Thermoresistance in black yeasts is associated with halosensitivity and HPP tolerance, but not with UV tolerance or sanitizer tolerance

Black yeasts can survive extreme conditions in food production owing to their polyextremotolerant character. However, significant strain-to-strain variation in black yeast thermoresistance has been observed. In this study, we assessed the variability in tolerance to nonthermal interventions among a collection of food-related black yeast strains. Variation in tolerance to UV light treatment, high pressure processing, sanitizers, and osmotic pressure was observed within each species. The two strains previously shown to possess high thermotolerance, Exophiala phaeomuriformis FSL-E2-0572 and Exophiala dermatitidis YB-734, were also the most HPP tolerant, but were the least halotolerant. Meanwhile, Aureobasidium pullulans FSL-E2-0290 was the most UV and sanitizer tolerant, but had been shown to have relatively low thermoresistance. Fisher’s exact tests showed that thermoresistance in black yeasts was associated with HPP tolerance and inversely with halotolerance, but no association was found with UV tolerance or sanitizer tolerance. Collectively, the relative stress tolerance among strains varied across interventions. Given this variation, a broad range of different food products are susceptible to black yeast spoilage. Additionally, different strains should be selected in challenge studies specific to the intervention. (1312/2000 characters)

Growth and division mode plasticity by cell density in marine-derived black yeasts

The diversity and ecological contribution of the fungus kingdom in the marine environment remain under-studied. A recent survey in the Atlantic (Woods Hole, MA, USA) brought to light the diversity and unique biological features of marine fungi. The study revealed that black yeast species undergo an unconventional cell division cycle, which has not been documented in conventional model yeast species such as Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast). The prevalence of this unusual property is unknown. Inspired by the findings in Woods Hole, I collected and identified >50 marine fungi species across 40 genera from the ocean surface, sediment, and macroalgal surface in the Pacific (Sugashima, Toba, Japan). The Sugashima collection largely did not overlap with the Woods Hole collection and included several unidentifiable species, further illustrating the diversity of marine fungi. Three black yeast species were isolated, two of which were commonly found in Woods Hole (Aureobasidium pullulans, Hortaea werneckii). Surprisingly, I observed that their cell division mode was dependent on cell density, and the previously reported unconventional division mode was reproduced only at a certain cell density. For all three black yeast species, cells underwent filamentous growth with septations at low cell density and immediately formed buds at high cell density. At intermediate cell density, two black yeasts showed rod cells undergoing septation at the cell equator, in a manner similar to S. pombe. In contrast, all eight budding yeast species showed a consistent division pattern regardless of cell density. In five budding yeast species, the mother cell formed a single bud at a time at an apparently random site, similar to S. cerevisiae. The other three budding yeast species possessed a fixed budding site. This study illustrates the plastic nature of the growth/division mode of marine-derived black yeast.

Intermediate thermoresistance in black yeast asexual cells variably increases with culture age, promoting survival and spoilage in thermally processed shelf-stable foods

Black yeasts are a functional group that has caused spoilage in cold-filled and hot-filled beverages as well as other water activity (a w )-controlled food products. We established quantitative thermoresistance parameters for the inactivation of 12 Aureobasidium and Exophiala isolates through isothermal experiments and a challenge study. Culture age (2-day vs. 28-day) variably affected the thermoresisitance among the black yeast strains. Variation in thermoresistance exists within each genus, but the two most resistant strains were the Exophiala isolates. The two most heat resistant isolates were E. phaeomuriformis FSL-E2-0572 with a D 60 -value of 7.69±0.63 min in 28-day culture and E. dermatitidis YB-734 with a D 60 -value of 16.32±2.13 min in 28-day culture. While these thermoresistance levels were, in some cases, greater than those for conidia and vegetative cells from other common food spoilage fungi, they were much more sensitive than the ascospores of heat resistant molds most associated with spoilage of hot-filled products. However, given that black yeasts have caused spoilage in hot-filled products, we hypothesized that this intermediate degree of thermoresistance may support survival following introduction during active cooling before package seals have formed. A challenge study was performed in an acidic (apple cider) and a w -controlled (maple syrup) product to evaluate survival. When apple cider was hot-filled at 82C, black yeast counts were reduced by 4.1-log CFU/ml 24 h after the heat treatment, but the survivors increased up to 6.7-log CFU/ml after two weeks. In comparison, the counts were below the detection limit after both 24 h and 14 days of shelf-life in both products when filled at their boiling points. This suggests that ensuring water microbial quality in cooling tunnels and nozzle sanitation may be essential in mitigating the introduction of these fungi.

Impact of black yeasts on the durability of polyurethane foam in tropical conditions in Vietnam

Polyurethane (PU) is a material that is widely used in industries. However, tropical climatic conditions in Vietnam often make the durability of PU lower than temperate climatic conditions. One of the causes of this problem is the biodegradation of microorganisms. In this study, biological characteristics of black yeast on PU is evaluated, thereby, initially assessing the impact of black yeast on the durability of PU in tropical conditions. Ten strains of black yeast were isolated from used PU foam samples in Vietnam. These black yeast strains were tested for enzyme activity including: urease activity, protease activity, polyurethanase activity. There were two strains selected to identify based on sequence analysis of the ITS1/ITS2 region, they belong to genus Aureobasidium. VN1Y3 was strain selected for biodegradability testing of PU foam samples. After 28 days of testing, the PU foam samples that have been infected with black yeasts have a change compared to the control in terms of color, surface, tensile strength, elongation. Research results have showed the impact of black yeast on the durability of PU.

Unconventional cell division cycles from marine-derived yeasts

Fungi have been found in every marine habitat that has been explored, however, the diversity and functions of fungi in the ocean are poorly understood. In this study, fungi were cultured from the marine environment in the vicinity of Woods Hole, MA, USA including from plankton, sponge and coral. Our sampling resulted in 36 unique species across 20 genera. We observed many isolates by time-lapse differential interference contrast (DIC) microscopy and analyzed modes of growth and division. Several black yeasts displayed highly unconventional cell division cycles compared to those of traditional model yeast systems. Black yeasts have been found in habitats inhospitable to other life and are known for halotolerance, virulence, and stress-resistance. We find that this group of yeasts also shows remarkable plasticity in terms of cell size control, modes of cell division, and cell polarity. Unexpected behaviors include division through a combination of fission and budding, production of multiple simultaneous buds, and cell division by sequential orthogonal septations. These marine-derived yeasts reveal alternative mechanisms for cell division cycles that seem likely to expand the repertoire of rules established from classic model system yeasts.

Development of an Improved Carotenoid Extraction Method to Characterize the Carotenoid Composition under Oxidative Stress and Cold Temperature in the Rock Inhabiting Fungus Knufia petricola A95

Black yeasts are a highly specified group of fungi, which are characterized by a high resistance against stress factors. There are several factors enabling the cells to survive harsh environmental conditions. One aspect is the pigmentation, the melanin black yeasts often display a highly diverse carotenoid spectrum. Determination and characterization of carotenoids depend on an efficient extraction and separation, especially for black yeast, which is characterized by thick cell walls. Therefore, specific protocols are needed to ensure reliable analyses regarding stress responses in these fungi. Here we present both. First, we present a method to extract and analyze carotenoids and secondly we present the unusual carotenoid composition of the black yeast Knufia petricola A95. Mechanical treatment combined with an acetonitrile extraction gave us very good extraction rates with a high reproducibility. The presented extraction and elution protocol separates the main carotenoids (7) in K. petricola A95 and can be extended for the detection of additional carotenoids in other species. K. petricola A95 displays an unusual carotenoid composition, with mainly didehydrolycopene, torulene, and lycopene. The pigment composition varied in dependency to oxidative stress but remained relatively constant if the cells were cultivated under low temperature. Future experiments have to be carried out to determine if didehydrolycopene functions as a protective agent itself or if it serves as a precursor for antioxidative pigments like torulene and torularhodin, which could be produced after induction under stress conditions. Black yeasts are a promising source for carotenoid production and other substances. To unravel the potential of these fungi, new methods and studies are needed. The established protocol allows the determination of carotenoid composition in black yeasts.

Development of an Improved Carotenoid Extraction Method and Characterisation of the Carotenoid Composition under Oxidative and Cold Stress in the Rock Inhabiting Fungus Knufia Petricola A95

Black yeasts are a highly specified group of fungi, which are characterized by a high resistance against stress factors. There are several factors enabling the cells to survive harsh environmental conditions. One aspect is the pigmentation, besides the melanin black yeasts often display a highly diverse carotenoid spectrum. Determination and characterization of carotenoids depend on an efficient extraction and separation, therefore especially for black yeast, characterized by thick cell walls specific protocols are needed to ensure analyses regarding stress responses in these fungi. Here we present both, a method to extract and analyze carotenoids and the unusual carotenoid composition of the black yeast Knufia petriola A95. Mechanical treatment combined with an acetonitrile extraction gave us very good extraction rates with a high reproducibility. The presented extraction and elution protocol allows the separation of the main carotenoids (7) in K. petricola A95 and should be suitable for the detection of additional carotenoids in other species. K. petricola A95 displays an unusual carotenoid composition, with mainly didehydrolycopene, torulene and lycopene. The pigment composition varied in dependency to oxidative stress but remained relatively constant if the cells were cultivated under low temperature. Black yeasts are a promising source for carotenoid production and other substances. To unravel the potential of these fungi new methods and studies are needed. The established protocol allows the determination carotenoid composition in black yeasts. Oxidative stress results in an adaptation in pigment composition in K. petricola A95. Future experiments have to be carried out to determine if didehydrolycopene functions as a protective agent itself or if it serves as a precursor for antioxidative pigments like torulene and torularhodin, which could be produced after induction under stress conditions.