Isolation and identification of molds and yeasts in medombae, a rice wine starter culture from Kompong Cham Province, Cambodia

Royal University of Agriculture, Phnom Penh, Cambodia Institute of Food Science and Technology, College of Agriculture, University of the Philippines Los Baños, College, Laguna – 4031, Philippines The National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baños, College, Laguna – 4031, Philippines Institute of Human Nutrition and Food, College of Human Ecology, University of the Philippines Los Baños, College, Laguna – 4031, Philippines


Introduction
A starter culture for rice fermentation is known as medombae in Cambodia. Spices, herbs, and a sweetener are ingredients commonly added also for dried starter preparation. Water is also added to the mixture and the previous starter was used as a source of inoculum at the rate of 1 to 2%. After mixing thoroughly, the mixture is being shaped into balls manually and placed on layers of rice husks or dried rice straw for 3 days at room temperature, sun-dried, and used as a starter for the production of alcoholic beverages such as rice wine. This technique of making dried starter culture may have originated in one place and later spread throughout Southeast Asia. On the other hand, milled rice or millet or other starch-based cereals are the main substrates for rice wine fermentation.
One of the major problems faced by commercial brewers of rice wine in Cambodia, as with the brewers of other indigenous beverages, is the variable quality of the product. Variability in quality is strongly correlated with the type of microorganisms present and quality control in the production of the traditional starter culture. Dung et al. (2005) developed a starter culture containing a defined mixed cultures of mold (Amylomyces rouxii) and yeast (Saccharomyces cerevisiae), and herbal extracts (from fennel and clove). However, starter culture in Cambodia is prepared using the traditional method, not the well-defined culture, and its production is limited only to some families because the recipe is kept secret and handed down from one generation to another. Thus, microorganisms present in starter culture is unknown. Hence, this investigation isolated and identified dominant and useful molds and yeasts in medombae from Kompong Cham province, Cambodia.

Isolation of microorganisms
Isolation of microorganism from medombae samples was carried out. Ten grams (10g) of the sample was added to 90 mL of 0.85% NaCl solution. Series of dilution was done and 1 mL of appropriate dilution was plated using the standard pour plating technique. Malt Yeast Extract Agar (MYA) medium containing 0.2% sodium propionate for yeasts; and Potato Dextrose Agar (PDA) medium containing tartaric acid for molds were used for plating. The petri dishes were incubated upside down at 30 o C for 48 hours and then the colonies of yeasts and molds were counted and reported as colony forming units/mL (CFU mL -1 ).
Different types of dominant colonies were picked up and transferred to MYA slant for yeasts, and PDA slant for molds.

Purification of cultures
Single colonies of representative isolates were purified following the dilution plating technique in the agar medium specified for a particular type of microorganisms. Separated colonies were transferred again to the agar slants. Purification was done by streaking on plated agar and repeated two or three times or until pure cultures were obtained, as confirmed by microscopic examination, are obtained.

Identification of microbial cultures
Purified microbial cultures were identified through morphological, cultural, physiological and biochemical tests following the methods described by Alexopoulus et al. (1996), Samson et al. (1995), and Frazier and Westhoff (1998and Kreger-van Rij (1984) and Lodder (1970) for yeast.

Results and discussion
Selection of representative colonies was based on the appearance of growth on PDA medium. Molds were identified through cultural and morphological test using an agar block technique. For yeasts, aside from the above tests, physiological and biochemical properties were also examined.

Identification of mold isolates
Molds were successfully screened using a modification of the screening techniques described by Alexopoulus et al. (1996), Samson et al. (1995), and Frazier and Westhoff (1998. Two dominant mold strains coded MA and MB ( Figure 1B) were chosen for identification.
MA mold isolate was observed white to creamishyellow cottony mycelia becoming brownish gray with aged; mycelium ≤10 mm in height; no soluble pigments and exudates produced; smooth, white to yellow on reverse side; ≤85 mm colony diameter; non-septated mycelium indicating that it belongs to Class Phycomycetes. Moreover, it has no sporangioles and characterized by the absence of stolon and rhizoids which are typical of Mucor spp.
Isolate MB is fast growing on PDA agar with cottony, aerial, white non-septate mycelium that turns grayish-white when aged; produces grayish-black spores and prominently forms rhizoid which is typical of Rhizopus spp. Cultural characteristics exhibited on different culture media (Table 1) as well as growth on Potato Dextrose Agar (PDA) at different temperatures were also used as the basis for the identification. Observation of growth was done for 7 days during incubation at 30 o C or until fruiting bodies/spores were observed.
Cultural and morphological characteristics of the mold strains using the agar block technique revealed that both MA and MB mold isolates were non-septated which is the typical property of Class Phycomycetes. Further, MA has no sporangioles and stolons and characterized by the absence of rhizoid which is typical of Mucor spp. On the other hand, MB strain had a discernible rhizoid. Figure 2 shows the simple key for differentiation of Mucor spp and Rhizopus spp. MB strain is closely related to R. oligosporus, R. stolonifer and R. oryzae. However, chlamydospores of the isolate are not very abundant unlike the R. oligosporus, thus this specie was deleted from the choices. Furthermore, incubation of the mold isolates to 37 o C showed good growth and this property differentiated the R. stolonifer from R. oryzae. Thus, the mold MB was identified as R. oryzae.

Identification of yeast isolates
Three yeast isolates, coded as Yo, Y D and Y 1 ( Figure  1C), were chosen for identification based on their cultural, morphological, and physiological properties following the methods described by Lodder (1970) and Kreger-Van Rij (1984).
The colony and cellular characteristics of yeasts are presented in Table 3.
The biochemical characteristics of Yo isolate were determined using the Vitek 2 identification system, and YST Card identification (Appendix A) and results revealed the identity as Candida tropicalis. On the other hand, the identity of Y D was determined through its morphological and cultural characteristic using agar technique (Appendix B). Moreover, its protein profile was compared to the reference and sample protein masses using BiomerieuxVitek MS (MALD-TOF) showing that Y D is a Saccharomyces cerevisiae. The biochemical and cellular characteristics of Y 1 isolate as described by Lodder (1970) and Kreger-Van Rij (1984) confirmed its identity as Saccharomycopsis spp. (Appendix C).
Results of various tests for identification of three yeast strains (Y o , Y D , and Y 1 ) isolated from medombae suggest that they are Candida tropicalis, Saccharomyces cerevisiae and Saccharomycopsis spp. based on the method of identification used, respectively (Figure 4). This study agreed with Tsuyoshi et al. (2005) and Thapa and Tamang (2004) who also identified the presence of S. cerevisiae in marcha. S. cerevisiae has been selected for the production of defined granulated starters for the production of high-quality Vietnamese rice wine (Dung 2004;Dung et al. 2005). In addition, Sm. fibuligera was also found as the most dominant yeast in marcha (Tamang and Sarkar 1995). Thapa and Tamang (2004) reported that saccharifying activities are mostly shown by Rhizopus spp. and Sm. fibuligera, whereas liquefying activities are shown by Sm. fibuligera and S. cerevisiae. Uchimura et al. (1990) isolated Saccharomycopsis in poo or phab (marcha of Bhutan). Yeast associated with ragi was Saccharomycopsis (Dwidjoseputro and Wolf 1970;Saono et al., 1974;Hadisepoetro et al., 1979;Hesseltine and Ray, 1988;Ardhana and Fleet, 1989;Yokotsuka, 1991). S. cerevisiae and Sm. fibuligera have also been reported to be present in bubod Dizon et al., 2009Dizon et al., , 2013; however, Sm. fibuligera is the dominant amylolytic yeast in bubod (Hesseltine and Kurtzman, 1990). Loogpang is an ethnic amylolytic starter from Thailand, which is commoly used to prepare alcoholic drinks and vinegar. Species of yeast present in loogpang are Sm. fibuligera FULL PAPER  and Saccharomyces (Dhamcharee, 1982;Uchinura et al., 1991). Sm. fibuligera of loogpang showed high glucoamylase activity (Sukhumavasi et al., 1975). Sm. fibuligera, S. cerevisiae and Candida tropicalis have been isolated in men (Dung et al., 2005(Dung et al., , 2006(Dung et al., , 2007. Sm. fibuligera and S. cerevisiae were also present in banh men (Thanh et al., 2008).