Article

Sakacin B, a bacteriocin produced by Lactobacillus sake isolated from Greek dry fermented sausages

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Abstract

When Lactobacillus sake 251, a strain isolated from naturally fermented Greek dry sausage was grown in MRS broth it excreted an antimicrobial factor that differed from organic acids and hydrogen peroxide. The substance was proteinaceous, heat stable and inhibitory towards various lactic acid bacteria of meat origin. This suggested that a narrow spectrum bacteriocin, designated sakacin B, was present in the broth. Sakacin B displayed a bactericidal mode of action on sensitive cells without causing cell lysis. It was secreted during late logarithmic phase and was stable within a pH range 2 to 9. In vitro production of sakacin B by the producer strain in a mixed culture caused a strong biocidal effect on growing indicator cells. Sakacin B was partially purified and found not to contain unusual amino acids. That it was a hydrophobic peptide was confirmed by SDS-PAGE electrophoresis. The molecular weight of sakacin B was estimated to be 6.3 kDa.

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... Some authors have reported their ability also to inhibit Gram-negative species (Blackburn et al., 1989; Lewus et al., 1991a; Stevens et al., 1991; Kalchayand et al., 1992; Arihara et al., 1996). The mode of action of some bacteriocins has been identified, but for many others only a general description of the bacteriostatic (Lewus et al., 1991b; Thompson et al., 1996) or bactericidal effects (van Belkum et al., 1991; Venema et al., 1993; Schved et al., 1993; Samelis et al., 1994; Enan et al., 1996) has been reported. ...
... Some authors have reported their ability also to inhibit Gram-negative species (Blackburn et al., 1989; Lewus et al., 1991a; Stevens et al., 1991; Kalchayand et al., 1992; Arihara et al., 1996). The mode of action of some bacteriocins has been identified, but for many others only a general description of the bacteriostatic (Lewus et al., 1991b; Thompson et al., 1996) or bactericidal effects (van Belkum et al., 1991; Venema et al., 1993; Schved et al., 1993; Samelis et al., 1994; Enan et al., 1996) has been reported. In the arid zones of Algeria, the milk preservation factories are unavailable, hence most raw goat's or ewes' milk is made into cheese, using goat's rennet for coagulation. ...
Article
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Lactic acid bacteria (LAB) from raw goat and ewes' milk, from arid and semi-arid zones in west Algeria were tested for the production of antimicrobial substances against Staphylococcus aureus in milk. Twenty samples of raw milk were collected from which 96 LAB strains were isolated and exhibited antagonistic effects on solid agar medium. Two strains exhibited inhibition towards S. aureus from their supernatant culture medium. A Lactococcus lactis subsp lactis biovar diacelylactis (Lc8) strain proved by far the most efficient, produced a heat stable bactericid substance with a proteinaceous nature, suggesting a bacteriocin. Lc8 inhibited S. aureus. in milk, so that no cell was counted after 48h of incubation. This strain acts either by decreasing the pH or by bacteriocin production which makes it a good candidate strain in cheese making and useful in preventing growth of S. aureus.
... Conversely with what observed in L. reuteri that can produce different interfering molecules ranging from 100 to 2700 Da (see introduction) and in L. lactis where a large variety of bacteriocins occur such as lactococcins [73], lacticin [74] and nisin [60], L. sakei mainly produces sakacins. The first discovered was sakacin A [75] but later other very similar bacteriocins like sakacin B [76], sakacin K [77], sakacin G [78], the chromosomally-encoded sakacin T and sakacin X [79], sakacin P [80], sakacin LSJ618 [81] and sakacin C2 [82] have been described. ...
Article
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Lactic acid bacteria (LAB) potential in the food industry and in the biotechnological sector is a well-established interest. LAB potential in counteracting especially food-borne infections has received growing attention, but despite being a road full of promises is yet poorly explored. Furthermore, the ability of LAB to produce antimicrobial compounds, both by ribosomal synthesis and by decrypting them from proteins, is of high value when considering the growing impact of multidrug resistant strains. The antimicrobial potential of 14 food-derived lactic acid bacteria strains has been investigated in this study. Among them, four strains were able to counteract Listeria monocytogenes growth: Lactococcus lactis SN12 and L. lactis SN17 by high lactic acid production, whereas L. lactis 41FLL3 and Lactobacillus sakei I151 by Nisin Z and Sakacin P production, respectively. Strains Lactococcus lactis MG1363, Lactobacillus rhamnosus 17D10 and Lactobacillus helveticus 4D5 were tested and selected for their potential attitude to hydrolyze caseins. All the strains were able to release bioactive peptides with already known antimicrobial, antihypertensive and opioid activities. These features render these strains or their bioactive molecules suitable for use in food as biocontrol agents, or as nutraceutical supplements to treat mild disorders such as moderate hypertension and children insomnia. These results highlight once again that LAB potential in ensuring food safety, food nutraceutical value and ultimately in favoring human health is still underexplored and underexploited.
... This is indicated in the neutral supernatant inhibitory assay results, with the greatest inhibitory activity against S. thypimurium. The synthesis of bacteriocin by producing bacteria occurs during an exponential phase trip [20] which usually follows the pattern of synthesis of primary metabolites. This production system is governed by extracromosomal plasmids [21]. ...
Article
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Bacteriocin is a protein compound which has bactericidal ability against pathogen bacteria. This research aims to study the inhibitory activity of bacteriocin produced from Lactobacillus SCG 1223 against Listeria monocytogenes, Salmonella thypimuruim and Escherchia coli. The bacteriocin produce from Lactobacillus SCG 1223 in the MRS broth media The experimental design used was Completely Randomized Design. The variations used in this design were percentage of inoculum (5%, 10%), medium pH (4, 6), incubation temperature (27°C, 40°C), and incubation time (4, 10, 14 hours). Result showed that bacteriocin from Lactobacillus SCG 1223 had wide spectrum toward L. monocytogenes, S. thypimuruim and E. coli. The highest bacteriocin activity toward L. monocytogenes produced by Lactobacillus SCG 1223 with 10% inoculum in media with initial pH 6, incubation temperature 27°C for 14 hour, toward S. thypimurium produced by Lactobacillus SCG 1223 with in media with initial pH 6, incubation temperature 40°C for 14 hour, and toward E. coli was 1085.81 AU/ml, produced by Lactobacillus SCG 1223 in MRS broth with initial pH 4, incubation temperature 40°C for 14 hour. This study is expected to find a new food preservative that can inhibit the growth of pathogenic bacteria and extend the shelf life of food. From the economic prospective of view, bacteriocin is very promising natural alternative biopreservatives.
... For example, the anionic peptide lactocin MXJ32 successfully bound to the cation-exchange column at pH 3.0 (Lü et al. 2014). Additionally, the anionic peptide sakacin B was purified by cation-exchange column at pH 5.2 (Samelis et al. 1994). Although a high purity enterocin T1 was obtained, the final recovery of peptide was quite low. ...
Article
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An enterocin-producing Enterococcus faecium T1 was isolated from Chinese Tibet cheese. The enterocin was purified by SP-Sepharose and reversed phase HPLC. It was identified as unique from other reported bacteriocins based on molecular weight (4629 Da) and amino acid compositions; therefore it was subsequently named enterocin T1. Enterocin T1 was stable at 80–100 °C and over a wide pH range, pH 3.0–10.0. Protease sensitivity was observed to trypsin, pepsin, papain, proteinase K, and pronase E. Importantly, enterocin T1 was observed to inhibit the growth of numerous Gram-negative and Gram-positive bacteria including Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Escherichia coli, Salmonella typhimurium, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Listeria monocytogenes. Take together, these results suggest that enterocin T1 is a novel bacteriocin with the potential to be used as a bio-preservative to control Pseudomonas spp. in food.
... Some authors have reported their ability also to inhibit Gram-negative species (Blackburn et al., 1989; Lewus et al., 1991a; Stevens et al., 1991; Kalchayand et al., 1992; Arihara et al., 1996). The mode of action of some bacteriocins has been identified, but for many others only a general description of the bacteriostatic (Lewus et al., 1991b; Thompson et al., 1996) or bactericidal effects (van Belkum et al., 1991; Venema et al., 1993; Schved et al., 1993; Samelis et al., 1994; Enan et al., 1996) has been reported. In the arid zones of Algeria, the milk preservation factories are unavailable, hence most raw goat's or ewes' milk is made into cheese, using goat's rennet for coagulation. ...
... The interactions of the induction factor with the food matrix (such as adsorption or inactivation) may have a great influence on bacteriocins production. On the other hand, the food matrix may also facilitate the concentration of the induction factor around cells or micro-colonies formed by the bacteriocinogenic culture in the food [18,51,52,126]. ...
Article
The interest on novel biological preservation methods has been increasing during recent years, supported by research indicating that antagonistic microorganisms and their antimicrobial metabolites may have some potential use as natural preservatives as a way not only to control the growth but also to inactivate undesired microorganisms in food. Biopreservation using lactic acid bacteria (LAB) and/or their antimicrobial metabolites represents an alternative for improving food safety. These antimicrobial properties of LAB were derived from competition for nutrients and the production of one or more antimicrobial active metabolites such as organic acids (mainly lactic and acetic acid), hydrogen peroxide and also other compounds, such as bacteriocins and antifungal peptides. The important contribution of probiotic LAB in food preservation has been attracting much attention because of the nutritional qualities of the raw material through an extended shelf life of food and their ability to inhibit spoilage and foodborne pathogens, which is interesting for the food industry. In this review, we compiled the available data on the commonly used preservative and present the current knowledge regarding the antimicrobial compounds, especially bacteriocins, the mechanisms of the action and recent applications of antimicrobial compounds in food. The use of competitive microbiota as a biotechnological tool for food preservation may lead to improve the optimization and quality assurance of food products while at the same time retaining the sensory qualities of the product such as color, flavor, texture and nutritional value.
... Up to now, several bacteriocins are known to be produced by L. sakei strains and their potential application in meat preservation has been studied. Examples are: curvacin A (Tichaczek, Nisen-Meyer, Nes, Vogel, & Hammes, 1992) identical to sakacin A (Shillinger & Lücke, 1989); sakacin P (Aasen et al., 2003;Tichaczek, Vogel, & Hammes, 1994;Urso, Rantsiou, Cantoni, Comi, & Cocolin, 2006) identical to bavaricin A (Larsen, Vogensen, & Josephsen, 1993); sakacin 674 (Holck, Axelsson, Hühne, & Kröckel, 1994), sakacin B (Samelis, Roller, & Metaxopolos, 1994), sakacin K (Hugas, Pages, Garriga, & Monfort, 1998), sakacin V18 (Cintas, Casaus, Fernandez, & Hernandez, 1998) and sakacin M (Sobrino et al., 1992) identical to lactocin S (Mortvedt, Nissen-Meyer, Sletten, & Nes, 1991); bavaricin MN (Kaiser & Montville, 1996), sakacin T (Aymerich et al., 1996), sakacin G (Simon, Fremaux, Cenatiempo, & Berjeaud, 2002), sakacin X (Vaughan, Eijsink, & Van Sinderen, 2003), sakacin Q (Mathiesen, Huehne, Kroeckel, Axelsson, & Eijsink, 2005) and sakacin 1 (Alves, Martinez, Lavrador, & De Martinis, 2006). All sakacins possess strong antilisterial activity and most of them belong to the Class IIa bacteriocins (Klaenhammer, 1988). ...
Article
Lactobacillus sakei ST22Ch, ST153Ch and ST154Ch were isolated from traditional pork product from Northwest of Portugal, and identified based on API50CHL, PCR with specific primers and 16s rDNA sequencing. RAPD-PCR analysis showed significant differences between isolates. The selected isolates inhibited the growth of Enterococcus spp., Listeria spp., Escherichia coli, Klebsiella spp., Pseudomonas spp., Staphylococcus spp., and Streptococcus spp. The mode of action of the bacteriocins was bactericidal, as observed against Enterococcus faecium. A reduction in antimicrobial activity was recorded after treatment of the bacteriocins with proteolytic enzymes, but not when they were exposed in presence of α-amylase, suggesting that they are not glycosylated. Maximal activity of bacteriocins was recorded during the early stationary phase and remained stable only for a short period, followed by a decrease. According to tricine/SDS-PAGE, the size of bacteriocins ST22Ch, ST153Ch and ST154Ch are approximately 3.0 kDa, 10.0 kDa and 3.0 kDa, respectively. Bacteriocins were heat tolerant and remained active after 2 h at 100 °C. Activity of bacteriocins was not affected by treatment with 1% Triton X-100, Tween 20, Tween 80, SDS, NaCl, urea and EDTA. In presence of 1% Triton X-114 bacteriocins were inactivated. PCR reactions targeting genes for enterocin A, enterocin P, sakacin P, sakacin G1 and sakacin G2 in the total DNA of L. sakei ST22Ch, ST153Ch and ST154Ch, generated positive results. Curvacin A gene was detected only in L. sakei ST154Ch DNA.
... For example, Lact. sake LV251 was recently found to produce a typical bacteriocin, sakacin B (Samelis et al. 1994b); however, its protein profile was identical to all other bacteriocin-negative Lact. sake strains (Fig. 2). ...
Article
J. SAMELIS, E. TSAKALIDOU, J. METAXOPOULOS AND G. KALANTZOPOULOS. 1995. To confirm the phenotypic characterization of 169 predominant sausage isolates of the Lactobacillus sake/curvatus group, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins of 29 representative strains was undertaken. The results of the electrophoretic analysis indicated that each species yielded a specific protein profile, identical to the respective type strain. It was shown that all melibiose-positive isolates belonged to Lact. sake, whether or not they were arginine- and maltose-positive, whereas all melibiose- and arginine-negative isolates were assigned to Lact. curvatus. A group which phenotypically appeared to lie between Lact. sake and Lact. curvatus, on the basis of arginine (+), melibiose (-) and maltose (-) reactions, was shown to be more closely related to Lact. sake. The SDS-PAGE method was valuable in distinguishing between species, but did not allow further differentiation of Lact. curvatus or Lact. sake strains displaying high heterogeneity in secondary physiological and biochemical properties.
... Enterococcal isolates were screened for antilisterial activity by a well diffusion assay (sAMELis et al., 1994b) that was slightly modified as follows: all isolates were grown in Mrs broth at 30°c; all indicator strains (table 2) were grown in brain Heart infusion (bHi) broth (LAb M, bury, Lancashire, uK) at 30°c for 18-22 h; tsAYE plates inoculated (0.2% v/v) with each of the indicator strains were used for the assay; on each plate the 6-mm diameter wells were filled with 50-60 µL of filter-sterilised Mrs culture supernatants of each isolate. initially, all isolates were tested against two indicator strains of Listeria monocytogenes, scott A and N-7143. ...
Article
A total of 86 enterococcal isolates from fully ripened Graviera, a traditional hard Greek cheese made from thermized (60°C; 30 sec) ewe/goat (90:10) milk without the use of commercial starters, were identified using phenotypic traits and sequencing of the 16S rDNA gene of representative strains. The antimicrobial potential of the isolates was also assessed. The en-terococci levels in the ripened cheeses were 7.8±0.2 log CFU/g. Enterococcus faecium (41 isolates) and E. durans (35 isolates) were the most prevalent species which share a 99% 16S rDNA homology. Five enterocin-producing (Ent +) E. faecium isolates inhibited in vitro growth of L. monocytogenes. None of the E. durans strains or other isolates produced enterocins. These results suggest that some Ent+ E. faecium strains may contribute to Listeria inhibition in Graviera cheese during ripening. However, before these isolates can be used as bio-protective adjuncts, their overall safety must be assessed in order to ensure a GRAS status at the E. faecium strain level.
... While the activity was detected clearly after electrophoresis, no bands were visible at all after Coomassie blue staining, or one diuse band was seen at the position of the inhibition zone. Similar observations were reported for sakacin B puri®cation (Samelis et al. 1994), where staining with Coomassie blue failed to develop a band at the position where inhibition of the indicator lawn was detected, when crude sakacin B was applied on a gel. Also lactacin F could not be detected by Coomassie dye staining (Muriana and Klaenhammer 1991a). ...
Article
Full-text available
Lactobacillus acidophilus LF221 produced bacteriocin-like activity against different bacteria including some pathogenic and food-spoilage species. Besides some lactic acid bacteria, the following species were inhibited: Bacillus cereus, Clostridium sp., Listeria innocua, Staphylococcus aureus, Streptococcus D. L. acidophilus LF221 produced at least two bacteriocins, acidocin LF221 A and acidocin LF221 B, which were purified by ammonium sulphate precipitation, ion-exchange chromatography, hydrophobic interaction and reverse-phase FPLC. The antibacterial substances were heat-stable, sensitive to proteolytic enzymes (trypsin, pepsin, pronase, proteinase K) and migrated as 3500- to 5000-Da proteins on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The sequences of 46 amino-terminal amino acid residues of peptide A and 35 of peptide B were determined. Among the residues identified, no modified amino acids were found. No significant homology was found between the amino acid sequences of acidocin LF221 A and other bacteriocins of lactic acid bacteria and 26% homology was found between acidocin LF221 B and brevicin 27. L. acidophilus LF221 may be of interest as a probiotic strain because of its human origin and inhibition of pathogenic bacteria, especially Clostridium difficile.
... When the same amount of partially-puri®ed bacteriocin preparation, suf®cient for detection of activity (25 ml), was applied to the part of the gel that was stained after electrophoresis, no visible bands were observed at the position of the inhibition zone. Inability of Coomassie Brilliant Blue to stain the active band has also been reported by workers handling other small-size bacteriocins such as lactacin F (Muriana and Klaenhammer 1991), carnosin LA44A (Van Laack et al. 1992), sakacin B (Samelis et al. 1994) and lacticin 481 (Piard et al. 1992). The puri®ed gassericin KT7 from the ®nal puri®cation step by HPLC reverse-phase chromatography was used for analysis of amino acid composition. ...
Article
A bacteriocin-producing Lactobacillus gasseri strain, KT7, was isolated from infant faeces. The supernatant fluid showed inhibitory activity not only against some lactic acid bacteria but also, against some pathogenic and food-spoilage species, including Clostridium, Listeria and Enterococcus. An antimicrobial peptide designated gassericin KT7 was isolated from Lactobacillus gasseri KT7. It was purified to homogeneity by a single four-step procedure: a crude supernatant fluid obtained from early stationary-phase culture in MRS medium was subjected to ammonium sulphate fractionation, CM-Sephadex cation-exchange chromatography, Phenyl-Sepharose hydrophobic chromatography and reverse-phase HPLC chromatography. Gassericin KT7 was sensitive to proteolytic enzymes, resistant to heat, active over a wide range of pH, and migrated as a 4.5-5.0 kDa peptide on SDS-PAGE. The bacteriocin was produced constitutively during exponential growth. It was bactericidal to sensitive cells and the bactericidal effect was not produced by cell lysis. The amino acid composition of the bacteriocin was determined and no modified amino acid was found among the residues identified.
... The growth values of this food borne pathogen were not influenced by plantaricin UGI negative variant. Similar results were published by Schillinger et al. and Samelis et al. [16,17]. ...
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The inhibition of Bacillus cereus ATCC 14579 viable growth by Lactobacillus plantarum UG1 bacteriocin (plantaricin UG1) in vitro and in food (pasteurized milk and minced meat) was studied. The inhibitory effect against B. cereus food-borne pathogen noticed in this study was due to plantaricin UG1, but not due to lactic acid produced by the L. plantarum UG1 culture. Plataricin UG1 negative clone did not affect viable growth of B. cereus in both broth and meat or pasteurized milk. The inhibitory effect of L. plantarum UG1 and its bacteriocion was apparently more in liquid systems (BHI broth & pasteurized milk) than in minced meat. The inhibitory effect of plantaricin UG1 against B. cereus was dependent on its concentration. The 22880 AU/ml concentration appeared to be an ideal preservative against B. cereus ATCC 14579 in liquid systems.
... curvatus often contain bacteriocin-forming strains (Hammes and Hertel, 1996). Up to now, several bacteriocins are known to be produced by Lb. sakei strains: sakacin A (Schillinger and Lücke, 1989), sakacin M (Sobrino et al., 1992), sakacin P (Tichaczek et al., 1992), sakacin 674 (Holck et al., 1994), sakacin B (Samelis et al., 1994), sakacin K (Hugas et al., 1995), and sakacin T (Aymerich et al., 2000). All sakacins possess strong antilisterial activity. ...
Article
Lactobacillus curvatus LTH 1174, a strain isolated from fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of temperature (20-38 degrees C) and pH (4.8-7.0) were investigated in vitro during laboratory fermentations using de Man, Rogosa and Sharpe (MRS) medium. A predictive, successfully validated model was set up to describe the influence of temperature and pH on the microbial behavior. Both cell growth and bacteriocin activity were influenced by changes in temperature and pH. The optimum temperature value for cell growth, 34.5 degrees C, did not correspond with the optimum temperature for curvacin A production (20-27 degrees C). Interestingly, the pH range for growth and curvacin A production was broad. Thus, Lb. curvatus LTH 1174 seems to be a promising bacteriocin-producing strain for use in European sausage fermentations that are performed at temperatures near 25 degrees C.
... Lactobacilli sausage isolates frequently produce bacteriocins or bacteriocin-like compounds, as has been shown for Lb. sakei (Sobrino et al., 1991;Tichaczek et al., 1992;Garriga et al., 1993;Samelis et al., 1994a;De Martinis and Franco, 1998;Aymerich et al., 2000b;Rosa et al., 2002;Tantillo et al., 2002), Lb. curvatus (Tichaczek et al., 1992;Sudirman et al., 1993;Mataragas et al., 2002), Lb. plantarum Rekhif et al., 1995;Enan et al., 1996;Aymerich et al., 2000b;Messi et al., 2001), Lb. brevis (Benoit et al., 1994), and Lb. casei (Vignolo et al., 1993). ...
Article
Starter cultures that initiate rapid acidification of the raw meat batter and that lead to a desirable sensory quality of the end-product are used for the production of fermented sausages. Recently, the use of new, functional starter cultures with an industrially or nutritionally important functionality is being explored. Functional starter cultures offer an additional functionality compared to classical starter cultures and represent a way of improving and optimising the sausage fermentation process and achieving tastier, safer, and healthier products. Examples include microorganisms that generate aroma compounds, health-promoting molecules, bacteriocins or other antimicrobials, contribute to cured meat colour, possess probiotic qualities, or lack negative properties such as the production of biogenic amines and toxic compounds. The vast quantity of artisan fermented sausages from different origins represents a treasure chest of biodiversity that can be exploited to create such functional starter cultures.
... Moreover, L. sakei and Lactobacillus curvatus isolates from meat often contain bacteriocinogenic strains. Up to now, several bacteriocins are known to be produced by L. sakei strains: sakacin A (Schillinger and Lucke, 1989), sakacin M (Sobrino et al., 1992), sakacin P (Tichaczek et al., 1994), sakacin 674 (Holck et al., 1994), sakacin B (Samelis et al., 1994), sakacin K (Hugas et al., 1995), Lactocin S (Skaugen et al., 1997), bavaricin MN (Kaiser and Montville, 1996), sakacin T (Aymerich et al., 2000), sakacin G (Simon et al., 2002), sakacin X (Vaughan et al., 2003) and sakacin Q (Mathiesen et al., 2005). All sakacins possess strong antilisterial activity. ...
Article
Bacteriocinogenic bacteria have been proposed to protect food products from Listeria contamination as bioprotective cultures. Lactobacillus sakei 2512 was demonstrated to inhibit the growth of Listeria on sliced cooked ham by challenge test. A liquid medium simulating ham, BHI5L200, was designed in order to select bioprotective strains for meat protection. Two strains were selected, from the 201 lactic acid bacteria screened, that produced bacteriocins at pH 5.8 in BHI5L200. The first one, Leuconostoc pseudomesenteroides 2733, produced a new bacteriocin which was purified and partially characterized. The second, Lactobacillus curvatus 2711, produced sakacin X and was shown to contain sakacin T and sakacin P structural genes. Co-culture experiments in BHI5L200 demonstrated that growth of Listeria was inhibited by L. sakei 2512 as well as by L. curvatus 2711.
Chapter
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Lactic acid bacteria (LABs) are Gram-positive, nonspore producing cocci or rods, catalase-negative, fastidious organisms with a poor pH tolerance (Kaban & Kaya, 2008). LABs are one of the most significant microorganisms utilized in food fermentations and for improving the flavour and texture of fermented foods (Hati, Mandal, & Prajapati, 2013). They are distinguished by the formation of lactic acid as the primary product from glucose, as well as the presence of growth inhibitors, such as bacteriocins, hydrogen peroxide, diacyl, and other compounds that hinder the expansion of food spoilage bacteria and pathogens (Alakomi et al., 2000). Lactic acid is a key end-product of certain bacteria, such as Bifidobacteria. Bifidobacterium belongs to the actinomycetes, whereas LAB belongs to the phylum Firmicutes of Gram-positive bacteria, which is linked to the class Bacilli. The G 1 C concentration of LAB DNA is low (König & Fröhlich, 2009).
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Latilactobacillus sakei is used extensively in industrial production and food fermentations. The species is primarily derived from fermented meat and vegetable products and is also found in human feces. Genomics and metabolomics have revealed unique metabolic pathways in L. sakei and molecular mechanisms underlying its competitive advantages in different habitats, which are mostly attributed to its flexible carbohydrate metabolism, cold tolerance, acid and salt tolerance, ability to cope with oxygen changes, and heme uptake. In recent years, probiotic effects of L. sakei and its metabolites have been identified, including the ability to effectively alleviate metabolic syndrome, inflammatory bowel disease, and atopic dermatitis. This review summarizes the genomic and metabolic characteristics of L. sakei and its metabolites and describes their applications, laying a foundation for their expanded use across the food and healthcare industries.
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A bacteriocin-producing lactic acid bacteria was isolated from Kimchi on MRS selective media with the use of Lactobacillus delbrueckii subsp. delbrueckii as an indicator strain. The strain YH-10 was identified as Lactococcus lactis subsp. lactis through the API test. The crude bacteriocin (freeze-dried 50% ammonium sulfate precipitate of culture supernatant) produced by the strain was named as lacticin YH-10. Lacticin YH-10 showed the growth inhibitory activity against Gram positive pathogenic bacteria and other lactic acid bacteria. The bacteriocin was inactivated by proteases such as protamex and aroase AP-10 and partially inactivated by amylase, proteinase K, trypsin, and papain. The lacticin YH-10 remained its activity with the treatment of heat at 100^{\circ}C for 60 min or the changes of pH 2 to 11. However, the activity was lost at high pH combined with the exposure to 100^{\circ}C. The bacteriocin production of the strain was started in the exponential phase and stopped in the stationary phase. The approximate molecular mass of the bacteriocin produced by the strain was approximate 14 kDa in the analysis on SDS-PAGE.
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Background: The performances of four autochthonous isolates of Lactobacillus plantarum were assessed to study the most important variables acting on acidification and propose a possible step-by-step approach for the validation at lab-scale. This main topic was addressed through three intermediate steps: (i) evaluation of acidification in liquid and solid media, as a function of salt, nitrites, nitrates, lactose, pepper and temperature; (ii) assessing acidification in a pork-meat preparation; iii) designing a protocol to improve the performances at sub-optimal temperatures. The concentration of the ingredients and the temperature were combined through a 3(k-p) Fractional Factorial Design. Acidification and viable count were assessed and modeled through a multi-factorial ANOVA. Results: In model systems acidification was affected by lactose and was maximum (ΔpH of ca. 2.8-3.0) in the combinations containing 0.4% lactose, 250 mg/kg nitrates or 150 mg/kg nitrites, 5% salt, and at 30 °C. Solid media caused a higher acidification. In the pork meat preparation, the effect of salt and nitrites was significant. At 10 °C the strains could not reduce pH, but this ability could be induced using an adaptation step. Conclusions: Acidification was affected by lactose in the model system, whereas in meat preparation the other variables were significant. In addition, a protocol to improve acidification at 10 °C was optimized.
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As consumer trends shift toward natural preservatives, it is becoming increasingly necessary to identify antimicrobials that can be used in food products to decrease proliferation of spoilage and pathogenic bacteria. The data presented identifies a novel plantaricin Q7. Lactic acid bacteria strains were isolated from food and of the 118 colonies that were screened, only one produced an inhibitory effect against Pseudomonas fluorescens AS 1.1802. Molecular and biochemical methods indicated this bacterium was Lactobacillus plantarum. Plantaricin Q7 was purified by absorption-desorption, CM-Fast Flow and Superdex Peptide 10/300 GL. Plantaricin Q7 became inactive following treatment with proteolytic enzyme, including proteinase K, pepsin, trypsin, papain, and pronase E. The plantaricin Q7 remained active following incubation at 121 °C for 20 min and following incubation pH ranging from 3.0 to 12.0 for 30 min. Pseudomonas putida AS1.1819, Pseudomonas aeruginosa CICC21636, Listeria monocytogenes ATCC19111, Escherichia coli ATCC25922, Salmonella typhimurium ATCC14028, Staphylococcus aureus, Shigella flexneri ATCC12022 and Shigella sonnei ATCC25931 were all sensitive to plantaricin Q7. The data presented suggests plantaricin Q7 can be applied to food products to inhibit the growth of numerous food spoilage bacteria and pathogens.
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Probiotic bacteria are defined as “live microbial feed supplement” that beneficially affect the host by improving intestinal microbial balance (1). Most of the probiotic strains are natural inhabitants of the intestines of humans and animals (2). The potential health benefits of probiotics have produced considerable interest in the U.S. with respect to the use of probiotics in food, pharmaceutical, and feed product applications (3–8). Currently, most probiotic bacteria are cultivated from the genera Lactobacillus and Bifidobacterium (9), but Enterococcus spp., Bacillus spp., and Streptococcus spp. also have great potential.
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Two strains of lactic acid bacteria, isolated from "lingüiça" (a typical Brazilian meat product) stored under refrigeration, produced antagonistic substances active against selected foodborne pathogens. The proteinaceous nature of the inhibitors was demonstrated and so they were classified as bacteriocins. The inhibition due to acid production and phages was ruled out. The bacteriocin produced by Leuconostoc sp was active against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. The bacteriocin produced by Lactobacillus sake was active against Listeria monocytogenes and Staphylococcus aureus. Gram negative bacteria were not inhibited by the bacteriocins produced.
Chapter
The increasing demands by consumers for foods and beverages prepared with natural preservatives, rather than with chemicals, has led to increased research into natural antimicrobials suitable for such use. A wide array of natural antimicrobials from various sources such as microorganisms, animals and plants has been investigated as potential preservatives. Some - such as nisin, natamycin, lysozyme and the lactoperoxidase system - are now playing a role in food and beverage preservative systems. Others are under evaluation. The use of single antimicrobials rarely provides an adequate effect, so most potential will be in the use of combinations of antimicrobials. Despite being natural, most natural additives and ingredients used as food preservatives will still require safety evaluation and legislation.
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The main purpose of this work is to give an overview on the chemical engineering aspects related with the production of probiotic cultures and bacteriocins. Firstly, some evidence of the potential of different Lactococci and Lactobacilli strains for prevention or treatment of different human diseases, or as growth promoters in farm animals is given. In addition, examples of different commercial dietary supplements containing probiotic lactoccocci and lactobacilli in combination or not with other probiotic bacteria are presented. Secondly, the main factors (cultivation method, culture media, and media composition) affecting the production of bacteriocins by lactoccocci and lactobacilli strains are showed. Examples of modelling procedures developed to describe the production of higher concentrations of biomass and bacteriocins in fed-batch cultures subjected or not to successive alkalizations are summarized. Finally, the corresponding mass balance equations performed in the latter cultures are presented to calculate the volumes of feeding substrates and the concentrations of nutrients (lactose, glucose, proteins, nitrogen, and phosphorous) added to the fermentation medium, as well as the concentrations of biomass and products in the fermentor just after each feeding.
Article
A novel bacteriocin, sakacin LSJ618, produced by the strain Lactobacillus sakei LSJ618 isolated from traditional Chinese fermented radish, was studied. L. sakei LSJ618 was identified by both phenotypical and physiological tests combined with 16S rDNA sequence analysis. Sakacin LSJ618 is sensitive to hydrolytic enzymes including lipase, is stable between pH 2–8, and is heat resistant (30 min at 121 °C). Sakacin LSJ618 exhibits inhibitory activity against food-spoiling bacteria and food-borne pathogens, including the Gram-positive Listeria monocytogenes, Staphylococcus aureus, Sarcina sp., Micrococcus luteus, and the Gram-negative Proteus sp. and Escherichia coli, but not against most of the lactic acid bacteria tested. Maximal production of bacteriocin was reached in the late stationary phase, and inhibitory activity declined within 26 h. The mode of action of sakacin LSJ618 was determined to be bactericidal, as evidenced by its action upon Micrococcus tetragenus. After partial purification by ammonium sulfate precipitation and Sephadex G-25 chromatography, the molecular weight of sakacin LSJ618 was determined to be 5.2 kDa by Tricine-SDS-PAGE. The identified properties of sakacin LSJ618 indicate that it is a novel bacteriocin with potential application as a biopreservative in the food industry.
Book
Ingredients in Meat Products presents the most up-to-date information regarding the utilization of non-meat ingredients in the manufacturing of processed meat products, in a way that is both comprehensive and practical. Emphasis has been placed on helping the reader attain a fundamental understanding of: (i) the basic chemical and physical properties of each of these groups of ingredients, as we understand them today; (ii) how these properties affect their functionality in meat systems; and (iii) how to take advantage of the ingredients' functional properties to maximize their application in real-life situations. Drawing on the knowledge and expertise of twenty expert contributors from North America and Europe, representing both academia and industry, the volume discusses all the major types of ingredients used in processed meat products today, both in the context of their traditional uses, as well as, when applicable, in the context of more novel-and in many instances commercially unexploited-applications. New, "next-generation, " ingredients are also discussed Ingredients in Meat Products is intended as a primary reference on the subject for university, industry, and government meat science researchers; graduate and undergraduate students with an interest in the topic; and meat and food industry product development, quality, production and marketing personnel. © Springer Science + Business Media, LLC 2009. All rights reserved.
Article
At a time when consumers are demanding the partial or complete removal of chemically synthesized preservatives from foods, there is also an increased demand for convenience foods with long shelf-lives. These consumer-led trends have fuelled a renewed interest in the development of ‘more natural’ preservatives for extending the shelf-life and maintaining the safety of foods. Although the antimicrobial properties of many compounds from plant, animal and microbial sources have been reported, their potential for use as natural food preservatives has not been fully exploited. In this paper, the possible uses of natural antimicrobial compounds as food preservatives, used either singly or in combination, are explored. Specific examples are given from a current transnational research project on Natural Antimicrobial Systems sponsored jointly by the European Commission and a consortium of eight food companies. The results of trials with a range of potential natural preservatives including lytic enzymes, bacteriocins from lactic acid bacteria and plant antimicrobials in laboratory media and in a variety of foods and beverages including apple juice, milk, hard-cooked cheese (Emmental) and fresh fruit slices are discussed.
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Several molecular taxonomic studies have revealed that many natural (wild) Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. lactis subspecies lactis cluster genotypically within subspecies cremoris and vice versa. Recently, we isolated two wild nisin-producing (Nis+) L. lactis strains, M78 and M104, of the lactis phenotype from Greek raw milk (J. Samelis, A. Lianou, A. Kakouri, C. Delbès, I. Rogelj, B. B. Matijašic, and M. C. Montel, J. Food Prot. 72:783–790, 2009); strain M78 possess a novel nisin A sequence (GenBank accession number HM219853). In this study, the actual subspecies identity of M78 and M104 isolates was elucidated, using 16S rRNA and acmA (encoding lactococcal N-acetylmuramidase) gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh (encoding lactate dehydrogenase) gene phylogenies. Except the acmA gene analysis, molecular tools revealed that isolates M78 and M104 clustered with strains of the cremoris genotype, including the LMG 6897T strain, while they were distant from strains of the lactis genotype, including the LMG 6890T strain. The two wild isolates had identical repetitive sequence-based PCR (rep-PCR), randomly amplified polymorphic DNA (RAPD), plasmid, and whole-cell protein profiles and shared high 16S rRNA (99.9%) and ldh (100%) gene sequence homologies. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG 6890T strain. To our knowledge, this is the first complete identification report on a wild L. lactis subsp. cremoris genotype of the lactis phenotype which is capable of nisin A production and, thus, has strong potential for use as a novel dairy starter and/or protective culture.
Chapter
The distinct sensory qualities and remarkable shelf-life characteristics of fermented sausages, as compared to cooked sausages, are largely due to acidification of the meat batter. Traditionally, acidification of the raw meat is the result of a microbial fermentation process, i.e. lactic acid production by lactic acid bacteria (LAB). Acidification is generally combined with protection from oxygen (stuffing into casings), extensive salting, and curing (Chap. 1), and with an ageing stage for product maturation. The latter stage can be absent, short, or long, depending on the type of product, and leads to drying, resulting in a lower water activity, as well as to a complex and desired flavor formation (Campbell-Platt & Cook, 1995; Lücke, 1998). Sometimes, smoking or heating is applied as a last step in the manufacturing process. Heating is common in the United States, where regulations require a core temperature of 58.3°C before selling the end-product (Lücke, 1998). The almost anaerobic environment and the low pH and water activity values that prevail in the sausage are to be considered as the main hurdles that inhibit undesirable microbial growth and lead to a relatively stable end-product. In the case of spontaneously fermented sausage or sausage prepared through back-slopping, LAB that cause acidification of the meat and, hence, start the fermentation process, originate from the raw material or production environment. However, LAB can also be added deliberately by the sausage manufacturer as a starter culture to the meat batter (Campbell-Platt & Cook, 1995; Hugas & Monfort, 1997). In contrast to spontaneous fermentation, where the manufacturer relies on the presence of a "house microbiota" (counts of 10 2 -10 3 LAB per gram of fresh batter), the addition of a starter culture leads to high initial LAB counts (10 6 -10 7 per gram of fresh batter). This enhances acidification, leads to a more standardized and predictable production process, shortens the development of firmness and the overall ripening time, and improves food safety (Lücke, 1998). As an alternative to the use of LAB starter cultures, some manufacturers prefer to apply chemical acidulants, mainly to shorten the production process. Best results have been obtained with glucono-delta-lactone (GdL) (Barbut, 2006). However, a disadvantage of chemical acidulants is that they generally induce rapid and poorly controlled acidification, leading to inhibition of flavor development. The present chapter focuses on fermentation and acidification ingredients that are applied in sausage technology. In particular, it will discuss starter cultures for sausage fermentation, including both classical and novel, functional starter cultures, as well as chemical acidulants.
Article
Two major aspects of fermented sausages are discussed in the article. After a brief history of enterohemorrhagicEscherichia coli with special regard to fermented meat products the basic experimental designs and results are tackled on the basis of which safety issue in this respect is thoroughly discussed. The results show a rather limited opportunity for meeting food safety requirements with short or medium time ripened raw fermented sausages while traditionally, long ripened sausages are in a much better position. Fermented meat products as probiotics are also discussed. After evolving the EHEC outbreaks caused by fermented sausages the possibilities for finding reliable methods and products have been narrowed and more research is needed to find optimum solution.
Article
Strain R1333, isolated from commercially available smoked salmon, was identified as Lactobacillus sakei based on biochemical tests, sugar fermentation reactions (API 50 CHL), PCR with species-specific primers and sequencing of the 16S rRNA gene. Strain R1333 produces a 3811 kDa class IIa bacteriocin, active against Streptococcus caprinus, Streptococcus macedonicus, Streptococcus spp., L. sakei, Lactococcus lactis subsp. lactis, Listeria innocua, Listeria ivanovii subsp. ivanovii and Listeria monocytogenes. The mode of activity against L. innocua 2030C and L. ivanovii subsp. ivanovii ATCC 19119 was bactericidal, resulting in cell lysis and enzyme- and DNA-leakage. The highest level of activity (1600 AU/mL) was recorded when cells were grown at 30°C in MRS broth (initial pH 6.5). Only 800 AU/mL was recorded when strain R1333 was grown in MRS without Tween 80. Lower levels of bacteriocin production were recorded when strain R1333 was grown in MRS at 20°C. Peptide R1333 adsorbs at low levels (200 AU/mL) to producer cells. Purification of bacteriocin R1333 was performed by 60% ammonium sulfate precipitation, followed by separation on a SepPak C(18) column and reverse-phase HPLC on a Nucleosil C(18) column with a linear gradient from 0.1% TFA to 90% acetonitryl. A molecular mass of 3811 kDa was determined by mass spectrometry. Based on mass spectrometry and sequencing of the PCR amplified fragment targeting the sakG gene, L. sakei R1333 is a potential producer of sakacin G. This is the first report of the identification of sakacin G produced by L. sakei isolated from smoked salmon.
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Within the project “Safety of traditional fermented sausages: Research on protective cultures and bacteriocins” the task of Work Package 5 (WP5) was to produce traditional fermented sausages inoculated with pathogens using selected protective cultures and/or bacteriocins. As a result from previous work packages, three strains of Lactobacillus sakei (I-151, I-154 and I-155) showed bacteriocin-based antimicrobial activity against Listeria monocytogenes only. The Task 5.1. was production of fermented sausages inoculated with L. monocytogenes, together with the three selected protective cultures. Three batches of the indigenous sausages were prepared according to original recipes. For each batch four groups of sausages were produced: a positive control group (sausages inoculated only with L. monocytogenes), and three groups of sausages inoculated with L. monocytogenes and one of the three protective strains of Lb. sakei (I-151, I-154 and I-155). A slight advantage of the strain I-151 over the other two was observed in antilisterial activity, which may suggests that the three investigated strains represented just three isolates of the same bacteria. This hypothesis was supported by results of genetic investigation of bacteriocin determinants of the isolates carried out in WP3, where it was verified that all the Italian strains are subclones of the same population. Having in mind these results, it may be suggested to use only Lb. sakei I-151 as a protective starter culture in further experimental investigation.
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The microbiological quality, safety, and composition of mixtures of ewe's and goat's milk (90:10) used for cheesemaking were evaluated before and after thermization at 60 and 67 degrees C for 30 s. Such mild thermal treatments are commonly applied to reduce natural contaminants of raw milk before processing for traditional hard Greek cheeses. Raw milk samples had an average total bacterial count of 7.3 log CFU/ml; most of these bacteria were lactic acid bacteria (LAB) and pseudomonads. The LAB flora of raw milk was dominated by enterococci (40.8%), followed by lactococci (20.4%), leuconostocs (18.4%), and mesophilic lactobacilli (10.2%). Enterococcus faecalis (30.1%) and Enterococcus faecium (13.7%) were the most common LAB isolates, followed by Enterococcus durans, Lactococcus lactis subsp. lactis, Lactobacillus plantarum, and Leuconostoc lactis. Thermization at 60 degrees C for 30 s was effective for reducing raw milk contamination by enterobacteria (5.1 log CFU/ml), coagulase-positive staphylococci (3.3 log CFU/ml), and Listeria (present in 25-ml samples) to safe levels, but it also reduced mesophilic lactococci, leuconostocs, lactobacilli, and selected enterococci (72.0%) in thermized milk. Thermization at 67 degrees C for 30 s had a major inactivation effect on all bacterial groups. Two nisin-producing L. lactis subsp. lactis strains (M78 and M104) were isolated from raw milk, but neither nisin-producing nor other bacteriocin-producing LAB strains were isolated from thermized milk. Thus, thermization treatments control harmful bacteria but also may have a negative impact on milk quality by reducing desirable LAB and the biodiversity of raw milk bacteria overall, inactivating potentially protective LAB strains and enhancing the ability of potentially pathogenic enterococci to grow in fresh cheese curds.
Article
A total of 348 lactic acid bacteria isolated from five batches of naturally fermented dry salami at various stages of ripening were characterised. The majority of the strains were assigned to two main phylogenetic groups of species: (i) the psychrotrophic, formerly called atypical, meat streptobacteria (169 strains) and (ii) a new genus Weissella (120), which was recently proposed (Collins et al., 1993) to include Leuconostoc paramesenteroides and some other closely related species. Meat streptobacteria were identified as Lactobacillus curvatus (88 strains) and L. sake (76), whereas 5 strains were indistinguishable and, thus designated L. sake/curvatus. Non-psychrotrophic streptobacteria were also isolated and identified as L. plantarum (34 strains), L. farciminis (10), L. coryniformis (1) and L. casei subsp. pseudoplantarum (1). The majority of the Weissella strains (86) were leuconostoc-like bacteria; four of them were identified as W. viridescens, 11 belonged to the newly described W. hellenica (Collins et al., 1993), another 11 resembled W. paramesenteroides, whereas 60 isolates were not classified to any species. The latter group comprised strains that produced D(L)-lactate. The remaining Weissella were gas-forming, arginine-positive rods assigned to W. minor (31) and W. halotolerans (3). Other species identified were Enterococcus faecium (10), Leuconostoc mesenteroides (1), L. brevis (1) and Pediococcus sp. (1). The main criteria used to distinguish between above species as well as their distribution on the five salami batches in relation to their succession with time and suitability as starters were discussed.
Article
A Lactobacillus sake strain (L. sake 2a), isolated from Brazilian sausage ('lingüiça'), possessed antilisterial activity. The inhibitory activity observed in culture media was due to a proteinaceous compound, as demonstrated by the sensitivity to proteolytic enzymes. Inhibition due to hydrogen peroxide, organic acids and lytic bacteriophages was ruled out. The inhibition of Listeria monocytogenes by L. sake 2a was also demonstrated in laboratory-prepared 'lingüiça'. After four weeks at 8 degrees C, counts of L. monocytogenes in the pork product co-inoculated with L. sake 2a were about 6 log10 lower than those in control samples containing only L. monocytogenes. These results suggest that L. sake 2a strain produces a bacteriocin-like substance that can be used to inhibit the growth of L. monocytogenes on Brazilian sausage.
Article
The effectiveness of enterocin CCM 4231 in controlling Listeria monocytogenes contamination in dry fermented Hornád salami was examined. Three independent salami treatments were conducted under pilot plant and laboratory conditions. Salamis were produced according to standard technological parameters and stages with ripening for 3 weeks. The reference samples consisted of the meat mixture without either L. monocytogenes or bacteriocin addition. The control sample (CS) consisted of the meat mixture with 1% of L. monocytogenes inoculum (10(8) cfu ml(-1)) added; while the experimental sample (ES) consisted of the same mixture with enterocin CCM 4231 (12800 AU g(-1)) added. Sampling was done on the first day of the experiment, before and after bacteriocin addition for ES, on the second day and after 1, 2 and 3 weeks. The enterocin addition resulted in the reduction of L. monocytogenes by 1.67 log cycle in the ES when compared to the CS immediately after addition of the bacteriocin. Although on the second day, the growth of L. monocytogenes in ES reached 3.38 cfu g(-1) (log 10), a difference of 1.72 log was found between the ES and the CS. After 1 week of ripening, the L. monocytogenes count in the CS reached 10(7) cfu g(-1); while in the ES the count was 10(4) cfu g(-1), a difference which was maintained after 2 and 3 weeks of ripening. However, bacteriocin activity in the ES could not be detected analytically. The meat mixture used did not contain Listeria.
Article
Enterocin was used to control the growth of Staphylococcus aureus strains SA1 and Oxford 209P in Sunar (milk nourishment for suckling babies) and during the yogurt-making process. Reduction by three orders of magnitude was noted in the growth of SA1 strain in Sunar milk nourishment between the enterocin-containing (ES) and the control samples (CS) at 1-d cultivation. An inhibitory effect of enterocin was observed when surviving of SA1 cells were checked 6 h after the start of cultivation (2 h after enterocin application; enterocin was applied after 4 h). Decrease in the count of Oxford 209P strain in yogurt was detected in ES after 1 d of storage in comparison with CS (10(3) and 10(0) CFU mL-1 g-1). Thus a decrease by three orders of magnitude was found between ES and CS at the time mentioned. On the other hand, no bacteriocin activity was detected in ES after 1 d. Activity was detected only immediately after enterocin addition to ES (400 AU/mL) as well as after 1 and 3 1/2 h (200 AU/mL). Although the slight regrowth of the indicator was obtained up to 1 week of yogurt storage, the difference between ES and CS persisted. The lowest pH of the final yogurt product was noted in the reference yogurt sample but differences among the pH values of yogurt samples were not significant.
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Lactic acid bacteria (134) from Italian sausages were tested for the production of antimicrobial substances (bacteriocins). Six percent of these showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus plantarum 35d in particular produced a bacteriocin of high activity (320 AU ml(-1)) and a wide range of antimicrobial activity including S. aureus, L. monocytogenes, and A. hydrophila. The bacteriocin withstood heating at 80 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The apparent molecular weight of the bacteriocin extracted with n-butanol was estimated to be 4.5 kDa.
Article
Lactic acid bacteria (LAB) have been used for centuries in the fermentation of a variety of dairy products. The preservative ability of LAB in foods is attributed to the production of anti-microbial metabolites including organic acids and bacteriocins. Bacteriocins generally exert their anti-microbial action by interfering with the cell wall or the membrane of target organisms, either by inhibiting cell wall biosynthesis or causing pore formation, subsequently resulting in death. The incorporation of bacteriocins as a biopreservative ingredient into model food systems has been studied extensively and has been shown to be effective in the control of pathogenic and spoilage microorganisms. However, a more practical and economic option of incorporating bacteriocins into foods can be the direct addition of bacteriocin-producing cultures into food. This paper presents an overview of the potential for using bacteriocin-producing LAB in foods for the improvement of the safety and quality of the final product. It describes the different genera of LAB with potential as biopreservatives, and presents an up-to-date classification system for the bacteriocins they produce. While the problems associated with the use of some bacteriocin-producing cultures in certain foods are elucidated, so also are the situations in which incorporation of the bacteriocin-producer into model food systems have been shown to be very effective.
Article
Lactic acid bacteria (LAB) commonly used in food as starter cultures are known to produce antimicrobial substances such as bacteriocins and have great potential as food biopreservatives. LAB isolated from traditional fermented foods (appam batter and pickles) were screened for bacteriocin production. Two lactobacilli, LABB and LABP (one from each source) producing bacteriocins were characterized. Both the bacilli were homo-fermentative, catalase negative and micro-aerophilic in nature. LABB was found to be a thermobacterium growing at 45 degrees C while LABP was a streptobacterium growing at 15 degrees C. Both were able to grow at pH 4.5-8.6 but were intolerant to high salt concentration. They failed to produce gas from glucose as well as ammonia from arginine. Among the sugars examined they could not ferment arabinose, raffinose, rhamnose or xylose. Additionally, LABB could not ferment esculin, gluconate or mannose. LABB is identified as Lactobacillus acidophilus while LABP as Lb. casei. Their bacteriocins showed a broad inhibitory spectrum against the indicator organisms tested. They were active below pH 8.0 and after autoclaving as well. There was a complete loss of activity when treated with proteolytic enzymes such as trypsin indicating the proteinaceous nature of the active molecules. SDS-PAGE of partially purified bacteriocins indicated the molecular mass of the bacteriocin as 3.8 and 4.5 kDa for LABB and LABP respectively.
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Two distinct regions of the Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6, each of which specified bacteriocin production as well as immunity, have been sequenced and analyzed by deletion and frameshift mutation analyses. On a 1.8-kb ScaI-ClaI fragment specifying low antagonistic activity, three open reading frames (ORFs) were present, which were organized in an operon. The first two ORFs, containing 69 and 77 codons, respectively, were involved in bacteriocin activity, whereas the third ORF, containing 154 codons, was essential for immunity. Primer extension analysis indicated the presence of a promoter upstream of the ORFs. Two ORFs were present on a 1.3-kb ScaI-HindIII fragment specifying high antagonistic activity. The first ORF, containing 75 codons, specified bacteriocin activity. The second ORF, containing 98 codons, specified immunity. The nucleotide sequences of both fragments upstream of the first ORFs as well as the first 20 bp of the first ORF of both bacteriocin operons appeared to be identical.
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An evaluation is described of the Pico-Tag system (Waters Associates) for the rapid, quantitative determination of amino-acids in food materials. The Pico-Tag method is an integrated technique for pre-column derivatization of amino-acids phenylisothiocyanate (PITC) followed by the separation of PTC-amino-acids by reversed-phase high-performance liquid chromatography. The PTC-amino-acids have a strong ultraviolet absorbance; detection is by measurement of this absorbance at 254 nm. There was good agreement between results obtained with this analyser and those obtained by the conventional ion-exchange chromatography method of post-column derivatization with ninhydrin. The Sep-pak method for sample clean-up was investigated for several of the foods analysed. Both total and free amino-acids may be successfully determined with the Pico-Tag system. The speed of analysis, repeatability and reproducibility of the chromatography (allowing automated data integration and collection from replicate injection), detection of primary and secondary amino-acids and general reliability of the system make the Pico-Tag method an excellent technique for amino-acid analysis of foods.
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The spectrum of inhibitory bacterial products includes antibiotics, metabolic products, lytic agents, enzymes, defective bacteriophages and bacteriocins. A precise definition of bacteriocins is not yet available. Bacteriocins, unlike bacteriophage, do not carry the genetic determinant necessary for self replication. The conditions to yield maximum production of bacteriocins are described. Some bacteria produce bacteriocin inhibitors and inactivators and thus escape the lethal action of their own bacteriocin. The known bacteriocins of Gram positive bacteria are tabulated together with their main physiochemical physicochemical The spectrum of activity has been studied and led to the establishment of bacteriocin typing procedures. It appears that bacteriocin, after adsorption to the bacterial envelope, acts as a lethal agent by a single hit process ('quantal' killing). There is a possibility that inhibitory organisms of low disease potential may eventually be used to control infections due to high pathogenicity organisms which would be a more specific method than the use of antibiotics. Finally, the bacteriocins of various Gram positive genera are briefly described. (Csonka - London)
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A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, was purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin UI49 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin UI49 belongs to the class of bacteriocins termed lantibiotics.
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Lactocin S, a bacteriocin produced by Lactobacillus sake L45, has been purified to homogeneity by ion exchange, hydrophobic interaction and reverse-phase chromatography, and gel filtration. The purification resulted in approximately a 40,000-fold increase in the specific activity of lactocin S and enabled the determination of a major part of the amino acid sequence. Judging from the amino acid composition, lactocin S contained approximately 33 amino acid residues, of which about 50% were the nonpolar amino acids alanine, valine, and leucine. Amino acids were not detected upon direct N-terminal sequencing, indicating that the N-terminal amino acid was blocked. By cyanogen bromide cleavage at an internal methionine, the sequence of the 25 amino acids (including the methionine at the cleavage site) in the C-terminal part of the molecule was determined. The sequence was Met-Glu-Leu-Leu-Pro-Thr-Ala-Ala-Val-Leu-Tyr-Xaa-Asp-Val-Ala-Gly-Xaa-Phe- Lys-Tyr-Xaa-Ala-Lys-His-His, where Xaa represents unidentified residues. It is likely that the unidentified residues are modified forms of cysteine or amino acids associated with cysteine, since two cysteic acids per lactocin S molecule were found upon performic acid oxidation of lactocin S. The sequence was unique when compared to the SWISS-PROT data bank.
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Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.
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Exudative fluids were collected from packages of five brands of all-beef wieners and inoculated to contain 10(4) to 10(5) CFU of a three-strain (Scott A, V7, and 101M) mixture of Listeria monocytogenes per ml. Listeriae were inactivated (decrease of 0.61 to 3.8 log10 CFU/ml) in all five exudates held at 4 degrees C for 29 days. L. monocytogenes grew (increase of 1.7 to 3.6 log10 CFU/ml) in two of five exudates held at 25 degrees C for 6 days. Exudate was inoculated with a derivative of Pediococcus acidilactici H (designated JBL1095) or treated with pediocin AcH (a bacteriocin) as a novel approach to control the growth of L. monocytogenes in wiener exudates. Initially, pediocin AcH caused rapid death (decrease of 0.74 log10 CFU/ml in 2 h) of L. monocytogenes in exudate held at 4 degrees C, but thereafter the inactivation was similar to that in control exudate (L. monocytogenes only) or exudate containing L. monocytogenes plus JBL1095. At 25 degrees C, L. monocytogenes grew in the presence of JBL1095 during the first 64 h of incubation, but thereafter the numbers of the pathogen decreased appreciably (5.84 log10 CFU/ml in 3 days). In the presence of pediocin AcH, there was a gradual decrease in numbers of L. monocytogenes throughout the storage period at 25 degrees C. These data indicate that added biopreservatives can potentiate and amplify the intrinsic listeriostatic or listericidal activity of wiener exudate.
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A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.
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Lacticin 481, a bacteriocin produced during the growth of Lactococcus lactis subsp. lactis CNRZ 481, was purified sequentially by ammonium sulfate precipitation, gel filtration, and preparative and analytical reversed-phase high-pressure liquid chromatography. Ammonium sulfate precipitations resulted in a 455-fold increase in total lacticin 481 activity. The entire purification protocol led to a 107, 506-fold increase in the specific activity of lacticin 481. On the basis of its electrophoretic pattern in sodium dodecyl sulfate-polyacrylamide gels, lacticin 481 appeared as a single peptide band of 1.7 kDa. However, dimers of 3.4 kDa also exhibiting lacticin activity were detected. Derivatives of the lacticin-producing strain which did not produce lacticin 481 (Bac) were sensitive to this bacteriocin (Bac) and failed to produce the 1.7-kDa band. Amino acid composition analysis of purified lacticin 481 revealed the presence of lanthionine residues, suggesting that lacticin 481 is a member of the lantibiotic family of antimicrobial peptides. Seven residues (K G G S G V I) were sequenced from the N-terminal portion of lacticin 481, and these did not shown any homology with nisin or other known bacteriocin sequences.
Article
A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.
Chapter
Many lactic acid bacteria have been shown to be strongly antagonistic against other bacteria and may possibly be used in some foods as “protective” cultures against spoilage bacteria and food-borne pathogens (e. g. Listeria monocytogenes). In most cases, the inhibition is caused by the formation of organic acids or hydrogen peroxide, but bacteriocins may also be involved. In the first part of this paper, a general overview on bacteriocins produced by lactic acid bacteria is given, and the second part presents results of personal studies that were performed to investigate the potential of a bacteriocin-producing strain of Lactobacillus sake to suppress growth of Listeria monocytogenes in minced meat and German-type spreadable sausage. It has been demonstrated that the bacteriocin was active against this pathogenic bacterium in both food systems.
Article
Garlic has been used for condiments and also for medicines to cure various diseases since ancient times. Many studies on the processing of garlic have been published, however, few of them were related with fermentation because of the antimicrobial action of the garlic. In this study, to conduct garlic fermentation, 4 lactic acid bacteria (LAB) strains with growth abilities in garlic medium were selected. Addition of various nitrogen, carbon, and mineral sources enerally did not improve the growth of experimental strains during garlic fermentation except for Lactobacillus casei KFRI 704 by yeast extract and Lactococcus lactis subsp. cremoris ATCC 19257 by mineral sources. High performance liquid chromatography (HPLC) analysis of 32 phenolic compounds during fermentation showed that formononetin was decreased time dependently. The concentrations of volatile compounds and alliin did not change during fermentation. The results of this study would provide the basic understanding of garlic fermentation by selected strains of LAB.
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
A numerical taxonomic study was performed on 52 Leuconostoc spp. strains isolated from chill-stored meats. Three clusters were observed; representative strains from these clusters, together with the type strains of previously described species, were examined by performing a cellular fatty acid analysis and deoxyribonucleic acid (DNA)-DNA hybridization. Cluster I1 contained seven strains, which were assigned to Leuconostoc mesenteroides subsp. mesenteroides on the basis of DNA relatedness and biochemical properties. Clusters I (30 strains) and I11 (15 strains) were shown to represent two new species, for which thc names Leuconostoc gelidum and Leuconostoc carnosum, respectively, are proposed. The type strains of L. gelidum and L. carnosum are strains NCFB 2775 and NCFB 2776, respectively. The genus Leuconostoc is presently composed of four species, viz., Leuconostoc mesenteroides (containing three subspecies, Leuconostoc mesenteroides subsp. mesenteroi-des, Leuconostoc me;renteroides subsp. dextranicum, and Leuconostoc mesenteroides subsp. cremoris), Leirconostoc paramesenteroides, Leuconostoc lacstis, and Leuconostoc oenos (8). This classification is based on phenotypic charac-teristics, including nutritional requirements (3, 5) , immuno-logical relationships of dehydrogenases (10, 13), and deoxy-ribonucleic acid (DNA) homologies (6, 14). The habitats of all of these species except Leuconostoc oenos are reported to be milk and dairy products and plant material; Leirconostoc oenos is isolated only from wine and related habitats (8, 21). However, there have been several reports of leuconostocs occurring among the dominant mi-crobial population on meats stored in vacuum packs or under modified gas atmospheres containing carbon dioxide (1, 11, 20, 22), where they may contribute to spoilage. Some isolates from meat have been identified by using an early classification scheme (3) as belonging to groups equivalent to Leirconostoc mesenteroides subsp. mesenteroides or Leu-conostoc paramesenteroides (1, 20). However, in our expe-rience many isolates are not identifiable as any of the previously described species. In this study we used numerical taxonomic techniques to group leuconostocs from stored packaged meats and deter-mined the relationships of the groups to the previously described species. Groups were also studied by performing a cellular fatty acid analysis and DNA homology experiments, which revealed two new species, for which the names Leirconostoc gelidum and Leitconostoc carnosum are pro-posed.
Article
Schemes for a rapid and simple identification of lactobacilli from meat and meat products are presented which also include the species only recently published (e.g. L. divergens and L. carnis), and which were verified by the investigation of 229 strains newly isolated from meat and meat products and of nine type strains.They are primarily based on differences in the fermentation of sugars and other easily determinable physiological characteristics, and therefore permit a rapid assignment of a new isolate to one of the Lactobacillus species. However, in most instances this identification has to be confirmed by determining additional characteristics (e.g. the whole range of fermented carbohydrates, types of murein etc.).
Article
A Lactobacillus sake strain L45 isolated from naturally fermented dry sausage, produced a bacteriocin designated lactocin S. The bacteriocin was moderately heat-stable and its activity was sensitive to proteases. Bacteriocin activity was found in the growth medium during the late exponential phase of growth and was directed against selected strains within the closely related genera Lactobacillus , Leuconostoc and Pediococcus. During propagation in liquid medium, non-bacteriocin-producing bacteria (Bac-) appeared with high frequency. Some isolates had also lost immunity to the bacteriocin (Imm-). Analyses of L45 Bac+ Imm+ isolates revealed two plasmids of about 50 kb (pCIM1) and 34 kb (pCIM2). All of the L45 Bac- Imm- variants had lost pCIM1. Three Bac- Imm+ isolates were found which still contained pCIM1. However, DNA restriction enzyme analyses disclosed differences between pCIM1 in the Bac+ Imm+ and Bac- Imm+ isolates. These experiments strongly suggest that the pCIM1 plasmids are involved in production of the bacteriocin and in immunity to the bacteriocin.
Article
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.
Article
The characteristics of 177 psychrotrophic lactic acid bacteria isolated from vacuum-packaged fresh beef have been studied. Eighteen isolates were identified as Leuconostoc mesenteroides and the remainder were lactobacilli. None of these could be identified down to a species level and they were considered to be atypical streptobacteria or atypical betabacteria. Atypical streptobacteria produced both isomers of lactic acid and did not ferment lactose and maltose. Atypical beta-bacteria produced only L(+) lactic acid. The nature of the isolates varied considerably from pack to pack. The API 50 lactobacillus identification system proved useful in studying these organisms.
Article
The main fermented meat products are fermented sausages in which lactic acid bacteria (LAB) are the essential agents of the ripening process. During indigenous fermentations Lactobacillus curvatus and L. sake are the dominating LAB. Their application as starter organisms ensures the dominance of the starter during the whole ripening process. The suppression of the competing fortuitous LAB depends on the quality of the raw materials and on technological factors. The physiological properties of lactic starters do not suffice to ensure a sensory quality which can be found in traditionally produced dry fermented sausages. Additional activities required are present in micrococci and yeasts which, therefore, are further components of starter culture preparations. Some strains of meat-borne lactobacilli exhibit the essential activities like nitrate reductase, nitrite reductase, catalase, lipase, and protease, respectively. To create the optimal starter cultures composed of lactobacilli, these activities have to be studied and optimized in strains of high competitiveness in the fermenting substrate.
Article
When chilled meat is packaged under vacuum or in a modified atmosphere with an elevated level of carbon dioxide, its microflora is dominated by lactic acid bacteria. Such new packaging technologies present opportunities for microbial control that may not only extend shelf life, but also enhance the microbiological safety of meats. Bacteriocins are antimicrobial proteins produced by certain bacteria; they act on target cells by various mechanisms, most of which are, as yet, unclear. This review summarizes the available information on the bacteriocins and bacteriocinogenic lactic acid bacteria from meats that could play a role in this innovative approach to meat preservation.
Article
Lactic acid bacteria produce a variety of antagonistic factors that include metabolic end products, antibiotic-like substances and bactericidal proteins, termed bacteriocins. The range of inhibitory activity by bacteriocins of lactic acid bacteria can be either narrow inhibiting only those strains that are closely related to the producer organism, or wide, inhibiting a diverse group of Gram-positive microorganisms. The following review will discuss biochemical and genetic aspects of bacteriocins that have been identified and characterized from lactic acid bacteria.
Article
Lactic acid bacteria can produce a variety of substances with antibacterial activity which are described in this article. Non-peptide antibacterial substances are distinguished from bacteriocins, which have a proteinaceous active site. Among the former, reuterin produced by Lactobacillus reuteri is a broad spectrum inhibitor active against Gram-positive and Gram-negative bacteria, yeasts, molds and protozoas. It is a glycerol derivative, β-hydroxypropionaldehyde. Bacteriocins can be produced by most lactic acid bacteria species and their spectrum of activity is generally restricted to organisms taxonomically close to the producer. The biochemical properties of bacteriocins, their structure and nature of their genetic determinants are highly variable. This literature review discusses the similarities and differences existing in this group of substances. Des bactéries lactiques sont capables de produire une variété de substances à activité antibactérienne décrites dans le présent article. On distingue les antibactériens non peptidiques des bactériocines dotées d'un site actif de nature protéique. Parmi les premiers, la reuterine produite par Lactobacillus reuteri est un antibiotique à large spectre, actif vis-à-vis des bactéries Gram-positives et Gram-négatives, des levures, des moisissures et des protozoaires. Il s'agit d'un dérivé du glycérol, le β-hydroxypropionaldéhyde. Les bactériocines peuvent être produites par la plupart des espèces de bactéries lactiques et leur spectre d'activité est restreint en général aux bactéries taxonomiquement proches du microorganisme producteur. Les propriétés biochimiques des bactériocines, leur structure et la nature de leur déterminant génétique sont d'une grande variabilité. Cette revue bibliographique fait le point des similitudes et des disparités qui existent au sein de ce groupe de susbstances.
Article
Lactic acid bacteria can produce a variety of antimicrobial compounds which may affect both the lactic acid bacteria themselves as well as undesirable or pathogenic strains. In this first section, we describe the biosynthesis, mode of action and activity spectra of these inhibitors. Metabolites of oxygen (hydrogen peroxide and free radicals) exhibit a bacteriostatic or bactericidal activity against lactic or non-lactic flora. When associated with the lactoperoxidase / thiocyanate system, hydrogen peroxide leads to the formation of inhibitory compounds which are bacteriostatic for lactic acid bacteria and bactericidal for Gram-negative bacteria. The antimicrobial activity of organic acids (lactic, acetic and formic) and of pH are closely linked. It appears that the non-dissociated fraction of these acids is the major inhibitory form. Thus, acetic acid whose pKa is higher than that of lactic acid exhibits the highest inhibitory activity. The antimicrobial activities of diacetyl, acetaldehyde and of the D isomers of amino acids are also described, although their effects are slight in usual lactic fermentations. Bacteriocins are the last type of inhibitory substance produced by some lactic acid bacteria. They will be described in a second section. Les bactéries lactiques sont capables de produire une variété de produits inhibiteurs dont les effets peuvent se répercuter sur la flore lactique elle-même mais aussi sur les flores indésirables ou pathogènes. Dans cette première partie, nous décrivons les mécanismes d'apparition, les modes d'action et les spectres d'activité de ces inhibiteurs. Les métabolites de l'oxygène (le peroxyde d'hydrogène et les radicaux libres) peuvent avoir des effets biologiques de nature bactériostatique ou bactéricide sur la flore lactique ou non lactique. Le peroxyde d'hydrogène associé au système lactoperoxydase / thiocyanate, catalyse la formation de produits inhibiteurs, bactériostatiques pour la flore lactique et bactéricides pour les bactéries Gram-négative. Les pouvoirs antibactériens des acides organiques et du pH sont intimement liés. Il semble que la fraction non dissociée de ces acides soit la forme inhibitrice majeure. De ce fait l'acide acétique de pKa supérieur au pKa de l'acide lactique, a un pouvoir inhibiteur supérieur. Les activités inhibitrices du diacétyle, de l'acétaldéhyde et des acides aminés de forme isomérique D sont décrites bien que leurs effets soient mineurs dans les fermentations lactiques habituelles. Les bactériocines produites par certaines bactéries lactiques seront décrites dans une seconde revue.
Article
A numerical taxonomic study was performed on named strains of Listeria, Erysipelothrix, Microbacterium thermosphactum, Lactobacillus, Streptococus, Propionibacterium, Kurthia and some possibly related bacteria using I43 unit characters covering a wide range of properties. The strains fell into six main clusters: (A) Listeria; (B) Microbacterium thermosphactum, Lactobacillus, Streptococcus; (C) Gemella, Erysipelothrix; (D) Kurthia and mainly aerobic corynebacteria; (E) Propionibacterium, Staphylococcus; (F) mainly Cellulomonas. The genus Listeria contained three subgroups corresponding to (I) Listeria monocytogenes, (2) Listeria grayi and (3) non-haemolytic listeria strains. Listeria murrayi did not appear to be sufficiently distinct from Listeria grayi to warrant separate species status. The genus Erysipelothrix was quite separate from the genus Listeria. Microbacterium thermosphactum was related to both the genera Listeria and Lactobacillus but formed a separate phenon that could appropriately be given generic rank. There were four distinct subgroups amongst the streptococci examined. Gemella strains appeared as a distinct phenon related to Erysipelothrix and the streptococci. The lactobacilli grouped into four phena largely corresponding to the subgenera Betabacterium, Streptobacterium and Thermobacterium and to the species Lactobacillus mali. Custers A, B, and C displayed a relatively close association to each other and it is recommended that all be included in the family Lactobacillaceae.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
Twenty Leuconostoc strains isolated from vacuum packaged Vienna-type sausages were screened for antagonistic activity against various Gram-positive organisms (including Listeria spp.). One of the three strains exhibiting inhibitory activity was chosen for further investigation. This strain was identified as Leuc. carnosum and the inhibitory substance produced was named carnosin. Carnosin was inactivated by trypsin but not by catalase or other non-proteolytic enzymes tested. Carnosin retained activity after heating at 100 degrees C for 20 min, whereas heating at 121 degrees C for 15 min resulted in complete loss of activity. Carnosin was active at pH values ranging from 2 to 9. Carnosin activity was not detectable until cells were in the late log-phase of growth. At low temperatures (4 degrees C), higher cell densities were required before carnosin activity could be detected. Carnosin was active against various lactic acid bacteria, Enterococcus faecalis and Enterococcus faecium and against Listeria spp. Difficulties in purification were reduced by growing Leuc. carnosum in a modified MRS medium, having 50% of the normal peptone concentration and no Tween or meat extract. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of partially purified carnosin indicated that it has a molecular mass between 2510 and 6000 Da. Yet, retention of activity after exhaustive dialysis suggested a molecular mass > 14kDa. It is hypothesized that carnosin forms large active complexes which can be dissociated to small (active) components.
Article
To determine whether pediocin is produced and has effective antilisterial activity during food fermentation, six sausage fermentation trials were conducted with antibiotic-resistant, pediocin-producing (Bac+) Pediococcus acidilactici PAC 1.0 (Strr Rifr) and an isogenic pediocin-negative (Bac-) derivative used as a control. Meat was inoculated (ca. 10(5) CFU/g) with a composite of five Listeria monocytogenes strains, each electrotransformed with pGK12 (Cmr Emr). P. acidilactici and L. monocytogenes populations were selectively enumerated by plating on media with antibiotics. This study indicated that the dry sausage fermentation process can reduce L. monocytogenes populations. Effective inactivation of L. monocytogenes was observed when the pH at the end of the fermentation portion of the process was less than 4.9. Pediocin was responsible for part of the antilisterial activity during the fermentation in each of the six trials. Furthermore, inhibition of L. monocytogenes during drying was enhanced in the presence of pediocin in the three trials in which L. monocytogenes could be detected throughout the drying process. Thus, pediocin production contributed to an increase in safety during both the fermentation and drying portions of sausage manufacturing.
Article
A new bacteriocin, termed lactococcin A (LCN-A), from Lactococcus lactis subsp. cremoris LMG 2130 was purified and sequenced. The polypeptide contained no unusual amino acids and showed no significant sequence similarity to other known proteins. Only lactococci were killed by the bacteriocin. Of more than 120 L. lactis strains tested, only 1 was found resistant to LCN-A. The most sensitive strain tested, L. lactis subsp. cremoris NCDO 1198, was inhibited by 7 pM LCN-A. By use of a synthetic DNA probe, lcnA was found to be located on a 55-kb plasmid. The lcnA gene was cloned and sequenced. The sequence data revealed that LCN-A is ribosomally synthesized as a 75-amino-acid precursor including a 21-amino-acid N-terminal extension. An open reading frame encoding a 98-amino-acid polypeptide was found downstream of and in the same operon as lcnA. We propose that this open reading frame encodes an immunity function for LCN-A. In Escherichia coli lcnA did not cause an LCN-A+ phenotype. L. lactis subsp. lactis IL 1403 produced small amounts of the bacteriocin and became resistant to LCN-A after transformation with a recombinant plasmid carrying lcnA. The other lactococcal strains transformed with the same recombinant plasmid became resistant to LCN-A but did not produce any detectable amount of the bacteriocin.
Article
Lactic acid bacteria isolated from Spanish dry fermented sausages were screened for antagonistic activities under conditions that eliminated the effects of low pH and hydrogen peroxide. From 720 isolates tested 119 were inhibitory to Lactobacillus fermentum CECT285. The isolates showing the largest inhibitory activity exhibited an antagonistic effect against several other lactobacilli and the selected foodborne pathogens Staphylococcus aureus and Listeria monocytogenes. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. The isolates were tentatively characterized as Lactobacillus sake. One of the isolates, L. sake 148 was chosen for further study. The compound excreted by L. sake 148 was active against various lactobacilli and several Gram-positive foodborne bacteria, but not against the Gram-negative bacteria tested. The antagonistic effects were almost eliminated by treatment with proteases, whereas they were heat resistant and bacteriostatic rather than bacteriocidal.
Article
Lactobacillus sake Lb 706 can release a bacteriocin inhibitory to Listeria monocytogenes. In MRS broth, viable counts decreased rapidly when Lact. sake Lb 706 was added, whereas growth of the listerias was not affected by a bacteriocin-negative variant of the same Lactobacillus strain. Inhibition of L. monocytogenes was also observed in pasteurized minced meat inoculated with Lact. sake Lb 706. The bacteriocin produced is apparently effective in meat. However, the effect of the bacteriocin producer was less evident in minced meat than in broth. In comminuted cured raw pork filled into casings (German-type 'fresh Mettwurst'), L. monocytogenes was able to grow at a pH of 6.3, but addition of Lact. sake Lb 706 prevented the growth of listerias during the first few days after manufacture. At normal pH (5.7) L. monocytogenes did not multiply and addition of Lact. sake Lb 706 reduced viable counts of listerias by about one log cycle. Lactobacillus sake Lb 706 therefore may have some potential as a protective culture in meat products.
Article
A heterofermentative lactic acid bacterium isolated from meat packaged under elevated CO2 levels was identified as Leuconostoc gelidum, based on the description of this new species by Shaw & Harding (1989). It grows well at refrigeration temperatures but not at 35 degrees C. The organism produces an inhibitory substance that is inactivated by protease and trypsin, but not by catalase or by heating at 62 degrees C, for 30 min. The bacteriocin-like inhibitory substance is produced early in the growth cycle, at 1, 5 and 25 degrees C. The inhibitory substance is active against a large number of closely related lactic acid bacteria, as well as a strain of Enterococcus faecalis and Listeria monocytogenes. There is initial evidence that the genetic information determining production of, and resistance to, the bacteriocin-like substance is plasmid mediated. Of the three plasmids found in this organism, loss of the 7.6 MDa plasmid resulted in loss of inhibitor production and resistance to the inhibitory substance. Loss of the 5.0 MDa plasmid did not result in a detectable phenotypic change in the organism.
Article
Harding, C.D. & Shaw, E.G. 1990. Antimicrobial activity of Leuconostoc gelidum against closely related species and Listeria monocytogenes. Journal of Applied Bacteriology69, 648–654. A newly isolated strain of Leuconostoc gelidum was evaluated for its ability to inhibit a wide spectrum of lactic acid bacteria, meat spoilage bacteria and food-related human pathogens, including Listeria monocytogenes. It was inhibitory to most of the lactobacilli, all the leuconostocs, and three strains of L. monocytogenes when tested both by direct and well diffusion methods. Cell-free extract retained activity after 60 min at 100d̀C but was sensitive to protease. Dialysis suggested a molecular weight in excess of 104 daltons. The inhibitory effect was bactericidal and rapid.
Article
Carnobacterium piscicola LV17 isolated from vacuum-packed meat produces bacteriocin(s) that is active against closely related lactic acid bacteria, Enterococcus spp., and a strain of Listeria monocytogenes but not against gram-negative bacteria. The bacteriocin has a bactericidal mode of action, is heat resistant, and is stable over a wide range of pH but is inactivated by proteolytic enzymes. Sensitive and resistant cells were shown to adsorb the bacteriocin, but cell death depended on contact of the bacteriocin with the cell membrane. Bacteriocin production is detected early in the growth cycle of the organism in APT broth, but it is not produced in APT broth adjusted to pH 5.5. Bacteriocin production and resistance to the bacteriocin produced are associated with two plasmids of 40 and 49 megadaltons. The possibility that two bacteriocins are produced is indicated because the inhibitory substances of the mutant strains containing either the 40- or 49-megadalton plasmids have different antimicrobial spectra.
Article
Commercially available polypeptide marker kits containing peptides generated by cyanogen bromide cleavage of either horse heart myoglobin or sperm whale myoglobin have been investigated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting on polyvinylidene difluoride membranes, and gas-phase sequencing. It could be shown that the molecular weights assigned to the SDS-PAGE bands by the companies are incorrect. Arranged in descending order, the marker kits are composed of the following polypeptide fragments from myoglobin: positions 1-153, 1-131, 56-153, 56-131, 1-55, and 132-153. A polypeptide comprising residues 1-14 was not found. According to these results the log Mr versus Rf plot used for calibration must be revised. For the separation of low molecular weight polypeptides and peptides a new gel system based on the theory of multiphasic zone electrophoresis combined with a modified Coomassie staining procedure is reported.
Article
An antimicrobial peptide designated pediocin AcH was isolated from Pediococcus acidilactici strain H. The pediocin AcH was purified by ion exchange chromatography. The molecular weight of pediocin AcH was determined by SDS-PAGE to be about 2700 daltons. Pediocin AcH was sensitive to proteolytic enzymes resistant to heat and organic solvents, and active over a wide range of pH. Pediocin AcH exhibited inhibition against several food spoilage bacteria and foodborne pathogens including Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes. It was bactericidal to sensitive cells and acted very rapidly. The bactericidal effect was not produced by either cell lysis or apparent loss of membrane permeability.
Article
The bacteriocin produced by Pediococcus acidilactici PAC 1.0, previously designated PA-1 bacteriocin, was found to be inhibitory and bactericidal for Listeria monocytogenes. A dried powder prepared from PAC 1.0 culture supernatant fortified with 10% milk powder was found to contain bacteriocin activity. An MIC against L. monocytogenes and lytic effects in broth cultures were determined. Inhibition by PA-1 powder occurred over the pH range 5.5 to 7.0 and at both 4 and 32 degrees C. In addition, inhibition of L. monocytogenes was demonstrated in several food systems including dressed cottage cheese, half-and-half cream, and cheese sauce.
Article
Physical factors leading to the separation of oligopeptides in the molecular weight range of 1,200 to 10,000 daltons by analytical-scale electrophoresis in polyacrylamide gel with sodium dodecyl sulfate are described. Increased acrylamide concentration, cross-linkage, and inclusion of 8 M urea to decrease gel porosity, increased gel length, and buffer ions of low mobility are factors which yield improved separation of such peptides. Electrophoretic mobilities of eleven peptides were linearly related to the logarithm of their molecular weights with a standard deviation of 18% in a system of improved resolution. The intrinsic charge and conformation of peptides were found to be relatively more important determinants of electrophoretic mobilities than for proteins larger than 10,000 daltons. Such determinants were relatively more important with four of the eleven peptides examined, leading to deviations from the log-linear slope greater than 18%. Because of the importance of intrinsic charge and conformation, the system, although allowing a first approximation in molecular weight determination, may also be applicable to peptide “mapping,” particularly for “insoluble” peptide mixtures with prominent hydrophobic association, such as encountered in cellular membranes, viruses, and proteolytic digests.
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.
Article
Parameters for production and purification of a bacteriocin produced by Lactobacillus acidophilus N2 are described. Production of lactacin B was pH dependent, with maximum activity detected in broth cultures maintained at pH 6. Lactacin B was purified by ion-exchange chromatography, ultrafiltration, and successive gel filtrations in the presence of 8 M urea and then 0.1% sodium dodecyl sulfate. The molecular weight of lactacin B was ca. 6,000 to 6,500, and the purified compound showed maximum absorbance at 211 nm. The activity of purified lactacin B was bactericidal to sensitive cells and restricted to members of the family Lactobacilliaceae, L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis. Characteristics identified for lactacin B indicated that it was a peptide and confirmed its identity as a bacteriocin.
Article
Shaw B. G. & Harding Charmaigne D. 1984. A numerical taxonomic study of lactic acid bacteria from vacuum packed beef, pork, lamb and bacon. Journal of Applied Bacteriology56, 25–40. A numerical taxonomic study using 79 unit characters has been performed on 100 isolates of lactic acid bacteria from refrigerated vacuum-packed beef, pork, lamb and bacon. Three clusters were observed at 78% S which contained all the strains apart from three unidentifiable streptobacteria, one Leuconostoc, and one strain of Pediococcus pentosaceus. One cluster (III) consisted of only one strain of Leuc. paramesenteroides and six unidentifiable Leuconostoc strains. The two largest clusters (I and II) were both composed entirely of streptobacteria. Cluster I contained 31 strains (G + C content 33–2–36–9 moles %) which were not identifiable with any described species. Cluster II contained 57 strains (G + C content 40–7–43–7 moles %) which were provisionally identified with Lactobacillus sake or Lact. bavaricus according to the lactic acid isomer produced. The division of nearly all the streptobacteria into two clearly defined clusters has resolved problems which have existed in the classification of lactic acid bacteria from vacuum-packed meat.
Article
The effect of starter culture and chemical acidulation on the growth and enterotoxigenesis of Staphylococcus aureus strain S-6 in Italian dry salami under commercial manufacturing conditions was studied. The experimental design included two levels of S. aureus (10 and 10/g), three levels of starter culture (0, 10, and 10/g), three levels of initial pH (pH(0)) (6.1, 5.5, and 4.8), two manufacturing plants, and three replications. S. aureus growth in the salami was affected significantly (P < 0.005) by pH(0), initial levels of S. aureus (staph(0)) and lactic acid bacteria (LAB(0)), day of fermentation, and by the interactions of pH(0) x day, pH(0) x LAB(0), LAB(0) x staph(0), pH(0) x staph(0), and pH(0) x location of fermentation. In general, the lower the pH(0) and the higher the LAB(0), the greater the inhibition of S. aureus. The LAB levels during the fermentation were affected significantly (P < 0.005) by pH(0), LAB(0), day of fermentation, location, LAB(0) x pH(0), and LAB(0) x day. Derived regression equations related level of S. aureus and LAB at any day of fermentation to a number of microbiological and chemical variables. Close similarity of observed and predicted levels of S. aureus and LAB growth demonstrated the usefulness of the experimental approach in evaluating the safety of a process. No detectable enterotoxin or thermonuclease was found at any stage of processing even when S. aureus reached levels of 10/g of salami.
Article
An atypical Leuconostoc paramesenteroides strain isolated from retail lamb produced a bacteriocin, leuconocin S, that was inactivated by alpha-amylase, trypsin, alpha-chymotrypsin, protease, and proteinase K but not by lipase or heat treatment at 60 degrees C for 30 min. Supernatants from culture broths produced two glycoprotein bands on sodium dodecyl sulfate-polyacrylamide gels; these had molecular weights of 2,000 and 10,000 and activity against Lactobacillus sake ATCC 15521. The crude bacteriocin preparation was bacteriostatic and dissipated proton motive force. Bacteriocin activity was produced over a wide pH range (5.2 to 7.9) on buffered agar medium, with an optimum pH of pH 6.15. The optimum pH for production in broth was 6.5 to 7.0.
Enhanced control of 1,istcriu mono-rytogenes by in .&-produced pediocin during dry fermented sausage production) 1,actic acid bac-teria in meat fermentation
  • P M Foegeding
  • A B Thomas
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Foegeding, P.M., Thomas, A.B., Pilkington, I l. I i. and Klaen-hammer, T.R. (1992) Enhanced control of 1,istcriu mono-rytogenes by in.&-produced pediocin during dry fermented sausage production. Applied and Encironmental Microbiology 58, 886890. Hammes, W.P., Hantleon, A. and Min, S. (1990) 1,actic acid bac-teria in meat fermentation. FEMS Microbiology Rez&ms 87, 165-174.
Purification and characterization of the Lactobacillus acidophilus bacteriocin, lac-tacin B Production of anti-microbial compounds by lactic acid bacteria (1992) A modified method to directly detect in SDS-PAGE the bacteriocin of Pediococcus ucidilactirz
  • S F Barefoot
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Barefoot, S.F. and Klaenhammer, T.R. (1984) Purification and characterization of the Lactobacillus acidophilus bacteriocin, lac-tacin B. Antimicrobial Agents and Chemotherapy 26, 328-334. Barnby-Smith, F.M., Roller, S.D., Woods, L.F.J., Barker, M.B., Nightingale, M. and Gibbs, P.A. (1989) Production of anti-microbial compounds by lactic acid bacteria. Research Repor'r No. 662, Leatherhead Food R.A. Bhunia, A.K. and Johnson, M.C. (1992) A modified method to directly detect in SDS-PAGE the bacteriocin of Pediococcus ucidilactirz. Letters in Applied Microbioloa 15, 5 7. Bhunia, A.K., Johnson, M.C. and Ray, B. (1988) Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus ucidilactici. Journai of Appiied Bactc-rtology 65, 261-268.
Bacte-riocins of Gram-positive bacteria Organization and nucleotide sequences of two lactococcal bacteriocin operons Characterization and partial purification of a bacteriocin pro-duced by Leuconostoc carnosum LA44A
  • J R Tagg
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  • L W Wannamaker
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Tagg, J.R., Dajani, AS. and Wannamaker, L.W. (1976) Bacte-riocins of Gram-positive bacteria. Bacteriological Reviews 40, Van Belkum, M.J., Hayema, B.J., Jeeninga, R.E., Kok, J. and Venema, G. (1991) Organization and nucleotide sequences of two lactococcal bacteriocin operons. Applied and Environmental Microbiology 57, 492498. Van Laack, R.L.J.M., Schillinger, U. and Holzapfel, W.H. (1992) Characterization and partial purification of a bacteriocin pro-duced by Leuconostoc carnosum LA44A. International Journal of Food Microbiology 16, 183-195.