Biomedical subjects

R Geisen

Publications and source records attributed to R Geisen.

14 recordsLinked to original sources

PubMed2005

Advances in the molecular diagnosis of ochratoxin A-producing fungi.

Ochratoxin A (OTA) is detected worldwide in various food and feed sources. The compound is produced by Penicillium nordicum and P. verrucosum, as well as by various species within the sections Nigri and Circumdati of the genus Aspergillus, with A. ochraceus and A. carbonarius known to be the predominant producers. Recently, various pairs of PCR primers based on AFLP, RFLP, RAPD and the calmodulin gene were developed to set up novel diagnostic approaches for OTA producers in the Aspergillus and Penicillium genera. Real-time PCR assays based on well-characterized genomic sequences in A. ochraceus and P. nordicum have also been set up. Since the application of such assays to the analysis of contaminated sample material was demonstrated in only a few cases, future studies should be focused on applying such methods in rapid, robust and user-friendly applications, and implementing them in HACCP concepts. The recent detection and characterization of OTA biosynthetic pathway genes in the Penicillium genus is an important step towards understanding what mechanisms influence production of the toxin in order to redesign production processes in the food and feed industry and to keep de-novo synthesis to a minimum.

Aspergillus↗

PubMed2001

Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing.

Seventy-six strains of Penicillium roqueforti used as starter cultures for mould ripened blue cheeses have been analysed for their RAPD genotype by using three different primers. A comparison of the RAPD patterns within each primer group revealed that the genetic constitution of the strains was similar, as most of the strains showed very similar overall patterns. Despite these similarities with each primer, distinct RAPD genotype groups could be identified. With one of the primers, it was possible to detect two heteropolymorphic DNA regions resulting in 13 different groups. With the other two primers, three or four groups could be identified. Between the groups of the different primers marked correspondence with respect to strain distribution could be observed, indicating that the polymorphisms detected by the primers were not independent. The RAPD patterns were compared to the production of secondary metabolites. A correlation was observed between the RAPD patterns of all primers and the production of mycophenolic acid. In addition, one of the primer (ari1) was able to distinguish between P. roqueforti strains producing larger or smaller numbers of metabolites.

Cheese↗

PubMed2001

Taxonomy and important features of probiotic microorganisms in food and nutrition.

Lactic acid bacteria are among the most important probiotic microorganisms typically associated with the human gastrointestinal tract. Traditionally, lactic acid bacteria have been classified on the basis of phenotypic properties, eg, morphology, mode of glucose fermentation, growth at different temperatures, lactic acid configuration, and fermentation of various carbohydrates. Studies based on comparative 16S ribosomal RNA sequencing analysis, however, showed that some taxa generated on the basis of phenotypic features do not correspond with the suggested phylogenetic relations. Thus, some species are not readily distinguishable by phenotypic characteristics. This is especially true for the so-called Lactobacillus acidophilus group, the Lactobacillus casei and Lactobacillus paracasei group, and some bifidobacteria, strains of which have been introduced in many probiotic foods, eg, the novel yogurt-like commodities. Consequently, modern molecular techniques, including polymerase chain reaction-based and other genotyping methods, have become increasingly important for species identification or for the differentiation of probiotic strains. Probiotic strains are selected for potential application on the basis of particular physiologic and functional properties, some of which may be determined in vitro. The classification and identification of a probiotic strain may give a strong indication of its typical habitat and origin. The species, or even genus name, may also indicate the strain's safety and technical applicability for use in probiotic products. Molecular typing methods such as pulsed-field gel electrophoresis, repetitive polymerase chain reaction, and restriction fragment length polymorphism are extremely valuable for specific characterization and detection of such strains selected for application as probiotics.

Bifidobacterium↗

PubMed2000

P. nalgiovense carries a gene which is homologous to the paf gene of P. chrysogenum which codes for an antifungal peptide.

Penicillium nalgiovense is related to P. chrysogenum. P. chrysogenum carries the paf gene (Penicillium antifungal peptide) with homology to the afp gene of Aspergillus giganteus. This gene codes for a peptide with antifungal activity. Based on the sequence of the published P. chrysogenum paf gene primers were generated. By the use of these primers a PCR product of the expected length could be isolated from strains of P. nalgiovense. This fragment was sequenced and compared to the sequence of the paf gene of P. chrysogenum. According to the results P. nalgiovense carries a gene (naf = P. nalgiovense antifungal peptide) which is highly homologous to the paf gene of P. chrysogenum. The gene also codes for a preproprotein with the same processing sites as the paf gene, suggesting that the mature product is also a 55 amino acid (aa) peptide. The naf gene has three amino acid exchanges compared to the paf gene, which however do not influence the amino acid sequence of the mature peptide. It also carries two introns at the same positions, however, the sequence differences between the introns are higher than between the coding regions. When P. nalgiovense were grown on plates containing other food-relevant fungi it showed weak antifungal activity.

Antifungal Agents↗

PubMed2000

Karyotype of Penicillium nalgiovense and assignment of the penicillin biosynthetic genes to chromosome IV.

The karyotype of Penicillium nalgiovense was determined by pulsed-field gel electrophoresis and compared to the karyotype of P. chrysogenum. Both species have four chromosomes, but they differ in the size of the chromosomes and in the overall size of the genome. The sizes of the P. nalgiovense chromosomes as determined by pulsed-field gel electrophoresis are: 9.1 Mb, 7.9 Mb, 5.4 Mb and 4.1 Mb which gives in summary a genome size of 26.5 Mb. This compares to 34.1 Mb for P. chrysogenum. The penicillin gene cluster was located by Southern hybridization on chromosome IV, the smallest chromosome of P. nalgiovense compared to chromosome 1, the largest chromosome of P. chrysogenum.

Chromosomes, Fungal↗

PubMed2000

Characterization of Leuconostoc gasicomitatum sp. nov., associated with spoiled raw tomato-marinated broiler meat strips packaged under modified-atmosphere conditions.

Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomato-marinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs). A mixed LAB population dominated by a Leuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product. Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentative Lactobacillus species were the other main organisms detected. An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called "protein swell." This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids. Protein swell has not previously been associated with any kind of meat product. A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant Leuconostoc species. In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L. gelidum was most similar to the spoilage-associated species. The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L. gelidum type strain. DNA-DNA reassociation, however, clearly distinguished the two species. The same strains showed only 22 and 34% hybridization with the L. gelidum type strain. These results warrant a separate species status, and we propose the name Leuconostoc gasicomitatum sp. nov. for this spoilage-associated Leuconostoc species.

Animals↗

PubMed1999

Inhibition of food-related pathogenic bacteria by god-transformed Penicillium nalgiovense strains.

Two strains of Penicillium nalgiovense, which carried the god gene of Aspergillus niger and had increased glucose oxidase (GOD) activity compared with the wild-type strain, were tested for their ability to suppress the growth of certain food-related pathogenic bacteria. In contrast to the wild type, which showed no antibacterial effect when grown in mixed culture with different bacteria, the two transformed strains were highly antagonistic. The strain that expressed higher amounts of GOD in general had higher inhibitory activity. Both strains showed antibacterial activity against Listeria monocytogenes, Salmonella Enteritidis, and Staphylococcus aureus. The inhibitory activity was dependent on the glucose concentration in the medium. S. aureus was completely inhibited at 1% glucose in the presence of the higher GOD-producing transformant. In contrast, if arabinose was used as a carbon source, no inhibition occurred. If catalase was added to the medium, the inhibitory activity of the transformants was completely inactivated, indicating that the hydrogen peroxide produced was responsible for the antibacterial activity of the transformants.

Antibiosis↗

PubMed1998

A PCR-ELISA for the detection of potential fumonisin producing Fusarium species.

A PCR-ELISA for the detection of potential fumonisin producing Fusarium species has been developed, using the ribosomal ITS1 sequence as target. For this purpose, the sequences of the ITS1 regions of different fumonisin producing Fusarium species have been determined and compared to the sequences of fumonisin non-producing species. In general, the ITS1 sequences were highly homologous. However, some minor sequence polymorphisms were detected, which differentiates potential fumonisin producing Fusarium species from non-producing species. By using these sequence differences, a PCR-ELISA for potential fumonisin producing Fusarium species was developed. All other ubiquitously occurring food-borne fungi tested showed negative results with this test.

Base Sequence↗

PubMed1997

Detection of aflatoxinogenic fungi in figs by a PCR reaction.

A PCR reaction was used to detect aflatoxinogenic Aspergillus flavus strains in contaminated figs. The reaction records the presence of three aflatoxin biosynthesis genes, namely the norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1) and sterigmatocystin-o-methyltransferase: (omt-A). The reaction gave a triplet pattern in the presence of DNA from A. flavus isolated from pure cultures. The reaction gave the same PCR products when pure fungal DNA was mixed with pure DNA isolated from figs, but the sensitivity was reduced by a factor of 10. The same set of bands was observed when isolated DNA from infected figs was used as template DNA but no signal was visible when DNA from uninfected figs was used as template.

Aflatoxins↗

PubMed1996

Genetically modified starter and protective cultures.

Modern approaches towards starter and protective culture improvement rely on advances in molecular biology. For most microorganisms used for food production, gene technological methods have been well developed. By recombinant DNA technology, 'tailor-made' starter and protective cultures may be constructed so as to combine technically desirable features. A single strain which normally would fail to accomplish a given 'task' may now be improved so as to meet a set of requirements necessary for a specific production or preservation process (e.g. wholesomeness, no off-flavour production, overproduction of bacteriocins or particular enzymes). In addition, undesirable properties (e.g. mycotoxin or antibiotic production by cheese moulds) may be eliminated by techniques such as 'gene disruption'.

Fermentation↗

PubMed1996

Transformation of Penicillium freii and a rapid PCR screening procedure for cotransformation events.

A strain of Penicillium freii was transformed to hygromycin B resistance using the pAN7-1 plasmid. The transformation frequency was approximately 100 transformants per microgram DNA per 1 x 10(7) protoplasts. Southern blotting revealed different types of integration into the genome of P.freii. When non-selectable plasmid DNA was added together with pAN7-1, this resulted in a cotransformation frequency of more than 60%. The cotransformation frequency was estimated using a facile DNA extraction method together with PCR.

Blotting, Southern↗

PubMed1995

Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes.

A review is presented on the present status of biological preservation of foods. Recent developments are discussed with respect to underlying mechanisms of inhibition by 'protective' cultures, and special reference is made to lactic acid bacteria (LAB) and their 'food-grade' safety. The role of bacteriocins, their limitations and potentiating role in biological systems, is also addressed. The use of enzymes (e.g. lysozyme) for food preservation is mainly restricted by economic factors, their inactivation by endogenous food components and their limited activity spectrum. Practical applications of protective cultures refer to particular food commodities that either constitute novel systems with respect to packaging and/or composition, or represent special hygienic risks. It is concluded that biological preservation cannot substitute GMP; it, however, offers an additional (and acceptable) processing parameter for improving the safety and assuring the quality of a given food.

Antibiosis↗

PubMed1994

Antagonistic activity of the food-related filamentous fungus Penicillium nalgiovense by the production of penicillin.

Defined strains of the genus Penicillium used as starter cultures for food and strains isolated from mold-fermented foods were analyzed for their ability to inhibit the growth of Micrococcus luteus DSM 348 used as an indicator organism. Most of the strains belonging to the species Penicillium nalgiovense showed antagonistic activity in agar diffusion assays. Penicillium camemberti and Penicillium roqueforti strains proved to be inactive in these tests. The inhibitory substance excreted by P. nalgiovense strains was totally inactivated when treated with beta-lactamase (penicillinase), indicating that a beta-lactam antibiotic is produced by these strains. This observation was verified by PCRs with primer sets specific to the [delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine] synthetase gene (pcbAB), the isopenicillin-N-synthase gene (pcbC), and the acyl coenzyme A:6-aminopenicillanic acid acyltransferase gene (penDE) from Penicillium chrysogenum using chromosomal DNA of the fungal strains as a template. These results indicate that penicillin biosynthesis is a characteristic often found in strains of P. nalgiovense. No specific PCR signal could be identified with DNA from P. camemberti and P. roqueforti.

Acyltransferases↗

PubMed1990

Two Penicillium camembertii mutants affected in the production of cyclopiazonic acid.

Penicillium camembertii was mutated and screened for cyclopiazonic acid-negative mutants. With a simple and rapid mini-extraction method for detection of cyclopiazonic acid production, we were able to isolate two strains which were affected in the production of this metabolite. One strain had completely lost the ability to synthesize detectable amounts of this secondary metabolite, whereas the other mutant produced 50 to 100 times less cyclopiazonic acid than the wild type. Also, the former strain had a changed morphology compared with the wild type. This morphological alteration appears to be coupled to the inability to produce cyclopiazonic acid because morphological revertants were able to synthesize cyclopiazonic acid to a level comparable to the wild type. The second mutant accumulated a new metabolite which was detectable by two-dimensional thin-layer chromatography. This new metabolite, however, appears not to be a direct precursor of cyclopiazonic acid.

Genes, Fungal↗