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Microbial Ecology in Health and Disease. 2009; 21: 211–220

ORIGINAL ARTICLE

Isolation and characterization of antimicrobial proteins produced by a potential probiotic strain of human Lactobacillus rhamnosus 231 and its effect on selected human pathogens and food spoilage organisms

P. S. AMBALAM1,5, J. B. PRAJAPATI2, J. M. DAVE3, BABOO M. NAIR4, ÅSA LJUNGH5 & B. R. M. VYAS1 1Department

of Biosciences, Saurashtra University, Rajkot, 2SMC College of Dairy Science, Anand Agricultural University, Society, Rajkot, India, 4Department of Applied Nutrition and 5Department of Medical Microbiology and Infectious Diseases, Lund University, Lund, Sweden Anand, 3Vrindavan

Abstract Objective: To study in vitro properties of potential probiotics and the antimicrobial activity of Lactobacillus rhamnosus 231 isolated from human faeces. Methods and Results: Lact. rhamnosus 231 isolated from human faeces tolerated bile salt (4%), phenol (0.5%), and NaCl (4%) and retained viability at low pH (2.5). The cell-free culture (CFC) filtrate and extracellular protein concentrate (EPC) of Lact. rhamnosus 231 contained antimicrobial substances active against Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes, Staphylococcus aureus, Salmonella spp., Helicobacter pylori, Campylobacter jejuni, Bacillus cereus, Bacillus megaterium, and Listeria monocytogenes. EPC contained a mixture of low molecular weight antimicrobial proteins, produced during log and stationary phases of growth against the test organisms. Thermostability of the antimicrobial proteins and their sensitivity to proteinase K was observed to be test organism specific. The antimicrobial activity was observed in the pH range 4.5–9 except against Ps. aeruginosa and Ent. aerogenes. These antimicrobial proteins are low molecular weight (4 kDa) anionic peptides as determined by tricine-SDS-PAGE and 2D gel. Periodic acid-Schiff’s (PAS) staining of gel confirmed the presence of carbohydrate moiety with low molecular weight peptides. The antimicrobial activity of the partially purified protein was determined against Staph. aureus 74B, H. pylori 33, H. pylori 17874, and C. jejuni CJE 33566. Conclusion: Human Lact. rhamnosus 231 exhibits in vitro properties of potential probiotic. CFC filtrate and EPC of Lact. rhamnosus 231 exhibit antimicrobial activity against potential human pathogens and food spoilage organisms. Antimicrobial proteins in EPC were partially purified and characterized. In vitro properties of potential probiotic and antimicrobial properties of Lact. rhamnosus 231 could be useful as food additive against human pathogens and removal of food contaminants in the target environment. Key words: Antimicrobial proteins, anti-Helicobacter activity, cell-free culture (CFC) filtrate, Lactobacillus rhamnosus 231, potential probiotic

Introduction Food-borne diseases are a major cause of morbidity and mortality, especially among children in low income communities where sanitation and hygiene are of very low standard. Poverty, lack of knowledge, poor methods of food preparation and storage, scarcity of potable water, and absence of health facilities are also contributing factors. Hence, the control of food spoilage organisms and food-borne pathogens is not easily attainable in such communities. Most common food-borne pathogens and food spoilage

organisms belong to the genera Salmonella, Campylobacter, Shigella, Escherichia, Pseudomonas, Enterobacter, Klebsiella, and Listeria (1). Helicobacter pylori is associated with gastritis, gastric and duodenal ulcers, and gastric malignancies. Recently, attention has been paid to study the interactions between H. pylori and probiotic lactobacilli (2). In such cases lactic acid fermentation could play an important role in decreasing the rate of morbidity and mortality caused by human pathogens and food spoilage organisms. Traditional lactic

Correspondence: B. R. M. Vyas, Department of Biosciences, Saurashtra University, Rajkot 360 005, India. Tel: +91 281 2586419. E-mail: brmvyas@hotmail. com (Received 20 November 2008; accepted 15 October 2009) ISSN 0891-060X print/ISSN 1651-2235 online © 2009 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS) DOI: 10.3109/08910600903429052

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fermentation is a spontaneous process initiated by the microorganisms normally present in the milk. It is efficient, less expensive, easily acceptable and adaptable, and applicable to low income households. Several nutritional and therapeutic properties of lactic acid bacteria (LAB) have been demonstrated. Probiotics are live microorganisms that actively enhance the health of consumers by improving the balance of microflora in the gut when ingested in sufficient numbers (3). Lactobacilli and bifidobacteria are most commonly used as LAB in probiotics. Functional properties of probiotic bacteria include antimicrobial activity against human pathogens and food spoilage organisms, hypocholesterolemic effect, antimutagenic and anticarcinogenic properties, improvement in lactose metabolism, and immunomodulation (1,4–8). Before probiotic bacteria can benefit human health, they must fulfill several criteria such as the ability to tolerate acid, bile salt, sodium chloride, and phenol so that they arrive alive in the intestine and compete with pathogenic microorganisms in the gastrointestinal tract (9). The mechanism(s) of the antibacterial activity of probiotic Lactobacillus strains appear to be

multifactorial (1). LAB produce a wide range of antimicrobial metabolites such as lactic acid, acetic acid, hydrogen peroxide, bacteriocin, other low molecular mass peptides, and fatty acids. In this paper, we report (i) the isolation, characterization, and identification of a human strain of Lactobacillus; (ii) the in vitro antimicrobial activity of cell-free culture filtrate and extracellular proteins against human pathogens and food spoilage organisms; and (iii) the production and partial characterization of these extracellular antimicrobial protein(s).

Material and methods Isolation of the Lactobacillus strain Lactobacillus strain of human origin was isolated from feces of a healthy human using de Mann-RogosaSharpe (MRS) agar (10), and preserved in 10% skimmed milk at 4°C. Culture was analyzed for Gram reaction, catalase production, and motility. Other strains and dehydrated media (Himedia, India and Oxoid, Basingstoke, Hampshire, UK) used in the present study are listed in Table I.

Table I. Antimicrobial spectrum of CFC filtrate and EPC of Lact. rhamnosus 231. Zone of inhibition (mm)∗ (n = 3) Strain 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

coli1

E. Ent. aerogenes1 K. pneumoniae1 Sh. dysenteriae1 Salm. paratyphi B (STB)1 Salm. typhimurium2 98 Salm. enteritidis3 13076 Ps. aeruginosa1105 Ps. aeruginosa4 705 Ps. aeruginosa4 618 Ps. aeruginosa3 H. pylori5 17874 H. pylori6 33 H. pylori4915 Camp. jejuni CJE 33566 B. megaterium7 B. subtilis7 B. cereus7 Micrococcus luteus7 Staph. aureus7 Staph. aureus8 Staph. aureus 4 74 B L. monocytogenes3 L. monocytogenes5

Media used

CFC filtrate

EPC

MacConkey agar EMB agar MacConkey agar SS agar SS agar SS agar Blood agar GPY agar GPY agar GPY agar GPY agar GAB agar GAB agar GAB agar TPY agar N agar N agar N agar N agar N agar Blood agar Blood agar Blood agar Blood agar

15  1.5 16  0.5 15  1 14  2 14  0.5 18  0.5 ND 18  1 ND ND ND ND ND ND ND 14  0.5 – 14  0.5 26  1 31  1.5 9.5  0.5 11  0.5 10  0.76 11  0.8

16  1 12  0.5 21  0.76 – 15  0.5 15  0.5 12  0.5 21  1 15  1 12  1 10  0.5 12  0.5 20  1 10  1 10  0.5 15  0.5 – 15  0.28 31  1 20  1 7.5  0.5 9.5  0.5 8.5  0.25 7.5  0.5

CFC filtrate, cell-free culture filtrate; EPC, extracellular protein concentrate; ND, not determined; –, no inhibition. 1Clinical strains were obtained from Government Hospital, Rajkot and Jamnagar, India. 2MTCC, (M Type Culture Center, India). 3ATCC (American Type Culture Center). 4University Hospital, Lund, Sweden. 5CCUG (Culture Collection of University of Gothenburg, Sweden). 6N. Figura, Pediatric Hospital, Siena, Italy. 7Christ College, Rajkot, India. 8NCTC (National Collection of Type Cultures, London, UK). ∗Including well  7 mm.

Antimicrobial proteins of Lactobacillus rhamnosus 231 Culture identification by API 50 CL medium The API 50 CH test kit and API CHL medium (BioMérieux, La Balme les Grottes, France) were used according to the manufacturer’s instructions for the identification of the isolates. Culture identification by 16S rDNA technique DNA extraction. DNA from Lact. rhamnosus 231 was extracted with QIAamp DNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored at –20°C for further analysis. PCR and DNA sequencing. The reaction mixture (25 μl) contained 13 reaction buffer II, 2.5 mmol.l–1 MgCl2, 0.8 mmol.l–1 dNTP, 0.5 μmol.l–1 forward primer Lac-F (agc agt agg gaa tct tcc aca a) and 0.5 μmol.l–1 reverse primer Lac-R (CAC CGC TAC ACA TGG AGT TCC ACT) (W.A. Al-Soud, personal communication), 0.05% casein and 0.05% formamide, 1 U GOLD DNA polymerase, and 5 μl extracted DNA. Sterile Millipore-filtered (Millipore, Bedford, MA, USA) deionized water (5 μl) was used as negative control. Amplification conditions were 94°C for 2 min/94°C for 30 s, 55°C for 30 s, and 72°C for 30s  30 cycles/72°C for 5 min. The 341 bp PCR products were electophoresed in 1% agarose gel that contained ethidium bromide (11) and were visualized by the use of a GelFotoSystem (Techtum Lab, Umeå, Sweden). PCR products were purified from agarose gels by Ultrafree-DNA centrifuge tubes (Millipore). Reaction mixture in a total volume of 10 μl contained 2 μl sequencing buffer, 1 μl Big Dye, 0.5 μl forward primer (1 μmol.l–1) and 5 μl purified PCR product. It was placed in a thin wall 0.2 ml PCR tube and subjected to thermal cycling in a PCR system using a hot lid to prevent evaporation. After thermal cycling, the content of each tube of the sequencing reactions was subjected to DNA precipitation by the ethanol/sodium acetate method (Applied Biosystems, Foster City, CA, USA). DNA sequencing was performed with an ABI Prism 310 DNA sequencer (Applied Biosystems) using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit version 3.1 (Applied Biosystems). Sequences were analyzed by using BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).The closest known relatives of the partial 16S rDNA sequences were determined using BLASTN 2.2.1 (http://www.ncbi.nlm.nih.gov.ludwig.lub.lu.se/blast/). Tolerance to pH, bile salt, NaCl, and phenol pH and bile tolerance (sodium taurocholate, Himedia, India) for Lact. rhamnosus 231 was evaluated as described by Jacobsen et al. (12). Briefly,

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MRS (pH 2.5 and 3.5), MRS (2 and 4% bile salt), and MRS (4 and 8% NaCl) were inoculated with test bacteria (108 cells.ml–1). After 4 h of incubation at 37°C, survival was checked by plating 100 μl onto MRS agar. After 24 h, tolerance to low pH, bile salt, and NaCl was checked by inoculating a loopful of culture from respective tubes to normal (original) MRS medium. Phenol tolerance was tested by cultivating the organisms in skimmed milk containing 0.4 and 0.5% phenol (9). All the tubes including control were incubated (37°C, 24 h) and observed for growth as development of turbidity. In the case of skimmed milk-phenol medium, clotting of milk was considered as positive growth. Preparation of cell-free culture filtrate and protein concentrate Cell-free culture (CFC) filtrate was obtained by harvesting 18 h old culture of Lact. rhamnosus 231 grown on MRS medium. The entire contents of the flask were centrifuged (10 000 g, 20 min, 4°C) and the supernatant was passed through a 0.45 μ Millipore filter. Proteins in the CFC filtrate were precipitated by ammonium sulfate (80% saturation) and held overnight at 4°C. The precipitates were separated by centrifugation (10 000 g, 20 min, 4°C) and dissolved in a minimum amount of acetate buffer (pH 4.5, 10 mmol.l–1). Clear supernatant obtained upon centrifugation (10 000 g, 10 min, 4°C) was used as extracellular protein concentrate (EPC). Antimicrobial activity of CFC filtrates and protein concentrate Antimicrobial activity of CFC filtrate and EPC was determined by well diffusion agar assay (13) against the organisms listed in Table I. Briefly, 100 μl of the 17 h culture of the test organism was spread on an agar plate (Table I). Wells measuring 7 mm were made; 100 μl of CFC filtrate or EPC was added to the wells and preincubated at 4°C for 2 h to allow diffusion of the sample before incubating the plates at 37°C for 24 h. MRS medium and buffers were used as controls. Growth curve and production profile of antimicrobial proteins (activity) produced by Lact. rhamnosus 231 Erlenmeyer flasks (250 ml) containing 100 ml MRS medium were sterilized, inoculated with 2% (v/v) of 24 h culture of Lact. rhamnosus 231 and incubated at 37°C. Samples removed at an interval of 4/6 h were analyzed for biomass (A600) and pH of the filtrate. Part of the supernatant was used to determine protein concentration according to Bradford (14) and the antimicrobial activity. EPC was prepared as described

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elsewhere from the other part of the supernatant and evaluated for protein concentration according to Bradford (14) and antimicrobial activity. Characterization of the antimicrobial activity of the protein concentrate EPC (1 ml) was treated with proteinase K (1 mg. ml–1 Sigma-Aldrich, St Louis, MO, USA) at 37°C for 6 h, followed by boiling the mixture for 5 min to inactivate enzyme (15). Inactivated buffered enzyme solution was used as control. The antimicrobial activity of the proteinase K-treated EPC, native EPC, and the controls were determined by well diffusion assay. Thermostability of the antimicrobial proteins present in EPC was evaluated by using heat-treated EPC (an aliquot of the protein concentrate in an Eppendorf tube held in boiling water bath for 30 min (15)) EPC in the antimicrobial activity assays. Residual antimicrobial activity of the cooled heat-treated samples and native EPC was tested by the agar diffusion assay. Antimicrobial activity of the EPC at various pH values was evaluated by using EPC prepared in the buffers as describe in the footnotes to Table III. Tricine-SDS-PAGE EPC was electrophoresed on 16.5/18% tricine-SDSpolyacrylamide gel (16). The electrophoresed gel was divided into two parts. One part was stained with Coomassie brilliant blue G-250 and silver staining technique (17), and the other part of the gel was assayed for antimicrobial activity against Ps. aeruginosa and B. cereus by gel overlayer method (18). Following fixation of the gel for 1 h in a solution of 20% isopropanol–10% acetic acid, the gel was washed for 2 h with deionized water, aseptically placed into a petri dish, and overlaid with N-agar inoculated with the described test organism for the agar diffusion assay. PAS staining of gel for glycoprotein Gels were stained by periodic acid-Schiff’s (PAS) base according to Doerner and White (19).

exclusion column (SEC), Superose 12 HR 10/30 previously equilibrated with acetate buffer (pH 4.5, 10 mmol.l–1) using FPLC (Amersham Pharmacia Biotech, Uppsala, Sweden). Isocratic elution was performed using the same buffer at 0.5 ml.min–1 flow rate. Five fractions were collected and concentrated by lyophilization. The antimicrobial activity of each fraction was determined against H. pylori 33 and 17874, C. jejuni 33566 and Staph. aureus 74B using agar diffusion assay. The protein profile of each fraction was determined by tricine-SDS-PAGE as described elsewhere. The antimicrobial activity of partially purified protein was quantified by taking the reciprocal of the highest dilution that exhibited a clear zone of inhibition and was expressed as activity units per ml (AU.ml–1) of culture media. The titer of the EPC, in AU.ml–1, was calculated as (1000/d)*D, where D is the highest dilution showing activity and d is the aliquot of EPC added to the well (20). 2D gel electrophoresis The first dimension isoelectric focusing was performed using IPGphore horizontal electrophoresis (Amersham Pharmacia Biotech). Immobiline dry strips (11 cm) with a pH range of 3–11 were rehydrated in 170 μl sample buffer containing 8 mol.l–1 urea, 2% (v/v) Nonidet P-40, 10 mmol.l–1 dithiothreitol, and 2% IPG buffer and 30 μl EPC sample. The proteins were focused for 12 h at 20°C, 500 V 60 min (~Vh 500), 1000 V 60 min (~Vh 1000), and 8000 V 120 min (~ Vh 16000). The electrofocused IPG strips were equilibrated with 6 ml of equilibration buffer (containing Tris-HCl (pH 6.8, 50 mmol.l–1), SDS (2% w/v), glycerol (26% v/v), bromophenol blue) for 2 15 min. Dithiothreitol (100 mg) was added to the first equilibration solution and 250 mg iodoacetamide was added to second equilibration solution. SDS-PAGE was then performed using 10–20% gradient crition gel (Biorad, Hercules, CA). Separation was performed at 25°C at 50 V until the bromophenol blue dye front reached the bottom of the gels. Gels were stained using Imperial protein stain kit (Pierce, Rockford, USA) according to the manufacturer’s instructions.

Protein purification Four hundred ml of an 18 h culture of Lact. rhamnosus 231 was centrifuged (10 000 g, 20 min, 4°C) and passed through a 0.45 μ Millipore filter. Solid ammonium sulfate was added to CFC filtrate to yield 80% saturation. The mixture was held overnight at 4°C and centrifuged (12 000 g, 4ºC, 30 min) to collect the precipitates. The precipitates were resuspended in acetate buffer (pH 4.5, 10 mmol.l–1) and loaded onto size

Nucleotide sequence accession no The 16S rRNA gene sequences of Lact. rhamnosus 231 has been deposited in GenBank under accession no. EF661653. Results Lact. rhamnosus 231 (LR 231), isolated from healthy human feces, is a gram-positive, catalase-negative,

Antimicrobial proteins of Lactobacillus rhamnosus 231 non-motile, and non-spore forming rod-shaped organism. Its ability to ferment ribose and growth at 15°C and 45°C indicate that it belongs to the group Streptobacterium. The identity of the LR 231 was established by API 50 CHL and 16S rDNA sequences analysis. Partial sequences of 16S rDNA exhibited 100% similarity with 16S rDNA sequences of Lact. rhamnosus and 99% similarity to that of Lact. paracasei spp. Viability of LR 231 decreased by one order of magnitude in the presence of 4% bile salt but it did not change at 2% bile salt concentrations. Viability was 96% after incubation for 4 h at pH 2.5 and 3.5 (Figure 1). Prolonged incubation (24 h) at low pH/2–4% bile salt concentration resulted in lysis of some of the cells as observed from the loss of the turbidity of the culture, while the other cells remained viable. LR 231 was able to grow in the presence of 4 and 8% NaCl. Viability of the cells growing in MRS medium containing 4 and 8% NaCl decreased to approximately 92% after 4 h. LR 231 grew in the presence of 0.4% phenol and remained viable in 0.5% phenol. Antimicrobial spectrum of CFC filtrate and EPC CFC filtrate and extracellular protein concentrate (EPC) of 18 h culture of LR 231 showed a wide spectrum of antimicrobial activity against grampositive organisms tested except Bacillus subtilis, as well as gram-negative organisms (Table I). The total protein in CFC filtrate and EPC was 154 and 1242 μg.ml–1, respectively. The antimicrobial activity of CFC filtrate was higher against Staphylococcus

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spp., Listeria monocytogenes, and Salmonella typhimurium in comparison with EPC, whereas the activity of the latter was higher or equivalent against B. cereus, B. megaterium, Micrococcus luteus, Escherichia coli, Klebsiella pneumoniae, and Ps. aeruginosa. EPC exhibited antimicrobial activity against Campylobacter jejuni 33566 and three strains of H. pylori CCUG 17874, G33 (phenotype cag+) and HP 915 (phenotype cag–) (Table I). All the three strains of H. pylori were inhibited, indicating that the cag phenotype is not associated with the sensitivity of H. pylori. EPC also exhibited antimicrobial activity against Salm. enteritidis and three strains of Ps. aeruginosa. MRS medium and acetate buffer did not show inhibition against the test organisms. Production of antimicrobial components Antimicrobial activity of CFC filtrate against the test organism increased with the growth of Lact. rhamnosus 231 and became stable when culture reached the stationary phase. A similar pattern of antimicrobial activity of CFC filtrate was observed against all the test organisms (Figure 2a, b). The antimicrobial spectrum of EPC against the test organisms changed with culture age. The concentration of protein in the CFC filtrate and EPC at 6 h interval was 37, 70, 34, 49, 38, 38, 27 and 373, 213, 279, 356, 297, 182, 303 μg.ml–1, respectively. The antimicrobial activity of EPC against L. monocytogenes (both strains), Camp. jejuni CJE 33566, and Staph. aureus 74B was higher during the early log phase but not in the case of CFC filtrate. This implies that the antimicrobial activity is due to extracellular proteins present in

Figure 1. Survival of Lact. rhamnosus 231 cells exposed in the presence or absence of bile salt (2 and 4%), NaCl (4 and 8%) and at pH (2.5 and 3.5) for 4 h at 37°C.

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P. S. Ambalam et al. After that the activity either varied marginally (against Camp. jejuni, both strains of L. monocytogenes), or decreased rapidly (against E. coli) or decreased slowly (against STB), whereas antimicrobial proteins against Staph. aureus and Ent. aerogenes are produced only during late exponential phase, and the activity does not change thereafter during the stationary phase. Antimicrobial proteins active against Ps. aeruginosa 105 were produced only in the stationary phase. Similar results were also observed in liquid assay (data not shown). Characteristics of antimicrobial protein(s)

Figure 2(a). Growth profile of Lactobacillus rhamnosus 231 in MRS medium at 37ºC.

CFC filtrate, which did not exhibit antimicrobial activity perhaps due to low protein concentration. The antimicrobial activity of EPC against E. coli, Camp. jejuni CJE 33566, Staph. aureus 74B, and L. monocytogenes CCUG 15528 was 10-fold higher than that of CFC filtrate. We also observed that the antimicrobial activity against Staph. aureus 74B was 100 AU.ml–1 throughout, L. monocytogenes was 100 AU.ml–1 up to 18 h, and the activity against Camp. jejuni CJE 33566 was 1000 AU.ml–1 up to 12 h and 100 AU.ml–1 up to 24 h. The antimicrobial activity against Staph. aureus, Ent. aerogenes, and Salm. paratyphi B (STB) appeared after 12 h of growth. On further growth, the activity against STB and E. coli decreased whereas activity remained stable against other test organisms. EPC showed antimicrobial activity against Ps. aeruginosa 105 after 20 h. This indicates that antimicrobial proteins active against Camp. jejuni CJE 33566, Listeria spp., E. coli, and STB are produced during early exponential phase.

The antimicrobial activity of heat-treated EPC varied between the test organisms (Table II). The antimicrobial activity of heat-treated EPC remained unchanged against Staph. aureus 74B, L. monocytogenes ATCC 35152, and Ps. aeruginosa ATTC 2785, indicating that the proteins produced by Lact. rhamnosus 231 that are active against these test organisms are thermostable. The heat treatment of EPC caused slight reduction in the antimicrobial activity against L. monocytogenes CCUG 15528 (5%) and Camp. jejuni CJE 33566 (15%). Antimicrobial activity of heat-treated EPC (in liquid assay) against E. coli, Shigella dysenteriae, Salm. paratyphi B, and Staph. aureus remained unchanged. Heat treatment caused complete loss of antimicrobial activity of EPC against Ps. aeruginosa 105. After treatment with proteinase K, the activity was lost completely against Ps. aeruginosa 605 and lost partially against H. pylori 33 (40%), Salm. enteritidis (25%), Camp. jejuni CJE 33566 (15%), and L. monocytogenes (5%), whereas the activity remained unchanged against Staphylococcus spp., Ps. aeruginosa 705, and Ps. aeruginosa ATCC 2785. The antimicrobial activity of EPC against Ps. aeruginosa, STB, and Ent. aerogenes was

Table II. Effects of proteinase K and temperature on the activity of EPC of Lact. rhamnosus 231. Zone of inhibition (mm)∗ n  3 Test organism Staph. aureus 74 B Ps. aeruginosa 705 Ps. aeruginosa ATCC 2785 Ps. aeruginosa 618 H. pylori 33 Staph. aureus NCTC 7428 Salm. enteritidis ATCC 13076 L. monocytogenes ATCC 35152 L. monocytogenes CCUG 15528 Camp. jejuni 33566

EPC 13 16 11 13 18 12 13 11 11 10.5

1  0.7  0.7 1 1 0 1 0 0 0

Proteinase K-treated EPC 13  1 16  0.7 11  0.7 – 11  01 12  0 10  2 10.5  0 10.5  0 90

EPC, extracellular protein concentrate; ND, not determined; –, no inhibition observed. well  7 mm.

∗Including

Heat-treated EPC 13  1 11  0.7 11  0.7 – ND ND ND 11  0 10.5  0 90

Proteinase K control – – – – – – – – – –

Antimicrobial proteins of Lactobacillus rhamnosus 231

217

Figure 2(b). Antimicrobial activity of CFC filtrate (A& C) and EPC (B&D) against different test organisms indicated as a legend of each graph. Antimicrobial activity was measured as zone of inhibition (mm) around punched hole (7 mm).

higher at pH 4.5 than at pH 7 and 9 (Table III), while for other test organisms the activity did not vary much at the pH values tested, indicating that the proteins exert antimicrobial activity over a broad pH range. Also, antimicrobial proteins at pH 4.5 showed activity at higher dilution (up to 1:100 dilution), which was not observed with antimicrobial proteins at pH 7 and 9. Buffer controls did not show any antimicrobial activity. Proteins present in EPC using 16.5 and 18% tricine-SDS-PAGE resolved into three bands, one diffuse band representing low molecular weight peptides and the other marking the presence of higher molecular weight proteins (Figure 3(1)). Glycoprotein staining with PAS reagent gave a diffuse reddish-pink band of low molecular weight peptides (Figure 3(1)). Gel overlayer confirmed that inhibition of the test organisms was due to the diffuse band of low molecular weight protein(s) (Figure 3(2)).

Native and denaturing gel showed similar profile of EPC, indicating that the low molecular weight diffuse band is a mixture of proteins and not a complex of proteins. Protein purification The purification table for EPC present in CFC filtrate is shown in Table IV. The first step was concentration of protein by ammonium sulfate precipitation (80%). After several runs of SEC (Figure 4), five fractions showing antimicrobial activity were collected. All the fractions showed activity (100 AU.ml–1), whereas the second and third fraction showed higher activity (1000 AU.ml–1) against Staph. aureus 74B. Only the second fraction of purified protein showed antimicrobial activity (100 AU.ml–1) against Camp. jejuni CJE 33566, H. pylori 33, and H. pylori 17874. The molecular weight of this peptide present in the

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Table III. Effect of pH on the activity of EPC of Lact. rhamnosus 231 against gram-positive and gram-negative bacteria. Zone of inhibition (mm)∗ n  2 Test organisms

CFC filtrate

E. coli

15 19 17 18 19.5 19 21

Salm. typhimurium 98 Salm. paratyphi A Salm. paratyphi B (STB) Ent. aerogenes Ps. aeruginosa105 Staph. aureus

1 2 0 2 0  0.7 1

pH 4.5#

pH 7

pH 9

19.5  0.7 16  1.4 18  1.4 18  1.4 18  1 20  0.7 15  1

 0.5  1.4  1.4  1.4  1.4 – 14  1

 0.5  1.4  0.7  0.7 – – 15  1.4

20 17 15 17 15

20 16 15 18

pH 4.5, 10 mmol.l–1 acetate buffer; pH 7, 10 mmol.l–1 phosphate buffer; pH 9, 10 mmol.l–1 Tris buffer. CFC filtrate, cell-free culture filtrate; –, no inhibition. ∗Including well = 7 mm.

second fraction as determined by tricine-SDS-PAGE (Figure 4) was 4 kDa. 2D gel electrophoresis The pI value of EPC before and after SEC was between 3 and 3.5 according to 2D gel analysis, and the molecular weight was approximately 4 kDa. The band stained yellow after silver staining of tricineSDS-PAGE and 2D gels. Discussion The present study reports the isolation of a human Lactobacillus strain with potential probiotic properties. The primary requirement for potential probiotic organisms is to survive during the passage through the acidic (pH 1.5–2.5) environment of the stomach. Lact. rhamnosus 231 (LR 231) survives at pH 2.5 for 4 h, a sufficiently long time for the cells to pass

Figure 3. Tricine-SDS-PAGE gels of partially purified extracellular protein(s) from Lactobacillus rhamnosus 231 cultures growing on MRS medium. (1) Lane (A) Low molecular weight standards (Bio-Rad, Hercules, CA), EPC stained with (lane B) “silver” and (lane C) “PAS” stain. (2) Inhibition of growth of Bacillus cereus (lane B) and Ps. aeruginosa 105 (lane C) by low molecular weight glycoprotein, which corresponds to the band observed in lane A.

through the stomach and reach their site of action in the intestine. In other studies (12,21), long-term exposure (24 h) to an acidic environment (>4 h) causes strong stress with significant loss of survival. However, LR 231 cells possess the ability to tolerate low pH upon prolonged incubation at pH 2.5. In another report, exposure of different LAB to pH 3.5 for 1 h induced de novo protein synthesis, of which some cross-reacted with heat-shock proteins. These heat-shock proteins can increase the stability of organisms and protect surface structures, like adhesins, from denaturation during passage in the GI tract (22). Lact. rhamnosus 231 possesses the ability to tolerate 4% bile, which is important for survival of probiotics in the GI tract. It is difficult to suggest a precise concentration to which a selected strain should be bile tolerant (12). Different concentrations have been used depending on the type of bile salt used in the studies (23,24). LR 231 possesses the ability to grow in the presence of 0.4% phenol and remained viable in 0.5% phenol, a toxic metabolite produced by intestinal bacteria during putrefaction in the GI tract (9). LR 231 also possesses the ability to grow in the presence of high NaCl concentration. The ability of LR 231 cells to survive in the presence of bile, NaCl, and phenol can help them to colonize, grow, and elicit the beneficial effect to the host. Lactobacilli exert antimicrobial activity against gram-negative and gram-positive organisms by producing lactic acid, acetic acid, and other organic acids, and hydrogen peroxide, strain-specific metabolites or non-lactic acid molecules (1,25–28). Lact. rhamnosus 231 shows a broad inhibitory spectrum against gram-negative and gram-positive bacteria, including human pathogens and food spoilage organisms. This antimicrobial activity is associated with CFC filtrate and extracellular antimicrobial protein(s) present in CFC filtrate. Lact. rhamnosus 231 produces strain- and/or genusspecific metabolites as evidenced by the variation in antimicrobial activity of EPC against different strains

Antimicrobial proteins of Lactobacillus rhamnosus 231

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Table IV. Purification table of extracellular antimicrobial proteins produced by Lact. rhamnosus 231.

Purification stage

Volume (ml)

Activity (AU.ml–1)

Total activity (AU)

Total Protein (mg)

Specific activity (AU.mg–1)

CFC filtrate Ammonium sulfate precipitate

400 15

10 100

4000 1500

61.6 18.6

64 80

Size exclusion chromatography (SEC) 1st fraction 2nd fraction 3rd fraction 4th fraction 5th fraction

5 25 25 20 5

100 1000 1000 100 10

500 25000 25000 2000 50

2.7 9.5 5.5 2.6 0.1

183 2631 4310 769 476

of Ps. aeruginosa, Salmonella spp., Staphylococcus spp., and L. monocytogenes. The antimicrobial extracellular proteins are produced during exponential and stationary phases. Changes in the antimicrobial activity spectrum of the EPC during different phases of growth provide evidence that the EPC is a mixture of antimicrobial proteins and its composition changes with the age of the culture. To our knowledge this is the first report of changing antimicrobial spectra of extracellular proteins produced by Lact. rhamnosus. The antimicrobial extracellular proteins identified are anionic having pI 3.0–3.5, low molecular weight (4 kDa) proteins. EPC shows antimicrobial activity over a broad pH range except against a few test organisms. Strain-specific thermostability and sensitivity to proteinase K digestion of antimicrobial proteins provide further evidence that the antimicro-

Figure 4. Superose 12HR 10/30 chromatogram of the EPC from 18 h old cultures of L. rhamnosus 231 growing on MRS medium. Inset shows Tricine-SDS-PAGE of partially purified glycopeptides eluted during SEC chromatography. Lane 1. Low molecular weight standards (Bio-Rad, Hercules, CA), lane 2. EPC after ammonium sulfate precipitation, lane 3 to 7, five pooled fractions 4-6, 7-8, 9-10, 11-13 and 14-16, respectively of EPC eluted during size exclusion chromatography.

bial activity of EPC is represented by a mixture of proteins that are heat/proteinase K sensitive and/or resistant. These antimicrobial proteins contain carbohydrate and lipid moieties evidenced in the PAS base staining for glycoproteins and the yellow band observed in the silver staining of PAGE, which is the characteristic of proteins containing lipid moieties and sialoglycoproteins (29,30). Polar lipid moieties were also detected by GC-MS in the extracellular protein concentrate (data not shown). The antimicrobial activity of EPC and partially purified extracellular low molecular weight protein(s) of Lact. rhamnosus 231 against different strains of H. pylori and Camp. jejuni CJE 33566 could be useful, especially in the prevention of infections caused by antibiotic-resistant strains of H. pylori and Campylobacter. There is only one report describing a role of proteins in anti-H. pylori activity (31). Servin (1) pointed to the existence of the least characterized fourth class of complex bacteriocin. Bacteriocins are proteinaceous antimicrobial compounds, also produced by LAB, that exhibit a bactericidal effect against taxonomically closely related organisms. However, the antimicrobial proteins of Lact. rhamnosus 231 reported here are distinct from bacteriocins produced by other Lactobacillus spp., as they exhibit a broad spectrum of activity against gram-positive and gram-negative organisms. These broad antimicrobial spectra produced by LR 231 are potentially valuable in topical treatment, bio-control, feed additives, and other applications that aim at eradicating gram-positive and gram-negative pathogens or nonpathogenic contaminants in the targeted environment. Activity of EPC at neutral and alkaline pH suggests that the antimicrobial activity of inhibitory protein is independent of acid. Lact. rhamnosus produces other antimicrobial metabolites, as evidenced from the antimicrobial activity of CFC filtrate even when EPC (independently) was less active. This evidence suggests the multifactorial nature of the antimicrobial activity and possibly a synergistic effect. A role of other metabolites remains to be identified.

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We conclude that Lact. rhamnosus 231 is a potential candidate for probiotic product preparation as evidenced by its ability to (i) resist low pH; (ii) grow in the presence of bile salt, NaCl, and phenol; and (iii) produce extracellular low molecular weight antimicrobial proteins active against human pathogens and food spoilage organisms, although in vivo trials are required to assess benefits provided to the host. Further work on the purification of these antimicrobial proteins, their mode of action, and antigenic properties is in progress. Acknowledgments This study was supported by SASNet-Lund University, Sweden, Science Research Council of Sweden, and an ALF-grant from Lund University Hospital. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Servin AL. Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMS Microbiol Rev 2004;28:405–40. 2. Felley C, Michetti P. Probiotics and Helicobacter pylori. Best Pract Res Clin Gastroenterol 2003;17:785–91. 3. Fuller R. A review: probiotics in man and animals. J Appl Bacteriol 1989;66:365–78. 4. Commane D, Hughes R, Shortt C, Rowland I. The potential mechanisms involved in the anticarcinogenic action of probiotics. Mutat Res 2005;591:276–89. º´ , Wadstrom T. Lactic acid bacteria as probiotics. 5. Ljungh A Curr Issues Intest Microbiol 2006;7:73–89. º´ . Lactobacillus 6. Lorca GL, Wadstrom T, Valdez GF, Ljungh A acidophilus autolysins inhibit Helicobacter pylori in vitro. Curr Microbiol 2001;42:39–44. 7. Ashar MN, Prajapati JB. Verification of hypocholesterolemic effect of fermented milk on human subjects with different cholesterol level. Folia Microbiol 2000;45:263–8. 8. Lebeer S, Vanderleyden J, De Keersmaecker SCJ. Genes and molecules of lactobacilli supporting probiotic action. Microbiol Mol Biol Rev 2008;72:728–64. 9. Khedekar CD. Characterization of human strains of Lactobacillus acidophilus isolates for their suitability in preparation of milk beverage and their antibacterial cum therapeutic ability. MSc (Dairy Microbiology) thesis, Gujarat Agriculture University, Anand, 1988. 10. De Man JC, Rogosa M, Sharpe ME. A medium for the cultivation of Lactobacilli. J Appl Bacteriol 1960;23:130–5. 11. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 2001. p. 5.4–5.13. 12. Jacobsen CN, Rosenfeldt Nielsen V, Hayford AE, Moller PL, Michaelsen KF, Paerrgaard A, et al. Screening of probiotic activities of 47 strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of 5 selected strains in humans. Appl Environ Microbiol 1999;65:4949–56.

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