Development of Wide-Spectrum Hybrid Bacteriocins for Food Biopreservation

The food industry demands new procedures and methods to produce minimally processed, ready to eat food with intact nutritional, taste, and flavor properties. The biopreservation and the use of both bacteriocins produced by lactic acid bacteria (LAB) and bacteriocinogenic strains as an alternative to substitute chemical antimicrobial for food preservation became increasingly important in the last two decades. When the new proposed natural preservatives techniques are applied, probiotics food can be obtained and, simultaneously, foodborne pathogens and spoilage contaminants can diminish. However, bacteriocins produced by LAB have a narrow antibacterial spectrum and are inactive against Gram-negative bacteria like Salmonella and the emergent enterohemorrhagic Escherichia coli. Knowing the mechanism of action and the structural features of microcins synthesized by Gram-negative bacteria and with potent antimicrobial activity against the mentioned microorganism, the proposal is to obtain hybrid peptides (microcin–bacteriocin) with broad antimicrobial spectrum. This review explains how the inability of bacteriocins to cross the outer membrane of Gram-negative bacteria unable them to act on the bacteria. It will also be discussed how a hybrid bacteriocin can be obtained.     

Incidence and antibiotic susceptibility of bacteriocin‐producing lactic acid bacteria from dairy products

Screening for bacteriocin production by strains of lactic acid bacteria (LAB) from local dairy products in Iran resulted in the detection of 10 bacteriocin‐producing strains. Among 105 isolated, 10 bacteriocin producers were phenotypically and genotypically identified as Enterococcus spp. The antimicrobial compounds produced by these novel strains were inactivated by trypsin, proteinase k. These bacteriocins also were active in a wide range of pH and temperature values, and inhibited not only the closely related LAB, but also Listeria monocytogenes.     

Characterization of semipurified enterocins produced by Enterococcus faecium strains isolated from raw camel milk

Food safety has become an issue of great interest worldwide. Listeria monocytogenes is a food-borne pathogen that causes listeriosis and is difficult to control in the dairy industry. The use of lactic acid bacteria (LAB) and their antimicrobial substances against Listeria is promising in food applications. Here, we report the isolation from raw camel milk of LAB displaying antilisterial activity. Two isolates were selected for their secretion of bacteriocin(s) and identified by 16S rRNA sequencing as Enterococcus faecium S6 and R9. The produced bacteriocins were partially purified by ammonium sulfate precipitation and then biochemically characterized. Antimicrobial activity was estimated to be 6,400 and 400 AU (arbitrary units)/mL for E. faecium S6 and R9, respectively. The proteinaceous nature of the bacteriocins was confirmed via enzymatic reactions. Moreover, lipolytic and glycolytic enzymes completely inactivated the antimicrobial effect of the bacteriocins. These bacteriocins were heat-resistant and stable over a wide range of pH (2.0 to 10.0). To confirm its inactivation by lipolytic and glycolytic enzymes, the bacteriocin of E. faecium S6 was further purified by gel filtration, which suggested the existence of carbohydrate and lipid moieties. In addition, enterocin-coding genes were identified by PCR, showing DNA fragments corresponding in size to enterocins A, B, and P for E. faecium S6 and to enterocins B and P for E. faecium R9. In conclusion, these results indicate that partially purified bacteriocins from E. faecium S6 and R9 may be beneficial in controlling Listeria in the dairy industry.     

Characterisation of anti-Listeria monocytogenes bacteriocins from Enterococcus faecium strains isolated from dairy products

Bacteriocin-producing lactic acid bacteria were isolated from 34 samples of dairy products. Nine bacteriocin producers were phenotypically and genotypically identified as Enterococcus faecium . By means of PCR-techniques, enterocin A was characterised in all of the nine bacteriocin-producing Enterococcus isolates. Enterocin-producing lactic acid bacteria were the most abundant in dairy products collected from different areas in Iran. Maximum bacteriocin production by Enterococcus faecium strains was detected in the stationary phase of growth. Bacteriocins produced by all isolates were found to have anti-listerial activity in sterile milk. The purified bacteriocins were identified as <6.5 kDa peptide by sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE). The molecular weights of bacteriocins were found to be the same in all strains. This bacteriocin might be useful as a natural preservative.     

ISOLATION AND PARTIAL CHARACTERIZATION OF A NOVEL BACTERIOCIN PRODUCED BY LACTOCOCCUS LACTIS SSP. LACTIS MC38

This work presents the isolation and partial characterization of a new lactococcal bacteriocin produced by Lactococcus lactis ssp. lactis MC38. The bacteriocin demonstrated broad spectrum of inhibition activity against both pathogenic and food spoilage organisms, and various lactic acid bacteria. This antimicrobial substance appeared to be proteinaceous because its activity was completely inactivated by proteinase K and α‐chymotrypsin. It was heat and pH stable. The apparent molecular mass of the purified bacteriocin, determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, was 8.0 kDa. The amino acid composition of the studied bacteriocin was found to be quite different from known lactococcal bacteriocins. The calculation of the number of amino acid residues in the bacteriocin molecule revealed that it contained 62 amino acids.     

Biochemical characterization of a bacteriocin‐like inhibitory substance produced by Enterococcus faecium MXVK29, isolated from Mexican traditional sausage

BACKGROUND: Enterococci are lactic acid bacteria that can produce bacteriocins, which may offer an additional hurdle to control the growth of food‐borne pathogens; moreover, these bacteriocins may have great potential as natural biopreservatives. The aim of this work was to characterize a bacteriocin‐like inhibitory substance (BLIS) with antilisterial activity produced by an enterococcal strain. RESULTS: The bacteriogenic strain was isolated from Mexican fermented sausages and identified as Enterococcus faecium with 99% sequence similarity. Maximal activity was detected at 16 h, where bacterial growth was in middle of the stationary phase. The producer strain was not inhibited by its own antimicrobial peptide. BLIS showed a strong anti‐Listeria activity and was inactivated by proteinase K. Heating (121 °C for 15 min) induced some inactivation, but thermotolerance was higher at acid pH values. The yield obtained with a pH‐mediated purification process was 32.7%, showing a band with an estimated molecular weight of 3.5 kDa. Automated N‐terminal Edman degradation showed the following sequence: YYGNGVTCGSHHCSVD. CONCLUSION: Biochemical characteristics of BLIS produced by E. faecium MXVK29 suggested that it belongs to Class IIa of the Klaenhammer classification and could be considered as a natural food preservative, although further studies need to be performed. Copyright © 2010 Society of Chemical Industry     

Enterococcus faecium P21: a strain occurring naturally in dry-fermented sausages producing the class II bacteriocins enterocin A and enterocin B

Enterococcus faecium P21 isolated from a Spanish dry-fermented sausage shows a narrow antimicrobial activity against closely related lactic acid bacteria and strong antimicrobial activity against spoilage and foodborne Gram-positive pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens and Clostridium botulinum. The antimicrobial activity is produced during growth in MRS broth at 32°C; it is heat resistant (20 min at 80–100°C) and abolished by protease treatment, and withstands exposure to pH 2–11, freeze-thawing, lyophilization and long term storage at −20°C. Purification of the antimicrobial activity by ammonium sulphate precipitation, gel filtration, and cation-exchange, hydrophobic interaction and reverse-phase chromatographies showed that the broad antimicrobial spectrum exerted by E. faecium P21 was indeed due to two peptide bacteriocins. Studies on theirN -terminal amino acid sequences and PCR and DNA analyses revealed that these bacteriocins are identical to the class II bacteriocins enterocin A and enterocin B. The genetic organization of the enterocin A (EntA) operon in E. faecium P21 shows that the bacteriocin structural gene (entA), the immunity gene (entiA) and the induction peptide gene (entF) are clustered and colinearly arranged. Strikingly, this organization was structurally different to that reported for the EntA-producer E. faecium CTC492.     

Optimization and Partial Purification of Bacteriocins from Enterococcus spp. Indigenous to Pakistan

This study was conducted to optimize bacteriocin producing enterococcal strains isolated from indigenous fermented dairy products of Pakistan. Isolates IJ-06, IJ-21, and IJ-31 were identified as Enterococcus faecium and IJ-11 was identified as Enterococcus faecalis. All of these enterococcal isolates were catalase, gelatinase negative, and non-hemolytic on sterile sheep blood. Bacteriocin production trials confirmed E. faecium IJ-06, IJ- 21, and IJ- 31 as the potential producer of bacteriocin showing antimicrobial activity in cell-free supernatant (CFS). E. faecalis IJ-11 displayed activity after partial purification. Optimization of culture conditions for the production of bacteriocin by the selected strains showed that maximum production was achieved at 35–37°C with 1% inoculum after 16 h of incubation and at pH 7–8. The molecular mass of the partially purified enterocins falls in the range of 4.5–4.7 kDa. These enterocins were close to class IIa of bacteriocins and were highly active against Listeria monocytogenes, Bacillus subtilis, Bacillus cereus and other closely related strains.     

提高乳酸菌细菌素合成量方法的研究进展

细菌素是某些细菌通过核糖体合成机制产生的蛋白质或多肽,能够抑制与其亲源关系相同或相近的微生物,某些细菌素在食品加工和发酵过程中能抑制致病菌和腐败菌。乳酸菌被认为是一般公认安全,其细菌素具有安全性高、稳定性好、抑菌谱广等优点,作为一种新型食品防腐剂备受关注,但商品化的乳酸菌细菌素十分有限,仅限于Nisin和Pediocin PA-1等少数几种,合成量低是细菌素在食品中应用受限的主要原因之一。从不同原料中筛选高产菌株、发酵培养基和发酵条件优化、诱变育种、原生质体融合、基因工程方法、群体感应系统调控六个方面,论述了增加乳酸菌细菌素合成量的方法,以期为实现乳酸菌细菌素的工业化生产提供一定的借鉴。     

Modelling contributes to the understanding of the different behaviour of bacteriocin-producing strains in a meat environment

Bacteriocins from lactic acid bacteria are potent antibacterial agents that enable the producer cells to selectively and efficiently inhibit a part of the competing background flora, including several spoilage bacteria and foodborne pathogens. The use of bacteriocinogenic lactic acid bacterium strains, either as starters or as protective cultures, is very limited in the food industry, possibly because many questions about the in situ functionality of such strains remain unanswered. A modelling approach was developed to study the functionality of lactic acid bacterium bacteriocin producers in a meat environment. The kinetic properties of three potential bacteriocin-producing starter cultures for sausage fermentation, namely Lactobacillus sakei CTC 494, Lactobacillus curvatus LTH 1174, and Lactobacillus amylovorus DCE 471 were compared in MRS, used as a meat simulation medium. Moreover, the application possibilities of the bacteriocin-producing Enterococcus faecium RZS C5 strain were evaluated. It turned out that growth and bacteriocin production by Lb. sakei CTC 494 and Lb. curvatus LTH 1174 are adapted to the meat environment, whereas Lb. amylovorus DCE 471 is kinetically not able to dominate the meat fermentation process. Furthermore, the use of bacteriocin-producing enterococci as functional cocultures seems to be attractive.     

Bacteriocins of lactic acid bacteria: Their potentials as food biopreservative

Abstract

Numerous strains of lactic acid bacteria used in the fermentation of foods are known to produce bacteriocins. In general, bacteriocins are a group of proteinaceous antimicrobial substances that inhibit the growth of closely related bacteria. However, some bacteriocins produced by lactic acid bacteria (LAB) exhibit a relatively broad antimicrobial spectrum and are active against several food‐spoilage and health‐threatening microorganisms. Many investigators have reported on the use of bacteriocins as food preservative to extend the shelflife of various foods. This review decribes the research that has been conducted on bacteriocinogenic lactic acid bacteria— isolated from a wide variety of foods and in some instances of animal origin—and the characteristics of bacteriocins. Special emphasis is placed on their potentials for use as food preservative and on their physicochemical nature, antibacterial spectrum, and genetic behavior.     

Biochemical and genetic evidence for production of enterocins A and B by Enterococcus faecium WHE 81

Enterococcus faecium WHE 81, isolated from cheese, has been reported to produce a bacteriocin called “enterocin 81” [J. Appl. Microbiol. 85 (1998) 521.]. Purification of “enterocin 81” was carried out using ammonium sulfate precipitation, desalting on ODP-90 reverse-phase column, and purification through SP Sepharose HP cation exchange and C₂/C₁₈ reverse-phase chromatographies. The antimicrobial was eluted from the C₂/C₁₈ column as four individually active fractions, designated A81, B81, C81 and D81. The purification procedure used proved particularly efficient for the bacteriocin in fraction D81, with a yield of 46%, while only 3.8% the bacteriocin in fraction B81 could be collected. MALDI-TOF mass spectrometry of the bacteriocins in fractions B81 and D81 showed respective masses of 4833.0 and 5462.2 Da. Amino acid sequencing of the two peptides revealed two class-II bacteriocins whose sequences were similar to those of enterocin A and enterocin B, respectively. Using proper primers, chromosomal fragments of 212 and 216 bp enclosing bacteriocin structural genes were PCR-amplified. Cloning of the amplicons and their sequencing revealed two genes with sequences identical to the structural genes of enterocins A and B, respectively. It was therefore clearly established that E. faecium WHE 81 produces bacteriocins respectively identical to enterocins A and B. Our results, combined with data from previous reports, suggest that the two bacteriocins may be widespread among enterococcal strains and may play an important role in controlling the growth of pathogens and other undesirable bacteria in certain fermented food products.     

Antibacterial activity of carnobacteria

This is the first report on bacteriocin production of the genusCarnobacterium. A total of 37 strains of carnobacteria were screened for antagonistic activities against other micro-organisms, including enterotoxigenic and pathogenic bacteria such asStaphylococcus aureus andListeria monocytogenes. Eighteen strains have been found to produce bacteriocins or bacteriocin-like substances primarily active against other carnobacteria. The cell-free culture supernatants exhibited inhibitory activity exclusively on bacteria closely related to the producers.L. monocytogenes was inhibited only by a supernatant that was concentrated tenfold. All other Gram-positive and Gram-negative bacteria tested in an agar well diffusion assay were not sensitive to the bacteriocins. The inhibitory activity was eliminated upon treatment with trypsin but was not affected by heating (10 min, 100°C). A bactericidal mode of action was demonstrated by usingCarnobacterium piscicola LV 61.     

Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants

Antimicrobial behavior of lactic acid bacteria (LAB) has been explored since many years to assess their ability to produce bacteriocin, a natural preservative, to increase the shelf life of food. This study aims to characterize bacteriocin producing strains of lactic acid bacteria isolated from acidic to slightly acidic raw vegetables including tomato, bell pepper and green chili and to investigate their potential to inhibit food related bacteria. Among twenty nine LAB screened for antimicrobial activity, three exhibited antagonism against closely related bacterial isolates which was influenced by varying temperature and pH. They were identified up to strain level as Lactococcus lactis subsp. lactis TI-4, L. lactis subsp. lactis CE-2 and L. lactis subsp. lactis PI-2 based on 16S rRNA gene sequence. Their spectrum of inhibition was observed against food associated strains of Bacillus subtilis and Staphylococcus aureus. Moreover, L. lactis subsp. lactis PI-2 selected on the basis of higher antimicrobial activity was further evaluated for bacteriocin production which was detected as nisin A and nisin Z. These findings suggest the possible use of L. lactis strains of vegetable origin as protective cultures in slightly acidic as well as slightly alkaline food by the bio-preservative action of bacteriocins.     

Preliminary characterization of bacteriocin-like substances from lactic acid bacteria isolated from organic leafy vegetables

This study presents the characterization of new strains of lactic acid bacteria (LAB) from organic vegetables. Forty-five strains of LAB isolated from vegetables were investigated by its antimicrobial activity against taxonomically related microorganisms. Genetic identification of selected LAB was performed by means of PCR method. These strains were Enterococcus faecium, Lactococcus lactis, Enterococcus hirae and Enterococcus canis. Bacteriocin-like substances were active against Gram-positive bacteria and Gram-negative foodborne pathogens (Listeria monocytogenes and Escherichia coli, respectively). The antimicrobial activity of LAB strains was inactivated by the addition of proteases, thus confirming the proteinaceous nature of the inhibition. In all four strains the bacteriocin activity was stable after extended refrigerated storage and freezing-thawing cycles. This fact suggests that bacteriocin produced by the four LAB strains may find application as biopreservatives in minimally processed vegetables.     

Antimicrobial‐Producing Lactic Acid Bacteria Isolated from Raw Barley and Sorghum

From a total of four thousand presumed lactic acid bacteria, obtained from raw, unmalted sorghum and barley, 308 isolates were shown to exhibit inhibitory activity against the indicator strain Listeria innocua 4202. Six of these inhibitor‐producing isolates were selected for further study on the basis of their relatively wide antimicrobial spectrum, which showed that these producers inhibited several Gram‐positive bacteria, including a range of beer spoiling bacteria. The proteinaceous nature, anti‐microbial activity against closely related species, heat resistance and pH stability of the inhibitory substances produced by these six bacteria identified these compounds as bacteriocins. All six isolates were shown to secrete the inhibitory compounds into the cell free supernatants. Bacteriocins produced by five of the six producers were purified to homogeneity. Further analytic data was obtained for three of the inhibitory compounds by means of mass spectroscopy and/or N‐terminal amino acid sequencing.     

Production of bacteriocins by <Emphasis Type="Italic">Enterococcus</Emphasis> spp. isolated from traditional,Iranian, raw milk cheeses,and detection of their encoding genes

Strong bacteriocins, or bacteriocins with a wide range of activity against pathogens and spoilage microorganisms, are actively sought for use as natural food preservatives. This work reports the inhibitory activity of 96 enterococcal isolates from two Iranian, raw milk cheeses against five indicator organisms (including Listeria innocua). Forty-eight isolates inhibited at least one indicator in spot agar assays. Of these, 20 isolates corresponding to 15 different strains were shown to produce bacteriocin-like substances in liquid cultures. PCR analysis revealed the genes coding for enterocins (enterococcal bacteriocins) A, B, P or X, or their combinations, in all but one of these 15 strains. In addition, the gene coding for enterocin 31 was detected in two strains. No amplification was obtained in one strain when using specific primers for all 13 bacteriocin genes sought. Three different enterocin genes were identified in most strains and four in one strain. Although the concomitant production of bacteriocins is still to be verified, producers of multiple enterocins could be of great technological potential as protective cultures in the cheese industry.     

Diversity of bacteriocins produced by lactic acid bacteria isolated from raw milk

Bacteriocin-producing lactic acid bacteria were isolated from 298 samples of raw ewes', goats’ or cows’ milk. Eighty-two bacteriocin producers were phenotypically and genotypically identified as L. lactis subsp. lactis (59 isolates), L. lactis subsp. cremoris (2 isolates), L. lactis subsp. lactis biovar diacetylactis (6 isolates), E. faecalis (7 isolates), E. faecium (1 isolate), L. paracasei subsp. paracasei (4 isolates), L. plantarum (1 isolate) and Leuconostoc spp. (2 isolates). By means of PCR-techniques, nisin was characterized in 39 of the 67 bacteriocin-producing lactococci and lacticin 481 in 23 isolates, some of which presented antilisterial activity. Enterocin AS-48 was produced by four enterococcal isolates. Four non-identified bacteriocins produced by 16 isolates showed a broad inhibitory spectrum. Nisin-producing lactic acid bacteria were the most abundant, but lacticin 481-producing lactococci and AS-48-producing enterococci were found at relatively high rates.     

Succession of dominant and antagonistic lactic acid bacteria in fermented cucumber: insights from a PCR-based approach

The goal of the investigation was to study the succession of major groups of lactic acid bacteria (LAB) and their antagonism in salt-fermented cucumber using PCR. In a direct detection method as well as a short enrichment process, PCR enabled detection of Leuconostoc and Lactobacillus during early hours of fermentation. Subsequently, Lactobacillus and Pediococcus emerged as the dominant genera. Nucleic acid sequence of culture-independent clones confirmed the detection of Pediococcus as a dominant genera emerging during late stages of fermentation. PCR also revealed time-dependent emergence of mesentericin, pediocin and plantaricin A producers and accounted for the LAB succession in the fermenting samples. A total of 328 LAB isolates were obtained collectively from 30 cucumber samples, of which PCR could identify an overwhelming 186 Lactobacillus isolates followed by 113 Pediococcus and 29 Leuconostoc isolates, respectively. Based on antimicrobial assay against target strain Leuconostoc mesenteroides NRRL B640, 28% of the LAB were bacteriocin producers, of which pediocin producers were substantial, followed by plantaricin A and mesentericin producers. The bacteriocins elaborated by the isolates were active against a large number of Gram-positive target LAB strains and pathogenic bacteria including Bacillus cereus, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.     

High‐Throughput Isolation of Bacteriocin‐Producing Lactic Acid Bacteria,with Potential Application in the Brewing Industry

Lactic acid bacteria (LAB) were isolated from malted cereals by means of a high‐throughput screening approach and investigated for antimicrobial activity against a range of beer‐spoiling bacteria. Putative bacteriocin‐producing strains were identified by 16S rRNA analysis and the inhibitory compounds were partially characterized. Following determination of the inhibitory spectra of the strains, an unspeciated Lactobacillus sp. UCC128, with inhibitory activity against a range of beer‐spoiling strains was subjected to further characterization. A bacteriocin was purified from this strain and analyzed by mass spectrometry to determine the weight of the protein. The result indicated that the bacteriocin was highly similar to pediocin AcH/PA‐1 from Pediococcus acidilactici. The bacteriocin‐producers identified in this study have the potential to be used in the brewing industry to enhance the microbiological stability of beer in conjunction with hurdles already in place in the brewing process.