Structure, Biosynthesis, and Antibacterial Mechanism of Lantibiotics Produced by Lactic Acid Bacteria

Korean kimchi is known for its myriad of lactic acid bacteria (LAB) with diverse bioactive compounds. This study was undertaken to isolate an efficient antifungal LAB strain among the isolated kimchi LABs. One thousand and four hundred LABs isolated from different kimchi samples were initially screened against Aspergillus niger. The strain exhibiting the highest antifungal activity was identified as Lactobacillus plantarum YML007 by 16S rRNA sequencing and biochemical assays using API 50 CHL kit. Lact.plantarum YML007 was further screened against Aspergillus oryzae, Aspergillus flavus, Fusarium oxysporum and other pathogenic bacteria. The morphological changes during the inhibition were assessed by scanning electron microscopy. Preliminary studies on the antifungal compound demonstrated its proteinaceous nature with a molecular weight of 1256·617Da, analysed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF). The biopreservative activity of Lact.plantarum YML007 was evaluated using dried soybeans. Spores of A.niger were observed in the negative control after 15days of incubation. However, fungal growth was not observed in the soybeans treated with fivefold concentrated cell-free supernatant of Lact.plantarum YML007. The broad activity of Lact.plantarum YML007 against various food spoilage moulds and bacteria suggests its scope as a food preservative. After screening 1400 kimchi bacterial isolates, strain Lactobacillus plantarum YML007 was selected with strong antifungal activity against various foodborne pathogens. From the preliminary studies, it was found that the bioactive compound is a low molecular weight novel protein of 1256·617Da. Biopreservative potential of Lact.plantarum YML007 was demonstrated on soybean grains, and the results point out YML007 as a potent biopreservative having broad antimicrobial activity against various foodborne pathogens.

The antagonistic effect of mixed lactic acid bacterial cultures against foodborne pathogens (Staphylocuccus aureus ATCC 25923, Shigella boydii clinical isolate, Pseudomonas aeruginosa ATCC 25853) was evaluated in pasteurized ayib stored at room temperature. The lactic acid bacteria (LAB) were tested for acid tolerance at pH 2.0, 2.5, 3.0 and 3.5 for three and six h and for bile tolerance for 24 and 48 h at 0.3% (w/v) bile salt concentration. Their antimicrobial effect on selected foodborne pathogens was assessed by co-culturing assays in laboratory medium. The effect of mixed LAB cultures against the foodborne pathogens tested was followed in ayib stored at ambient conditions for 9 days. Only 11 LAB isolates were isolated upon their survival at a pH value of 3.5. Out of the 11 LAB isolates selected from a total of 60 based on their survival at pH 3.5, 6 isolates showed survival at pH 2.5 and pH 3.0 for 3 h with survival rates of 1.03-22% and 5-100%, respectively. The same 6 LAB isolates displayed tolerance to 0.3% bile salt concentration for up to 48 h. In the presence of acid-bile tolerant LAB isolates as compared to the control (without any LAB bacteria), Ps. aeruginosa was inhibited by all six to varying degree while, Sh. boyidii and S. aureus were inhibited by five of the LAB in laboratory medium. The mixed LAB culture was inoculated in to pasteurized ayib which was stored at ambient temperature for nine days and completely eliminated Ps. aruginosa, S. aureus and Sh. boyidii from day 5, 6, and 7, respectively. The result indicates that the mixed acid-bile tolerant LAB cultures eliminated the test pathogens in both laboratory medium and in ayib. The mixed acid-bile tolerant LAB culture could possibly be used as candidate potential protective starter culture for preparation of ayib. Key words: Ayib, lactic acid bacteria, foodborne pathogens, acid-bile tolerance, inhibition

Lantibiotic bovicin HJ50 is produced by Streptococcus bovis HJ50 and acts as the extracellular signal to autoregulate its own biosynthesis through BovK/R two-component system. Bovicin HJ50 shows a linear N-terminal and glubolar C-terminal structure, and the sensor histidine kinase BovK contains eight transmembrane segments lacking any extensive surface-exposed sensory domain. The signal recognition mechanism between bovicin HJ50 and BovK is still unknown. We performed saturated alanine scanning mutagenesis and other amino acid substitutions on bovicin HJ50 using a semi-in vitro biosynthesis. Results of the mutants inducing activities indicated that several charged and hydrophobic amino acids in ring B of bovicin HJ50, as well as two glycines were key residues to recognize BovK. Circular dichroism analyses indicated that both glycines contributed to bovicin HJ50 structural changes in the membrane. Biotin-labeled bovicin HJ50 could interact with the N-terminal sensor of BovK, and several charged residues and a conserved hydrophobic region in the N-terminal portion of BovK sensor domain were important for interacting with the signal bovicin HJ50. By combining the results, we suggested a mechanism of bovicin HJ50 recognizing and activating BovK mainly through electrostatic and hydrophobic interactions.

The production of antagonistic substances by lactic acid bacteria (LAB) is well known and LAB are widely used in food preserving. Among antagonistic substances bacteriocins are very interesting. The interest for use of bacteriocins of LAB as natural antimicrobial compounds has appeared mainly because some of them are able to inhibit certain Gram-positive spoilage bacteria and food borne pathogens like Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus and Clostridium butyricum. Bacteriocins of LAB could not fully substitute traditional preserving factors in food, but offer an additional hurdle. They can be introduced into foods as starter cultures, protective cultures or as additives in semi-purified, purified or pre-fermented form. Application of bacteriocins in foods is limited because of some characteristics: narrow activity spectrum, inactivation by proteolytic enzymes, binding to food components, too low "in situ" production, influence of intrinsic factors (pH, Aw), resistant strains. Some of those obstacles could be overcome by combining more bacteriocins or by genetically modifying bacteriocin producer. Until now only nisin and pediocins are industrially used, but many others are being tested on experimental level. Some promising examples of food preservation using bacteriocins or bacteriocins producing cultures are presented.

Fermentation of various food stuffs by lactic acid bacteria is one of the oldest forms of food biopreservation. Bacterial antagonism has been recognized for over a century, but in recent years, this phenomenon has received more scientific attention, particularly in the use of various strains of lactic acid bacteria (LAB). Certain strains of LAB demonstrated antimicrobial activity against foodborne pathogens, including bacteria, yeast and filamentous fungi. Furthermore, in recent years, many authors proved that lactic acid bacteria have the ability to neutralize mycotoxin produced by the last group. Antimicrobial activity of lactic acid bacteria is mainly based on the production of metabolites such as lactic acid, organic acids, hydroperoxide and bacteriocins. In addition, some research suggests other mechanisms of antimicrobial activity of LAB against pathogens as well as their toxic metabolites. These properties are very important because of the future possibility to exchange chemical and physical methods of preservation with a biological method based on the lactic acid bacteria and their metabolites. Biopreservation is defined as the extension of shelf life and the increase in food safety by use of controlled microorganisms or their metabolites. This biological method may determine the alternative for the usage of chemical preservatives. In this study, the possibilities of the use of lactic acid bacteria against foodborne pathogens is provided. Our aim is to yield knowledge about lactic acid fermentation and the activity of lactic acid bacteria against pathogenic microorganisms. In addition, we would like to introduce actual information about health aspects associated with the consumption of fermented products, including probiotics.

Lactic acid bacteria (LAB) found in Ethiopian traditional fermented foods and beverages have potential antagonistic effects against foodborne pathogens due to their capacity to produce various antimicrobial metabolites. This study evaluated the antagonistic activity of LAB isolated from Ethiopian traditional fermented foods and beverages against foodborne pathogens and characterized their antimicrobial substances. A total of 180 traditional fermented foods and beverages were collected, and the antagonistic activities of LAB were evaluated against selected foodborne pathogens. The effects of pH, temperature, enzymes, and food additives on the antagonistic effects of cell-free supernatant produced by LAB were investigated. LAB identification and characterization were conducted using an integrated phenotypic approach and MALDI TOF MS spectrum analysis, and data were analyzed using one-way ANOVA and Tukey post hoc analysis. A total of 956 LAB were isolated, of which seventeen (17 LAB) isolates of Pediococcus pentosaceus (Pc. pentosaceus (n = 7)), Pediococcus acidilactici (Pc. acidilactici (n = 2)), Enterococcus faecium (Ec. faecium (n = 6)), and Lactococcus lactis (Lc. lactis (n = 2)) were screened for antagonistic activity based on their ability to produce bacteriocins, probiotic activity, and preservative potential. Pc. pentosaceus JULABB16, Pc. pentosaceus JULABB01, and Ec. faecium JULABBr39 showed strong antagonistic activity against all pathogens, with mean inhibition zone diameters ranging from 23.50 to 35.50mm. Lc. lactis, Pc. pentosaceus, Pc. acidilactici, and Ec. faecium produced bioactive metabolites that were sensitive to proteolytic enzymes and capable of withstanding high temperatures (80-100°C) and acid concentrations (pH 2-10). The CFS produced by Lc. lactis, Pc. pentosaceus, Pc. acidilactici, and Ec. faecium showed the most impending antagonistic activity against all pathogens. The bioactive substances produced by LAB isolates had promising effects against food spoilage and pathogenic bacteria, making them potential natural food preservatives.

Bacteriocin producing bacteria are commonly found in meat products to enhance theirshelf-life. In the present study, bacterial species were isolated from meat samples (beef) from differentlocalities of Lahore, Pakistan. MRS agar medium was used to isolate lactic acid bacteria (LAB) throughspread and streak methods (incubated for 72 h at 37 °C). Identification of bacteriocinogenic LAB strainswas done by using staining techniques, morphology based characteristics and biochemical tests. Thesestrains were BSH 1b, BSH 3a, BIP 4a, BIP 3a, BIP 1b and BRR 3a. Antibacterial activity of LAB wasperformed against food borne pathogens viz., Escherichia coli and Staphylococcus aureus through paperdisc diffusion method. Three bacterial strains showed maximum inhibition and characterised by ribotypingviz., BIP 4a was identified as Lactobacillus curvatus, BIP 3a was Staphylococcus warneri and BIP 1b wasLactobacillus graminis. Optimum pH 5-6.5 and 30-37 °C temperature for isolated bacterial strains wasrecorded. Protein concentration measured was 0.07 mg/mL for BSH 1b, 0.065 mg/mL for BSH 3a,0.057 mg/mL for BIP 4a, 0.062 mg/mL for BIP 1b, 0.065 mg/mL for BIP 3a and for BRR 3a 0.078 mg/mL,respectively. Bacteriocin of all isolates except BIP 3a was found to be sensitive towards pepsin and resistanttowards Rnase. Bacteriocin production was stable at between pH 5.0 and 6.0 and resistant temperaturewas 40 °C. It was concluded that lactic acid bacteria (LAB) from meat can be helpful as antibacterialagents against food-borne bacterial pathogens because of thermostable producing bacteriocin.

Background: Lactic acid bacteria (LAB) are important organisms recognized for fermentative ability as well as health and nutritional benefits. A large number of bacteriocins from LAB have been characterized and a number of studies have indicated the potential usefulness of bacteriocin in food preservative. The objective of this study was to evaluate the antagonistic effects and bacteriocinogenic activity of LAB isolated from Sorghum bicolor-based ‘ogi’ against selected food borne bacteria from cabbage samples. Methodology: Five samples of Sorghum bicolor-based ‘ogi’ and 5 samples of suspected infected cabbage heads were randomly collected using sterile water proof material from Abakpa main market, Abakaliki, and processed at the Applied Microbiology Laboratory of Ebonyi State University, for isolation of LAB and food borne pathogen by conventional culture and biochemical identification tests. Antagonistic effects of LAB and its bacteriocinogenic activity were determined by agar well diffusion test. Results: Three different Lactobacillus species designated A, B, and C, were isolated from the Sorghum bicolorbased ‘ogi’ and 5 bacterial species were isolated from cabbage heads; Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella, and Shigella species. The Lactobacillus species had inhibitory effect against S. aureus, E. coli, and Shigella species with inhibition zone diameters (IZD) of 19 mm, 10 mm, and 10 mm respectively. The crude bacteriocin extracts from the Lactobacillus species showed higher inhibitory activity against tested bacterial isolates at 10-1 (0.1ml) than at 10-2 dilution (0.01ml), and the inhibitory activity was higher at pH 2 than pH 6 and 7, with no activity at pH 8. Conclusion: This study showed that LAB and its extracted bacteriocin demonstrated in vitro inhibitory activity against food borne pathogens isolated from cabbage heads. There is the need to further characterize the active components of the bacteriocin for possible commercial use as preservatives and potential source of new antimicrobial agent. Keywords: Lactic acid bacteria, bacteriocin, cabbage, fermented food, ‘ogi’

The function of lactic acid bacteria and brine solutions on biogenic amine formation by foodborne pathogens in trout fillets

Lactic acid bacteria (LAB) have been extensively explored as potential biopreservants. They could produce substances with antimicrobial properties such as bacteriocins and organic acids which can also be the cause of antagonistic activity shown by LAB. Thus, the objective of this study is to assess the antagonistic activity of LAB isolated from fermented Oreochromis niloticus against foodborne pathogens and to determine the potential of LAB as a surface decontaminant of raw chicken breast and Tilapia fish fillet. The antagonistic activity of LAB has been shown to affect Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus. When LAB was introduced to the mixed cultures of E. coli, S. typhimurium, and S. aureus, the growth of those pathogens drastically reduced and this has proven that LAB grows stronger and more stable while eliminating the pathogens. LAB and their cell-free supernatant (CFS) were also introduced into the raw chicken breast and fresh Tilapia fish fillet, where E. coli growth was recorded. Both cell cultures and CFS of LAB showed inhibition of E. coli on chicken breast and Tilapia fish fillet in the range of 0.16 to 1.28 log10 reduction and 0.12 to 1.12 log10 reduction, respectively. In conclusion, the results above suggest that LAB isolated from fermented O. niloticus has the potential to be a surface decontaminant. Additionally, both LAB and their CFS can also be used as biopreservative for both chicken breast and fish fillet due to a very good antagonistic activity shown by the LAB toward the foodborne pathogens.

Bacteriocins from lactic acid bacteria (LAB) are natural antimicrobial peptides or proteins with interesting potential applications in food preservation and health care. The present study was aimed to isolate bacteriocinogenic LAB from dairy products, fermented food and environmental waste samples. One thirty five colonies of LAB were isolated and screened for bacteriocin production by agar overlay method. Among them, Lactobacillus acidophilus NCIM5426 isolated from home made paneer showed maximum zone of inhibition i.e 22-24mm in diameter against food borne ( Listeria monocytogenes , Staphylococcus aureus ) and human pathogens ( Escherichia coli , Salmonella typhi ). The antibacterial compound from the culture supernatant was found to be proteinaceous in nature and therefore, identified as bacteriocin. The bacteriocin of L. acidophilus NCIM5426 was found to be heat-stable (121°C for 15 min) and active over a wide pH range of 4.0-10.0. It showed stability (60%) for 30 days at room temperature (30-32°C). Addition of surfactants (EDTA, SDS, Hexadecyl trimethylamonium bromide) up to 1% to crude bacteriocin showed increase in antibacterial activity where as metal ions (Calcium Chloride, Zinc Sulphate and Mercuric Chloride) in low concentration (0.5-1mgL -1 ) decreased the activity. The bacteriocin was purified to its homogeneity by ammonium sulfate precipitation followed by gel filtration chromatography and HPLC. Molecular weight of bacteriocin was found to be 5.6 and 5.8 KDa by SDS page and LC/MS respectively. Production and activity of bacteriocin was significant (50%) even at higher salt (NaCl) concentration i.e 3%. Our present study demonstrates the possibility of using L. acidophilus NCIM5426 or its bacteriocin as a biopreservative in dairy industry.

Bacterial peptides of low molecular weight displaying antagonism towards other bacterial members are referred to as bacteriocins. Bacteriocins are an essential member of the broadly classified group of lantibiotics which finds several commercial uses. This work comprises of the determination of structure of the most important class of bacteriocins – the leucocin group. Structure elucidation was performed using homology modelling approach and the modeled structure was validated using their Ramachandran calculations. Ligand interaction pockets were identified by calculating the Delaunay triangulation number and structural properties of amino acids were documented. Structure elucidations of such lantibiotics are important for future endeavours towards analyses of protein protein interactions. Key WordsLantibiotics, bacteriocin, homology modelling, Pocket Identification Introduction Molecules isolated from bacteria that have demonstrated bactericidal activity are called bacteriocins. Ribosomally synthesized peptide bacteriocins from Gram-positive bacteria can be subdivided into two major classes. Bacteriocins of class I are characterized by having modified amino acid residues (e.g., lantibiotics) and bacteriocins of class II are characterized by not possessing modified amino acid residues (e.g., small heat stable non-lantibiotics). These two classes are the most studied due to their abundance and their potential use for industrial applications. They are similar in size (approx 2060 amino acids), mostly cationic, and possess a hydrophobic domain and/or amphiphilic region, which may relate to their activity on membranes. The defining characteristic of lantibiotics is that they contain the unusual amino acids lanthionine or -methyllanthionine. Generally, type A lantibiotics are characterized by being strongly cationic (with 2 to 7 net positive charge), having molecular masses more than 2100 Da, and having rigid ring conformations separated by areas of flexibility [1]. It is currently believed that their primary bactericidal activity is mediated through the formation of voltage-dependent membrane channels. By contrast, type B lantibiotics are characterized by being neutral or slightly anionic with a 0 to –1 net charge, having molecular masses of less than 2100 Da, and having a more compact globular structure. The main function lies in the inhibition of the essential enzymes. Two peptide bacteriocins are unique as, two genes encoding each of the peptides are located next to each other and both of the peptides are required for bactericidal activity. As their bactericidal activity resembles type I lantibiotics they are often classified as the type I class [2]. Lantibiotics are produced by a large number of Gram positive bacteria such as Streptococcus and Streptomyces to attack other gram positive bacteria and as such they are considered a member of the bacteriocins [3]. Lantibiotics are well studied because of the commercial use of these bacteria in the food industry for making dairy products such as cheese. Posttranslational modification events and their extents are the basis for the classification of the bacteriocins. The lantibiotics are a class of more extensively modified bacteriocins, also called Class I. Bacteriocins for which disulfide bonds are the only modification to the peptide are Class II bacteriocins [2]. Nisin and epidermin are members of a family of lantibiotics that bind to a cell wall precursor lipid component of target bacteria and disrupt cell wall production. The duramycin families of lantibiotics bind phosphoethanolamine in the membranes of its target cells and seem to disrupt several physiological functions. The name Lantibiotics was introduced in 1988 as an abbreviation for Lanthionine-containing peptide antibiotics”. In spite of this naming, Lantibiotics are not classed as antibiotics. One reason for this is that they are constructed ribosomally while antibiotics are constructed by enzyme action. The first structures of these antimicrobial agents were produced by pioneering work by Gross and Morell in the late sixties and early seventies, thus marking the formal introduction of Lantibiotics. Since then Lantibiotics such as Nisin have been used auspiciously for food preservation and have yet to encounter significant bacterial resistance. These attributes of lantibiotics have led to more detailed research into their structures and biosynthetic pathways. Type A Lantibiotics are long flexible molecules – e.g., Nisin, subtilin, epidermin. Subgroup AI includes Mutacin II, subgroup AII includes Mutacin I & III. Type B Lantibiotics are globular eg mersacidin, actagardine, cinnamycin. The biosynthesis is interesting. They are synthesized with a leader polypeptide sequence which is only removed during the transport of the molecule out of the synthesizing cell. They are synthesized by ribosome’s, which distinguishes them from antibiotics which are synthesized by enzymes. Lantibiotics show substantial specificity for some components (eg lipid II) of bacterial cell membranes especially of Gram positive bacteria. Type A kill rapidly by pore formation, type B inhibit peptidoglycan biosynthesis. They are Structural analysis of Leucocine – an essential bacteriocin International Journal of Bioinformatics Research, ISSN: 0975–3087, Volume 2, Issue 2, 2010 2 active in very low concentrations. Lantibiotics are produced by Gram-positive bacteria and show strong antimicrobial action towards a wide range of other Gram-positive bacteria. As such they have become attractive candidates for use in food preservation (by inhibiting pathogens that cause food spoilage) and the pharmaceutical industry (to prevent or fight infections in humans or animals). Bacteriocins are antibacterial compounds that are produced from lactic acid bacteria and their lethality lies in the fact that they are able to create pores in the bacterial membrane thus causing lysis of the target cell. Examples of bacteriocins are nisin and leucocin. Nisin inhibits the growth of most gram-positive bacteria, particularly sporeformers (e.g., Clostridium botulinum. This bacteriocin has been approved for use as a food preservative in the United States since 1989. Leucocin is inhibitory to the growth of Listeria monocytogenes. Lactic acid bacteria are also of economic importance in the preservation of agricultural crops. A popular method of crop preservation utilizes what is termed silage. In this method crop plants are exposed to lactic acid bacteria which results in the lowering of the surface pH and prevents the growth of other microorganisms [4]. Materials and Method The sequences of the lantibiotics were obtained from the Genbank nucleotide collection at the NCBI [5] home page. All the available sequences were primarily downloaded as a batch file and then were subjected to the analyses. Primarily the sequences were subjected to homology analysis through BLASTp to retrieve the nearest homologues. All the sequences were then collected to generate a pool of 300 sequences. From this pool each sequence was selected and was subjected to BLAST search against the Protein Databank (PDB) [6]. This resulted in the derivation of several suitable templates on which the structure could be modeled on. The best template was found to be that of Bacteriocin Carnobacteriocin B2 (1RY3). Using this template the structures were then modeled using Modeller 9.2. Once the modeled structure was obtained then the rotamer and Ramachandran angles were tested at the Mol probity server and were substantiated using the VADAR program. The Delaunay triangulation score of each of the residues were then estimated and then best six binding pockets were identified using the CASTp

Chapter 8 - Ecofriendly Synthesis of Metal/Metal Oxide Nanoparticles and Their Application in Food Packaging and Food Preservation

Chapter 11 - Lactic acid bacteria and bacteriocins as biopreservatives

Fermented tilapia (Tilapia nicoliticus) is one of the famous fermented food in Malaysia. Lactic acid bacteria (LAB) which well known as GRAS (Generally Regarded as Safe) are present in most fermented foods and they are well-known non-pathogenic bacteria that play an important role in everyday life. Apart from LAB, spices have also been used for centuries across different regions of the world to improve aroma, flavour and food preservative. This research was aimed to explore a potential natural food preservative using LAB isolated from fermented Tilapia nicoliticus incorporated with various spices (9% turmeric, 6% chilli and 9% black pepper) against foodborne pathogens. The isolation of LAB in different media (MRS, MRS+CaCO3, M17 and Tomato Juice Agar) showed the highest LAB count on day-9 and day-15 during the fermentation period in fermented Tilapia incorporated with black pepper, turmeric and chilli. The highest antimicrobial activity by LAB against Bacillus cereus was observed in fermented tilapia incorporated with black pepper. On the other hand, fermented fish incorporated with chilli showed the highest antimicrobial activity against Staphylococcus aureus, Escherichia coli and Salmonella enterica serovar Typhimurium. Higher antimicrobial activity was detected in fermented Tilapia in the presence of LAB together with the spices, in comparison to the presence of LAB alone, suggesting synergistic effects between LAB in fermented fish with spices could enhance stronger antimicrobial activities against food pathogens and therefore, served as a natural food preservative.