Polyamine determination in different strains of the yeast Debaryomyces hansenii by high pressure liquid chromatography

Polyamines (putrescine, spermidine and spermine) participate in many biochemical processes that are indispensable for cell differentiation and proliferation. In the present study, polyamine types were determined in 13 Debaryomyces hansenii strains isolated from different sources by high pressure liquid chromatography. Very low putrescine levels were observed in the strains DhhBCS007, DhfBCS001 and DhfBCS002. Spermidine and spermine were the most prevalent amines detected in all the yeasts, ranging from 7.71 to 18.42 mg/kg of spermidine in the strain L2 and 9.39–18.96 mg/kg for spermine in strain DhhCBS004. D. hansenii strain L2 and CBS004 isolated from citrus fruit and marine water, respectively, were the main polyamine-secreting yeasts. The results will contribute to our ability to select the most appropriate strains for use, as dietary complement (probiotic) in the animal food industry, including aquaculture.     

变性梯度凝胶电泳法初步解析茯砖茶渥堆发酵过程中细菌群落结构

为解析茯砖茶渥堆发酵过程中细菌群落结构和种类,对渥堆过程中不同时间段细菌16S rDNA 的V3可变区进行扩增,对细菌变性梯度凝胶电泳（denaturing gradient gel electrophoresis,DGGE）图谱中条带进行克隆、测序和序列比对。结果表明：黑毛茶在渥堆过程中以渥堆24 h为分界点,前后各自细菌群落结构相似,但前后的差异较大;从16S rDNA 的V3可变区比对结果证明黑毛茶渥堆过程中有诺卡氏菌属、新鞘脂菌属、短波单胞菌属、韦龙氏假单胞菌属、突那梭菌属、克雷伯氏菌属、乳杆菌属及不可培养的ε-变形菌、腐败螺旋菌属、黏球菌属、根瘤菌属和未知分类地位的不可培养细菌6 种。采用DGGE指纹图谱能更全面、更真实地反映黑毛茶渥堆发酵过程中细菌群落的结构和多样性变化。     

Ammonia production and its possible role as a mediator of communication for Debaryomyces hansenii and other cheese-relevant yeast species

Ammonia production by yeasts may contribute to an increase in pH during the ripening of surface-ripened cheeses. The increase in pH has a stimulatory effect on the growth of secondary bacterial flora. Ammonia production of single colonies of Debaryomyces hansenii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Geotrichum candidum was determined on glycerol medium (GM) agar and cheese agar. The ammonia production was found to vary, especially among yeast species, but also within strains of D. hansenii. In addition, variations in ammonia production were found between GM agar and cheese agar. Ammonia production was positively correlated to pH measured around colonies, which suggests ammonia production as an additional technological parameter for selection of secondary starter cultures for cheese ripening. Furthermore, ammonia appeared to act as a signaling molecule in D. hansenii as reported for other yeasts. On GM agar and cheese agar, D. hansenii showed ammonia production oriented toward neighboring colonies when colonies were grown close to other colonies of the same species; however, the time to oriented ammonia production differed among strains and media. In addition, an increase of ammonia production was determined for double colonies compared with single colonies of D. hansenii on GM agar. In general, similar levels of ammonia production were determined for both single and double colonies of D. hansenii on cheese agar.     

Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production

The spoilage yeast Brettanomyces/Dekkera can persist throughout the winemaking process and has the potential to produce off-flavours that affect the sensory quality of wine. The main objective of this study was to select different strains of Brettanomyces bruxellensis isolated from red wines and to compare their volatile phenol production. From a collection of 63 strains, eight strains of B. bruxellensis were selected for volatile phenol production after the application of molecular techniques such as ISS–PCR, PCR–DGGE and REA–PFGE. All strains showed three large chromosomes of similar size with PFGE. However, unique restriction profiles of the chromosomes were visible after NotI digestion that clearly distinguished the strains. All strains were capable of producing large quantities of 4-ethylphenol and 4-ethylguaiacol from p-coumaric acid and ferulic acid, respectively in synthetic media. However, the diversity among strains for volatile phenol production differed between synthetic media and wine with regard to the maximum production levels of 4-ethylphenol and 4-ethylguaiacol. This study illustrated the diversity of B. bruxellensis strains that occur during winemaking.     

Effect of selected strains of Debaryomyces hansenii on the volatile compound production of dry fermented sausage “salchichón”

Different biotypes of Debaryomyces hansenii, characterized by mitochondrial DNA (mtDNA) restriction analysis, were inoculated in dry fermented sausages to evaluate their influence as single starter culture on volatile compound generation throughout the ripening process. Similar evolution of physicochemical parameters and microbial population was observed in both uninoculated and inoculated sausages. The tested biotypes modified the volatile compound profile of sausages specially in esters, branched alcohols and aldehydes. The biotype of D. hansenii with the E mtDNA restriction pattern is the most suitable to be used as starter culture since it produced volatile compounds involved in flavour development of dry-cured meat products such as 3-methylbutanol, 3-methylbutanal and 2-propanone. Moreover, the use of D. hansenii strains with the B, C2 and E mtDNA restriction patterns, as a mixed starter culture, should be also considered to generate low amount of sulphur compounds in dry-cured meat products.     

The Influence of Debaryomyces hansenii, Candida deformans and Candida zeylanoides on the aroma formation of dry-cured “lacón”

The volatile profile of dry-cured “lacón” that has been inoculated with three different yeasts were determined and compared with a non-inoculated dry-cured “lacón”. Yeasts (Debaryomyces hansenii, Candida deformans and Candida zeylanoides) that were used as starter cultures in the present study were selected among yeasts that were isolated from native dry-cured “lacón” at different stages of ripening process. These starters were spread on dry-cured “lacón” surface in order to test their capacity to contribute on the generation of volatile compounds. A total of forty two volatile compounds were detected by dynamic headspace sampling followed by gas chromatography–mass spectrometry analysis. Significant differences (P < 0.001) on the volatile profiles of different batches were found in comparison with non-inoculated samples, showing the highest total area values for the inoculated ones. Esters were the most abundant chemical family in all batches studied except for C. zeylanoides batch, which showed greater amount of hydrocarbons than esters. The second more abundant family was hydrocarbons for control and C. deformans batches (147.6 and 445.24 × 10⁶ area units, respectively), alcohols for D. hansenii (363.77 × 10⁶ area units) and esters for C. zeylanoides (248.33 × 10⁶ area units). However, the aldehyde compound group in control batch samples was found to be significantly higher than in the inoculated ones (P < 0.001). Among inoculated batches, D. hansenii batch showed the lowest hexanal content (14.42 × 10⁶ area units) in comparison with non-inoculated batch (105.99 × 10⁶ area units). Among all batches studied, D. hansenii batch presented the highest area values for esters, alcohols, linear hydrocarbons, ketones, acids and furans; control batch for aldehydes and C. zeylanoides batch for branched hydrocarbons. Therefore, the study showed that every yeast strain produced a specific volatile profile which was also different from that of the control dry-cured “lacón”.     

Microbial succession of Debaryomyces hansenii strains during the production of Danish surfaced-ripened cheeses

Surface-ripened cheeses of the Danbo type were analyzed for the presence of yeasts with special emphasis on Debaryomyces hansenii. Samples were taken from pasteurized milk, brine, and inoculation slurries and from cheese surfaces during ripening at a Danish dairy. D. hansenii was found to be the dominant yeast species throughout the ripening period, whereas other yeast species such as Trichosporon spp., Rhodotorula spp., and Candida spp. were found in minor concentrations during early stages of cheese ripening. Mitochondrial DNA RFLP was used to show that several strains of D. hansenii were present from the onset of ripening. Thereafter, a microbial succession among the strains took place during the ripening. After 3 d of ripening, only one strain was found. This particular strain was found to be dominant in 16 additional batches of surface-ripened cheeses. We investigated the cause of the observed microbial succession by determining the variation in strains with regard to their ability to grow on lactate and at different pH and NaCl concentrations. The strains were shown to vary in their ability to grow on lactate. In a full factorial design at three levels with factor levels close to the actual levels on the cheese surface, differences in pH and NaCl tolerances were observed. The dominant strain was found to be better adapted than other strains to the environmental conditions existing in surface-ripened cheeses during production [e.g., lactate as the main carbon source, pH 5.5 to 6.0 and NaCl concentrations of 7 to 10% (wt/vol)].     

Comparison of volatile compounds produced in model cheese medium deacidified by Debaryomyces hansenii or Kluyveromyces marxianus

The aroma of a deacidified cheese medium is the result of the overall perception of a large number of molecules belonging to different classes. The volatile compound composition of (60%) cheese medium (pH 5.8) deacidified by Debaryomyces hansenii (DCM(Dh)) was compared with the one deacidified by Kluyveromyces marxianus (DCM(Km)). It was determined by dynamic headspace extraction, followed by gas chromatography separation and quantification as well as by mass spectrometry identification. Whatever the media tested, a first class of volatile compounds can be represented by the ones not produced by any of the yeasts, but some of them are affected by K. marxianus or by D. hansenii. A second class of volatile compounds can be represented by the ones produced by K. marxianus, which were essentially esters. Their concentrations were generally higher than their thresholds, explaining the DCM(Km) global fruity odor. A third class can be represented by the ones generated by D. hansenii, which were essentially methyl ketones with fruity, floral (rose), moldy, cheesy, or wine odor plus 2-phenylethanol with a faded-rose odor. The impact of methyl ketones on the DCMDh global flavor was lower than the impact of 2-phenylethanol and even negligible. Therefore, the global faded-rose odor of D. hansenii DCM can be explained by a high concentration of 2-phenylethanol.     

Comparison of molecular and metabolomic methods as characterization tools of Debaryomyces hansenii cheese isolates

Debaryomyces hansenii is one of the yeast species most frequently isolated from cheese and salty foods, however little is known about the phenotypic and molecular variability of its strains. In order to explore the possibilities of a large study on its biodiversity, some D. hansenii strains were selectively isolated from pecorino cheese sampled in ten different Italian regions. All isolates were identified as D. hansenii on the basis of conventional and molecular taxonomic analysis. The D1/D2 domain sequences of the 26S-rDNA did not show any variation, confirming the extreme homogeneity of this species. PCR-duplex-RAPD banding patterns analyzed with PCoA showed interesting clustering related to the geographic areas of isolation, although some overlapping between strains derived from different districts could be observed. A FTIR (Fourier Transform Infrared Spectroscopy) metabolomic fingerprint produced groupings weakly related to those observed with RAPD and less associated with the isolation locales. The discriminatory power of metabolomic fingerprint was able to discriminate strains otherwise considered identical. This preliminary study showed that, in spite of the homogeneity at the 26S-rDNA level, the D. hansenii strains exhibit high molecular and metabolomic variability somehow linked to the places of isolation. Further studies will be necessary to better investigate on the link between terroir and strain variability, as well as on the relation between genotypic and metabolomic fingerprints.