070 - Rha'idah Madihah - Jurnal Internasional - Tekfer
070 - Rha'idah Madihah - Jurnal Internasional - Tekfer
070 - Rha'idah Madihah - Jurnal Internasional - Tekfer
Department of Food Science and Technology, College of Agriculture, Ferdowsi University of Mashhad,
Mashhad, Iran
Article history Abstract
Received: 30 August 2013 “Tarkhineh” is a traditional fermented food produced from a mixture of spontaneously fermented
Received in revised form: butter milk and wheat flour in Iran. Nine samples of Tarkhineh were collected from different
9 June 2014
rural areas in Kermanshah province, Iran. The isolates were grouped and identified using a
Accepted: 13 June 2014
combination of phenotypic and genotypic methods including repetitive extragenic palindromic
Keywords polymerase chain reaction (REP-PCR) fingerprinting, biochemical methods and carbohydrate
profiling and then evaluation the probiotic properties of them. According to the results these 54
Tarkhineh isolates belonged to Lactobacillus plantarum (19), Lactobacillus fermentum (17), Lactobacillus
Lactobacillus pentosus (9), Lactobacillus brevis (8) and Lactobacillus diolivorans (1) that profile bonding
Probiotic characteristics
from rep-PCR showed Lactobacillus plantarum have a high intra-species diversity. Media of pH
rep-PCR
= 2.0–7.0 and bile salt concentrations of 0.0-0.5% were used as stress conditions. Antibacterial
activity of the probiotic Lactobacillus was determined by means of the spot-on-lawn method.
In conclusion, our results showed that 3 strains have potential probiotic value that two of them
was Lactobacillus fermentum and one of them was Lactobacillus plantarum.
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*Corresponding author.
Email: [email protected]
2488 Vasiee et al./IFRJ 21(6): 2487-2492
MRS broth culture to pH 6 with 1 N NaOH and Table 1. Species and numbers of majority cultured
adding suspension of bile salts to obtain different microorganisms identified through different formulation
concentration (0.0, 0.1, 0.2, 0.3, 0.4, and 0.5%). of Tarkhineh using molecular methods and sequencing
Viable bacteria were determined after 24 and 48 h Species
Formulation
F1 F2 F3 Media Total
incubation at 37oC (Barakat et al., 2001). Lactobacillus plantarum 7 9 3 MRS 19
Lactobacillus fermentum 2 14 1 MRS 17
Lactobacillus pentosus 2 7 - MRS 9
Antibacterial activity Lactobacillus brevis
Lactobacillus diolivorans
4
-
4
-
-
1
MRS
MRS
8
1
The antibacterial activity of isolates was Formulation: F1: Tarkhineh with butter milk, F2: Tarkhineh with
High-fat yogurt, F3: Tarkhineh with Low-fat yogurt
determined by agar spot-on-lawn method. The
Table 2. Morphological, physiological and biochemical
indicator bacteria used in this study were Escherichia characteristics of isolated Lactobacillus
coli TISTR 780, Listeria innocua ATCC 33090 Biochemical Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus
DNA extraction
DNA extraction of isolates was performed Figure 2. (A): REP-PCR typing profiles obtained with
according to procedure of Denazist extraction DNA primer BoxA2R among the 19 Lb. plantarum strains or
isolates from Tarkhineh. (B): dendogram of similarity of
kit, Iran.
the different typing patterns clustered by the UPGMA
method using the Simple Matching coefficient.
Typing of isolates
All Lb. plantarum isolates were grouped In total, 54 isolates from Tarkhineh were identified
by rep-PCR typing using primer BoxA2R (5′- from the counting plates of MRS-agar (Table. 1).
ACGTGGTTTGAAGAGATTTTCG-3′) according These 54 isolates were classified into 5 groups based
to Edalatian et al. (2012). PCR amplifications were on biochemical tests and carbohydrate fermentations.
performed in Labcycler Gradient 011-101 (Sensquest Table 2 showed the biochemical tests of Lactobacillus
Goettingen, Germany). Amplification products from spp. in Tarkhineh. Among 54 Lactobacillus strains
rep-PCR were subjected to electrophoresis in 1.5% which were isolated from Tarkhineh, 19, 17, 9, 8
agarose gels in 1x TBE buffer for at 90 min and 75V and 1 isolates belonged to Lactobacillus plantarum,
(Tamang et al., 2005). Bands were visualized under Lactobacillus fermentum, Lactobacillus pentosus,
UV light after staining with DNA Green Viewer and Lactobacillus brevis and Lactobacillus diolivorans,
photographed. Pattern similarity was expressed via respectively. The Lb. plantarum and Lb. diolivorans
the simple matching (SM) coefficient, and patterns strains were not able to produce CO2 from glucose,
were clustered by the Un-weighted pair group method indicating they were homofermentative LAB. This
using arithmetic averages (UPGMA) (Edalatian et LAB group was only able to ferment glucose to lactic
al., 2012). acid. Whereas, Lb. fermentum, Lb. brevis and Lb.
pentosus were in the group of hetero-fermentative.
Results and Discussion LAB which they were able to ferment glucose to lactic
acid, ethanol/acetic acid, and CO2. Lb. plantarum, Lb.
Identification and typing of LAB species brevis and Lb. diolivorans strains were able to grow
All 54 strains isolated from Tarkhineh were at 10oC, but no growth at 45oC. While, Lb. fermentum
identified as Lactobacillus based on their morphology, and Lb. pentosus were able to grow at 45oC, but none
Gram staining, non-motility and catalase reaction. of the colonies grew at 10oC (Kam et al., 2012).
2490 Vasiee et al./IFRJ 21(6): 2487-2492
and 1% because its lag time is the shortest. Afsharian, Director of microbiology lab, and Nafiseh
Daavati, PhD student of food microbiology, for their
Detection of antibacterial activity sincere assistance for this study.
Lactic acid bacteria are well known producer
of antimicrobial compounds especially bacteriocins References
which have high antimicrobial activity (Aween et al.,
2012). Lactobacillus are known for their production Allameh, S.K., Daud, H., Yusoff, F.M., Saad, C.R. and Ideris,
of various antimicrobial compounds. The production A. 2012. Isolation, identification and characterization
of these compounds by intestinal microflora is one of of Leuconostoc mesenteroides as a new probiotic from
the most important mechanisms responsible for the intestine of snakehead fish (Channa striatus). African
Journal of Biotechnology 11(16): 3810-3816.
antagonistic phenomenon and therefore it is essential
Aween, M.M., Hassan, Z., Muhialdin, B.J., Noor, H.M.
to examine this property in isolates that candidates for and El-jamel, Y.A. 2012. Evaluation on Antibacterial
probiotic (Bilkova et al., 2011). The good probiotics Activity of Lactobacillus acidophilus strains isolated
should present their antimicrobial actions particularly from Honey. American Journal of Applied Sciences
to the pathogens in the GI system (Klayruang et al., 9(6): 807-817.
2008). In this study, Staphylococcus aureus, Listeria Barakat, O.S., Ibrahim, G., Tawfik, N., El-Kholy, W. and
innocua and Escherichia coli were used as the test Gad El-Rab, A. 2011. Identification and probiotic
bacteria because they are occasionally found as food characteristics of Lactobacillus strains isolated from
borne microorganisms that might cause gastroenteritis. traditional Domiati cheese. International Journal of
Three potentially probiotic Lactobacillus isolates Microbiology Research 3: 59-66.
Begley, M., Gahan, C.G. and Hill, C. 2005. The interaction
were subjected to antibacterial activity assay. The
between bacteria and bile. Federation of European
results are shown in table 3. The isolate no. B20 Microbiological Societies 29(4): 625-651.
showed the most antibacterial potency to S. aureus Behringer, M., Williams, G. and Oyarzabal, O. 2011.
and E. coli whereas the isolate no. B2 demonstrated Typing of Campylobacter jejuni and Campylobacter
the highest potency to L. innocua. The production of coli isolated from live broilers and retail broiler meat
organic acid and hydrogen peroxide by Lactobacilli by flaA-RFLP, MLST, PFGE and REP-PCR. Journal
was reported to inhibit both gram positive and gram of Microbiological Methods 84: 194–201.
negative bacteria, whereas bacteriocin affects only Bilkova, A., Sepova, H.K., Bukovsky, M. and Bezakova,
the growth of gram positive bacteria (Klayruang et L. 2011. Antibacterial potential of Lactobacilli isolated
al., 2008). from a lamb. Veterinarni Medicina 7(56): 319–324.
Boke, H., Aslim, B. and Alp, G. 2010. The role of resistance
to bile salts and acid tolerance of exopolysaccharides
Conclusion
(EPSS) produced by yogurt starter bacteria. Archives
of Biological Sciences 62(2): 323-328.
The present study concluded that Lactobacillus Dhanasekaran, D., Saha, S., Thajuddin, N. and
spp. was predominant in microflora of Tarkhineh. Panneerselvam, A. 2008. Probiotic effect of
Although the original Ingredient of this food is butter Lactobacillus isolates against bacterial pathogens in
milk, also we have used low and high-fat yogurt Claris orientalis. Facta Universitatis Series: Medicine
to investigate microbial populations and probiotic and Biology 15(3): 97-102.
characteristics and compare with the original sample Edalatian, M.R., Najafi, M.B.H., Mortazavi, A., Alegría,
that made with butter milk. Tarkhineh where local A., Nassiri, M.R., Bassami, M.R. and Mayo, B. 2012.
butter milk and high-fat yogurt was used, in terms Microbial diversity of the traditional Iranian cheeses
Lighvan and Koozeh, as revealed by poly-phasic
of population, diversity and probiotic characteristics
culturing and culture-independent approaches. Dairy
was richer than low-fat yogurt. According to the Science and Technology 92(1): 75-90.
results of acid test, bile salt tolerance and antibacterial Fernandez, M., Boris, S. and Barbes, C. 2003. Probiotic
activity we can conclude these 3 isolates, including properties of human Lactobacilli strains to be used
Lb. fermentum (2 isolate) and Lb. plantarum (1 in the gastrointestinal tract. Journal of Applied
isolate), have good Characteristics to be considered Microbiology 94(3): 449-455.
as probiotic bacteria. However, other tests such as Gevers, D., Huys, G. and Swings, J. 2001. Applicability
antibiotic resistance and adhesion properties better to of rep-PCR fingerprinting for identification of
be done. Lactobacillus species. Federation of European
Microbiological Societies 205(1): 31-36.
Acknowledgments Hassanzadazar, H., Ehsani, A., Mardani, K. and Hesari,
J. 2012. Investigation of antibacterial, acid and bile
tolerance properties of Lactobacilli isolated from
The authors thank Professor Mohammd Reza Koozeh cheese. Veterinary Research Forum 3 (3):
Nassiri, Department of Animal Science, Shahnaz 181-185.
2492 Vasiee et al./IFRJ 21(6): 2487-2492
Hoque, M., Akter, F., Hossain, K., Rahman, M., Billah, Food Biotechnology 9(12) 12-28.
M. and Islam, K. 2010. Isolation, Identification and Tamang, J.P., Tamang, B., Schillinger, U., Franz, C.M.,
Analysis of probiotic properties of Lactobacillus spp. Gores, M. and Holzapfel, W.H. 2005. Identification
from selective regional yoghurts. World Journal of of predominant Lactic Acid Bacteria isolated from
Dairy and Food Sciences 5(1): 39-46. traditionally fermented vegetable products of the
Hutari, A., Jaseem, W.S., Hamid, A.A. and Yusoff, Eastern Himalayas. International Journal of Food
W.M.W. 2011. Screening of Lactobacillus strains Microbiology 105(3): 347-356.
against Salmonella both isolated from Malaysian free-
range chicken intestine for use as probiotic. Sains
Malaysiana 40(10): 1115-1122.
Iñiguez-Palomares, C., Pérez-Morales, R. and Acedo-
Félix, E. 2007. Evaluation of probiotic properties in
Lactobacillus isolated from small intestine of piglets.
Revista Latinoamericana de Microbiología 49(3-4):
46-54.
Kam, W., Wan-Aida, W. and Sahilah, A. 2012. Identification
of predominant Lactobacillus species in liquid
sourdough fermentation. International Food Research
Journal 19(4): 1739-1743.
Kesmen, Z., Yetiman, A.E., Gulluce, A., Kacmaz, N.,
Sagdic, O., Cetin, B. and Yetim, H. 2012. Combination
of culture-dependent and culture-independent
molecular methods for the determination of lactic
microbiota in Sucuk. International Journal of Food
Microbiology 153(3): 428-435.
Kisworo, D., Elegado, F.B. and Barraquio, V.L. 2008.
Phenotypic and genotypic characterization of probiotic
bacteria isolated from probiotic dairy products in the
Philippines. Philippine Journal of Science 137(1): 77-
83.
Klayraung, S., Viernstein, H., Sirithunyalug, J. and
Okonogi, S. 2008. Probiotic properties of Lactobacilli
isolated from Thai traditional food. Scientia
Pharmaceutica 76(3): 485-503.
Liu, C., Zhang, Z.Y., Dong, K., Yuan, J.P. and Guo, X.K.
2009. Antibiotic resistance of probiotic strains of
Lactic Acid Bacteria isolated from marketed foods
and drugs. Biomedical and Environmental Sciences
22(5): 401-412.
Mourad, K. and Nour-Eddine, K. 2006. In vitro preselection
criteria for probiotic Lactobacillus plantarum strains
of fermented olives origin. International Journal of
Probiotics and Prebiotics 1(1): 27-32.
Sahadeva, R., Leong, S., Chua, K., Tan, C., Chan, H., Tong,
E., Wong, S. and Chan, H. 2011. Survival of commercial
probiotic strains to pH and bile. International Food
Research Journal 18(4): 1515-1522.
Sengun, I.Y., Nielsen, D.S., Karapinar, M. and Jakobsen,
M. 2009. Identification of Lactic Acid Bacteria
isolated from Tarhana, a traditional Turkish fermented
food. International Journal of Food Microbiology
135(2): 105-111.
Setyawardani, T., Rahayu, W., Maheswari, R. and Palupi, N.
2011. Identification and Characterization of Probiotic
Lactic Acid Bacteria Isolated from Indigenous Goat
Milk. Animal Production 13(1): 57-63.
Sieladie, D.V., Zambou, N.F., Kaktcham, P.M., Cresci,
A. and Fonteh, F. 2011. Probiotic properties of
Lactobacilli strains isolated from raw cow milk in the
western highlands of Cameroon. Innovative Romanian