http://informahealthcare.com/ijf ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, 2014; 65(5): 582–588 ! 2014 Informa UK Ltd. DOI: 10.3109/09637486.2014.880666
IN VITRO AND ANIMAL STUDIES
Some probiotic and antibacterial properties of Lactobacillus acidophilus cultured from dahi a native milk product Talat Mahmood, Tariq Masud, and Asma Sohail
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Department of Food Technology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan
Abstract
Keywords
In this study, different strains of Lactobacillus acidophilus from dahi were analyzed for certain probiotic and antibacterial properties. Initially, these strains were confirmed by the amplification of 16S rRNA regions and then screened for antibacterial activities against food borne pathogens. The phenotypic relationship between apparent antibacterial activity and cell wall proteins were established by cluster analysis. It was observed that those strains, which have prominent bands having size 22–25 kDa possess antibacterial activity. On the basis of wide spectrum of killing pattern, a strain LA06FT was further characterized that showed no change in its behavior when subjected to the antibiotic protected environment and grow well in acidbile conditions. The bacteriocin produced by this strain has specific antibacterial activity of 5369.13 AU mg 1. It remained stable at 60–90 C and pH range of 4.5–6.5 while proteolytic enzymes inactivate the bacteriocin that confirm its proteinic nature having molecular weight of 8.5 kDa.
Antibacterial activity, dahi, Lactobacillus acidophilus, plasmid profile, probiotic culture, SDS-PAGE
Introduction Dahi is one of the fermented milk products in south Asian countries like Bangladesh, India and Pakistan and supposed to be a natural remedy for gastrointestinal problems. This character is due to the presence of probiotic lactic acid bacteria (LAB) like Lactobacillus delbrueckii ssp., L. bulgaricus, L. helveticus, L. acidophilus and S. thermophiles. Among them, L. acidophilus presents the predominant group of LAB that has technological advantages over other bacteria (Ivanova et al., 2000). They can be used as starter culture in fermented dairy products and also enhance their shelf life through anti-microbial peptides with some novel features (Dewan & Tamang, 2007). In addition to acidification of milk, the production of some secondary metabolites like antibacterial peptides and other probiotic features were also related to these bacteria (Bhowmik et al., 2009). Application of these compounds to eradicate food pathogens without any toxic or other adverse effects has great significance as natural preservatives (Karthikeyan & Santhosh, 2009). Bacteriocins are mostly ribosomally synthesized peptides, having lethal effect against the pathogenic strains and this characteristic makes them parallel to chemical preservatives (Todorov, 2008). On the structural and molecular bases, bacteriocin of LAB are divided into four classes (Klaenhammer, 1993) and according to this classification, aciodocin belonged to ClassII non-lanthionines that show some resistant to heat and pH, but
Correspondence: Tariq Masud, Department of Food Technology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan. Tel: +92-51-9290694. E-mail: [email protected]
History Received 26 March 2013 Revised 17 October 2013 Accepted 31 December 2013 Published online 1 April 2014
sensitive to protolytic enzymes having low molecular mass (Mobarez et al., 2008). Role of cell proteins is highly important in the expression of antagonistic and probiotic characteristics (Floros et al., 2012). Many research work has been carried out to assess the antibacterial potential of LAB and the application of their metabolites as novel food preservatives (Ghazi et al., 2009). However, in this article we describe for the first time that SDS proteins with band size of 22–25 kDa play a major role in the development of the homological features. In the consequence, a single strain was identified with improved functional attributes and its bacteriocin was characterized. So, the present study was designed for the investigation of probiotic and antibacterial features of some wild strains of L. acidophilus with aims that new strains would help to introduce genetically diversifying bacteria of dahi having worth mentioning qualities. Furthermore, their antibacterial compound would be purified and could be applied as natural preservatives having no harm full effect on consumer since it was isolated from probiotic culture.
Materials and methods Isolation and identification of L. acidophilus Dahi samples were obtained from the local market. Producer used undefined starter, a part of mature product of previous batch. Thirteen L. acidophilus strains were obtained by inoculating on the selected media MRS (deMan Regosa and Sharp, England) additionally supplemented with 2% lactose. The microbial isolates were identified by morphological and biochemical testing. Lactobacillus acidophilus isolates were confirmed by the amplification of partial 16S rRNA regions (757 bp) using a specific primer.
DOI: 10.3109/09637486.2014.880666
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Amplification For the amplification of 16S rRNA regions of L. acidophilus, primers (GCTGAACCAACAGATTCAC and ACTACCAGGGT ATCTAATCC) were designed and obtained according to the sequence published in the National Center for Biotechnology Information (NCBI). Ten mircoliters of reaction mixture was prepared by adding 3.8 mL of nanopure water, 1.0 mL of 10 buffer, 0.2 mL of 5 mM dNTPs, 0.6 mL of 25 Mm MgCl2, 1 mL forward primer and 1 mL of reverse primer, 2 mL of DNA (diluted to 1: 49) and 0.4 mL of Taq polymerase. The PCR cycling programs used were: Temperature1 at 94 C for 2 min, Temperature2 at 94 C for 30 s, Temperature3 at 50 C for 30 s, Temperature4 at 72 C for 45 s and Temperature5 at 72 C for 20 min. PCR cycles were maintained at 94 C for 2 min, followed by 35 cycles of 94 C for 30 s, annealing temperature 50 C for 30 s, 72 C for 45 s and a final extension temperature of 72 C for 20 min. The ready samples were run on the agarose gel of 1.2% concentration in 1 TBE working buffer of pH 8.0 (5 stock buffer was prepared by dissolving 13.5 g Tris Base, 6.875 g boric acid in 20 mL of 0.5M EDTA). Horizontal mini prep. electrophoresis (WEALTEC Corp., Sparks, NV ) was used at 70 volts for 1.5 h. The sample was mixed with 3 mL bromophenol blue tracking dye and loaded into the well. For genomic DNA Hind III DNA MW marker of 23 130–564 bp was used while 100 Base Pair Plus DNA Ladder marker of 3000–100 bp was applied as standard for PCR. Ethidium bromide (0.5 mg/mL) was used for the illumination of band that was observed under UV light. Bacterial strains and growth media Different strains L. acidophilus were grown on the selected media of MRS. The indicator strains for antibacterial assay were Listeria monocytogenes ATCC 19115, grown on Listeria enrichment broth base (CM0863 Oxoid, Hampshire England), Staphylococcus aureus ATCC 6538, grown on Staphylococcus Medium No110 (Hampshire England), Pseudomonas aeruginosa ATCC 25923 and Escherichia coli-ATCC25922 (Brain Heart Infusion (BHI) medium Oxoid Ltd, Baingstoke, UK). The stocks of all strains were maintained in 20% (v/v) glycerol stored at 80 C. Screening of L. acidophilus isolates for anti-microbial activities The isolates of L. acidophilus were screened for anti-microbial activities by the paper disc method (Havoorver & Harlander, 1993). Testing strains were grown in MRS media for 24 h at 37 C. Cell-free supernatant of tested strains was collected by centrifugation at 12 000 g/10 min and pH was adjusted to 5.5. Ten microliters of sterilized supernatant was applied on the lawn of indicator strains (106 cfu/mL, compared to 0.50 O.D of MacFarland solutions) and anti-microbial activities were evaluated on the basis of clear zone formation (mm) around the disk. The bacteriocin bioassay was defined as the reciprocal of the highest dilution (2n) that resulted in inhibition of the indicator lawn. Thus, the AU/mL was defined as 2n 1000/10 mL. Whole cell protein analysis by SDS-PAGE of L. acidophilus Bacterial cell protein profile of the selected isolates was determined by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), as described by Laemmili (1970). Bacterial cell pallets were collected by centrifugation at 6000 g for 5 min and were washed twice with 200 mL of stacking gel buffer (0.5 M Tris base of pH 6.8) and finely re-suspended in 10 mL in the same buffer. A total of 80 mL of sample buffer (I M Tris buffer of 6.8 pH, 10% SDS, glycerol, b-mercaptoethanol, 0.005% bromophenol blue) and 6.5 mL of b-mercaptoethanol
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was added to the above mixture. Then heat shock was given by first boiling this mixture for 5 min, and then immediately cooling to ice temperature. A total of 5 mL of each sample and protein marker (116–14.4 kDa, Fermentas) was loaded on polyacrelamyde gel (4.5% stacking gel and 12.5% resolving gel). Selection and characterization of best-suited strain for bacteriocin production Hierarchical cluster analysis was performed to determine the similarity index among various strains and for the selection of single strain having maximum potential of anti-microbial activity. Characterization of LA06FT for probiotic potential Acid and bile tolerance of L. acidophilus LA06FT Bile tolerance was determined in MRS broth containing 1–3% bile salt-LP0055 (Oxide, Hampshire, England), as reported by Brashears et al. (1998). In vitro acid tolerance by the selected strain was determined by adjusting the pH of growth media (1.5–10.5). Growth was monitored by change in OD600 nm and pH set at 6.2 was used as control, as recommend by manufacturers. Antibiotic susceptibility test of L. acidophilus LA06FT The disk susceptibility test was done according to the Bauer– Kirby method, as reported by Jan Hudzicki (2009). The test strain was screened for possible resistance against 10 selected antibiotics (Difco laboratories, MI), used in Hospitals to treat the infections. The antibiotics included were Nalidixic acid (30 mg), Ampicillin (30 mg), Penicillin (10 AU), Amoxicillin 30 mg, Ciprofloxacine 5 mg; Tetracycline (30 mg); Vancomycin (30 mg), Erythromycin (15 mg), Gentamicin (10 mg) and Sulphamethoxazol (25 mg). Characterization of bacteriocin produced by LA06FT Effect of acidification, heating and enzymes treatment on the activity of bacteriocin of LA06FT Bacteriocin containing supernatant was adjusted to pH of 2.5, 3.5, 4.5, 6.5, 7.5, 8.5, 9.5 and 10.5 with 6 N HCl or NaOH to check the effect of acidification as described by Oh et al. (2000) and then tested for anti-antibacterial activities. Similarly, cell-free supernatant was heated to 60, 70, 90, 100 and 120 C to determine the thermostability as described by Todorov & Dicks (2005) and then tested for anti-antibacterial activities. Bacteriocin containing cell-free supernatant was treated with 0.1 mg mL 1 of proteinase k, lipase, a-amylase and trypsin (Ivanova et al., 2000) and then tested for anti-antibacterial activities. Purification of bacteriocin of LA06FT Purification of bacteriocin of LA06FT was carried out in a threestep procedure: first, bacteriocin containing cell-free supernatant was obtained from cultured broth by centrifugation and sterilized by microfiltration; second, antibacterial peptides were precipitated by ammonium sulfate; and third, dialysis through 12 000 kDa molecular weight cut-off membranes followed by freeze drying of partially purified bacteriocin. Molecular sizing of bacteriocin of LA06FT To measure the molecular weight of partially purified bacteriocin produced by LA06FT, Tricine SDS-PAGE was used as illustrated by Batdorj et al. (2006). The acrylamide was applied at a concentration of 16.5%. Sample was loaded on two vertical parts along with a standard marker. When running, complete gel was cut into two parts. The piece of gel having standard marker side
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was placed in the solution of coomassie blue for staining. Other part of the gel having only sample was properly washed with deionizer water and used to measure its anti-microbial activity on the lawn of indicator strain. Statistical analysis Hierarchical cluster analysis was applied and dendrogram was constructed using Package for the Social Sciences (SPSS) statistical package version 12.0 (SPSS Inc., Chicago, IL) for the screening of bacteriocin producing strains and for SDS protein profile of these strains. Variance in bacteriocin activity and probiotic potential were analyzed using a two-factor factorial experiment on M-Stat C Statistical software according to the procedures described by Steel et al. (1996).
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Isolation and identification of L. acidophilus Thirteen isolates of L. acidophilus were obtained from the dahi samples. All isolates were identified using classical microbiological techniques (data not shown) and further confirmed by amplification 16S rRNA region by species-specific primers of L. acidophilus. The results obtained for PCR products are presented in Figure 1. It was observed that all strains have PCR products of approximately 750–760 bp. Lactobacillus acidophilus has been isolated from dahi as in the study by Maqsood et al. (2008), who reported that dahi was a rich source of probiotic lactobacilli. Antagonistic properties of L. acidophilus Thirteen strains of L. acidophilus were tested for anti-bacterial activity against food-borne pathogens (Table 1). It was observed that six strains (TLA01FT, TLA04FT, TLA05FT, LA08FT, LA06FT and LA07FT) have more prominent inhibitory pattern. Hierarchical cluster analysis was carried out to determine the similarity index among the various strains of L. acidophilus (Figure 2) and LA06FT was selected for further characterization since this strain was effective against both Gram-positive and Gram-negative bacteria. Previously many researchers reported that bacteriocin produced by L. acidophilus was capable to kill both Gram-negative and Gram-positive bacteria. Results were in agreement with the finding of Karthikeyan & Santhosh, (2009), who reported that bacteriocin produced by L. acidophilus was effective against Gram-positive and Gram-negative bacteria.
3000 2000 1500 1200 1000 900 800 700 600 500 400 300 200 100
Figure 1. PCR amplification of 16S RNA region of selected strains of L. acidophilus. Line 1: Marker of 100 bp, Line 2: TLA01FT, Line 3: TLA02FT, Line 4: TLA03FT, Line 5: TLA04FT, Line 6: TLA05FT, Line 7: TLA06FT, Line 8: TLA07FT, Line 9: TLA08FT, Line 10: TLA09FT, Line 11: TLA10FT, Line 12: LA05FT, Line 13: LA06FT, Line 14: LA07FT.
Int J Food Sci Nutr, 2014; 65(5): 582–588
Oh et al. (2000) screened five strains of L. acidophilus for bacteriocin production that were effective against the Gram-positive bacteria like B. cereus, L. monocytogenes and S. aureus. SDS protein profiling of L. acidophilus Protein profile of SDS-cell wall protein was applied for phenotypic relationship between the selected antibacterial activity and the presence or absence of particular bands in cell wall proteins (Figure 3). It was evident from electrophoregram that all bacteriocin producing strains of L. acidophilus produced a protein band of 22 kDa, and this band was absent in non-bacteriocin producing strains. Studies related to electrophoretic protein revealed that different strains of L. acidophilus have both major (60, 45, 43, and 35 kDa) and minor bands (97, 68, 47, 31 and 18 kDa) as reported by Teanpaisan & Dahlen, (2006). However, numbers, size and position of these bands were highly affected by change in ecological and growth conditions that were distressing phenotypic characteristics and not the genotype (Kim et al., 2005). Probiotic characteristics of L. acidophilus LA06FT Effect of bile salt on the pH and growth of L. acidophilus LA06FT Since probiotic bacteria pass through alimentary tract, checking its tolerance for bile and acid are very important phenomena and the results recorded for change in pH and OD are presented in Table 2. It was observed that LA06FT could grow in the presence of 3% bile salt concentration, however it reduced significantly after 6 h. Previous data about the bile tolerance also support our results. Heng et al. (2004) found that L. acidophilus can survive at 2% concentration of bile salt. The results of the present study are in agreement with the findings of Succi et al. (2005), who reported that L. rhamnosus remained active in 2% bile salt concentration. While considering these bacteria as probiotics, they should become a part of the normal microbial flora in the intestine, survive the gastrointestinal passage, and should be able to adhere and colonize in the intestinal tract (Oh et al., 2000). Acid tolerance of L. acidophilus LA06FT In vitro acid tolerance by LA06FT is presented in Table 3. It was observed that high and low initial pH of growth media result in the reduction of bacteriocin activity. At pH 2.5 and 3.5, bacteriocin activities reduced to 100%. However, it remained viable at this low pH, that is human stomach pH. Maximum bacteriocin-specific activity was at pH 6.5, 4.5 and 7.5. This phenomenon has been observed by other researchers (Zamfir et al., 2000) who reported that the bacteriocin produced by L. acidophilus is highly pH dependent and can effectively control the growth of other organisms below pH 6. Therefore, for optimizing bacteriocin production in vitro, the LAB should be grown at the pH values between 5.5 and 7.0 (Todorov & Dicks, 2005). Antibiotic susceptibility test Antibiotic susceptibility test was performed to determine the safety level of selected strain. Results obtained for different antibiotics are presented in Table 4. On the basis of inhibition zone, three groups of antibiotics were identified: first, LA06FT showed resistance to Nalidixic acid, Ciprofloxacine, Gentamicin and Vancomycin (0 to 12 mm); second, Sulphamethoxazol and Erythromysin (12 to 16 mm) showed intermediate behaviour; and third, strain was sensitive to Ampicillin, Penicillin, Tetracycline and Amoxicillin (17 to 33 mm). Then these strains were grown in antibiotic-protected environment to assess the
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Table 1. Screening of different isolates of L. acidophilus for antibacterial activities. Indicator strains S.No
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1 2 3 4 5 6 7 8 9 10 11 12 13
Name of strains L. acidophilus TLA01FT L. acidophilus TLA02FT L. acidophilus TLA03FT L. acidophilus TLA04FT L. acidophilus TLA05FT L. acidophilus TLA06FT L. acidophilus TLA07FT L. acidophilus TLA08FT L. acidophilus TLA09FT L. acidophilus TLA10FT L. acidophilus LA05FT L. acidophilus LA06FT L. acidophilus LA07FT
E. coli ATCC 25922
S. aureus ATCC 6538
P. aeruginosa ATCC 25923
L. monocytogenes ATCC 19115
+++
+++
++
++
+ ++++
++ +
+++
+++ ++++
+
+++
++++
+++
++++ +++
++++
++++
++++ +++
Inhibition zone is mean of three readings. Inhibition zone is ranked as no visible inhibition ( ), visible inhibition (+), diameter of inhibition zone 4 mm (++), diameter of inhibition zone between 4 to 8 mm (+++) and diameter of inhibition zone 8 to12 mm (++++).
Figure 2. Hierarchical cluster analysis of different strains of L. acidophilus for similarity index among them for their anti-bacterial activity showing that first group (TLA10FT, LA04FT, TLA02FT, TLA07FT, TLA09FT, TLA03FT and TLA06FT) have above 80% similarity index.
susceptibility towards the antibiotics. It was observed that LA06FT did not change its behavior towards the antibiotics, so this strain was found to be safe. The results of the present study were supported by the earlier work of Gupta et al. (1996), they examined seven strains of L. acidophilus for antibiotics susceptibility test and found that some strains are sensitive to various antibiotics (Ampicillin, Erythromycin, Tetracycline), but these strains show resistance to Norfloxacin and Nalidixic acid. In general, it was noted that resistance pattern is species specific (Ortu et al., 2007). Similarly, Kastner et al. (2006) observed antibiotics resistance in 27 probiotic cultures which were not their intrinsic feature. Nature of the resistance might vary according to
the habitats from where these strains were isolated (Bernardeau et al., 2008). Technological characteristics of L. acidophilus LA06FT Acidification, heating and enzymes treatment on the activity of bacteriocin of LA06FT Bacteriocin of LA06FT appeared to be stable over wide range of pH (4.5–7.5) and fairly heat stable between 60 and 90 C for 20 min, as illustrated in Figure 4(a). Similarly, bacteriocin of LA06FT was treated with proteolytic and non-proteolytic enzymes to determine the proteineous nature (Figure 4b). It was
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Int J Food Sci Nutr, 2014; 65(5): 582–588
(a) KDa
observed that bacteriocin was highly sensitive to proteolytic enzymes like proteinase K and had no effect on lipase and amylase. These characteristics made it a suitable bio-preservative for acidic and low acidic food products wherein heat treatment are commonly applied as previously reported by Mobarez et al. (2008) and Karthikeyan & Santhosh (2009). The heat stability shown by bacteriocin ruled out the possibility that this inhibitory activity was due to the action of bacteriophages since phages cannot survive at 121 C for 15 min. Some contradictions were also noted among the various studies like Batdorj et al. (2006), where strong heat stability of bacteriocin was reported even at 121 C; it might be due the presence of the large number of stable cross linkages or due to high cysteine contents. However, in regard to enzymes, acidocin-LA06FT was similar to acidocin CH5 (Chumchalova et al., 2004) and acidocin DSM (Deraz et al., 2005) and closely matched with the findings of Batdorj et al. (2006), who reported that bacteriocin was completely inactivated by proteolytic enzymes since bacteriocin contains cleavage-sites suitable for their binding.
116.0 62.2 45.0
35.0
25.0 18.4 14.4
65 66
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(b)
Table 4. Antibiotic susceptibility test of strain L. acidophilus LA06FT. S. No 1 2 3 4 5 6 7 8 9 10
Figure 3. (a) SDS-PADE of cell wall proteins of probiotic strains of L. acidophilus, line 1: protein marker of 14.4 to 116 kDa, line 2: TLA01FT, line 3: TLA04FT, line 4: TLA05FT, line 5: LA08FT, line 6: LA06FT and line 7: LA07FT. L. acidophilus strains produce 6 prominent bands of 80, 62.2, 45, 35, 22 and 14.4 kDa and minor bands of 68, 43, 41, 39, 25 and 18 kDa. (b) SDS-PAGE of non-probiotic strains of L. acidophilus.
Antibiotics
Zone size (mm)
Patron
Nalidixic acid (30 mg) Ampicillin (30 mg) Ciprofloxacine (5 mg) Tetracycline (30 mg) Penicillin (10 U) Sulphamethoxazol (25 mg) Erythromycin (15 mg) Gentamicin (10 mg) Amoxicillin (30 mg) Vancomycin (30 mg)
0.0 ± 0.00 26.0 ± 1.00 10.0 ± 0.25 30.0 ± 0.50 32.0 ± 0.45 14.0 ± 0.33 16.0 ± 1.55 12.0 ± 1.43 33.0 ± 1.81 0.0 ± 0.00
Resistant Sensitive Resistant Sensitive Sensitive Resistant Intermediate Resistant Sensitive Intermediate
The results are expressed in term of inhibition zone around the discs and mentioned it resistant (0 12 mm), intermediate (13 16 mm) and sensitive (17 33 mm). Standard error was calculated at LSD value 0.0783.
Table 2. Effect of different concentration of bile salts on the growth of L. acidophilus LA06FT in terms of change in pH and OD. Optical density (OD) Intervals 0h 1h 2h 4h 6h
Control g
0.04 (0.00) 0.046fg (13.04) 0.08efg (51.81) 0.18a (78.84) 0.30b (86.71)
1% g
0.03 (0.00) 0.05fg (41.18) 0.07efg (58.90) 0.15cd (80.0) 0.20c (58.0)
2% fg
0.05 (0.00) 0.07efg (30.26) 0.08efg (40.45) 0.10def (48.54) 0.11def (51.82)
pH 3% defg
0.09 (0.00) 0.123de (26.83) de 0.13 (31.30) 0.12de (29.01) 0.12de (28.00)
Control e
6.00 (0.00) 5.87f (2.17) 5.83f (2.83) 5.50g (8.33) 5.01h (16.50)
1% c
6.20 (0.00) 6.15cd (0.81) 6.14cd (0.97) 6.08de (1.97) 5.79f (6.61)
2% b
3%
6.36 (0.00) 6.21c (2.36) 6.15cd (3.30) 6.07de (4.56) 6.05de (4.87)
a
6.50 6.21c 6.21c 6.20c 6.20c
(0.00) (4.46) (4.46) (4.62) (4.62)
Means followed by same alphabets are non-significant for p50.05. For pH, DMR test was determined at LSD values of 0.1044 and standard deviation 0.0361 and for OD. DMR test was determined at LSD values of 0.05218 and standard deviation 0.01826. Control: Growth of tested strain on MRS without bile salt.
Table 3. Acid tolerance of L. acidophilus LA06FT. Initial pH
Final pH
Change in pH
OD 600 nm
Bacteriocin activity (AU/mL)
Reduction (%)
Specific activity (AU/OD)
2.50 ± 0.01 3.50 ± 0.03 4.50 ± 0.02 5.50 ± 0.07 6.50 ± 0.05 7.50 ± 0.07
2.50 ± 0.01 3.10 ± 0.01 3.80 ± 0.05 4.00 ± 0.04 4.00 ± 0.04 3.85 ± 0.03
0.00 0.40 0.70 1.50 2.50 3.50
0.25 ± 0.03 1.20 ± 0.01 1.40 ± 0.01 2.00 ± 0.02 1.80 ± 0.07 1.70 ± 0.07
000.0 ± 0.00 000.0 ± 0.00 200.0 ± 20.0 800.0 ± 34.0 400.0 ± 26.0 100.0 ± 11.0
100.0 ± 5.0 100.0 ± 5.0 75.0 ± 2.5 0.0 ± 0.0 50.0 ± 1.9 87.5 ± 2.9
0.00 ± 0.0 0.00 ± 0.0 142.86 ± 8.1 470.59 ± 7.2 222.20 ± 12.2 50.00 ± 3.1
Results were compared against control pH of 6.2 of MRS media. OD, final pH and bacteriocin activity was measured after 24 h incubation. Results were means of three replications. Values ± corresponded to standard error.
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Purification of bacteriocin of LA06FT Purification of bacteriocin produced by L. acidophilus LAFT06 was carried out in a three-step procedure and purification scheme is summarized in Table 5. Cell-free supernatant was the first level of purification that was the result of centrifugation and filtration indicating 100% recovery at a specific activity of 5369.13 AU mg 1. Previously, many researchers determined purification levels of bacteriocin containing supernatant. Chumchalova et al. (2004) determined the purification of acidocin CH5 produced by L. acidophilus CH5 and observed that bacteriocin activity and specific activity were 3200AU mL 1 o
Temerature tolerance ( C)
(a) 60
70
80
90
100
120 900
12
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and 2900 AU mg 1, respectively. Similarly, Batdorj et al. (2006) used centrifugation as the first level of purification and found a specific activity of 256AU mg 1 for cell-free supernatant. At the second level, acidocin LA06FT was purified to homogeneity by ammonium sulfate precipitation (ASP), most widely used techniques for the purification of bacteriocin, producing total activity of 160 000 AU resulting in 80% concentration of bacteriocin pellets. Results were different from the earlier findings of Deraz et al. (2005), who applied 40% ammonium sulfate for the precipitation of bacteriocin produced by L. acidophilus DSM20079 and got 94% recovery, since the solubility of bacteriocin varies according to the ionic strength of salt. At the third level, partially purified bacteriocin by ASP was freeze dried and it was observed that antibacterial activity increased to 3200 AU/mL that was two times higher than the ammonium sulfate precipitation. The results were almost similar to that reported by Burianek & Yousef (2000).
600 AU/ ml
Inhibition zne (mm)
Molecular sizing of bacteriocin of LA06FT
700 8
500 6 400 4
300 200
2 100 0
0 2.5
3.5
4.5
5.5
6.5
7.5
8.5
9.5
10.5
Acid tolerance (pH)
(b) 900
Molecular weight of partially purified bacteriocin was determined by SDS-PAGE. Results revealed acidocin LA06FT had single band of proximately 8.5 kDa (Figure 5). Zone of inhibition corresponding to the position of bacteriocin also confirmed the identified bacteriocin. The present results coincided with previous studies on molecular characterization of bacteriocin produced by L. acidophilus, which stated that most of the bacteriocins were of low molecular weight (Zamfir et al., 2000). Research finding were also in line with the results of Todorov & Dicks (2005) who found that bacteriocin produced by the L. pentosus had low molecular weight, they also studied its antibacterial behavior.
10
800
9
700
8
600
7 6
500
5
400
4
kDa Zone (mm)
AU/ ml
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800 10
62.2
3
45.0
200
2
35.0
100
1
0
0 amylase Trypsin Enzymes
(b)
116.0
300
Lipase
(a)
25.0
Proteinase-k
Figure 4. (a) Temperature ( C) and acid tolerance (pH) by the bacteriocin produced by LA06FT; (), effect of heating in the bacteriocin activity (AU mL 1); (m), effect of different pH on the bacteriocin activity. (b) Effect of different enzymes on the activity of bacteriocin produced by LA06FT. Enzymes were used at the concentration of 0.1 mg mL 1. (), bacteriocin activity (AU mL 1); (m), Zone of inhibition (mm).
18.4 14.4
8.5
Figure 5. Molecular weight (8.5 kDa) of purified bacteriocin LA06FT determined by SDS gel electrophoresis (a) molecular weight of bacteriocin and marker (b) gel was overlaid on the lawn of S. aurues to determine the bacteriocin of purified bacteriocin.
Table 5. Steps for the purification of bacteriocin produced by LA06FT.
Purification stages Centrifugation and filtration Ammonium sulphate ppt Freeze drying a
Volume (mL)
Activity (AU/mL)
Total activity (AU)a
Protein (mg/mL)b
Total protein (mg)c
Specific activity (AU/mg)d
Purification factore
Recovery (%)f
250 100 5
800 1600 3200
200 000 160 000 16 000
0.149 0.097 0.012
37.25 9.70 0.06
5369.13 16 494.85 266 666.67
1.00 3.07 49.67
100.00 80.00 8.00
Protein concentration was determined by the Lowry method. Bacteriocin activity can be calculated by well diffusion method. c Total activity (AU) and total protein (mg) was determined by the multiplication of volume by activity (AU/mL) and protein (mg/mL). d Specific activity was determined by dividing total activity with total protein concentration (AU/mg). e Purification factor was determined by dividing the subsequent value of specific activity by specific activity of cell-free supernatant. f Percentage recovery was determined by dividing the total activity of subsequent step by total activity of cell-free supernatant. b
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Conclusion Lactobacillus acidophilus has been found in many traditional fermented milk products. The rapid growth and compatible behavior towards primary starter species ensure its survival in yogurt-like products. Therefore, it is important to study technological and therapeutic properties of this bacterium. Present study provides an insight regarding its probiotic potential and possible safety concerns. Similarly, bacteriocin producing L. acidophilus LA06FT share same homology with the other reported bacteriocin. Although bacteriocin production is an inherited trail, supplementing the growth medium with growth limiting factors could enhance bacteriocin production. In this consequence, purification of these compounds would be further helpful to meet the thirst of new and more effective bacteriocins.
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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
References Batdorj B, Dalgalarrondo M, Choist Y, Pedroche J, Metro F, Prevost H, Chobert JM, Haertle T. 2006. Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag. J Appl Microbiol 101:837–848. Bernardeau M, Vernoux JP, Henri-Dubernet S, Gue´guen M. 2008. Safety assessment of dairy microorganisms: the Lactobacillus genus. Int J Food Microbiol 1:126:278–285. Bhowmik D, Dubey J, Mehra S. 2009. Probiotic efficiency of S. platensisstimulating growth of lactic acid bacteria. World J Dairy Food Sci 4:160–163. Brashears MM, Gilliland SE, Buck LM. 1998. Bile salt deconjugation and cholesterol removal from media by Lactobacillus casei1. J Dairy Sci 81:2103–2110. Burianek L, Yousef AE. 2000. Solvent extraction of bacteriocin from liquid cultures. Appl Microbiol Lett 31:193–197. Chumchalova J, Stiles J, Josephsen J, Plockova M. 2004. Characterization and purification of acidocin CH5, a bacteriocin produced by L. acidophilus CH5. J Appl Microbiol 96:1082–1089. Deraz SF, Karlsson EN, Hedstrom M, Andersson MM, Mattiasson B. 2005. Purification and characterization of acidocin D20079, a bacteriocin produced by L. acidophilus DSM 20079. J Biotechnol 117:343–354. Dewan S, Tamang J. 2007. Dominant lactic acid bacteria and their technological properties isolated from the Himalayan ethnic fermented milk products. Antonie van Leeuwenhoek 92:343–352. Floros G, Hatzikamari M, Litopoulou-Tzanetaki E, Tzanetakis N. 2012. Probiotic and technological properties of facultatively hetero fermentative Lactobacilli from Greek traditional cheeses. Food Biotechnol 26: 85–105. Ghazi F, Henni DE, Benmechernene Z, Kihal M. 2009. Phenotypic and whole cell protein analysis by SDS-PAGE for identification of dominants lactic acid bacteria isolated from Algerian raw milk. World J Dairy Food Sci 4:78–87. Gupta PK, Mital BK, Garg SK. 1996. Characterization of Lactobacillus acidophilus strains for use as dietary adjunct. Int J Food Microbiol 29: 105–109. Havoorver DG, Harlander SK. 1993. Screening methods for detecting bacteriocins activity. Bacteriocins of lactic acid bacteria. San Diego (CA): Academic Press.
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Heng NCK, Burtenshaw GA, Jack RW, Tagg JR. 2004. Sequence analysis of pDN571, a plasmid encoding novel bacteriocin production in M-type 57 Streptococcus pyogenes. Plasmid 52:225–229. Ivanova I, Kabadjova P, Pantev S, Danova S, Dousse X. 2000. Detection purification and characterization of a noval bacteriocin substance produced by Lactobacillus lactis subsp. lactis B14 isolated from bozabulgarian traditional cereals beverage. Biocatal: Vestnik Maskov Univ Kimia 41:47–53. Jan Hudzicki. 2009. Kirby-Bauer disk diffusion susceptibility test protocol. Reported by American Society for Microbiology. Available at: http://www.microbelibrary.org/component/resource/laboratory-test/ 3189-kirby-bauer-disk-diffusion-susceptibility-test-protocol. Accessed on 02 March 2013. Karthikeyan V, Santhosh SW. 2009. Study of bacteriocin as a food preservative and the L. acidophilus strain as probiotic. Pak J Nutr 8: 335–340. Kastner S, Perreten V, Bleuler H. 2006. Antibiotic susceptibility patterns and resistance genes of starter cultures and probiotic bacteria used in food. Syst Appl Microbiol 29:145–155. Kim TW, Song HS, Kim HY. 2005. Distribution of dominant Bifidobacteria in the intestinal microflora of Korean adults and seniors, identified by SDS-PAGE of whole cell proteins and 16S rDNA sequence analysis. J Microbiol 15:388–394. Klaenhammer TR. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol Rev 12:39–86. Laemmili UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. Maqsood SF, Hasan Masud T, Imran M. 2008. Preliminary characterization of bacteriocin produced by Lactobacillus acidophilus TS1 isolated from traditional dahi. Annals Microbiol 58:617–622. Mobarez AM, Doust RH, Sattari M, Mantheghi N. 2008. Antimicrobial effects of bacteriocin like substance produced by L. acidophilus from traditional yoghurt on P. aeruginosa and S. aureus. J Biological Sci 8: 221–224. Oh S, Kim SH, Worobo RW. 2000. Characterization and purification of a bacteriocin produced by a potential probiotic culture, Lactobacillus acidophilus 30SC. J Dairy Sci 83:2747–2752. Ortu S, Felis GE, Marzotto M, Deriu A, Molicotti P, Sechi LA, Dellaglio F, Zanetti S. 2007. Identification and functional characterization of Lactobacillus strains isolated from milk and Gioddu, a traditional rdinian fermented milk. Int Dairy J 10:1016–1025. Steel R, Torrie J, Dickey D. 1996. Principles and procedures of statistics: a biometrical approach, 3rd ed. New York: McGraw Hill Book Co. Succi M, Tremonte P, Reale A, Sorrentino E, Grazia L, Pacifico S, Coppola R. 2005. Bile salts and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. FEMS Microbiol Lett 244:129–137. Teanpaisan R, Dahlen G. 2006. Use of polymerase chain reaction techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis for differentiation of oral Lactobacillus species. Oral Microbiol Immunol 21:79–83. Todorov SD. 2008. Bacteriocin production by L. Plantarum AMA-K isolated from AMASI, a Zimbabwean fermented milk product and study of the adsorption of bacteriocin ama-k to listeria sp. Brazil J Microbiol 39:178–187. Todorov SD, Dicks LMT. 2005. Effect of growth medium on bacteriocin production by Lactobacillus plantarum ST194BZ, a strain isolated from Boza. Food Technol Biotechnol 43:165–173. Zamfir M, Callewaert R, Cornea PC, Savu L, Vatafu I, DeVuyst L. 2000. Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801. J Appl Microbiol 87:923–931.
IN VITRO AND ANIMAL STUDIES
Some probiotic and antibacterial properties of Lactobacillus acidophilus cultured from dahi a native milk product Talat Mahmood, Tariq Masud, and Asma Sohail
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Department of Food Technology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan
Abstract
Keywords
In this study, different strains of Lactobacillus acidophilus from dahi were analyzed for certain probiotic and antibacterial properties. Initially, these strains were confirmed by the amplification of 16S rRNA regions and then screened for antibacterial activities against food borne pathogens. The phenotypic relationship between apparent antibacterial activity and cell wall proteins were established by cluster analysis. It was observed that those strains, which have prominent bands having size 22–25 kDa possess antibacterial activity. On the basis of wide spectrum of killing pattern, a strain LA06FT was further characterized that showed no change in its behavior when subjected to the antibiotic protected environment and grow well in acidbile conditions. The bacteriocin produced by this strain has specific antibacterial activity of 5369.13 AU mg 1. It remained stable at 60–90 C and pH range of 4.5–6.5 while proteolytic enzymes inactivate the bacteriocin that confirm its proteinic nature having molecular weight of 8.5 kDa.
Antibacterial activity, dahi, Lactobacillus acidophilus, plasmid profile, probiotic culture, SDS-PAGE
Introduction Dahi is one of the fermented milk products in south Asian countries like Bangladesh, India and Pakistan and supposed to be a natural remedy for gastrointestinal problems. This character is due to the presence of probiotic lactic acid bacteria (LAB) like Lactobacillus delbrueckii ssp., L. bulgaricus, L. helveticus, L. acidophilus and S. thermophiles. Among them, L. acidophilus presents the predominant group of LAB that has technological advantages over other bacteria (Ivanova et al., 2000). They can be used as starter culture in fermented dairy products and also enhance their shelf life through anti-microbial peptides with some novel features (Dewan & Tamang, 2007). In addition to acidification of milk, the production of some secondary metabolites like antibacterial peptides and other probiotic features were also related to these bacteria (Bhowmik et al., 2009). Application of these compounds to eradicate food pathogens without any toxic or other adverse effects has great significance as natural preservatives (Karthikeyan & Santhosh, 2009). Bacteriocins are mostly ribosomally synthesized peptides, having lethal effect against the pathogenic strains and this characteristic makes them parallel to chemical preservatives (Todorov, 2008). On the structural and molecular bases, bacteriocin of LAB are divided into four classes (Klaenhammer, 1993) and according to this classification, aciodocin belonged to ClassII non-lanthionines that show some resistant to heat and pH, but
Correspondence: Tariq Masud, Department of Food Technology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan. Tel: +92-51-9290694. E-mail: [email protected]
History Received 26 March 2013 Revised 17 October 2013 Accepted 31 December 2013 Published online 1 April 2014
sensitive to protolytic enzymes having low molecular mass (Mobarez et al., 2008). Role of cell proteins is highly important in the expression of antagonistic and probiotic characteristics (Floros et al., 2012). Many research work has been carried out to assess the antibacterial potential of LAB and the application of their metabolites as novel food preservatives (Ghazi et al., 2009). However, in this article we describe for the first time that SDS proteins with band size of 22–25 kDa play a major role in the development of the homological features. In the consequence, a single strain was identified with improved functional attributes and its bacteriocin was characterized. So, the present study was designed for the investigation of probiotic and antibacterial features of some wild strains of L. acidophilus with aims that new strains would help to introduce genetically diversifying bacteria of dahi having worth mentioning qualities. Furthermore, their antibacterial compound would be purified and could be applied as natural preservatives having no harm full effect on consumer since it was isolated from probiotic culture.
Materials and methods Isolation and identification of L. acidophilus Dahi samples were obtained from the local market. Producer used undefined starter, a part of mature product of previous batch. Thirteen L. acidophilus strains were obtained by inoculating on the selected media MRS (deMan Regosa and Sharp, England) additionally supplemented with 2% lactose. The microbial isolates were identified by morphological and biochemical testing. Lactobacillus acidophilus isolates were confirmed by the amplification of partial 16S rRNA regions (757 bp) using a specific primer.
DOI: 10.3109/09637486.2014.880666
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Amplification For the amplification of 16S rRNA regions of L. acidophilus, primers (GCTGAACCAACAGATTCAC and ACTACCAGGGT ATCTAATCC) were designed and obtained according to the sequence published in the National Center for Biotechnology Information (NCBI). Ten mircoliters of reaction mixture was prepared by adding 3.8 mL of nanopure water, 1.0 mL of 10 buffer, 0.2 mL of 5 mM dNTPs, 0.6 mL of 25 Mm MgCl2, 1 mL forward primer and 1 mL of reverse primer, 2 mL of DNA (diluted to 1: 49) and 0.4 mL of Taq polymerase. The PCR cycling programs used were: Temperature1 at 94 C for 2 min, Temperature2 at 94 C for 30 s, Temperature3 at 50 C for 30 s, Temperature4 at 72 C for 45 s and Temperature5 at 72 C for 20 min. PCR cycles were maintained at 94 C for 2 min, followed by 35 cycles of 94 C for 30 s, annealing temperature 50 C for 30 s, 72 C for 45 s and a final extension temperature of 72 C for 20 min. The ready samples were run on the agarose gel of 1.2% concentration in 1 TBE working buffer of pH 8.0 (5 stock buffer was prepared by dissolving 13.5 g Tris Base, 6.875 g boric acid in 20 mL of 0.5M EDTA). Horizontal mini prep. electrophoresis (WEALTEC Corp., Sparks, NV ) was used at 70 volts for 1.5 h. The sample was mixed with 3 mL bromophenol blue tracking dye and loaded into the well. For genomic DNA Hind III DNA MW marker of 23 130–564 bp was used while 100 Base Pair Plus DNA Ladder marker of 3000–100 bp was applied as standard for PCR. Ethidium bromide (0.5 mg/mL) was used for the illumination of band that was observed under UV light. Bacterial strains and growth media Different strains L. acidophilus were grown on the selected media of MRS. The indicator strains for antibacterial assay were Listeria monocytogenes ATCC 19115, grown on Listeria enrichment broth base (CM0863 Oxoid, Hampshire England), Staphylococcus aureus ATCC 6538, grown on Staphylococcus Medium No110 (Hampshire England), Pseudomonas aeruginosa ATCC 25923 and Escherichia coli-ATCC25922 (Brain Heart Infusion (BHI) medium Oxoid Ltd, Baingstoke, UK). The stocks of all strains were maintained in 20% (v/v) glycerol stored at 80 C. Screening of L. acidophilus isolates for anti-microbial activities The isolates of L. acidophilus were screened for anti-microbial activities by the paper disc method (Havoorver & Harlander, 1993). Testing strains were grown in MRS media for 24 h at 37 C. Cell-free supernatant of tested strains was collected by centrifugation at 12 000 g/10 min and pH was adjusted to 5.5. Ten microliters of sterilized supernatant was applied on the lawn of indicator strains (106 cfu/mL, compared to 0.50 O.D of MacFarland solutions) and anti-microbial activities were evaluated on the basis of clear zone formation (mm) around the disk. The bacteriocin bioassay was defined as the reciprocal of the highest dilution (2n) that resulted in inhibition of the indicator lawn. Thus, the AU/mL was defined as 2n 1000/10 mL. Whole cell protein analysis by SDS-PAGE of L. acidophilus Bacterial cell protein profile of the selected isolates was determined by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), as described by Laemmili (1970). Bacterial cell pallets were collected by centrifugation at 6000 g for 5 min and were washed twice with 200 mL of stacking gel buffer (0.5 M Tris base of pH 6.8) and finely re-suspended in 10 mL in the same buffer. A total of 80 mL of sample buffer (I M Tris buffer of 6.8 pH, 10% SDS, glycerol, b-mercaptoethanol, 0.005% bromophenol blue) and 6.5 mL of b-mercaptoethanol
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was added to the above mixture. Then heat shock was given by first boiling this mixture for 5 min, and then immediately cooling to ice temperature. A total of 5 mL of each sample and protein marker (116–14.4 kDa, Fermentas) was loaded on polyacrelamyde gel (4.5% stacking gel and 12.5% resolving gel). Selection and characterization of best-suited strain for bacteriocin production Hierarchical cluster analysis was performed to determine the similarity index among various strains and for the selection of single strain having maximum potential of anti-microbial activity. Characterization of LA06FT for probiotic potential Acid and bile tolerance of L. acidophilus LA06FT Bile tolerance was determined in MRS broth containing 1–3% bile salt-LP0055 (Oxide, Hampshire, England), as reported by Brashears et al. (1998). In vitro acid tolerance by the selected strain was determined by adjusting the pH of growth media (1.5–10.5). Growth was monitored by change in OD600 nm and pH set at 6.2 was used as control, as recommend by manufacturers. Antibiotic susceptibility test of L. acidophilus LA06FT The disk susceptibility test was done according to the Bauer– Kirby method, as reported by Jan Hudzicki (2009). The test strain was screened for possible resistance against 10 selected antibiotics (Difco laboratories, MI), used in Hospitals to treat the infections. The antibiotics included were Nalidixic acid (30 mg), Ampicillin (30 mg), Penicillin (10 AU), Amoxicillin 30 mg, Ciprofloxacine 5 mg; Tetracycline (30 mg); Vancomycin (30 mg), Erythromycin (15 mg), Gentamicin (10 mg) and Sulphamethoxazol (25 mg). Characterization of bacteriocin produced by LA06FT Effect of acidification, heating and enzymes treatment on the activity of bacteriocin of LA06FT Bacteriocin containing supernatant was adjusted to pH of 2.5, 3.5, 4.5, 6.5, 7.5, 8.5, 9.5 and 10.5 with 6 N HCl or NaOH to check the effect of acidification as described by Oh et al. (2000) and then tested for anti-antibacterial activities. Similarly, cell-free supernatant was heated to 60, 70, 90, 100 and 120 C to determine the thermostability as described by Todorov & Dicks (2005) and then tested for anti-antibacterial activities. Bacteriocin containing cell-free supernatant was treated with 0.1 mg mL 1 of proteinase k, lipase, a-amylase and trypsin (Ivanova et al., 2000) and then tested for anti-antibacterial activities. Purification of bacteriocin of LA06FT Purification of bacteriocin of LA06FT was carried out in a threestep procedure: first, bacteriocin containing cell-free supernatant was obtained from cultured broth by centrifugation and sterilized by microfiltration; second, antibacterial peptides were precipitated by ammonium sulfate; and third, dialysis through 12 000 kDa molecular weight cut-off membranes followed by freeze drying of partially purified bacteriocin. Molecular sizing of bacteriocin of LA06FT To measure the molecular weight of partially purified bacteriocin produced by LA06FT, Tricine SDS-PAGE was used as illustrated by Batdorj et al. (2006). The acrylamide was applied at a concentration of 16.5%. Sample was loaded on two vertical parts along with a standard marker. When running, complete gel was cut into two parts. The piece of gel having standard marker side
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was placed in the solution of coomassie blue for staining. Other part of the gel having only sample was properly washed with deionizer water and used to measure its anti-microbial activity on the lawn of indicator strain. Statistical analysis Hierarchical cluster analysis was applied and dendrogram was constructed using Package for the Social Sciences (SPSS) statistical package version 12.0 (SPSS Inc., Chicago, IL) for the screening of bacteriocin producing strains and for SDS protein profile of these strains. Variance in bacteriocin activity and probiotic potential were analyzed using a two-factor factorial experiment on M-Stat C Statistical software according to the procedures described by Steel et al. (1996).
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Isolation and identification of L. acidophilus Thirteen isolates of L. acidophilus were obtained from the dahi samples. All isolates were identified using classical microbiological techniques (data not shown) and further confirmed by amplification 16S rRNA region by species-specific primers of L. acidophilus. The results obtained for PCR products are presented in Figure 1. It was observed that all strains have PCR products of approximately 750–760 bp. Lactobacillus acidophilus has been isolated from dahi as in the study by Maqsood et al. (2008), who reported that dahi was a rich source of probiotic lactobacilli. Antagonistic properties of L. acidophilus Thirteen strains of L. acidophilus were tested for anti-bacterial activity against food-borne pathogens (Table 1). It was observed that six strains (TLA01FT, TLA04FT, TLA05FT, LA08FT, LA06FT and LA07FT) have more prominent inhibitory pattern. Hierarchical cluster analysis was carried out to determine the similarity index among the various strains of L. acidophilus (Figure 2) and LA06FT was selected for further characterization since this strain was effective against both Gram-positive and Gram-negative bacteria. Previously many researchers reported that bacteriocin produced by L. acidophilus was capable to kill both Gram-negative and Gram-positive bacteria. Results were in agreement with the finding of Karthikeyan & Santhosh, (2009), who reported that bacteriocin produced by L. acidophilus was effective against Gram-positive and Gram-negative bacteria.
3000 2000 1500 1200 1000 900 800 700 600 500 400 300 200 100
Figure 1. PCR amplification of 16S RNA region of selected strains of L. acidophilus. Line 1: Marker of 100 bp, Line 2: TLA01FT, Line 3: TLA02FT, Line 4: TLA03FT, Line 5: TLA04FT, Line 6: TLA05FT, Line 7: TLA06FT, Line 8: TLA07FT, Line 9: TLA08FT, Line 10: TLA09FT, Line 11: TLA10FT, Line 12: LA05FT, Line 13: LA06FT, Line 14: LA07FT.
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Oh et al. (2000) screened five strains of L. acidophilus for bacteriocin production that were effective against the Gram-positive bacteria like B. cereus, L. monocytogenes and S. aureus. SDS protein profiling of L. acidophilus Protein profile of SDS-cell wall protein was applied for phenotypic relationship between the selected antibacterial activity and the presence or absence of particular bands in cell wall proteins (Figure 3). It was evident from electrophoregram that all bacteriocin producing strains of L. acidophilus produced a protein band of 22 kDa, and this band was absent in non-bacteriocin producing strains. Studies related to electrophoretic protein revealed that different strains of L. acidophilus have both major (60, 45, 43, and 35 kDa) and minor bands (97, 68, 47, 31 and 18 kDa) as reported by Teanpaisan & Dahlen, (2006). However, numbers, size and position of these bands were highly affected by change in ecological and growth conditions that were distressing phenotypic characteristics and not the genotype (Kim et al., 2005). Probiotic characteristics of L. acidophilus LA06FT Effect of bile salt on the pH and growth of L. acidophilus LA06FT Since probiotic bacteria pass through alimentary tract, checking its tolerance for bile and acid are very important phenomena and the results recorded for change in pH and OD are presented in Table 2. It was observed that LA06FT could grow in the presence of 3% bile salt concentration, however it reduced significantly after 6 h. Previous data about the bile tolerance also support our results. Heng et al. (2004) found that L. acidophilus can survive at 2% concentration of bile salt. The results of the present study are in agreement with the findings of Succi et al. (2005), who reported that L. rhamnosus remained active in 2% bile salt concentration. While considering these bacteria as probiotics, they should become a part of the normal microbial flora in the intestine, survive the gastrointestinal passage, and should be able to adhere and colonize in the intestinal tract (Oh et al., 2000). Acid tolerance of L. acidophilus LA06FT In vitro acid tolerance by LA06FT is presented in Table 3. It was observed that high and low initial pH of growth media result in the reduction of bacteriocin activity. At pH 2.5 and 3.5, bacteriocin activities reduced to 100%. However, it remained viable at this low pH, that is human stomach pH. Maximum bacteriocin-specific activity was at pH 6.5, 4.5 and 7.5. This phenomenon has been observed by other researchers (Zamfir et al., 2000) who reported that the bacteriocin produced by L. acidophilus is highly pH dependent and can effectively control the growth of other organisms below pH 6. Therefore, for optimizing bacteriocin production in vitro, the LAB should be grown at the pH values between 5.5 and 7.0 (Todorov & Dicks, 2005). Antibiotic susceptibility test Antibiotic susceptibility test was performed to determine the safety level of selected strain. Results obtained for different antibiotics are presented in Table 4. On the basis of inhibition zone, three groups of antibiotics were identified: first, LA06FT showed resistance to Nalidixic acid, Ciprofloxacine, Gentamicin and Vancomycin (0 to 12 mm); second, Sulphamethoxazol and Erythromysin (12 to 16 mm) showed intermediate behaviour; and third, strain was sensitive to Ampicillin, Penicillin, Tetracycline and Amoxicillin (17 to 33 mm). Then these strains were grown in antibiotic-protected environment to assess the
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Table 1. Screening of different isolates of L. acidophilus for antibacterial activities. Indicator strains S.No
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1 2 3 4 5 6 7 8 9 10 11 12 13
Name of strains L. acidophilus TLA01FT L. acidophilus TLA02FT L. acidophilus TLA03FT L. acidophilus TLA04FT L. acidophilus TLA05FT L. acidophilus TLA06FT L. acidophilus TLA07FT L. acidophilus TLA08FT L. acidophilus TLA09FT L. acidophilus TLA10FT L. acidophilus LA05FT L. acidophilus LA06FT L. acidophilus LA07FT
E. coli ATCC 25922
S. aureus ATCC 6538
P. aeruginosa ATCC 25923
L. monocytogenes ATCC 19115
+++
+++
++
++
+ ++++
++ +
+++
+++ ++++
+
+++
++++
+++
++++ +++
++++
++++
++++ +++
Inhibition zone is mean of three readings. Inhibition zone is ranked as no visible inhibition ( ), visible inhibition (+), diameter of inhibition zone 4 mm (++), diameter of inhibition zone between 4 to 8 mm (+++) and diameter of inhibition zone 8 to12 mm (++++).
Figure 2. Hierarchical cluster analysis of different strains of L. acidophilus for similarity index among them for their anti-bacterial activity showing that first group (TLA10FT, LA04FT, TLA02FT, TLA07FT, TLA09FT, TLA03FT and TLA06FT) have above 80% similarity index.
susceptibility towards the antibiotics. It was observed that LA06FT did not change its behavior towards the antibiotics, so this strain was found to be safe. The results of the present study were supported by the earlier work of Gupta et al. (1996), they examined seven strains of L. acidophilus for antibiotics susceptibility test and found that some strains are sensitive to various antibiotics (Ampicillin, Erythromycin, Tetracycline), but these strains show resistance to Norfloxacin and Nalidixic acid. In general, it was noted that resistance pattern is species specific (Ortu et al., 2007). Similarly, Kastner et al. (2006) observed antibiotics resistance in 27 probiotic cultures which were not their intrinsic feature. Nature of the resistance might vary according to
the habitats from where these strains were isolated (Bernardeau et al., 2008). Technological characteristics of L. acidophilus LA06FT Acidification, heating and enzymes treatment on the activity of bacteriocin of LA06FT Bacteriocin of LA06FT appeared to be stable over wide range of pH (4.5–7.5) and fairly heat stable between 60 and 90 C for 20 min, as illustrated in Figure 4(a). Similarly, bacteriocin of LA06FT was treated with proteolytic and non-proteolytic enzymes to determine the proteineous nature (Figure 4b). It was
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(a) KDa
observed that bacteriocin was highly sensitive to proteolytic enzymes like proteinase K and had no effect on lipase and amylase. These characteristics made it a suitable bio-preservative for acidic and low acidic food products wherein heat treatment are commonly applied as previously reported by Mobarez et al. (2008) and Karthikeyan & Santhosh (2009). The heat stability shown by bacteriocin ruled out the possibility that this inhibitory activity was due to the action of bacteriophages since phages cannot survive at 121 C for 15 min. Some contradictions were also noted among the various studies like Batdorj et al. (2006), where strong heat stability of bacteriocin was reported even at 121 C; it might be due the presence of the large number of stable cross linkages or due to high cysteine contents. However, in regard to enzymes, acidocin-LA06FT was similar to acidocin CH5 (Chumchalova et al., 2004) and acidocin DSM (Deraz et al., 2005) and closely matched with the findings of Batdorj et al. (2006), who reported that bacteriocin was completely inactivated by proteolytic enzymes since bacteriocin contains cleavage-sites suitable for their binding.
116.0 62.2 45.0
35.0
25.0 18.4 14.4
65 66
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(b)
Table 4. Antibiotic susceptibility test of strain L. acidophilus LA06FT. S. No 1 2 3 4 5 6 7 8 9 10
Figure 3. (a) SDS-PADE of cell wall proteins of probiotic strains of L. acidophilus, line 1: protein marker of 14.4 to 116 kDa, line 2: TLA01FT, line 3: TLA04FT, line 4: TLA05FT, line 5: LA08FT, line 6: LA06FT and line 7: LA07FT. L. acidophilus strains produce 6 prominent bands of 80, 62.2, 45, 35, 22 and 14.4 kDa and minor bands of 68, 43, 41, 39, 25 and 18 kDa. (b) SDS-PAGE of non-probiotic strains of L. acidophilus.
Antibiotics
Zone size (mm)
Patron
Nalidixic acid (30 mg) Ampicillin (30 mg) Ciprofloxacine (5 mg) Tetracycline (30 mg) Penicillin (10 U) Sulphamethoxazol (25 mg) Erythromycin (15 mg) Gentamicin (10 mg) Amoxicillin (30 mg) Vancomycin (30 mg)
0.0 ± 0.00 26.0 ± 1.00 10.0 ± 0.25 30.0 ± 0.50 32.0 ± 0.45 14.0 ± 0.33 16.0 ± 1.55 12.0 ± 1.43 33.0 ± 1.81 0.0 ± 0.00
Resistant Sensitive Resistant Sensitive Sensitive Resistant Intermediate Resistant Sensitive Intermediate
The results are expressed in term of inhibition zone around the discs and mentioned it resistant (0 12 mm), intermediate (13 16 mm) and sensitive (17 33 mm). Standard error was calculated at LSD value 0.0783.
Table 2. Effect of different concentration of bile salts on the growth of L. acidophilus LA06FT in terms of change in pH and OD. Optical density (OD) Intervals 0h 1h 2h 4h 6h
Control g
0.04 (0.00) 0.046fg (13.04) 0.08efg (51.81) 0.18a (78.84) 0.30b (86.71)
1% g
0.03 (0.00) 0.05fg (41.18) 0.07efg (58.90) 0.15cd (80.0) 0.20c (58.0)
2% fg
0.05 (0.00) 0.07efg (30.26) 0.08efg (40.45) 0.10def (48.54) 0.11def (51.82)
pH 3% defg
0.09 (0.00) 0.123de (26.83) de 0.13 (31.30) 0.12de (29.01) 0.12de (28.00)
Control e
6.00 (0.00) 5.87f (2.17) 5.83f (2.83) 5.50g (8.33) 5.01h (16.50)
1% c
6.20 (0.00) 6.15cd (0.81) 6.14cd (0.97) 6.08de (1.97) 5.79f (6.61)
2% b
3%
6.36 (0.00) 6.21c (2.36) 6.15cd (3.30) 6.07de (4.56) 6.05de (4.87)
a
6.50 6.21c 6.21c 6.20c 6.20c
(0.00) (4.46) (4.46) (4.62) (4.62)
Means followed by same alphabets are non-significant for p50.05. For pH, DMR test was determined at LSD values of 0.1044 and standard deviation 0.0361 and for OD. DMR test was determined at LSD values of 0.05218 and standard deviation 0.01826. Control: Growth of tested strain on MRS without bile salt.
Table 3. Acid tolerance of L. acidophilus LA06FT. Initial pH
Final pH
Change in pH
OD 600 nm
Bacteriocin activity (AU/mL)
Reduction (%)
Specific activity (AU/OD)
2.50 ± 0.01 3.50 ± 0.03 4.50 ± 0.02 5.50 ± 0.07 6.50 ± 0.05 7.50 ± 0.07
2.50 ± 0.01 3.10 ± 0.01 3.80 ± 0.05 4.00 ± 0.04 4.00 ± 0.04 3.85 ± 0.03
0.00 0.40 0.70 1.50 2.50 3.50
0.25 ± 0.03 1.20 ± 0.01 1.40 ± 0.01 2.00 ± 0.02 1.80 ± 0.07 1.70 ± 0.07
000.0 ± 0.00 000.0 ± 0.00 200.0 ± 20.0 800.0 ± 34.0 400.0 ± 26.0 100.0 ± 11.0
100.0 ± 5.0 100.0 ± 5.0 75.0 ± 2.5 0.0 ± 0.0 50.0 ± 1.9 87.5 ± 2.9
0.00 ± 0.0 0.00 ± 0.0 142.86 ± 8.1 470.59 ± 7.2 222.20 ± 12.2 50.00 ± 3.1
Results were compared against control pH of 6.2 of MRS media. OD, final pH and bacteriocin activity was measured after 24 h incubation. Results were means of three replications. Values ± corresponded to standard error.
Lactobacillus acidophilus
DOI: 10.3109/09637486.2014.880666
Purification of bacteriocin of LA06FT Purification of bacteriocin produced by L. acidophilus LAFT06 was carried out in a three-step procedure and purification scheme is summarized in Table 5. Cell-free supernatant was the first level of purification that was the result of centrifugation and filtration indicating 100% recovery at a specific activity of 5369.13 AU mg 1. Previously, many researchers determined purification levels of bacteriocin containing supernatant. Chumchalova et al. (2004) determined the purification of acidocin CH5 produced by L. acidophilus CH5 and observed that bacteriocin activity and specific activity were 3200AU mL 1 o
Temerature tolerance ( C)
(a) 60
70
80
90
100
120 900
12
587
and 2900 AU mg 1, respectively. Similarly, Batdorj et al. (2006) used centrifugation as the first level of purification and found a specific activity of 256AU mg 1 for cell-free supernatant. At the second level, acidocin LA06FT was purified to homogeneity by ammonium sulfate precipitation (ASP), most widely used techniques for the purification of bacteriocin, producing total activity of 160 000 AU resulting in 80% concentration of bacteriocin pellets. Results were different from the earlier findings of Deraz et al. (2005), who applied 40% ammonium sulfate for the precipitation of bacteriocin produced by L. acidophilus DSM20079 and got 94% recovery, since the solubility of bacteriocin varies according to the ionic strength of salt. At the third level, partially purified bacteriocin by ASP was freeze dried and it was observed that antibacterial activity increased to 3200 AU/mL that was two times higher than the ammonium sulfate precipitation. The results were almost similar to that reported by Burianek & Yousef (2000).
600 AU/ ml
Inhibition zne (mm)
Molecular sizing of bacteriocin of LA06FT
700 8
500 6 400 4
300 200
2 100 0
0 2.5
3.5
4.5
5.5
6.5
7.5
8.5
9.5
10.5
Acid tolerance (pH)
(b) 900
Molecular weight of partially purified bacteriocin was determined by SDS-PAGE. Results revealed acidocin LA06FT had single band of proximately 8.5 kDa (Figure 5). Zone of inhibition corresponding to the position of bacteriocin also confirmed the identified bacteriocin. The present results coincided with previous studies on molecular characterization of bacteriocin produced by L. acidophilus, which stated that most of the bacteriocins were of low molecular weight (Zamfir et al., 2000). Research finding were also in line with the results of Todorov & Dicks (2005) who found that bacteriocin produced by the L. pentosus had low molecular weight, they also studied its antibacterial behavior.
10
800
9
700
8
600
7 6
500
5
400
4
kDa Zone (mm)
AU/ ml
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800 10
62.2
3
45.0
200
2
35.0
100
1
0
0 amylase Trypsin Enzymes
(b)
116.0
300
Lipase
(a)
25.0
Proteinase-k
Figure 4. (a) Temperature ( C) and acid tolerance (pH) by the bacteriocin produced by LA06FT; (), effect of heating in the bacteriocin activity (AU mL 1); (m), effect of different pH on the bacteriocin activity. (b) Effect of different enzymes on the activity of bacteriocin produced by LA06FT. Enzymes were used at the concentration of 0.1 mg mL 1. (), bacteriocin activity (AU mL 1); (m), Zone of inhibition (mm).
18.4 14.4
8.5
Figure 5. Molecular weight (8.5 kDa) of purified bacteriocin LA06FT determined by SDS gel electrophoresis (a) molecular weight of bacteriocin and marker (b) gel was overlaid on the lawn of S. aurues to determine the bacteriocin of purified bacteriocin.
Table 5. Steps for the purification of bacteriocin produced by LA06FT.
Purification stages Centrifugation and filtration Ammonium sulphate ppt Freeze drying a
Volume (mL)
Activity (AU/mL)
Total activity (AU)a
Protein (mg/mL)b
Total protein (mg)c
Specific activity (AU/mg)d
Purification factore
Recovery (%)f
250 100 5
800 1600 3200
200 000 160 000 16 000
0.149 0.097 0.012
37.25 9.70 0.06
5369.13 16 494.85 266 666.67
1.00 3.07 49.67
100.00 80.00 8.00
Protein concentration was determined by the Lowry method. Bacteriocin activity can be calculated by well diffusion method. c Total activity (AU) and total protein (mg) was determined by the multiplication of volume by activity (AU/mL) and protein (mg/mL). d Specific activity was determined by dividing total activity with total protein concentration (AU/mg). e Purification factor was determined by dividing the subsequent value of specific activity by specific activity of cell-free supernatant. f Percentage recovery was determined by dividing the total activity of subsequent step by total activity of cell-free supernatant. b
588
T. Mahmood et al.
Conclusion Lactobacillus acidophilus has been found in many traditional fermented milk products. The rapid growth and compatible behavior towards primary starter species ensure its survival in yogurt-like products. Therefore, it is important to study technological and therapeutic properties of this bacterium. Present study provides an insight regarding its probiotic potential and possible safety concerns. Similarly, bacteriocin producing L. acidophilus LA06FT share same homology with the other reported bacteriocin. Although bacteriocin production is an inherited trail, supplementing the growth medium with growth limiting factors could enhance bacteriocin production. In this consequence, purification of these compounds would be further helpful to meet the thirst of new and more effective bacteriocins.
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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
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