Isolation, Characterization, and Identification of Yeasts Associated with Foods from Assiut City, Egypt

The objective of this work was to isolate, characterize physiologically, and identify yeasts associated with foods from Assiut city, Egypt. Fifty-two colonies of yeasts associated with orange, mandarin, tomato, squash, sobia drink, mango juice, sugarcane juice, yogurt and buttermilk samples, collected from Assiut City, Egypt, were isolated. Out of which, Eleven isolates were selected randomly and subjected to morphological, biochemical studies and molecular identification techniques employing sequence of internal transcribed spacer (ITS) regions and partial D1/D2 large-subunit domains of the 26S ribosomal RNA. Identified yeasts were belonged to six genera and species; four species belonged to ascomycetes: Debaryomyces hansenii (five isolates), Saccharomyces cerevisiae (one isolate), Candida tropicalis (one isolate), and Pichia (one isolate). In addition, two yeasts species belonged to basidiomyces: Rhodotorula mucilaginosa (two isolates), and Trichosporon dulcitum (one isolate). In spite of low frequency of yeasts isolates on the tested food, mango juice and buttermilk showed the higher sources for incidences during this study.


INTRODUCTION
Yeasts are unicellular eukaryotes that belong to the Kingdom of Fungi and play various roles in affecting the quality and safety of food products. They are ubiquitous, and commonly spoilage fruits, vegetables and other plant materials, in addition to, an association with soil and insects [1]. Decaying fruits are an important microhabitat for several yeast species [2]. Colonization of fast growing, fermentative, low assimilative profiles of apiculate yeasts: (Kloeckera, Hanseniaspora, and Saccharomycodes) initiate severe deterioration of fruits [3]. Oranges and mangoes usually rotted by spoilage of a wide variety of fermentative or weakly non-fermenting yeasts [4,5]. Basidiomycetous genera such as Cryptococcus and Sporobolomyces yeasts were dominated on leaves of mango trees [6]. In addition, ascomycetous yeasts associated the wounded parts of the phylloplane [7].
Molecular identification techniques employing sequencing the internal transcribed spacer (ITS) regions and partial D1/D2 large-subunit domains of the 26S ribosomal RNA (rRNA) have become the most frequently and convenient method for identification of yeasts isolates. This study aimed to isolate yeasts associated with milk products, fruits, and juices from Assiut city, Egypt. The yeasts were identified morphologically, physiologically as well as by molecular techniques.

Samples and Isolation
Three samples from each food source; orange, mandarin, tomato, squash, sobia drink, mango juice, sugarcane juice, yogurt, and buttermilk were collected from the local markets in Assiut city, Egypt. The isolation was achieved according to Lodder and Kreger [14], and Kurtzman et al. [15], where the samples were settled at room temperature (25~30ºC) for 48 hours (h), then used for isolation. Liquid samples were direct spread and streaked on yeast peptone dextrose agar (YPD), as well of a 1/10 w/v of solid samples/ sterile water were homogenized and used for direct plating techniques during this study. A medium composed of 10 g/L of yeast extract, 20 g/L of peptone, 20 g/L of dextrose, 20 g/L of agar and added ampicillin (100 mg/L), and chloramphenicol (100 mg/L) to prevent bacterial contamination, and then the plates were incubated at 28±2ºC for 10 days. Purification of yeast colonies were achieved by streaked methods. The cell/colony morphologies of the purified yeasts isolates were investigated and monitored under microscope. Yeast cultures were maintained on 2% YPD slants at 4ºC for short period storage. In addition, equal volumes of propagated yeast cultures and 80% glycerol were mixed well and stored at 80ºC for long time preservation.

Identification of Yeast Strains
Selected yeast isolates were identified based on their morphological, biochemical properties according to Kurtzman et al. [15]; Walt and Yarrow [16] While, molecular techniques according to Nisiotou et al. [17].

Morphological characteristics
The strains were first checked for their morphological aspects and growth patterns on solid and liquid media. Characteristic features of textures (mucoid, fluid or viscous, butyrous); elevation (flat or raised); color (yellow, orange and red); surface (glistening or dull, smooth, rough, and sectored) and margin (entire, undulating, lobed, and filaments) were investigated. The cells of a young actively growing culture from 2~3 days at 25°C were stained by lacto phenol-cotton blue and investigated microscopically to determine the shape of cells, budding (Mono, Bi, and multipolar) or fission and conidial formation. Moreover, the formation of compact, coherent, flocculent, or mucoid sediment, a ring, floating islets or a pellicle of yeasts was examined. Additionally, formation of germ tubes was checked using 24 h cultivated yeasts and incubated at 37°C for 2~3 h in blood serum then investigated under light microscope according to Kurtzman et al. [15].

Physiological and biochemical characteristics
The ability of eleven yeasts isolates to utilize and grow aerobically on carbon and nitrogen as a sole source of energy was studied. Several carbon sources (D-glucose, D-galactose, lactose, maltose, sucrose, raffinose, soluble starch, Dribose, L-rhamnose, D-xylose, glycerol, Dmannitol, ethanol, and methanol) used in this study. While the nitrogen sources used were nitrate, nitrite, and L-lysine. Assimilation tests were achieved by replica plate method, where a set of plates contained different carbon or nitrogen source in a carbon or nitrogen basal agar medium were inoculated by yeasts isolates. The growth of colonies of negative control plates (without carbon or nitrogen sources) was compared with plates supplemented with carbon or nitrogen sources after 24~48 h of incubation [15].
On the other hand, fermentation abilities of yeast isolate to ferment 2% sugar solutions of (glucose, lactose, maltose, raffinose, galactose, sucrose, and xylose) and 4% of raffinose was tested. Pure yeast isolates were cultivated on YPD liquid medium for 48 h and then the cell pellets were collected by centrifugation. Fermentation was started by addition of sugar solutions to cell pellets and resuspended prior to insert Durham tubes (inverted without any air in aseptic condition) to collect any gas produced during fermentation. Thereafter, the tubes were incubated at 25°C for up to 28 days. The fermentation abilities were inspected at frequent intervals for accumulated gas in the Durham tubes, as well as changes in indicator (phenol red) color were investigated according to Kurtzman et al. [15].
Moreover, other physiological characteristics were performed to examine the osmotic pressure of yeast isolates such as growth abilities on 50, 60% w/v glucose, and 10% NaCl plus 5% glucose. In addition to, the ability of growth at 37°C, urea hydrolysis, starch formation, tolerance to 1% acetic acid. Furthermore, acid production from glucose of Custer's chalk medium at 25°C after incubation of two weeks was carried out according to the methods of Kurtzman and Fell [18].

Molecular identification
Purified yeast isolates were identified based on sequences of rRNA gene of large subunit (LSU) according to Nisiotou et al. [17] as the following: Genomic DNA was extracted from overnight grown yeast cells in broth YPD medium at 28°C and 150 rpm. Yeasts cells pellets were collected by centrifugation and resuspended in a lysis buffer (2% Triton X-100, 1% sodium dodecyl sulfate, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, and 1 mM EDTA) with glass beads and mixture of phenol, chloroform, isoamyl alcohol (25:24:1). Thereafter, vigorously vortex the mixture to lysis yeast cell walls and DNA precipitated by chilled absolute alcohol as described previously [19].

Morphological Characterization
The morphological characters of selected yeast isolates were summarized in Table 1.

Assimilation tests
All selected yeast isolates assimilated glucose and maltose and were unable to assimilate methanol as a sole carbon source. Only isolate Y.5 well-assimilated citrate and xylose as reflected in the results shown in Table 2. Isolates Y.5 and Y.16 negatively and poorly assimilated sucrose, respectively, while the other isolates showed a well assimilation. Lactose efficiently assimilated by Y.7~10, 13 and 16 whereas the rest was not. For starch, only Y.5 strongly assimilated it from among the eleven-tested yeasts isolates while, Y.7~10 and 13 were weakly dissociation abilities (Table 2).

Fermentation tests
Seven sugars (glucose, lactose, maltose, raffinose, galactose, sucrose, D-xylose) were used during study fermentation abilities of yeasts isolates. Isolate Y.2 was able to ferment all sugars except xylose and lactose, while Y. 5 and 6 fermented only glucose. On the other hand, Y.14, 15 and 16 were unable to ferment any sugar. Yeasts isolates Y.7~10 and 13 were efficient to ferment glucose and less for galactose, while unable to ferment the rest of sugars ( Table 3). The results of additional physiological characteristics were summarized in Table 4.

Molecular Identification
Eleven yeasts isolates were identified, based on molecular identification techniques using amplified fragments of ITS and D1/D2 domains of large subunit, which belonged to six genus: D. hansenii (five isolates), S. cerevisiae (one isolate), C. tropicalis (one isolate), Pichia kudriavzevii (one isolate), R. mucilaginosa (two isolates), and Trichosporon dulcitum (one isolate). In the phylogenetic tree, all strains  (Fig. 1). Their accession numbers were coordinated with identified names and listed in Table 5 and Fig. 1.

DISCUSSION
Isolation of yeasts from natural resources is the most successful technique to obtain yeast isolates that have abilities to utilize and ferment various exogenous compounds [21]. Isolation of yeast species associated with different food samples collected from Assiut city, Egypt, was investigated. The species S. cerevisiae, C. tropicalis, P. kudriavzevii, D. hansenii, R. mucilaginosa, T. dulcitum were isolated. The results showed that all these species were found in lower frequency. This could be due to that normal yeast flora on fresh, undamaged fruits are generally low, presence of preservatives for juices, and presence of lactic acid bacteria for milk products. D. hansenii was associated only with fruit samples and mango juice, whereas R. mucilaginosa was isolated only from mango juice.       The complex identification yeasts by a conventional methodology of biochemical that requires 60~90 tests for correct species identification [22]. Nonetheless, these methodology are necessary for detect any special physiological character. Furthermore, due to high accuracy of identification by molecular techniques, it was used in parallel with biochemical tests for the identification of yeast species. The obtained results from morphological characteristics of the isolated colonies coupled with the biochemical tests were highly significant in yeast identification and correlated with molecular technique identification.
D. hansenii showed the most frequented strain where five isolates of D. hansenii isolated from different sources (fruit samples and mango juice). It has an ability to assimilate all tested sources of carbon except methanol, in addition to, ferment glucose and galactose. Galactose is a 'non-conventional' nutrient for yeasts, which however can be used as a sole carbon source when glucose is absent from the medium. Thus, the ability of the yeast cells to assimilate galactose indicated the expression of the GAL genes [23]. As a consequent of that, D. hansenii may has GAL genes. It has caused spoilage of fruit, juice, marzipan, and canned figs [24,25]. It has been isolated from a variety of fruit [24], soft drinks [26], raw tomatoes [27], sugarcane [28] and been reported in high numbers in yoghurt [29]. It proved to be genetically and biochemically interesting yeasts with considerable biotechnological promise [30].
R. mucilaginosa is the second most frequented strain. The results in consistent with Haridy [31], where Rhodotorula spp. are common strains isolated from dairy products. The presence of R. mucilaginosa in the mango juice signified cross-contamination during juice preparation because Rhodotorula species have been reported as a saprophyte skin, vaginal and respiratory specimens [32]. Contamination may also occur through contaminated containers because all members of the Rhodotorula genus, which has affinity to synthetic materials in general [33]. The incidence of R. mucilaginosa in the mango juice is very dangerous because it has been reported to cause Onychomycosis, which is a dermatological problem in immunocompetent patient [34].
P. kudriavzevii was associated only with yogurt during our study. It initially classified as a strain of Issatchenkia orientalis, but Kreger-van Rij [35] noted that this strain (CBS 5459) differed from the others in being sorbose-positive and citratenegative. An isolated strain has an ability to assimilate glucose, galactose, maltose, sucrose, ethanol and glycerol, in addition to, ferment only glucose. P. kudriavzevii gained much attention recently in the biotechnology. Recombinant P. kudriavzevii VTT C-79090T had been able to produce up to 171 g L −1 of D-xylonate from 171 g L −1 D-xylose at low pH [36]. Yuangsaard et al. [37] reported that newly isolated thermotolerant Pichia kudriavzevii had been produced 7.86% (w/v) ethanol from cassava starch hydrolysate, which adjusted to 18% glucose at 40°C within 24 h. Moreover, the ability of P. kudriavzevii to utilize glycerol makes it an excellent source for biodiesel production. Recently, Thiru et al. [38] have carried out a detailed investigation on the production of biodiesel from crude glycerol using Cryptococcus curvatus. S. cerevisiae was isolated from sugarcane juice, C. tropicalis from Egyptian sobia drink, and T. dulcitum from buttermilk. Mendoza et al. [39] stated that Rhodotorula, Pichia, Candida, and Saccharomyces were frequently isolated from pasteurized fruit juices while; Candida and Saccharomyces spp. have often been reported as spoilage-causing organisms in citrus juices [40,41].

CONCLUSION
This study reported the occurrence of yeast species associated with dairy products, fruits, and juices collected from Assiut City, Egypt. Eleven yeast isolates belonged to six genera were successfully identified. Four species belonged to ascomycetes: D. hansenii (five isolates), S. cerevisiae (one isolate), C. tropicalis (one isolate), and P. kudriavzevii (one isolate). In addition, two yeasts species belonged to basidiomyces: R. mucilaginosa (two isolates), and T. dulcitum (one isolate). The biochemical identification was highly consistent with molecular identification techniques using amplified fragments of ITS and D1/D2 domains of large subunit. Dairy products, fruits, juices were commonly associated by different yeasts and it can be used for isolation of different genera and species.