Isolation and Molecular Identification of Rhodotorula mucilaginosa from Local Sources and Carotenoid Production Using Fruit Waste Extract

2.1 Purification of pigmented yeast isolates

Each stained yeast colony was individually transferred to dishes containing yeast malt extract agar (YMA) medium, prepared according to the method described in Section 3, using a sterile inoculation ring. The colonies were then streaked onto the agar surface and incubated at 30 ℃ for 72 hours. The inoculation and purification steps were repeated several times until pure colonies with uniform morphological characteristics were obtained. Their purity was verified by examining the external appearance of the colonies, along with microscopic examination of the cells after staining with methylene blue [11].

2.2 Preservation of yeast isolates

The isolates previously grown on YMA medium were transferred onto YMA agar slants in test tubes. The cultures were incubated at 30 ℃ for 48 h and subsequently stored at 5 ℃. The isolates were subcultured monthly to maintain their viability [12].

The samples were grouped and coded according to their sources, and the letters used in the coding system were selected by the researcher in accordance with the experimental procedures.

2.3 Isolation and purification of yeast isolates

A total of 67 samples were collected for the isolation of pigmented yeasts. The samples included flowers from different plants (41 samples), tree leaves (3 samples), soil (5 samples), spoiled fruits (6 samples), pickles (3 samples), homemade sweets (5 samples), milk (2 samples), and cheese (2 samples), which were collected from different areas of Mosul City and Telkaif District. The samples were prepared by taking 25 g of solid samples or 25 mL of liquid samples and adding them to 225 mL of peptone water in a 500 mL conical flask, followed by incubation at 30 ℃ for 3 h to activate the yeasts present in the samples. Thereafter, 0.1 mL of each sample was transferred to a Petri dish, and Potato Dextrose Agar (PDA) medium supplemented with the antibiotic chloramphenicol at a concentration of 250 mg per 1,000 mL of growth medium was added to prevent bacterial growth. The plates were incubated in an inverted position at 30 ℃ for 48 h [13].

Four plates were inoculated for each replicate, and representative colonies were selected based on differences in pigmentation and the uniformity of colony morphology observed on the plates. Subsequently, individual pigmented yeast colonies were transferred using a sterile inoculating loop onto plates containing YMA medium and streaked on the agar surface. The plates were incubated at 30 ℃ for 72 h, and the purification process was repeated until pure colonies with similar morphological characteristics and microscopic appearance were obtained after staining with methylene blue [14].

2.4 Preparation of fruit waste extract medium

The extract was prepared using wastes obtained from local juice shops in Mosul City, which included orange and pomegranate residues generated after juice processing. Briefly, 1 kg of orange pulp and pomegranate arils were combined at a ratio of 1:1 with 2 L of water and heated at 100 ℃ for 30 min. The mixture was then cooled and centrifuged at 5000 rpm for 15 min, followed by filtration. The resulting filtrate was collected and sterilized by passage through a 0.22 µm membrane filter [15].

2.5 Determination of sugars

Sugars in the fruit waste extract (FWE) were determined using two methods: the first method was used to estimate reducing sugars using the colorimetric 3,5-dinitrosalicylic acid (DNS) method [16], Standard solutions were prepared using pure glucose at concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, and 120 mg/mL. One milliliter of each standard solution was transferred into a test tube and mixed with 1 mL of DNS reagent. Two drops of 0.1 M sodium hydroxide solution were then added to the mixture. The tubes were heated to boiling in a water bath for 5 min, subsequently cooled, and then supplemented with 10 mL of distilled water. After thorough mixing, the absorbance was measured using a spectrophotometer at a wavelength of 540 nm. A standard calibration curve was then constructed using the corresponding glucose concentrations to determine the glucose concentration in the fruit waste extract. 1 mL of the filtrate was taken and subjected to the same procedure described above. The glucose concentration in the sample was subsequently estimated using the prepared standard calibration curve (Figure 1).

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Figure 1. Standard calibration curve for glucose determination

The second method was the phenol–sulfuric acid method, which was used to determine total sugars, including reducing and non-reducing sugars [17]. The procedure was carried out by transferring 1 mL of glucose standard solutions at different concentrations ranging from 32 to 192 µg/mL into test tubes. One milliliter of 5% phenol solution was added to each tube and mixed thoroughly. Subsequently, 5 mL of concentrated sulfuric acid was added to the mixture, and the tubes were shaken vigorously. The tubes were then placed in a water bath at 25–30 ℃ for 30 min. Absorbance was measured at 490 nm using a spectrophotometer to construct the standard calibration curve (Figure 2).

For the determination of carbohydrates in the fruit waste extract (FWE), 1 mL of the extract was taken, the required dilutions were prepared using distilled water, and the procedure described above was then applied.

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Figure 2. Standard calibration curve for total carbohydrate determination

2.6 Preparation of yeast inoculum

The inoculum used throughout the different screening stages was prepared according to the method described in reference [18]. Ten loopfuls were taken from a 48-h-old pure culture grown on YMA and incubated at 30 ℃. The inoculum was transferred to a 250 mL flask containing 50 mL of YMB, previously adjusted to pH 5.0. The culture was then incubated in a shaking incubator at 100 rpm for 24 h at 30 ℃, with the inclusion of an additional screening stage. During the primary screening, the inoculum was prepared in Yeast Malt Extract Broth (YMB), whereas yeast extract–malt extract–fruit waste extract (YMF) medium was used during the secondary screening. The YMF medium was prepared by dissolving 3 g of malt extract, 3 g of yeast extract, and 5 g of peptone in 100 mL of distilled water. The resulting basal mixture was autoclaved and aseptically supplemented with 900 mL of filter-sterilized FWE. The pH was adjusted to 5.0. The yeast isolates were then grown in YMF medium under static conditions until reaching an optical density of 1.0 at 570 nm (OD₅₇₀ = 1.0) to acclimatise the isolates to the presence of FWE in the cultivation medium. In the final stage of screening, the same YMF medium was used with pH adjusted to 5.0. Cultures were incubated in a shaking incubator at 30 ℃ and 100 rpm for 24 h. The yeast growth was then determined after measuring the absorbance at 570 nm, and the incubation was further continued under the same conditions for another 7 days.

2.7 Quantitative screening of isolates for carotenoid production

The YMB and YMF media used for the primary and secondary screening, respectively, were adjusted to pH 5.0. A flask-to-medium volume ratio of 5:1 was maintained, and the inoculum size was set at 10% (v/v). The cultures were incubated under static conditions at 30 ℃ for eight days.

Growth and pigment production were measured by reading the absorbance of diluted culture medium (1:9, v/v) with distilled water after 2 and 8 days of incubation. For each reading, the same uninoculated culture medium diluted in the same way was used as the spectrophotometric blank, and the absorbance readings were corrected accordingly. Absorbance was measured at 450 nm for estimation of cellular pigment production and 570 nm for estimation of growth. The results were expressed as the corrected absorbance value × 10, and the pigment production index and growth index were calculated as follows:

Pigmentation ratio $=\frac{A_{450}(8 d)}{A_{450}(2 d)}$   (1)

Growth ratio $=\frac{A_{570}(8 d)}{A_{570}(2 d)}$   (2)

where, A₄₅₀(8d) and A₄₅₀(2d) are the absorbance readings at 450 nm after 8 and 2 days, respectively, whereas A₅₇₀(8d) and A₅₇₀(2d) are the corresponding absorbance readings at 570 nm.

This approach was used to select the most promising isolates with the greatest ability to grow and produce carotenoids during the primary and secondary screening stages, and the tables were designed accordingly.

2.8 Determination of total carotenoids

The carotenoid content was determined using a β-carotene calibration curve prepared at concentrations of 2, 4, 6, and 8 µg/ml. The amount of carotenoids in the samples was subsequently calculated from the resulting standard curve. The β-carotene stock standard solution was prepared by accurately weighing 0.1 g of β-carotene standard and transferring it into a 100 mL volumetric flask. The standard was dissolved in a small volume of hexane, and after complete dissolution, the volume was made up to the calibration mark with hexane. The resulting solution was designated as solution A. Subsequently, 1 mL of solution A was transferred into another 100 mL volumetric flask, and the volume was made up to the calibration mark with hexane. The solution was mixed thoroughly and designated as solution B. Each milliliter of solution B contained 10 µg of β-carotene standard. A series of standard solutions with concentrations of 2, 4, 6, 8, and 10 µg/mL was then prepared from solution B using hexane, as follows:

1. 2 µg/mL: 1 mL of solution B + 4 mL of hexane.
2. 4 µg/mL: 2 mL of solution B + 3 mL of hexane
3. 6 µg/mL: 3 mL of solution B + 2 mL of hexane
4. 8 µg/mL: 4 mL of solution B + 1 mL of hexane
5. 10 µg/mL: undiluted solution B

Subsequently, the absorbance was measured at 450 nm, as shown in Figure 3, and the following equation was applied to calculate the total carotenoid content:

$M=\frac{R F V_s \times 1000}{V_c}$   (3)

where,

M = total carotenoid content (µg/L).

R = spectrophotometric absorbance reading at 450 nm.

F = conversion factor, equivalent to 4.80 µg carotene/mL solvent.

Vₛ = total volume of solvent used for carotenoid extraction (mL).

V_c = volume of the culture used for carotenoid extraction (mL).

The β-carotene standard calibration curve used for carotenoid determination is shown in Figure 3.

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Figure 3. Standard calibration curve for β-carotene determination

The total carotenoid concentration was converted from micrograms per liter (µg/L) to micrograms per gram of dry cell weight (µg/g) by dividing the carotenoid concentration obtained from the above equation by the dry biomass concentration expressed in grams per liter (g/L). The total carotenoid content was calculated using the equation presented above.

Carotenoid extraction was carried out according to the method [19], with slight modifications aimed at reducing the use of organic solvents in accordance with Sustainable Development Goal 12 (Responsible Consumption and Production). The modification was supported by recent studies demonstrating the effectiveness of ultrasound treatment in cell wall disruption and improvement of extraction efficiency [20]. A 50 mL aliquot of the fermentation culture was centrifuged at 3500 rpm for 15 min. The resulting supernatant was discarded, and the cell pellet was washed repeatedly with distilled water until the wash water became colorless. After removing the wash supernatant, a hexane–acetone solvent mixture (1:1, v/v) was added at a ratio of 10 mL of solvent per gram of cell pellet, and the mixture was thoroughly homogenized. Cell disruption was then performed by ultrasonication at a frequency of 30 kHz for 60 min at 40 ℃. The mixture was subsequently kept overnight in the flask to allow greater penetration of the solvent into the cells and thereby enhance extraction efficiency.

The sample was then centrifuged, and the solvent-containing top layer was collected and filtered using suitable filter paper. The extraction process was repeated several times until the cells were completely decolorized. The cell residue was discarded, while the filtrate was collected, and a 15% sodium chloride solution was added to aid in the purification and separation of the hexane layer. The mixture was transferred to a separation funnel, and the layers were separated to obtain the pure solvent layer. Finally, the purified hexane layer was collected, and its absorbance was measured at 450 nm using a spectrophotometer.

2.9 Determination of dry cell weight

The dry weight of the cells was estimated using 50 mL of fermentation culture. The sample was centrifuged at 3500 rpm for 15 minutes. The sedimented cells were collected and washed twice with distilled water, discarding the supernatant after each centrifugation. The washed biomass was then dried at 85 ℃ until its weight stabilized, and the dry weight of the cells was expressed in grams per liter of culture.

2.10 Analysis of carotenoids produced by the selected isolate using high-performance liquid chromatography

Carotenoids were analyzed using a SYKAM high-performance liquid chromatography (HPLC) system, Germany, equipped with a C18-ODS column (250 × 4.6 mm, 5 µm) and a UV–Visible detector. The column temperature was maintained at 30 ℃. Detection was performed at 485 nm. The mobile phase consisted of acetonitrile, isopropanol, and ethyl acetate at a volumetric ratio of 20:40:40 (v/v/v), with a flow rate of 1 mL/min. The injection volume was 0.1 mL (100 µL), and the total chromatographic run time was 20 min [21].

2.11 Molecular identification

The isolate was identified by extracting deoxyribonucleic acid (DNA) from yeast isolate H1 using a specific DNA extraction kit supplied by Geneaid and following the manufacturer’s protocol. The ITS region was amplified and sequenced using the forward primer ITS1F and the reverse primer ITS4; the size of the amplified DNA fragment produced by PCR was compared with a standard DNA ladder of different molecular sizes to estimate the approximate size of the amplified ITS region and to evaluate the efficiency and specificity of the amplification process using agarose gel electrophoresis [22]. The PCR cycling conditions used in this study are presented in Table 1.

Table 1. Polymerase chain reaction (PCR) cycles [23]

This study demonstrated the preliminary ability to isolate carotenoid-producing pigmented yeasts from different local sources, including flowers, soil, and local sweets. Among the tested isolates, the soil-derived isolate H1 showed the highest biomass and carotenoid production under the experimental conditions when cultivated in YMF medium containing FWE. Molecular identification based on ITS-region analysis identified the selected isolate as Rhodotorula mucilaginosa. In addition, the close similarity between the HPLC retention time of the extracted pigment and that of the β-carotene standard suggested that β-carotene was a major component of the produced carotenoids. Preliminary results show that the local isolate R. mucilaginosa H1 is capable of producing carotenoids under the experimental conditions established in this study. However, determining its suitability for practical applications or industrial production requires further studies, including evaluating pigment stability, detecting solvent residues, verifying its safety for food use and its practical efficacy, as well as optimizing production conditions and exploring the possibility of scaling up the process.

The ITS sequence of the local isolate H1 was submitted to the GenBank database of the National Center for Biotechnology Information (NCBI) under submission number SUB16176960 and was still under review at the time of manuscript preparation.