitinase activity were compared. The activities of crude chitinases of 10 bacterial isolates from different environments were estimated in terms of (1) the release of p-nitrophenol from the chromogenic chito-oligosaccharide analogues, p-nitrophenyl-beta-D-N,N'-diacetylchitobiose, p-nitrophenyl-N-acetyl-beta-D-glucosamine and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, (2) the release of reducing sugars from chitin and (3) the formation of clearing zones on chitin agar. When crude chitinase from Bacillus pabuli was used the hydrolysis of p-nitrophenyl-beta-D-N,N'-diacetylchitobiose correlated well with the release of reducing sugars from chitin and the formation of clearing zones on chitin agar. However, when the activity of crude chitinases from the different bacterial isolates were compared no agreement was found between the hydrolysis of p-nitrophenyl-beta-D-N,N'-diacetylchitobiose and the release of reducing sugars from chitin or the formation of clearing zones on chitin agar. It was concluded that the assay with chromogenic p-nitrophenyl chito-oligosaccharide analogues is not well suited for studies that compare the chitinase activity of different bacteria.

Volatile metabolites produced by six fungal species compared with other indicators of fungal growth on cereal grains.

Six fungal species, Penicillium brevicompactum, P. glabrum, P. roqueforti, Aspergillus flavus, A. versicolor, and A. candidus, were inoculated on moistened and autoclaved wheat and oat grains. They were cultivated in glass vessels provided with an inlet and outlet for air. Air was passed through the vessels to collect volatile fungal metabolites on porous polymer adsorbents attached to the outlet. Samples were collected at two fungal growth stages. Adsorbed compounds were thermally desorbed, separated by gas chromatography, and identified by mass spectrometry. Differences in the production of volatile metabolites depended more on the fungal species than on the grain type. The fungal growth stage was not an important factor determining the composition of volatiles produced. 3-Methylfuran was produced in similar amounts regardless of the fungal species and substrate (oat versus wheat). The production of volatile metabolites was compared with the production of ergosterol and CO2 and the number of CFU. The production of volatile metabolites was more strongly correlated with accumulated CO2 production than with actual CO2 production and more strongly correlated with ergosterol contents of the grain than with numbers of CFU.

Fungal volatiles as indicators of food and feeds spoilage.

Fungal growth leads to spoilage of food and animal feeds and to formation of mycotoxins and potentially allergenic spores. Fungi produce volatile compounds, during both primary and secondary metabolism, which can be used for detection and identification. Fungal volatiles from mainly Aspergillus, Fusarium, and Penicillium have been characterized with gas chromatography, mass spectrometry, and sensory analysis. Common volatiles are 2-methyl-1-propanol, 3-methyl-1-butanol, 1-octen-3-ol, 3-octanone, 3-methylfuran, ethyl acetate, and the malodorous 2-methyl-isoborneol and geosmin. Volatile sesquiterpenes can be used for taxonomic classification and species identification in Penicillium, as well as to indicate mycotoxin formation in Fusarium and Aspergillus. Developments in sensor technology have led to the construction of "electronic noses" (volatile compound mappers). Exposure of different nonspecific sensors to volatile compounds produces characteristic electrical signals. These are collected by a computer and processed by multivariate statistical methods or in an artificial neural network (ANN). Such systems can grade cereal grain with regard to presence of molds as efficiently as sensory panels evaluating grain odor. Volatile compound mapping can also be used to predict levels of ergosterol and fungal colony-forming units in grain. Further developments should make it possible to detect individual fungal species as well as the degree of mycotoxin contamination of food and animal feeds.