Malolactic fermentation in lingonberry juice and its use as a preservative.

Lingonberry is a common wild berry that is often sold as jams and beverages. It naturally contains high amounts of the weak acid preservative benzoic acid making it an interesting ingredient for shelf-life extension. Despite this, their use as a raw ingredient is limited by the inherently intense sour taste. This study aimed to improve the taste of lingonberry juice by subjecting it to malolactic fermentation in order to reduce the sourness, and to investigate the benzoic acid in lingonberries as a natural preservative in juice blends by determining the microbial stability. After initial screening of lactic acid bacteria, a Lactiplantibacillus plantarum strain was used as the starter for subsequent investigations. Upon raising the pH, all malic acid was completely converted to lactic acid after seven days. The fermented juice was mixed with blackcurrant juice in different proportions. Challenge tests of the blends showed Listeria monocytogenes could not grow in any juice samples, while Candida albicans only grew in the pure blackcurrant juice. Aspergillus brasiliensis growth was delayed in all samples containing benzoic acid in a concentration-dependent manner. The sourness and astringency were substantially reduced in the juice with added L. plantarum compared to the unfermented juice.

Do Multi-year Applications of Bacillus thuringiensis subsp. israelensis for Control of Mosquito Larvae Affect the Abundance of B. cereus Group Populations in Riparian Wetland Soils?

Bacillus thuringiensis subsp. israelensis (Bti) is a soil-borne bacterium affiliated to the Bacillus cereus group (Bcg) and has been used in biocontrol products against nematoceran larvae for several decades. However, knowledge is limited on whether long-term Bti application can affect the structure of indigenous communities of Bcg and the overall abundance of Bti. Using species- and group-specific quantitative PCR assays, we measured the Bcg- and Bti-abundances in riparian wetlands in the River Dalälven floodplains of central Sweden. On five occasions during one vegetative season, soil samples were collected in alder swamps and wet meadows which had been treated with Bti for mosquito larvae control during the preceding 11 years, as well as in untreated control sites and well-drained forests in the same area. The average abundance of Bcg in alder swamps was around three times higher than in wet meadows. Across all sites and habitats, the Bti treatments had no effect on the Bcg-abundance, whereas the Bti-abundance was significantly higher in the treated than in the control sites. However, for individual sampling sites, abundances of Bti and Bcg were not correlated with the number of Bti applications, indicating that added Bti possibly influenced the total population of Bti in the short term but had only a limited effect in the longer term. The findings of this study increase the understanding of the ecology of Bti applications for mosquito control, which can facilitate environmental risk assessment in connection with approval of microbiological control agents.

Chromosome-Directed PCR-Based Detection and Quantification of Bacillus cereus Group Members with Focus on B. thuringiensis Serovar israelensis Active against Nematoceran Larvae.

Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification of B. thuringiensis serovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use of B. thuringiensis serovar israelensis in the environment.

Cryo-electron microscopy specimen preparation by means of a focused ion beam.

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Trehalose synthesis in Aspergillus niger: characterization of six homologous genes, all with conserved orthologs in related species.

BACKGROUND: The disaccharide trehalose is a major component of fungal spores and is released upon germination. Moreover, the sugar is well known for is protective functions, e.g. against thermal stress and dehydration. The properties and synthesis of trehalose have been well investigated in the bakers' yeast Saccharomyces cerevisiae. In filamentous fungi, such knowledge is limited, although several gene products have been identified. RESULTS: Using Aspergillus niger as a model fungus, the aim of this study was to provide an overview of all genes involved in trehalose synthesis. This fungus has three potential trehalose-6-phosphate synthase encoding genes, tpsA-C, and three putative trehalose phosphate phosphatase encoding genes, tppA-C, of which two have not previously been identified. Expression of all six genes was confirmed using real-time PCR, and conserved orthologs could be identified in related Aspergilli. Using a two-hybrid approach, there is a strong indication that four of the proteins physically interact, as has previously been shown in S. cerevisiae. When creating null mutants of all the six genes, three of them, ΔtpsA, ΔtppA and ΔtppB, had lower internal trehalose contents. The only mutant with a pronounced morphological difference was ΔtppA, in which sporulation was severely reduced with abnormal conidiophores. This was also the only mutant with accumulated levels of trehalose-6-phosphate, indicating that the encoded protein is the main phosphatase under normal conditions. Besides ΔtppA, the most studied deletion mutant in this work was ΔtppB. This gene encodes a protein conserved in filamentous Ascomycota. The ΔtppB mutant displayed a low, but not depleted, internal trehalose content, and conidia were more susceptible to thermal stress. CONCLUSION: A. niger contains at least 6 genes putatively involved in trehalose synthesis. Gene expressions related to germination have been quantified and deletion mutants characterized: Mutants lacking tpsA, tppA or tppB have reduced internal trehalose contents. Furthermore, tppA, under normal conditions, encodes the functional trehalose-6-phosphate-phosphatase.

Functional analysis of the C-II subgroup killer toxin-like chitinases in the filamentous ascomycete Aspergillus nidulans.

Chitinases are hydrolytic enzymes responsible for chitin polymer degradation. Fungal chitinases belong exclusively to glycoside hydrolases family 18 and they are categorized into three phylogenetic groups (A, B and C), which are further divided into subgroups (A-II to A-V, B-I to B-V and C-I to C-II). Subgroup C chitinases display similarity with the α/ß-subunit of the zymocin yeast killer toxin produced by Kluyveromyces lactis, suggesting a role of these enzymes in fungal-fungal interactions. In this study, we investigated the regulation and function of 4 Aspergillus nidulans subgroup C-II killer toxin-like chitinases by quantitative PCR and by constructing gene deletion strains. Our results showed that all 4 genes were highly induced during interactions with Botrytis cinerea and Rhizoctonia solani, compared to self-interactions. In addition, chiC2-2 and chiC2-3 were also induced during contact with Fusarium sporotrichoides, while none of these genes were induced during interactions with Phytophthora niederhauserii. In contrast, no difference in expression levels were observed between growth on glucose-rich media compared with media containing colloidal chitin, while all genes were repressed during growth on R. solani cell wall material. Phenotypic analysis of chitinase gene deletion strains revealed that B. cinerea biomass was significantly higher in culture filtrate derived from the ΔchiC2-2 strain compared to biomasses grown in media derived from A. nidulans wild type or the other chitinase gene deletion strains. The analysis also showed that all chitinase gene deletion strains displayed increased biomass production in liquid cultures, and altered response to abiotic stress. In summary, our gene expression data suggest the involvement of A. nidulans subgroup C-II chitinases in fungal-fungal interactions, which is further proven for ChiC2-2. In addition, lacking any of the 4 chitinases influenced the growth of A. nidulans.

The lactic acid bacteria metabolite phenyllactic acid inhibits both radial growth and sporulation of filamentous fungi.

BACKGROUND: Food spoilage caused by molds is a severe problem. In food and feed, e.g. dairy products, sourdough bread and silage, lactic acid bacteria are used as starter cultures. Besides lactic and acetic acid, some strains produce other low molecular weight compounds with antifungal activities. One of these metabolites is phenyllactic acid (PLA), well known for its antifungal effect. The inhibitory effect of PLA has only partially been investigated, and the objective of this study was to elucidate in detail the antifungal properties of PLA. RESULTS: We investigated the outgrowth of individual conidia from Aspergillus niger, Cladosporium cladosporioides and Penicillium roqueforti, and observed the morphologies of resulting colonies on solid media using different acid concentrations. We found that PLA inhibits molds similar to weak acid preservatives. Furthermore, it has an additional activity: at sub-inhibitory concentrations, fungal colonies displayed slower radial growth and inhibited sporulation. The L isoform of PLA is a more potent inhibitor than the D form. Increased expression of phiA was observed during PLA treatment. This gene was initially identified as being induced by Streptomyces-produced macrolide antibiotics, and is shown to be a structural protein in developed cells. This suggests that PhiA may act as a general stress protectant in fungi. CONCLUSION: From a food protection perspective, the results of this study support the usage of lactic acid bacteria strains synthesizing PLA as starter cultures in food and feed. Such starter cultures could inhibit spore synthesis, which would be beneficial as many food borne fungi are spread by airborne spores.

Intracellular trehalase activity is required for development, germination and heat-stress resistance of Aspergillus niger conidia.

The disaccharide trehalose is known as a stress protectant in several kinds of organisms, including fungi, where it is a major carbohydrate in resting structures, e.g. asexual conidia. The gene encoding the enzyme responsible for degradation of intracellular trehalose, treB, was deleted and the phenotype was analyzed in terms of morphology, trehalose content during conidial outgrowth and stress tolerance. The mutant conidiophores produced fewer and less viable spores, and during early stages of germination the internal levels of trehalose were higher compared to the wild type. When subjecting the mutant to various stresses (weak acid and salt), no increased sensitivity could be observed, but in line with previous observations, e.g. in Aspergillus nidulans, Aspergillus niger ΔtreB spores in a very early stage of germination were less sensitive to heat stress. In contrast, when subjecting resting spores to 55 °C, an intact treB gene was essential for survival. This finding suggests that trehalose mobilization is required to facilitate cell recovery after heat-induced damage.

A site-specific focused-ion-beam lift-out method for cryo Transmission Electron Microscopy.

The focused-ion-beam (FIB) is the method of choice for site-specific sample preparation for Transmission Electron Microscopy (TEM) in material sciences. A lamella can be physically lifted out from a specific region of a bulk specimen with submicrometer precision and thinned to electron transparency for high-resolution imaging in the TEM. The possibility to use this tool in life sciences applications has been limited by the lack of lift-out capabilities at the cryogenic temperatures often needed for biological samples. Conventional cryo-TEM sample preparation is mostly based on ultramicrotomy, a procedure that is not site-specific and known to produce artifacts. Here we demonstrate how a cooled nanomanipulator and a custom-built transfer station can be used to achieve cryo-preparation of TEM samples with the FIB, enabling high-resolution investigation of frozen-hydrated specimens in the TEM.

Functional analysis of glycoside hydrolase family 18 and 20 genes in Neurospora crassa.

Glycoside hydrolase family 18 contains hydrolytic enzymes with chitinase or endo-N-acetyl-ß-D-glucosaminidase (ENGase) activity, while glycoside hydrolase family 20 contains enzymes with ß-N-acetylhexosaminidase (NAGase) activity. Chitinases and NAGases are involved in chitin degradation. Chitinases are phylogenetically divided into three main groups (A, B and C), each further divided into subgroups. In this study, we investigated the functional role of 10 Neurospora crassa genes that encode chitinases, 2 genes that encode ENGases and 1 gene that encode a NAGase, using gene deletion and gene expression techniques. No phenotypic effects were detected for any of the studied group A chitinase gene deletions. Deletion of the B group member chit-1 resulted in reduced growth rate compared with the wild type (WT) strain. In combination with the presence of a predicted glycosylphosphatidylinositol anchor motif in the C-terminal of chit-1, indicating cell wall localization, these data suggest a role in cell wall remodeling during hyphal growth for chit-1. Deletion of the ENGase gene gh18-10 resulted in reduced growth rate compared with WT, increased conidiation, and increased abiotic stress tolerance. In addition, Δgh18-10 strains displayed lower secretion of extracellular proteins compared to WT and reduced levels of extracellular protease activity. The connection between gh18-10 ENGase activity and the endoplasmic reticulum associated protein degradation process, a stringent quality control of glycoprotein maturation, is discussed. N. crassa group C chitinase genes gh18-6 and gh18-8 were both induced during fungal-fungal interactions. However, gh18-6 was only induced during interspecific interactions, while gh18-8 displayed the highest induction levels during self-self interactions. These results provide new information on functional differentiation of fungal chitinases.

Formulation and stabilisation of the biocontrol yeast Pichia anomala.

The yeast Pichia anomala has antifungal activities and its potential in biocontrol and biopreservation has previously been demonstrated. To practically use an organism in such applications on a larger scale the microbe has to be formulated and stabilised. In this review we give an overview of our experience of formulating and stabilising P. anomala strain J121 in a wider perspective. The stabilisation techniques we have evaluated were liquid formulations, fluidised bed drying, lyophilisation (freeze-drying) and vacuum drying. With all methods tested it was possible to obtain yeast cells with shelf lives of at least a few months and in all cases the biocontrol activity was retained. Fluidised bed drying was dependent on the addition of cottonseed flour as a carrier during the drying process. In liquid formulations a sugar, preferentially trehalose, was a required additive. These two kinds of microbial stabilisation are easily performed and relatively inexpensive but in order to keep the cells viable the biomaterial has to be stored at cool temperatures. However, there is room for optimization, such as improving the growth conditions, or include preconditioning steps to enable the cells to produce more compatible solutes necessary to survive formulation, desiccation and storage. In contrast, lyophilisation and vacuum drying require a lot of energy and are thus expensive. On the other hand, the dried cells were mostly intact after one year of storage at 30°C. Inevitably, the choice of formulation and stabilisation techniques will be dependent also on the intended use.

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain.

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce ('biopreservation') has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers' yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.

Comparative molecular evolution of trichoderma chitinases in response to mycoparasitic interactions.

Certain species of the fungal genus Trichoderma are potent mycoparasites and are used for biological control of fungal diseases on agricultural crops. In Trichoderma, whole-genome sequencing reveal between 20 and 36 different genes encoding chitinases, hydrolytic enzymes that are involved in the mycoparasitic attack. Sequences of Trichoderma chitinase genes chi18-5, chi18-13, chi18-15 and chi18-17, which all exhibit specific expression during mycoparasitism-related conditions, were determined from up to 13 different taxa and studied with regard to their evolutionary patterns. Two of them, chi18-13 and chi18-17, are members of the B1/B2 chitinase subgroup that have expanded significantly in paralog number in mycoparasitic Hypocrea atroviridis and H. virens. Chi18-13 contains two codons that evolve under positive selection and seven groups of co-evolving sites. Chi18-15 displays a unique codon-usage and contains five codons that evolve under positive selection and three groups of co-evolving sites. Regions of high amino acid variability are preferentially localized to substrate- or product side of the catalytic clefts. Differences in amino acid diversity/conservation patterns between different Trichoderma clades are observed. These observations show that Trichoderma chitinases chi18-13 and chi18-15 evolve in a manner consistent with rapid co-evolutionary interactions and identifies putative target regions involved in determining substrate-specificity.

The decarboxylation of the weak-acid preservative, sorbic acid, is encoded by linked genes in Aspergillus spp.

The ability to resist anti-microbial compounds is of key evolutionary benefit to microorganisms. Aspergillus niger has previously been shown to require the activity of a phenylacrylic acid decarboxylase (encoded by padA1) for the decarboxylation of the weak-acid preservative sorbic acid (2,4-hexadienoic acid) to 1,3-pentadiene. It is now shown that this decarboxylation process also requires the activity of a putative 4-hydroxybenzoic acid (3-octaprenyl-4-hydroxybenzoic acid) decarboxylase, encoded by a gene termed ohbA1, and a putative transcription factor, sorbic acid decarboxylase regulator, encoded by sdrA. The padA1,ohbA1 and sdrA genes are in close proximity to each other on chromosome 6 in the A. niger genome and further bioinformatic analysis revealed conserved synteny at this locus in several Aspergillus species and other ascomycete fungi indicating clustering of metabolic function. This cluster is absent from the genomes of A. fumigatus and A. clavatus and, as a consequence, neither species is capable of decarboxylating sorbic acid.

Experimental setups and considerations to study microbial interactions.

Within ecosystems microorganisms coexist and interact. Knowledge of these interactions is of great importance in the fields of ecology, food production, and medicine. Such interactions often involve the synthesis of antibiotic secondary metabolites. Different kinds of s molecules or direct contacts are other forms of microbial interactions. Recently, modern molecular methods such as microarrays and proteomics have been employed to investigate such interactions. In this chapter, the use of proteomics for studies of microbial interactions is discussed. The choice of experimental setup is dependent on the aims of the specific study. One aspect of competition between microbes can be simulated by treatment of one microbe with antibiotics produced by a competing microbe. A more complicated approach involves cocultivation of the competitors, but in order to reveal species-specific protein patterns it is advisable to keep the organisms separated. Alternative techniques are to monitor alterations in the proteomes between the wild-type and mutant strains. The mutant can be either natural or created using random or targeted mutagenesis. Generally, a proteomic study will reveal proteins with both expected and surprising changes in abundance upon competition, but also previously unknown proteins are likely to be identified. A proteomic approach is usually insufficient to obtain a complete data set describing microbial interactions. Therefore, it is essential to follow up identification of proteins with changed abundance by, e.g., the creation of knockout strains for phenotypic analyses. Despite the limitations, proteomics is a useful method, and an important complement to other approaches for studies of microbial interactions.

Auxotrophy for uridine increases the sensitivity of Aspergillus niger to weak-acid preservatives.

Weak-acid preservatives such as sorbic acid are added to foods to prevent fungal spoilage. The modes of action of weak-acid preservatives are only partially understood and, in this paper, further insight is presented into the mechanisms by which weak acids inhibit the growth of fungi. Uridine-requiring strains of Aspergillus niger were shown to be more sensitive to weak acids (including sorbic, acetic and benzoic acids) than wild-type (WT) strains. In contrast, sensitivity to other, non-acidic, antifungal substances was similar in mutant and WT strains. By complementing a pyrG(-) strain of A. niger with an intact pyrG gene, WT-like resistance to weak-acid preservatives was restored. Using (14)C-labelled uridine, sorbic acid was shown to completely inhibit uridine uptake in germinating conidia in a non-competitive manner. It is therefore proposed that the additional weak-acid sensitivity of the pyrG(-) strains was caused by weak-acid inhibition of uridine uptake. Several other auxotrophic strains of A. niger were screened for sensitivity to acetic, sorbic and decanoic acids. Strains auxotrophic for either adenine or uridine were found to have enhanced sensitivity but, in contrast, amino acid auxotrophs showed resistance comparable to that of the WT. Uridine auxotrophs of Saccharomyces cerevisiae were not more sensitive to weak acids compared to WT strains. In conclusion, this study describes a previously unknown mechanism of action of weak acids against the filamentous fungus A. niger, which may fundamentally affect our understanding of the preservation of food against spoilage fungi.

Biological preservation of plant derived animal feed with antifungal microorganisms: safety and formulation aspects.

During storage of moist animal feed, growth of detrimental fungi causing spoilage, or being mycotoxigenic or pathogenic, is a severe problem. Addition of biopreservative yeasts or lactic acid bacteria can significantly reduce this problem. However, their use requires several careful considerations. One is the safety to the animal, humans and the environment, tightly connected to legal aspects and the need for pre-market authorisation when supplementing feed with microorganisms. Although both yeasts and lactic acid bacteria are considered comparatively safe organisms due to low production of toxic metabolites, it is of great importance to understand the mechanisms behind the biopreservative abilities. Another important issue concerns practical aspects, such as the economic production of large amounts of the organisms and the development of a suitable formulation giving the organisms a long shelf life. These aspects are discussed and a recommendation of this review is that both safety and formulation aspects of a specific microbe should be considered at an early stage in the selection of new organisms with biopreservation potential.

Optimisation and comparison of liquid and dry formulations of the biocontrol yeast Pichia anomala J121.

The biocontrol yeast Pichia anomala J121 can effectively reduce mould growth on moist cereal grains during airtight storage. Practical use of microorganisms requires formulated products that meet a number of criteria. In this study we compared different formulations of P. anomala. The best way to formulate P. anomala was freeze-drying. The initial viability was as high as 80%, with trehalose previously added to the yeast. Freeze-dried products could be stored at temperatures as high as 30 degrees C for a year, with only a minor decrease in viability. Vacuum-drying also resulted in products with high storage potential, but the products were not as easily rehydrated as freeze-dried samples. Upon desiccating the cells using fluidised-bed drying or as liquid formulations, a storage temperature of 10 degrees C was required to maintain viability. Dependent on the type of formulation, harvesting of cells at different nutritional stresses affected the initial viabilities, e.g. the initial viability for fluidised-bed-dried cells was higher when the culture was fed with excess glucose, but for freeze-drying it was superior when cells were harvested after depletion of carbon. Using micro-silos we found that the biocontrol activity remained intact after drying, storage and rehydration for all formulations.

Oxygen and carbon source-regulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala.

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, ADH1 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 nine-fold 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.

Co-cultivation of antifungal Lactobacillus plantarum MiLAB 393 and Aspergillus nidulans, evaluation of effects on fungal growth and protein expression.

The fungal inhibitory effects of strain Lactobacillus plantarum MiLAB 393, producing broad-spectrum antifungal compounds, were evaluated. A co-cultivation method was set up to monitor effects on fungal growth and protein expression of growing Aspergillus nidulans with L. plantarum MiLAB 393. The effects of inhibitory metabolites produced by L. plantarum MiLAB 393, cyclo(l-Phe-l-Pro), lactic acid and 3-phenyllactic acid, were also investigated by addition of pure compounds to the growth medium of A. nidulans. The co-cultivation strongly affected the morphology of the fungal mycelium and decreased the biomass to 36% of control. Co-cultivation with Lactobacillus coryniformis MiLAB 123 gave only marginal morphological changes and minor biomass reduction, suggesting specific effects of L. plantarum MiLAB 393. The amount of several A. nidulans-proteins was increased during co-cultivation and by all of the inhibiting substances. This study shows that the growth of A. nidulans is inhibited during co-cultivation with L. plantarum MiLAB 393 and that the expression of fungal proteins is altered.