In vitro study of antioxidant and antibacterial activities ... - Springer Link

54 downloads 0 Views 1021KB Size Report
square), AG2 (multiplication sign), AG20 (filled circle), and AG21 (emp- ty circle) in the presence of hydrogen peroxide. Fig. 4 Survival of lactobacilli K-series ...
Folia Microbiol DOI 10.1007/s12223-017-0531-x

In vitro study of antioxidant and antibacterial activities of Lactobacillus probiotic spp. Z. Pourramezan 1 & R. Kasra Kermanshahi 1 & M. Oloomi 2 & A. Aliahmadi 3 & H. Rezadoost 4

Received: 22 July 2016 / Accepted: 10 May 2017 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2017

Abstract This study investigated the influence of aeration and minimal medium conditions on antioxidant and antibacterial activities of 21 probiotic Lactobacillus strains isolated from dairy products. The probiotic potential of the isolates was evaluated by pH and bile tolerance. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to confirm the phenotypic identification of isolates. Antioxidant producer isolates were screened by resistance to reactive oxygen species (ROS). The antioxidant and antibacterial activities of extracellular materials after 48 h fermentation with antioxidative strains were determined using 2,2diphenyl-1-picrylhydrazyl (DPPH) and broth microdilution assays, respectively. The results indicate that the antioxidant capacity of supernatants was increased by using of both minimal medium and agitation. The antibacterial activity was increased in minimal medium, but there has nearly no change in the antibacterial properties by using both agitation and

* Z. Pourramezan [email protected] * R. Kasra Kermanshahi [email protected]; [email protected]

1

Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

2

Molecular Biology Unit, Pasteur Institute of Iran, Pasteur Ave, Tehran 13164, Iran

3

Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran

4

Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran

minimal medium. The maximum antibacterial activity was observed during mid-exponential phase until the beginning of the early-stationary phase, but the maximum antioxidant activity was detected at the stationary growth phase. There is a significant relationship between antioxidant and antibacterial activities of the cell-free probiotic extracts, and their production rates are closely related to the fermentation type. The bioactive materials from probiotics could be extracted in a large amount at an appropriate time under a suitable condition.

Introduction Natural products have been a major source of therapeutic molecules. They have been used as alternative healthcare treatment and also in discovery of new drugs (Kusuma et al. 2014). The search for natural bioactive compounds (NBCs) with potential for the prevention and treatment of human diseases is an important issue in many industries and laboratories (Ajikumar et al. 2008). One of these NBCs could be the antioxidant and antibacterial materials extracted from probiotics. Over the last two decades, some studies on health benefits of probiotics have been published. These literature have focused on health benefits of probiotics such as treatment and prevention of diseases, such as cancer, urogenital tract infections, allergy, inflammatory diseases, bowel syndromes, diarrhea (Reid et al. 2003), lactose maldigestion, hypertension, cardiovascular disease, diabetes, Alzheimer’s disease, neurological disorders, and obesity (Woo et al. 2014). There is increasing evidence to suggest that various degenerative diseases could be the consequence of cellular injury caused by free radicals (Lobo et al. 2010). Some probiotics, especially lactobacilli, possess antioxidant activities and are able to

Folia Microbiol

ƒFig. 1

Dendrogram showing clustering and relationships of Lactobacillus reference and isolated strains based on 20 biochemical and physiological analyses. The isolates were grouped by means of the Jaccard correlation coefficient and unweighted pair group method algorithm (UPGMA) cluster analysis with Bionumerics software version 7.5

decrease the risk of accumulation of ROS during ingestion of food (Kim et al. 2005). Another important trait of probiotic strains is antimicrobial activity against pathogens (Sindhu et al. 2014). The production of natural bioactive compounds by probiotics was shown to be dependent on the growth environment (Annuk et al. 2003). There is a need to improve fermentation conditions, detection, and separation of the effective materials that would revolutionize the screening of NBCs from probiotics. The improved growth condition could provide an unknown signal that activates an unidentified gene cluster leading to the production of a new metabolite and/or increase the production of the desired compounds (Zerikly and Challis 2009). The growth phase of the probiotics during fermentation could have effects on the antioxidant and antibacterial productions. Wang et al. (2013) reported that antioxidant activity began at early exponential growth phases and maximum activity was reached at the stationary phase. It has been found that the oxidative stress and exhaustion of an essential nutrient limit the growth of a bacterial culture which then enters into stationary growth phase (Hussain et al. 2013). Therefore, it was hypothesized that by using starvation (minimal medium) and oxidative stress (aeration), some silent gene clusters activated and antioxidant production could be induced genetically. However, to the best of our knowledge, the oxidative stress response and survival capacity of different Lactobacillus species under aerobic growth conditions and the comparison between optimal and hostile environments for antioxidant and antibacterial activities have not been investigated yet. In summary, in this study, some Lactobacillus probiotic was isolated from dairy products. These isolates were identified by using colonial, cell morphology, physiological characteristics, and carbohydrate fermentation profiles. As random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) has proved to be an informative method suitable for the study of a large number of strains in a short time (Nigatu et al. 2001), this method was used to confirm the phenotypic identification. The antioxidant and antibacterial activities of extracellular material from these strains were determined by DPPH and broth microdilution assays, respectively. Finally, the effect of aeration and starvation

Folia Microbiol Fig. 2 Dendrogram showing normalized agarose gel electrophoresis products for the reference and 12 isolated strains of Lactobacillus species using the GelCompar II version 6.6 program. Patterns were grouped with the unweighted pair group method algorithm (UPGMA) based on the Pearson product moment correlation coefficient

during fermentation were investigated on their antioxidative and antibacterial activities. This study is the first attempt to approach stationary phase adaptation of lactobacilli strains using a true minimal medium called Plantarum Minimal Medium (PMM5) and oxidative stress (aeration).

Strains of Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 11303, Lactobacillus acidophilus ATCC 4356, Lactobacillus plantarum ATCC 8014, Lactobacillus casei

spp. casei PTCC 1608, Lactobacillus rhamnosus PTCC 1607, and Lactobacillus fermentum PTCC 1638 were purchased from Iranian Research Organization for Science and Technology (IROST). The samples of dairy product (yoghurt, camel doogh, and cow milk kefir) were collected from different areas of Iran. The isolation, purification, and preservation of the isolates were done according to the method described by Federici et al. (2014). The strains were named with the first letter of their isolation niches (Y, AG, and K stand for yoghurt, Agh-Ghala camel doogh, and cow milk kefir, respectively) and numerical after the letters are used to indicate the order in which they were isolated. For antioxidant and antibacterial activity determination, the isolates were anaerobically cultured in Man-Rogosa-Sharpe (MRS) broth in 37 °C (Merck, Germany) and then for possible

Fig. 3 Survival of lactobacilli AG-series isolates including AG12a (empty triangle), AG32a (filled triangle), AG32b (empty square), AG1 (filled square), AG2 (multiplication sign), AG20 (filled circle), and AG21 (empty circle) in the presence of hydrogen peroxide

Fig. 4 Survival of lactobacilli K-series isolates including K1C (empty triangle), K2 (empty circle), K3 (multiplication sign), K4 (filled triangle), K5 (filled circle), and K6 (filled square) in the presence of hydrogen peroxide

Materials and methods Samples, microorganisms, and culture media

Folia Microbiol Fig. 5 Survival of lactobacilli Yseries isolates including YA1a (empty square), YA1b (filled circle), YA2 (multiplication sign), YA3 (filled square), and Ym (filled triangle) in the presence of hydrogen peroxide

increase production, PMM5 broth (Wegkamp et al. 2010) and aeration (150 rpm) were used (Federici et al. 2014). The probiotic potential of the isolates was evaluated by pH and bile tolerance according to Dunne et al. (2001) and Ryan et al. (2008).

Phenotypic and genotypic identification Biochemical tests The isolates were tested for their ability to grow in MRS broth at 10 and 42 °C by incubating for 7 days and 24–48 h, respectively. Oxidase, motility test, gas from glucose, arginine hydrolysis, esculin hydrolysis, nitrate reduction test, gelatin hydrolysis, citrate utilization test, carbohydrate fermentation tests, and growth in nutrient broth (NB) and in the presence of 2.0, 4.0, and 8.0% NaCl were assessed for all isolates (Vos et al. 2009). Numerical analysis of physiological and carbo-

Fig. 6 Survival of standard reference strains of lactobacilli including L. casei spp. casei PTTC 1708 (filled square), L. fermentum PTCC 1638 (filled circle), L. rhamnosus PTCC 1607 (filled triangle), L. acidophilus ATCC 4356 (empty circle), and L. plantarum ATCC 8014(empty triangle) in the presence of hydrogen peroxide

hydrate fermentation results was done using Bionumerics Software version 7.5 (Applied Maths). DNA extraction and RAPD-PCR analysis Lactobacilli were grown overnight in 5 mL MRS broth. The DNA was isolated by using of Gene Transfer Pioneers (GTP, Iran) kit according to the manufacturer’s instruction. The extracted genomes were quantified using a Picodrop Spectrophotometer (Picodrop, UK). Genotypic differentiation was approached by RAPD-PCR comparing the profiles with those of lactobacilli reference strains. Each RAPD-PCR analyses were performed using a single 10-mer random primer PAP3 (5′-TGGATTGGTC-3′) (Séré et al. 2007) or Coc (5′-AGCAGCGTGG-3′) (Federici et al. 2014) (Bioneer, korea). The method of Séré et al. (2007) was used to perform PCR in a Mastercycler (Eppendorf, Germany), and RAPDPCR products were analyzed by electrophoresis run on

Folia Microbiol Table 1 Zone of growth inhibition (mm) of isolates around paper disk contain 15 μL of 10 mmol/L paraquat

Screening of antioxidative isolates by resistance against ROS

Isolates

Zone of growth inhibition (mm)a

L. durianis strain AG1

7.5 ± 0.5

L. suebicus strain AG2 AG12b1b L. aviarius strain AG12b2

7.5 ± 0.5 7.5 ± 0.5

L. parakefiri strain AG32a AG32b

10 ± 0.5 10 ± 0.5

L. parakefiri strain AG33d L. suebicus strain AG21

7 ± 0.25 11 ± 0.5 13 ± 1.5 12 ± 0.5

Three resistance methods against ROS (such as application of hydrogen peroxide, superoxide anions and hydroxyl radicals) were used for screening of the antioxidative strains. The resistance of lactobacilli in the presence of hydrogen peroxide was examined according to the method of Kim et al. (2005). Briefly, the overnight cultures of isolates were suspended at the level of 107 CFU/mL in isotonic saline and incubated with 0.4 mmol/L hydrogen peroxide. At 1 h intervals, the number of viable cells was estimated by plating 1 mL of suspensions onto the MRS agar plates and incubated for 48 h. Resistance of lactobacilli to superoxide anions generated by paraquat (1,1′-dimethyl-4,4′-bipyridinium, M-2244, Sigma) was tested using the disk diffusion assay according to the method described by Kullisaar et al. (2002). Resistance to hydroxyl radicals was examined by the method of Kim et al. (2005). Briefly, 107 CFU/mL strains were incubated with the solution containing 10 mmol/L THA and 0.01 mmol/L CuSO4.5H2O in SPB. Hydroxyl radicals were generated via the Fenton reaction by addition of 1.0 mmol/L hydrogen peroxide in the suspension.

7.75 ± 0.25

K3b K4b L. pentosus strain K5 L. manihotivorans strain YA1b

11 ± 0.5 8±1

L. kimchii strain YA2 L. alimentarius strain Ym

10 ± 0.5 16 ± 0.5

L. acidophilus ATCC 4356 L. plantarum ATCC 8014

17 ± 0.75 16 ± 1.5

a

The data represent the mean ± standard deviation of three independent experiments

b

Isolates that were not identified by biochemical and molecular methods

Cell-free culture supernatant preparation 2.0% agarose gels, stained with ethidium-bromide, and photographed. Conversion, normalization, and numerical analysis of the patterns were carried out with the GelCompar II version 6.6 software (Applied Maths). Similarity coefficients for pairs of tracks were calculated by using Pearson’s correlation coefficient, and strains were grouped by using the unweighted pair group method with arithmetic averages (UPGMA) for construction of the dendrogram (Nigatu et al. 2001).

Fig. 7 The survival of the lactobacilli AG-series isolates including AG12a (filled circle), AG1 (empty circle), AG2 (filled square), AG12b2 (empty triangle), AG32a (filled diamond), AG32b (multiplication sign), AG12b1 (plus sign), AG20 (empty square), and AG21 (filled triangle) in the presence of hydroxyl radical

The methanol extract of cell-free culture supernatants was prepared according to the method described by Saadatzadeh et al. (2013). Determination of antioxidant activity The antioxidant activity of supernatant was determined via DPPH assay according to the method described by Li et al. (2012). Then, the absorbance was read against a blank at

Folia Microbiol Fig. 8 The survival of the lactobacilli K-series isolates including K1C (filled triangle), K2 (filled square), K3 (empty circle), K4 (empty triangle), K5 (empty square), and K6 (filled circle) in the presence of hydroxyl radical

Fig. 9 The survival of the lactobacilli Y-series isolates including YA1a (filled circle), YA1b (filled triangle), YA2 (filled square), YA3 (empty circle), and Ym (empty square) in the presence of hydroxyl radical

517 nm using a PowerWave XS2 Microplate spectrophotometer (Bio-Tek Instruments Inc., USA). Triplicate measurements were performed, and the radical scavenging activity of the samples was calculated as follows:

Fig. 10 The Survival of standard reference strains of lactobacilli including L. casei spp. casei PTTC 1708 (multiplication sign), L. fermentum PTCC 1638 (empty triangle), L. rhamnosus PTCC 1607 (filled square), L. acidophilus ATCC 4356 (empty square), and L. plantarum ATCC 8014 (filled circle) in the presence of hydroxyl radical

Inhibition of DPPH radical ð%Þ    ¼ Acontrol −Asample =Acontrol  100

Folia Microbiol Table 2 DPPH scavenging activity of extracellular methanol extract of all isolates after 48 h anaerobic cultivation in MRS broth at 37 °C Probiotic strains

IC50 (μg/mL)a

Table 3 Minimal medium and aeration effects on the antioxidant production of different probiotic lactobacilli by DPPH assay of extracellular materials of 48 h anaerobic and aerobic fermentation in PMM5 broth

L. durianis strain AG1

925.21

Probiotic strains

L. suebicus strain AG2 L. hilgardii strain AG12a

Anaerobic IC50 (μg/mL)a

Aerobic (150 rpm) IC50 (μg/mL)a

AG12b1b

1292.88 1445.13 1134.48

L. durianis strain AG1

768.73

686.69

L. suebicus strain AG2

975.87

406.27

L. aviarius strain AG12b2

929.4

L. suebicus strain AG20 L. suebicus strain AG21

881.82 899.14

535.27 963.98

512.02 877.91

L. parakefiri strain AG32a L. durianis strain AG32b

1535.4 1508.56

L. hilgardii strain AG12a AG12b1b L. aviarius strain AG12b2 L. suebicus strain AG20

675.25 532.81

664.71 541.28

L. casei strain K1C

880.96 917.81 1334.35 1286.97

L. suebicus strain AG21 L. parakefiri strain AG32a

590.798 914.07

531.6 326.73

L. durianis strain AG32b

912.08

740.52

L. casei strain K1C K2b K3b K4b L. pentosus strain K5 K6b L. crispatus strain YA1a L. manihotivorans strain YA1b

492.01 725.63 1003.78 1078.53 768.26 744.21 793.35 860.53

266.82 711.23 960.52 987.26 674.21 692.60 697.64 765.26

L. kimchii strain YA2 L. rhamnosus strain YA3

712.80 701.86

589.6 635.26

L. acidophilus ATCC 4356 L. casei spp. casei PTTC 1708 L. rhamnosus PTCC 1607

848.27 846.56 866.041

790.47 373.46 787.91

L. plantarum ATCC 8014 L. fermentum PTCC 1638

755.87 823.64

685.26 726.43

K2b K3b K4b L. pentosus strain K5 K6b L. crispatus strain YA1a L. manihotivorans strain YA1b

885.2 909.70 1131.76 1139.87

L. kimchii strain YA2 L. rhamnosus strain YA3 L. acidophilus ATCC 4356 L. casei spp. casei PTTC 1708

1323.88 906.28 1005.4 1330.59

L. rhamnosus PTCC 1607

1013.56

L. plantarum ATCC 8014 L. fermentum PTCC 1638

984.26 980.87

a

The data are means for three independent experiments carried out in triplicate

b

Isolates that were not identified by biochemical and molecular methods

a

The data are means for three independent experiments carried out in triplicate

b

where Ablank is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound. Sample concentration providing 50% inhibition (IC50) was calculated from the graph plotted of inhibition percentage against samples concentration.

Antibacterial assay In vitro antibacterial activity of products was assessed against S. aureus ATCC 6538 and E. coli ATCC 11303 strains by broth microdilution susceptibility tests according to the standard protocol (Balouiri et al. 2016). All experiments were done in triplicate, and chloramphenicol was used as standard antibiotic.

Isolates that were not identified by biochemical and molecular methods

Results Phenotypic and genotypic identification Twenty-one strains, which were isolated from yoghurt, camel dough, and cow milk kefir, were considered as Lactobacillus spp. based on their bacillus shape, positive Gram reactions, non-motility, absence of catalase activity, and spore formation. Both nitrate reduction test and gelatin hydrolysis were negative for all isolates. All the strains could grow at 37 °C, pH 3.0, and in 0.15 and 0.3% dexocycholate. All of the isolates were able to ferment arabinose. With the exception of Ym isolate, all isolates fermented galactose. UPGMA dendrogram generated by using of physiological traits and sugar fermentation profile is shown at Fig. 1.

Folia Microbiol Table 4 Antibacterial activity of methanol cell-free extract of MRS broth anaerobically fermented with different probiotic lactobacilli for 48 h by microdilution susceptibility tests after 24 h incubation at 37 °C

Probiotic isolates

Escherichia coli ATCC 11303

Staphylococcus aureus ATCC 6538

MIC (mg/mL)

MBC (mg/mL)

MIC (mg/mL)

MBC (mg/mL)

L. durianis strain AG1

128

128

128

256

L. suebicus strain AG2

128

128

128

256

L. hilgardii strain AG12a AG12b1a

32

64

64

64

128

128

128

256

L. aviarius strain AG12b2 L. suebicus strain AG20

128 64

128 64

256 64

256 128

L. suebicus strain AG21 L. parakefiri strain AG32a

64 64

128 128

256 256

256 512

L. durianis strain AG32b

32

32

32

64

32 64 128 256

64 128 128 256

64 128 128 256

64 128 256 256

128 128 128 128

256 128 128 128

128 256 128 128

256 256 256 128

L. kimchii strain YA2 L. rhamnosus strain YA3 L. rhamnosus PTCC 1607

32 32 64

32 32 64

32 64 64

64 128 128

L. fermentum PTCC 1638 L. plantarum ATCC 8014 L. acidophilus ATCC 4356

128 128 64

128 128 64

128 128 64

128 256 128

64

64

64

128

L. casei strain K1C K2a K3a K4a L. pentosus strain K5 K6a L. crispatus strain YA1a L. manihotivorans strain YA1b

L. casei spp. casei PTTC 1708 a

Isolates that were not identified by biochemical and molecular methods

Amplified products using Coc primer ranged in size from 500 to 3000 bp, and each strain gave a slightly different RAPD pattern, indicative of the genotypic diversity among Lactobacillus strains (Fig. 2). According to the results, the species of Lactobacillus parakefiri, Lactobacillus suebiscus, L. rhamnosus, L. casei, and Lactobacillus durianis can be discriminated on the basis of RAPD-PCR patterns using Coc primer and were correctly grouped with the corresponding biochemical tests. But Lactobacillus hilgardii, Lactobacillus pentosus, Lactobacillus alimentarius, Lactobacillus kimchii, Lactobacillus manihotivorans, Lactobacillus crispatus, and Lactobacillus aviarius were not correctly grouped on the basis of Coc primer RAPD-PCR patterns. Strains with RAPD identification in disagreement with phenotypical identification were analyzed by sugar fermentation profile. However, a definitive identification of K2, K3, K4, K6, and AG12b1 isolates was not achieved by phenotypic and genotypic methods. Strain AG12b1 has no similarity with other isolates by both sugar fermentation and RAPD profiles.

Screening of antioxidative isolates by resistance against ROS. Results in Figs. 3, 4, 5, and 6 were shown that all lactobacilli strains except L. parakefiri strain AG33d survived after 6 h in the presence of 1.0 mmol/L hydrogen peroxide. Superoxide-dependent growth inhibition was established in the case of some strains that had an inhibition zone around paper disk contain paraquat, and the zone of growth inhibition (mm) was measured that is shown in Table 1. Other strains could withstand superoxide anions. In the presence of highly damaging hydroxyl radicals that were produced via Fenton reaction, L. parakefiri strain AG33d did not survive at all and the antioxidative lactobacilli strains survived for at least 300 min (Figs. 7, 8, 9, and 10). Determination of antioxidant and antibacterial activities Since L. parakefiri strain AG33d and L. alimentarius strain Ym were highly susceptible to ROS, these strains were not used for further investigation.

Folia Microbiol Table 5 Antibacterial activity of methanol cell-free extract of PMM5 broth anaerobically fermented with different probiotic lactobacilli for 48 h by microdilution susceptibility tests after 24 h incubation at 37 °C

Probiotic isolates

Escherichia coli ATCC 11303

Staphylococcus aureus ATCC 6538

MIC (mg/mL)

MBC (mg/mL)

MIC (mg/mL)

MBC (mg/mL) 50.72

L. durianis strain AG1

25.36

25.36

50.72

L. suebicus strain AG2

25.36

25.36

50.72

50.72

L. hilgardii strain AG12a AG12b1a

8.87

8.87

17.74

17.74

25.36

25.36

50.72

50.72

L. aviarius strain AG12b2 L. suebicus strain AG20

25.36 6.90

25.36 6.90

50.72 13.79

50.72 27.58

L. suebicus strain AG21 L. parakefiri strain AG32a

6.90 6.90

6.90 6.90

13.79 13.79

27.58 27.58

L. durianis strain AG32b

23.09

46.18

23.09

46.18

L. casei strain K1C K2a K3a K4a L. pentosus strain K5 K6a L. crispatus strain YA1a L. manihotivorans strain YA1b

25.32 25.32 50.64 101.28

25.32 50.64 50.64 101.28

25.32 50.64 50.64 101.28

50.64 101.28 101.28 202.56

50.64 50.64 101.28 101.28

101.28 101.28 101.28 101.28

101.28 101.28 101.28 101.28

202.56 202.56 202.56 202.56

L. kimchii strain YA2 L. rhamnosus strain YA3 L. rhamnosus PTCC 1607

38.97 101.28 25.36

77.94 101.28 25.36

77.94 101.28 50.72

155.88 202.56 50.72

L. fermentum PTCC 1638 L. plantarum ATCC 8014 L. acidophilus ATCC 4356

8.87 8.87 25.36

8.87 8.87 25.36

17.74 17.74 50.72

17.74 17.74 50.72

L. casei spp. casei PTTC 1708

25.36

25.36

50.72

50.72

a

Isolates that were not identified by biochemical and molecular methods.

For determination of antioxidant activity of samples, butylated hydroxytoluene (BHT) (1 mg/mL) was used as standard antioxidant in all tests. The IC50 of BHT equals to 4.82 μg/mL that was calculated using MS Excel software (y = 12.22x − 8.97, R2 = 0.97). IC50 values of lyophilized cell-free extracellular methanol extract of all probiotic isolates after 48 h anaerobic cultivation in MRS and PMM5 broths at 37 °C are presented in Tables 2 and 3. The most effective DPPH radical savaging activity was exhibited by L. casei strain K1C, L. suebicus strain AG20, L. pentosus strain K5, L. suebicus strain AG21, and L. rhamnosus strain YA3, respectively. The minimum inhibitory concentration (MIC) (μg/mL) of chloramphenicol against S. aureus ATCC 6538 and E. coli ATCC 11303 was 2 and 0.5, respectively .Supernatants obtained from all antioxidative isolates exhibited varying degrees of inhibitory activity against E. coli and S. aureus (Table 4). A large increase in antibacterial and antioxidant activity was observed in minimal medium (PMM5) compared to

enrichment medium (MRS) (Tables 3 and 5). A slight increase in antioxidant production was observed in culture agitated at 150 rpm compared to that obtained in non-agitated PMM5 (Table 3) and MRS broth, but the antibacterial properties decrease by the agitation (Table 6).

Discussion All isolated Lactobacillus strains showed good bile tolerance at 0.15 and 0.3% concentration of bile and pH 3. Similar results were reported by Kõll et al. (2008). It is difficult to identify a microorganism only by using the changes in pH as an indicator of growth in the presence of different sugars. The most accurate results were obtained when both phenotypic and genotypic attributes were used to identify the species (Fitzsimons et al. 1999). For this purpose, we used RAPD-PCR to confirm the phenotypic identification of strains and species affiliation determination. The results of current study show that informative patterns were obtained

Folia Microbiol Table 6 Antibacterial activity of methanol cell-free extract of PMM5 broth aerobically (150 rpm) fermented with different probiotic lactobacilli by microdilution susceptibility tests after 24 h at 37 °C

Probiotic isolates

Escherichia coli ATCC 11303

Staphylococcus aureus ATCC 6538

MIC (mg/mL)

MBC (mg/mL)

MIC (mg/mL)

MBC (mg/mL) 130.88

L. durianis strain AG1

130.88

130.88

130.88

L. suebicus strain AG2

130.88

130.88

261.76

261.76

L. hilgardii strain AG12a AG12b1a

54.41

54.41

108.82

108.82

130.88

130.88

261.76

261.76

L. aviarius strain AG12b2 L. suebicus strain AG20

130.88 31.86

130.88 63.73

261.76 63.73

261.76 127.46

L. suebicus strain AG21 L. parakefiri strain AG32a

63.73 63.73

63.73 63.73

63.73 63.73

127.46 127.46

L. durianis strain AG32b

46.17

46.17

46.17

128.34

L. casei strain K1C K2a K3a K4a L. pentosus strain K5 K6a L. crispatus strain YA1a L. manihotivorans strain YA1b

65.44 65.44 261.76 261.76

130.88 130.88 261.76 261.76

261.76 261.76 261.76 261.76

261.76 261.76 523.52 523.52

261.76 261.76 130.88 130.88

261.76 261.76 130.88 130.88

261.76 261.76 130.88 130.88

523.52 523.52 130.88 261.76

L. kimchii strain YA2 L. rhamnosus strain YA3 L. rhamnosus PTCC 1607

311.78 54.41 108.82

623.56 54.41 108.82

623.56 108.82 217.64

623.56 108.82 217.64

L. fermentum PTCC 1638 L. plantarum ATCC 8014 L. acidophilus ATCC 4356

130.88 130.88 65.44

130.88 130.88 130.88

130.88 130.88 130.88

261.76 261.76 261.76

L. casei spp. casei PTTC 1708

65.44

130.88

130.88

261.76

a

Isolates that were not identified by biochemical and molecular methods

using Coc primer, and the majority of the strains studied showed unique patterns and could be easily distinguished from each other. On the other hand, the determined genotypes were completely consistent with the phenotypes and biochemical identification of the isolates. Antioxidant activity of microorganisms is one of the reasons for their increased resistance to reactive oxygen species (ROS). It has been reported that the antioxidant strains have significantly high resistance to ROS compared with the lowantioxidant strains (Kullisaar et al. 2002) that is augmented by findings of the present study. Kullisaar et al. (2002) stated that the antioxidative lactobacilli survived for 34 min in the presence of hydroxyl radicals. This study has provided remarkable evidence that all isolates could survive until 300 min and have some potent mechanisms to decline the effects of ROS. This high tolerance toward hydroxyl radicals is probably related to a wide variety of Lactobacillus species that were used. According to a recent study by Saadatzadeh et al. (2013), using lyophilized probiotic extract is an innovation strategy for developing probiotic products. Extraction

temperature is an important factor which might be effective in chemical constituents. The IC50 value increased significantly by increasing the temperature during extraction, (Motamed et al. 2014), so the lyophilization before methanol extraction of probiotic supernatants seems to be a suitable way of extraction. Literature reveals that Lactobacillus spp. showed a broad inhibitory spectrum against the indicator organisms tested (Ryan et al. 2008). Lactobacillus sake and L. plantarum strains from meat and meat products (Toksoy et al. 1999); L. paracasei subsp. paracasei and L. acidophilus strains isolated from infant feces (Xanthopoulos et al. 2000); and L. rhamnosus, L. casei, and L. lactis subsp. lactis (Nyanzi et al. 2015) had good inhibitory activity against the pathogens that is in agreement with the findings of the present study. In addition, the antibacterial activity of methanol extracts from extracellular material of L. durianis, L. kimchii, L. suebicus, and L. hilgardii strains against E. coli and S. aureus was confirmed in the current study. Erdogrul and Erbilir (2006) demonstrated that L. casei showed weak antibacterial activity against E. coli and

Folia Microbiol

S. aureus that is in contrast with results of the present study that L. casei strain K1C has the strongest antibacterial effect. In this study, it was shown that the inhibitory activity of the tested isolates supernatants was slightly less active against S. aureus as compared to that obtained against E. coli, indicating that S. aureus might be less sensitive, which is in contrast with the results of Sindhu et al. (2014). In this study, it was also illustrated that different strains of one species might show different resistance against ROS and antioxidative and antibacterial activities. According to the results, L. suebicus strain AG20 in comparison with L. suebicus strain AG2 had higher resistance toward ROS and inhibition of DPPH radical and lower MIC (Figs. 3 and 7; Tables 1, 2, and 4). Resistance to hydrogen peroxide and hydroxyl radicals demonstrated a relation to inhibition of DPPH radicals since L. suebicus strain AG20, the most active strain against hydroxyl radical scavenging activity, has the least IC 50 (881.82) and relatively low MIC (64 mg/mL) that is in contrast with the results of Kim et al. (2005) that stated that resistance to hydrogen peroxide seemed to have no relation to inhibition of lipid peroxidation. Environmental factors are the key parameters which have significant influence on the growth rate and level of production of antioxidant and antibacterial materials. The lactobacilli strains isolated in this study are microaerophiles and grow both in the anaerobic and aerobic conditions. Kullisaar et al. (2002) stated that if there is more oxygen in a medium, there will be more oxygen-free radicals generated and the antioxidative activity of the lactobacilli can be elevated. According to the results of current study, the antioxidant activities increased at aerobic conditions. Therefore, the preferred condition for antioxidant production was at a minimal medium cultured aerobically and incubated at 37 °C, 150 rpm for 48 h. The present results show that the antibacterial production was not affected by agitation that is in agreement with the previous reports by Abbasiliasi et al. (2011). There was no significant difference in the antibacterial production under anaerobic and aerobic conditions. Wang et al. (2013) reported that antioxidant activity began at early exponential growth phases; maximum activity was reached at the stationary phase. Hussain et al. (2013) demonstrated that bacterial cells enter stationary phase within 24 to 48 h of inoculation depending on the broth used. In this study, 48 h-fermented broths were used for further analysis, which were at the mid-stationary and late-stationary growth phases of strains cultured in MRS and PMM5 broths, respectively. Cheigh et al. (2002) stated that the production of bacteriocin is growth-associated because production occurred during mid-exponential phase and increased until it reaches a maximal level at the end of exponential phase or at the beginning of the early-stationary phase where the maximal biomass was

observed. It is supposed that some of the antibacterial properties of the isolates are related to bacteriocin production, because the antibacterial activity was at maximum level in MRS broth after 48 h of fermentation (stationary phase), but the maximum antibacterial production in PMM5 medium was at 24 to 42 h that is during mid-exponential phase and end of the stationary growth phase of bacteria in this medium. Messens et al. (2003) mentioned that the loss of bacteriocin activity may be due to degradation by endogenous protease induced during the growth phase. Therefore, for antibacterial properties, it is recommended that the fermented medium during mid-exponential phase until the beginning of the earlystationary phase was harvested. The current study provided the evidence that most of the Lactobacillus species isolated from yoghurt, cow milk kefir, and camel dough have the potential of antimicrobial and antioxidant activity which indicated the potential health benefits of these dairy products. Therefore, these strains could be useful as starter cultures to equip antioxidants in food or as natural preservatives and may be a promising material for medicinal purposes, applied microbiology, and scientific food industry. Acknowledgements The author would like to thank Department of Biology, Medicinal Plants, and Drug Research Institute, Shahid Beheshti University, for the laboratory facility provided during this study. This research is a Ph.D. dissertation that was funded by Alzahra University. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

References Abbasiliasi S, Ramanan RN, Ibrahim TAT, Mustafa S, Rosfarizan M, Daud HHM, Ariff AB (2011) Effect of medium composition and culture condition on the production of bacteriocin-like inhibitory substances (BLIS) by Lactobacillus paracasei LA07, a strain isolated from Budu. Biotechnol Biotechnol Equip 25:2652–2657 Ajikumar P, Tyo K, Carlsen S, Mucha O, Phon T, Stephanopoulos G (2008) Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. Mol Pharm 5:167–190 Annuk H, Shchepetova J, Kullisaar T, Songisepp E, Zilmer M, Mikelsaar M (2003) Characterization of intestinal lactobacilli as putative probiotic candidates. J Appl Microbiol 94:403–412 Balouiri M, Sadiki M, Ibnsouda SK (2016) Methods for in vitro evaluating antimicrobial activity: a review. J Pharm Anal. doi:10.1016/j. jpha.2015.11.005 Cheigh CI, Choi HJ, Park H, Kim SB, Kook MC, Kim TS, Hwang JK, Pyun YR (2002) Influence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi. J Biotechnol 95:225–235 Dunne C, O’Mahony L, Murphy L, Thornton G, Morrissey D, O’Halloran S, Feeney M, Flynn S, Fitzgerald G, Daly C, Kiely B, O’Sullivan GC, Shanahan F, Collins JK (2001) In vitro selection

Folia Microbiol criteria for probiotic bacteria of human origin: correlation with in vivo findings. Am J Clin Nutr 73:386S–392S Erdogrul Ö, Erbilir F (2006) Isolation and characterization of Lactobacillus bulgaricus and Lactobacillus casei from various foods. Turk J Biol 30:39–44 Federici S, Ciarrocchi F, Campana R, Ciandrini E, Blasi G, Baffone W (2014) Identification and functional traits of lactic acid bacteria isolated from Ciauscolo salami produced in Central Italy. Meat Sci. doi: 10.1016/j.meatsci.2014.05.019 Fitzsimons NA, Cogan TM, Condon S (1999) Phenotypic and genotypic characterization of non-starter lactic acid bacteria in mature cheddar cheese. Appl Environ Microbiol 65(8):3418–3426 Hussain MA, Knight MI, Britz ML (2013) Understanding the starvation adaptation of Lactobacillus casei through proteomics. Asian J Agric Food Sci 1:264–275 Kim HS, Chae HS, Jeong SG, Ham JS, Im SK, Ahn CN, Lee JM (2005) In vitro antioxidative properties of lactobacilli. Asian Australas J Anim Sci. doi:10.5713/ajas.2006.262 Kõll P, Mändar R, Marcotte H, Leibur E, Mikelsaar M, Hammarström L (2008) Characterization of oral lactobacilli as potential probiotics for oral health. Oral Microbiol Immunol. doi:10.1111/j.1399-302X. 2007.00402.x Kullisaar T, Zilmer M, Mikelsaar M, Vihalemm T, Annuk H, Kairane C, Kilk A (2002) Two antioxidative lactobacilli strains as promising probiotics. Int J Food Microbiol. doi:10.1016/S0168-1605(01) 00674-2 Kusuma IW, Murdiyanto Arung ET, Syafrizal KY (2014) Antimicrobial and antioxidant properties of medicinal plants used by the Bentian tribe from Indonesia. Food Sci Human Wellness 3:191–196 Li WJ, Cheng XL, Liu J, Lin RC, Wang GL, Du SS, Liu ZL (2012) Phenolic compounds and antioxidant activities of Liriope muscari. Molecules. doi:10.3390/molecules17021797 Lobo V, Patil A, Phatak A, Chandra N (2010) Free radicals, antioxidants and functional foods: impact on human health. Pharmacogn Rev 4: 118–126 Messens W, Verluyten J, Leroy F, de Vuyst L (2003) Modelling growth and bacteriocin production by Lactobacillus curvatus LTH 1174 in response to temperature and pH values used for European sausage fermentation processes. Int J Food Microbiol 81:41–52 Motamed SM, Motlagh SS, Bagherzadeh H, Forouz SA, Tafazoli H (2014) Evaluation of antioxidant activity of Ruta graveolens L. extract on inhibition of lipid peroxidation and DPPH radicals and the effects of some external factors on plant extract’s potency. Res J Pharm 1:45–50

Nigatu A, Ahrné S, Molin G (2001) Randomly amplified polymorphic DNA (RAPD) profiles for the distinction of Lactobacillus species. Antonie Van Leeuwenhoek. doi:10.1023/A:1010290403124 Nyanzi R, Shuping DSS, Jooste PJ, Eloff JN (2015) Antibacterial and antioxidant activity of extracts from probiotic bacteria. J Food Res 4(5):122–132 Reid G, Jass J, Sebulsky MT, McCormick JK (2003) Potential uses of probiotics in clinical practice. Clin Microbiol Rev 16(4):658–672 Ryan KA, Jayaraman T, Daly P, Canchaya C, Curran S, Fang F, Quigley EM, O’Toole PW (2008) Isolation of lactobacilli with probiotic properties from the human stomach. Lett Appl Microbiol. doi:10. 1111/j.1472-765X.2008.02416.x Saadatzadeh A, Fazeli MR, Jamalifar H, Dinarvand R (2013) Probiotic properties of lyophilized cell free extract of Lactobacillus casei. Jundishapur J Nat Pharm Prod 8(3):131–137 Séré Y, Onasanya A, Afolabi A, Mignouna HD, Akator K (2007) Genetic diversity of the blast fungus, Magnaporthe grisea (Hebert) Barr, in Burkina Faso. Afr J Biotechnol 6(22):2568–2577 Sindhu CS, Sindhu A, Khetarpaul N (2014) Antimicrobial activity of probiotic fermented barley based food products. Int J Microbial Resour Technol 2(2):23–27 Toksoy A, Beyatli Y, Aslim B (1999) Sucuk ve Sosislerden izole Edilen Lactobacillus plantarum Suşlarinin Bazi Metabolik ve Antimikrobiyal Aktivitelerinin İncelenmesi. Turk J Vet Anim Sci 23(6):533–540 Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman W (2009) Bergey’s manual of systematic bacteriology, 2d edn. Springer, New York, pp 465–510 Wang Z-R, Sheng J-P, Tian X-L, Wu TT, Liu W-Z, Shen L (2013) Antioxidant activities of Bacillus simplex XJ-25 isolated from sand biological soil crusts and its properties. Afr J Microbiol Res 7:2198– 2204 Wegkamp A, Teusink B, De Vos WM, Smid EJ (2010) Development of a minimal growth medium for Lactobacillus plantarum. Lett Appl Microbiol 50:57–64 Woo J-Y, Gu W, Kim K-A, Jang S-E, Han MJ, Kim D-H (2014) Lactobacillus pentosus var. plantarum C29 ameliorates memory impairment and inflammaging in a D-galactose-induced accelerated aging mouse model. Anaerobe 27:22–26 Xanthopoulos V, Litopoulou-Tzanetaki E, Tzanetakis N (2000) Characterization of Lactobacillus isolates from infant faeces as dietary adjuncts. Food Microbiol. doi:10.1006/fmic.1999.0300 Zerikly M, Challis GL (2009) Strategies for the discovery of new natural products by genome mining. Chembiochem 10:625–633