ISSN 10683674, Russian Agricultural Sciences, 2012, Vol. 38, No. 3, pp. 239–241. © Allerton Press, Inc., 2012.

Original Russian Text © S.A. Yurik, M.L. Filipenko, E.A. Khrapov, V.I. Semenikhin, 2012, published in Doklady Rossiiskoi Akademii Sel’skokhozyaistvennykh Nauk, 2012, No. 3,
pp. 45–47.

MICROBIOLOGY

Identification of Streptococcus thermophilus Used in Starter Cultures

in the Production of Hard Cheeses
S. A. Yurika, M. L. Filipenkob, E. A. Khrapovb, and V. I. Semenikhina
a
Siberian and Far Eastern Institute of Experimental Veterinary Science, Novosibirsk oblast, 630501 Russia
b
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk Russia
email: vsemenikhin@mail.ru
Received June 14, 2011

Abstract—A prototype developed by the method of revealing Streptococcus thermophilus in PCR with the aid
of synthetic oligonucleotide primers in starter cultures in the production of hard cheeses has specificity and
makes it possible to effectively reveal and identify S. thermophilus strains and cultures used in the dairy pro
cessing industry.

Keywords: oligonucleotide primers, PCR, Streptococcus thermophilus

DOI: 10.3103/S1068367412030196

Several methods are presently used for typing
Streptococcus thermophilus strains and isolates, the
main one being the method based on the accumula
tion of bacteria in a nutrient medium. To isolate lactic
acid streptococci, a suspension of a sample is prepared
on physiological solution and sterile milk is inocu
lated. The culture is heated at 40°C until the milk cur
dles. A study of the biological and biochemical prop
erties and comparison of the data obtained with the
data of differential Table 17.16 of Bergey’s Manual of
Determinative Bacteriology [1] allow typing the investi
gated bacteria and determining species affiliation. The
shortcomings of the given method include the need to
conduct numerous biochemical differentiating tests,
length of the testing process, and high cost.
Restriction fragment length polymorphism (RFLP)
is used for taxonomy, genetic mapping, and assess
ment of species affiliation. However, in the opinion of
the authors [2], this method cannot be recommended
for assessing species affiliation and degree of phyloge
netic relationship of S. thermophilus strains.
Another technique for determining species affilia
tion is the method of genotyping S. thermophilus by the
16S rNA gene. In this case, synthesis and sequencing
of the 16S rNA gene sequence are carried out. Then
the sequence of the given gene is compared with the
sequences characteristic for S. thermophilus from the
GenBank database. Among the shortcomings of this
method are the need for sequencing amplicons, com
paring each with the GenBank database, length of the
testing process and high cost, as well as the fact that
alignment of nucleotide sequences of investigated cul
tures with respect to this gene reveals 97–100% simi
larity not only to reference strains but also to Strepto
coccus salivarius, Streptococcus vestibularis, Enterococ
cus faecium, and Uncultured bacterium.
Methods based on revealing fragments of the
genome of various bacteria in biological material by
means of the polymerase chain reaction are presently
recognized as the most sensitive and specific. This
method allows detecting the pathogen at its very low
concentrations and shortening the length of investiga
tions by 8–10 times compared with bacteriological
methods. In connection with this, the purpose of our
investigations was to develop a test system for revealing
and identifying S. thermophilus with the aid of specific
synthetic oligonucleotide primers in PCR.
METHOD
We investigated 15 S. thermophilus cultures isolated
in different years in the Microbiology Laboratory of
the Siberian Cheese Industry Research Institute (Sib
NIIS), Siberian Branch, Russian Agricultural Acad
emy (Barnaul) from raw milk obtained from the Altai
and Barnaul industrial complexes and from dry bacte
rial starters from the Siberian Branch of the AllRus
sian Dairy Industry Research Institute (Omsk),
Ukrainian Meat and Dairy Industry Research Insti
tute, as well as obtained in different years from the col
lection of the AllRussian Butter and Cheese Industry
Research Institute (Uglich).
To extract DNA using 10% CTAB, we took suspen
sions of S. thermophilus strains and isolates cultured
and accumulated on nutrient broth from hydrolyzed
milk.
The search for specific primers was carried out on
the basis of a selected DNA fragment characteristic

239
240 YURIK et al.

1 2 3 4 5 6 7 8 M K– Bank Search.html). Final selection of primers was

based on the following criteria: high level of similarity
of DNA fragment to DNA of various S. thermophilus
strains, content of GC bases not less than 50% and
don’t form dimers, and are complementary to the
DNA sequence at the boundaries of the specific frag
ment. The sequences of oligonucleotide primers Stt1F
and Stt2R were determined using a DNA sequence
alignment algorithm in Alignment Service V.4.0 and
GENCNER programs [3], and the OLIGO 4.0 pro
gram was used for analyzing primers with respect to
level of free energy. Chemical synthesis of specific
primers was accomplished by the amidophosphate
method on an ASM102U automatic synthesizer
(Biosset Ltd, Novosibirsk) in the Department of
Fragment of investigated S. thermophilus DNA in PCR
Chemistry of Natural Compounds, State Research
with primers Stt1F and Stt2R: (1) 91; (2) 101; (3) 282; Center of Virology and Biotechnology Vektor. The
(4) 1244; (5) 1244M; (6) 1254; (7) 1294; (8) Dat; (M) 100 concentration of oligonucleotide primers in the
bp DNA Ladder (400 × 2) marker; (K–) negative control. mother liquor was determined by the spectrometric
method.
only for S. thermophilus during an analysis of complete The polymerase chain reaction was set up on BIS
genomes of reference S. thermophilus strains M105 thermal cyclers (Novosibirsk). From the results
we judged the size of the synthesized cDNA fragment
MG18311, CNRZ1066, and LMD9 given in the Gen migrating in 1.0% agarose gel at current strength 35–
Bank database (http://www.ncbi.nlm.nih.gov/Gen 40 mA during 30–40 min. As markers we used a 100 bp
DNA ladder (400 × 2). The results obtained were doc
umented by a digital camera and amplicon sequencing
Results of investigations to determine specificity of reaction was done with respect to two DNA strands with the use
with primers Stt1F and Stt2R
of the conventional Maniatis–Fritsch–Sambrook [4]
PCR with primers Stt1F and Maxam–Gilbert [5] procedures.
Culture
and Stt2R The analysis was considered positive if the size of
Staphylococcus aureus Negative the amplicon corresponded to the expected 655 bp
fragment size.
Staphylococcus albus ''
Streptococcus pyogenes ''
Streptococcus epidermitis '' RESULTS AND DISCUSSION
Escherihia coli '' Extraction of S. thermophilus DNA. During extrac
Salmonella Dublin '' tion from the bacterial culture, the cells were sedi
Proteus vulgaris ''
mented into a platelet by centrifugation for 1–2 min at
5000–7000 rpm. The supernatant was removed. We
Streptococcus thermophilus 1244 Positive added 50 µl of culture to 300 µl of 10% CTAB heated
(SibNIIS collection) to 80°C and stirred and incubated it at 80°C for
Streptococcus thermophilus 1254 '' 40 min. Then it was cooled to room temperature and
(SibNIIS collection)
extracted with a phenol:chloroform (1 : 1) mixture and
then with chloroform. We transferred the water phase
Streptococcus thermophilus D/I '' containing DNA to another test tube, added 1/10 vol
(SibNIIS collection) ume of 3 M sodium acetate, pH 4.8, and two volumes
Streptococcus thermophilus 282 '' of ethanol, and placed it in a freezer for 1 h at –20°C.
(SibNIIS collection)
Then it was centrifuged for 10 min at 13000 rpm, the
sediment was washed with 70% ethanol, dried at 37°C
Streptococcus thermophilus 101 '' for 25–30 min, and dissolved in 30–50 µl of auto
(SibNIIS collection) claved bidistilled water.
Streptococcus thermophilus CK9 '' Conduction of polymerase chain reaction. A PCR
(SibNIIS collection) mixture was prepared for each investigated DNA sam
Streptococcus thermophilus 91 '' ple: 10× standard buffer, pH 8.8; 2.5 mM dNTP; 0.15 µg
each of primers Stt1F and Stt2R; 0.5 units of Taq DNA
(SibNIIS collection) polymerase; 2 µl of DNA sample and up to 25 µl of
Distilled water Negative autoclaved bidistilled water.

RUSSIAN AGRICULTURAL SCIENCES Vol. 38 No. 3 2012

 IDENTIFICATION OF Streptococcus thermophilus
Temperature regime of conducting PCR. The ampli
fication program consisted of the following tempera
ture regimes: one cycle of heating the reaction mixture
at 95°C for 3 min; then 35 cycles of denaturation at
94°C for 0.2 min, annealing at 60°C for 0.2 min,
extension at 72°C for 0.4 min, and complete synthesis
at 72°C for 0.8 min.
Determination of the size of PCR products. The PCR
products were analyzed by electrophoresis in 0.5 ×
Trisborate buffer prepared from 5x TBE buffer. Elec
trophoresis was carried out at a voltage of 10 V/cm of
gel length until the stain penetrated at least 2.0–2.5 cm
of the gel from the start. The results of electrophoresis
were taken into account by examining the gel in ultra
violet light with wavelength 254 nm on a transillumi
nator. The PCR results were considered positive if the
PCR product corresponded to a 655 bp DNA frag
ment size (figure).
Determination of PCR specificity. The results of
investigations to determine the specificity of the reac
tion with primers Sttt1F and Stt2R (table) showed that
positive analyses of PCR products are obtained only
when a DNA fragment characteristic only for S. ther
mophilus was used as the template. The analyses were
negative when DNA of other bacteria was used.
To confirm the specificity of the PCRtested DNA
fragment characteristic only for S. thermophilus, a
fragment was turned out on a DNA template of
S. thermophilus strain 1244 (SibNIIS, Barnaul) and
sequenced. The nucleotide sequence of the DNA frag
ment being synthesized was analyzed by alignment
methods with other published sequences of complete
RUSSIAN AGRICULTURAL SCIENCES Vol. 38 No. 3
241
genomes of S. thermophilus reference strains
LMG18311, CNRZ1066, and LMD9 in the GenBank
database (http://www.ncbi.nlm.nih.gov/Search.html). It
was established that the examined nucleotide
sequence of S. thermophilus strain 1244 coincided with
the nucleotide sequence when the aforementioned
S. thermophilus reference strains were used as the tem
plate.
Thus, the developed method of revealing S. ther
mophilus in PCR by means of synthetic oligonucle
otide primers has specificity and allows effective reve
lation and identification of S. thermophilus strains and
cultures used in the dairy processing industry.
REFERENCES
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Krieg, N., Sneath, P., Staley, J., and Williams, S.
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2. Botina, S.G., Comparative Analysis of Genome Sizes
of Streptococcus thermophilus Strains, Botina, S.G.,
Piksasova, O.V., Tsygankov, Yu.D., and Sukhodolets, V.V.,
Genetika, 2007, vol. 43, no. 7, pp. 891–897.
3. Resenchuk, S.M., Alignment Service: Creation and
Processing of Alignments of Sequences of Unlimited
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