Institut für Lebensmittelwissenschaft und Biotechnologie

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Ecological studies of the Lactobacillus biota in the human digestive tract and adaptation of intestinal lactobacilli to the sourdough ecosystem
(2005) Dal Bello, Fabio; Hertel, Christian
Among the bacteria inhabiting the human gut, lactobacilli have received considerable attention, due to their putative health promoting effects (Reid, 1999; Vaughan et al., 1999). Cultivation of lactobacilli is considered to be reliable and numerous studies using plating on selective media have been performed to investigate these bacteria in intestinal ecosystems (Tannock, 1995; Reuter, 2001). Recently, the application of PCR-DGGE in combination with primers specific for lactic acid bacteria (LAB) detected species which are not considered to be intestinal inhabitants but food-associated, such as Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides and Pediococcus pentosaceus (Walter et al., 2001; Heilig et al., 2002). Remarkably, these species could not be recovered by traditional bacteriological culture on Rogosa SL agar (Walter et al., 2001). In Chapter III, different cultivation media, as well as new incubation conditions were applied to overcome these difficulties. Human faecal samples were plated on selective and non-selective media and incubated under standard condition (37°C, anaerobiosis) for faecal LAB as well as alternative condition (30°C, 2% O2). PCR-DGGE analyses of resuspended bacterial biomass (RBB) obtained from agar plates revealed that the species composition of the recovered LAB was affected stronger by the incubation condition than by the used medium. It was observed that food-associated LAB such as L. sakei and Lc. mesenteroides, hitherto not described as intestinal inhabitants, are more easily selected when the alternative incubation condition is used. Identification of randomly picked colonies grown under the alternative condition on Rogosa SL agar showed that L. sakei is one of the predominant food-associated LAB species in faecal samples, reaching counts of up to 106 CFU per gram faeces. Comparison of the results of bacteriological culture with those obtained by PCR-DGGE analysis of the RBB showed that investigation of RBB is a fast and reliable method to gain insight into the species composition of culturable LAB in faeces. Examination of the faecal Lactobacillus populations over longer periods has revealed marked variation in the complexity and stability of these populations among human subjects (Vanhoutte et al., 2004, Walter et al., 2001). Ecological studies indicate that most Lactobacillus species found in the human gastrointestinal tract (GIT) are likely to be transient (allochthonous), originating from either the oral cavity or food (reviewed in Bibiloni et al., 2004). In order to investigate if oral lactobacilli constitute a part of the faecal Lactobacillus biota, the Lactobacillus biota of saliva and faeces of three human subjects were investigated and compared at two time-points in a three months interval (Chapter IV). The species composition of the Lactobacillus biota of human saliva and faeces was found to be subject-specific and fluctuated to some degree, but the species Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus vaginalis were detected at both time-points in saliva and faecal samples of individual subjects. RAPD-PCR analysis indicated that several strains of these species were present both in the oral cavity and in the faecal samples of the same subject. Oral isolates of the species L. gasseri and L. vaginalis showing identical RAPD types were found to persist over time, suggesting that these species are autochthonous to the oral cavity. The results of Chapter IV, together with recently published data (reviewed in Bibiloni et al., 2004), give strong evidence that some lactobacilli found in human faeces are allochthonous to the intestine and originate from the oral cavity. Lactobacilli have been detected in diverse environments and have been the subject of considerable research due to their commercial use in the food industry (reviewed in Hammes and Hertel, 2003). Several Lactobacillus species are commonly detected in both fermented food and the human GIT, but the genetic background for this ecological versatility is poorly understood. Lactobacillus reuteri is a dominant member of the microbiota of type II sourdough fermentations (Meroth et al., 2003) and is considered one of the truly autochthonous Lactobacillus species in humans (Reuter, 2001). The in vivo expression technology (IVET) developed by Walter et al. (2003) was used to identify genes (so-called ivi genes) of the sourdough isolate L. reuteri LTH5531 that show elevated levels of expression during growth of this organism in a type II sourdough fermentation (Chapter V) and during passage through the GIT of mice (Chapter VI). Thirty-eight induced fusions were found to be highly expressed during the sourdough fermentation (Chapter V), and 29 genes could be identified on the basis of the available sequence information. Four genes encoded stress-related functions (e.g. acid and general stress response) reflecting the harsh conditions prevailing during sourdough fermentation. Further eight genes were involved in acquisition and synthesis of amino acids and nucleotides, indicating their limited availability in sourdough. The remaining genes were either part of functionally unrelated pathways or encoded hypothetical proteins. The identification of a putative proteinase and a component of the arginine deiminase pathway are of technological interest, as they are potentially involved in the formation of aroma precursors. Remarkably, IVET with the genomic library that was successfully used in the sourdough study (Chapter V) did not detect ivi promoters when LTH5531 inhabited the GIT of mice (Chapter VI). With IVET, active promoters are selected by expression of an "essential growth factor" (in our system the erythromycin resistance mediated by ErmGT) that allows the organism to colonize and/ or grow in the ecosystem (Rainey, 1999, Walter et al., 2003). Expression of ivi promoters in particular ecosystems must therefore be permanent and strong in order to allow comparable growth rates of ivi clones and clones bearing constitutive promoters, especially in the GIT, where inactive bacteria are washed out. The findings of Chapter V and VI indicate that L. reuteri LTH5531 does not possess strongly expressed "GIT inducible" genes, while possessing 38 ones specifically induced in sourdough. Ivi genes are more likely to contribute to the ecological performance of an organism in a specific environment than genes expressed equally in a broad range of habitats (Rainey, 1999, Gal et al, 2003, Walter et al., 2005). Therefore, traits encoded by ivi genes are likely to be adaptive and the extent of their expression would be shaped by natural selection to improve ecological fitness. The presence of thirty-eight "sourdough specific" ivi fusions in L. reuteri LTH5531 probably reflects the long term adaptation of LTH5531 to the sourdough environment, just as ivi genes detected in strain 100-23 reflect adaptation of this GIT isolate to the rodent GIT (Walter et al., 2003). Indeed, LTH5531 was isolated from an experimental sourdough that had been inoculated with an industrial starter. This industrial starter has been propagated over several years, giving the organisms present sufficient time to adapt. In accordance with this, by using RAPD-PCR, Meroth et al. (2003) showed that strain LTH5531 was present in a commercial type II sourdough starter collected 10 years prior isolation of LTH5531, thus indicating that this strain has adapted to the sourdough environment for at least 10 years. The results of Chapter V clearly demonstrated that knowledge of gene expression and metabolic activities of bacteria during food fermentations can be obtained by applying IVET. The results collected provide an important molecular basis on which improved starter strains can be developed for industrial exploitation. Moreover, the results of Chapter VI show the importance of working with highly adapted, autochthonous strains in studies of microbial ecology in order to reveal the adaptive interactions responsible for the ecological success of these bacteria in their natural environment or during food fermentations.
Development of a genetically defined diploid yeast strain for the application in spirit production
(2005) Schehl, Beatus; Heinisch, Jürgen
Yeast strains of the species Saccharomyces cerevisiae currently in use for the production of consumable alcohols such as beer, wine and spirits are genetically largely undefined. This prevents the use of standard genetic manipulations, such as crossings and tetrad analysis, for strain improvement. Furthermore, it complicates the application of the majority of modern methods developed in yeast molecular biology. In this work two haploid laboratory strains with suitable auxotrophic markers were used for the construction of a genetically well defined, prototrophic diploid production strain. This strain was tested for its fermentative and sensory performances in comparison to commercially available yeasts. Different fruit mashes were fermented, subjected to distillation and analysed for fermentation parameters including growth, sugar utilization, ethanol production and generation of volatile compounds, higher alcohols, uretahne and glycerol. All spirits produced were tested for their sensory performances and the data obtained statistically consolidated. Our results clearly demonstrate that this laboratory strain does not display any disadvantage compared with commercial yeasts in spirit production for any of the parameters tested, yet it offers the potential to apply both classical breeding and modern molecular genetic techniques adjusting yeast physiology to special production schemes.
Effekt der Überproduktion von Enzymen des Glucosestoffwechsels auf das Wachstum und die Alkoholbildung in der Hefe Saccharomyces cerevisiae
(2006) Emili, Markus; Heinisch, Jürgen
The wine-, beer- and baker's yeast Saccharomyces cerevisiae is the major source in world wide alcohol production. Regarding the research in bioethanol production, the work presented here was aimed to examine the effect of the in vivo overproduction of all enzymes contributing to the conversion of glucose to ethanol in the yeast Saccharomyces cerevisiae with the prospect of increasing ethanol formation. S. cerevisiae is probably the best studied eucaryotic organism with respect to both classical and molecular genetics. It turned out to be of great advantage that two different multi-copy-vectors could be employed in these studies. Each of them was used in the first part of the work to insert half of the set of genes intended for overexpression. The first genes were inserted by restriction and ligation and later on a combination of the PCR-technique, with which the genomic fragments of interest were amplified, and the efficient homologous recombination in vivo was used. With these methods, the gene encoding a hexose transporter (HXT1), all the genes encoding glycolytic enzymes (HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH1 bzw. TDH2, PGK1, GPM1, ENO2, PYK1), as well as the genes encoding enzymes needed for the conversion of pyruvate to ethanol (PDC1, ADH1), were cloned. Following the isolation from yeast, the plasmids were amplified in E. coli and characterized by restriction analysis. The measurement of specific enzyme activity in crude extract of yeast transformants with such plasmids showed a slight overproduction (factor 1,5 to 3,0) for all enzymes, except for glyceraldehyde-3-phosphate dehydrogenase. For HXT1, an increased mRNA level (factor 14 in contrast to the control) was taken as evidence for overproduction. In the enzymatic determinations a clear tendency showing a lower overproduction with an increasing number of genes on the plasmids was observed. These findings suggest a negative feedback on glycolytic flux regulation. The the growth rates obtained in the second part of the work also showed a clear reduction with increasing numbers of plasmid-encoded genes. Regarding the physiological parameters, no changes in the coefficients for glucose consumption and ethanol formation could be found in comparison to a wild-type control, and the yield remained basically unchanged as well. Interestingly, abolishing the ATP-inhibition of phosphofructokinase by expression of a mutant allele of PFK1, resulted in a faster growth of transformants with an otherwise isogenic background. This result indicates the physiological relevance of the allosteric regulation at this essential glycolytic step. A lack of enzyme activity in one of the glycolytic steps in deletion mutants normally leads to growth inhibition on hexoses. On this basis, the construction of a yeast strain was initiated with the objective to obtain stable multi-copy transformants simply by growing cells on different sugars as carbon sources. In detail, this was done by crossing a strain carrying a pgi1-deletion with a strain carrying a pyk1-deletion followed by sporulation and tetrad dissection. Preliminary data with intermediate strain constructs indicate a clear increase in plasmid stability after growing cells on complex media. From the results of this thesis, valuable insights into the regulation of the glycolytic flux in vivo can be deduced, which may serve as a basis for ongoing research on the improvement of ethanol formation by yeast.
Hydrostatic high pressure treatment of casein to generate defined particle and gel structures
(2006) Merel-Rausch, Eva; Hinrichs, Jörg
The focus of the work was to study the influence of pressure treatment conditions on pressure-induced casein structures in detail. The influence of process parameters like pressure build-up, pressure level, holding time and release rate but also temperature, ionic strength and casein concentration were determined. This work showed that the structure formation of casein under high pressure treatment depends on numerous factors. Sols but also gels can be formed and could be used for different applications particularly with the choice of the release rate and the milieu conditions, even if pressure conditions and casein concentration are kept constant.
Improving food safety of sprouts and cold-smoked salmon by physical and biological preservation methods
(2007) Weiss, Alexander; Hammes, Walter
The safety of raw, ready-to-eat foods is of paramount importance and is in the focus of the food industry, consumers as well as food scientists. To improve the food safety status of the products, efficient decontamination as an important processing step and/or the use of protective microorganisms as biocontrol agents are promising approaches. In our work we successfully used these approaches for raw sprouts and cold-smoked salmon as examples for RTE foods. Therefore the set goals have been successfully performed and essential scientific knowledge has been contributed. The results have been published and are described in the following in form of the respective abstracts. Thermal seed treatment to improve the food safety status of sprouts: Alexander Weiss and Walter P. Hammes. 2003. Thermal seed treatment to improve the food safety status of sprouts. (Journal of Applied Botany. 77: 152-155) Efficacy of heat treatment in the reduction of salmonellae and Escherichia coli O157:H? on alfalfa, mung bean and radish seeds used for sprout production: Weiss Alexander and Hammes, Walter P. 2005. Efficacy of heat treatment in the reduction of salmonellae and Escherichia coli O157:H? on alfalfa, mung bean and radish seeds used for sprout production. (Eur. Food Res. Tech. 211, 187-191) Characterization of the microbiota of sprouts and their potential for application as protective cultures: Alexander Weiss, Christian Hertel, Silke Grothe, Diep Ha and Walter P. Hammes 2006. Characterization of the microbiota of sprouts and their potential for application as protective cultures. (Applied and Environmental Microbiology. Submitted for publication) Lactic acid bacteria as protective cultures against Listeria spp.on cold-smoked salmon: Weiss Alexander and Hammes, Walter P. 2006. Lactic acid bacteria as protective cultures against Listeria spp.on cold-smoked salmon. (Eur. Food Res. Tech. 222, 343-346)
The impact of milk properties and process conditions on consistency of rennet-coagulated curd and syneresis of rennet curd grains
(2008) Thomann, Stephan; Hinrichs, Jörg
Although cheesemaking is an ancient art, modern cheese production relies on the implementation of innovative technology and tailor-made starter bacteria to remain competitive in the production of commodity-type cheeses such as soft and semi-hard cheese. Any intervention in the cheesemaking procedure, i.e. in milk composition, milk treatment and microbial fermentation, affects textural properties of curd at cutting and finally syneresis. The latter is the key step in cheesemaking since the degree of syneresis determines the moisture content of the raw cheese, by which ripening as well as rheological properties and sensory are affected. This work aimed to investigate the syneresis of rennet curd grains in order to generate a kinetic model for predicting syneresis. On the one hand, the experiments covered the implementation of EPS-(exopolysaccharide producing) cultures in the manufacture of soft cheese and likewise the investigation of the cheesemaking potential of Dahlem Cashmere goat?s milk. On the other hand, the interrelated effects of homogenization, microfiltration and pH on rheological properties of rennet-induced milk gels, on syneresis and finally on cheese composition, yield and functionality were to study. Three mathematical models were compared for their suitability describing syneresis and providing kinetic parameters. The kinetic parameters obtained by a linearised model gave best curve fittings to the experimental data with high coefficient of correlation (r² > 0.99). Furthermore, the model provides a parameter (RWRmax) that gives information about the endpoint of syneresis. From this value, interpretation about the curd structure and the interaction of milk composition and physical factors on syneresis is possible. Fermentation media inoculated with non-EPS-producing Streptococcus thermophilus and EPS-producing strains of Lactococcus lactis subsp. cremoris and Lactobacillus sakei were added in a concentration from 5 % to 10 % (w/w) to the milk prior to soft cheese manufacture. The cheesemaking experiments showed that the addition of fermentation media with EPS-cultures retarded syneresis, accelerated microbial fermentation and finally caused ripening problems. By means of model experiments regarding syneresis and influence of pH value, the manufacture of soft cheese was technologically adapted. The approach demonstrated that soft cheese manufacture was yet feasible and moisture content of the raw cheese was increased by the addition of fermentation media, inoculated with EPS-cultures. Analysis of variance revealed that syneresis was significantly affected by homogenization, MF and pH. It was shown that milk composition and MF markedly influenced the endpoint of syneresis, RWRmax. Curd grains made from skim milk had the highest RWRmax value. It is assumed, that differences in curd microstructure due to fat globule distribution and content affect syneresis since cutting was performed at equal curd firmness. The experiments demonstrate that homogenization and MF can be combined to reach curd firmness and syneresis which are in accordance with values in conventional cheesemaking. Combination of homogenization and MF was promising on cheese yield, and based on the results and experience gained in this study, a new and simplified process for semi-hard cheesemaking was invented. It was shown, that the adjusted cheese yield and component recovery increased due to the interaction of homogenization and MF. Hence, the combination of homogenization and MF in cheese manufacture is promising.
Herstellung und Charakterisierung einer rekombinanten, sequenzspezifischen Protease zur Generierung bioaktiver Peptide
(2009) Hug, Thomas; Fischer, Lutz
The aim of the present thesis was the production and biochemical characterization of a sequence specific microbial protease. This enzyme should be applied in food protein hydrolyzation in order to generate bioactive peptides. To determine the substrate specificity of serine protease PRT1 from Xanthomonas campestris pv. campestris this strain was cultivated in a shaking flusk. After dialysis of the culture broth and addition of 1,10-phenantroline for metalloprotease PRT2 inactivation an enzyme activity of 0,34 nkat Caseine/mL was detected. The conversion of several chromogenic peptide substrates revealed that PRT1 does not offer a clear substrate specificity. The lysyl endopeptidase LysC from Lysobacter enzymogenes ssp. enzymogenes was obtained by cultivation of the wild type strain (ATCC 27796). In a bioreactor (1 L scale) a maximum protease activity of 0,084 nkat Tos-Gly-Pro-Lys-pNA/mL in the culture broth was detected after 45 h. The LysC gene was amplified by PCR using genomic template DNA and was cloned into the E. coli expression vector pET20b(+), leading to no detectable recombinant protease when expressed in E. coli BL21(DE3). Thus for the heterologous expression a synthetic gene construct was applied which was formerly described in literature. It contained a short N-terminal pro-sequence (MGSK) and a codon usage adapted to E. coli. The bioreactor cultivation (5 L scale) of E. coli BL21(DE3) pET20b-MGSK-LysC led to LysC inclusion bodies. The solubilization of the inclusion bodies and the following enzyme renaturation using L-arginine as an unspecific folding additive resulted in a maximum protease activity of 0,06 nkat Tos-Gly-Pro-Lys-pNA/L Culture 5h after IPTG induction. To increase the yield of recombinant protease activity the influence of the LysC propeptides on the in vitro renaturation of the protease was investigated. For this purpose both pro-peptide DNAs were sequenced, cloned and heterologously expressed in E. coli BL21(DE3). The addition of C-terminal and N-terminal propeptide to the LysC renaturation led to a maximum of 27fold (1.56 Μkat Tos-Gly-Pro-Lys-pNA/L Culture) LysC activity increasion compared to the L-arginine renaturation. As an alternative food-grade expression system the in the literature already established system of Lactobacillus plantarum NC8, L. sakei Lb790 and the E. coli-Lactobacillus shuttle vector pSIP409 was tested. However, the shaking flusk cultivations of neither L. plantarum NC8 pSIP409-MGSK-LysC nor L. sakei Lb790 pSIP409-MGSK-LysC led to detectable recombinant lysyl endopeptidase. As a possible reason therefor the different codon usage of E. coli and Lactobacillus was assumed. So expression experiments were performed using point mutated variants of the beta-galctosidase gene from Kluyveromyces lactis. It could be shown that the exchange of the serine codons tca/agt and tcc had a significant effect on the resulting enzyme activity. The exchange of three codons led to a decreation of beta-galactosidase activity of 38 %. The characterization of the recombinant lysyl endopeptidase LysC confirmed the high substrate specificity for lysine residues at P1 position. The pH and temperature optimum was 8.5 and 45°C, respectively. At 4°C and pH 9 the enzyme was stable for at least 20.5 h, whereas at 45°C only 40 % residual activity were detected after 1 h. An inhibiting effect on LysC was demonstrated for Ba2+, NH+ and PMSF. Hydrolysis of bovine caseine by LysC for generated the ACE inhibiting peptides EMPFPK, FALPQYLK, NMAINPSK and ALNEINQFYQK as well as the antioxidative VLPVPQK, which all were unambiguously identified by LC-ESI-MS/MS. Performing appropriate in vitro assays, the radical scavenging acticvity (IC50 = 4,85 Μg/mL), lipoxygenase inhibition (IC50 = 23,6 Μg/mL) and ACE inhibition (IC50 = 2,78 Μg/mL) of the caseine hydrolysate were quantified.
Isolierung universell einsetzbarer und mikrobiologisch stabiler Sauerteigstarterkulturen durch spontane Fermentationen mit Amaranth.
(2009) Sterr, Yasemin Arzu; Schmidt, Herbert
Spontaneous fermented sourdoughs prepared from five amaranth flours were screened for the presence of lactic acid bacteria (LAB) that predominate the autochthonous microbiota and thus may be suitable as starter cultures. The doughs were fermented with daily backslopping on the laboratory scale for 10 days with a dough yield of 200 at 30°C. Every 24 hours, the pH-values and total titratable acidity degrees were determined and samples were analyzed for the presence of LAB and yeasts by cultural methods. The identity of the isolates was traced during the fermentation with RAPD-PCR and two different primers, and the strains were identified by sequence analysis of the 16S rDNA genes. The strains Lactobacillus plantarum RTa12, L. sakei RTa14, and Pediococcus pentosaceus RTa11 were selected and applied as starters in further laboratory fermentations. All strains were predominant in repeated experiments, both, as single strains and in combination. During the first 24 h, L. plantarum RTa12 and P. pentosaceus RTa11 grew quite similar. The pH-value dropped earlier with P. pentosaceus RTa11, while both strains gave the same pH-values after 10 h of fermentation. In the challenge test with the autochthonous mikrobiota both strains overgrew the other LAB of the spontaneous fermented dough within the first eight hours, and were therefore considered dominant over the resident microbiota. Influences of the incubation temperature on the fermentation characteristics were mainly assessed in the viable cell counts, the pH-values and the titratable acidity degrees at 25°C. The pH-values for both strains were at high incubation temperatures (30 and 35°C) during the fermentation lower than at 25°C, respectively. However, after 24 h of fermentation both strains reached a pH-value of approximately 4.0 after 24 h. Further sugar, organic acid, mannitol and ethanol profiles of fermented doughs were determined with HPLC. Mainly analyzed metabolites in the doughs were glucose, sucrose, lactate, and acetate. To compare the potential starter cultures with commercial available startercultures, fermentations with two industrial startercultures were performed for 24 h at 30°C and a dough yield of 200. Both strains were able to compete with the commercial available starter cultures concerning viable cell counts, total titratable acidity and pH-values. Because of the dominance of both strains in sourdough fermentations with amaranth, the ability for acidification in a short time, the capacity to compete with the autochthonous mikrobiota, the robustness against lesser effects of the environment, e. g. variation of the temperature, and at least because of the ability to compete with commercial available startercultures, thus, the characterized strains L. plantarum RTa12 and P. pentosaceus RTa11 are interesting candidates as starter cultures for amaranth sourdoughs.
Molecular characterization of the interaction of lactobacilli with food environments and enterohemorrhagic Escherichia coli O157:H7
(2009) Hüfner, Eric; Hertel, Christian
The first part of this thesis focuses on the gene expression of Lactobacillus sakei and Lactobacillus reuteri in food fermentation using in vivo expression technology (IVET) and DNA microarray hybridization analysis, respectively. Both technologies allow the identification of regulated genes in a specific environment, which are likely to contribute to the ecological performance of the organism. Thus, the obtained results provide a basis for the development of new strategies to improve the fermentation process, as it was demonstrated by the development of an efficient method for the improvement of sausage fermentation using L. sakei. To obtain hygienically safe products, the function of starter cultures mostly relies on the ability to acidify and produce other antimicrobial principles. However, it was recently demonstrated that the interaction with pathogens also can take place on another level, apart from killing or growth inhibition. Lactobacilli have been shown to influence the virulence gene expression of enterohemorrhagic Escherichia coli (EHEC) via the bacterial communication system termed quorum sensing. The second part of the thesis explores the impact of quorum sensing between Lactobacillus reuteri strains and EHEC O157:H7 on EHEC virulence gene expression. By using a green fluorescent protein reporter gene assay, it was demonstrated for the first time that the transcription of the ler virulence regulator gene is significantly reduced by secreted substances of L. reuteri in a strain- and quorum sensing-dependent manner.
Dekontamination von pharmazeutischen Isolatoren mit verdampftem Wasserstoffperoxid : Charakterisierung von Einflussparametern und Optimierung des Maschinendesigns
(2010) Unger-Bimczok, Beatriz; Kottke, Volker
In the pharmaceutical industry sterile drugs, which can not be terminally sterilized, have to be prepared and handled under aseptic conditions. The application of isolator technology with physical separation of the process from the environment and the operator is commonly used. Prior to the aseptic processing, the inner isolator surfaces have to be treated with a sterilant to reduce the microbial contamination to a defined acceptable level. Today vaporized hydrogen peroxide (VPHP) is most commonly used for this purpose. Different parameters like hydrogen peroxide concentration, humidity, and condensation have an influence onto the microbicidal activity of the decontamination cycle. Also isolator design factors (e.g. material of construction, geometrical structure) can impact the inactivation results. The objective of the presented thesis was to investigate the mode of action of the VPHP and the relationship between different influencing cycle parameters in order to develop a recommendation for optimum decontamination conditions. An additional goal was to analyze the impact of different construction materials, surface finish and geometrical structures onto the inactivation efficiency of the sterilant to improve the design of aseptic processing machines regarding VPHP decontamination. For the studies an pharmaceutical isolator connected to a VPHP generator was used. Standard decontamination cycles with varying combinations of hydrogen peroxide and water concentration, cycle time and condensation levels were developed. Biological indicators (BIs) with defined initial spore population of Geobacillus stearothermophilus were exposed to the different VPHP cycles. By determination of inactivation kinetics for the microbial test challenge, the sporicidal activity for each set of cycle conditions was evaluated. The applied microbial methods were Most Probable Number (MPN) technique as well as the determination of decimal reduction times (D-values). BIs were not only tested when openly exposed to the sterilizing atmosphere, but also inside of defined gaps to challenge the penetration capability of the VPHP into small lumens under diffusive conditions. Different construction materials were inoculated with defined spore populations to investigate the resistance behaviour of the spores on varying surfaces. Supplementary the physico-chemical characteristics of the respective materials were analyzed in detail to draw conclusions regarding correlation of surface quality and inactivation properties. The results demonstrate that the decisive factor for a successful decontamination is the overall microscopic interaction with the bioburden on the surface. It is shown, that the microcondensation in the sub-visible range is effective for good inactivation performance and that further condensation in the visible range does not enhance the microbicidal activity. The data illustrate that the microbial inactivation is accelerated by increasing hydrogen peroxide concentration. An H2O2 level of 800 ppm ensures a sufficient deposition of sterilant onto the surface and results in excellent and reproducible kill. For sterilant levels > 800 ppm no further improvement in inactivation is detectable. It is shown that for openly exposed BIs a lower H2O2 level (400 ppm) can be compensated by higher humidity. The elevated water content in the decontamination atmosphere promotes the sterilant deposition. The higher the hydrogen peroxide level is, the more independent from humidity becomes the inactivation effect. For H2O2 levels of 800 ppm, the microbicidal activity of the VPHP is found to be independent from the water concentration. In contrast to the openly exposed BIs, for the inactivation of spores exposed under diffusive conditions inside of gaps, a lower hydrogen peroxide level can not be compensated by higher humidity. Solely the hydrogen peroxide concentration and the overall cycle duration are able to influence the decontamination success inside of the trenches. It is demonstrated that in principle complex structures can be decontaminated by the means of VPHP but the penetration capability is limited. The inactivation is impeded with decreasing gap cross section and with increasing gap depth. It is shown that different construction materials and surface textures have an impact onto the resistance behaviour of spores towards VPHP.
Neue Messtechniken und Simulationen für die Strömungsvorgänge und den örtlichen Stoffübergang in nicht-Newtonschen Fluiden
(2010) Zheng, Gongyuan; Kottke, Volker
Flow and transport phenomena in non-Newtonian fluids are of significant industrial interest. As a kind of static mixer, spacers play an important role in process engineering concerning problems of mixing, homogenizing and dispersing in connection with residence time behavior up to the enhancement of heat and mass transfer. However, the efficiency of such spacers strongly depends on fluid properties, the characteristic geometry and operating conditions. Additionally, it is very difficult to respond to changed operating conditions for such spacers and ill-designed feed spacers can hinder the realization of a high performance. Here, experimental and numerical methods were introduced for the investigation of flow phenomena and mass transfer for non-Newtonian fluids in spacer-filled channels.
Molekulargenetische Untersuchungen zur Expression des Typ III Effektors NleA 4795 von Shiga Toxin-produzierenden Escherichia coli
(2010) Schwidder, Maike; Schmidt, Herbert
Shiga toxin-producing E. coli (STEC) are the causative agents of foodborne infections in many countries and can lead to severe diseases like hemorrhagic colitis or the life-threatening hemolytic uremic syndrome. The bacteria colonize the human intestine where they normally cause the formation of characteristic ?attaching and effacing?-lesions. Essential for this effect is a pathogenicity island, termed as ?locus of enterocyte effacement? (LEE), that encodes the components of a type III secretion system and several effector proteins, which are translocated directly into the host cells by the TTSS machinery. In addition to the LEE-encoded effectors a large number of effector proteins have been identified which are encoded outside of the pathogenicity island. Among these is the ?non-LEE encoded effector A? (NleA), which is encoded on cryptic or inducible prophages and is widely distributed among pathogenic E. coli strains. In the present study, the expression and regulation of the nleA-variant nleA4795 of E. coli O84:H4 strain 4795/97 was investigated, which is located on the Shiga toxin-converting bacteriophage BP-4795. Therefore, different environmental conditions as well as certain regulatorproteins were tested on their influence on nleA4795-expression using a luciferase-reportersystem and the quantitative real-time PCR. Among the analyzed environmental factors, certain concentrations of NaCl and KCl were identified to activate nleA4795-expression, indicating an osmotic-based influence. The suggested induction of nleA4795 in preconditioned medium due to quorum sensing could not be confirmed, since none of the so far known autoinducers showed a positive influence on the expression. The increased expression of nleA4795 could be associated with a reduced amount of nutrients in subsequent investigations and therefore demonstrated a relation between nleA4795-expression and bacterial stress-response-systems. Furthermore, a possible correlation of nleA4795-expression with the induction of phage BP 4795 and Shiga toxin-expression was analyzed. Different from the expression of Shiga toxin, induction-experiments with norfloxacin showed no activation, but a strong repression of nleA4795-expression. Analysis of the regulatory level demonstrated that the expression of nleA4795 depends on the three LEE-encoded regulators Ler, GrlA und GrlR as well as on the Pch-regulators, which are encoded outside of the LEE. The non-LEE encoded regulator EtrA showed no influence on the expression of nleA4795. In addition, the regulator proteins Ler, GrlA and PchA were tested for direct binding to the nleA4795-promoterregion. Regulators GrlA and PchA showed no specific binding and were therefore classified as indirect regulators of nleA4795-expression. In contrast, regulator Ler showed a specific binding to different areas of the nleA4795-promoter region and thereby confirmed the integration of nleA4795 in the Ler-mediated circuit of LEE-regulation.
Verbesserung der Energie-, Stoff- und Emissionsbilanzen bei der Bioethanolproduktion aus nachwachsenden Rohstoffen
(2010) Fleischer, Sven; Senn, Thomas
In this thesis, a process was realized that uses starchy raw material (triticale) as well as lignocellulosic biomass (corn silage) in one ethanol production process. In contrast to other so called 2nd generation ethanol processes, which only use lignocellulosic material, the problem of the very low potential ethanol concentration (
Mikrobiologische und biochemische Analyse der Fermentationseigenschaften von Lactobacillus paralimentarius AL28 und Lactobacillus plantarum AL30 in Sauerteigen aus Pseudozerealien
(2011) Vogel, Antje; Schmidt, Herbert
Pseudocereals are absent of gluten and therefore are important for people having a gluten-intolerance. Today no commercial starter cultures are available for sourdough fermentations with pseudocereals. This PHD-Thesis shows results of the characterisation of L. paralimentarius AL28 and L. plantarum AL30 concerning an application in pseudocereal sourdoughs. The fermentation properties of the strains, applied as single strains and in combination, were assessed in laboratory scale fermentations with amaranth and buckwheat. The fermentation studies were performed with a dough yield of 200 and over a period of two to ten days at 30°C with daily refreshment step. The investigated strains acidified the sourdoughs fast within the first two propagation steps as single strains as well as in combination (approximately pH 4). In amaranth higher total titratable acidity (TTA) -values (TTA between 25 and 30) were measured than in buckwheat (TTA of 20). 16S rDNA / 28S rDNA-PCR sequencing and RAPD-PCR were applied to determine the bacterial and eucaryotic species affiliation, respectively, and to trace specific strains during the fermentation process. The analysed strains competed against the autochthonous microbiota with the result of suppression the majority of yeasts and moulds as well as strains of their own species within the first 12 h of sourdough fermentation. They also suppressed an autochthonous microbiota grown up to 10^8 cfu/g sourdough. Single strains as well as the combination of both strains dominated the microflora in all tested flours / fermentation batches. Both strains displayed reproducible results concerning their over-all fermentation characteristics. L. paralimentarius AL28 and L. plantarum AL30, respectively, dominated the LAB viable counts in all flours after 10 days of fermentation as single strains (>/= 68 % and >/= 98 % of LAB, respectively) as well as in combination. In the latter case strain L. plantarum AL30 was especially competitive in buckwheat (AL28:AL30 = 1:1), more than in amaranth (AL28:AL30 = 4:1). The strains were characterised by their short lag-phase of
Molekulare Interaktionen von Milchsäurebakterien mit enterohämorrhagischen Escherichia coli und humanen Darmepithelzellen
(2011) Stöber, Helen; Schmidt, Herbert
The interactions of 19 benign strains of lactic acid bacteria, bifidobacteria and staphylococci with five enterohemorrhagic Escherichia coli (EHEC) strains of different serotypes and virulence gene spectrum were investigated using a HT29 cell culture infection model. As a parameter for the infection the secretion of Interleukin 8 (IL-8) of the infected cells was analyzed by ELISA. None of the used benign strains induced an IL-8 secretion, whereas the infection with the EHEC strains leads ? independent of their virulence profile - to high amounts of IL-8. In coinfection assays with the pathogen EDL933 (O157:H7) and different test strains the secretion of IL-8 of the cultured cells was decreased by a few strains. With 12 of 19 tested strains, a weak reduction < 30 % of IL-8 secretion of HT29 cells after coinfection with EHEC O157:H7 strain EDL933 was observed. Six strains reduced the IL-8 secretion up to 60 % and the strain B. breve DSMZ 20083 decreased the IL-8 production about 73 %. Coinfection assays with different strains of one species (B. adolescentis DSMZ 20083 and DSMZ 20086 as well as L. johnsonii BFE 633 and DSMZ 10533) showed the strain specificity of the observed anti-inflammatory effect, due to different capabilities of IL-8 reduction. In further coinfection assays with different EHEC strains of the serotypes O103:H2, O26:H-, 0157:H- and O113:H21 different abilities of the benign strains to influence the infection with the different pathogen strains were noted. Therefore the protective anti-inflammatory effect is strain specific for the tested benign bacteria and also depends on the application of EHEC strains with different sero- and virulence types. Further investigations indicated the imperative of living bacteria for the observed protective effect; neither culture supernatant nor inactivated bacteria showed an effect on the IL-8 secretion of the EDL933 infected HT29 cells. The analysis of the cell culture supernatants 6 h after infection with different bacteria detected the production of lactic and acetic acid. The application of these acids in infection assays with EDL933 did not lead to an reduced IL-8 secretion of the infected cells. Therefore the production of organic acids did not explain the protective effect. The induction of IL-8 could not be traced back to the influence of a single virulence factor. Four PMK5 strains with deletions in different virulence genes induced similar IL-8 secretions in comparison to cells infected with the wild-type strain. Coinfection assays with the mutants and S. pasteuri LTH 5211 showed also similar IL-8 reductions than coinfection assays with the wild-type strain. It is to suppose that the anti-inflammatory effects of the benign bacteria do not influence a single virulence factor of the tested EHEC strains. As a second parameter the activation of the transcription factor ?Nuclear Factor kappa B? (NF-κB) of coinfected HT29 cells was monitored using a reporter-genassay. In comparison to the single EHEC-infection, the NF-κB activation was reduced by all tested lactic acid bacteria, bifidobacteria and S. pasteuri LTH 5211 in coinfection trials significantly. No strain-specificity and no pathogen-specificity could be observed. Interestingly, stimulation of the HT29 cells with benign bacteria led to inhibition of NF-κB activity, the measured values were less than the values of the negative control PBS. A gene expression analysis of toll-like receptors (TLRs), recognizing bacteria on cell surfaces and initiating the immune response, showed no regulation for TLR2. Infection with EDL933 led to down regulation of TLR4 and to up regulation of TLR9. Stimulation with L. rhamnosus GG, L. johnsonii DSMZ 10533 or L. fermentum DSMZ 20052 led neither to regulation of TLR4 nor TLR9. The benign bacteria did not influence the EHEC-induced TLR4 regulation in coinfection trials; in contrast the regulation of TLR9 was reduced significantly. The model described here is useful for screening basic effects of protective bacteria that are able to counteract EHEC-mediated effects on human cells and to study the molecular interaction between bacteria as well as between bacteria and human cultured cells.
Investigations on the mechanisms of sterilization by non-thermal low-pressure nitrogen-oxygen plasmas
(2011) Roth, Stefan; Hertel, Christian
Plastic based materials are increasingly used for packaging of pharmaceuticals (especially biologicals), food or beverages and production of medical devices. Their heat sensitivity requires safe and efficient non-thermal methods for decontamination. Plasma technology has the potential to provide a suitable means since it works at low temperatures and ? in contrast to conventional methods like application of ionizing radiation or ethylene oxide exposure ? is safe to operate, is free of residues and does not alter the bulk properties of the materials. Plasmas can generate various agents potentially active in decontamination like ultra-violet (UV) radiation, radicals and other reactive particles. To acquire an approval for plasma technology as a novel sterilization method, its process safety has to be proven. The research community has proposed hypotheses and models on its mechanisms of action, which are at least partially speculative. Still little is known about the details of the biologic effects of the combination of the various plasma agents on the components of microbial cells or spores. Especially, the question remains open which components of a cell or spore are the primary targets, and which of the agents are most effective in the inactivation process. The acquisition of such knowledge is necessary to identify parameters suitable to control, monitor, and assess the safety of plasma sterilization processes. The aims of the presented work are to elucidate which components of a cell or spore are the primary targets in low-pressure plasma sterilization, and which of the putative agents contained in the plasma are most effective in the inactivation process. To accomplish this, in the presented work suitable microbiological methods were established and the inactivation of bacterial spores and cells and fungal conidia by microwave induced low-pressure low-temperature nitrogen-oxygen plasmas was investigated. Moreover, two strategies were pursued that have hitherto not been applied in published plasma sterilization studies: (i) Using spores of Bacillus subtilis mutants to identify structural components serving as targets for sterilization with plasma and (ii) characterizing the response of Deinococcus radiodurans R1 cells to plasma treatment and identify repair processes during recovery from plasma induced damages in viable cells. Plasmas producing a maximum of UV emission were most effective in inactivating bacterial cells and spores. The inactivation followed a biphasic kinetics consisting of a log-linear phase with rapid inactivation followed by a slow inactivation phase. A continuous model fit was applied to the experimental data allowing reliable calculation of decimal reduction values for both phases. Cells of D. radiodurans were found to be more resistant than spores of B. subtilis. For B. subtilis spores, in the course of plasma treatment damage to DNA, proteins and spore membranes were observed by monitoring the occurrence of auxotrophic mutants, inactivation of catalase (KatX) activity and the leakage of dipicolinic acid, respectively. Spores of the wild-type strain showed highest resistance to plasma treatment. Spores of mutants defective in nucleotide excision repair (uvrA) and small acid-soluble proteins (ΔsspA ΔsspB) were more sensitive than those defective in the coat protein CotE or spore photoproduct repair (splB). Exclusion of reactive particles and spectral fractions of UV radiation from access to the spores revealed that UV-C radiation is the most effective inactivation agent in the plasma, whereby the splB and ΔcotE mutant spores were equally and slightly less sensitive, respectively, than the wild-type spores. The extent of damages in the spore DNA as determined by quantitative PCR correlated with the spore inactivation. Spore inactivation was effectively mediated by a combination of DNA damage and protein inactivation. DNA was identified to be the primary target for spore inactivation by UV radiation emitted by the plasma. Coat proteins were found to constitute a protective layer against the action of the plasma. For the investigation of the recovery from plasma-induced damages, cells of D. radiodurans R1 were subjected to short plasma treatments with various plasmas. A part of the survivors was sublethally injured as determined by their ability to form colonies on standard medium but not on stress medium and by the observation of a prolonged lag phase. Incubation of the cells in a recovery medium after plasma treatment allowed a part of the survivors to recover their ability to grow on stress medium. This recovery strongly depended on transcriptional and translational processes and cell wall synthesis, as revealed by addition of specific inhibitors to the recovery medium. Genes involved in DNA repair, oxidative stress response and cell wall synthesis were induced during recovery, as determined by quantitative RT-PCR. Damage to chromosomal DNA caused by plasma agents and in-vivo repair during recovery was directly shown by quantitative PCR. Plasmas with less UV radiation emission were also effective in killing D. radiodurans cells but resulted in less DNA damage and lower induction of the investigated genes. The response of D. radiodurans to plasma indicated that DNA, proteins and cell wall are primary targets of plasma, whose damage initially leads to the cells' death. Protein oxidation was more important for the killing of D. radiodurans cells than of B. subtilis spores. Thus, the plasma process parameters must regard the expected contaminating biological material in order to obtain a high-level sterilization. The results provide new insight into the interaction of non-thermal low-pressure plasmas with microorganisms. This knowledge supports the definition of suitable parameters for novel plasma sterilization equipment to control process safety. For example, monitoring the UV intensity below 280 nm and spectrometric online measurement of bands related to excited reactive gas particle species during the process is recommended.
Untersuchungen zur spezifischen Genexpression von enterohämorrhagischen Escherichia coli (EHEC) in der Lebensmittelmatrix
(2012) Kroj, Andrea; Schmidt, Herbert
Ground beef as a high risk food is known to be an cause of human infection with Shiga toxin-producing E. coli (STEC). The pathogens infect humans by the ingestion of undercooked ground meat and cause severe diseases like hemorrhagic colitis or the life-threatening hemolytic uremic syndrome. E. coli O157:H7 strain EDL933 as a representative of enterohemorrhagic E. coli (EHEC), a subgroup of STEC, was analysed for in vivo induced genes in ground beef with the help of the in vivo expression technology. It could be demonstrated that the promoter selection vector pKK232-8, which contains a promoterless chloramphenicol resistance gene, is not a suitable vector for a study of gene expression in this matrix. The detection of in vivo expressed genes using the alcohol-soluble and bacteriostatic antibiotic was not possible. Therefore, the promoter selection vector pAK-1 was developed. The new vector system was based on a water-soluble and bactericidal kanamycin resistance gene for selection. In the present study, the vector was established and used for analysis of the gene expression in ground meat. 20 in vivo induced genes that were expressed during growth in ground meat under elevated temperature conditions at 42°C could be detected. Eight genes were associated with energy and nucleotide metabolism, macromolecule synthesis, transport and stress response of the cell. The major part of 12 genes was attributed to a putative or unknown function. Predominantly, identified genes could not be associated with virulence or stress response of the cell. The results of this study, using the in vivo expression technology, showed that genes which are expressed under specific conditions in ground meat could be detected with the help of the chosen method. A first insight into the gene expression of strain EDL933 in ground beef could be acquired. During further investigations a comparison of the fitness of 23 E. coli strains belonging to serogroups O26, O103 and O157 was realized. The isolates originating from foods, patients with HUS and animals were compared in ground beef. The determined differences showed strain-specificity and temperature-specificty. The fitness of the strains varied dependent on the chosen temperatures at 15, 20 and 37 degrees. The analysis of the strains based on ten virulence factors showed that the observed differences could not be attributed to the presence or the number of virulence genes. A correlation between the fitness and the production of a bacteriocin could not be found.
Jahresbericht 2011 / Institut für Lebensmittelwissenschaft und Biotechnologie
(2012)
Impact of process parameters on the sourdough microbiota, selection of suitable starter strains, and description of the novel yeast Cryptococcus thermophilus sp. nov.
(2013) Vogelmann, Stephanie Anke; Hertel, Christian
The microbiota of a ripe sourdough consists of lactic acid bacteria (LAB), especially of the genus Lactobacillus, and yeasts. Their composition is influenced by the interplay of species or strains, the kind of substrate as well as the process parameters temperature, dough yield, redox potential, refreshment time, and number of propagation steps (Hammes and Gänzle, 1997). As taste and quality of sourdough breads are mainly influenced by the fermentation microbiota, intense research has been focused on determination of sourdough associated species and search for new starter cultures. In recent years, economic competition pressure and new consumer demands have led to steady research for new cereal products, especially with health benefit or for people suffering from celiac disease. For these reasons, alternative cereals like oat and barley (both toxic for celiac disease patients) as well as the celiac disease compatible cereals rice and maize, sorghum and millets, the pseudocereals amaranth, quinoa and buckwheat as well as cassava got into the focus of interest. However, information about the microbiota of sourdoughs fermented with buckwheat, amaranth, quinoa, oat or barley is not available except for the following recent studies: a study about the microbiota of amaranth sourdoughs by Sterr et al. (2009), a study about barley sourdough by Zannini et al. (2009), a study about oat sourdoughs by Huettner et al. (2010) and a study about buckwheat and teff sourdoughs by Moroni et al. (2011). The microbiota of sourdoughs from the other mentioned cereals as well as cassava was multiply characterised but not systematically. Fermentation conditions were partly not clearly defined, and identification of species was often based on physiological criteria only, known to be insufficient for the exact classification of LAB. Thus, in this thesis, the influence of the process parameters substrate, temperature, refreshment time, amount of backslopping dough as well as the interplay between the different species or strains were examined and potential starter strains were selected. In Chapter III, the effect of the substrate on the sourdough microbiota was examined and suitable starter cultures for fermentation of non-bread cereals and pseudocereals were selected. Eleven different flours from wheat, rye, oat, barley, millet, rice, maize, amaranth, quinoa, buckwheat and cassava were inoculated with a starter mixture containing numerous LAB and yeasts. Sourdoughs were fermented at 30 °C and refreshed every 24 hours until the microbiota was stable. Species were identified by PCR-DGGE as well as bacteriological culture and RAPD-PCR, followed by 16S/26S rRNA sequence analysis. In these fermentations, the dominant yeast was Saccharomyces cerevisiae; Issatchenkia (I.) orientalis was only competitive in the quinoa and the maize sourdough. No yeasts were found in the buckwheat and the oat sourdough. The dominant LAB species were Lactobacillus (L.) paralimentarius in the pseudocereal sourdoughs, L. fermentum, L. helveticus and L. pontis in the cereal sourdoughs, and L. fermentum, L. plantarum and L. spicheri in the cassava sourdough. Competitive LAB and yeasts were inserted as starters for a further fermentation using new flours from rice, maize, millet and the pseudocereals. After ten days of fermentation, most of the starter strains were still dominant, but L. pontis and L. helveticus could not compete with the other species. It is remarkable that from the numerous starter strains which all were adapted to or isolated from sourdoughs, only a few were competitive in these fermentations; but if, then in most cases in a lot of different flours. In Chapter IV, the effects of the exogenous process parameters substrate, refreshment time, temperature, amount of backslopping dough as well as competing species on the two microbial associations L. sanfranciscensis ? Candida (C.) humilis and L. reuteri ? L. johnsonii ? I. orientalis were examined. Both associations had previously been found to be competitive in sourdough (Kline and Sugihara, 1971a; Nout and Creemers-Molenaar, 1987; Gobbetti et al., 1994a; Garofalo et al., 2008; Böcker et al., 1990; Meroth et al., 2003a). 28 sourdough batches were fermented under defined conditions until the microbiota was stable. Dominant LAB and yeasts were characterized by bacteriological culture, RAPD-PCR and 16S/26S rRNA gene sequence analysis. The process parameters for the association L. sanfranciscensis ? C. humilis could be defined as follows: rye bran, rye flour or wheat flour as substrate, temperatures between 20 and 30 °C, refreshment times of 12 to 24 hours and amounts of backslopping dough from 5 to 20 %. In addition, the association was predominating against all competing lactic acid bacteria and yeasts. The association L. reuteri ? L. johnsonii ? I. orientalis was competitive at temperatures of 35 to 40 °C, refreshment times of 12 to 24 hours and the substrates rye bran, wheat flour and rye flour, but only with sufficient oxygen supply. Cell counts of I. orientalis fell rapidly under the detection limit when using high amounts of doughs (small ratio of surface to volume) and refreshment times of 12 hours. The fermentations depicted in Chapter III and IV give new information about the influence of process parameters on the sourdough microbiota. The studies show that the sourdough microbiota is markedly influenced by the process parameters and kind and quality of substrate. The competitiveness of a single LAB or yeast is strain specific. Interactions between microorganisms also play an important role. However, for the search for suitable starter strains, it would be beneficial to know the reasons, why a single LAB or yeast strain is better adapted to specific process parameters or substrates than others. One of the starter sourdoughs used for fermentation I described in Chapter III was a sourdough made from cassava flour, inoculated with several LAB. No yeast had been inserted, but several yeasts were isolated from the ripe sourdough, which are supposed to originate from the cassava flour. An unknown yeast species constituted 10 % of the isolated yeasts which is described as novel species Cryptococcus thermophilus sp. nov. in Chapter V. This yeast is characterized by budding on small neck-like structures, no fermentative ability, growth at 42 °C and without vitamins, a major ubiquinone of Q-10, as well as the production of green or blue fluorescent substances in the growth medium. It is distinct from related species by the ability to assimilate raffinose and cadaverine, the inability to assimilate soluble starch, xylitol, galactitol, butane-2,3-diol, sodium nitrite and lysine, and the inability to produce starch-like substances. The closest relatives are the yeasts belonging to the Cryptococcus humicola complex.
Sensory and consumer-oriented studies on the effect of fat in different food matrices : a comparison between yoghurt, vanilla custard, Lyon-style and liver sausages
(2013) Tomaschunas, Maja; Busch-Stockfisch, Mechthild
The number of overweight and obese people all over the world increases and overweight and obesity promote the risk for a number of diseases. From the viewpoint of the consumer, it is important to change eating habits and to enhance the extent of physical activity in today’s sedentary lifestyles. From the viewpoint of the industry, the amount of fat in foods may be reduced. However, the degree of liking for a food is often related to its fat content because of the various effects of fat on sensory properties. The effect of fat depends on the food matrix and furthermore, consumers expect the presence of different properties as well as different intensities of certain properties depending on the food. Consequently, a detailed sensory approach is needed to successfully develop foods reduced in fat. Selection of samples for this study based on popularity, on differences in the food matrix as well as on the fact of belonging to the category of meat or rather dairy products, because current data showed that an increased fat intake amongst others arises from an increased consumption of meat and dairy products. Selection resulted in plain stirred yoghurt (0.1 to 12.0% fat) and starch-based vanilla custard (0.1 to 15.8% fat) as well as Lyon-style sausages (3.0 to 25.0% fat) and liver sausages (3.0 to 30.0% fat). Technologies to reduce or rather to substitute fat were adapted to each food matrix, applying innovative approaches. For each food matrix, samples with varying fat content were produced and were evaluated in terms of sensory properties using descriptive analysis, and consumers’ acceptability using hedonic tests. Afterwards, descriptive and hedonic data were statistically correlated. Therefore, the present work on the one hand aimed to apply adapted innovative technologies to reduce or rather to substitute fat in different food matrices and to survey their acceptability. On the other hand, the effects of fat and fat reduction on sensory properties and liking as well as the various drivers of liking and disliking were aimed to be examined and contrasted. Concerning yoghurt, the results showed an increasing effect of fat on attributes creamy (flavor and texture), viscous (appearance and texture) as well as fatty mouth feel. Consumers preferred yoghurts with medium fat (3.5 to 6.0%) and also high fat (12.0%) contents. Liking was driven by attributes sour, aromatic, astringent and partially by descriptors creamy, viscous and fatty mouth feel. Contrariwise, graininess and yellowness as well as too high intensities in attributes creamy, viscous and fatty mouth feel led to rejection. Substituting fat by means of adding whey protein did not enhance liking, but increasing protein did. Finally, the results showed that medium protein contents (4.5%) and high casein-to-whey protein ratios (80/20) could lead to accepted low-fat yoghurts. Regarding vanilla custard, fat increased intensities in attributes thick (appearance and texture), creamy (flavor and texture), sticky and fatty, whereas yellowness, surface shine, jelly, cooked and vanilla flavor, as well as harmonious were decreased by fat. Low to medium fat custards (1.5 to 8.6%) showed best liking scores and attributes vanilla and cooked flavor, harmonious, vegetable fat flavor, sticky, fatty and creamy texture were found to drive liking. On the opposite, custards high in jelly texture and partially too high in thickness, whiteness and creamy flavor were disliked. The addition of a vegetable fat cream led to well accepted medium fat (2.9%) vanilla custards. In Lyon-style sausages, fat exerted an increasing effect on attributes meat flavor, aftertaste meat flavor, greasy and juicy, and a decreasing effect on red color intensity, spicy, spicy aftertaste, raspy throat, coarse and firm. Regarding liver sausages, fat increased scores in attributes greasy, creamy texture, lumpy (appearance and texture), foamy, off-flavor and sweet. Contrariwise, it decreased red color intensity, odor attributes spicy, liver and metallic as well as flavor descriptors spicy, liver, aftertaste, peppery, bitter and metallic and also texture properties firm and furred tongue. For both types of sausage, preferences were mainly found for medium fat contents (10.0 and 17.0%), but consumers partially also liked sausages high in fat (25.0 or rather 30.0%) and low in fat (3.0%). No clear drivers of liking could be detected for the sausages. The results showed that the addition of inulin, citrus fiber and partially rice starch led to a successful imitation of fat or rather to acceptable fat-reduced sausages which are furthermore fiber enriched. The current study gives an interesting overview of the various effects of fat depending on the food matrix. It furthermore gives evidence for the successful development of an assortment of popular fat reduced meat and dairy products.

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