To Örebro University

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.

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 > DG18. 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 degreesC than at 5 degreesC or 25 degreesC for up to 42 weeks, although the difference in mean counts of yeasts stored at - 18 degreesC and 25 degreesC was only 0.78 log(10) cfu/ml of rehydrated suspension. TGYC was equal to PCAC and superior to the other four media in recovering yeasts stored at - 18 degreesC, 5 degreesC, or 25 degreesC 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.

The potential Use Of yeasts to control grain-storage molds was evaluated by coculturing the yeast Pichia anomala with Penicillium roqueforti and Aspergillus candidus on agar plates, using different temperatures, water activities (a(w)), and nutrient concentrations. Addition of 10 ppm cycloheximide to malt-extract agar inhibited Pichia anomala completely without affecting mold growth, making it possible to quantify the inhibition as a reduction in colony-forming units (cfu). For A. candidus, numbers of cfu and hyphal lengths were reduced at an initial yeast concentration of 10(4) cells/plate and reduced below detection limit at 10(8) cells/plate. A clear reduction in growth of Penicillium roqueforti was only observed at 10(8) yeast cells/plate. The antagonistic effect was generally more pronounced at low (6, 15-degrees-C) and high (30, 37-degrees-C) temperatures than at ambient ones. Pichia anomala inhibited growth of both molds more strongly in a substrate-rich medium than in a medium with a low substrate content. In water agar (low substrate concentration) the degree of inhibition of Penicillium roqueforti was larger at 0.96 a(w) than at 0.98 a(w).

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.

The influence of pH, temperature and carbon source (glucose and maltose) on growth rate and ethanol yield of Dekkera bruxellensis was investigated using a full-factorial design. Growth rate and ethanol yield were lower on maltose than on glucose. In controlled oxygen-limited batch cultivations, the ethanol yield of the different combinations varied from 0.42 to 0.45 g (g glucose)(-1) and growth rates varied from 0.037 to 0.050 h(-1). The effect of temperature on growth rate and ethanol yield was negligible. It was not possible to model neither growth rate nor ethanol yield from the full-factorial design, as only marginal differences were observed in the conditions tested. When comparing three D. bruxellensis strains and two industrial isolates of Saccharomyces cerevisiae, S. cerevisiae grew five times faster, but the ethanol yields were 0-13% lower. The glycerol yields of S. cerevisiae strains were up to six-fold higher compared to D. bruxellensis, and the biomass yields reached only 72-84% of D. bruxellensis. Our results demonstrate that D. bruxellensis is robust to large changes in pH and temperature and may have a more energy-efficient metabolism under oxygen limitation than S. cerevisiae.

Aerobic long-term incubations (40-wk) were employed to measure the potentially mineralizable nitrogen (N₀) in five 30-yr old cropping systems. The cropping systems consisted of: (1) bare fallow; (2) cropping with no additions; (3) cropping with 80 kg N ha^-1 y^-1 as Ca(NO₃)₂; (4) cropping with 80 kg N ha^-1 yr^-1 as Ca(NO₃)₂ plus 1800kg C ha^-1 yr^-1 as straw; and (5) cropping with 80 kg N ha^-1 yr^-1 plus 1800 kg C ha^-1 yr^-1 as farmyard manure. The amounts of N mineralized during the 40-wk incubations were between 93 and 168 μg g^-1 (302-543 kg N ha^-1 down to 25cm depth) with the lowest value for the fallow and the highest for the farmyard manure treatment. Microbial biomass-C and -N were measured on four occasions during the incubations. The biomass-C showed a rapid decrease to week 4 (to 36% of the initial mass), a slower decrease to week 9 (to 23% of initial mass) and a very slow decline to the final determination at the end of the incubation (to 8% of initial mass). The biomass-N displayed a similar pattern. Two related models were employed to describe the kinetics of N-mineralization during incubation: (1) a two-component first-order; and (2) a simplified special case of the two-component model. In all cases except the straw-amended soil, the simplified two-component model offered the best description of the curves of accumulated mineral-N. The available fraction, N_a, of soil organic-N had mineralization rate constants similar to those for mineralization of microbial biomass.

Penicillium roqueforti was recently reclassified into the three species P. roqueforti, Penicillium carneum, and Penicillium paneum based on differences in ribosomal DNA sequences and secondary metabolites, e.g. mycotoxins. This is the first report on interaction between these closely related mould species under stress conditions. The yeast Pichia anomala (J121) inhibits growth of P. roqueforti in grain stored in malfunctioning airtight storage systems. The ability of P. anomala to inhibit all three species of the P. roqueforti group was examined in separate experiments as well as the competition between the three mould species when co-cultured with or without the yeast in non-sterile wheat grain (a(w) 0.95) under restricted air supply. Mould growth was analysed by dilution plating after 14 days and the individual colonies identified by random amplified polymorphic DNA (RAPD) fingerprinting. When co-culturing the P. roqueforti group in wheat without P. anomala all three species were able to grow to the same extent. Also, when co-culturing all species of the P. roqueforti group together with P. anomala, the growth response of the three species was very similar. Al yeast levels of 10(4) CFU g(-1),grain a pronounced inhibition was observed and at 10(5) CFU g(-1) grain a fungicidal effect was detected, indicating a potentiated effect of P. anomala when co-culturing the three mould species.

The Penicillium roqueforti group has recently been split into three species, P, roqueforti, Penicillium carneum, and Penicillium paneum, on the basis of differences in ribosomal DNA sequences and secondary metabolite profiles. We reevaluated the taxonomic identity of 52 livestock feed isolates from Sweden, previously identified by morphology as P. roqueforti, by comparing the sequences of the ribosomal internal transcribed spacer region. Identities were confirmed with random amplified polymorphic DNA analysis and secondary metabolite profiles. Of these isolates, 48 were P. roqueforti, 2 were P. paneum, and 2 were Penicillium expansum. No P. carneum isolates were found, The three species produce different mycotoxins, but no obvious relationship between mold and animal disease was detected, based on medical records, P. roqueforti appears to dominate in silage, but the ecological and toxicological importance of P. carneum and P. paneum as feed spoilage fungi is not clear. This is the first report of P. expansum in silage.

The metabolite production of lactic acid bacteria JAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml(-1) for two of the three test organisms).

This qualitative study reports on lactic acid bacteria (LAB), yeasts and moulds isolated from three artisanal Swedish cellar-stored goat cheeses aged for 1, 3 and 5 months. Starter culture LAB dominated in the younger cheeses, and Leuconostoc pseudomesenteroides, common in raw goats' milk, had persisted from the unpasteurised milk into all the cheeses. Non-starter LAB dominated in the 5 month cheese, in particular, Lactobacillus sakei, a meat-associated LAB not previously isolated from cheese. Debaryomyces hansenii, and Penicillium and Mucor species were dominant among the yeasts and moulds, respectively. The cheese rind was not formed primarily from Penicillium species as in traditional cheeses such as Camembert - rather, mycelium from Mucor mucedo contributed to rind formation. Mould species known to produce sterigmatocystin, aflatoxins or ochratoxin A in cheese were not isolated in this study; growth of mycotoxigenic Aspergilli may have been inhibited by the cool conditions in the earth-cellar (4-6 degrees C).

An electronic nose was used to classify grain samples based on their smell and to predict the degree of moldy/musty odor. A total of 235 samples of wheat, barley and oats, which had been odor classified by at least two grain inspectors, were used. Headspace samples from heated grain were pumped through chambers containing metal oxide semiconductor field effect transistor (MOSFET) sensors, SnO2 semiconductors and an infrared detector monitoring CO2. The sensor signals were evaluated with a pattern-recognition software program based on artificial neural networks. The samples were divided into either the four classes moldy/musty, acid/sour, burnt, or normal or the two classes good and bad according to the inspectors descriptions. They were also assigned a score describing their intensity of moldy/musty odor. The electronic nose correctly classified approximate to 75% of the samples when using the four-class system and approximate to 90% when using the two-class system. These values exceeded the corresponding percentages of agreement between two grain inspectors classifying the grain.

The fungal-specific lipid ergosterol correlates with fungal biomass and often also with the degree of mycotoxin contamination of cereals. We compared the ability of a near-infrared reflectance (NIR) instrument with a broad wavelength range (400-2500 nm) and a near-infrared transmittance (NIT) instrument with a narrower wavelength range (850-1050 nm) to predict the ergosterol content of naturally infected barley samples. The two instruments were equally good at predicting ergosterol content in Swedish samples (r(2) = 0.81 and 0.83 for NIT and NIR, respectively). The NIT instrument was then used for samples from three countries (Sweden, Ireland, UK). This model had about the same root mean-squared error (approximate to 5 mg of ergosterol/kg, db, of grain) as the dataset with only Swedish samples, although the r(2) value was lower (0.58). The investigation has shown that it is possible to predict ergosterol content in whole barley samples using NIR or NIT instrumentation, and acceptable models can be obtained using different barley cultivars and samples from different countries and harvest years. This should make it possible to routinely predict the fungal biomass at grain terminals.

Adsorption of the volatile fungal metabolites 2-methylfuran, 3-methyl-1-butanol, and 1-octen-3-ol to wheat grains, and their subsequent desorption, were investigated. Adsorption was performed both dynamically and statically. In the dynamic system, volatile compounds in a N2 flow were led through 400 g of wheat in a glass column. In the static system, 400 g of wheat was stored in an airtight glass vessel containing the volatile compounds in the atmosphere. Three desorption procedures were compared: an N2 flow at 20-degrees-C, an N2 flow at 50-degrees-C, and extraction with supercritical CO2. 3-Methyl-1-butanol and 1-octen-3-ol were efficiently adsorbed and could also readily be desorbed to considerably higher extents at 50-degrees-C than at 20-degrees-C. The supercritical CO2 extraction was more efficient than N2 desorption in extracting volatile compounds, but because of the smaller sample sizes (1 g), the amounts extracted per gram of grain were lower than the amounts extracted with N2 desorption at 50-degrees-C. The adsorbed amount of each volatile compound was calculated as the difference between content in the N2 stream before passage through the wheat and its content after passage. Desorption by means of a N2 stream led to the recovery of about 5% of 2-methylfuran, 35% of 3-methyl-1-butanol, and 100% of 1-octen-3-ol.

Ten Penicillium and Aspergillus species, four with a strong musty off-odor and six reference fungi without any characteristic odor, were cultivated on oatmeal agar for 5 days in cultivation vessels provided with an inlet and an outlet for air. Samples of volatile metabolites were collected on a porous polymer adsorbent attached to the outlet from day 2 through day 5 after inoculation. Adsorbed compounds were desorbed thermally and analyzed with GC/MS and a combined GC and sensory analysis, the GC sniff technique. Multivariate analysis of GC/MS and fungal odor data revealed strong associations between 6 of 65 volatile compounds and musty off-odor. The GC sniff technique showed that five of these, dimethyl disulfide, 1-octen-3-ol, 2-methylisoborneol, and two C11H18 compounds, had prominent off-odors. In addition, geosmin, 1-methoxy-3-methylbenzene, and methylphenol were produced in large amounts by some off-odorous fungi and contributed to their unpleasant odor. 3-Methylfuran, 2-methyl-1-propanol, and 3-methyl-1-butanol were much more commonly produced than the off-odorous compounds. Both odorous and other volatile metabolites could be detected after 2 days of fungal growth. The production of odorous metabolites was enhanced at the time of sporulation.

Penicillium aurantiogriseum Dierckx was cultivated on six agar substrates (barley meal agar, oat meal agar, wheat meal agar, malt extract agar, Czapek agar, and Norkrans agar) and on oat grain for 5 days in cultivation vessels provided with an inlet and an outlet for air. Volatile metabolites produced by the cultures were collected on a porous polymer adsorbent by passing an airstream through the vessel. Volatile metabolites were collected between days 2 and 5 after inoculation. CO₂ production was simultaneously measured, and after the cultivation period ergosterol contents and the numbers of CFU of the cultures were determined. Alcohols of low molecular weight and sesquiterpenes were the dominant compounds found. During growth on oat grain the production of 8-carbon alcohols and 3-methyl-1-butanol was higher and the production of terpenes was lower than during growth on agar substrates. The compositions of the volatile metabolites from oat grain were more similar to those from wheat grain, which was used as a substrate in a previous investigation, than to those produced on any of the agar substrates. Regarding the agar substrates, the production of terpenes was most pronounced on the artificial substrates (Czapek agar and Norkrans agar) whereas alcohol production was highest on substrates based on cereals. The production of volatile metabolites was highly correlated with the production of CO₂ and moderately correlated with ergosterol contents, whereas no correlation with the numbers of CFU was found. Thus, the volatile metabolites formed and the ergosterol contents of fungal cultures should be good indicators of present and past fungal activity.

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.

Lactobacillus plantarum FST 1.7 was screened for in vitro antimicrobial activity and was shown to be active against spoilage moulds and bacteria. Isolation of antimicrobial compounds from cell-free supernatant identified lactic acid, phenyllactic acid and the two cyclic dipeptides cyclo ((L)-Leu-(L)-Pro) and cyclo ((L)-Phe-(L)-Pro) as the major components responsible for this activity. L. plantarum FST 1.7 was tested for the ability to produce the antifungal compounds during sourdough fermentation and to produce bread of good quality and increased shelf-life. A rheofermentometer was used to examine the gaseous release and development characteristics of the dough. A range of parameters was determined including pH, TTA and specific loaf volume. The results were compared with those obtained using Lactobacillus sanfranciscensis, a chemically acidified and a non-acidified dough. The quality of sourdough and bread produced using L. plantarum FST 1.7 was comparable to that obtained using common sourdough starters, e.g. L. sanfranciscensis. Sourdoughs and breads were evaluated for the ability to retard growth of Fusarium culmorum and Fusarium graminearum two fungi found on breads. Sourdough and bread produced with strain FST 1.7 showed consistent ability to retard the growth of both Fusarium species, thus indicating that L. plantarum FST 1.7 has also the potential to improve the shelf-life of wheat bread.

The yeast Pichia anomala inhibits the spoilage mold Penicillium roqueforti in laboratory experiments with high-moisture wheat in malfunctioning airtight storage. The ability of P. anomala to prevent mold growth during 14 months of grain storage was evaluated in outdoor silos with different air permeabilities. Freshly harvested wheat in 160-kg portions was inoculated with 10(2) colony-forming units (cfu) g(-1) P. roqueforti, alone or together with 10(4) cfu, g(-1) P. anomala. During the first month P. anomala increased to about 10(6) cfu g(-1) in the treated silos to reach 10(7) cfu g(-1) after 9 months. Naturally occurring P. anomala in the untreated silos increased from 10(2) to about 10(3) cfu g(-1) during the first month and reached the same level as the treated silos after 9 months. Oxygen levels were reduced below the detection limit within 1 day, while carbon dioxide levels increased to 80-90% during the first month. P. roqueforti did not grow in wheat treated with P. anomala, regardless of silo permeability, but had increased to 10(5) cfu g(-1) in the untreated silos after 14 months of storage.

The biocontrol yeast Pichia anomala inhibits the growth of a variety of mold species. We examined the mechanism underlying the inhibition of the grain spoilage mold Penicillium roqueforti by the biocontrol yeast P. anomala J121 during airtight storage. The biocontrol effect in a model grain silo with moist wheat (water activity of 0.96) was enhanced when complex medium, maltose, or glucose was added. Supplementation with additional nitrogen or vitamin sources did not affect the biocontrol activity of the yeast. The addition of complex medium or glucose did not significantly influence the yeast cell numbers in the silos, whether in the presence or absence of P. roqueforti. Mold growth was not influenced by the addition of nutrients, if cultivated without yeast. The products of glucose metabolism, mainly ethanol and ethyl acetate, increased after glucose addition to P. anomala-inoculated treatments. Our results suggest that neither competition for nutrients nor production of a glucose-repressible cell wall lytic enzyme is the main mode of action of biocontrol by P. anomala in this grain system. Instead, the mold-inhibiting effect probably is due to the antifungal action of metabolites, most likely a combination of ethyl acetate and ethanol, derived from glycolysis. The discovery that sugar amendments enhance the biocontrol effect of P. anomala suggests novel ways of formulating biocontrol yeasts.

The yeast Pichia anomala J121 inhibits spoilage by Penicillium roqueforti in laboratory and pilot studies with high-moisture wheat in malfunctioning airtight storage. We tested the biocontrol ability of an additional 57 yeast species in a grain mini silo system. Most yeast species grew to CFU levels comparable to that of P. anomala J121 after 14 days of incubation (> 10(6) CF U g(-1)). Of the 5 8 species, 38 (63 strains) had no mold-inhibitory effects (Pen. roqueforti levels > 10(5) CFU g(-1)). Among these were 11 species (18 strains) that did not grow on the wheat grain. Several of the non-inhibiting yeast species have previously been reported as biocontrol agents in other postharvest environments. Weak inhibitory activity, reducing Pen. roqueforti levels to between 10(4) and 10(5) CFU g(-1), was observed with 11 species (12 strains). Candida silvicola and Pichia guillermondii reduced Pen. roqueforti to <10(4) CFU g(-1). Candida fennica, Candida pelliculosa, Candida silvicultrix, P. anomala (29 strains), Pichia burtonii, Pichia farinosa and Pichia membranifaciens strongly inhibited Pen. roqueforti (< 10(3) CFU g(-1)) in the mini silos, but none had higher biocontrol activity than P. anomala strain J121. This report is the first of biocontrol activity of C fennica and C silvicultrix. The ability of 27 yeast species to grow to high CFU values without inhibiting mold growth suggests that nutrient competition may not be the main mode of action of P. anomala J121.

The zygomycete Rhizopus oligosporus is traditionally used to ferment soybean tempeh, but it is also possible to ferment other legumes and cereals to tempeh, The traditional soybean tempeh harbours a multitude of microorganisms with potentially beneficial or detrimental effects on quality. Lactic acid bacteria (LAB) have positive effects on the safety of soybean tempeh, but the effects of LAB on R. oligosporus growth have not been investigated. We have developed a cereal grain tempeh by fermenting pearled barley with R. oligosporus ATCC 64063. Four LAB species, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri and Lactococcus lactis were assessed for their growth abilities and their effects on R. oligosporus growth during barley tempeh fermentation. Growth of LAB was assayed as colony forming units (cfu), while growth of R. oligosporus was measured as ergosterol content and hyphal length. The two fungal measurements highly correlated (r=0.83, P < 0.001, n = 90). The ergosterol content of fungal mycelia ranged from 11.7 to 30.1 mg/g fungal dry matter. L. plantarum multiplied from 4.8 to 7.4 log cfu/g dry tempeh and L. fermentum increased from 4.4 to 6.8 log cfu/g during 24 h incubation at 35 degrees C. L. reuteri and L. lactis had significantly slower growth, with increases from 4.8 to 5.6 log cfu/g and 5.0 to 5.4 log cfu/g, respectively. The growth of R. oligosporus and the final pH (4.9) in barley tempeh were not significantly influenced by any of the LAB investigated.

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.

Rhizopus oligosporus Saito can ferment soybeans or cereal grains to tempeh, a sliceable cake with improved nutritional properties. Volatiles produced by different R. oligosporus strains grown on malt extract agar (MEA), barley and soybean were investigated. The effect of co-cultivation with Lactobacillus plantarum on the production of volatiles was also studied. Volatile compounds were collected in situ by headspace diffusion and identified by GC-MS. The ten R. oligosporus strains that had different colony morphologies on MEA produced very similar volatile profiles, except for slight variations among the minor volatile compounds (e.g. sesquiterpenes). Likewise, practically no differences in volatile profiles were observed between three of the strains grown on soybeans. In contrast, the R. oligosporus volatile profile on soybean was different from that on barley from the same strain. Co-cultivation with L. plantarum did not influence volatile production by R. oligosporus. The dominant compounds produced on all three Substrates were ethanol, acetone, ethyl acetate, 2-butanone, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-methyl-1-butanol. Acetaldehyde and 2-methyl-propanal were also produced on MEA and barley, while 2-pentanone, methyl acetate, 2-butanol and 3-methyl-3-buten-1-ol were observed on soybeans. Ethanol, 2-methyl-1-butanol and 3-methyl-1-butanol were the most abundant volatile compounds produced on MEA and barley, while 2-butanone was the dominant volatile metabolite on soybean. The mushroom odour compounds, 3-octanone and 1-octen-3-ol, were only detected from soybean and soybean tempeh.

Barley tempeh was produced by fermenting barley kernels with Rhizopus oligosporus. The potential of the yeasts Saccharomyces cerevisiae (three strains), S. boulardii (one strain), Pichia anomala (one strain) and Kluyveromyces lactis (one strain) to grow together with R. oligosporus during barley tempeh fermentation was evaluated. All yeast strains grew during the fermentation and even during cold storage of tempeh (P < 0.01). The growth of yeasts slightly increased the ergosterol contents, but did not influence amino acid contents and compositions, and did not reduce phytate contents. Slight increases of vitamins B-6 and niacinamide, and slight decreases of B, and biotin were observed. Quantification of fungal growth is difficult during mixed species fermentations because ergosterol is found in all fungal species, and colony-forming-unit (cfu) estimations are not reliable for R. oligosporus and other sporulating fungi. Therefore, we developed a quantitative real-time PCR method for individually quantifying S. cerevisiae and R. oligosporus growth in barley tempeh. The PCR results were highly correlated with the ergosterol content of R. oligosporus and with the number of cfu of S. cerevisiae. Thus, real-time PCR is a rapid and selective method to quantify yeasts and R. oligosporus during mixed species fermentation of inhomogenous substrate such as barley tempeh.

The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions. Growth and metabolite formation of P. anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis. Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively. HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step. Biomass production was higher in treatment (a), whereas the specific ethanol production rate during growth on glucose was similar in both treatments. This implies that oxygen availability affected the respiration and not the fermentation of the yeast. Following glucose depletion, ethanol was oxidized to acetate in treatment (a), but continued to be produced in (b). Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (b). Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments. The levels of these metabolites were generally significantly higher in treatment (b) than in (a), indicating their importance for P. anomala during severe oxygen limitation/anaerobic conditions.

We amplified, sequenced and studied the transcriptional regulation of genes of the alcoholic fermentation pathway in the biocontrol and non-Saccharomyces wine yeast, Pichia anomala. Two ADH isogenes, PaADH1 and PaADH2, and one PDC gene, PaPDC1, were amplified from genomic P. anomala DNA by a two-step PCR approach, using degenerated primers against conserved regions of the respective genes for cloning core regions, and PCR-based gene walking for cloning the respective 5' and 3'-ends. According to sequence analysis, ADHI and PDC1 are most likely cytoplasmatic proteins, while ADH2 is most probably localized in the mitochondria. PaADH1 was expressed during aerobic growth on glucose, ethanol and succinate, but was ninefold upregulated in response to oxygen limitation when grown on glucose. The gene seems to be involved in both production and consumption of ethanol. Only low expression of PaADH2 was detected during growth on glucose and ethanol, but it was highly expressed during growth on the non-fermentable carbon source succinate and repressed by the addition of glucose. PaPDC1 was expressed during aerobic growth on glucose and was upregulated four-fold in response to oxygen limitation. PaPDC1 expression was lower in cells grown on ethanol and succinate than on glucose and was up- regulated two- and four-fold, respectively, after glucose addition. Our results demonstrate that transcription of genes of the fermentative pathway is regulated by hypoxia and carbon source but posttranscriptional regulation may play a major role in regulating the metabolic flux.

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose(-1)) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.

The ascomycetous yeast Pichia anomala J121, inhibits mould growth in malfunctioning airtight storage systems for moist animal feed grain. Extensive studies of P. anomala J121 have given detailed knowledge of growth physiology and limiting environmental factors, which is necessary to understand the inhibitory activity. Our main objective is to identify the mechanisms behind the inhibitory activity. We have two non-exclusive working hypothesis: ^I)P. anomala produces antifungal substances and ^II)P. anomala out-competes the mould for space, nutrients, and oxygen. We have found that volatile metabolites restrict radial growth and sporulation of moulds in mouth-to-mouth assays where agar plates are placed facing each other with the yeast inoculated on the upper plate and the mould on the lower. Previous studies of P. anomala have shown that it produces high quantities of ethyl acetate - a mould-inhibitory substance. We are working to identify homologous genes in P. anomala J121 to the acetyltransferase encoding genes ATF1 and ATF2 in Saccharomyces cerevisiae. Another approach has been to identify intra- and extracellular metabolites during aerobic and oxygen limited growth. Intracellular metabolites were identified by Magic Angle Spinning-High Resolution-Nuclear Magnetic Resonance (MAS-HR- NMR) that allows analysis of living cells. Extracellular metabolites were analysed with HPLC. Glycerol, well known for its role during osmotic stress, is accumulated in response to oxygen stress.

The yeast Pichia anomala J121 prevents mold spoilage and enhances preservation of moist grain in malfunctioning storage systems. Development of P. anomala J121 as a biocontrol agent requires in-depth knowledge about its physiology. P. anomala J121 grew under strictly anaerobic conditions, at temperatures between 3degreesC and 37degreesC, at pH values between 2.0 and 12.4, and at a water activity of 0.92 (NaCl) and 0.85 (glycerol). It could assimilate a wide range of C- and N-sources and produce killer toxin. A selective medium containing starch, nitrate, acetic acid, and chloramphenicol was developed for P. anomala. P. anomala was equally sensitive as Candida albicans to common antifungal compounds. Growth ability at a range of environmental conditions contributes to the competitive ability of the biocontrol yeast P. anomala J121.

A diploid and a haploid strain of Pichia anomala were tested for their biocontrol ability against the spoilage mould Penicillium roqueforti in glass tubes filled with grain at two water activities (a(w)). At a(w) 0.98, the two yeast strains grew and inhibited mould growth equally well and showed similar patterns of ethyl acetate production, reaching maximum values of 10-14 mug ml(-1) headspace. At a(w) 0.95, both growth and biocontrol performance of the haploid strain were reduced. Ethyl acetate formation was also substantially reduced, with maximum headspace concentrations of 4 mug ml(-1). We conclude that ethyl acetate is a major component of the anti-mould activity. The inhibitory effect of ethyl acetate was confirmed in a bioassay where the pure compound reduced biomass production of P. roqueforti.

Pichia anomala inhibits growth of mould during airtight storage of moist cereal grain for animal feed. The cereal grain is stored in large airtight containers where the anaerobic environment prevents growth of mould. Air can leak into the system due to the removal of grain or technical difficulties in keeping completely anaerobic conditions. This subsequently enables growth of spoilage moulds. Addition of P. anomala cells inhibits the growth of mould making the system more robust [1]. We analysed the physiological basis of the biocontrol activity. P. anomala is a Crabtree negative yeast but in contrast to other Crabtree negative organisms it can grow under anaerobic conditions [2]. The ability to switch between respiratory and fermentative growth in response to O₂-availability is essential for its survival in the airtight system and for its biocontrol activity. End products of the sugar metabolism had inhibitory effects on mould growth. Addition of glucose to a model biocontrol system enhanced biocontrol activity without increasing yeast biomass, suggesting the involvement of a product of glycolysis in biocontrol. Sugar consumption, production of ethanol and other metabolites and the activity of key enzymes were investigated in cells grown under defined conditions of oxygen and nutrient supply. The impact of the different parameters on biocontrol activity is discussed. [1] Druvefors et al. (2002) FEMS Yeast Res. 2: 389-394 [2] Fredlund et al. (2002) FEMS Yeast Res. 2: 395-402

Streptomyces halstedii K122 was previously found to produce antifungal compounds on solid substrates that inhibit radial growth of fungi among Ascomycetes, Basidiomycetes, Deuteromycetes, Oomycetes, and Zygomycetes, and strongly affected hyphal branching and morphology. During growth of S. halstedii K122 in submerged culture, no antifungal activity could be detected. However, cultivation of S. halstedii in thin (1 mm) liquid substrate layers in large surface-area tissue culture flasks caused intense growth and sporulation of S. halstedii K122, and the biologically active compounds could be extracted from the mycelium with methanol. Antifungal compounds were purified using C18 solid phase extraction and silica gel column chromatography, and identified as bafilomycins B1 and C1, using 2D NMR and FAB MS. Production of bafilomycins, which are specific inhibitors of vacuolar ATPases, has not been reported from S. halstedii previously. Minimum inhibitory concentrations (MIC) of bafilomycins BI and C1, amphotericin B, and nikkomycin Z were determined at pH 5.5 and 7.0 for the target fungi Aspergillus fumigatus, Mucor hiemalis, Penicillium roqueforti, and Paecilomyces variotii. Penicillium roqueforti was the most sensitive species to all the compounds investigated. The MIC values for amphotericin B were 0.5-4 mu g.mL(-1) for the fungi tested, and pH did not affect the toxicity. The MIC values for nikkomycin Z ranged from <0.5 mu g.mL(-1) for Mucor hiemalis to >500 mu g.mL(-1) for Aspergillus fumigatus, and pH had no influence on toxicity. Bafilomycins B1 and C1 were equally active against the fungal species tested, with MIC values in the range of <0.5-64 mu g.mL(-1). All fungi were more sensitive to both bafilomycin B1 and C1 at pH 7.0 than at pH 5.5.

Chitinolytic bacteria are used as biocontrol agents of plant pathogenic fungi. They might also potentially inhibit growth of molds, e.g., Aspergillus spp. and Penicillium spp., in stored plant material. We isolated chitinolytic bacteria from airtight stored cereal grain and evaluated their antifungal capacity. Between 0.01 and 0.5% of the total aerobic counts were chitinolytic bacteria. Gram-negative bacteria, mainly Pseudomonadaceae, constituted approximately 80% of the chitinolytic population. Gram-positive isolates belonged predominantly to the Corynebacterium-Arthrobacter group, Streptomyces, and Bacillus. Chitinolytic activity was evaluated using culture filtrates from chitin-grown isolates as the release of p-nitrophenol from p-nitrophenyl N,N'-diacetylchitobiose and as the formation of clearing zones on chitin agar. No correlation between chitinolytic activity and antifungal effects was found when challenging Penicillium roqueforti Dierckx with bacterial isolates on chitin agar in a dual culture bioassay. Fungal hyphae frequently grew seemingly unaffected through the bacterial colony of a high chitinase producer on colloidal chitin. Only 4% of the chitinolytic isolates had strong effects on fungal growth. Among these, Streptomyces halstedii (K122) and Streptomyces coelicolor (K139) inhibited growth of a broad range of fungi. Streptomyces halstedii affected hyphal morphology and decreased the radial growth rate of all fungi investigated. These effects were not caused by volatile metabolites, polyenes, or N-carbamoyl-D-glucosamine.

The chitinolytic properties of Bacillus pabuli KI 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 a(w) > 0.99. Chitinase production was induced when B. pabuli KI 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.

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(l) 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.

Fast-growing Zygomycetes, most notably Rhizopus oligosporus, are traditionally used in many food fermentations, for example, for soybean tempeh production. R. oligosporus is considered to belong to the Rhizopus microsporus group. Certain R. microsporus strains have been reported to produce either the pharmaceutically active rhizoxins or the highly toxic rhizonins A and B. In this study was investigated the formation of secondary metabolites by R. microsporus, R. oligosporus, and Rhizopus chinensis grown on a wide range of different semisynthetic and natural substrates. Liquid chromatography, combined with photodiode array detection and high-resolution mass spectrometric techniques, was used to identify secondary metabolites. Growth on maize, brown rice, and Pharma agar gave both the highest amounts and the maximum diversity of rhizoxins and rhizonins. Rhizoxins were produced by all four R. microsporus strains, whereas only one strain produced rhizonins. The six R. oligosporus and four R. chinensis strains investigated did not produce any of these two classes of metabolites.

The fungus Rhizopus oligosporus (R. microsporus var. oligosporus) is traditionally used to make tempe, a fermented food based on soybeans. Interest in the fungus has steadily increased, as it can also ferment other substrates, produce enzymes, and treat waste material. R. oligosporus belongs to the R. microsporus group consisting of morphologically similar taxa, which are associated with food fermentation, pathogenesis, or unwanted metabolite production (rhizonins and rhizoxins). The ornamentation pattern, shape, and size of sporangiospores of 26 R. microsporus group strains and two R. oryzae strains were studied using low-temperature SEM (LT-SEM) and LM. This study has shown that: (1) LT-SEM generates images from well-conserved sporangiophores, sporangia, and spores. (2) Robust spore ornamentation patterns can be linked to all different taxa of the R. microsporus group, some previously incorrectly characterized as smooth. Ornamentation included valleys and ridges running in parallel, granular plateaus, or smooth polar areas. Distribution of ornamentation patterns was related to spore shape, which either was regular, ranging from globose to ellipsoidal, or irregular. Specific differences in spore shape, size, and ornamentation were observed between Rhizopus taxa, and sometimes between strains. (3) R. oligosporus has a defect in the spore formation process, which may be related to the domesticated nature of this taxon. It had a high proportion, 10-31 %, of large and irregular spores, and was significantly differentiated from other, natural Rhizopus taxa as evaluated with partial least squares discriminant analysis. it is remarkable that the vehicle of distribution, the sporangiospore, is affected in the strains that are distributed by human activity. This provides information about the specificity and speed of changes that occur in fungal strains because of their use in (food) industry.

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.

To determine the influence of barley roots on microorganisms and N-transfonning processes in soil, numbers of nitrifiers and potential nitrification and denitrification rates were measured every week for 5 wks. The barley plants were grown in growth chambers in which the root-containing soil layer (A) was separated from three outer soil layers (B, C, D). The numbers and biomass of bacteria, numbers of flagellates and amoebae, total and FDA-active hyphal lengths, microbial biomass carbon and respiration were also determined.

The numbers of ammonium oxidizers were positively correlated with root biomass but did not differ significantly between soil layers. Potential ammonium oxidation was stimulated in the root-layer, while potential nitrite oxidation was stimulated in the B- and C-layers.

The denitrification activity (measured anaerobically in the presence of excess No^- ₃) was positively correlated with root biomass in the A-layer. Denitrification activity in the B-layer was positively correlated with the water content of the soil. When roots grew near the nets separating the root layer from the other layers, denitrification activity was stimulated in the next layer (B).

We propose that nitrite oxidation in the root zone partly depends on the reduction of nitrate. This would explain why nitrite-oxidizer numbers were usually several orders of magnitude higher than ammonium-oxidizer numbers.

Bacterial numbers decreased between wks 1 and 5. Increases in bacteria, naked amoebae and flagellates in all layers between wks 2 and 3 indicated that bacteria were produced until wk 3. There were no signs of bacterial production after wk 3.

The total length of hyphae and the length of FDA-active hyphae were not significantly different between layers. However, both of these parameters, as well as total microbial biomass carbon and respiration, were consistently highest in the A-layer.

Interactions of IIb-elements, Zn, Cd and Hg, with three common soil fungi, Trichoderma harzianum, Penicillium spinulosum and Mortierella isabellina, have been studied. The accumulation of the metals by the fungi was studied as a function of pH at constant ionic strength and at concentration levels of the metals representative of natural systems. Two stages of fungal activity were considered in the experiments. The fungi generally exhibited high affinity for metal ions indicated by distribution coefficients (log K-d, in 1 kg(-1)) of about 3.5 +/- 1, 2.5 +/- 1 and 4 +/- 1 for Zn, Cd and Hg, respectively. The pH-dependence of the accumulation as well as the isotherms at constant pH were similar between the fungi, and the maximum capacities were at least 50 mmoles kg(-1) mycelium (dw). Metal accumulation by starved mycelia was almost independent of pH, while non-starved mycelia in two cases accumulated more metals at low pH. Calculations of the distribution of metals in a model soil system of inorganic and organic constituents as well as fungal biomass indicated that the amounts of metal associated to the fungi are negligible at neutral pH. However, due to the ability of these fungi to accumulate metals independently of pH, the fraction of metals associated to fungal biomass at low pH may be significant, and, in some cases, predominant. This illustrates that the effects of fungi on metal distribution in soil should not be neglected, e.g. during a progressing acidification.

Fungi are capable of accumulating metals and, in soil, such accumulation may influence metal speciation and transport. The interactions between a common soil fungus, Trichoderma harzianum, and IIb elements were studied in the present investigation. The accumulation of the metals zinc, cadmium and mercury by starved and non-starved mycelium at different pH was determined by a batch technique using radioactive tracers; uptake of the metals was found to be large, with respective distribution coefficients of about 10(3.5), 10(2.5) and 10(4.0) for zinc, cadmium and mercury, respectively. Metal accumulation by a starved system was largely independent of pH in the range 3-9, where in a non-starved system an increased accumulation of zinc (at 10(-8) m) was observed at low pH (3-5). Potentiometric titrations performed on the two systems revealed significant differences in acid capacities, i.e. values close to zero for the starved system and 500-800 meq kg-1 for the non-starved system. The maximum metal uptake was at least 50 mmol kg-1 at pH 6.5 (calculated from adsorption isotherms). The present findings suggests that in the non-starved system a metabolite is produced and then released when the pH is within a certain range.

The capacity of chitin (from crab shells) and of fungal cell walls from Trichoderma harzianum to accumulate zinc, cadmium and mercury was studied as well as the effects of adsorbed metals on the enzymatic hydrolysis by Novozym 234 of the two substrates. The total adsorbing capacity with respect to these metals was estimated to be at least 10 mmol kg(-1) chitin (dry weight) and 50 mmol kg(-1) fungal cell walls (dry weight), respectively, at pH 6.1. Enzymatic digestion of fungal cell walls preloaded with mercury and cadmium was significantly reduced, while zinc did not cause any significant inhibition. The effect of metal complexation by chitin on the enzymatic digestion was not as pronounced as for fungal cell walls. This could reflect the fact that chitin sorbed a lower total amount of metals. The inhibitory effect of metals on the enzymatic hydrolysis was caused by the association of the metals with the two substrates and not by the presence of free metals in solution.

Verrucine F (3), a quinazoline similar to verrucines A (1) and B (2), contains one anthranilic acid residue, one L-glutamine residue, and one alpha,beta-unsaturated phenylalanine residue, as determined by NMR, MS, and chemical methods. Compounds I and 3, but not 2, were produced by Penicillium verrucosum J255 and eight additional P. verrucosum strains. Verrucines were typically more concentrated in the inner (older) parts of the colony, and 3 peaked 4-8 days after 1, but at 10-fold lower concentrations (similar to 200 mu g/g). Verrucine F (3) formed spontaneously from 1 in buffered water solutions. probably by oxidation at C-1 followed by water elimination.

Xeromyces bisporus can grow on sugary substrates down to 0.61, an extremely low water activity. Its genome size is approximately 22Mb. Gene clusters encoding for secondary metabolites were conspicuously absent; secondary metabolites were not detected experimentally. Thus, in its dry' but nutrient-rich environment, X.bisporus appears to have relinquished abilities for combative interactions. Elements to sense/signal osmotic stress, e.g. HogA pathway, were present in X.bisporus. However, transcriptomes at optimal (approximate to 0.89) versus low a(w) (0.68) revealed differential expression of only a few stress-related genes; among these, certain (not all) steps for glycerol synthesis were upregulated. Xeromyces bisporus increased glycerol production during hypo- and hyper-osmotic stress, and much of its wet weight comprised water and rinsable solutes; leaked solutes may form a protective slime. Xeromyces bisporus and other food-borne moulds increased membrane fatty acid saturation as water activity decreased. Such modifications did not appear to be transcriptionally regulated in X.bisporus; however, genes modulating sterols, phospholipids and the cell wall were differentially expressed. Xeromyces bisporus was previously proposed to be a chaophile', preferring solutes that disorder biomolecular structures. Both X.bisporus and the closely related xerophile, Xerochrysium xerophilum, with low membrane unsaturation indices, could represent a phylogenetic cluster of chaophiles'.

Little is known about the mould, Xeromyces bisporus, unique in its strong xerophilicity and ability to grow at water activity (a(w)) 0.62, lower than for any other known organism. The linear growth rates of one fast and one slow-growing strain of X. bisporus were assessed at 20, 25, 30 and 37 degrees C on solid agar media containing a mixture of glucose and fructose to reduce a(w) to 0.94, 0.88, 0.84, 0.80, 0.76 and 0.66. Growth rates of xerophilic species closely related to X. bisporus, viz. Chrysosporium Mops, C. xerophilum and Monascus eremophilus, were also assessed. Optimal conditions for growth of both X. bisporus strains were approx. 0.84 a(w) and 30 degrees C, despite FRR 2347 growing two- to five-fold faster than CBS 185.75. X. bisporus FRR 2347 even grew well at 0.66 a(w) (0.48 mm/day). C. Mops and C xerophilurn were more tolerant of high a(w) than X. bisporus. and could be differentiated from each other based on: the faster growth of C. xerophilum; its preference for temperatures >= 30 degrees C and a(w) >= 0.94 (c.f. <= 25 degrees C and similar to 0.88 a(w) for C Mops); and its ability to grow at 0.66 a(w), which is the lowest a(w) reported to date for this species. M. eremophilus grew slowly (max. 0.4 mm/day) even in its optimal conditions of similar to 0.88 a(w) and 25 degrees C. To investigate the competitive characteristics of X. bisporus at low a(w), both X. bisporus strains were grown in dual-culture with xerotolerant species Aspergillus flavus and Penicillium roqueforti, and xerophilic species A. penicillioides, C. Mops, C. xerophilum and Eurotium chevalieri, on glucose-fructose agar plates at 0.94, 0.84, 0.80 and 0.76 a(w) and at 25 degrees C. Growth rates and types of interactions were assessed. Excretion of inhibitory substances acting over a long-range was not observed by any species; inhibitors acting over a short-range that temporarily slowed competitors' growth or produced a protective zone around the colony were occasionally observed for A. penicillioides, C. Mops and C. xerophilum. Instead, rapid growth relative to the competitor was the most common means of dominance. The xerotolerant species. A. flavus and P. roqueforti were dominant over X. bisporus at 0.94 a(w). E. chevalieri was often dominant due to its rapid growth over the entire a(w) range. At a(w) < 0.80, X. bisporus was competitive because it grew faster than the other species examined. This supports the concept that its ideal environmental niche is sugary foods with low a(w).

Dairy propionibacteria are widely used in starter cultures for Swiss type cheese. These bacteria can ferment glucose, lactic acid, and glycerol into propionic acid, acetic acid, and carbon dioxide. This research examined the antifungal effect of dairy propionibacteria when glycerol was used as carbon source for bacterial growth. Five type strains of propionibacteria were tested against the yeast Rhodotorula mucilaginosa and the molds Penicillium commune and Penicillium roqueforti. The conversion of (13)C glycerol by Propionibacterium jensenii was followed with nuclear magnetic resonance. In a dual culture assay, the degree of inhibition of the molds was strongly enhanced by an increase in glycerol concentrations, while the yeast was less affected. In broth cultures, decreased pH in glycerol medium was probably responsible for the complete inhibition of the indicator fungi. NMR spectra of the glycerol conversion confirmed that propionic acid was the dominant metabolite. Based on the results obtained, the increased antifungal effect seen by glycerol addition to cultures of propionibacteria is due to the production of propionic acid and pH reduction of the medium.

Large amounts of food and feed are lost every year due to spoilage by moulds and yeasts. Biopreservation, i.e. the use of microorganisms as preservatives instead of chemicals, has gained increased interest. Lactic acid bacteria and propionibacteria might be particularly useful due to their important role in many food fermentations. Knowledge of the antifungal effects of the organic acids produced by these bacteria is necessary to understand their inhibitory activity. We evaluated the antifungal activity of the type strains of five dairy propionibacteria, Propionibacterium acidipropionici, P. jensenii, P. thoenii, P. freudenreichii subsp. freu denreichii and P. freudenreichii subsp. shermanii against eight food- and feedborne moulds and yeasts. A dual culture system assayed the inhibitory activity on three different agar media, sodium lactate (SL), de Man Rogosa Sharp (MRS) and MRS without acetate (MRS-ac). The amounts of organic acids produced during growth of propionibacteria in liquid SL, MRS and MRS-ac were also determined. The minimal inhibitory concentration (MIC) values of propionic, acetic and lactic acid were established for all fungi at pH 3, 5 and 7. Propionic acid, followed by acetic acid, was the most potent antifungal acid. Inhibition at pH 7 generally required concentrations above 500 mM for all three acids, at pH 5 the MIC values for propionic and acetic acids were 20-120 mM and above 500 mM for lactic acid. At pH 3, the MIC values were, with one exception, below 10 mM for both propionic and acetic acid and above 160 mM for lactic acid. The yeast Pichia anomala was the fungus most resistant to organic acids. The propionibacteria exhibited a pronounced species variation in antifungal activity on MRS (+/- acetate) agar, with P. thoenii being the most potent. Four of the five propionibacteria species produced more propionic and acetic acid in liquid SL medium than in MRS (+/- acetate) broth. However, when SL agar was used as the growth medium, none of the propionibacteria inhibited fungal growth.

Antifungal compounds from cultures of five type strains of dairy propionibacteria, as well as from the cultivation medium, were studied. Cell-free supernatants and medium were fractionated by C-18 solid phase extraction. The aqueous 95% acetonitrile fractions were analyzed by GC-MS or subjected to reversed-phase HPLC, to identify, quantify or isolate antifungal substances. The resulting HPLC fractions were screened for antifungal activity against the mold Aspergillus fumigatus and the yeast Rhodotorula mucilaginosa. Active fractions were further separated by HPLC and the structures of the compounds were determined by spectroscopic and chromatographic methods. All five strains produced 3-phenyllactic acid, at concentrations ranging from 1.0 mu g mL(-1) (Propionibacterium freudenreichii ssp. shermanii) to 15.1 mu g mL(-1) (Propionibacterium thoenii), and at Ld-ratios ranging from 2 : 3 (Propionibacterium acidipropionici) to 9 : 1 (Propionibacterium freudenreichii). A number of active compounds found in cultures of propionibacteria were also present in noninoculated growth medium: two antifungal diketopiperazines, cyclo(L-Phe-L-Pro) and cyclo(L-Ile-L-Pro), and seven antifungal linear peptides. Three of the linear peptides corresponded to sequences found in the medium component casein, suggesting their origin from this component, whereas the diketopiperazines were suggested to be formed from medium peptides by heat treatment.