Identification and lipolytic activity of yeasts isolated from foods and wastes

ABSTRACT Thirty-three yeasts were isolated from palm oil industrial wastes and traditional fermented foods in Thailand. Based on the analysis of the sequences of the D1/D2 region of the large subunit ribosomal RNA gene (LSU rDNA) and their phenotypic characteristics, they were identified as Pichia kudriavzevii (11 isolates), Candida ethanolica (1 isolate), Clavispora lusitaniae (2 isolates), Ogataea polymorpha (1 isolate), Hanseniaspora opuntiae (1 isolate), Lodderomyces elongisporus (1 isolate), Saccharomyces cerevisiae (2 isolates), C. tropicalis (5 isolates), Yarrowia lipolytica (2 isolates), Magnusiomyces ingens (1 isolate), and Magnusiomyces capitatus (3 isolates), Trichosporon insectorum (1 isolate), and Trichosporon asteroides (2 isolates). Seven strains, T. insectorum 4E-1D, M. capitatus 5E-1T and 5E-2D, T. asteroides 8E-1T and 8E-1D, and Y. lipolytica Fy-12 and Fy-13, showed high lipolytic activity ranged from 5.21 ± 0.09 to 45.68 ± 2.37 U/mL. Moreover, these seven strains exhibited good lipolytic activity after culturing in the medium containing palm oil (11.79 ± 0.67 to 28.19 ± 4.84 U/mL) and soy oil (9.14 ± 1.08 to 22.97 ± 0.69 U/mL) as lipase inducers. The result of this study suggests that the palm oil industrial wastes and Thai fermented foods could be promised as the invaluable sources of lipolytic yeasts.

Lipases have been found in animals, plants and microorganisms. However, enzymes from microbial sources are currently receiving more attention because of their interesting characteristics, such as stability in an organic solvent and high substrate specificity (Salihu et al. 2012). Microorganisms are recognised as sources of extracellular lipases, especially yeasts and filamentous fungi such as Candida spp., Yarrowia lipolytica, Rhodotorula spp., Pichia spp., Saccharomycopsis crataegensis, Torulospora globosa, Trichosporon asteroides, Mucor spp. and Aspergillus spp. (Sharma et al. 2001;Vakhlu and Kour 2006;Ertugrul et al. 2007). Furthermore, some of the yeast strains are considered as non-pathogenic, the processes for lipase production based on yeasts have been classified as GRAS (generally regarded as safe) (Wang et al. 2007).
Naturally, yeasts and moulds have ability to produce lipases are found in several habitats including waste of vegetable oil, dairy product industries, seeds, and deteriorated food (Singh and Mukhopadhyay 2012). The present study aimed to isolate and analyse the diversity of lipolytic yeasts from foods and wastes in Thailand. We also carried out the hydrolysis of various substrates by crude lipases produced by the selected isolates. To our knowledge, this is the first report on the lipolytic yeasts associated with fermented foods and the palm oil industrial wastes in Thailand.

Isolation of lipolytic yeasts
The palm oil industrial wastes including palm cake, palm shell and palm fibre were collected from 2 factories, Eastern Palm Oil Co., Ltd. and Suksomboon Co., Ltd. in Chonburi province and fermented food samples: fermented beef (Mam-Neua), fermented pork (Mam-Moo), fermented pork sausage, fermented fish (Pla-Som) and fermented rice (Khao Kab), were collected from the markets in Bangkok, Thailand. One gram of each sample was enriched in a tube containing 10 mL lipolytic medium (0.3% (w/v) yeast extract, 0.3% (w/v) peptone, 0.01% (w/v) CaCl 2 · 2H 2 O and 0.5% (v/v) Tween 20) supplemented with 200 mg·L −1 chloramphenicol (modified from Barrow and Feltham 1993) and incubated at 30°C for 3 days. After cultivation, the enrichment samples were streak on the lipolytic agar medium supplemented with 200 mg·L −1 chloramphenicol and the plates were incubated at 30°C for 3 days. Representative yeast colonies were picked up based on colonial characteristics and purified by single colony streaking method on yeast extract-malt extract (YM) agar medium [0.3% (w/v) yeast extract, 0.3% (w/v) malt extract, 0.5% (w/v) peptone, 1% (w/v) glucose and 1.5% (w/v) agar]. The yeast isolates were maintained on YM agar slant at 4°C, freeze in YM broth supplemented with 10% (v/v) glycerol at −80°C, and lyophilised for long-term preservation.

Phenotypic characteristics
The morphological characteristics were performed as described by Yarrow (1998) and Kurtzman et al. (2011). For physiological characteristics, Yeast identification system API 20 C (bioMérieux, Marcy 1ʹ Etoile, France) was used according to the manufacturer's instructions. The kit system allows the determination of the assimilation of 19 carbon sources for clinical isolates of pathogenic yeasts. The strips were incubated at 30°C for 48 h (24 to 48 h was recommended).

26S rDNA sequence and phylogenetic analysis
A loopful of yeast cells was suspended in 100 μL lysis buffer in a 1.5 mL microcentrifuge tube (Raeder and Broda 1985) and boiled in a water bath or metal heated block for 15 min. After boiling, 100 μL of 2.5 M potassium acetate (pH 7.5) was added, keep on ice for 2 h, and centrifuged at 14,000 rpm for 5 min. The upper layer was extracted twice with 100 μL chloroform/isoamyl alcohol [24:1 (v/v)] and DNA in the upper layer was precipitated with cold isopropanol, dried and dissolved in 30 μL MilliQ water. The D1/ D2 domain of the large subunit ribosomal RNA gene (LSU rDNA D1/D2) was amplified using polymerase chain reaction (PCR) with primers F63 (5ʹ-GCATATCAATAAGCGGAGGAAAAG-3ʹ) and LR3 (5ʹ-GGTCCGTGTTTCAAGACGG-3ʹ) (O'Donnell 1993). The PCR condition was performed according to the methods of Kutzman and Robnett (1988). The PCR product was checked by agarose gel electrophoresis and purified using a GenepHlow TM PCR clean up kit (Geneaid, New Taipei, Taiwan). The purified PCR product was sequenced at Macrogen (Korea). The sequences were compared with available sequence data using a BLASTN search on NCBI GenBank database (Altschul et al. 1997) and were aligned with sequences of related species retrieved from GenBank using the multiple alignment software CLUSTAL_X version 1.8 (Wanapat and Rowlinson 2007). The phylogenetic distances were calculated with the Kimura-2-parameter model (Kimura 1980) and tree topologies were inferred by using the neighbour-joining (Saitou and Nei 1987), which were constructed using MEGA 7.0 software (Kumar et al. 2016). The confidence values of the branch node were evaluated by using the bootstrap resampling method with 1,000 replications (Felsenstein 1985).

Screening of lipolytic activity
Thirty-three yeast isolates were cultured on YM agar medium at 30°C for 48 h. After cultivation, each isolate was standardised the turbidity with McFarland standard solution No. 2 to obtain approximately 1 × 10 6 CFU/mL in 0.85% NaCl and used as inoculum. One millilitres of the inoculum was added into 10 mL of the production medium containing 0.5% (w/v) yeast extract, 0.5% (w/v) peptone, 0.2% (w/v) glucose, 0.01% (w/v) MgSO 4 · 7H 2 O, 0.1% (w/v) K 2 HPO 4 and 1% (v/v) Tween 20 as a lipase inducer, pH 7.0, incubated by shaking (180 rpm) at 30°C for 72 h. The culture was centrifuged at 14,000 rpm for 10 min and the supernatant was analysed for lipolytic activity.

Induction of lipolytic activity with palm and soy oils in liquid cultures
The seven selected isolates, 4E-1D, 5E-1T, 5E-2D, 8E-1T, 8E-1D, Fy-12 and Fy-13, were grown at 30°C for 48 h in 500 mL Erlenmeyer flask containing 100 mL YM broth. Each isolate was standardised the turbidity with McFarland standard solution No. 2 to obtain approximately 1 × 10 6 CFU/mL in 0.85% NaCl and used as an inoculum. The inoculum (1%) was inoculated and cultivated in 100 mL of the production medium supplemented with 1% (v/v) of palm oil or soy oil under shaking condition at 30°C for 72 h. Cells were removed by centrifuging at 14,000 rpm for 10 min and the cultured supernatant were quantitatively determined for enzyme activity.

Determination of lipolytic activity
The lipolytic activity was determined spectrophotometrically using p-nitrophenyl butyrate (p-NPB) as substrate (Fickers et al. 2003;Gomes et al. 2011). The substrate was prepared by dropwise addition of 0.1 mL of solution A (20.9 mg of p-NPB was dissolved in 10 mL absolute ethanol) into 10 mL of solution B (0.1 M sodium-potassium phosphate buffer, pH 7.2 containing 0.1 M NaCl) by using a volumetric flask, the final concentration of the substrate was 0.1 mM. The culture broth (20 μL) was added into 180 μL of the substrate and incubated for 60 minutes at 37°C. The release of p-nitrophenol was measured at 405 nm using a microplate reader (Wallac 1420, PERKINELMER, USA). The production medium was used as a control. One Unit of lipase activity was defined as the amount of enzyme releasing 1 μmole fatty acid per minute at the assay condition.

Statistical analysis
All experiments were conducted in triplicate and statistical analysis was performed by SPSS 21.0 software (IBM, U.S.A). Analysis of variance was carried out using the ANOVA procedure by the Duncan method to determine significant (p ≤ 0.05) differences between the means.

Results
A total of 33 yeast isolates were characterised by the phenotypic characteristics based on the colony morphology and carbon utilisation. According to the taxonomic keys of Kurtzman et al. (2011), the isolated yeast genera were phenotypically identified as Pichia, Candida, Magnusiomyces, Clavispora, Saccharomyces, Yarrowia, Trichosporon, Hanseniaspora, Lodderomyces and Ogataea. Their differential colonial appearance and carbon utilisation such as adonitol, L-arabinose, xylitol, D-xylose, D-lactose, etc. are listed in Table 1. Accurately, the identification of the species level was done with the molecular method using the analysis of the D1/D2 domain of the 26S rRNA gene sequences (Table 2) and the neighbour-joining phylogenetic analysis (Figure 1).
In addition, the two isolates of the genus Magnusiomyces, M. capitatus strains 5E-1T and 5E-2D, and the two strains of the genus Yarrowia, Y. lipolytica strains Fy-12 and Fy-13 also showed significant lipolytic activity. These results were in accordance with several studies as described previously. Salgado et al. (2019) revealed that M. capitatus strain JT5 isolated from olive mill wastewaters exhibited 3.96 U/mL of lipase activity after cultured in the production medium containing olive oil as a carbon source. Y. lipolytic was wellknown as the lipase-producing yeast. Corzo and Revah (1999) had studied and found that Y. lipolytica strain 681 produced 31.9 and 30.9 U/mL in the production medium supplemented with corn oil and olive oil, respectively. Additionally, Domínguez et al. (2003) reported that the high lipolytic activity level of Y. lipolytica strain CECT 1240 was obtained with the liquid culture containing sunflower oil (58 U/mL), olive oil (49 U/mL) and tributyrin (33 U/mL). This study suggested that Trichosporon, Magnusiomyces and Yarrowia strains could be further studied and used for lipase production.

Conclusion
The present study showed that Thai fermented foods and the palm oil industrial wastes could be employed as a valuable source for the isolation of lipolytic yeasts. Thirty-three isolates can be divided into 2 groups including ascomycetous (30 isolates) and basidiomycetous (3 isolates) yeasts. The seven isolates, Trichosporon insectorum 4E-1D, Magnusiomyces capitatus 5E-1T and 5E-2D, Trichosporon asteroides 8E-1T and 8E-1D, and Yarrowia lipolytica Fy-12 and Fy-13, exhibited a significant level of lipolytic activity in Tween 20 liquid culture and were chosen for studying the lipolytic activity in palm and soy oils. Each selected strain showed varying levels of lipolytic activity in both palm and soy oils ranging from 9.14 to 28.19 U/mL. The further study focuses on the optimisation of lipase production by the best lipase producer, T. insectorum strain 4E-1D, as well as the characterisation and purification of lipase from the liquid medium which may provide for the commercial exploitation.