Klaus Gori

AI-2 signalling is induced by acidic shock in probiotic strains of Lactobacillus spp.

Survival and ability to respond to various environmental stresses such as low pH are important factors for lactobacilli for their function as probiotics. LuxS-mediated quorum sensing mechanism, which is based on the production of universal signal molecule called autoinducer-2 (AI-2), regulates important physiological traits and a variety of adaptive processes in different bacteria. The aim of this study was to investigate the effect of acidic stress on LuxS-mediated quorum sensing (AI-2 signalling) in four probiotic strains of different Lactobacillus species. Initially, the production of AI-2-like molecule was investigated in four strains of Lactobacillus spp. at standard growth conditions using Vibrio harveyi bioluminescence assay. Species variation in AI-2 activity was observed. AI-2 activity started at early-exponential growth phase and increased during the mid-exponential phase concomitant with the reduction of pH, reaching maximum at late exponential phase (L. rhamnosus GG) or at stationary phase (L. salivarius UCC118, L. acidophilus NCFM and L. johnsonii NCC533). Acidic shock experiments were conducted on L. rhamnosus GG and L. acidophilus NCFM after exposure to different acidic shocks (pH 5.0, 4.0 and 3.0) and to pH 6.5 as control, measuring AI-2 activity and transcription of the luxS gene. AI-2 activity increased by lowering the pH in a dose dependent manner and was negatively influenced by acid adaptation. In both species, the luxS gene was repressed after exposure to pH 6.5 as control. However, after acidic shock (pH 4.0) a transient response of luxS gene was observed and the transcription augmented over time, reaching a maximum level and decreased subsequently. Acid adaptation of cells attenuated the transcription of this gene. Based on the observations done in the present study, the luxS gene appears to have a clear role in acidic stress response in probiotic lactobacilli. This might be important in the survival of these bacteria during the passage through the gastrointestinal tract, and further influence the cell-to-cell communication among bacteria in the intestinal microbiota.

Isolation and Identification of the Microbiota of Danish Farmhouse and Industrially Produced Surface-Ripened Cheeses

For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene, respectively. At the end of ripening, the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria (LAB) starter cultures Lactococcus lactis subsp. lactis and Leuconostoc mesenteorides as well as non-starter LAB including different Lactobacillus spp. The cheese from the industrial dairy was almost exclusively dominated by Lb. paracasei. The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp. and/or Brachybacterium spp. Brevibacterium spp. was found to be subdominant compared to other bacteria on the farmhouse cheeses, and no Brevibacterium spp. was found on the cheese from the industrial dairy, even though B. linens was used as surface-ripening culture. Moreover, Gram-negative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses. The surface yeast microbiota consisted primarily of one dominating species for each cheese. For the farmhouse cheeses, the dominant yeast species were Yarrowia lipolytica, Geotrichum spp. and Debaryomyces hansenii, respectively, and for the cheese from the industrial dairy, D. hansenii was the dominant yeast species. Additionally, denaturing gradient gel electrophoresis (DGGE) analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study. Furthermore, DGGE bands corresponding to Vagococcus carniphilus, Psychrobacter spp. and Lb. curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripening.

Expression of the GPD1 and GPP2 orthologues and glycerol retention during growth of Debaryomyces hansenii at high NaCl concentrations

The highly NaCl‐tolerant yeast Debaryomyces hansenii produces and obtains high levels of intracellular glycerol as a compatible solute when grown at high NaCl concentrations. The effect of high NaCl concentrations (4%, 8% and 12% w/v) on the glycerol production and the levels of intra‐ and extracellular glycerol was determined for two D. hansenii strains with different NaCl tolerance and compared to one strain of the moderately NaCl‐tolerant yeast Saccharomyces cerevisiae. Initially, high NaCl tolerance seems to be determined by enhanced glycerol production, due to an increased expression of DhGPD1 and DhGPP2 (AL436338) in D. hansenii and GPD1 and GPP2 in S. cerevisiae; however, the ability to obtain high levels of intracellular glycerol seems to be more important. The two D. hansenii strains had higher levels of intracellular glycerol than the S. cerevisiae strain and were able to obtain high levels of intracellular glycerol, even at very high NaCl concentrations, indicating the presence of, for example, a type of closing channel, as previously described for other yeast species. Copyright

Intracellular pH homeostasis plays a role in the NaCl tolerance of Debaryomyces hansenii strains

The effects of NaCl stress on cell area and intracellular pH (pHi) of individual cells of two Debaryomyces hansenii strains were investigated. Our results show that one of the strains was more NaCl tolerant than the other, as determined by the rate of growth initiation. Whereas NaCl stress caused similar cell shrinkages (30–35%), it caused different pHi changes of the two D. hansenii strains; i.e., in the more NaCl-tolerant strain, pHi homeostasis was maintained, whereas in the less NaCl-tolerant strain, intracellular acidification occurred. Thus, cell shrinkage could not explain the different intracellular acidifications in the two strains. Instead, we introduce the concept of yeasts having an intracellular pKa (pKa,i) value, since permeabilized D. hansenii cells had a very high buffer capacity at a certain pH. Our results demonstrate that the more NaCl-tolerant strain was better able to maintain its pKa,i close to its pHi homeostasis level during NaCl stress. In turn, these findings indicate that the closer a D. hansenii strain can keep its pKa,i to its pHi homeostasis level, the better it may manage NaCl stress. Furthermore, our results suggest that the NaCl-induced effects on pHi were mainly due to hyperosmotic stress and not ionic stress.

Debaryomyces hansenii strains differ in their production of flavor compounds in a cheese‐surface model

Flavor production among 12 strains of Debaryomyces hansenii when grown on a simple cheese model mimicking a cheese surface was investigated by dynamic headspace sampling followed by gas chromatography‐mass spectrometry. The present study confirmed that D. hansenii possess the ability to produce important cheese flavor compounds, primarily branched‐chain aldehydes and alcohols, and thus important for the final cheese flavor. Quantification of representative aldehydes (2‐Methylpropanal, 3‐Methylbutanal) and alcohols (2‐Methyl‐1‐propanol, 3‐Methyl‐1‐butanol, and 3‐Methyl‐3‐buten‐1‐ol) showed that the investigated D. hansenii strains varied significantly with respect to production of these flavor compounds. Contrary to the alcohols (2‐Methyl‐1‐propanol, 3‐Methyl‐1‐butanol, and 3‐Methyl‐3‐buten‐1‐ol), the aldehydes (2‐Methylpropanal, 3‐Methylbutanal) were produced by the D. hansenii strains in concentrations higher than their sensory threshold values, and thus seemed more important than alcohols for cheese flavor. These results show that D. hansenii strains may have potential to be applied as cultures for increasing the nutty/malty flavor of cheese due to their production of aldehydes. However, due to large strain variations, production of flavor compounds has to be taken into consideration for selection of D. hansenii strains as starter cultures for cheese production.

Production of Bread, Cheese and Meat

Historic references to fermentation of dough for baking and fermentation of beer originate from the Sumerians and the Babylonians and, under the Pharaohs in ancient Egypt, the brewing of beer was a trade (Jorgensen 1948). At that time, the fermentation of bread was achieved by using a mixture of yeast and lactic acid bacteria maintained in a dough medium. After each fermentation, a portion of the dough was retained for starting the next batch or a close connection with beer brewing was established so that surplus yeast from breweries was used for production of bread. These same methods are still used in certain regions in Africa and probably other parts of the world where ancient technologies have survived and can be experienced today. In the industrialised part of the world, these methods remained in use, and did not change until late in the eighteenth century when yeast was first propagated for direct use in bread making in the Netherlands by the so-called Dutch method, which had a very low efficiency. As a result of the work of Louis Pasteur and the Danish botanists, Emil Christian Hansen and Alfred Jorgensen and others in the late nineteenth century, the role of oxygen in yeast propagation was realised, the anaerobic condition of fermentation (“life without oxygen”) was understood, Saccharomyces cerevisiae was described and the use of pure cultures was introduced. This was a very significant breakthrough for the industrialised production of baker’s yeast. A similar process improvement followed in 1920, with the introduction of the “fed-batch” process. In this process, sugar is fed incrementally during yeast propagation, avoiding repressions and leading to increased biomass production. It forms the basis of commercial processes used today for manufacturing baker’s yeast, and has developed into a highly centralised industry offering a cheap bulk commodity This is contrary to the historical development of other industrial yeast cultures like brewer’s yeast (Jorgensen 1948). For reviews on the history of baker’s yeast, see Rose and Vijayalakshmi (1993) and Jenson (1998).

Relationship between growth and pH gradients of individual cells of Debaryomyces hansenii as influenced by NaCl and solid substrate

Aims:  To examine the relationship between the growth and pH gradients of Debaryomyces hansenii at a single‐cell level.