Isolation And Identification Of Sporobolomyces Sp. YB 2013 And Detection Of The Pigments Produced By The Strain

Ballistosporous yeast is a kind of basidiomycetous fungi, which consist of genera Sporobolomymces, Sporidiobolus, Bullera and so on, the main characteristic of them is catapulting ballistospores. They esist widely in nature. Reproductive mode of ballistosporous yeasts mostly is budding when cultivated artificially, meanwhile, they can catapult ballistospore, but the ability of ballistospores production is closely related to the culture environment. The early research of ballistosporous yeasts mainly focused on ecological distribution and classification. Recent years found that ballistosporous yeasts not only can produce the metabolites, such as pigments, esters, sugars and more, but also be applied to vitamin synthesis and pollution control. Therefore, ballistosporous yeasts, which can be applied in the field of food, medicine, environmental protection and so on, get more and more attention of people.In this study, we have isolated a strain YB2013 from mulberry leaf surface from Gongxian county, Yibin city, Sichuan Province, China, who possessed the ability to accumulate pigments. After synthetically analyzed the results of morphological researches and molecular biological datas, the strain was identified Fungi, Dikarya, Basidiomycota, Pucciniomycotina, Agaricostilbomycetes, mitosporic Agaricostilbomycetidae, Sporobolomyces, and named Sporobolomyces sp. YB2013. Besides, we got three pigments after extraction and purification the fermented production of this strain by used thin-layer chromatography and silica gel column chromatography, then we adopted high performance liquid chromatography and mass spectrometry technology to detect those pigments, finally, the results shown that they all belonged to the carotenoid family.The main contents and results of this study are as follow:1. Isolation and identification of Sporobolomyces sp. YB2013In this study, we have isolated a strain YB2013 from mulberry leaf surface in Yibin, Sichuan Province, China, which strain possessed the ability to accumulate pigments. Study shown that the strain YB2013 can form round colonies on Sabouraud’s solid medium, with smooth, sticky surface and neat edge. The pigments mainly distributed in the inside of the yeast cell, meanwhile, there was a kind of special smell when opened the lid of petri dish. The cell of strain YB2013 was oval when observed under the microscope, with obvious budding characteristics, like general yeast (as Saccharomyces cerevisiae).When was cultured upside down, strain YB2013 could form white print on the lid of petri dish, which corresponding with the colonies. The white print was consist of ballistospores, shaped kidney or bean under the microscope. We found that the ability to catapult spore of strain YB2013 would weaken or even vanish in the process of continuous subculture, but strain YB2013 would recover some catapulting spore ability when co-cultured with other bacteria or fungi.The proliferation curve of strain YB2013 contained five stages in liquid culture: the incubation period, the first logarithmic growth phase, the plateau phase, the second logarithmic growth phase and the extinction period. Strain YB2013 with two logarithmic growth phases was different with classic microbial growth curve, speculated that this phenomenon was related to the production of ballistospores. In addition to, we found this strain could form swollen cells, speculated this way may be a mechanisms to stress resistance.According to the morphological characteristics and some related literature, we preliminarily conjectured that strain YB2013 was a ballistosporous yeast. In order to further clarify its taxonomic status, we amplified and sequenced the 18S rDNA, ITS rDNA and 26S rDNA of this strain, respectively. Then analyzed the sequencing results used BLAST on the NCBI and phylogenetic tree. Finally, strain YB2013 was identified and named Sporobolomyces sp. YB2013.2. Extraction, purification and detection of pigments from strain YB2013In this study, we have extracted, purified and preliminary detected the pigments produced by Sporobolomyces sp. YB2013. The study results showed that strain YB2013 predominantly accumulate carotenoid. We cultured strains YB2013 in liquid medium at first, then got bacteria precipitation by centrifugation. Next, absolute ethyl alcohol was applied to extract the bacteria precipitation and concentrated the extracting solution, then double distilled water chloroform mixture (v:v=1:1) was applied to extract the concentrated solution again, by this way, we would get the crude extraction of pigments. The crude extraction was determined by thin layer chromatography (petroleum ether: acetic ether=20:0.1, v/v), results shown that there was almost five components in this crude extraction, three components with relatively high content of them was researched: the yellow pigment a, the light red pigment b and the red pigment c, with the Rfa around 0.67, Rfb around 0.32, Rfc around 0, respectively. Next, the crude extraction of pigments was further purified by column chromatograph, then vacuum concentrated and freeze-dried this three components respectively. TLC was applied to detect again, the result showed that the Rf value of pigment a and (3-carotene standard was similar, and almost each component turned blue after spraying sulfuric acid ethanol chromogenic agent(v:v=1:9), then we preliminarily identified pigments from strain YB2013 belong to carotenoid. Liquid chromatography-mass spectrometry analysis indicated that pigment a was β-carotene, pigment b and pigment c maybe belong to the carotenoid family.