Johan Schnürer

Lactobacillus plantarum MiLAB 393 Produces the Antifungal Cyclic Dipeptides Cyclo(l-Phe-l-Pro) and Cyclo(l-Phe-trans-4-OH-l-Pro) and 3-Phenyllactic Acid

ABSTRACT We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay. Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species. No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C18 SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C18 column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(l-Phe-l-Pro), cyclo(l-Phe-trans-4-OH-l-Pro) and 3-phenyllactic acid (l/d isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against A. fumigatus and P. roqueforti were 20 mg ml−1 for cyclo(l-Phe-l-Pro) and 7.5 mg ml−1 for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(l-Phe-l-Pro) and cyclo(l-Phe-trans-4-OH-l-Pro) by lactic acid bacteria is reported here for the first time.

MICROBIAL BIOMASS AND ACTIVITY IN AN AGRICULTURAL SOIL WITH DIFFERENT ORGANIC MATTER CONTENTS

Changes in soil fertility caused by various organic and N-fertilizer amendments were studied in a long-term field trial mostly cropped with cereals. Five treatments were included: (I) fallow, (II) cropping with no C or N addition, (III) cropping with N-fertilization (80 kg ha −1 yr−1), (IV) cropping with straw incorporation (1800kg Cha−1 yr−1) and N-fertilization (80 kg ha−1yr−1), and (V) cropping with addition of farmyard manure (80 kg N + 1800kg Cha−1yr−1). The treatments resulted in soil organic matter contents ranging from 4.3% (I) to 5.8% (V). Microbial biomass and activity were determined by chloroform fumigation, direct counting of fungi (fluorescein diacetate (FDA)-staining and Jones-Mollison agar-film technique) and bacteria (acridine orange staining), most probable number determinations of protozoa, esterase activity (total FDA hydrolysis) and respiration. Both biomass estimates and activity measurements showed a highly significant correlation with soil organic matter. Microbial biomass C ranged from 230 to 600 μg C g−1 dry wt soil, as determined by the fumigation technique, while conversions from direct counts gave a range from 380 to 2260 μg C. Mean hyphal diameters and mean bacterial cell volumes decreased with decreasing soil organic matter content.

Lactobacillus coryniformis subsp. coryniformis Strain Si3 Produces a Broad-Spectrum Proteinaceous Antifungal Compound

ABSTRACT 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°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 (NH4)2SO4 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.

Broad and complex antifungal activity among environmental isolates of lactic acid bacteria

More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dual-culture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined. Several of the strains showed strong inhibitory activity against the moulds A. fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa. Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited. Several isolates showed reduced antifungal activity after storage and handling. The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis. Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates. The degree of fungal inhibition was not only related to production of lactic or acetic acid. In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.

Antifungal 3-hydroxy fatty acids from Lactobacillus plantarum MiLAB 14

ABSTRACT We report the identification and chemical characterization of four antifungal substances, 3-(R)-hydroxydecanoic acid, 3-hydroxy-5-cis-dodecenoic acid, 3-(R)-hydroxydodecanoic acid and 3-(R)-hydroxytetradecanoic acid, from Lactobacillus plantarum MiLAB 14. The concentrations of the 3-hydroxy fatty acids in the supernatant followed the bacterial growth. Racemic mixtures of the saturated 3-hydroxy fatty acids showed antifungal activity against different molds and yeasts with MICs between 10 and 100 μg ml−1.

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.

Microbial biomass as a fraction of potentially mineralizable nitrogen in soils from long-term field experiments

Abstract Aerobic long-term incubations (40-wk) were employed to measure the potentially mineralizable nitrogen (N 0 ) 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(NO 3 ) 2 ; (4) cropping with 80 kg N ha −1 yr −1 as Ca(NO 3 ) 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.

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.

Effects of moisture on soil microorganisms and nematodes: a field experiment

The effects of soil moisture changes on bacteria, fungi, protozoa, and nematodes and changes in oxygen consumption were studied in a field experiment. In one plot the soil was drip-irrigated daily for 10 days, while an adjacent plot experienced one rainfall and was then allowed to dry out. Oxygen consumption was the parameter measured which responded most rapidly to changes in soil moisture content. Lengths of fluorescein diacetate-active hyphae paralleled oxygen consumption in both plots. Total hyphal length was not affected by one rainfall but increased from 700 mg−1 dry weight soil to more than 1,600 m in less than 10 days in the irrigated plot. In the rain plot, bacterial numbers doubled within 3 days and declined during the following period of drought. In the irrigated plot, numbers increased by 50% and then remained constant over the duration of the study. Only small changes in protozoan numbers were observed, with the exception of the last sampling date in the irrigated plot when large numbers of naked amoebae were recorded 2 days after a large natural rainfall. Nematode numbers, especially obligate root feeders, increased in both treatments. The increases were caused by decoiling rather than growth. The results indicate that fungal respiration was dominating, while bacteria, lacking a suitable source of energy, were less active, except for the first days.

Metabolite Profiles of Lactic Acid Bacteria in Grass Silage

ABSTRACT The metabolite production of lactic acid bacteria (LAB) 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).