Identification of Yeasts and Evaluation of their Distribution in

Taiwanese Kefir and Viili starters

Sheng-Yao Wang12, Hsi-Chia Chen2, Je-Ruei Liu23, Yu-Chun Lin2, Ming-Ju Chen24

1 Experimental Farm, National Taiwan University, Taipei, Taiwan, R.O.C.

2Department of Animal Science and Technology, National Taiwan University, Taipei,

Taiwan, R.O.C.

3Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, R.O.C.

4Research Center of Food and Biomolecules, National Taiwan University, Taipei,

Taiwan, R.O.C.

Corresponding author: Dr. Ming-Ju Chen, Department of Animal Science and

Technology, National Taiwan University, No. 50 Lane 155 Sec. 3. Keelung Rd., Taipei

106, Taiwan, R.O.C.

Tel: 886-2-33664169, Fax: 886-2-27336312

Email: [email protected]

Running title: Identification of Yeasts in Kefir and Viili Cultures Abstract

The objective of the present study was to investigate yeast communities in kefir grains and viili starters in Taiwan through conventional microbiological cultivation, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing methods. In addition, the direct identification potential of culture-independent DGGE techniques was also evaluated in this study. Results indicated that a combination of culture-dependent methods with PCR-DGGE and sequencing could successfully identify four yeast strains from both types of cultures in Taiwan. Kluyveromyces marxianus, Saccharomyces turicensis and Pichia fermentans were found in Taiwanese kefir grains with a distribution of 76%, 22% and

2%, respectively, while Kluyveromyces marxianus, Saccharomyces unisporus and

Pichia fermentans were identified in viili starters corresponding to 58%, 11% and

31% of the total cell counts respectively. Furthermore, the culture-free method was also applied to identify the yeast strains using DGGE. Only two yeast strains, Klu. marxianus and Saccharomyces turicensis were found in kefir grains and two, including Klu. Marxianus and Pichia fermentans, in viili starters. These results suggest that in samples containing multiple species, PCR-DGGE may fail to detect some species. Sequences of yeast isolates reported in this study have been deposited in the GenBank database under Accession Nos. DQ139802, AF398485, DQ377652, and AY007920.

Keywords: yeast; kefir grain; viili; microbiological cultivation; DGGE

2 Introduction

Of cultural different origins, sources and processing methods, soured milk has diversified into a variety of products, such as dahi, dadih, kefir, koumiss, långfil and viili (Mistry, 2004; Chen et al., 2006; Dharmawan et al., 2006). Viili is a viscous mesophilic fermented milk that originated in Scandinavia, which is claimed to have various functional benefits and health improving potential (Kitazawa et al., 1991;

Nakajima et al., 1992; Kitazawa et al., 1993; Kitazawa et al., 1996; Raus-Madiedo et al., 2006). This cultured milk beverage results from the microbial action of lactic acid bacteria and a surface-growing yeast-like fungus, Geotrichum candidum (Boutrou and

Guéguen, 2005), present in milk that forms a velvety surface on viili (Leporanta,

2003). Kefir originated in the Caucasus Mountains of Russia centuries ago and has likewise been credited with various health-promoting properties (Liu et al., 2002; Liu et al., 2006a; Liu et al., 2006b). This cultured milk beverage is results from the microbial action of a wide community of microorganisms present in kefir grains in milk. Kefir has a uniform, creamy consistency and a slightly acidic taste caused mostly by lactic acid, along with some effervescence due to carbon dioxide, a minute

(<2%) concentration of alcohol due to the action of yeast cells present in the grains, and a variety of aromatic substances including acetaldehyde, acetoin and diacetyl which impart its characteristic flavor.

In order to better understand the fermentation process and to evaluate the health benefits of both fermented products, researchers have identified the various bacteria and yeasts in viili starters and kefir grains (Lin et al., 1999; Simova et al., 2002;

Leporanta, 2003; Shurtleff and Aoyagi, 2004; Witthuhn et al., 2004). Successful isolation and identification of microorganisms depends on the selection of suitable

3 growth media. However, such media are not necessarily suited to the growth of all microorganisms present in kefir grains or viili starters. Fujisawa et al. (1988) observed that Lactobacillus kefiranofaciens grew on KPL agar, but not on BL and

MRS agars. Farnworth and Mainville (2003) compared MRS, KPL, and Rogosa-CW to lactic acid whey medium and found that lactic acid whey medium yielded better growth rates for the lactobacilli present in kefir grains. In addition, some studies revealed that many of the microorganisms isolated are closely related and therefore challenging to isolate and identify.

Although the majority of identification techniques are culture-dependent methods, culture-independent methods, which do not require the microorganisms to be cultivated in media, have important advantages over their culture-dependent analogs. Over the past decade, culture-independent identification techniques based on genotype have multiplied, with varied techniques displaying differences in discriminatory power, reproducibility and work-load. Of those techniques, PCR combined with Denaturing Gradient Gel Electrophoresis (DGGE) has proven to be a useful method for analyzing complex microbial populations that does not require prior separation of individual inhabitants (Muyzer and Smalla, 1998). This method has been used successfully to evaluate bacterial composition of probiotic products (Fasoli et al.,

2003), to identify probiotics in South African products (Theunissen et al., 2005), to profile the yeast populations in raw milk (Cocolin et al., 2002), to investigate yeast populations associated with Ghanaian coca (Nielsen et al., 2005), to determine yeast strains involved in fermentation of Coffea Arabica in East Africa (Masoud, et al.,

2004), and to differentiate Lactobacillus species present in the gastrointestinal tract

(Walter et al., 2000). On the other hand, certain studies (Felske et al., 1998; Fasoli et

4 al., 2003; Theunissen et al., 2005) indicated that PCR-DGGE did not reveal microorganisms present at a level lower than 1% of the total microbial population in complex microflora.

Since safety and quality control are crucial for kefir and viili products, investigation of their microbiological profiles is important. Moreover, the presence of yeasts in viili starters and kefir grains plays a key role in the fermentation process and in forming the flavor and aroma of these fermented milks. Although many yeast strains, have been identified in kefir grains and in the final products (Farnworth and

Mainville, 2003) based on phenotypic properties (Rohm et al., 1992; Pintado et al.,

1996), restriction length polymorphism (RFLP), and DNA/DNA hybridization, none of these strains were determined by PCR-DGGE. Thus the purpose of this study was to identify strains of yeast and to study their distribution in kefir grains and viili starters in Taiwan by a combination of culture-dependent, PCR-DGGE and sequencing methods. In addition, the direct identification potential of the culture-independent DGGE techniques was also evaluated for this study.

Materials and Methods

Experimental design

For this paper the research is presented in two phases—culture-dependent and culture-independent methods—with a description of procedures each of which involves several steps. A flowchart depicting the entire procedure for identification and determining distribution of yeasts in kefir grains and viili starters is shown in

Figure 1. Most steps in the flowchart will be explicated in the sections that follow.

Kefir grains and viili starters

Kefir grains and viili starters were collected from Shinchu and Taipei

5 respectively, two cities in northern Taiwan (Lin et al., 1999). In the laboratory, they were propagated at 20º C for 20 hours with twice-or thrice-weekly transfers in sterilized milk, and kept at 4º C for short-term and –80º C for long-term storage.

Isolation and enumeration of microorganisms

Ten grams each of kefir grains and viili starters were homogenized in 90mL of sterile saline solution in a Stomacher (Laboratory blender stomacher 400, Seward,

England) until no grain particles were observed. Concentrations of the viable yeasts in suspensions were obtained by serial plating dilutions. Yeasts and molds were examined on potato dextrose agar (PDA, Difco, Detroit, MI, USA), with 100 ppm chlortetracycline (Sigma, St. Louis, MO, USA) to inhibit the growth of other bacteria.

The plates were incubated at 25º C for three days (Lin et al., 1999). The colonies resulting from samples were counted. The Harrison disc method was used to select colonies from each plate, which were picked up and purified by streaking on the same medium.

Reference strains

The reference strains used for this study, including Kluyveromyces marxianus var. marxianus (BCRC 20330), Scaccharomyes cerevisia (BCRC 21685), Saccaromyces turicensis (BCRC 22968), Saccharomyces unisporus (BCRC 21975) and Pichia fermentans var. fermentans (BCRC 22090), were obtained from the Bioresource

Collection and Research Center of the Food Industry Research and Development

Institute in Hsinchu, Taiwan.

The Harrison disc method

The Harrison disc method was adopted from Harrigan (1998). This method was used to determine the prevalent microbes that developed on each dilution and to select

6 representative colonies from each plate in a random statistical manner for further purification and identification. The Harrison disc method can calculate the distribution of various microorganisms present in a sample.

DNA isolation

Ten grams each of kefir grains and viili starters were homogenized in a

Stomacher as previously described. One gram each of kefir grain solution, viili starter solution, or their isolated yeasts was kept at 4º C and 5000×g for 10 min to collect cells. The pellets were suspended in 500μL sorbitol reaction solution [1M sorbitol

(Merck, Darmstadt, Germany), 100mM EDTA (Merck), 14 mM β-mercaptoethanol

(Merck), 200 U lyticase (Merck)] and incubated at 30º C for 30 min prior to centrifuging at 5000×g for 5 min. The pellets were subjected to DNA extraction using the Blood and Tissue Genomic DNA Extraction Miniprep System

(VIOGENE-BIOTEK Co., Taipei, Taiwan).

API 20C system

The API 20C system (bioMérieux, Marcy l’Etoile, France) performs 21 assimilation tests for carbohydrates and includes a database with 47 different species.

All yeast identification procedures were conducted in accordance with the manufacturer’s instructions. The reactions were examined visually and determined to be positive or negative based on the presence or absence of turbidity in the carbohydrate wells.

Denaturing gradient gel electrophoresis

1. DNA amplification

The DNA amplification was modified using the method of Cocolin et al. (2002).

The D1 region of the 26S rRNA gene was amplified by PCR using the primers

7 NL1GC (5’-GCG GGC CGC GCG ACC GCC GGG ACG CGC GAG CCG GCG

GCG GGC CAT ATC AAT AAG GGG AGG AAA AG-3’) (the GC clamp is underlined) and a reversed primer LS2 (5’-ATT CCC AAA CAA CTC GAC TC-3’)

(Cocolin et al., 2000). PCR was carried out on a total volume of 50μL containing 20 mM Tris HCl (Sigma), 10 mM KCl (Sigma), 2 mM MgCl2 (Merck), 0.1 mM dNTPs

(Promega, Madison, WI, USA), 0.2 mM of the primers, 1.25 U Taq-polymerase

(Yeastern Biotech, Taipei, Taiwan) and 1μL of the extracted DNA.

The PCR products were generated using an initial denaturation step of five minutes at 95º C followed by 30 cycles of denaturation at 95º C for 60 seconds, annealing at 52º C for 45 seconds, and elongation at 72º C for 60 seconds. A final chain extension was done for eight minutes at 72º C. Amplified products were run on a 2% agarose gel (Nippon Gene Co., Tokyo, Japan), stained with ethidium bromide

(Fluka & Riedel, St. Gallen, Switzerland) and visualized under UV light.

2. Denaturing gradient gel electrophoresis

The PCR fragments were separated by denaturing gradient gel electrophoresis

(DGGE) using the BioRad DCode Universal Mutation Detection System (Bio-Rad

Laboratories, Hercules, CA, USA). Separation of the PCR amplicons was obtained by the direct application of 20μL of PCR products onto 9% (w/v) polyacrylamide gels

(Bio-Rad) in 50x TAE buffer containing a linear denaturant gradient of between 30% and 55% (100% corresponds to 7 M urea (Merck) and 40% w/v formamide (Merck).

Electrophoresis was performed with a constant voltage of 200V at 60º C for 3.5 hours, the gel was stained with ethidium bromide (Fluka & Riedel) for 15 minutes and the fragments were visualized under UV light. The DGGE reference markers were amplicons obtained from five yeast species in equal amounts.

8 Sequencing of PCR-amplified 26S rDNA region

The identification was performed by sequencing the 5’ end of the 26S rDNA encompassing the D1 and D2 expansion domains using the primers NL1 (5’-GCAT

ATC AAT AAG GGG AGG AAA AG-3’) and a reversed primer NL4

(5’-GGTCCGTGTTTCAAGACGG-3’) (O’Donnell, 1993). All PCR products amplified with primers designed for yeasts were sequenced for species-specific confirmation. The PCR products were cleaned with the Concert Rapid PCR

Purification system (QIAquick Gel Extraction Kit, Qiagen Inc., Valencia, CA, USA) and DNA concentration was checked on a spectrophotometer. These DNA products were sent for sequencing. The DNA sequences were aligned in Vector NTI Advance 9

(Invitrogen Co., Carlsbad, CA, USA) and a hierarchy of similar sequences were obtained.

Results and discussion

Culture-dependent and culture-independent methods

The yeast counts for kefir grains and viili starters were 7.36 log CFU/g and 7.43 log CFU/g, respectively. Using the Harrison disc method 124 colonies were isolated in kefir grains and 91 colonies in viili starters. These were classified by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). PCR-DGGE profiles (Figure 2) indicated that three different yeast stains (named HY1, HY2 and

HY3) were found in kefir grains, and another three strains (named TY1, TY2 and

TY3) were observed in viili starters.

Since many plating procedures are partially selective and exclude part of the microbial community, DNA of yeast strains in kefir grains and viili starters were extracted and directly identified by PCR-DGGE. Results (Figure 3) indicated that

9 kefir grains contained HY1 and HY2 stains. HY3 located previously by a culture-dependent method was not identified. Similarly, only two yeast strains were found in viili starters (TY1 and TY3); TY2 was not discovered.

Culture-independent methods directly using PCR-DGGE did not require microorganisms to be cultivated in specific media, thus one might expect to find more yeast strains in kefir grains and viili starters using PCR-DGGE. On the contrary, fewer varieties of yeast strains were identified directly by PCR-DGGE than were found by a combination of culture-dependent methods. Possible explanations might be that the cell numbers of certain yeast strains were lower than the detection limit of

DGGE or that high quantities of competitor templates were present (Theunissen et al.,

2005). Cocolin et al. (2000) proved that the presence of a DGGE band represents a yeast population above a minimum threshold value of 103 cells /ml and thus identifies only the predominant yeast populations in these starters. Moreover, species present at higher populations in the mixture will give greater amounts of template DNA, and, therefore have a higher probability of detection (Prakitchaiwattana et al., 2004).

Identification of isolates

For further identification of yeast strains isolated from both cultures, the API

20C system, PCR-DGGE and DNA sequencing methods were applied. The API 20C system performs 21 assimilation tests for carbohydrates. Results in Table 1 indicate that, except for HY3 and TY3, yeasts isolated from both starters could digest

D-galactose. On the other hand, only HY1 from kefir grains and TY1 from viili starters could metabolize D-lactose. Both HY1 and TY1 showed very similar assimilation of carbohydrates and were identified as Candida famata, while TY3 and

HY3 were determined to be Rhodotorula minua. HY2 and TY2 were not identified by

10 this system, however. The reports on the accuracy of identification for the API 20C system have varied from 88% (Davey et al., 1995) to 99% (Fenn et al., 1994).

Although this method is effective for identifying relatively common yeasts, its application is more limited for accurate identification of less frequently recovered taxa

(Ramani et al., 1998). Furthermore, visual interpretation of this method was sometimes difficult and required greater experience.

The results of yeast identification by DGGE are shown in Figure 4. The expected

250-bp (including 40 GC clamp) PCR fragments were successfully amplified from all reference and sample strains. As reported, Kluyveromyces marxianus var. marxianus

(lane 1), Scaccharomyes cerevisia (lane 2), Saccaromyces turicensis (lane 3),

Saccharomyces unisporus (lane 4), and Pichia fermentans var. fermentans (lane 5), gave specific patterns in the DGGE gel that could be easily used for identification purposes. Pichia fermentans var. fermentans presented several DGGE bands due to the amplification of multiple copies of the ribosomal genes that would allow precise species identification by DGGE, as previously described by Cocolin et al. (2001).

The DGGE patterns obtained by PCR-based DGGE analysis of kefir grains

(lanes 7-9) and viili starters (lanes 10-12) are also shown in Figure 4. Band positions in the unknown sample lane were compared visually with reference band positions.

Results indicated that kefir grains contained Kluyveromyces marxianus var. marxianus (HY1, lane 7) and Saccharomyces turicensis (HY2, lane 8), whereas viili starters included Kluyveromyces marxianus var. marxianus (TY1, lane 10) and

Saccharomyces unisporus (TY2, lane 11). All strains belonging to the same species showed the same migration in the gel. The DGGE patterns of HY3 and TY3 were very similar to the patterns of Pichia fermentans var. fermentans (lane 5), forming

11 several lower bands but the migration of bands was different. Further identification of

HY3 and TY3 was necessary.

In order to verify the PCR-DGGE results, the PCR-amplified D1/D2 domain of

26S rDNA region was sequenced. After alignment was carried out in BLAST, 6 sequences generated from species-specific primers for identification of yeasts isolated from kefir grains and viili starters showed 99-100% homology (Table 2) with the sequences retrieved from Genbank accession numbers. No differences were observed between results from DNA sequencing and PCR-DGGE detecting yeast strains, but a distinct result was found using API. These four yeasts (Kluyveromyces marxianus,

Saccharomyces turicensis, Pichia fermentans, Saccharomyces unisporus) identified by PCR-DGGE and sequencing were not listed in the API 20C data base, but the biocodes of these isolates generated patterns similar to other strains listed in the API

20C and yielded a false identification. In addition, although HY3 and TY3 were determined to be Pichia fermentans, the subspecies varied from the reference strain.

In spite of this, it was possible to unequivocally identify different species from the same genera with DGGE analysis due to the different patterns obtained in the gel

(Cocolin et al., 2002). In our study, the specific migrations allowed for easy and rapid identification at the subspecies level, of Pichia fermentans and Pichia fermentans var. fermentans.

The Kluyveromyces marxianus isolated from our kefir grains was also found in kefir grains from Austria (Rohm et al., 1992) and Switzerland (Wyder and Puhan,

1997). Other stains identified Saccharomyces turicensis and Pichia fermentans in

European kefir grains as well (Wyder et al., 1999). Farnworth and Mainville (2003) reviewed the results from different original kefir grains and concluded that the list of

12 microorganisms in kefir grains even from different parts of the world would not be very extensive, as a contaminant species would probably not survive due to the production of compounds by the symbiotic flora of kefir. On the other hand, Witthuhn et al. (2004) isolated and characterized the microbial populations of eight different kefir grains from South Africa and none of these yeast stains were identified in

Taiwanese kefir. These differences could be explained mainly by the different origins and identification methods. Most traditional viili cultures also contain yeast strains

(Kontusaari et al., 1985; Shurtleff and Aoyagi, 2004), but these authors did not specify the various strains.

Distribution

The Harrison disc method used for random statistical selection of representative colonies allowed for the calculation of the percentage distribution of the yeast found in kefir grains and viili starters. Figure 5 depicts the percentage of the prevalent yeast population present in kefir grains and viili starters. Kluyveromyces marxianus accounted for 76% of the total isolates, constituting the most dominant yeast found in kefir grains, followed by Saccharomyces turicensis (22%) and Pichia fermentans

(2%). In viili starter samples, Kluyveromyces marxianus accounted for 58% of the total isolates, followed by Pichia fermentans (31%) and Saccharomyces unisporus

(11%). Since Kluyveromyces marxianus can utilize both lactose and galactose as its carbon source (Table 1), this yeast can multiply well in milk. This may explain why this strain was the primary yeast in both culture samples. Both Pichia fermentans and

Saccharomyces unisporus, the least common yeasts isolated in kefir grains and viili starters respectively, could not be identified by culture-independent methods using

PCR-DGGE.

13

Conclusion

Briefly, combination of culture-dependent method with PCR-DGGE and sequencing could successfully identify four yeast strains from both cultures in Taiwan.

Kluyveromyces marxianus, Saccharomyces turicensis and Pichia fermentans were found in Taiwanese kefir grains with 76%, 22% and 2% distribution, respectively, while Kluyveromyces marxianus, Saccharomyces unisporus and Pichia fermentans were identified in viili starters with 58%, 11% and 31% distribution, correspondingly.

Furthermore, the culture-free method was also applied to identify the viili and kefir yeasts using DGGE. Pichia fermentans in kefir grains and Saccharomyces unisporus in viili starter were not identified. These results suggested that, in samples containing multiple species, PCR-DGGE might fail to detect some species.

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19

Figure 1 Schematic representation of the experimental approaches used for the analysis of the yeasts in Taiwanese kefir grains and viili starters.

20

Figure 2. Classification of yeast strains isolated from kefir grains (A) and viili starters (B) by PCR-DGGE.

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Figure 3. DGGE profiles of the PCR products obtained from the DNA extracted directly from kefir grains and viili starters. Lane 1, HY1 strain; Lane 2, HY2 strain; Lane 3, HY3 strain; Lane 4, TY1 strain; Lane 5, TY2 strain; Lane 6, TY3 strain; Lane 7 and 8, viili starter; Lane 9 and 10, kefir grain.

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Figure 4. DGGE profiles of the yeast strains using a denaturing gradient from 30% to 55% of urea and formamide. Lane 1, Kluyveromyces marxianus var. marxianus BCRC 20330; Lane 2, Scaccharomyes cerevisia BCRC 21685; Lane 3, Saccaromyces turicensis BCRC 22968; Lane 4, Saccharomyces unisporus BCRC 21975; Lane 5, Pichia fermentans var. fermentans BCRC 22090; Lane 6, Mixed reference strains; Lane 7, HY1 starin; Lane 8, HY2 strain; Lane 9, HY3 strain; Lane 10, TY1 strain; Lane 11, TY2 strain; Lane 12, TY3 strain.

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Figure 5. The percentage of the prevalent yeast population present in kefir grains (A) and viili starters (B) determined using Harrison’s disc method and PCR-DGGE.

24 Table 1 Identification of yeast strains isolated from starters by biochemical method Sample kefir grains viili starters Strain No. HY1 HY2 HY3 TY1 TY2 TY3 D-Glucose ＋ ＋ ＋ ＋ ＋ ＋

Glycerol ＋ － ＋ ＋ － ＋

Calcium-2-keto-gluconate ＋ － ＋ ＋ － ＋

L-Arabinose ＋ － － ＋ － －

D-Xylose ＋ － ＋ ＋ － ＋

Adonitol ＋ － － ＋ － －

Xylitol ＋ － － ＋ － －

D-Galactose ＋ ＋ － ＋ ＋ －

Inositol － － － － － －

D-Sorbitol ＋ － － ＋ － － Methyl-α ＋ ＋ － ＋ － －

D-Glucopyranoside

N-Acetyl-Gluco-samine ＋ ＋ ＋ ＋ － ＋

D-Cellobiose ＋ － ＋ ＋ － ＋

D-Lactose ＋ － － ＋ － －

D-Maltose ＋ ＋ － ＋ － －

Sucrose ＋ ＋ － ＋ － －

D-Trehalose － ＋ － ＋ － －

D-Melezitose ＋ － － ＋ － －

D-Raffinose ＋ ＋ － ＋ － －

Identified to be Candida -- Rhodotorula C. -- Rhodotorula (API kit) famata minua famata minua ID % 99.9 92.8 98.2 92.8

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Table 2. Sequences information from the 26S rDNA PCR product obtained from the yeast strains isolated from kefir grains and viili starters Strain Number Closest relative % Identitya Accession number kefir grain HY1 Kluyveromyces marxianus 100 DQ139802 HY2 Saccharomyces turicensis 99 AF398485 HY3 Pichia fermentans 99 DQ377652 viili starter TY1 Kluyveromyces marxianus 100 DQ139802 TY2 Saccharomyces unisporus 99 AY007920 TY3 Pichia fermentans 99 DQ377652 aIdentical nucleotides percentage in the sequence obtained from the agarose band and the sequence obtained found in NCBI.

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