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.
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.
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.
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.
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.
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.
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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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.
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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
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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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