Debaryomyces Hansenii Strains Research Articles

Two Debaryomyces hansenii strains as starter cultures for improving the nutritional and sensory quality of dry-cured pork belly

Dry-cured meat products are gaining attention owing to their distinctive sensory characteristics and health benefits. In this study, two Debaryomyces hansenii strains were investigated for their potential as starter cultures for dry-cured pork belly products. After preliminary screening, these D. hansenii strains, namely, S20 and S26, both exhibiting with excellent aroma-producing capacity in a dry-cured meat model, were selected as single-strain starter cultures. For comparison, a non-inoculated control was also evaluated. In S20- and S26-inoculated pork belly, yeast dominated the microbiota and improved microbiological safety by suppressing Enterobacteriaceae growth. Compared with the non-inoculated control, the inoculated pork belly yielded higher hardness and redness (a*) values. Starter culture inoculation accelerated proteolysis in pork belly, improving the content of total free amino acids (TFFAs) and several essential free amino acids (Thr, Val, Met, Ile, Leu, and Phe) at the end of processing. Moreover, the inoculated samples exhibited higher levels of fat oxidation-derived aldehydes as well as esters, acids, alcohols and other compounds than the non-inoculated control at the end of the 95-day ripening period. Overall, these findings provide new insights into the application of D. hansenii isolated from dry-cured ham to dry-cured pork belly.

Debaryomyces hansenii Strains from Traditional Chinese Dry-Cured Ham as Good Aroma Enhancers in Fermented Sausage

In some countries, yeasts are still not allowed in the production of commercially fermented sausages. Therefore, further research is needed on producing fermented meat products using different strains of yeasts. In this study, two strains of Debaryomyces hansenii (D. hansenii Y61 and Y67) were inoculated in fermented sausages to study their effects as starter cultures. The inoculation of D. hansenii strains affected ripening by decreasing the pH and aw. The sausages inoculated with Y61 and Y67 exhibited decreases in lipid oxidation of 40.70% and 36.04%, respectively, and Enterobacteriaceae counts of 50% and 100%, respectively. The inoculating yeasts Y61 and Y67 increased the lightness (L*) and redness (a*) of fermented sausages. The D. hansenii-inoculated sausages had higher levels of free amino acids and fatty acids, which improved the digestibility, sensory value, and safety of these sausages. Moreover, the total amount of ester compounds increased by 87.14% and 83.31% in the Y61- and Y67-inoculated groups, respectively, which contributed to the aroma. Better sensory attributes were also found in the sausages inoculated with Y61 and Y67 D. hansenii. Native D. hansenii Y61 and Y67 are, therefore, good starter cultures for fermented sausage production. Together, the results provide data supporting future research and the use of yeast-fermented sausages.

Purification, characterization of a novel α-glucosidase from Debaryomyces hansenii strain MCC 0202 and chromatographic separation for high purity isomalto-oligosaccharides production

This study characterized a novel intracellular α-glucosidase, produced by Debaryomyces hansenii. The enzyme was purified through ultra-filtration, ammonium sulfate precipitation, chromatography Q-Sepharose and Sephacryl S-200 column. SDS-PAGE analysis confirmed the molecular weight of the purified protein to be 125 kDa. The enzyme exhibited maximum activity at pH 4.5and 50 °C and stability at pH 4.0 – 6.0 at 30 – 60 °C. The optimum isomalto-oligosaccharides (IMOs) bio-synthesis temperature using 400 g/L maltose was evaluated to be 35 °C with an optimum enzyme unit determined as 4.34 U g−1. The enzyme exhibits maximum maltose utilization of 78%, with or without inorganic salts, and was inhibited by ZnCl2, CoCl2 and CuSO4. Using maltose, the Km and Vmax values were 7.55 µM and 909 µmol/ml/mg respectively. In 3 L bioreactor, 4.34 U of enzyme produced IMOs with 47% purity utilizing 400 g/L maltose at 35 °C for 14 h. Furthermore, low purity IMOs was passed through cationic ion exchange resins to obtain high purity IMOs by separating glucose and maltose. This pioneering study produced high purity IMOs with 89% purity and 60% yield with greater conversion efficiency using a purified enzyme with a higher concentration of maltose and chromatographic method, which were confirmed by HPLC.

Exploring Biocontrol of Unwanted Fungi by Autochthonous Debaryomyces hansenii Strains Isolated from Dry Meat Products.

The exploration of alternatives to the use of chemical preservatives in food is a topic that has attracted great attention. The implementation of regulations associated with the reduction of these elements directly affects the production of cured meat products, with the premise of looking for more “natural” alternatives. From a previously identified collection of 24 strains of Debaryomyces hansenii, isolated from dry meat products of the “Valle de los Pedroches” (Córdoba), a screening was carried out to determine which strains had inhibitory potential against a battery of fungi belonging to the genera Aspergillus, Penicillium, and Candida. After a series of general trials, four strains showing the greatest potential were selected by a streak inhibition assay performed at several concentrations of NaCl. The inhibitory activity of the selected D. hansenii strains was later evaluated by measuring their fungal antagonistic diffusible and volatile compound production following radial inhibition and mouth-to-mouth approaches, respectively. Growth aspects, sporulation, and morphology changes were also considered during these assays. The results support ideas already raised in previous studies, such as the presence of D. hanseniii could imply a reduction of pathogenic fungi in food. Autochthonous yeast strains inhibited not only the mycelial growth, but also sporulation, which strengthens the biocontrol activity of this yeast. Our results show that, under certain conditions, all tested D. hansenii strains were able to alter the growth/development of fungi, being especially evident in the cases of Penicillium expansum and Aspergillus niger. Finally, our research can facilitate the future comparison of results in this area, since we contributed to standardize the methodology described to date, we quantified the number of yeast cells and spores used during the experiments, we homogenized growth conditions for both, yeasts, and molds, and applied an image analyzer software to quantify the growth of the studied microorganisms in solid media.

The Role of Plasma Membrane Pleiotropic Drug Resistance Transporters in the Killer Activity of Debaryomyces hansenii and Wickerhamomyces anomalus Toxins.

The killer strains of Debaryomyces hansenii and Wickerhamomyces anomalus species secrete antimicrobial proteins called killer toxins which are active against selected fungal phytopathogens. In our research, we attempted to investigate the role of plasma membrane pleiotropic drug resistance (PDR) transporters (Pdr5p and Snq2p) in the mechanism of defense against killer toxins. Saccharomyces cerevisiae mutant strains with strengthened or weakened pleiotropic drug resistance due to increased or reduced number of mentioned PDR efflux pumps were tested for killer toxin susceptibility. The present study demonstrates the influence of the Snq2p efflux pump in immunity to W. anomalus BS91 killer toxin. It was also shown that the activity of killer toxins of D. hansenii AII4b, KI2a, MI1a and CBS767 strains is regulated by other transporters than those influencing W. anomalus killer toxin activity. In turn, this might be related to the functioning of the Pdr5p transporter and a complex cross-talk between several regulatory multidrug resistance networks. To the best of our knowledge, this is the first study that reports the involvement of PDR transporters in the cell membrane of susceptible microorganisms in resistance to killer yeasts’ toxins.

Biocontrol activity of Debaryomyces hansenii against blue mold on apple and pear during cold storage

To provide fruit throughout the whole year, maintain quality and reduce spoilage, apples and pears are stored at low temperature. However, the development of rots, caused mainly by Penicillium expansum, cannot be avoided. To prevent fruit losses, biological control has been proposed as a potential alternative. In this work, 16 psychrotrophic, non-pectinolytic Debaryomyces hansenii strains were evaluated in a bioassay for their potential biocontrol against P. expansum rots in apples and pears. Isolates with different degrees of biocontrol effectiveness were further investigated in vitro to elucidate mechanisms of antagonism that may have contributed to biocontrol. No correlation between any of the studied mechanisms and biocontrol activity could be established. One of the isolates, designated F9D, was selected due to its ability to reduce rot incidence in more than 95% in apples and 85% in pears. This strain could be a good candidate for the development of a yeast-based formulation to protect both types of fruit. An ISSR-PCR method was developed for typing the selected strain. This molecular marker could be a useful tool to follow the fate of the strain applied on fruit.

Bioprocesses with Reduced Ecological Footprint by Marine Debaryomyces hansenii Strain for Potential Applications in Circular Economy.

The possibility to perform bioprocesses with reduced ecological footprint to produce natural compounds and catalyzers of industrial interest is pushing the research for salt tolerant microorganisms able to grow on seawater-based media and able to use a wide range of nutrients coming from waste. In this study we focused our attention on a Debaryomyces hansenii marine strain (Mo40). We optimized cultivation in a bioreactor at low pH on seawater-based media containing a mixture of sugars (glucose and xylose) and urea. Under these conditions the strain exhibited high growth rate and biomass yield. In addition, we characterized potential applications of this yeast biomass in food/feed industry. We show that Mo40 can produce a biomass containing 45% proteins and 20% lipids. This strain is also able to degrade phytic acid by a cell-bound phytase activity. These features represent an appealing starting point for obtaining D. hansenii biomass in a cheap and environmentally friendly way, and for potential use as an additive or to replace unsustainable ingredients in the feed or food industries, as this species is included in the QPS EFSA list (Quality Presumption as Safe—European Food Safety Authority).

Debaryomyces hansenii Strains Isolated From Danish Cheese Brines Act as Biocontrol Agents to Inhibit Germination and Growth of Contaminating Molds.

The antagonistic activities of native Debaryomyces hansenii strains isolated from Danish cheese brines were evaluated against contaminating molds in the dairy industry. Determination of chromosome polymorphism by use of pulsed-field gel electrophoresis (PFGE) revealed a huge genetic heterogeneity among the D. hansenii strains, which was reflected in intra-species variation at the phenotypic level. 11 D. hansenii strains were tested for their ability to inhibit germination and growth of contaminating molds, frequently occurring at Danish dairies, i.e., Cladosporium inversicolor, Cladosporium sinuosum, Fusarium avenaceum, Mucor racemosus, and Penicillium roqueforti. Especially the germination of C. inversicolor and P. roqueforti was significantly inhibited by cell-free supernatants of all D. hansenii strains. The underlying factors behind the inhibitory effects of the D. hansenii cell-free supernatants were investigated. Based on dynamic headspace sampling followed by gas chromatography-mass spectrometry (DHS-GC-MS), 71 volatile compounds (VOCs) produced by the D. hansenii strains were identified, including 6 acids, 22 alcohols, 15 aldehydes, 3 benzene derivatives, 8 esters, 3 heterocyclic compounds, 12 ketones, and 2 phenols. Among the 71 identified VOCs, inhibition of germination of C. inversicolor correlated strongly with three VOCs, i.e., 3-methylbutanoic acid, 2-pentanone as well as acetic acid. For P. roqueforti, two VOCs correlated with inhibition of germination, i.e., acetone and 2-phenylethanol, of which the latter also correlated strongly with inhibition of mycelium growth. Low half-maximal inhibitory concentrations (IC50) were especially observed for 3-methylbutanoic acid, i.e., 6.32–9.53 × 10–5 and 2.00–2.67 × 10–4 mol/L for C. inversicolor and P. roqueforti, respectively. For 2-phenylethanol, a well-known quorum sensing molecule, the IC50 was 1.99–7.49 × 10–3 and 1.73–3.45 × 10–3 mol/L for C. inversicolor and P. roqueforti, respectively. For acetic acid, the IC50 was 1.35–2.47 × 10–3 and 1.19–2.80 × 10–3 mol/L for C. inversicolor and P. roqueforti, respectively. Finally, relative weak inhibition was observed for 2-pentanone and acetone. The current study shows that native strains of D. hansenii isolated from Danish brines have antagonistic effects against specific contaminating molds and points to the development of D. hansenii strains as bioprotective cultures, targeting cheese brines and cheese surfaces.

Effect of Debaryomyces hansenii and the antifungal PgAFP protein on Alternaria spp. growth, toxin production, and RHO1 gene expression in a tomato-based medium

Tomato fruit is susceptible to Alternaria spp. spoilage, which poses a health risk due to their mycotoxin production. Biopreservation relies on the use of whole microorganisms or their metabolites to manage spoilage microorganisms including filamentous fungi. However, the use of treatments at fungistatic level might activate intracellular pathways, which can cause an increment in mycotoxin accumulation. The objective of this work was to evaluate the effect of two strains of Debaryomyces hansenii and the antifungal protein PgAFP at 10 and 40 μg/mL. Both growth and production of two of the most common mycotoxins (tenuazonic acid and alternariol monomethyl ether) by Alternaria tenuissima sp.-grp. and Alternaria arborescens sp.-grp. on a tomato-based matrix, were analysed at 12 °C. Additionally, the impact of these biocontrol agents on the stress-related RHO1 gene expression was assessed. All treatments reduced mycotoxin accumulation (from 27 to 92% of inhibition). Their mode of action against Alternaria spp. in tomato seems unrelated to damages to fungal cell wall integrity at the genomic level. Therefore, the two D. hansenii strains (CECT 10352 and CECT 10353) and the antifungal protein PgAFP at 10 μg/mL are suggested as biocontrol strategies in tomato fruit at postharvest stage.

Rapid PCR Method for the Selection of 1,3-Pentadiene Non-Producing Debaryomyces hansenii Yeast Strains.

To prevent microbial growth and its consequences, preservatives such as sorbic acid or its salts, commonly known as sorbates, are added to foods. However, some moulds and yeasts are capable of decarboxylating sorbates and producing 1,3-pentadiene. This is a volatile compound with an unpleasant “petroleum-like “odour, which causes consumer rejection of the contaminated products. In this work, we studied the production of 1,3-pentadiene in 91 strains of the yeast Debaryomyces hansenii, and we found that nearly 96% were able to produce this compound. The sequence of the FDC1Dh gene was analysed showing differences between 1,3-pentadiene producer (P) and non-producer (NP) strains. A specific PCR assay with degenerated primers based on the gene sequence was developed to discern NP and P strains. It was tested on D. hansenii strains and on some physiologically related species frequently isolated from foods, such as D. fabrii, D. subglobosus and Meyerozyma guillermondii. This method could be applied for the selection of NP D. hansenii strains, useful in biotechnological food production and as a biocontrol agent.

Evaluation of the efficacy of Debaryomyces hansenii as protective culture for controlling Listeria monocytogenes in sliced dry-cured ham

The anti-Listeria monocytogenes activity of 30 Debaryomyces hansenii strains was evaluated to be used as protective cultures in sliced dry-cured ham. Some of the yeast strains exhibited inhibitory potential against the L. monocytogenes strains S2, S4-2, S12-1 and S7-2 belonging to the serotypes 1/2a, 1/2b, 1/2c and 4b, respectively, when they were co-inoculated in a dry-cured ham-based medium at 0.95 and 0.93 water activity (aw). The antagonistic activity varied with the yeast and bacterium strains and the aw. The most active strain (D. hansenii 258H) was selected for assessing its activity on slices of dry-cured ham. D. hansenii 258H showed a limited action and even a promotion of the L. monocytogenes growth in the slices of dry-cured ham. In addition, the upregulation of some key virulence genes of L. monocytogenes and the unsuitability appearance on the slices of dry-cured ham inoculated with D. hansenii 258H suggest that such yeast is not a good candidate to be used as protective culture against L. monocytogenes in sliced dry-cured ham. Such effect should be considered even if D. hansenii is not used as a protective culture due to its presence as native microbial population in dry-cured ham.

Inoculation with a terroir selected Debaryomyces hansenii strain changes physico-chemical characteristics of Iberian cured pork loin

Debaryomyces hansenii Lr1, previously isolated from naturally fermented cured pork loin (“lomo embuchado”), was used to inoculate Iberian pork loin under four different conditions. In all cases, specifically inoculated D. hansenii yeasts grew on the surface of the product and affected its physico-chemical and sensory characteristics. Inoculated pork loin kept higher water activity and pH and possessed lower sodium content than control samples at the end of the 90 days ripening period. Moreover, inoculation with Lr1 yeast did not change the general profile of fatty acids but modified the levels of volatile and aroma compounds by decreasing aldehydes and increasing esters and alcohol compounds. Principal Component Analysis (PCA) of the different physico-chemical parameters that were determined showed a clear separation between the samples indicating that they were different. A tendency in consumers acceptance to prefer cured pork loin specifically inoculated with the higher amounts of yeasts used in this study was observed.

Impact of a selected Debaryomyces hansenii strain's inoculation on the quality of Sardinian fermented sausages

Dominant yeast species in Salsiccia Sarda, a traditional fermented sausage produced in Sardinia (Italy), were evaluated through the monitoring three typical production processes. Six different species were identified by molecular techniques, but Debaryomyces (D.) hansenii proved to be dominant. A D. hansenii strain was selected according to its technological features and used in three experimental sausage productions at farm scale with the aim to evaluate its antifungal effect. In all cases, two batches were inoculated with a previously selected autochthonous starter cultures (Lactobacillus plantarum and Staphylococcus xylosus), whereas two batches were left to spontaneous fermentation. D. hansenii was inoculated on the sausages surface by brushing after the sausages drying, by immersion in a yeast suspension after the stuffing, or, alternatively, casings were dipped in a yeast suspension before the dough stuffing. Microbial counts in the sausages core did not appear to be affected by D. hansenii application, while outcomes obtained for casings appeared soundly diversified. Brushing on the sausages surface at the onset of fermentation proved to be the best approach to treat sausages. Yeast inoculation exerted a noteworthy anti-mould effect, independently of the mode of application and, on the other hand, did not affect the overall quality and typical features of the product.

Biocontrol of aflatoxigenic Aspergillus parasiticus by native Debaryomyces hansenii in dry-cured meat products

Dry-cured meat products, such as dry-cured ham or dry-fermented sausages, are characterized by their particular ripening process, where a mould population grows on their surface. Some of these moulds are hazardous to the consumers because of their ability to produce mycotoxins including aflatoxins (AFs). The use of native yeasts could be considered a potential strategy for controlling the presence of AFs in dry-cured meat products. The aim of this work was to evaluate the antagonistic activity of two native Debaryomyces hansenii strains on the relative growth rate and the AFs production in Aspergillus parasiticus. Both D. hansenii strains significantly reduced the growth rates of A. parasiticus when grown in a meat-model system at different water activity (aw) conditions. The presence of D. hansenii strains caused a stimulation of AFs production by A. parasiticus at 0.99 aw. However, at 0.92 aw the yeasts significantly reduced the AFs concentration in the meat-model system. The relative expression levels of the aflR and aflS genes involved in the AFs biosynthetic pathway were also repressed at 0.92 aw in the presence of both D. hansenii strains. These satisfactory results were confirmed in dry-cured ham and dry-fermented sausage slices inoculated with A. parasiticus, since both D. hansenii strains significantly reduced AFs amounts in these matrices. Therefore, both tested D. hansenii strains could be proposed as biocontrol agents within a HACCP framework to minimize the hazard associated with the presence of AFs in dry-cured meat products.

Probiotic effects of marine Debaryomyces hansenii CBS 8339 on innate immune and antioxidant parameters in newborn goats.

Several marine Debaryomyces hansenii strains have shown probiotic effects on aquatic animals, and D. hansenii-derived β-glucans have recently provided immunostimulant effects on goat leukocytes. This study assessed the probiotic effects of live yeast D. hansenii CBS 8339 on newborn goats administered orally, and subsequently challenged in vitro with Escherichia coli. D. hansenii CBS 8339 demonstrated the capacity to survive gastrointestinal tract conditions (bile salts and acid pH tolerance) and adhere to goat intestine. Twelve Saanen × Nubian crossbred newborn goats (2.9 ± 0.47kg) were fed with a controlled diet or D. hansenii (0.7g/kg body weight per day)-supplemented milk for 30days. Blood samples of newborn goats were taken at days 15 and 30, and peripheral blood leukocytes were isolated for bacterial challenge, and immunological and antioxidant analyses. Despite cell viability was higher in leukocytes of goat kids fed with the yeast supplement, protection against E. coli challenge was not significantly affected. On the other hand, at day 15, oral administration of D. hansenii enhanced respiratory burst and catalase activity and increased superoxide dismutase activity after challenge. In contrast, at day 30, administration of the yeast supplement increased peroxidase activity and enhanced nitric oxide production and catalase activity after challenge. Finally, the yeast-supplemented diet upregulated the expression of the receptor genes TLR (2, 4, 6), modulator genes Raf.1, Syk, and Myd88, transcription factor gene AP-1, and cytokine genes IL-1β and TNF-α only at day 15 in leukocytes from unchallenged goat kids. These results demonstrated that a short time (15days) of orally administering the probiotic D. hansenii CBS 8339 to newborn goats stimulated innate immune and antioxidant parameters and the expression of immune-related gene signaling pathways.

Purification, characterization and in vivo biocontrol efficiency of killer toxins from Debaryomyces hansenii strains

Nowadays, the biological control of various yeast and mold pathogens that cause diseases in humans, animals, and plants is an increasing of interest. The discovery of novel agents allows prevention of infectious diseases and post-harvest losses reported every year. In the study, we aimed to investigate the production, purification, and characterization as well as in vivo biocontrol efficiency of killer toxins produced by Debaryomyces hansenii strains TEM8 and TEM17. The molecular mass of the killer toxins was 31.5 kDa and they showed high stability at pHs between 2.5 and 5.5 and up to 37 °C. Their internal amino acid sequences matched the DEHA2G18766g (CAG90862.1) from D. hansenii CBS767, which is similar to Saccharomyces cerevisiae YGR282C BGL2 endo-beta-1,3-glucanase. The yeasts and their purified killer toxins significantly inhibited the growth of plant pathogenic fungi Alternaria brassicicola, Alternaria citri, Aspergillus niger and Rhizopus stolonifer in fruits. The findings of this paper have recommended these yeast strains and their toxins as effective biocontrol agents against fungi that cause post-harvest diseases.

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.

Effect of starter cultures on survival of Listeria monocytogenes in Čajna sausage

The aim of the study was to evaluate the survival of Listeria monocytogenes during the production of Čajna sausage with short maturation time. Sausage batter was inoculated with three different serotypes 4b and serotype 1/2a of L. monocytogenes. Control sausages were without any starter culture added; the second batch was inoculated with strains of Lactobacillus sakei, Staphylococcus carnosus and Staphylococcus xylosus, and the third batch was inoculated with strains of Debaryomyces hansenii, Lactobacillus sakei, Pediococcus acidilactici, Pediococcus pentosaceus, Staphylococcus carnosus and Staphylococcus xylosus. After 18 days of ripening, L. monocytogenes was not detected in any of the sausages, but during this fermentation and drying, the numbers of this pathogen was lower in the sausages inoculated with starter cultures.

Screening of Debaryomyces hansenii Strains for Flavor Production under a Reduced Concentration of Nitrifying Preservatives Used in Meat Products.

A total of 15 Debaryomyces hansenii strains from different food origins were genetically characterized and tested on a culture medium resembling the composition of fermented sausages but different concentrations of nitrifying preservatives. Genetic typing of the D. hansenii strains revealed two levels of discrimination: isolation source or strain specific. Different abilities to proliferate on culture media containing different concentrations of nitrate and nitrite, as sole nitrogen sources and in the presence of amino acids, were observed within D. hansenii strains. Overall metabolism of amino acids and generation of aroma compounds were related to the strain origin of isolation. The best producers of branched aldehydes and ethyl ester compounds were strains isolated from pork sausages. Strains from cheese and llama sausages were good producers of ester compounds and branched alcohols, while vegetable strains produced mainly acid compounds. Nitrate and nitrite reduction affected in different ways the production of volatiles by D. hansenii.

Debaryomyces hansenii up regulates superoxide dismutase gene expression and enhances the immune response and survival in Pacific red snapper (Lutjanus peru) leukocytes after Vibrio parahaemolyticus infection

Application of yeast is increasing to improve welfare and promotes growth in aquaculture. The halotolerant yeast Debaryomyces hansenii is normally a non-pathogenic yeast with probiotic properties and potential source of antioxidant enzymes as superoxide dismutase. Here, first, we characterized the sequence features of MnSOD and icCu/ZnSOD from Pacific red snapper, and second, we evaluated the potential antioxidant immune responses of the marine yeast Debaryomyces hansenii strain CBS004 in leukocytes which were then subjected to Vibrio parahaemolyticus infection. In silico analysis revealed that LpMnSOD consisted of 1186 bp, with an ORF of 678 bp encoding a 225 amino acid protein and LpicCu/ZnSOD consisted of 1090 bp in length with an ORF of 465 bp encoding a 154 amino acid protein. Multiple alignment analyzes revealed many conserved regions and active sites among its orthologs. In vitro assays using head-kidney and spleen leukocytes immunostimulated with D. hansenii and zymosan in response to V. parahaemolyticus infection reveled that D. hansenii strain CBS004 significantly increased transcriptions of MnSOD and icCu/ZnSOD genes. Flow cytometry assay showed that D. hansenii was able to inhibit apoptosis caused by V. parahaemolyticus in the Pacific red snapper leukocytes and enhanced the phagocytic capacity in head-kidney leukocytes. Immunological assays reveled an increased in superoxide dismutase and peroxidase activities, as well as, in nitric oxide production and reactive oxygen species production (respiratory burst) in fish stimulated with D. hansenii. Finally, our results. These results strongly support the idea that marine yeast Debaryomyces hansenii strain CBS004 can stimulate the antioxidant immune mechanism in head-kidney and spleen leukocytes.