Probiotic Characteristics of Lactobacillus spp. Isolated - J-Stage

Food Sci. Technol. Res., 18 (1), 91 – 98, 2012

Probiotic Characteristics of Lactobacillus spp. Isolated from Fermented Milk Product Dahi Aijaz Hussain Soomro1* and Tariq Masud2 1

Institute of Food Sciences and Technology, Sindh Agriculture University Tando Jam, Pakistan

2

Department of Food Technology, PMAS-Arid Agriculture University, Rawalpindi, Pakistan

Received February 19, 2011; Accepted September 28, 2011 The aim of this study was to evaluate the probiotic potential of 35 strains of Lactobacillus spp. isolated from dahi a traditional fermented milk product. In order to select the candidate probiotic strains Lactobacillus spp. isolated belonged to the L. delbrueckii subsp. bulgaricus, L. acidophilus, L. casei and L. helveticus were examined for antimicrobial activity against selected pathogens, acid and bile tolerance and antibiotic susceptibility. It was observed that L. acidophilus LA 02 produced antimicrobial activities against the test strains and tolerated pH 2 and 3 and survived the bile salt concentration of 0.1, 0.2 and 0.3 %. It was resistant to vancomycin, erythromycin and chloramphenicol. This is the first report describing the probiotic properties of dairy isolates of Lactobacillus spp. in Pakistan. Keywords: acid, bile tolerance, antibiotic resistance, Lactobacillus acidophilus, probiotic culture, dahi

Introduction Traditionally fermented milks have been popular since ancient times. Dahi similar to yogurt is a popular traditional fermented milk product of Pakistan having its own microbiological properties. It is produced from buffalo milk by a traditional method using an indigenous non-descriptive starter culture (previously made dahi) containing unknown mixture of lactic acid bacteria (LAB). It is believed that dahi assists in digestion and cures intestinal disorders including diarrhea and dysentery. Historically, the major use of food fermentation by lactic acid bacteria (LAB) has been to prevent spoilage. However, the emergence of new pathogens, the resurgence of old ones, and development of antibiotic resistance have caused an increase in research on a group of LAB known as probiotics that could be potentially used to combat bacterial infections. Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host (WHO, 2001). Due to their perceived health benefits probiotic bacteria have been increasingly included in yoghurts and fermented milks during the past two decades. Probiotics, mainly members of genera Lactobacillus and Bifidobacterium, are normal resi*To whom correspondence should be addressed. E-mail: [email protected]

dents of the complex ecosystem of the gastrointestinal tract (Mitsuoka, 1992). These probiotic organisms are considered to be having prevention against gastrointestinal infections; by adherence to host cells, to exclude or reduce pathogenic bacteria and to produce acids, hydrogen peroxide and bacteriocins antagonistic to the growth of pathogens, and this ultimately reinforces the body’s natural defense mechanisms (Klein et al., 1998). Selection of promising probiotic bacteria must fulfill certain standards, and many in vitro tests are performed when screening for probiotic candidates. The main criteria for selecting probiotic strains are their acid and bile tolerance, survival through the gastrointestinal tract, ability to adhere to gut epithelial tissue, temporary colonization, antagonism against pathogens and good technological properties (O’Sullivan, 2001; Tuomola et al., 2001). As, for the safety assessment of new probiotic lactobacilli isolates, a careful evaluation of their antibiotic resistance patterns is also recommended (Danielsen and Wind, 2003). The aim of this study was to evaluate the probiotic potential of 35 strains of Lactobacillus spp. isolated from dahi a traditional fermented milk product of Pakistan region, in order to select the candidate probiotic strains for further investigation.

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Materials and Methods Isolation and identification of Lactobacillus species from dahi The study was conducted at Department of Food Technology, University of Arid Agriculture Rawalpindi, Pakistan. Fifty dahi samples were collected randomly from markets of Rawalpindi, Pakistan and were subjected to the study. The streak plate method was used to isolate the Lactobacillus spp. on solid selective media. A loopful of each dahi sample was streaked on MRS agar (Oxoid, Basingstoke, UK) plates and incubated at 37℃ for 24 − 48 h. After incubation the culture was observed for growth, single and isolated colonies were picked and sub-cultured on MRS agar (Oxoid, Basingstoke, UK) media and incubated at 37℃ for 24 − 48 h to obtain a pure culture of the isolates, simultaneously the smears were prepared and stained with Gram’s stain and examined under microscope for the staining character and morphology of the isolates. The identification was performed according to the morphological, cultural and biochemical characteristics as described by Collins and Lyne (1980). Bacterial isolates and growth conditions The isolated bacteria, of which strains were identified as 15 Lactobacillus delbrueckii subsp. bulgaricus, 10 Lactobacillus acidophilus, 5 Lactobacillus casei and 5 Lactobacillus helveticus were maintained in MRS broth (Oxoid, Basingstoke, UK) with 20% glycerol and stored at −20℃. Before use the isolates were propagated twice in MRS broth (Oxoid, Basingstoke, UK) at 37℃ under anaerobic conditions. All the indicator bacterial strains Escherichia coli, Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus and Salmonella enterica subsp enterica serovar Typhi (Salmonella Typhi) were provided by department of Pathology Army Medical College Rawalpindi and maintained at 4℃ in Nutrient agar (Oxoid, Basingstoke, UK) slants, and 24 h before start of the experiment were transferred to fresh broth. Detection of antimicrobial activity For detection of antimicrobial activity the paper disc method was used (Ohmomo et al., 2000). The Lactobacillus strains were grown anaerobically in MRS broth (Oxoid, Basingstoke, UK) at 37℃ for 16 h. The supernatants were collected by centrifugation at 13000 × g for 20 min at 4℃, the pH of the supernatants were adjusted to 6.5 with 1M NaOH and filter sterilized using a 0.45 μm porosity filters (Sartorius, Germany). 100 μL of indicator strains grown in Nutrient broth (Oxoid, Basingstoke, UK) were mixed with 3.5 ml of soft agar and were overlaid on the Nutrient agar (Oxoid, Basingstoke, UK) plates. The plates were incubated at 37℃ for 2 h. Then sterile filter paper discs measuring 6 mm diameter, thin type with an adsorbed aliquot of 20 μL of cell-free filter sterilized supernatant were placed on overlaid soft agar plates containing target strain. After incubation at 37℃ for 18 h the inhibitory activity was evalu-

A.H. Soomro & T. Masud

ated based on the formation of a clear zone around the paper disk. Acid tolerance Activated cultures of Lactobacillus strains were examined for acid tolerance at pH 2 and 3 according to the method described by Charteris et al. (1998a). Aliquots of 0.1ml were removed at 0 h, 1.5 h and 3 h for determination of viable counts. Total viable counts were determined by the pour plate method in MRS agar (Oxoid, Basingstoke, UK) after serial dilutions in buffered peptone-water (Oxoid, Basingstoke, UK) (0.1% bacteriological peptone, 0.85% NaCl). Plates were incubated anaerobically at 37℃ for 48 h. Bile tolerance For the determination of bile tolerance activated cultures were centrifuged at 2500 × g for 5 min and the pellet that was resuspended in 0.5 mL MRS broth (Oxoid, Basingstoke, UK) was used as inoculum. A volume of 5 ml MRS broth with 0.1 − 0.3% (w/v) oxgall (Difco) was inoculated with 50 μL of the inoculum. Aliquots of 0.1 mL were removed at 0 h and 3 h of incubation at 37℃ for total viable counts determination. Antibiotic susceptibility profile Antibiotic susceptibility was detected using the agar disk diffusion method as described by Bauer et al. (1966). All the tests were performed in Mueller-Hinton agar (Oxoid, Basingstoke, UK) media. Antibiotic discs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, kanamycin, vancomycin and penicillin (Oxoid, Basingstoke, UK) were applied to the surface of agar plates that has previously been air dried and were incubated at 37℃ overnight to determine the susceptibility to antibiotics. Results Detection of antimicrobial activity Among the isolated Lactobacillus spp. 15 isolates were identified phenotypically as L. delbrueckii subsp. bulgaricus, 10 L. acidophilus, 5 L. helveticus and 5 L. casei from dahi. These were screened for the production of antimicrobial activity against E. coli, B. cereus, E. faecalis, S. aureus and S. Typhi as indicator strains by using paper disc method. Ten isolates including 5 strains of L. acidophilus, 3 strains of L. delbruekii subsp. bulgaricus and 2 strain of L. casei (Table 1) showed antimicrobial activity against the indicator strains. Of these isolates, L. acidophilus LA 02 exhibited maximum zone of inhibition towards the gram-positive and gram-negative indicator bacteria including E. coli (11.5 mm), B. cereus (8.0 mm), E. faecalis (11.5mm), S. aureus (10.5 mm) and S. Typhi (8.0mm), followed by L. casei LC 04 that showed inhibition zones of 8, 7.5, 8, 7.5 and 4.5 mm against E. coli, B. cereus, E. faecalis, S. aureus and S. Typhi, respectively.

Probiotic Characteristics of Lactobacillus spp. Isolated from Fermented Milk Product Dahi

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Table 1. Antimicrobial activity produced by culture supernatants o f Lactobacillus isolates against test organisms. Isolate

E. coli

B. cereus

E. faecalis

S. aureus

S. Typhi

− + − − + − − − − +

− − − − − − − − − −

− ++ − − − − − − − +

− − − − − − − − − −

− − − − ++ − − − − −

− ++ − − ++ ++++ + ++ − −

− − − − + +++ − + − −

− − − − ++ ++++ + ++ − −

− ++ − − + ++++ ++ − − −

− + − − − +++ − + − −

− − − − −

− − − − −

− − − − −

− − − − −

− − − − −

− +++ − + −

− +++ − − −

− +++ − − −

− +++ − − −

− ++ − − −

L. delbrueckii subsp. bulgaricus Lb 1 Lb2 Lb 3 Lb 4 Lb 5 Lb 6 Lb 7 Lb 8 Lb 9 Lb 10 L. acidophilus LA J2 M1 M4 M6 ID6 LA 02 LA 05 LA10 F6 F11 L. helveticus LH 2 LH 3 LH 4 LH 5 LH 10 L. casei LC F76 LC 04 LC8 LC12 LC22

−, no visible inhibition, +, zone 1-3 mm, ++, zone 4-5 mm, +++, zone 6-8 mm, ++++, zone more than 8 mm

The other strains including L. acidophilus M1, ID 6, LA 05, LA 10, L. delbruekii subsp. bulgaricus Lb 2, Lb 5, Lb 10 and L. casei LC 12 showed limited inhibitory activity against indicator strains. Moreover, all the L. helveticus strains did not show any inhibition against the indicator bacteria. Acid tolerance Ten Lactobacillus strains exhibiting antimicrobial activity were further screened for acid tolerance. Most of the tested strains exhibited survival at pH 3 (Table 2). No significant reduction of the viable counts were observed after 1.5 h of incubation as compared to the initial inoculum, in all the tested strains. Whereas, after 3 h of incubation at pH 3 the viable counts of the majority of the strains were reduced. Five Lactobacillus strains (Lb2, Lb10, M1,

ID6, and LC 12) exhibited a significant reduction, while the viable counts of the strain LC12 were very low. On the contrary, the viability of the Lactobacillus strains was very low at pH 2 and only the L. acidophilus LA 02 was tolerant after 1.5 and 3h of incubation. After 3 h of incubation all the other strains exhibited significant reduction as compared to the initial inoculum. Bile tolerance Resistance to bile is an important characteristic that enables Lactobacillus to survive and grow in the intestinal tract. Only viable count of strain LA 02 was increased as compared to the inoculum, indicating that this strain was able to grow in the presence of 0.1 and 0.2 % bile. The survival of the isolated strains in oxgall showed that Lb 2,

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A.H. Soomro & T. Masud Table 2. Survival of Lactobacillus isolates under different pH conditions after 1.5 and 3 h incubation. The values are viable cell count log10 (CFU mL-1). Isolate

pH 2

pH 3

0h

1.5 h

Lb 2 Lb 5 Lb 10

7.66 ± 0.20 6.50 ± 0.26 7.20 ± 0.10

3.53 ± 0.15 3.56 ± 0.30 3.12 ± 0.15

M1 ID6 LA 02 LA 05 LA10

7.80 ± 0.17 7.66 ± 0.15 7.83 ± 0.05 7.56 ± 0.15 8.16 ± 0.20

3.20 ± 0.10 3.23 ± 0.15 6.90 ± 0.10 3.13 ± 0.15 4.06 ± 0.20

L. acidophilus 2.66 ± 0.15 2.03 ± 0.15 5.86 ± 0.05 2.80 ± 0.20 3.06 ± 0.25

4.96 ± 0.15 3.36 ± 0.15

L. casei 3.63 ± 0.11 2.20 ± 0.17

LC 04 LC12

7.40 ± 0.20 7.66 ± 0.11

3h

0h

1.5 h

3h

5.00 ± 0.15 5.23 ± 0.15 5.00 ± 0.20

3.20 ± 0.20 4.80 ± 0.20 3.86 ± 0.15

7.86 ± 0.15 7.63 ± 0.15 7.76 ± 0.05 7.66 ± 0.15 7.96 ± 0.11

5.60 ± 0.20 5.46 ± 0.05 7.26 ± 0.15 5.56 ± 0.15 6.23 ± 0.15

4.53 ± 0.15 4.10 ± 0.10 5.23 ± 0.15 4.16 ± 0.15 5.00 ± 0.10

7.83 ± 0.11 7.63 ± 0.15

6.36 ± 0.15 5.96 ± 0.15

5.36 ± 0.23 2.56 ± 0.20

L. delbrueckii subsp. bulgaricus 2.16 ± 0.11 7.60 ± 0.10 2.56 ± 0.20 6.70 ± 0.20 2.66 ± 0.15 7.30 ± 0.10

Each value in the table represents the mean value ± standard deviation (SD) from three replications.

Table 3. Survival of Lactobacillus isolates under different bile concentrations after 3 h incubation. The values are viable cell count log10 (CFU mL-1). Oxgall concentration (%) Isolate

0.1 0h

0.2 3h

0h

0.3 3h

0h

3h

7.36 ± 0.10 7.00 ± 0.26 6.90 ± 0.20

0 0 0

Lb 2 Lb 5 Lb10

7.39 ± 0.07 6.85 ± 0.08 7.10 ± 0.10

L. delbrueckii subsp. bulgaricus 3.58 ± 0.06 7.48 ± 0.14 2.58 ± 0.09 2.54 ± 0.07 6.95 ± 0.14 0 0 7.00 ± 0.34 0

M1 ID6 LA 02 LA 05 LA10

7.74 ± 0.11 7.69 ± 0.10 7.88 ± 0.10 7.67 ± 0.27 8.17 ± 0.16

3.71 ± 0.34 3.99 ± 0.19 8.20 ± 0.10 5.00 ± 0.20 4.90 ± 0.06

L. acidophilus 7.76 ± 0.11 7.58 ± 0.31 7.65 ± 0.05 7.48 ± 0.07 7.66 ± 0.02

0 2.80 ± 0.10 7.98 ± 0.01 3.00 ± 0.15 2.95 ± 0.16

7.68 ± 0.11 7.76 ± 0.50 7.68 ± 0.04 7.65 ± 0.06 7.78 ± 0.20

0 0 6.82 ± 0.12 2.20 ± 0.20 2.89 ± 0.11

LC 04 LC 12

7.54 ± 0.12 7.62 ± 0.26

7.04 ± 0.14 4.08 ± 0.24

L. casei 7.62 ± 0.20 7.75 ± 0.19

6.21 ± 0.10 0

7.72 ± 0.09 7.50 ± 0.32

5.54 ± 0.25 0

Each value in the table represents the mean value ± standard deviation (SD) from three replications.

Lb 5, Lb 10, M1, ID6 and LC 12 were not able to survive in the presence of 0.3 % bile (Table 3). Whereas, in 0.3% bile strain LA 02 survived the most but still the viable count was slightly reduced. Antibiotic susceptibility profile Results (Table 4) showed that all the L. delbrueckii subsp. bulgaricus strains were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin and penicillin whereas vancomycin

resistance was observed in Lb5, Lb6, Lb7 and Lb10. The L. acidophilus strains were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin and penicillin except LA J2 and LA02 were resistant to chloramphenicol and erythromycin. Among the L. helveticus all were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin and penicillin. All L. casei strains showed susceptibility to tetracycline, chloramphenicol, erythromycin and ampicillin.


However, the susceptiblity to β-lactam penicillin was observed in L. casei LC12 and LC22. None of the tested strains were found sensitive to the nucleic acid synthesis inhibitor ciprofloxacin and protein synthesis inhibitor kanamycin.

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Antagonistic properties of probiotic strains are essential in order to prevent the infection and ⁄ or invasion of pathogenic bacteria. In this screening of the antimicrobial activity of the lactobacilli five different pathogens E. coli, B. cereus, E. faecalis, S. aureus and S. typhi were selected as indicator strains (Table 1). Among the tested strains L. acidophilus LA 02 and L. casei LC 04 produced antagonistic effect against all of the indicator strains with L. acidophilus LA 02 showing the strongest activity. These results are in agreement with the

Discussion The study aims to assess probiotic characteristics of Lactobacillus spp., such as antimicrobial ability, acid and bile tolerance and antibiotic susceptibility of the isolates.

Table 4. Antibiotic Susceptibility Profile of Lactobacillus isolates. Isolate

Antibiotics used (μg/ disc) K 30

Te 30

Lb 1 Lb2 Lb 3 Lb 4 Lb 5 Lb 6 Lb 7 Lb 8 Lb 9 Lb 10

R R R R R R R R R R

S S S S S S S S S S

LA J2 M1 M4 M6 ID6 LA 02 LA 05 LA10 F6 F11

R R R R R R R R R R

S S S S S S S S S S

LH 2 LH 3 LH4 LH 5 LH10

R R R R R

LC F76 LC 04 LC8 LC12 LC22

R R R R R

C 30

E 15

AMP 10

CIP 5

P 10

Va 30

L. delbrueckii subsp. bulgaricus S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S

R R R R R R R R R R

S S S S S S S S S S

S S S S R R R S S R

R S S S S R S S S S

L. acidophilus R S S S S R S S S S

S S S S S S S S S S

R R R R R R R R R R

S S S S S S S S S S

R R R R R R R R R R

S S S S S

S S S S S

L. helveticus S S S S S

S S S S S

R R R R R

S S S S S

R R R R R

S S S S S

S S S S S

L. casei S S S S S

S S R S S

R R R R R

R R R S S

R R R R R

Whereas, S: Susceptible, R: Resistant, AMP 10: Ampicillin, CIP 5: Ciprofloxacin, C 30: Chloramphenicol, E 15: Erythromycin, Te 30: Tetracycline, K 30: Kanamycin, P 10: Penicillin, VA: Vancomycin

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findings of Zamfir et al. (1999) who were of the view that L. acidophilus IBB 801 showed inhibitory spectrum against E. coli and Salmonella panama. Similarly, Jacobsen et al. (1999) have reported that 20 out of 47 strains of Lactobacillus spp. inhibited the growth of both gram-positive and negative pathogenic bacteria. Furthermore, Fernandez et al. (2003) observed a strong inhibition expressed by two human strains of Lactobacillus spp. against the foodborne pathogens. The determination of the acid tolerance of starter and probiotic bacteria is important in order to predict strain performance during gastric transit. L. acidophilus, L. casei and L. delbrueckii subsp. bulgaricus strains seem to exhibit a large variation in their tolerance to an acid environment. The strains of L. acidophilus group have been reported to tolerate 2 h of incubation at pH 2 (Pereira and Gibson 2002), this is in agreement with the observation in present study that L. acidophilus LA 02 showed acid tolerance after 1.5 h at pH 2 and 3. As far as L. delbrueckii subsp. bulgaricus is concerned, the strains exhibited a moderate reduction at pH 2 for 3 h is some what in accordance to the findings of Pereira and Gibson (2002) that L. delbrueckii subsp. bulgaricus JCM 1002 exhibited a slight reduction after 2 h of incubation at pH 2. Generally, it was well documented that strains of L. acidophilus exhibit more acid and bile resistance than other lactic acid bacteria (Gilliland, 1979). The pH value 2 and 3 used in the present study for the selection of potentially probiotic strains is very selective and even though pH 3 is not the most common pH value of the human stomach, however, it assures the isolation of the acidtolerant strains. It was observed in this study that pH 2.0 allowed the L. acidophilus LA 02 to survive. Bile tolerance is one of the most essential criteria for a strain to be used as a probiotic culture (Gilliland, 1979). Bile salts are surface-active chemicals produced in the liver from the catabolism of cholesterol. Bile acids have been shown to inhibit microorganisms and their inhibitory activity is greater than organic acids (Robins-Browne et al., 1981). Generally, LAB isolated from the intestine, do not grow well in the media containing 0.15% bile acids. However, some LAB such as L. acidophilus and L. casei, are capable of surviving in the presence of bile due to their ability to deconjugate bile acids. Bile resistance and the ability of LAB to inhabit the intestinal tract appear to be correlated (Gilliland and Speck, 1977; Hood and Zottola, 1988). Most of the L. acidophilus strains are sensitive in the presence of 0.3% bile, as reported by other researchers (Gupta, et al., 1996; Jacobsen, et al., 1999). In this study two L. acidophilus strains were inhibited, and only three strains survived the 0.3 % bile concentration. From the two L. casei strains only one strains LC 04 exhibited decreased viable count in the presence of oxgall. How-

A.H. Soomro & T. Masud

ever, L. delbrueckii subsp. bulgaricus strains have been reported as sensitive to bile salts (Jacobsen, et al., 1999) which is in agreement with our results for the three strains tested. A key requirement for probiotic strains is that they should not carry transmissible antibiotic resistance genes. Ingestion of bacteria carrying such genes is undesirable as horizontal gene transfer to recipient bacteria in the gut could lead to the development of new antibiotic-resistant pathogens (Saarela, et al., 2000). In the present study all the strains were found susceptible to the inhibitors of cell wall synthesis ampicillin, with the exception of L. casei LC8 that was found resistant to this antibiotic. The results are in accordance with the data reported from previous studies indicating that most Lactobacillus strains are susceptible to β-lactams, especially to penicillin except L. casei LCF76, LC04 and LC8 (Charteris et al., 1998b; Danielsen and Wind, 2003). In this study the Lactobacillus strains were resistant to vancomycin, except L. delbrueckii subsp. bulgaricus Lb1, Lb2, Lb3, Lb4, Lb8 and Lb9. The vancomycin resistance in Lactobacillus spp. such as L. acidophilus, L. helveticus and L. casei has been reported to be intrinsic, chromosomally encoded and not inducible or transferable (Klein, et al., 2000; Saarela, et al., 2002). It was also observed that these lactobacilli strains were resistant to the inhibitors of protein synthesis kanamycin. Previous studies have also reported high level of resistance to aminoglycosides for all investigated lactobacilli (Charteris et al., 1998b; Danielsen and Wind, 2003; Arici et al., 2004). Lactobacilli may have a natural reduced susceptibility to aminoglycosides, perhaps due to low cell membrane permeability (Charteris et al., 1998b). All the strains were found susceptible to tetracycline, as the tetracycline resistance in Lactobacillus is considered as an acquired characteristic, which can be conjugally transferred to other genera of lactic acid bacteria and become a public health safety problem for potential probiotic strains (Gevers et al., 2003). The strains L. acidophilus LAJ2 and LA02 were resistant to chloramphenicol and erythromycin, while L. casei LC04 was resistant to chloramphenicol. Chloramphenicol and erythromycin are associated with acquired resistance (Testore et al., 2002). None of the tested strains were sensitive to the inhibitor of nucleic acid synthesis such as ciprofloxacin. High prevalence of ciproflaxin resistance among lactobacilli strains has been previously reported and it was believed to be a potentially natural occurring characteristic of lactobacilli (Katla et al., 2001; Μandar et al., 2001; Danielsen and Wind, 2003). Whereas, it was further observed that the differences in the degree of inhibition with various antibiotics were possibly due to their different mode of action on the cell components such as the cell wall, protein and DNA synthesis, DNA gyrase and RNA polymerase (Neu, 1992). Nowadays antibiotic resistance has


become a serious problem in treatment of infections caused by a variety of microorganisms due to the indiscriminate use of antibiotics in human and veterinary medicine, as well as in animal growth promoters. In conclusion, our results showed that L. acidophilus LA 02 has potential probiotic value and have the best characteristics to fulfill the criteria of a probiotic strain. Based on the overall performance of this bacterium it can be suggested that L. acidophilus LA 02 could be used as adjunct culture in yoghurt and as probiotic culture for dairy industry. In addition, it is recommended that this strain be further analyzed according to the selection criteria like stimulation of the immunological system and adhesion to the mucosa and/or intestinal epithelium.

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Gupta, P.K., Mital, B.K. and Garg, S.K. (1996). Characterization of Lactobacillus acidophilus strains for use as dietary adjunct. Inter. J. Food Microbiol., 29, 105-109. Hood, S.K. and Zottola, E.A. (1988). Effect of low pH on the ability of Lactobacillus acidophilus to survive and adhere to human intestinal cells. J. Food Sci., 53, 1514-1516. Jacobsen, C.N., Rosenfeldt-Nielsen, V., Hayford, A.E., Moller, P.L., Michaelsen, A., Paerregaard, K.F., Sandstrom, B., Tvede, M. and Jakobsen, M. (1999). Screening of pro79 biotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Appl. Environ. Microbiol., 65, 4949-4956. Katla, A.K., Kruse, H., Johnsen, G. and Herikstad, H. (2001). Antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products. Inter. J. Food Microbiol., 67, 147-52.

Acknowledgements

The authors acknowledge the enabling role of

Klein, C., Pack, A., Bonaparte, C. and Reuter, G. (1998). Taxonomy

the Higher Education Commission Islamabad, Pakistan and appreci-

and physiology of probiotic lactic acid bacteria. Inter. J. Food

ate its financial support through “Development of S&T Manpower through Indigenous PhD (300 Scholars).”

Microbiol., 41, 103-125. Mandar, R., Loivukene, K., Hutt, P., Karki, T. and Mikelsaar, M. (2001). Antibacterial susceptibility of intestinal lactobacilli of

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