Probiotics & Antimicro. Prot. DOI 10.1007/s12602-015-9186-6

Probiotic Potential of Lactobacillus Strains Isolated from Sorghum-Based Traditional Fermented Food K. Poornachandra Rao • G. Chennappa • U. Suraj • H. Nagaraja • A. P. Charith Raj M. Y. Sreenivasa



Ó Springer Science+Business Media New York 2015

Abstract Sorghum-based traditional fermented food was screened for potential probiotic lactic acid bacteria. The isolates were identified by biochemical, physiological and genetic methods. Species identification was done by 16s rRNA sequence analysis. The functional probiotic potential of the two Lactobacillus species viz., Lactobacillus plantarum (Lact. plantarum) and Lactobacillus pentosus (Lact. pentosus) was assessed by different standard parameters. The strains were tolerant to pH 2 for 1 h and resistant to methicillin, kanamycin, vancomycin and norfloxacin. Two (Lact. plantarum COORG-3 and Lact. pentosus COORG8) out of eight isolates recorded the cell surface hydrophobicity to be 59.12 and 64.06 %, respectively. All the strains showed tolerance to artificial duodenum juice (pH 2) for 3 h, positive for bile salt hydrolase test and negative for haemolytic test. The neutralized cell-free supernatant of the strains Lact. pentosus COORG-4, Lact. plantarum COORG-1, Lact. plantarum COORG-7, Lact. pentosus COORG-8 and Lact. plantarum COORG-3 showed good antibiofilm activity. Lact. pentosus COORG-8 exhibited

74 % activity against Pseudomonas aeruginosa-MTCC 7903 and Lact. plantarum COORG-7 showed 68 % inhibition of biofilm against Klebsiella pneumonia MTCC 7407. Three (Lact. plantarum COORG-7, Lact. pentosus COORG-5 and Lact. pentosus COORG 8) out of eight isolates exhibited a good antimicrobial activity against Listeria monocytogenes and five isolates (Lact. pentosus COORG 2, Lact. plantarum COORG 1, Lact. plantarum COORG 4, Lact. pentosus COORG 3 and Lact. plantarum COORG 6) are active against Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella pneumonia, Enterococcus faecalis. The study also evaluated the cholesterol lowering property of the Lactobacillus strains using hen egg yolk as the cholesterol source. The cholesterol in hen egg yolk was assimilated by 74.12 and 68.26 % by Lact. plantarum COORG 4 and Lact. pentosus COORG 7, respectively. The results of the present study suggest that the Lactobacillus strains isolated and characterized from sorghum-based fermented product may be used as probiotic strains for therapeutic applications.

K. Poornachandra Rao  G. Chennappa  H. Nagaraja  M. Y. Sreenivasa (&) Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India e-mail: [email protected]

Keywords Fermented food  Lactobacillus plantarum  Lactobacillus pentosus  Probiotics  Biofilm

K. Poornachandra Rao e-mail: [email protected]

Introduction

U. Suraj NRC on DNA Finger Printing, NBPGR, PUSA Campus, New Delhi 110012, India A. P. Charith Raj Laboratory of Plant Pathology, Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho 3, Hirosaki, Aomori Ken 036-8561, Japan

Traditionally, fermented foods have been consumed for thousands of years for their beneficial properties such as increased shelf life, enhanced flavour and texture of the final product. There is a rich source of traditional fermented foods, consumed by millions of people. Most of these traditional fermented foods are enriched with beneficial bacteria named Probiotics. The probiotic potential of the lactic

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acid bacteria (LAB) from traditional fermented foods has been well documented [1, 4, 10, 22, 39]. Several different traditional fermented foods like, fermented cereal-based foods [40, 41] dahi, gundruk, sinki, iniziang sang, iromba, fermented rai, kanjika and handua [14], chicken and calves [7] and other traditional fermented food products have been well documented for their probiotic potential [36, 41, 46, 51]. From all these studies, a number of LAB species such as Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus sakei, Lactococcus lactis, Leuconostoc species, Enterococcus faecium, Streptococcus thermophillus, Bifidobacteria, Weissella species have been isolated and identified. Some of the LAB species such as novel Lactobacillus casei, Lactobacillus plantarum with potential properties were also identified from the fermented food products [25, 27, 31]. Recent investigations on the antibiofilm activities of the LAB are of prime importance, because of its antimicrobial properties for the control of pathogen and spoilage microorganisms that produce biofilms. The efficacy of the LAB species to control the biofilm formation by various human pathogens has been well documented. Slama et al. [44] reported that the LAB strains isolated from Tunisian traditional fermented food showed a significant reduction in the biofilm formation by Listeria species. Similarly, Das et al. [13], Lakhtin et al. [26], Maldonado et al. [29], Tahmourespou and Kermanshahi [45] also reported that the efficacy of the different LAB strains isolated from different fermented food products showed for their potential ability to reduce the biofilm formation by human pathogens. Probiotic LAB has the significant role in reducing the blood serum cholesterol. Several reports have highlighted that consumption of probiotic LAB in the form of dairy products results in the decreased serum cholesterol levels [20, 33, 43]. Other studies have shown that some LAB lowered both total cholesterol and low-density lipoprotein (bad cholesterol) in the serum [28, 48]. However, the studies also concluded that the ability of removing cholesterol depends on the source of isolation and species of LAB [3, 28, 51]. Despite of the wide range of LAB isolated from several fermented foods with diverse probiotic aspects, still there are many traditional fermented foods that have not been exploited for potential probiotic applications. Several traditional fermented foods of South India such as Dhokla, Appam, Sauerkraut, Kinema, Kallappam, Koozh and Mor Kuzhambu were screened for Probiotic potential [22]. However, in India, there is a limited knowledge on the probiotic potential of microbial community associated with cereal-based fermented especially traditional sorghum-based fermented foods. Kunene et al. [25] studied on the origin of LAB from a sorghum-based fermented weaning food. Some of the other traditional fermented sorghum foods documented for the exploitation of probiotics include ‘Injera’ an undeniable national food of

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Euthopians and ‘Kisra’ prepared from fermented dough of sorghum [6], sorghum malt-based fermented milk beverage [20], Hussuwa [50] a semisolid sorghum-based fermented food. In the present study, sorghum-based traditional fermented food was obtained from the northern part of Karnataka, as there is a rich treasure of fermented foods and many of them are unexploited. The sorghum-based fermented food is traditionally prepared and consumed by the households in Gulbarga region (Northern part of Karnataka). Jood et al. [21] reported that the LAB in the sorghum-based fermented foods not only promotes the improvement of nutritional quality of the product by improving the vitamin B-complex, essential amino acids but also enhance the potential health benefits of the host by producing antimicrobial compounds, lowering cholesterol and maintaining good microbial balance of the gastrointestinal tract. For the first time in Karnataka, sorghum-based fermented food has been screened for its probiotic potential. The LAB isolated from traditional fermented foods is much accepted, and it has promising probiotic properties, but it has to be well studied by both in vitro and in vivo methods to explore their significant health benefits. With this background, the present study aimed at the isolation, screening, characterization, identification and in vitro evaluation of probiotic potential of lactic acid bacteria from sorghum-based traditional fermented food that has not been exploited in Karnataka.

Materials and Methods Isolation of LAB Sorghum-based traditional fermented food was obtained from the households of Gulbarga region of Karnataka in a sterile container and stored under 4 °C. The sample was collected from 3 day fermented concoction for the screening of the lactic acid bacteria. LAB strains were isolated by serial dilution and spread plate method on MRS agar plates supplemented with 0.25 % (w/v) L-cystine as described by Ozgun and Vural [34]. The plates were incubated at 37 °C for 48 h under anaerobic conditions. The well grown, discrete colonies were enumerated for total colony forming unit, and sub-cultured strains were stored at -20 °C in 40 % glycerol for further studies. Biochemical Characterization of LAB Biochemical and morphological characters were studied as per the standard protocol of Cappuccino and Sherman [8]. The isolates were tested for Gram staining, catalase test, arginine hydrolysis, bile salt hydrolase, carbohydrate fermentation test with different carbohydrates viz, glucose, L-arabinose, D-fructose, maltose, mannitol, raffinose,

Probiotics & Antimicro. Prot. D-xylose and sorbitol (Hi Media). All the isolates were tested for their survival ability at different temperature (15, 37 and 45 °C) and Salinity (4 and 6.5 % NaCl concentrations).

DNA Extraction and PCR Amplification The isolates were grown in 5 mL MRS broth supplemented with L-cystine in a rotary shaker at 37 °C overnight. The DNA was extracted as per the protocol described by Chennappa et al. [11]. The DNA was purified and amplified using primer 27 F (AGAGTTTGATCMTGGCTCAG) and 519 R (GWATTACCGCGGCKGCTG). PCR amplification reactions were carried out in a 25 lL reaction mixture. 1 lL of the DNA was amplified with 2.5 lL of 109 PCR buffer, 2.5 lL of 25 mM MgCl2, 2.0 lL of 2 mM dNTPs, 1.0 lL of 20 pmol primer 27 F, 1.0 lL of 20 pmol primer 519 R, 0.125 lL of LA Taq and water up to 14.875 lL. PCR conditions were as follows: initial denaturation at 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing for 30 s at 53 °C, extension at 74 °C for 2 min, followed by a final extension at 74 °C for 13 min. Amplified product was confirmed by agarose gel (1 %) electrophoresis and documented using Syngene G-box gel documenting system. Identification and Nucleotide Sequence Accession Number The sequences were confirmed with NCBI BLAST database for the identity. The phylogenetic tree was constructed with online software MEGA 5.1 using Maximum Likelihood NJ method. All the sequences of Lactobacillus strains were deposited in NCBI-GenBank along with location of the isolates. Accession numbers are KF247233, KF247234, KF247235, KF247236, KF247237, KF247238, KF247239 and KF247240.

Antibiotic Susceptibility Test Antibiotic susceptibility of the isolated strains was tested against eight selected antibiotics using Himedia antibiotic discs Vancomycin (VA) -30 mcg, Methycillin (MET) -5 mcg, Norfloxacin (NX) -10 mcg, Bacitracin (B) -10 U, Kanamycin (K) -30 mcg, Amoxycllin (AMC) -30 mcg, Penicillin G (P) -10 U and Erythromycin (ERY) -15 mcg by Kirby Bauer Disc Diffusion method. Test LAB of 24-h culture was inoculated onto MRS agar plates by swabbing using sterile cotton swabs. Antibiotic discs were placed on the inoculated plates. After incubation at 37 °C for 24 h, inhibition zones around the discs were measured and susceptibility was compared to the reference chart of zone size interpretative chart for antibiotics as per CLSI (2011). Antimicrobial Activity Antimicrobial activity is one of the important tests for the assessment of probiotics. Neutralized cell-free supernatant (CFS) was used to test the production of antimicrobial compounds by well diffusion method [32]. Eight different pathogenic bacteria were used as the indicator strains including Escherichia coli-MTCC 7416, Staphylococcus aureus-MTCC 3160, Bacillus subtilis-MTCC 121, Pseudomanas aeruginosa-MTCC 7903, Klebsiella pneumoniaeMTCC-7407, Listeria monocytogenes (clinical isolate), Enterococcus faecalis-MTCC 6845 and Enterobacter aerogenes-MTCC 7325. 18-h-old cultures of indicator strains were swabbed on the Muller Hinton agar (Hi Media) plates. Test LAB-CFS of 50 lL was added to the wells and allowed to diffuse and incubated at 37 °C for 24 h. The diameter (mm) of the inhibition zone was measured and expressed as (Mean ± standard deviation) to determine the antimicrobial activity. Cell Surface Hydrophobicity

In Vitro Tests for Probiotic Potential of LAB Strains Acidic pH Tolerance The pH tolerance of the strains was tested according to the procedure described by Sahadeva et al. [39]. Phosphate buffer saline (PBS) solution was prepared for acid tolerance studies by adjusting the pH (2, 3 and 6) using 1 M HCl. 1 mL of test LAB sample of 106 cells mL-1 was inoculated into the three test tubes containing 9 mL of PBS solution with pH 2, 3, 6, respectively, mixed thoroughly and incubated for 0, 90 and 180 min. After incubation, plating was done and plates were incubated anaerobically at 37 °C for 48 h. Acid tolerance was determined by comparing the CFU mL-1 in all the MRS agar plates with control plates.

In vitro cell surface hydrophobicity was determined by microbial adhesion to hydrocarbons (MAHC) assay as described by the Kalyaraung et al. [23]. Briefly, overnight cultures of the isolates were centrifuged at 6,0009g for 10 min using cold centrifuge (Eppendorf) and resuspended in 50 mM PBS buffer. 1.0 mL of n-Hexane was added to 3 mL of bacterial suspension (Ao) and vortex for 2 min. The tubes were kept undisturbed for 30 min at 37 °C to separate the two phases. Aqueous layer (A) was removed, and OD was measured at 600 nm using spectrophotometer (Thermo Scientific). Hydrophobicity was calculated as the percentage decrease in the optical density of the initial aqueous bacterial suspension due to cells partitioning into a hydrocarbon layer. The percentage of cell surface hydrophobicity (% H) of the strain adhering to n-Hexane was

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calculated using the equation % H = [(Ao - A)/ Ao) 9 100]. Same procedure was followed for Xylene. The results were compared with the standard culture of Lactobacillus plantarum MTCC 9483. Resistance to Artificial Duodenum Juice

24 h. Egg yolk cholesterol removal was measured at 4, 8, 16 and 24 h following incubation. After incubation, the cells were centrifuged at 5,400g for 15 min at 4° C. The amount of cholesterol in the supernatant was determined by the modified colorimetric method of Rudel and Morris [37]. The percentage of cholesterol removal was estimated using the formula:

To evaluate the resistance of the isolates to stimulated gastric juice, 1 mL of the overnight culture was suspended in 9 mL of the artificial duodenum juice (NaCl-1.28 g, KCl-0.239 g, NaHCO3-6.4 g, Bile salt-0.3 %, pepsin0.1 g, sterile dis H2O-1,000 mL and pH-2.5) and incubated at 37 °C for the 0, 1, 2 and 3 h. Sodium phosphate buffer was used as control. After incubation, viable cell count was measured as CFU mL-1 formed by serial dilution and plate count method [16].

Results

Haemolytic Activity

Isolation and Biochemical Characterization of LAB

The isolates were tested for the haemolytic activity by using the procedure described by Argyri et al. [3]. All isolates were streaked on blood agar plates containing 5 % (w/v) human blood and incubated for 48 h at 37 °C. The plates were examined for b-haemolysis, a-haemolysis or non-haemolytic (No zones around colonies).

A total of 32 LAB strains were isolated from the sorghumbased fermented food analysed in the present study. The colonies which change the pH of the media to acidic (yellow) were preliminary screened. Eight isolates which showed antimicrobial activity were selected for further study. The isolates of gram positive and catalase negative were considered as presumptive LAB. Biochemical characterization reveals that the isolates were homo-fermentative producing only acid, but no gas from glucose and negative for arginine hydrolysis. The isolates were able to ferment L-arabinose, D-fructrose, maltose, mannitol, raffinose, D-xylose and sorbitol. The optimum growth was seen at 37 °C and 3 % NaCl (Table 1).

Antibiofilm Activity of LAB Inhibition of Biofilm formation assay was carried out in a sterile 96-well flat-bottomed microtitre plate. Two bacterial pathogens i.e. Pseudomonas aeruginosa (MTCC 7903) and Klebsiella pneumoniae (MTCC 7407) were grown in Tryptic Soy Broth media (Hi Media) and allowed for the biofilm formation. After incubation, the media were poured out from the wells and washed with sterile PBS buffer. Then, 200 lL of CFS was added to each well following the incubation for 24 h. Crystal violet staining was carried out [30] for assessing the biofilm formation. The culture without CFS was served as control. The results were expressed as percentage of biofilm inhibition [18]. % I ¼ OD Control  OD Sample= OD Control  100 where I (%) is the percentage of biofilm inhibition, OD control is the optical density in control and OD sample is the optical density in sample with LAB-CFS.

Percentage of cholesterol removed ¼ ½ðamount of cholesterol of the control amount of cholesterol of the inoculated groupÞ = amount of cholesterol of the controlÞ  100:

Identification by 16S rRNA Sequencing and Phylogenetic Analysis PCR amplification was carried out using 27 F and 519 R primers, and the amplicon size was approximately 550 bp. Sequence analysis of the amplicons confirmed that the four strains (COORG 1, COORG 4, COORG 5 and COORG 6) showed highest similarity (100 %) to Lact. plantarum and remaining four isolates (COORG 2, COORG 3, COORG 7 and COORG 8) were similar to Lact. pentosus. The sequences were deposited in NCBI-GenBank and obtained Accession Numbers (Fig. 1). In Vitro Tests for Probiotic Potential of LAB Strains: S

In Vitro Cholesterol Removal Using Egg Yolk Acidic pH Tolerance MRS broth was prepared by supplementing 4 % commercial fresh hen egg yolk as the cholesterol source, as described by Hua et al. [19]. 100 lL of 18-h culture broth of LAB strains was inoculated and then incubated at 37° C for

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Tolerance to acidic pH is one of the pre-requisite for the validation of probiotics. The strains COORG 3 and COORG 7 showed tolerance to pH 2 for 3 h of incubation.

Probiotics & Antimicro. Prot. Table 1 Morphological, physiological and biochemical characterization of the isolated LAB strains Tests

Isolates COORG-1

COORG-2

COORG-3

COORG-4

COORG-5

COORG-6

COORG-7

COORG-8

Morphology

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Gram staining

?

?

?

?

?

?

?

?

Catalase test

-

-

-

-

-

-

-

-

Arginine hydrolysis

-

-

-

-

-

-

-

-

10

?

?

?

-

-

?

?

-

37 45

? ?

? -

? -

? -

? ?

? ?

? ?

? -

Growth at different temperature

Growth at different NaCl concentration 3%

?

?

?

?

?

?

?

?

5%

?

?

?

-

-

-

?

-

7%

-

-

-

-

-

?

-

-

Bile salt (0.3 % oxgal) hydrolysis

?

?

?

?

?

?

?

?

Gas from Glucose

-

-

-

-

-

-

-

-

Glucose

?

?

?

?

?

?

?

?

Maltose

?

?

?

?

?

?

?

?

Mannitol

?

?

?

?

?

?

?

?

Arabinose

?

?

?

?

?

?

?

?

Lactose

?

?

?

?

?

?

?

?

Xylose

-

-

?

-

?

-

-

-

Sorbitol Raffinose

-

-

-

-

? -

-

-

-

Carbohydrate fermentation

100 Lactobacillus plantarum strain COORG - 4

Lactobacillus plantarum strain COORG - 6

49

Lactobacillus pentosus strain COORG - 7 94

Lactobacillus pentosus strain COORG - 2 Lactobacillus pentosus strain COORG - 8 Lactobacillus plantarum strain COORG - 1

91 50

Lactobacillus pentosus strain COORG - 3 Lactobacillus plantarum starin COORG - 5

0.05

Fig. 1 Lactobacillus isolated from sorghum-based fermented food and identified as species viz., Lactobacillus plantarum and Lactobacillus pentosus by Dendrogram based on BLAST algorithm and neighbouring tree method

However, the bacterial count of the strains COORG 1 and COORG 2 has significantly decreased when compared to control (pH 6) for 3 h incubation. The viability of the strains decreased as the incubation time increases with decrease in pH of the media. The Lactobacillus strain COORG 2 showed the significantly decreased viable cell

number from 8.6 9 106 to 1.98 9 106 CFU mL-1 at 0 to 3 h of incubation at pH 2, however, COORG 3, COORG 4 and COORG 7 showed slight reduction in the viable cell numbers. At pH 3 for 1–3 h of incubation, all the strains showed minimal reduction did not find any significant differences in viable cell count (Table 2).

123

123

10.94 ± 2.82 10.12 ± 1.41 9.68 ± 1.41 8.84 ± 2.82 4.66 ± 1.24 6.18 ± 1.14 7.62 ± 4.24 8.22 ± 1.21 a

n=3

Mean ± SD of log value of viable cell count

2.22 ± 1.41 5.44 ± 2.12 8.62 ± 1.21 COORG-8

7.28 ± 1.41

10.64 ± 1.24

10.78 ± 2.82 9.86 ± 1.41

9.98 ± 1.21 9.06 ± 2.82

8.78 ± 1.21 7.98 ± 1.41

8.74 ± 2.12 4.24 ± 1.21

4.18 ± 2.82 5.52 ± 2.12

5.86 ± 2.82 7.14 ± 4.24

6.96 ± 4.24 7.94 ± 2.82

8.24 ± 2.82 2.28 ± 1.41

3.96 ± 1.41 5.22 ± 2.12

4.98 ± 2.82 6.82 ± 2.12

6.88 ± 2.82

8.64 ± 1.41

7.96 ± 1.21

COORG-6

COORG-7

10.44 ± 2.12 10.48 ± 2.12 9.50 ± 1.41 9.66 ± 1.41 8.98 ± 2.14 8.96 ± 2.14 8.68 ± 1.14 8.46 ± 2.12 3.98 ± 2.82 4.16 ± 4.24 6.26 ± 2.41 5.98 ± 1.41 7.74 ± 1.41 7.68 ± 2.82 8.62 ± 2.82 8.46 ± 1.41 2.36 ± 1.41 2.44 ± 2.41 5.72 ± 2.82 5.24 ± 2.82 8.42 ± 2.82 8.76 ± 1.41 COORG-4 COORG-5

7.46 ± 1.21 6.34 ± 2.12

9.98 ± 1.14

10.26 ± 1.41 9.46 ± 1.41 9.12 ± 2.82 8.92 ± 1.41 4.22 ± 2.14 5.24 ± 1.41 6.82 ± 2.82 7.96 ± 1.41 4.08 ± 4.24 5.44 ± 2.12 7.92 ± 2.12 COORG-3

6.86 ± 1.41

9.14 ± 1.41

9.24 ± 1.41 8.86 ± 2.82

8.98 ± 1.41 8.84 ± 2.82

8.46 ± 4.24 4.86 ± 1.41

4.68 ± 1.41 6.12 ± 1.21

6.28 ± 2.82 7.42 ± 4.24

7.28 ± 2.82 8.62 ± 2.21

8.46 ± 1.14 2.02 ± 2.82

1.98 ± 4.24 5.42 ± 2.21

5.38 ± 1.41 6.84 ± 2.12

6.88 ± 1.41

8.66 ± 1.41

2h 1h

Log CFU mL-1 a

Isolates

Hexane and Xylene when compared to the standard culture (Table 4). Among the isolates, strain COORG 6 showed optimum cell surface hydrophobicity (62.6 %) with n-Hexane and strain COORG 5 showed maximum hydrophobicity (63.6 %) with Xylene. However, least hydrophobicity was observed with the strain COORG 1 (30.4 %) to n-Hexane and strain COORG 4 showed 34.6 % with Xylene as least hydrophobicity. In total, strain COORG 6 showed 59 % of adhesion ability followed by COORG 5 and COORG 8 with 58.5 and 56.4 % adhesion, respectively, and strains COORG 1 and COORG 4 exhibited least adhesion property (36 %) when compared to standard Lact. plantarum strain (56.2 %).

Table 2 Tolerance of LAB isolates to acidic pH

Cell Surface Hydrophobicity

8.64 ± 2.12

1h 0h 0h

3h

pH 3 pH 2

1h

2h

3h

The antimicrobial activity of all the isolates was tested against entero-pathogenic bacteria. The isolates have the significant antimicrobial activity against all the indicator strains. The zone of inhibition was measured in mm and it ranged from 4 to 17. The strains COORG 5, COORG 7 and COORG 8 showed good antimicrobial activity (15, 17 and 16 mm, respectively) against L. monocytogenes, which is an important food pathogen and exhibited activity against Enterococcus faecalis (15, 14 and 17 mm, respectively) which is an enteric pathogen. The strains COORG 1(against B. subtilis), COORG 2 (against E. coli), COORG 3 and COORG 4 (S. aureus) displayed inhibition to only one each of the eight pathogens studied. The strain COORG 6 displayed moderate activity against all the pathogens. However, among all the strains, COORG 5, COORG 7 and COORG 8 showed significant antimicrobial activity against almost all the indicator strains (Table 3). The activity may be due to the bacteriocin-like inhibitory substances (BLIS) produced by the potential Lactobacillus strains.

COORG-1

Antimicrobial Activity

0h

pH 6 (Control)

2h

3h

The antibiotic resistance profile was carried out with eight important antibiotics. The results were obtained by comparing the reference chart of performance standards for Antimicrobial Disc Susceptibility Tests. All the isolates were sensitive to penicillin, amoxycillin and erythromycin. The strains COORG 4 and COORG 6 showed resistance to bacitracin. Furthermore, all the isolates were resistant to methicillin, kanamycin, vancomycin and norfloxacin. However, the strains COORG 4 and COORG 6 were intermediate to amoxicillin, and strains COORG 5 and COORG 7 were intermediate to bacitracin. Sensitivity to antibiotics is one of the criteria for the evaluation of probiotics.

COORG-2

Antibiotic Susceptibility Test

10.02 ± 4.24

Probiotics & Antimicro. Prot.

Probiotics & Antimicro. Prot. Table 3 Antimicrobial activity of the isolated LAB strains Sl. No.

Antimicrobial activity of the isolates (CFS at pH 7)a

Indicator strain

COORG-1

COORG-2

COORG-3

COORG-4

COORG-5

COORG-6

COORG-7

COORG-8

1

E. coli

13 ± 1.41

14 ± 2.82

12 ± 2.12

11 ± 1.41

16 ± 2.82

10 ± 8.48

17 ± 1.41

12 ± 2.82

2

S. aureus

10 ± 2.82

13 ± 1.41

15 ± 1.41

14 ± 141

13 ± 1.41

11 ± 2.82

14 ± 2.82

14 ± 2.12

3

B. subtilis

15 ± 1.41

10 ± 2.82

08 ± 2.82

10 ± 2.82

15 ± 1.41

10 ± 4.24

14 ± 2.82

13 ± 2.82

4

Pseudomanas aeruginosa

09 ± 1.41

06 ± 1.41

07 ± 2.21

09 ± 1.41

13 ± 1.41

12 ± 1.41

16 ± 2.82

15 ± 1.41

5

K. pneumonia

08 ± 1.44

08 ± 2.21

10 ± 2.21

09 ± 1.41

14 ± 2.82

11 ± 1.41

12 ± 1.41

12 ± 2.82

6

L. monocytogenes

09 ± 1.41

13 ± 1.41

11 ± 1.41

07 ± 2.12

15 ± 1.41

09 ± 2.82

17 ± 1.41

16 ± 1.41

7 8

Enterobacter aerogenes Enterococcus fecalis

10 ± 2.82 04 ± 1.41

04 ± 2.82 12 ± 2.82

06 ± 2.82 11 ± 1.41

10 ± 2.82 11 ± 4.24

10 ± 2.12 15 ± 1.41

11 ± 3.56 08 ± 2.82

13 ± 1.41 14 ± 2.82

15 ± 1.41 17 ± 4.24

Mean ± SD of diameter of inhibition zones in mm

Cell surface hydrophobicity n-Hexanea

Xyleneb

% Adhesionc

1

L. plantarum COORG-1

30.4 ± 1.2

42.2 ± 1.4

36.3

2

L. plantarum COORG-4

38.6 ± 1.4

34.6 ± 1.2

36.6

3

L. plantarum COORG-5

52.7 ± 2.2

63.6 ± 2.4

58.5

4

L. plantarum COORG-6

62.6 ± 1.6

55.4 ± 1.6

59.0

5

L. pentosus COORG-2

46.8 ± 1.8

58.4 ± 1.2

52.6

6

L. pentosus COORG-7

52.4 ± 1.6

43.6 ± 2.2

48.0

7

L. pentosus COORG-8

48.6 ± 2.2

64.2 ± 1.2

56.4

8

L. pentosus COORG-3

36.4 ± 1.4

48.2 ± 1.2

42.3

9

Lactobacillus plantarum MTCC 9483

56.8 ± 2.2

55.6 ± 1.6

56.2

Tolerance to Artificial Duodenum Juice

0h 1h

CFU ml-1

6

2h 3h

4 2

8 G

7

6

O

O R

G

O

O R

C

C

C

O

O R

G

G

5

4

O R O

C

C

O

O R

G

G

3

2

O R O

O

O R

G

G

1

0

C

None of the Lact. plantarum and Lact. pentosus strains was significantly affected by the artificial duodenum juice for 1 and 2 h incubation. However, there was a decrease in the viability of the LAB strains after 3 h incubation. The strains COORG 3 and COORG 7 showed reduction in their viable count of 1.5 9 106 and 2.2 9 106 CFU mL-1, respectively after 3 h incubation. Among all the strains, COORG 1, COORG 2 and COORG 8 exhibited tolerance to artificial duodenum juice with more viable number than the minimal viable number 3.5 9 106 CFU mL-1. The least tolerance to artificial duodenum juice was observed with the strains COORG 3 and COORG 7 (Fig. 2).

8

C

Average values of a and b

Isolate

O R

c

Sl. No.

O

Table 4 Cell surface hydrophobicity of the LAB strains

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a

Fig. 2 Tolerance of Lactobacillus strains to artificial duodenum juice at 0, 1, 2 and 3 h of incubation

Haemolytic Activity In Vitro Antibiofilm Activity Haemolytic activity is assessed based on the presence or absence of clearing zone around the growth of the Lactobacillus strains on the blood agar plates. The results of the study revealed that none of the isolates induced haemolysis. Hence, the strains are non-haemolytic.

Based on the OD values, the biofilm eradication percentage was calculated. The strains COORG 7 and COORG 8 showed 74 and 58 % inhibition, respectively, of Pseudomonas biofilm, and the strains COORG 8, COORG 4,

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%Inhibition of Klebsiella Biofilm

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Egg Yolk Cholesterol Removal from the Media

80 60 40 20 0

Fig. 4 Inhibition of Klebsiella biofilm by neutralized LAB-CFS

Egg yolk cholesterol removal (%)

The cholesterol levels of 24-h LAB strains were varied and the cholesterol removal ratio (p \ 0.05) ranged from 24 to 68 %. Lact. pentosus strain COORG-7 reduced more cholesterol when compared to the other strains from the media supplemented with egg yolk as cholesterol source. Two LAB strains (COORG 7 and COORG 4) showed sharp growth patterns in the initial duration of the incubation. Cholesterol removal percentage was utmost with 74.12 % by Lact. plantarum strain COORG 4 followed by Lact. pentosus strain COORG 7 with 68.26 % assimilation (Fig. 5). The other strains showed negligible amount of cholesterol removal. Minimum percentage of the cholesterol removal was showed by Lact. plantarum strain COORG 5.

100

C on tr C o O O l R G C 1 O O R G C 2 O O R G C 3 O O R G C 4 O O R G C 5 O O R G C 6 O O R G C 7 O O R G 8

COORG 7 and COORG 1 showed 78, 59, 58, and 58 % inhibition, respectively, of Klebsiella biofilm, and the strains COORG 5 showed 24 % inhibition, respectively, for Pseudomonas biofilm and Klebsiella biofilm (Figs. 3, 4). The neutralized LAB-CFS was effective in eradicating the biofilm formation of two strong biofilm formers. Four of the LAB strains COORG 1, COORG 4, COORG 7 and COORG 8 showed good antibiofilm activity against Pseudomonas aeruginosa (MTCC 7903) and Klebsiella pneumoniae (MTCC 7407).

90 80 70 60

0

50

4

40

8

30

16

20

24

10 0 10-

COORG COORG COORG COORG COORG COORG COORG COORG 1 2 3 4 5 6 7 8

Fig. 5 Egg yolk cholesterol removal (%) by LAB strains at 0, 4, 8, 16 and 24 h of incubation

Discussion

100 80 60 40 20

C

O

on tr o O l R G C 1 O O R G C 2 O O R G C 3 O O R G C 4 O O R G C 5 O O R G C 6 O O R G C 7 O O R G 8

0

C

%Inhibition of Pseudomonas Biofilm

The present study was conducted to know the potential health benefits of the Lactobacillus strains isolated from sorghum-based traditional fermented food. All the isolates were gram positive, catalase negative and arginine hydrolysis negative. The isolates have the ability to ferment

Fig. 3 Inhibition of Pseudomonas biofilm by neutralized LAB-CFS

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glucose, maltose, mannitol, arabinose and lactose. Lactobacillus strains isolated from other fermented foods, such as fermented broccoli stems ‘Yan-tsai-shin’ [10], sorghumbased infant weaning cereal [25] showed similar kind of reports. The phylogenetic studies were carried out using BLAST algorithm revealed Dominant LAB species identified from the study are Lact. plantarum (COORG 4, COORG 1, COORG 6, COORG 5) and Lact. pentosus (COORG 2, COORG 7, COORG 8, COORG 3). The 16S rRNA sequence profiling is one of the cost-effective tools and enables the researchers to identify the complex microbial communities up to species level at exceptional depth and resolution. Phylogenetic analysis was studied using the neighbour joining tree method to analyse the homology of the isolates Lact. plantarum and Lact. pentosus. The probiotic potential of the LAB strains has been carried out. In the present study, the resistance of the Lactobacillus strains to acidic pH environment represent the selection of acid tolerant strains. Similar results were reported by Turchi et al. [47], where the Lactobacillus strains showed tolerance to acidic pH (pH 2). The evaluation of antibiotic resistance profile was to promote the safety evaluation and development of potential probiotic

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LAB. The significance of the isolate Lact. pentosus (COORG 4 and COORG 6) was the resistance to bacitracin at a concentration of 10 U, when compared to the previous report of Charteris et al. [9] which showed resistance of LAB to bacitracin at 10 mcg. All the isolates are susceptible to penicillin, amoxycillin and erythromycin, a macrolide antibiotic, but resistant to commonly used antibiotics supporting the previous reports [5, 15]. The resistance profile was calculated based on zone size interpretative chart for antibiotics as per CLSI (2011). The natural resistance of the isolates to clinically important antibiotics may provide a way for the development of antibiotic/probiotic combination therapies for conditions like diarrhoea, female urogenital tract infection and infective endocarditis [9]. The isolates showed a good antimicrobial activity against E. coli, S. aureus, P. aeruginosa, K pneumoniae, B. subtilis and L. monocytogenes which are major food borne pathogens and of special concern with regard to food safety due to its psychrotrophic and ubiquitous characteristics [24]. The isolates showed antimicrobial activity against K. pneumonia, which is reported as the causal agent of several food borne diseases [35]. The Lactobacillus strains showed antimicrobial activity against Enterococcus faecalis, Enterobacter aerogenes. The antimicrobial activity differed among all the LAB strains. Certain LAB showed activity against certain indicator strains. The cellfree supernatant (CFS) was neutralized to deactivate the acids and to exclude the activity due to organic acids. Hence, the activity may be due to bioactive substances such as bacteriocin-like inhibitory substances (BLIS), biosurfactants and other relavant molecules. Similar results were also reported with Lact. plantarum that showed antimicrobial activity against E. coli and Salmonella [42] and Francois et al. [17] also observed the antimicrobial activity of Lact. plantarum against K pneumoniae, Pseudomonas sp and Enterococcus faecalis. Belicova et al. [5] reported the inhibitory activity of Lactobacillus plantarum against L. monocytogenes. Yang et al. [49] observed strong antifungal activity of Lactobacillus species against Penicillium and Aspergillus sps., which was on contrary to the isolates of the present study. Cell surface hydrophobicity has been conducted to know the ability of LAB strains to adhere the gut region, as it is one of the criteria to be a potential probiotic strain. Microbial adhesion to hydrocarbon assay (MAHC) was conducted to know the in vitro cell surface hydrophobicity of the Lactobacillus strains isolated in the present study along with the Lactobacillus strain (MTCC 9483) as control. The result of MAHC assay was that the strain COORG 6 recorded maximum (59 %) of adhesion and the least (36.3 %) adhesion in COORG 1 strain. Our results are in correlation with the results obtained by Collado et al. [12]

and Duary et al. [14], where 44.2 % of maximum adhesion and 25.06 % of minimum adhesion was reported by Lact. plantarum species. Tolerance to artificial duodenum juice is one of the criteria for the evaluation of survival potential of probiotic Lactobacillus strains under stimulated gastric conditions. The Lactobacillus strains in the present study showed the tolerance to artificial duodenum juice. It is observed that the tolerance of the LAB strains correlates/inversely proportional with the incubation time. When the incubation time increased from 0 to 3 h, the CFU mL-1 of the LAB strains has been decreased. Three strains (COORG 1, COORG 2 and COORG 8) among the eight were exhibited maximum tolerance to stimulated gastric juice. Several reports on the survival ability of Lactobacillus under stimulated gastric juice have been documented supporting the present study [16, 38, 47]. The Lactobacillus strains of the present study could resist the stimulated gastric conditions. Testing the haemolytic activity is utmost important to determine the virulence factors of the Lactobacillus strains. The results of the present study revealed that all the isolates were negative for haemolytic activity supporting the previous studies of Anas et al. [2]. On contrary, Argyri et al. [3] reported four strains of Lact. pentosus to exhibit a-haemolysis. Our study clarifies that the isolates were non-haemolytic and non-virulent ultimately non-pathogenic and can be suggested to use as probiotics. Formation of pathogenic biofilms is a critical issue in the field of food processing and in the clinical fields like nosocomial infections. The Lactobacillus has been proved to be one of the effective tools in controlling the pathogenic biofilms. The results of the present study support the use of LAB-CFS to treat the pathogenic biofilms with a maximum eradication of 74 %. Similar results of antibiofilm activities were reported by Mathkhury et al. [30] using supernatant of Lact. acidophillus. Slama et al. [44] reported the antibiofilm activities of Lact. plantarum against Listeria monocytogenes. The neutralized LAB-CFS was very effective in eradicating the biofilm-forming pathogenic bacteria. The possible mechanism behind the antibiofilm effects of neutralized LAB-CFS is that the production of bacteriocin or other related compounds. To be a significant probiotic strain, it should possess some of the important health benefits other than survival under gastric conditions. The results of the present study clarifies that the Lactobacillus strains has the ability to lower the cholesterol from the medium, which depicts the cholesterol lowering ability of the host. One strain COORG 4 has displayed a maximum cholesterol lowering ability of 74 %. However, the cholesterol removal was growth associated and strain dependent. The results from the graph

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indicated that the cholesterol assimilation values increased with the bacterial growth. As the incubation time increased, the cholesterol removal has decreased. The maximum cholesterol removal was seen during the lag phase of the incubation. The results of the present study were in accordance with study of Liu et al. [28], and cholesterol assimilation was strain dependent. The present work aims to increase the knowledge on exploration of potential probiotic strains from traditional fermented foods which are unique to Karnataka state. The results of the present study concludes that the isolated LAB strains represented promising probiotic properties like cell surface hydrophobicity, acid tolerance, antimicrobial activity, resistance to stimulated gastric conditions. In view of this, Lact. plantarum and Lact. pentosus could be considered for the preliminary selection of potential probiotics strains. The antibiofilm activity of the isolates was also taken into account as one of the probiotic property. Cholesterol lowering ability of the LAB strains in the study concluded that the activity is strain dependent and growth assisted. The isolated LAB strains showed promising probiotic properties under invitro conditions; however, further studies are necessary to confirm the beneficial role of these strains for human health. Acknowledgments We are thankful to the University Grants Commission, New Delhi for providing financial support in the form of Major Research Project (No. F. 40-134/2011 (SR) dated 4th July 2011). We thank Prof. S M Gadad, Gulbarga University for providing the sorghum-based fermented product. We also thank Rakshith D, Research Scholar, Department of Microbiology, University of Mysore for his assistance in the construction of Phylogenetic tree. Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this study.

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Probiotic potential of lactobacillus strains isolated from sorghum-based traditional fermented food.

Sorghum-based traditional fermented food was screened for potential probiotic lactic acid bacteria. The isolates were identified by biochemical, physi...
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