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

Abstract

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.

Raman spectroscopic detection of carotenoids in cattle skin

“…S2 †). The obtained absorbance scan does not match with melanin scan 43 and in excellent agreement with the Flieger et al 44 report. Based on these considerations, the study was performed for carotenoid determination in cattle skin using Raman spectroscopy and HPLC.…”

Identification, Quantification and Kinetic Study of Carotenoids and Lipids in Rhodotorula toruloides CBS 14 Cultivated on Wheat Straw Hydrolysate

“…Levels of torulene were below the LOQ. Torulene and torularhodin compounds produced by R. toruloides were identified and quantified using their respective commercial reference standards, unlike some previous studies, where they were quantified using either β-or γ-carotene as the reference standards [20,46].…”

“…Usually, a spectrophotometric technique, based on a standard curve with different concentrations of one individual carotenoid, is used to identify the total carotenoid content. In one study, torulene and torularhodin were quantified based on a β-carotene standard curve and related to the specific pigment extinction coefficients [20]. In another study, torulene was prepared as a crude pigment, isolated and purified from a torulene-producing yeast, and used as a standard for quantification in yeast samples [21].…”

Hyphal morphology and substrate porosity -rather than melanization- drive penetration of black fungi into carbonate substrates

“…A melanized strain was already assayed in vitro, under controlled conditions, for its penetration patterns within different carbonate and silicate rock coupons, showing a high penetration rate (after four months: from few hundreds of microns to some millimetres, depending on the lithology), making it suitable for similar laboratory experiments [16]. Moreover, the development of an efficient toolkit for the genetic modification of K. petricola, including the deletion of genes regulating the production of protective pigments, as melanins, but also carotenoids [44][45][46], has further enforced its suitability as a model organism to understand biodeterioration processes on heritage surfaces and thus address conservation and control strategies.…”