alexa Novel Antibacterial Polypeptide Laparaxin Produced by <em>Lactobacillus paracasei</em> Strain NRRL B-50314 Via Fermentation | OMICS International
ISSN:2157-7463
Journal of Petroleum & Environmental Biotechnology

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Novel Antibacterial Polypeptide Laparaxin Produced by Lactobacillus paracasei Strain NRRL B-50314 Via Fermentation

Siqing Liu1*, Brian J Wilkinson2, Kenneth M Bischoff1, Stephen R Hughes1, Joseph O Rich1 and Michael A Cotta3
1RPT Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University St, Peoria, IL 61604, USA
2School of Biological Sciences, Illinois State University, Normal, IL 61791-4120 USA
3BER Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University St, Peoria, IL 61604, USA
Corresponding Author : Dr. Siqing Liu
RPT Research Unit
National Center for Agricultural Utilization Research
Agricultural Research Service
United States Department of Agriculture
1815 N. University St, Peoria, IL 61604, USA
E-mail:[email protected]
Received April 11, 2012; Accepted April 25, 2012; Published April 27, 2012
Citation: Liu S, Wilkinson BJ, Bischoff KM, Hughes SR, Rich JO, et al. (2012) Novel Antibacterial Polypeptide Laparaxin Produced by Lactobacillus Paracasei Strain NRRL B-50314 Via Fermentation. J Pet Environ Biotechnol 3:121. doi:10.4172/2157-7463.1000121
Copyright: © 2012 Liu S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

This study reports the production and characterization of a novel antibacterial polypeptide, designated laparaxin, which is secreted by Lactobacillus paracasei NRRL B-50314. Crude laparaxin has antibacterial activity against a wide variety of Gram-positive bacteria, including: lactic acid bacteria (Lactococcus lactis and Lactobacillus buchneri), food-borne pathogens (Listeria monocytogenes), gastrointestinal pathogens (Enterococcus faecalis), and opportunistic pathogens (Staphylococcus aureus methicillin-sensitive (MSSA) and methicillin resistant (MRSA) strains, a hetero-vancomycin-intermediate methicillin resistant strain (Hetero VISA also MRSA) MM66, and homogeneous vancomycin intermediate (Homo VISA). Using L. lactis as an indicator strain, the inhibitory activity of crude laparaxin was detected originally in early log phase, and the activity maximizes at the early stationary phase and remains stable after prolonged incubation. Laparaxin activity is stable after 30 min of incubation at 94°C. Higher concentrations of inhibitory activity are produced when glucose, fructose and sucrose are used as carbon-sources in growth media. Crude laparaxin has potential applications in food and feed industries, as well as in clinical and veterinary medicine.

Keywords
Anaerobe; Fermentation; Lactobacillus; Antibacterial; Polypeptide
Introduction
Conventional antibiotics are not easily degraded, and residues can accumulate in the environment which promotes the emergence of multi-drug resistant strains [1]. In clinical medicine, the emergence of drug-resistant strains limits the effectiveness of conventional antibiotic therapy, which could be life-threatening to patients if treatment is nonresponsive to available drugs.
In the fuel ethanol fermentation industry, where fermenting microbes are used to convert biomass sugars to fuels and chemicals, bacterial contamination of the fermentors often lead to down time of the production facilities and increased operational cost [2]. To prevent undesired bacteria from growing and competing for nutrients with fermenting microbes, antimicrobial agents have been used in some commercial fermentation tanks. A disadvantage of applying conventional antibiotics is that they do not degrade easily, such that remaining residues may accumulate along the production process and can contribute to the emergence of multi-drug resistant bacterial strains. Recently, bacterial strains with multidrug resistance to virginiamycin and penicillin were reported in dry-grind ethanol plants [3].
New antibacterial agents and control strategies are needed to prevent and control the prevalent occurrence of bacterial infection/ contamination and multi-drug resistance and to reduce or replace conventional antibiotics [1]. This is of particular interest for the ethanol industry because the major fermentation byproduct DDGS has been sold as animal feed. Biodegradable agents with bactericidal activities are promising safe alternatives to synthetic antibiotics and need to be explored in preventing and controlling bacterial infections.
In nature, many microorganisms produce various compounds with anti-bacterial properties. One group of these compounds, bacteriocins, consists of bactericidal proteins with a mechanism of action similar to ionophore antibiotics. Bacteriocins have been described as proteinaceous compounds produced by bacteria that have a biologically active protein moiety and bactericidal action which can inhibit or eliminate the growth of sensitive bacterial species [4]. Bacteriocins are often active against species which are closely related to the producer. Their widespread occurrence from complex microbial communities such as the intestinal tract, oral mucosa, or other epithelial surfaces suggests that bacteriocins may have a regulatory role in terms of population dynamics within bacterial ecosystems.
Most bacteriocins have been identified among lactic acid producing bacteria, which include Lactobacillus species, Bifidobacterium species, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus acidilactici, Sporolactobacillus inulinus, Streptococcus thermophilus, etc [5]. These species are in wide use throughout the fermented dairy, food and meat processing industries. They are Gram-positive, nonsporulating, catalase-negative organisms devoid of cytochromes. They are anaerobic but are aerotolerant, fastidious, acid-tolerant, and strictly fermentative with lactic acid as the major endproduct of sugar fermentation [6]. Their role in the preservation and flavour characteristics of foods has been well documented [7]. Most of the bacteriocins produced by this group are active only against other lactic acid bacteria, but several display antibacterial activity towards more phylogenetically distant Gram-positive bacteria and, under certain conditions, Gram-negative bacteria [8].
The lantibiotic peptide nisin produced by Lactococcus lactis is the best known and well characterized bacteriocin [9-11]. Nisin is desired because it is a biodegradable antibacterial agent and is safe to use as a food preservative in processed dairy products. However, since nisin cannot be synthesized artificially, the only route of production is via fermentation, involving complicated post-translational modifications, thus, it remains an expensive product. To date, pediocin is the only other bacteriocin which has been used as a food preservative in processed dairy products [12].
With zero tolerance for bacterial contamination in food and feed processing, plus generally restricted antibiotic usage, commercial production of new bacteriocins for applications in food, feed and medicine has drawn more attention [13,14]. In reality, there is a need to develop new bacteriocins with a wide range of antibacterial activities, especially against bacteria that are antibiotic resistant.
In this paper, we report the production of a biodegradable polypeptide laparaxin by a novel bacterial isolate Lactobacillus paracasei that shows antibacterial activities against several antibiotic resistant Staphylococcus aureus strains. The biodegradable bactericidal agents have potential health benefits to human beings and the environment. Anticipated applications of laparaxin also exist in the biofuel industry to control bacterial contamination during fermentation. Additional studies in fermentation efficiency, substrate utilization, media formulation and scale-up fermentation are needed to demonstrate potential for commercial production.
Materials and Methods
Bacterial strains and growth conditions
Lactobacillus strains [6,15] were maintained on MRS plates (Becton Dickinson, Sparks, MD) under anaerobic conditions (BBL GasPak anaerobic system, Becton Dickinson, Franklin Lakes, New Jersey) and grown in MRS broth at 30°C without shaking. Staphylococcus aureus strains [16,17] were grown in tryptic soy broth (TSB) (Becton Dickinson, Sparks, MD) or in Mueller-Hinton broth containing CaCl2 (50 mg L-1) (MHBc) (Becton Dickinson, Sparks, MD) at 37°C with shaking at 210 rpm. Listeria monocytogenes strain 10403S [18] and Entercoccus faecalis CK111 [19] were grown in brain heart infusion (BHI) broth (Becton Dickinson, Sparks, MD) at 37°C with shaking at 210 rpm.
For carbon source utilization tests of Lactobacilli, a simplified MRS medium (designated MRSsi) was developed by varying concentrations of several components in MRS [20]. MRSsi contains the following per liter: 5 g of casamino acids, 5 g of peptone, 5 g of yeast extract, 0.5 ml of Tween 80, 0.05 g of MnSO4 • 4H2O, 0.2 g of MgSO4 • 7H2O, 0.1 g of CoCl2 • 6H2O, and 5 g of sodium acetate. MK media contains the following per liter: 10 g beef extract, 5 g tryptone, 5 g yeast extract, 2 g ammonium citrate, 0.05 g of MnSO4 • 4H2O, 0.1 g of MgSO4 • 7H2O, 2 g K2HPO4 , 20 g of K2HPO4, and 5 g sodium acetate. The pH of the media was adjusted to 6.5. Concentrated sugars were autoclaved separately and added prior to use. Growth was monitored by measuring the A600 periodically and fermentation products were analyzed via HPLC as previously described [21].
Production of crude antibacterial polypeptide
The L. paracasei NRRL B-50314 strain was inoculated in 3 ml of MRS broth and incubated overnight. About 2.5 ml of the culture was then transferred to 500 mls MRS in a close capped media bottle and incubated at 30°C for 24 hours. The bacterial cells were removed by centrifugation and the supernatant was filtered using a Nalgene disposable bottle top filter (pore size 0.2 μm) (Nalgene, Rochester, NY). Aliquots of the filtrate were stored at -20°C. This filtered supernatant from culture broth of L. paracasei NRRL B-50314 was used as crude laparaxin for growth inhibition and gel-overlay assays.
Growth inhibition/antimicrobial activity assay
The bacterial strains used as indicator organisms for gel overlay assays are listed in Table 1. A single colony of the desired indicator bacterium was inoculated in 3 ml of broth as specified above and grown overnight at 30°C. Briefly, 50 μl of indicator bacterial cells of late log phase culture were mixed with 8 ml of melted top agar (0.75 % agar in MRS), which had been cooled to 55°C, and poured immediately onto pre-warmed (room temperature) MRS plates.
Meanwhile, individual samples containing 15 μl of crude laparaxin and 15 μl loading dye were denatured at 90°C for 3 min and subjected to SDS PAGE on 8-16% Tris-HCl gels (Ready gels, BioRad Laboratories, Hercules, CA 94547) at 80 volts for 15 min, followed by 130 volts for 60 min. The gels were washed with ddH2O for 15 min. The single lanes were excised with a clean razor blade and placed onto the above mentioned fresh solidified top agar containing indicator cells. The plates were left in the hood for 30 min, and then incubated at 30°C or 37°C overnight. The width and height of the clearing zones of each indicator strains were recorded. The gel overlay images were acquired using a digital camera (BioDoc-It System, UVP, Inc. Upland, CA).
Antibacterial activities were also measured by inhibition of growth of the indicator strain Lactococcus lactis LM0230 in 96 well plates. Each well contained 260 μl of MRS broth, 5 μl of fresh overnight culture of L. lactis, and 15 μl of crude laparaxin. The mixture was incubated for 5 hours at 30°C without shaking, after which, growth inhibition of the indicator strain was measured spectrophotometrically at 600 nm with a microplate reader (Spectra Max M5, Molecular Devices, Sunnyvale, CA). The antibacterial activity was calculated against a positive control where MRS media was used to replace the crude laparaxin, and presented as the percentage of inhibition based on A600 of the indicator strain relative to control samples.
16S rDNA sequencing
Genomic DNA of L. paracasei NRRL B-50314 was isolated using the Gram-positive DNA purification kit (Epicentre, Madison, Wisconsin). Genomic PCRs were performed by using 16s rDNA primers [20], and the PCR fragment was sequenced using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit and the ABI Prism 310 DNA sequencer (Perkin-Elmer, Foster City, California). Sequence analyses were performed with the SDSC biology workbench (http://www. sdsc.edu/Research/biology/) and through the National Center for Biotechnology Information, NCBI (http://www.ncbi.nlm.nih.gov/).
Conversion of various sugars to laparaxin and other fermentation products
Fermentations of L. paracasei NRRL B-50314 were performed in 2 liter fermentors (Biostat B, B. Braun International, Germany), at constant pH (6.0) controlled using 4M NaOH and 4M phosphoric acid. Fermentations were carried out by 2% inoculation at 30°C with 100 rpm stirring. Samples were taken periodically during the course of fermentation. The concentrations of residual sugars and fermentation products, including lactate, acetate, and ethanol, were measured by HPLC using a 300 mm Aminex HPX-87H column (Bio Rad, Richmond, CA) and a refractive index detector (G1362A, Agilent Technologies, Palo Alto, CA). Samples were run at 65°C and eluted at 0.6 ml min-1 with 5 mM sulfuric acid [20].
Results and Discussion
Isolation and identification of bacterial contaminants
The L. paracasei strain was isolated from a laboratory culture tube of Lactobacillus buchneri which showed decreased turbidity after 24 hrs compared to other similar L. Buchneri cultures. This particular culture tube was initially suspected to be infected by phage, but later attempts to isolate phage particles were unsuccessful. The culture was then plated out on MRS to test for contamination. Single colonies from the MRS plate were washed by vortexing with sterile water, plated out and re-streaked on MRS. Six individual colonies were inoculated in MRS broth and grown overnight. These cultures were centrifuged and supernatant filtered and used for antibacterial activity test. Simply, 50% of filtered supernatant of overnight cultures were mixed with 50% of the indicator L. buchneri culture at A600 0.2. Among 6 isolates, one was capable of delaying growth of L. buchneri. This isolate was found closely related to L. Paracasei subsp. tolerans by sequence analysis of 16S rDNA. The new strain was deposited in the ARS Culture Collection as L. paracasei NRRL B-50314.
Bactericidal activities
To further confirm the growth tests, a gel overlay technique was used to test for antibacterial activities in cell-free culture supernatants of NRRL B-50314. A SDS-PAGE gel slice was placed over a freshly seeded lawn of indicator bacterium within a thin layer of top agar across the surface of an agar plate. The growth of bacteria distributed through the top agar produces a homogeneously turbid lawn after overnight incubation except where antibacterial agents are applied. The inhibition of bacterial growth can reduce or eliminate the turbidity of the lawn near the agent, thus, the antibacterial activity is judged by the width of the zone of inhibition around it. Figures 1a and 1b show inhibition of L. lactis and E. faecalis respectively by crude laparaxin. It is interesting to note that in addition to highly active low molecular weight laparaxin, there are other higher molecular weight proteins (Figure 1A) that can prevent L. Lactis growth. The strength of the crude laparaxin over different bacterial species including drugresistant pathogens is presented in Table 1. These results indicated that the crude laparaxin exhibited a broad spectrum of inhibition including pathogens Listeria monocytogenes 10403S, Staphylococcus aureus COL (a MRSA strain), S. aureus SH1000, plus several other antibiotic resistant species such the Gram-positive bacterium E. faecalis (Table 1).
Next, production of crude laparaxin was assessed over a 24 hour period. Laparaxin is produced in early exponential phase and reaches its highest level at the stationary phase (Figure 2). Since this experiment was performed in a culture bottle without pH control, the final pH dropped to around 4.0. It is interesting to note that lower inhibitory activity was detected in the pH controlled bioreactor at pH 6.0 when compared to that produced in the bioreactor at pH 5.0 (data not shown). The production of laparaxin was also examined using a range of carbon/energy sources including fructose, mannose, lactose, sucrose, maltose, and cellobiose using both MK and MRSsi media. Strain B-50314 grows well in most of these carbohydrates except cellobiose and xylose. With a 2% inoculum, the maximum A600 reached up to 7.0 in 24 hours when fructose, glucose and sucrose were used (data not shown). When the two types of media were compared, cells grown in MRSsi produced more laparaxin than in MK. Of the carbohydrates tested, glucose, fructose and sucrose were the best carbon-sources for laparaxin production (Figure 3, Table 2).
The crude laparaxin was subjected to high temperature (94°C) treatments ranging from 30 min to 120 min. When compared with a control sample stored at 4°C for one week, heat treatment for greater than 60 min decreased inhibitory activity significantly, but did not abolish the inhibitory activity. The growth inhibition activity of crude laparaxin remained effective for 30 min of high temperature (94°C) treatment (Figure 4).
Other fermentation products from L. Paracasei NRRL B-50314
In addition to the polypeptide laparaxin which was produced and secreted into culture broth, the other major fermentation product of L. paracasei NRRL B-50314 is lactate. The strain can produce lactate from various substrates including glucose, fructose, sucrose, lactose, mannose and maltose (Table 2). The strain does not degrade cellobiose and is unable to use xylose, arabinose, and ribose.
Summary
In this study, we reported the discovery of antibacterial activity (designated laparaxin) produced and secreted into the culture broth by a novel strain of L. paracasei B-50314. The crude laparaxin inhibits growth of several Gram-positive bacterial pathogens, and the antimicrobial effect is due to a specific polypeptide (laparaxin) as indicated by protein gel analyses. The strength of laparaxin activity varies against different indicator strains, and appeared most potent against S. aureus 209P, DU4916S MRSA strains and L. buchneri B-30929. The laparaxin is temperature stable, and remains active after 30 min of incubation at 94°C. Of the six carbon mono- and disaccharides tested, glucose, fructose and sucrose are the best growth substrates for laparaxin production.
Acknowledgements
We thank Jacqueline Zane and Song Yang for their excellent technical assistance. We would like to express our appreciation to Karen Hughes for editing this manuscript.
References

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