Antibacterial metabolites of lactic acid bacteria: their diversity and properties

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.     

Microbial production of bacteriocins: Latest research development and applications

Bacteriocins are low molecular weight peptides secreted by the predator bacterial cells to kill sensitive cells present in the same ecosystem competing for food and other nutrients. Exceptionally few bacteriocins along with their native antibacterial property also exhibit additional anti-viral and anti-fungal properties. Bacteriocins are generally produced by Gm+, Gm– and archaea bacteria. Bacteriocins from Gm?+?bacteria especially from lactic acid bacteria (LAB) have been thoroughly investigated considering their great biosafety and broad industrial applications. LAB expressing bacteriocins were isolated from fermented milk and milk products, rumen of animals and soil using deferred antagonism assay. Nisin is the only bacteriocin that has got FDA approval for application as a food preservative, which is produced by Lactococcus lactis subsp. Lactis. Its crystal structure explains that its antimicrobial properties are due to the binding of NH₂ terminal to lipid II molecule inhibiting the peptidoglycan synthesis and carboxy terminal forming pores in bacterial cell membrane leading to cell lysis. The hinge region connecting NH₂ and carboxy terminus has been mutated to generate mutant variants with higher antimicrobial activity. In a 50 ton fermentation of the mutant strain 3807 derived from L. lactis subsp. lactis ATCC 11454, 9,960?IU/mL of nisin was produced. Currently, high purity of nisin (>99%) is very expensive and hardly commercially available. Development of more advanced tools for cost-effective separation and purification of nisin would be commercially attractive. Chemical synthesis and heterologous expression of bacteriocins ended in low yields of pure proteins. At present, bacteriocins are almost solely applied in food industries, but they have a great potential to be used in other fields such as feeds, organic fertilizers, environmental protection and personal care products. The future of bacteriocins is largely dependent on getting FDA approval for use of other bacteriocins in addition to nisin to promote the research and applications.     

Novel bacteriocins from lactic acid bacteria (LAB): various structures and applications

Bacteriocins are heat-stable ribosomally synthesized antimicrobial peptides produced by various bacteria, including food-grade lactic acid bacteria (LAB). These antimicrobial peptides have huge potential as both food preservatives, and as next-generation antibiotics targeting the multiple-drug resistant pathogens. The increasing number of reports of new bacteriocins with unique properties indicates that there is still a lot to learn about this family of peptide antibiotics. In this review, we highlight our system of fast tracking the discovery of novel bacteriocins, belonging to different classes, and isolated from various sources. This system employs molecular mass analysis of supernatant from the candidate strain, coupled with a statistical analysis of their antimicrobial spectra that can even discriminate novel variants of known bacteriocins. This review also discusses current updates regarding the structural characterization, mode of antimicrobial action, and biosynthetic mechanisms of various novel bacteriocins. Future perspectives and potential applications of these novel bacteriocins are also discussed.     

Exploration of antimicrobial potential in LAB by genomics

A tremendous flow of information has been created through various genome sequencing projects worldwide. So far, 128 bacterial genome sequences have been completed and 391 are under way. Many of these bacteria, including several lactic acid bacteria (LAB), are used in the production and preservation of food and feed. The major antimicrobial and biopreservative substance produced by LAB is organic acid; however, some LAB produce additional antimicrobial compounds. Among these, the bacteriocins have demonstrated great potential as food preservatives. Additionally, antimicrobial compounds different from the bacteriocins have recently been identified, of which several display strong antifungal activity. The information obtained from genomics and related technologies will have great impact on the future identification and development of new antimicrobial agents. Developments will include the identification of pathways for the production of antimicrobials and genome mining for new antimicrobial peptides.     

Gassericin A: a circular bacteriocin produced by Lactic acid bacteria Lactobacillus gasseri

During the recent years extensive efforts have been made to find out bacteriocins from lactic acid bacteria (LAB) active against various food spoilage and pathogenic bacteria, and superior stabilities against heat treatments and pH variations. Bacteriocins isolated from LAB have been grouped into four classes. Circular bacteriocins which were earlier grouped among the four groups of bacteriocins, have recently been proposed to be classified into a different class, making it class V bacteriocins. Circular bacteriocins are special molecules, whose precursors must be post translationally modified to join the N to C termini with a head-to-tail peptide bond. Cyclization appears to make them less susceptible to proteolytic cleavage, high temperature and pH, and, therefore, provides enhanced stability as compared to linear bacteriocins. The advantages of circularization are also reflected by the fact that a significant number of macrocyclic natural products have found pharmaceutical applications. Circular bacteriocins were unknown two decades ago, and even to date, only a few circular bacteriocins from a diverse group of Gram positive organisms have been reported. The first example of a circular bacteriocin was enterocin AS-48, produced by Enterococcus faecalis AS-48. Gassereccin A, produced by Lactobacillus gasseri LA39, Reutericin 6 produced by Lactobacillus reuteri LA6 and Circularin A, produced by Clostridium beijerinickii ATCC 25,752, are further examples of this group of antimicrobial peptides. In the present scenario, Gassericin A can be an important tool in the food preservation owing to its properties of high pH and temperature tolerance and the fact that it is produced by LAB L. gasseri, whose many strains are proven probiotic.     

Sensitivity to the two‐peptide bacteriocin lactococcin G is dependent on UppP,an enzyme involved in cell‐wall synthesis

Most bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor‐mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two‐peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G‐resistant mutants and performed whole‐genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non‐sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactis uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.     

Purification and partial characterization of bacteriocin produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> ssp. <Emphasis Type="Italic">lactis</Emphasis> LL171

Lactic acid bacteria (LAB) are possessing ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this regard, novel bacteriocin compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was purified by ammonium sulphate precipitation, dialysis and gel filtration. The molecular weight (≈3.4 kDa) of obtained bacteriocin was confirmed by SDS-PAGE, which revealed single peptide band. Molecular identification of LAB strain isolated from Tulum Cheese was conducted using 16S rDNA gene sequencing as Lactococcus lactis ssp. lactis LL171. The amino acid sequences (KKIDTRTGKTMEKTEKKIELSLKNMKTAT) of the bacteriocin from Lactococcus lactis ssp. lactis LL171 was found unique and novel than reported bacteriocins. Further, the bacteriocin was possessed the thermostable property and active at wide range of pH values from 1 to 11. Thus, bacteriocin reported in this study has the potential applications property as food preservative agent.     

Bacteriocinogenic LAB Strains for Fermented Meat Preservation: Perspectives,Challenges, and Limitations

Over the last decades, much research has focused on lactic acid bacteria (LAB) bacteriocins because of their potential as biopreservatives and their action against the growth of spoilage microbes. Meat and fermented meat products are prone to microbial contamination, causing health risks, as well as economic losses in the meat industry. The use of bacteriocin-producing LAB starter or protective cultures is suitable for fermented meats. However, although bacteriocins can be produced during meat processing, their levels are usually much lower than those achieved during in vitro fermentations under optimal environmental conditions. Thus, the direct addition of a bacteriocin food additive would be desirable. Moreover, safety and technological characteristics of the bacteriocinogenic LAB must be considered before their widespread applications. This review describes the perspectives and challenges toward the complete disclosure of new bacteriocins as effective preservatives in the production of safe and “healthy” fermented meat products.     

Characterization of some bacteriocins produced by lactic acid bacteria isolated from fermented foods

Lactic acid bacteria (LAB) isolated from different sources (dairy products, fruits, fresh and fermented vegetables, fermented cereals) were screened for antimicrobial activity against other bacteria, including potential pathogens and food spoiling bacteria. Six strains have been shown to produce bacteriocins: Lactococcus lactis 19.3, Lactobacillus plantarum 26.1, Enterococcus durans 41.2, isolated from dairy products and Lactobacillus amylolyticus P40 and P50, and Lactobacillus oris P49, isolated from bors. Among the six bacteriocins, there were both heat stable, low molecular mass polypeptides, with a broad inhibitory spectrum, probably belonging to class II bacteriocins, and heat labile, high molecular mass proteins, with a very narrow inhibitory spectrum, most probably belonging to class III bacteriocins. A synergistic effect of some bacteriocins mixtures was observed. We can conclude that fermented foods are still important sources of new functional LAB. Among the six characterized bacteriocins, there might be some novel compounds with interesting features. Moreover, the bacteriocin-producing strains isolated in our study may find applications as protective cultures.     

Linear DNA plasmids of yeasts

Abstract Proteinaceous antimicrobial compounds are produced by a diversity of species ranging from bacteria to humans. This review focuses on the mode of action of pore-forming bacteriocins produced by Gram-positive bacteria. The mechanism of action of specific immunity proteins, which protect the producer strains from the lethal action of their own products (producer self-protection), are also discussed.     

Mechanistic action of pediocin and nisin: recent progress and unresolved questions

Nisin and pediocin PA-1 are examples of bacteriocins from lactic acid bacteria (LAB) that have found practical applications as food preservatives. Like other natural antimicrobial peptides, LAB bacteriocins act primarily at the cytoplasmic membranes of susceptible microorganisms. Studies with in vivo as well as in␣vitro membrane systems are directed toward understanding how bacteriocins interact with membranes so as to provide a mechanistic basis for their rational applications. The dissipation of proton motive force was identified early on as the common mechanism for the lethal activity of LAB bacteriocin. Models for nisin/membrane interactions propose that the peptide forms poration complexes in the membrane through a multi-step process of binding, insertion, and pore formation. This review focuses on the current knowledge of: (1) the mechanistic action of nisin and pediocin-like bacteriocins, (2) the requirement for a cell factor such as a membrane protein, (3) the influence of membrane potential, pH, and lipid composition on the of specificity and efficacy of bacteriocins, and (4) the roles of specific amino acids and structural domains of the bacteriocins in their action. Received: 3 April 1998 / Received last revision: 27 July 1998 / Accepted: 29 July 1998     

PCR detection of the structural genes of nisin Z and lacticin 481 in Lactococcus lactis subsp. lactis INIA 415, a strain isolated from raw milk Manchego cheese

A Lactococcus strain with strong antimicrobial activity was isolated from raw milk Manchego cheese during a survey on the production of bacteriocins by lactic acid bacteria present in raw milk cheeses. It was identified as Lactococcus lactis subsp. lactis, phenotypically by its morphological and physiological characteristics and genotypically by a PCR technique. When tested for tolerance to known bacteriocins produced by lactococci, it was shown to be resistant to nisin A and nisin Z. The presence of genes encoding nisin and lacticin 481 was revealed by PCR techniques with specific probes. Sequences of the respective PCR amplified fragments matched sequences reported for nisin Z and lacticin 481.     

Bacteriocins from lactic acid bacteria and their potential in the preservation of fruit products

Bacteriocins produced by lactic acid bacteria (LAB) are well-recognized for their potential as natural food preservatives. These antimicrobial peptides usually do not change the sensorial properties of food products and can be used in combination with traditional preservation methods to ensure microbial stability. In recent years, fruit products are increasingly being associated with food-borne pathogens and spoilage microorganisms, and bacteriocins are important candidates to preserve these products. Bacteriocins have been extensively studied to preserve foods of animal origin. However, little information is available for their use in vegetable products, especially in minimally processed ready-to-eat fruits. Although, many bacteriocins possess useful characteristics that can be used to preserve fruit products, to date, only nisin, enterocin AS-48, bovicin HC5, enterocin 416K1, pediocin and bificin C6165 have been tested for their activity against spoilage and pathogenic microorganisms in these products. Among these, only nisin and pediocin are approved to be commercially used as food additives, and their use in fruit products is still limited to certain countries. Considering the increasing demand for fresh-tasting fruit products and concern for public safety, the study of other bacteriocins with biochemical characteristics that make them candidates for the preservation of these products are of great interest. Efforts for their approval as food additives are also important. In this review, we discuss why the study of bacteriocins as an alternative method to preserve fruit products is important; we detail the biotechnological approaches for the use of bacteriocins in fruit products; and describe some bacteriocins that have been tested and have potential to be tested for the preservation of fruit products.     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.     

Lantibiotics, class I bacteriocins from the genus Bacillus

Antimicrobial peptides exhibit high levels of antimicrobial activity against a broad range of spoilage and pathogenic microorganisms. Compared with bacteriocins produced by lactic acid bacteria, antimicrobial peptides from the genus Bacillus have been relatively less recognized despite their broad antimicrobial spectra. These peptides can be classified into two different groups based on whether they are ribosomally (bacteriocins) or nonribosomally (polymyxins and iturins) synthesized. Because of their broad spectra and high activity, antimicrobial peptides from Bacillus spp. may have great potential for applications in the food, agricultural, and pharmaceutical industries to prevent or control spoilage and pathogenic microorganisms. In this review, we introduce ribosomally synthesized antimicrobial peptides, the lantibiotic bacteriocins produced by members of Bacillus. In addition, the biosynthesis, genetic organization, mode of action, and regulation of subtilin, a well-investigated lantibiotic from Bacillus subtilis, are discussed.     

The continuing story of class IIa bacteriocins.

Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.     

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates

AIMS: The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from human intestine. METHODS AND RESULTS: A total of 111 LAB were isolated from human adult stool and screened for their bacteriocin production. Neutralized cell-free supernatants from Lactococcus lactis subsp. lactis MM19 and Pediococcus acidilactici MM33 showed antimicrobial activity. The antimicrobials in the supernatant from a culture of L. lactis inhibited Enterococcus faecium, various species of Lactobacillus and Staphylococcus aureus; while those in the supernatant from a culture of P. acidilactici inhibited Enterococcus spp., some lactobacilli and various serotypes of Listeria monocytogenes. The antimicrobial metabolites were heat-stable and were active over a pH range of 2-10. The antimicrobial activities of the supernatants of both bacteria were inhibited by many proteases but not by catalase. The plate overlay assay allowed an approximation of size between 3.5 and 6 kDa for both antimicrobial substances. CONCLUSIONS: As the antagonistic factor(s) produced by L. lactis MM19 and P. acidilactici MM33 were sensitive to proteolytic enzymes, it could be hypothesized that bacteriocins were involved in the inhibitory activities. Inhibition spectrum and biochemical analysis showed that these bacteria produced two distinct bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: We are the first to isolate bacteriocin-producing strains of Pediococcus and Lactococcus from human intestine. These strains might be useful for control of enteric pathogens.     

The structure of pyogenecin immunity protein, a novel bacteriocin-like immunity protein from Streptococcus pyogenes

Background  

Many Gram-positive lactic acid bacteria (LAB) produce anti-bacterial peptides and small proteins called bacteriocins, which enable them to compete against other bacteria in the environment. These peptides fall structurally into three different classes, I, II, III, with class IIa being pediocin-like single entities and class IIb being two-peptide bacteriocins. Self-protective cognate immunity proteins are usually co-transcribed with these toxins. Several examples of cognates for IIa have already been solved structurally. Streptococcus pyogenes, closely related to LAB, is one of the most common human pathogens, so knowledge of how it competes against other LAB species is likely to prove invaluable.     

Interactive effects of pH and temperature on the bacteriocin stability by response surface analysis

The combined influence of pH and temperature on bacteriocins produced by three lactic acid bacteria, Pediococcus pentosaceus MMZ26, Enterococcus faecium MMZ17 and Lactococcus lactis MMZ25, isolated from Tunisian traditional dry fermented meat was studied using a second order orthogonal factorial design and response-surface methodology (RSM). This method allows estimating the interactive effects of pH and temperature on the stability of each bacteriocin. The high heat stability of the three bacteriocins was demonstrated, with optimum values at light acidic pH around 5.0, temperature below 90°C and short incubation times. This study contributes to a better understanding of relation between bacteriocins production and stability in order to enhance their, in situ, application as a food and feed biopreservative in fermented and/or heated food products.     

Characterization of bacteriocins produced by strains of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> isolated from Minas cheese

Interest in obtaining bacteriocin-producing strains of lactic acid bacteria (LAB) from different sources has been increasing in recent years due to their multiple applications in health and food industries. This study focused on the isolation and characterization of metabolically active populations of bacteriocinogenic LAB and the evaluation of their antimicrobial substances as well as of some nutritional requirements of them. One hundred and fifty colonies of LAB from artisanal cheeses produced in Minas Gerais state (Brazil) were isolated and screened for their antimicrobial activity. According to their activity against Listeria monocytogenes, ten strains were selected and subsequently identified using biochemical and molecular techniques including 16s rRNA amplification and sequencing as Enterococcus faecalis, Lactobacillus spp., and Pediococcus pentosaceus. Antimicrobial substances produced by four of the selected strains, P. pentosaceus 63, P. pentosaceus 145, P. pentosaceus 146, and P. pentosaceus 147, were biochemically characterized, and presented sensitivity to proteolytic enzymes (suggesting their proteinaceous nature) and to extreme pH. Antimicrobial activity showed stability after treatment with lipase, catalase, α-amylase, and chemicals. Growth kinetics of the P. pentosaceus selected showed maximal bacteriocin production at 37 °C during the end of the exponential growth phase (25,600 AU/mL) and stable production during 24 h of incubation. Dextrose, maltose, and a mixture of peptone, meat extract, and yeast extract increased bacteriocin production. This study demonstrated that dairy products provide a good alternative for obtaining LAB, with the ability to produce antimicrobial substances such as bacteriocins that have potential use as biopreservatives in food.