Determination of Antifungal Effect of Edible Coatings Containing Williopsis saturnus var. saturnus Against Yeast and Mold Growth on Kashar Cheese.

One of the most important problems of Kashar cheese producers is mold and yeast spoilage during storage. Williopsis saturnus var. saturnus killer yeast has been reported to have an antagonistic effect on mold and yeast reproduction. In this study, the antifungal effect of a whey protein concentrate (WPC) coating containing W. saturnus was investigated in Kashar cheese. WPC with or without W. saturnus (7 log CFU/g) or W. saturnus without coating was applied on the surface of Kashar cheese samples and stored at 4 °C for 56 days. Microbiological, chemical, physical and sensory properties of cheese samples were assessed. After 56 days of storage, the numbers of lactic acid bacteria were not affected by WPC containing W. saturnus; however, the population of W. saturnus increased by 1 log CFU/g. Application of W. saturnus reduced the growth of other yeasts and molds (P < 0.05). Chemical, physical and sensory properties of all coated cheeses remained unchanged. In a conclusion, use of WPC coatings containing W. saturnus can potentially minimize mold and yeast spoilage of cheese during storage.

Wine aroma evolution throughout alcoholic fermentation sequentially inoculated with non- Saccharomyces/Saccharomyces yeasts.

Nine non-Saccharomyces yeasts belonging to 6 species (Torulaspora delbrueckii, Metschnikowia pulcherrima, Lachancea thermotolerans, Zygosaccharomyces bailii, Williopsis pratensis and Candida zeylanoides) and two mixed inoculum of T. delbrueckii and L. thermotolerans were screened for aroma formation and fermentative behaviour in sequential inoculations with Saccharomyces cerevisiae. The main differences in aromatic composition within wines were detected in the first stages of vinification between S. cerevisiae and non-Saccharomyces species, but not within the latter species. Most of the wines made with non-Saccharomyces in sequential fermentation with S. cerevisiae tended to produce higher ethanal and glycerol and lower volatile acidity than those inoculated only with S. cerevisiae. However, no significant differences were found in alcohol content. The addition of S. cerevisiae tended to standardise the wines and only those made with T. delbrueckii and L. thermotolerans, both alone and together, showed different aromatic profiles. Wines elaborated with non-Saccharomyces yeasts that quickly decreased in tanks showed characteristics similar than those made only with S. cerevisiae. Therefore, sequential inoculation of non-Saccharomyces/Saccharomyces is a useful tool to modulate wine characteristics, but only with species which remain longer in tanks. These findings can be useful to carry out selection processes within these species.

Antifungal, Mechanical, and Physical Properties of Edible Film Containing Williopsis saturnus var. saturnus Antagonistic Yeast.

The molding of food products causing health risks is a main problem in the food industry. In this study, as an alternative solution for preventing mold growth, an antifungal edible film was developed by incorporating Williopsis saturnus var. saturnus (0; 3; 7; and 9 logs CFU/cm2 ) into whey protein concentrate (WPC) based films. Antifungal properties of the films against Penicilium expansum and Aspergillus niger were analyzed using the disc diffusion method. Physical (barrier, solubility, color), mechanical (tensile strength and percent elongation) properties of the films as well as the survival of W. saturnus in the film were assessed during 28 days of storage at 23 °C. According to the results, the viability of W. saturnus (7 and 9 logs CFU/cm2 ) in WPC films stored for 28 days under vacuum or non-vacuum decreased to 36% and 60%, respectively. In addition, films containing W. saturnus decreased the viability of P. expansum and A. niger by 29% and 19%, respectively. Adding yeast did not change the tensile strength (P > 0.05), but significantly decreased % elongation and increased water vapor and oxygen permeability and water solubility (P < 0.05). In conclusion, this study showed that the developed films may be useful for inhibiting mold growth on foods.

A novel non-dairy beverage from durian pulp fermented with selected probiotics and yeast.

This study investigated the effects of sequential inoculation (Seq-I) of Bifidobacterium animalis subsp. lactis or Lactobacillus casei with yeast Williopsis saturnus on durian pulp fermentation. Seq-I of W. saturnus following B. animalis subsp. lactis did not bring about any significant differences compared to the B. animalis subsp. lactis monoculture due to the sharp early death of W. saturnus soon after inoculation. However, Seq-I of W. saturnus significantly enhanced the survival of L. casei and improved the utilization of fructose and glucose compared to L. casei monoculture. In addition, there were significant differences in the metabolism of organic acids especially for lactic acid and succinic acid. Furthermore, Seq-I produced significantly higher levels of volatile compounds including alcohols (ethanol and 2-phenylethyl alcohol) and acetate esters (2-phenylethyl acetate, isoamyl acetate and ethyl acetate), which would positively contribute to the flavour notes. Although the initial volatile sulphur compounds were reduced to trace levels after fermentation, but the durian odour still remained. This study suggests that the use of probiotics and W. saturnus to ferment durian pulp could act as a potential avenue to develop a novel non-dairy durian-based functional beverage to deliver probiotics.

Relationship between salt tolerance and nanoparticle synthesis by Williopsis saturnus NCIM 3298.

This work describes cell associated and extracellular synthesis of nanoparticles by the yeast, Williopsis saturnus. The yeast was able to grow in the absence and presence of sodium chloride (NaCl) and form nanoparticles in a cell associated manner. The content of melanin, a stress-associated pigment was found to be progressively greater in the presence of increasing concentrations of NaCl. With higher quantities of melanin (extracted from yeast cells grown in the presence of 4% of NaCl), smaller sized nanoparticles were obtained. This is the first report on understanding the relationship between halotolerance, production of a stress-related pigment (melanin) and synthesis of nanoparticles with antioxidant properties by using W. saturnus as a model system. The cell free extracts derived from cultures grown in the absence of NaCl were able to mediate extracellular synthesis of gold and silver nanoparticles and the biomolecule mediating nanoparticle synthesis was identified to be a glycolipid. Extracellularly synthesized gold nanoparticles displayed good catalytic activity and rapidly mediated the reduction of 4-nitrophenol to 4-aminophenol.

Enhancement of bifidobacteria survival by Williopsis saturnus var. saturnus in milk.

The viability of three strains of probiotic Bifidobacterium lactis that were inoculated into UHT milk was examined with and without the presence of the yeast, Williopsis saturnus var. saturnus NCYC 22, in polypropylene tubes at 30 °C. The B. lactis viable cell count for strains HN019 and BB-12 remained above 6.0 Log cfu/ml, while strain B94 had 5.7 Log cfu/ml after six weeks of incubation in the presence of the co-inoculated yeast. Incubating the bifidus milk without added yeast under anaerobic condition did not improve the survival of B. lactis HN019, indicating that oxygen removal may not be responsible for W. saturnus NCYC 22's viability enhancing property. The addition of yeast supernatant or non-viable yeast also did not show any stabilising effects, suggesting that physical contact and/or interaction between viable W. saturnus and B. lactis plays an important role in sustaining the viability of the probiotic. W. saturnus NCYC 22 could increase the survival of B. lactis in bifidus milk under ambient temperature regardless of the initial concentration of yeast cells inoculated due to yeast growth. This study demonstrated the viability enhancing effect of viable W. saturnus NCYC 22 on B. lactis HN019, which could help towards extending the shelf-life of dairy beverages containing probiotic bifidobacteria.

Effects of fermentation temperature and aeration on production of natural isoamyl acetate by Williopsis saturnus var. saturnus.

Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Williopsis saturnus var. saturnus is a yeast which can produce isoamyl acetate by esterification of amyl alcohols with acetyl coenzyme A via fermentation. The evaluation of this kind of production as an alternative way to obtain natural banana flavour could be possible, if the levels produced were high enough to make a commercial product. In this study, the effects of temperature (15°C and 25°C) and aeration (aerobic, semiaerobic, and anaerobic) on the production of isoamyl acetate by Williopsis saturnus var. saturnus from sugar beet molasses were examined. According to the results obtained, isoamyl acetate production rate and specific productivity were higher at 25°C than at 15°C and at semiaerobic condition than aerobic and anaerobic conditions. Williopsis saturnus var. saturnus showed a production rate of 0.703 mg (-1) h(-1) and a specific productivity of 0.0297 mg L(-1) cell(-1) h(-1) isoamyl acetate with semiaerobic condition at 25°C. The maximum amount of isoamyl acetate reached with these conditions was 118 mg/L.

Evidence for a transient peroxynitro acid in the reaction catalyzed by nitronate monooxygenase with propionate 3-nitronate.

Nitronate monooxygenase is a flavin-dependent enzyme that catalyzes the denitrification of propionate 3-nitronate (P3N) and other alkyl nitronates. The enzyme was previously known as 2-nitropropane dioxygenase, until its reclassification in 2010 by the IUBMB. Physiologically, the monooxygenase from fungi protects the organism from the environmental occurrence of P3N, which shuts down the Krebs cycle by inactivating succinate dehydrogenase and fumarase. The inhibition of these enzymes yields severe neurological disorders or death. Here, we have used for the first time steady-state and rapid kinetics, viscosity and pH effects, and time-resolved absorbance spectroscopy of the enzyme in turnover with P3N and the substrate analogue ethyl nitronate (EN) to elucidate the mechanism of the reaction. A transient increase in absorbance at ∼300 nm, never reported before, was seen during steady-state turnover of the enzyme with P3N and oxygen, with no concomitant changes between 400 and 600 nm. The transient species was not detected when oxygen was absent. Anaerobic reduction of the enzyme with P3N yielded anionic flavosemiquinone and was fast (e.g., ≥1900 s(-1)). Steady-state kinetics demonstrated that oxygen reacts before the release of the product of P3N oxidation from the enzyme. No pH effects were seen with P3N on kcat/Km, kcat/Koxygen, and kcat; in contrast, with EN, the kcat/Km and kcat decreased with increasing pH defining two plateaus and a pKa ∼ 8.0. Solvent viscosity at the pH optima suggested product release as being partially controlling the overall rate of turnover with the physiological substrate and its analogue. A mechanism that satisfies the kinetic results is proposed.

Yeast ratio is a critical factor for sequential fermentation of papaya wine by Williopsis saturnus and Saccharomyces cerevisiae.

The growth kinetics and fermentation performance of Williopsis saturnus and Saccharomyces cerevisiae at ratios of 10:1, 1:1 and 1:10 (W.:S.) were studied in papaya juice with initial 7-day fermentation by W.saturnus, followed by S. cerevisiae. The growth kinetics of W. saturnus were similar at all ratios, but its maximum cell count decreased as the proportion of S. cerevisiae was increased. Conversely, there was an early death of S. cerevisiae at the ratio of 10:1. Williopsis saturnus was the dominant yeast at 10:1 ratio that produced papaya wine with elevated concentrations of acetate esters. On the other hand, 1:1 and 1:10 ratios allowed the coexistence of both yeasts which enabled the flavour-enhancing potential of W.saturnus as well as the ethyl ester and alcohol-producing abilities of S. cerevisiae. In particular, 1:1 and 1:10 ratios resulted in production of more ethyl esters, alcohols and 2-phenylethyl acetate. However, the persistence of both yeasts at 1:1 and 1:10 ratios led to formation of high levels of acetic acid. The findings suggest that yeast ratio is a critical factor for sequential fermentation of papaya wine by W.saturnus and S. cerevisiae as a strategy to modulate papaya wine flavour.

Fermentation of three varieties of mango juices with a mixture of Saccharomyces cerevisiae and Williopsis saturnus var. mrakii.

This study was carried out to ascertain the behavior and fermentation performance of mixed yeasts in mango juices of three varieties. Saccharomyces cerevisiae MERIT.ferm and Williopsis saturnus var. mrakii NCYC500 at a ratio of 1:1000 were simultaneously inoculated into juices of three mango (Mangifera indica L.) varieties (R2E2, Harum Manis and Nam Doc Mai). Both yeasts grew well in all juices and there was no early growth arrest of either yeast, but there was late death of W. saturnus var. mrakii NCYC500 in the Nam Doc Mai juice. Fructose, glucose and sucrose were consumed to trace levels in all juices. Changes in citric, tartaric, malic, acetic and succinic acids varied with mango varieties. While the changes of major volatiles were similar in all varieties, there were significant varietal differences in the volatile composition of the resultant mango wines. The volatiles, especially most of the terpenes, of the juices decreased drastically and new volatiles such as ß-citronellol were formed. R2E2 wine had more fruity, sweet and creamy notes, and retained more of its original character due to a higher retention of ketones/lactones. Harum Manis wine had the lowest aroma intensity with more green and terpenic notes associated with higher levels of residual terpenes than the other two varieties. Nam Doc Mai wine possessed the highest aroma intensity with winey, yeasty, fruity and floral notes attributed to higher amounts of alcohols, acetate esters and ethyl esters. These findings may help develop different styles of mango wine.

A novel activity for fungal nitronate monooxygenase: detoxification of the metabolic inhibitor propionate-3-nitronate.

Nitronate monooxygenase (NMO; E.C. 1.13.12.16) oxidizes alkyl nitronates to aldehydes and nitrite. Although the biochemistry of the enzyme from fungal sources has been studied extensively, the physiological role is unknown. The ability of NMO to detoxify propionate-3-nitronate was tested by measuring growth of recombinant Escherichia coli containing the gene encoding for the enzyme in either the absence or presence of the nitronate and its conjugate acid 3-nitropropionate. The mixture propionate-3-nitronate/3-nitropropionate is toxic to E. coli cells lacking expression of NMO, but the toxicity is overcome through either induction of the gene for NMO or through addition of exogenous enzyme to the cultures. Both Williopsis saturnus and Neurospora crassa were able to grow in the presence of 0.4mM propionate-3-nitronate and 19.6mM 3-nitropropionate, while a knockout mutant of N. crassa lacking NMO was inhibited by concentrations of propionate-3-nitronate and 3-nitropropionate >0.3 and 600µM, respectively. These results strongly support the conclusion that NMO functions to protect the fungi from the environmental occurrence of the metabolic toxin.

Volatile sulphur compounds and pathways of L-methionine catabolism in Williopsis yeasts.

Volatile sulphur compounds (VSCs) are important to the food industry due to their high potency and presence in many foods. This study assessed for the first time VSC production and pathways of L: -methionine catabolism in yeasts from the genus Williopsis with a view to understanding VSC formation and their potential flavour impact. Five strains of Williopsis saturnus (var. saturnus, var. subsufficiens, var. suavolens, var. sargentensis and var. mrakii) were screened for VSC production in a synthetic medium supplemented with L: -methionine. A diverse range of VSCs were produced including dimethyl disulphide, dimethyl trisulphide, 3-(methylthio)-1-propanal (methional), 3-(methylthio)-1-propanol (methionol), 3-(methylthio)-1-propene, 3-(methylthio)-1-propyl acetate, 3-(methylthio)-1-propanoic acid (methionic acid) and ethyl 3-(methylthio)-1-propanoate, though the production of these VSCs varied between yeast strains. W. saturnus var. saturnus NCYC22 was selected for further studies due to its relatively high VSC production. VSC production was characterised step-wise with yeast strain NCYC22 in coconut cream at different L: -methionine concentrations (0.00-0.20%) and under various inorganic sulphate (0.00-0.20%) and nitrogen (ammonia) supplementation (0.00-0.20%), respectively. Optimal VSC production was obtained with 0.1% of L: -methionine, while supplementation of sulphate had no significant effect. Nitrogen supplementation showed a dramatic inhibitory effect on VSC production. Based on the production of VSCs, the study suggests that the Ehrlich pathway of L: -methionine catabolism is operative in W. saturnus yeasts and can be manipulated by adjusting certain nutrient parameters to control VSC production.

Purification, characterization and gene cloning of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2.

As the killer toxin produced by Williopsis saturnus WC91-2 could kill many sensitive yeast strains, including the pathogenic ones, the extracellular killer toxin in the supernatant of cell culture of the marine yeast strain was purified and characterized. The molecular mass of the purified killer toxin was estimated to be 11.0 kDa according to the data from SDS-PAGE. The purified killer toxin had killing activity, but could not hydrolyze laminarin. The optimal conditions for action of the purified killer toxin against the pathogenic yeast Metschnikowia bicuspidate WCY were the assay medium with 10% NaCl, pH 3-3.5 and temperature 16 °C. The gene encoding the killer toxin from the marine killer yeast WC91-2 was cloned and the ORF of the gene was 378 bp. The deduced protein from the cloned gene encoding the killer toxin had 125 amino acids with calculated molecular weight of 11.6 kDa. It was also found that the N-terminal amino acid sequence of the purified killer toxin had the same corresponding sequence deduced from the cloned killer toxin gene in this marine yeast, confirming that the purified killer toxin was indeed encoded by the cloned gene.

Disruption of the gene encoding ß-1, 3-glucanase in marine-derived Williopsis saturnus WC91-2 enhances its killer toxin activity.

As the ß-1, 3-glucanase produced by the marine-derived Williopsis saturnus WC91-2 could inhibit the activity of the killer toxin produced by the same yeast, the WsEXG1 gene encoding exo-ß-1, 3-glucanase in W. saturnus WC91-2 was disrupted. The disruptant WC91-2-2 only produced a trace amount of ß-1, 3-glucanase but had much higher activity of killer toxin than W. saturnus WC91-2. After the disruption of the WsEXG1 gene, the expression of the gene was significantly decreased from 100% in the cells of W. saturnus WC91-2 to 27% in the cells of the disruptant WC91-2-2 while the expression of the killer toxin gene in W. saturnus WC91-2 and the disruptant WC91-2-2 was almost the same. During 2-l fermentation, the disruptant WC91-2-2 could produce the highest amount of killer toxin (the size of the inhibition zone was 22 ± 0.7 mm) within 36 h when the cell growth reached the middle of the log phase.

Williopsis saturnus yeast killer toxin does not kill Streptococcus pneumoniae.

Streptococcus pneumoniae is an important human bacterial pathogen, and the increase in antibiotic resistance demands the development of new antimicrobial compounds. Several reports have suggested that yeast killer toxins show activity against bacteria and we therefore investigated the activity of K9 killer toxin from the yeast Williopsis saturnus var. mrakii NCYC 500 against S. pneumoniae. However, no inhibition of bacterial growth was observed with concentrated K9 preparations in agar diffusion assays and in liquid culture. Although cell morphology was slightly affected by K9 treatment, no effect on cellular viability was detectable, and K9 had no stimulatory effect on cell lysis induced by ß-lactams or Triton X-100. This indicated that K9 did not contribute to cell wall damage. Moreover, flow cytometry was used as a sensitive assessment of integrity of cells exposed to killer toxin. No significant damage of S. pneumoniae cells was evident, although minor changes in fluorescence suggested that K9 killer toxin may interact with bacterial surface components.

Cloning of exo-ß-1,3-glucanase gene from a marine yeast Williopsis saturnus and its overexpression in Yarrowia lipolytica.

The exo-ß-1,3-glucanase structural gene (WsEXG1 gene, accession number: FJ875997.2) was isolated from both the genomic DNA and cDNA of the marine yeast Williopsis saturnus WC91-2 by inverse PCR and RT-PCR. An open reading frame of 1,254 bp encoding a 417 amino acid protein (isoelectric point: 4.5) with calculated molecular weight of 46.2 kDa was characterized. The promoter of the gene (intronless) was located from -28 to -77 and had one TATA box while its terminator contained the sequence AAGAACAATAAACAA from +1,386 to +1,401. The protein had the Family 5 glycoside hydrolase signature IGLELLNEPL and a fragment with the sequence of NLCGEWSAA, where the Glu-310 (E) was considered to be the catalytic nucleophile. The WsEXG1 gene was overexpressed in Yarrowia lipolytica Po1h and the recombinant WsEXG1 was purified and characterized. The molecular weight of the purified rWsEXG1 was 46.0 kDa. The optimal pH and temperature of the purified rWsEXG1 were 5.0°C and 40°C, respectively. The purified rWsEXG1 had high exo-ß-1,3-glucanase activity. Therefore, the recombinant ß-1,3-glucanase may have highly potential applications in food and pharmaceutical industries.

Profile of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus.

This study investigated the formation and utilization of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus. Time-course papaya juice fermentations were carried out using pure cultures of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 and a mixed culture of the two yeasts at a ratio of 1:1000 (R2:NCYC2251). Changes in S. cerevisiae cell population, Brix, sugar consumption and pH were similar in the mixed culture and in the S. cerevisiae monoculture. There was an early growth arrest of W. saturnus in the mixed culture fermentation. A range of volatile compounds were produced during fermentation including fatty acids, alcohols, aldehydes and esters and some volatile compounds including those initially present in the juice were utilized. The mixed culture fermentation of S. cerevisiae and W. saturnus benefited from the presence of both yeasts, with more esters being produced than the S. cerevisiae monoculture and more alcohols being formed than the W. saturnus monoculture. The study suggests that papaya juice fermentation with a mixed culture of S. cerevisiae and W. saturnus may be able to result in the formation of more complex aroma compounds and higher ethanol level than those using single yeasts.

A new methodology to obtain wine yeast strains overproducing mannoproteins.

Yeast mannoproteins are highly glycosylated proteins that are covalently bound to the beta-1,3-glucan present in the yeast cell wall. Among their outstanding enological properties, yeast mannoproteins contribute to several aspects of wine quality by protecting against protein haze, reducing astringency, retaining aroma compounds and stimulating growth of lactic-acid bacteria. The development of a non-recombinant method to obtain enological yeast strains overproducing mannoproteins would therefore be very useful. Our previous experience on the genetic determinants of the release of these molecules by Saccharomyces cerevisiae has allowed us to propose a new methodology to isolate and characterize wine yeast that overproduce mannoproteins. The described methodology is based on the resistance of the killer 9 toxin produced by Williopsis saturnus, a feature linked to an altered biogenesis of the yeast cell wall.

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation.

AIM: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. METHODS AND RESULTS: For this study, fermentations were performed in sterilized grape must at 18 degrees C. Inoculum level was 5 x 10(6) cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml(-1). It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. CONCLUSION: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.

beta-1,3-glucanase inhibits activity of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2.

The marine-derived Williopsis saturnus WC91-2 was found to produce very high killer toxin activity against the pathogenic yeast Metschnikowia bicuspidata WCY isolated from the diseased crab. It is interesting to observe that the purified beta-1,3-glucanase from W. saturnus WC91-2 had no killer toxin activity but could inhibit activity of the WC91-2 toxin produced by the same yeast. In contrast, the WC91-2 toxin produced had no beta-1,3-glucanase activity. We found that the mechanisms of the inhibition may be that the beta-1,3-glucanase competed for binding to beta-1,3-glucan on the sensitive yeast cell wall with the WC91-2 toxin, causing decrease in the amount of the WC91-2 toxin bound to beta-1,3-glucan on the sensitive yeast cell wall and the activity of the WC91-2 toxin against the sensitive yeast cells. In order to make W. saturnus WC91-2 produce high activity of the WC91-2 toxin against the yeast disease in crab, it is necessary to delete the gene encoding beta-1,3-glucanase.