Walter P. Hammes

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the generaLactobacillus, Pediococcus, Leuconostoc, andWeissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such asLactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strainLactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.

Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis

ABSTRACT Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a bakers yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the bakers yeast, respectively.

Characterization of the bacteriocins curvacin A from Lactobacillus curvatus LTH1174 and sakacin P from L. sake LTH673

Summary Two bacteriocin producing strains, one of Lactobacillus curvatus and one of L. sake have been isolated employing a catalase-containing bacteriocin-screening-medium for lactobacilli. Both bacteriocins were produced in the late exponential growth phase, and were not only active against closely related lactobacilli but also against the opportunistic food pathogens Listeria monocytogenes and Enterococcus faecalis . The inhibitory compounds were only slightly affected by heat treatment but distroyed by proteinase K and trypsin. Both bacteriocins were purified to homogeneity by ammonium sulfate precipitation, cation exchange, hydrophobic interaction and reversed phase chromatography. The bacteriocins from L. curvatus LTH1174 and L. sake LTH673 were termed curvacin A and sakacin P, respectively. Amino acid composition analysis and automated protein sequencing revealed that curvacin A and sakacin P are small peptides of 38–41 and 41 amino acid residues, respectively. No unusual amino acids were detected. In the N-terminal region curvacin A and sakacin P share the similar segment — Tyr-Gly-Asn-Gly-Val —. No sequence similarity was detected to previously characterized bacteriocins indicating that these bacteriocins are novel.

The formation of biogenic amines by fermentation organisms

ZusammenfassungDas Potential zur Bildung von biogenen Aminen (BA) wurde bei 523 Stämmen, die 35 Spezies aus dem Verwandtschaftsbereich der Lebensmittelfermentationsorganismen zugehörten, untersucht. Zu diesem Zweck wurden ruhende Zellen in Phosphatpuffer (pH 5,5) verwendet und die Bildung der folgenden BA verfolgt: Putrescin, Cadaverin, Histamin, Tyramin, 2-Phenylethylamin. Bei Spezies der GeneraLactococcus, Leuconostoc, Pediococcus, Streptococcus und einigenLactobacillus spp., darunterL. pentosus undL. sake fehlte dieses Potential. Eine deutliche Bildung von BA wurde bei Stämmen von Carnobakterien,Lactobacillus buchneri, L. curvatus, L. reuten undStaphylococcus carnosus beobachtet. Weniger ausgeprägt war die Aminbildung beiLactobacillus alimentarius, L. brevis, L. bavaricus, L. delbrueckii ssp.lactis, Micrococcus spp. undStaphylococcus piscifermentans. Ein Potential bei Spezies, die bei der Fermentation von Milch, Wein und Sauerkraut bekanntermaßen erwünscht sind, wurde nur bei wenigen Stämmen gefunden. In Anbetracht der Bedeutung bestimmter Spezies als Starterkulturen bei Lebensmittelfermentationen und der möglichen Anwendung als Schutzkulturen oder Probiotika ist die mögliche Bildung von BA durch Auswahl geeigneter Stämme ausreichend zu berücksichtigen.AbstractA total of 523 strains representing 35 species related to food fermentation organisms of practical importance were investigated for their potential for formation of biogenic amines (BA). The investigation was performed with resting cells in phosphate buffer (pH 5.5) and the formation of the following BAs was followed: putrescine, cadaverine, histamine, tyramine and 2-phenylethylamine. No potential was observed in species ofLactococcus, Leuconostoc, Pediococcus, Streptococcus and severalLactobacillus spp., such asL. pentosus andL. sake. A remarkable potential to form BA was observed in strains of carnobacteria,Lactobacillus buchneri, L. curvatus, L. reuteri, Staphylococcus carnosus and, to a lesser extent, inL. alimentarius, L. brevis, L. bavaricus, L. delbrueckii ssp.lactis, Micrococcus spp. andS. piscifermentans. In well known species with a practical function in the fermentation of dairy products, wine or cabbage a potential was observed for few strains only. In view of their role as starters in food fermentation, or their potential use in protective cultures and as probiotics, BA formation by the organisms has to be taken into consideration by selecting appropriate strains.

Effect of ecological factors on the inhibitory spectrum and activity of bacteriocins

The effect of food components and ecological factors on the activities of nisin, sakacin P and curvacin A was evaluated. Lactobacillus curvatus, Listeria innocua, Salmonella and Escherichia coli including E. coli O157:H7 were used as target organisms. Lecithin, casein, and divalent cations were antagonists of the bacteriocins at 0.1%, 0.1% and 10 mmol l(-1), respectively. A decrease in pH as well as the presence of EDTA, propyl-parabene or NaCl at concentrations of 0-1 mmol y(-1), 0-0.16 g l(-1), and 0-6% (w/w), respectively, increased the activity of all bacteriocins. These compounds as well as a pH < 5.5 rendered the Gram-negative target organisms sensitive against bacteriocins. Of practical importance is the respective effect of NaCl at concentrations > 5% which are achieved in fermentation and ripening processes, e.g. in production of fermented sausages. A characteristic response was observed for each of the bacteriocins. It is suggested that bacteriocins of lactic acid bacteria are effective against a wide range of microorganisms including E. coli O157:H7 if applied in combination with other preservative principles prevailing in foods.

Identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis

ABSTRACT Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and bakers yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum. In sourdough A (traditional process with rye flour), C. humilis dominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40°C, respectively, S. cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant. Isolates identified as C. humilis and S. cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the bakers yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S. uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S. cerevisiae. The last four species were also detected in sourdoughs A, B, and C.

Characterization of Reutericyclin Produced by Lactobacillus reuteri LTH2584

ABSTRACT Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains ofEscherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C8 chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. Höltzel, M. G. Gänzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766–2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.

Identification of lactobacilli from sourdough and description of Lactobacillus-pontis sp-nov.

The microflora of sourdough preparations was investigated by examining the physiological characteristics, whole-cell protein patterns, and 16S rRNA sequences of Lactobacillus isolates. Strains isolated from sourdough were placed in the species Lactobacillus brevis, Lactobacillus sanfrancisco, and Lactobacillus reuteri. 16S rRNA sequences were determined for L. brevis, Lactobacillus fructivorans, Lactobacillus fermentum, L. sanfrancisco, and L. reuteri, and oligonucleotide probes for fast specific identification of these sourdough lactobacilli were deduced. The physiological characteristics, protein patterns, and 16S rRNA sequences of these organisms were compared with data for other sourdough lactobacilli and additional reference strains. Strains of a Lactobacillus species were isolated from rye sourdough; these strains may account for most of the flora in sourdough made from wheat or rye. These organisms were differentiated from other sourdough lactobacilli by their protein pattern, 16S rRNA sequence, G + C content, and physiological properties. The 16S rRNA sequence of this species was determined, and we constructed a phylogenetic tree which reflected the relationships of this species to other lactobacilli. This organism is closely related to L. reuteri. A new Lactobacillus species, Lactobacillus pontis, is proposed. The type strain is L. pontis LTH 2587 (= DSM 8475 = LMG 14187). We describe a general strategy in which a polyphasic approach was used to characterize a new species.

Histamine and tyramine degradation by food fermenting microorganisms

Microorganisms suitable for food fermentation were examined with regard to their potential to degrade histamine and tyramine. Out of 64 lactic acid bacteria evaluated in this study, 27 degraded histamine and one tyramine, respectively, with low activity. Among 32 strains of Brevibacterium linens and coryneform bacteria, 21 exhibited histamine and tyramine oxidase activity. None of 20 strains of Staphylococcus carnosus tested degraded histamine or tyramine. One strain out of nine strains of Geotrichum candidum degraded tyramine slightly. Among 44 strains of Micrococcus sp. examined, 17 degraded either one or two biogenic amines. In this study Micrococcus varians (M. varians) LTH 1540 exhibited the highest tyramine oxidase activity of all strains tested and was therefore investigated in detail. The enzyme was found to be located in the cytoplasm and was not membrane bound. The reaction end product p-hydroxyphenyl acetic acid was detected by HPLC analysis. An activity staining for the amine oxidase in a native polyacrylamide gel based on the formation of H2O2 during amine oxidation was developed. Resting cells of the strain exhibited optimal tyramine oxidase activity at a pH of 7 at 37-40 degrees C. The enzyme in the cell free extract had a pH optimum between 7-8. The enzyme activity was decreased by NaCl, glucose and hydralazine. Phenylethylamine and tryptamine were oxidized at lower concentrations than tyramine. The potential for amine degradation was not found to be associated with that of formation of biogenic amines, as 23 microorganisms with the ability to metabolise biogenic amines exhibited no decarboxylase activity toward histidine, tyrosine, phenylalanine, lysine or ornithine.

A 23S rDNA-targeted polymerase chain reaction-based system for detection of Staphylococcus aureus in meat starter cultures and dairy products.

A polymerase chain reaction-based system for detection of Staphylococcus aureus was developed. The system consisted of the following components: (i) selective enrichment, (ii) DNA isolation, (iii) amplification of DNA with primers targeted against the 23S rRNA gene, and (iv) evaluation of the specificity of the polymerase chain reaction by Southern hybridization and nested polymerase chain reaction. The method achieved a high degree of sensitivity and unambiguity as required for the detection of contaminants in food starter preparations. The method permitted detection of Staphylococcus aureus in preparations of meat starter cultures containing Staphylococcus carnosus either alone or in combination with lactobacilli, pediococci, and/or Kocuria varians. Detection limits were sufficiently low to show within 12 h the presence of 10(0) CFU of S. aureus in starter preparations containing 10(10) CFU of S. carnosus. The system was also applied to dried skim milk and cream. For detection without selective enrichment, a protocol was developed and permitted detection of 120 CFU of S. aureus in 1 ml of cream within 6 h. With nested polymerase chain reaction, the detection limit was decreased by one order of magnitude.