Isolation and identification of yeasts from local fruits in Thua Thien Hue province, Vietnam

. In this study, a total of 30 yeast isolates were recovered from local fruits in Thua Thien Hue province. Genetic characterization based on the ITS sequences identified isolates belonging to 3 species including Saccharomyces cerevisiae, L achancea fermentati , and Clavispora fructus , with high sequence homology (over 99%) compared to published sequences in the GenBank. All identified S. cerevisiae isolates could grow well at 30°C and ferment several sugar including fructose, galactose, sucrose, mannose, maltose, and raffinose with different performances, but were inhibited at temperature higher than 35°C. The strains also grew well in the medium containing 5% ethanol (v/v) and 200 g/L glucose, but their growth ability was decreased gradually with an increase in ethanol and glucose concentrations. Interestingly, D14 strain was able to grow in the medium supplemented with 12% of ethanol, and 500 g/L of glucose at 45°C, while D7 strain could utilize both mannitol and glycerol at a low level. Our results also indicated that some strains have relatively high sedimentation efficiency, which are favorable conditions for beer fermentation and biomass recovery. The isolated yeast strains with good tolerance properties may provide a potential source of valuable raw materials for applications


Introduction
Yeasts play an essential role in various fermentation processes such as baking and brewing, while the ethanol released by yeast, carbon dioxide is of utmost need for the rising of flour dough, maturation, and creation of flavor [1].Fermentation is a relatively complex process in which numerous adverse conditions may damage yeast cells, for example, osmotic pressure, ethanol concentration, and high temperature [2].
Therefore, tolerance to high temperatures is one of the most desirable characteristics of yeasts, which is of interest in the fermentation industry.In particular, this property is beneficial for reducing cooling costs, increasing conversion rates of sugar to ethanol, and reducing contamination by other strains, resulting in an increase in fermentation productivity [3].Thermotolerant yeasts isolated from nature have gained considerable attention since they can grow and ferment efficiently in uncomfortable conditions [4][5][6].Particularly, these properties are usually not found in S. cerevisiae which has been widely used for industrial production at optimal temperatures of around 25-35°C.For example, some isolated yeasts can grow and ferment at temperatures above 40°C, such as Kluyveromyces marxianus [7], Pichia kudriavzevii [8], and Candida tropicalis [9].New S. cerevisiae strains from different sources are expected to have different phenotypic and genotypic profiles in comparison with traditional strains used in the industry [10].The wild yeast S. eubayanus has been used in the industrial production of lager beer [11].Similarly, the S. uvarum strain isolated from apple chicha also exhibited a good potential for the production of commercial cider without any apparent flavour defects [12].Many other yeast species are also emerging as candidates for industrial production of food and beverages [13].
Vietnam has a long history of agriculture and diverse ecosystems along whole regions grant the existence of more than 13,000 plant species, belonging to 3,500 genera and 500 families, 60% of which are indigenous origin that constitute a potential source of yeast strains [14].Among them, fruits harbour a complicated community of yeast species associated with spontaneous ethanol ic fermentation [15].Thua Thien Hue province, The aim of this study is to isolate some S. cerevisiae strains that can tolerate high temperature, ethanol content, and exhibit flocculation from local fruits in Thua Thien Hue province.

2
Materials and Methods

Materials
Fruit samples (mango, orange, pineapple, and mangosteen) without growth stimulants and pesticides were collected from the farms in Thua Thien Hue province, Vietnam.These samples were preserved in sterile plastic bags and left to naturally ferment at room temperature (25-30C) for 2-5 days.

Yeast isolation
Yeasts were isolated in accordance with a previous study of Nguyen [16] with some modifications.Briefly, ground 1 g of fermented fruit (non-peels) in 0.

Morphological and phenotypical characteristics
One hundred and twenty yeast-like colonies were isolated from the collected fruits on YEPDA plates.The isolated yeast strains exhibited identical morphology, such as whitish or cream color and round shape, but there were differences in the surface and margin of colony morphology.This result is consistent with the description by Kurtzman et al. [17], who reported that yeasts exhibited a range of colors from white-cream to tan, besides varying in texture, surface, elevation, and margin.
Based on the morphology, color, and dimension of colonies, 30 typical isolates were selected and divided into 6 groups, as described in Table 1.Colony morphology with rough, smooth, or flat outside was captured by a camera ProgRes® CT3 CMOS (Germany) (Fig. 2), while oval or ellipse of yeast cell shape was observed by a microscope (Olympus) with 100x magnification (Fig. 3).isolates showed 99% identity to C. fructus (Table 2).

High temperature tolerance
According to literatures, the optimal temperature for S. cerevisiae growth is usually ranged from 25°C to 30°C [20,21].At high temperatures, yeast cells face to stress which induces increasing membrane fluidity, changing in protein structures and functions that lead to growth inhibition or cell death [22,23].In this study, S. cerevisiae isolates grew well at temperatures ranging from 20 to 30°C (Fig. 5A).But the growth rate of all isolates significantly declined when temperature reached 35°C or could not grow at 45°C, except D14 and X30 strains that were able to grow up to 45°C.Similar results were also observed in the study of Techaparin [24] who found some S. cerevisiae isolated from Mekong region exhibited moderate growth at 45°C, but others could not.
Nasir A [25] also reported that S. cerevisiae strains isolated from fruit sources (pineapple and orange) were highly thermotolerant as growing well up to 40°C.
As stated in the study of Sree [20], yeast can grow at temperatures as high as 40°C, being a thermotolerant yeast.Therefore, D14 and X30 isolates are thermotolerant yeasts and may be suitable for industrial Fig. 5. applications.

Ethanol tolerance
The effect of ethanol on the growth of S. cerevisiae isolates was also examined, the results shown in KKU-VN35 isolated from agricultural products exhibited tolerance to 13% ethanol [24].The range of ethanol tolerance of S. cerevisiae isolated from palm wine [26] and sugar cane [27] was also determined from 7-12%.In contrast, a higher ethanol tolerance (16%) of S. cerevisiae was found in the study of Tsegaye [28].According to Coulibaly [29], the higher ethanol tolerance exhibited by these strains could be due to their greater capability to consume ethanol in the presence of oxygen, as the ethanol tolerance of yeasts greatly depends on mitochondria.Ethanol resistance is an extremely complex mechanism involved in multiple physiological processes that each rely on many different genes, in addition to combining alleles and mutations sophisticatedly can lead to improve ethanol tolerance [30].

Sugar fermentation
All species of the genus Saccharomyces can utilize glucose as a sole carbon, and the distinct ability is up to each strain [31,32].As illustrated in Fig. 6, all isolates were capable to use efficiently glucose with concentrations in the range of 20 to 100 g/L after 24 h of incubation.However, the increase in glucose concentration, from 200 to 400 g/L, inhibited yeast cell growth.Attractively, the D14 isolate could grow in a medium containing up to 500 g/L glucose, which means that the cell membranes of the D14 strain could endure great osmotic tension.Ortiz-Zamora [33] reported that the yeast isolated from agricultural sources (grape juice, sugarcane molasses, and cane juice) had a good adaptation to 200 g/L glucose and remarkable growth inhibition at glucose levels ranging from 25 to 40% (w/v, equivalent to 250 to 400 g/L), depending on the strain.Thatipamala [34] suggested that sugar inhibition is related to instantaneous biomass yield and typically begins at concentrations above 150 g/L glucose, and the specific growth rate was found to decrease linearly with further increase in substrate content.The ability to facilitate different carbon sources of isolated S. cerevisiae was also tested.As results shown in Table 3, all isolates were able to ferment fructose, galactose, sucrose, mannose, maltose, and raffinose.In contrast, manitol was only fermented by D7 and M19, while C5, D7, and X30 could use glycerol as a sole carbon source.
However, the assimilation of glycerol or mannitol at low levels in these isolates was consistent with the result of Swinnen [35] located in the North Central Coast region of Vietnam, has a transitional climate between two regions of the North and South of Vietnam, scorching in the dry season and humid cold in the rainy season.Therefore, this is one of the most abundant places in fruit biodiversity.

Finally, pellet was
Fig. 1 based on ITS1/2 sequences in the Saccharomyces Genome Database (SGD).The PCR program was pre-denaturation at 95°C for 5 min,

Fig. 5B indicated
Fig.5Bindicated that all isolates grew well at 2% ethanol (v/v) when compared to the control (without ethanol supplementation).But the growth of yeast was significantly affected when

Fig. 6 .
Fig. 6.The growth of yeast strains by glucose concentration in YPD medium after 24 h

3 . 5 Flocculation
Yeast flocculation is a crucially known phenomenon in the brewing industry that may enhance the survival of yeast cells in an environment with limited nutrient conditions [38].In the phase of pre-final fermentation, single cells begin to gather up dense clusters and settle at the bottom of the reaction tank.The flocculation of yeast can facilitate the filtration process and biomass recovery of byproducts, concurrently reduce the toxicity and increase the sweetness of beer [38,39].To determine the flocculation ability of isolated S. cerevisiae, 5 mL of overnight cultures were vigorously vortexed for 1 min and transferred to glass tubes for sedimentation analysis.As results shown in Fig. 7A, cells of isolates C5, D14, and N22 markedly formed macroscopic flocs (clusters of cells), which sedimented efficiently after 10 min; the sedimentation of X30 was less efficient, while C1, D7 and M49 isolates exhibited poor flocculation.Based on the formula described in the method section, the sedimentation rates of C5, D14, N22 and X30 isolates were 68.3, 61.2, 76.6, and 43.3%, respectively, as shown in Fig. 7B.Similar findings were also observed in previous studies [26,40], who found the flocculation efficiency of some S. cerevisiae varied between 58 and 93.1%.Since flocculation ability varies among strains and could be the result of differences in the expression of flocculin genes, further studies need to be carried out to understand the molecular mechanism that controls this event.

Table 1 .
Characteristics of yeast colonies in each group

Table 2 .
GenBank accession numbers for the ITS nucleotide sequences of isolated yeast strains who found that S.