food biopreservation
1.3 Bacteriocins of Gram-positive bacteria and the nature of bacteriocin-like inhibitory substance (BLIS)bacteriocin-like inhibitory substance (BLIS)
In this laboratory we first proposed use of the acronym BLIS (bacteriocin-like inhibitory substance) as a term of convenience to denote inter-bacterial inhibition that appears likely to be due to the production of bacteriocin(s), but prior to confirmation of the genetic and molecular identity of the inhibitory agent(s).
Bacteriocins of Gram-positive bacteria have recently been classified into four major divisions:
(a) Class I: post-translationally modified small (<10 kDa) peptides (the lantibiotics)
(b) Class II: non-modified small peptides (c) Class III: large (> 10 kDa) proteins and (d) Class IV: cyclic peptides (Heng et al. 2007).
Examples of bacteriocins found in Classes I–IV and their sub-divisions are presented in Figure 1.1. It seems prudent to regard bacteriocin classification schema as works in progress since the range of molecular entities potentially classifiable as bacteriocins is continuing to expand both in numbers and in compositional heterogeneity. Bacteriocins are composed of peptides or peptide-complexes, typically comprise between 30 and 60 amino acid residues, and are released in bioactive forms extracellularly. Many act on the bacterial cytoplasmic membrane, disrupting the proton motive force by forming pores in the phospholipid bi-layer (Cintas et al. 2001; Ammor et al. 2006). Other modes of action described include the inhibition of protein synthesis, peptidoglycan formation and spore germination; and interference with sodium and potassium transport (Upreti 1994;
Chatterjee et al. 2005).
The bacteriocins of LAB are generally ineffective against Gram-negative bacteria due to the possession by such organisms of an outer membrane (Gänzle et al. 1999). Exposure to certain sub-lethal stresses may however render the outer membrane permeable to bacteriocins such as nisin and pediocin and under these conditions killing activity has been demonstrated (Kalchayanand et al. 1992).
Some bacteriocinogenic LAB have also been found to have limited direct inhibitory activity against Gram-negative bacteria. For example, in a study that used simple agar diffusion assays to screen over 10,000 LAB from poultry production environments for activity against Campylobacter jejuni, 2% of tested isolates were found to be inhibitory (Stern et al. 2005). Similarly, propionin PLG-1, a heat-labile 10 kDa bacteriocin produced by the dairy bacterium Propionibacterium thoenii, has been reported to be inhibitory toward C. jejuni (Barefoot and Nettles 1993) and bacteriocin-like inhibitory activity against both Campylobacter and Helicobacter pylori has been reported in lactobacilli from the human gut (Strus et al. 2001).
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Some bacteria can produce more than one bacteriocin and multiply-bacteriocinogenic strains are, for example, especially common in the species Streptococcus salivarius, Streptococcus uberis and Streptococcus mutans (Table 1.1). Bacteriocin-producing S. salivarius harbour megaplasmids (160–220 kb), some of which have been shown to encode as many as five different bacteriocins. Streptococcus uberis 42 produces both nisin U (a Class I [lantibiotic]
bacteriocin) and uberolysin (a Class IV [cyclic] bacteriocin). Streptococcus mutans UA140 produces the lantibiotic mutacin I and a Class II bacteriocin (mutacin IV). Conversely, the same bacteriocin can sometimes be produced by strains of different LAB species (Table 1.2). For example, the bioactive forms of the lantibiotics SA-FF22 (Tagg and Wannamaker 1978) and macedocin (Georgalaki et al. 2002) are identical peptides, initially shown to be produced by Streptococcus pyogenes and more recently by Streptococcus macedonicus respectively. Highly-homologous SA-FF22-like peptides are also known to be Fig. 1.1 Gram-positive bacteriocin classes and sub-divisions. Based on Cotter et al.
(2005) with modifications by Heng et al. (2007).
Identifying new protective cultures and culture components 7
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X produced by strains of Streptococcus salivarius and Streptococcus equisimilis
(Wescombe 2002). Similarly, sakacin A and curvacin A are the same molecule produced by Lactobacillus sakei and Lactobacillus curvatus respectively (Axelsson and Holck 1995; Axelsson 2007 – pers. comm.). The kinetics of production of a particular bacteriocin may also differ according to the host strain.
For example, sakacin A is produced throughout the growth of L. sakei, but curvacin A is only produced in the late logarithmic growth-phase by L. curvatus (Holck et al. 1992; Vogel et al. 1993).
The naming of bacteriocins lacks formal guidelines but is generally based upon either the species or generic designation of the original source bacterium.
Examples of bacteriocins named for their species of origin are the salivaricins, ubericins, and curvacin, whereas the staphylococcins and lactocins display their generic heritage. Since a variety of bacteriocins may be produced by bacteria belonging to a single species, additional designations are required in order to more precisely specify each particular bacteriocin molecule. Once again, a variety Table 1.1 Examples of LAB that produce more than one bacteriocin
Producer Bacteriocin Reference
C. piscicola LV17 Carnobacteriocin A, B2, BM1 Quadri et al. (1994) Worobo et al. (1994) E. faecium CTC492 Enterocin A, B Nilsen et al. (1998) L. plantarum C11 Plantaricin EF, JK Anderssen et al. (1998)
L. sakei 5 Sakacin 5X, P, T Vaughan et al. (2001)
S. uberis 42 Nisin U, uberolysin Wirawan et al. (2007)
S. mutans UA140 Mutacin I, IV Qi et al. (2001)
S. mutans K8 Mutacin K8, IV Robson et al. (2007)
S. salivarius 9 Salivaricin 9, A4 Wescombe et al. (2009) S. salivarius K12 Salivaricin A2, B Hyink et al. (2007)
Table 1.2 Examples of the same bacteriocin produced by different LAB species
Bacteriocin Producer species Reference
SAFF22a (macedocin) S. pyogenes Jack et al. (1994)
S. macedonicus Georgalaki et al. (2002)
Sakacin-A (curvacin-A) L. sakei Axelsson and Holck
L. curvatus (1995)
Salivaricin A1a S. pyogenes Wescombe et al.
S. dysgalactiae subsp. equisimilis (2006b) S. agalactiae
Pediocin PA-1 Pediococcus (several spp.) Miller et al. (2005) L. plantarum
a Similar peptides are also known to be produced by strains of S. salivarius and S. dysgalactiae subsp. equisimilis.
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of conventions have been adopted, but we favour the allocation of successive letters of the alphabet (e.g., salivaricin A and salivaricin B were the first bacteriocins characterized from the species S. salivarius). For even more precise specification of a particular bacteriocin the strain designation of the producer bacterium can be included within the bacteriocin name (e.g., streptococcin A-FF22 is a bacteriocin produced by S. pyogenes strain FF22 – Tagg and Wannamaker 1978).
Bacteriocins that have only minor conservative differences in the amino acid sequences of their propeptide components resulting in no significant change to their (a) secondary structure, (b) activity spectrum and (c) the specific cross-immunities of their respective producer strains are more appropriately referred to as natural variants (Heng et al. 2007). For example, nisin Z, nisin Q and nisin U are natural variants of the first-described nisin A (Wirawan et al. 2006).
Most small bacteriocins are active over a wide pH range and their high iso-electric points allow them to interact, under physiological pH conditions, with the anionic surface of bacterial cells (Oscáriz and Pisabarro 2001). This feature, combined with their generally highly hydrophobic nature, has enabled purification procedures to be developed based on hydrophobic interaction, cation exchange and reversed phase chromatography resins (Oscáriz and Pisabarro 2001). Small bacteriocins tend also to be heat-stable due to their content of di-sulphide and thioether bonds, which limit the potential for un-folding under heat stress conditions. Consequently, small bacteriocins tend to retain their activity after autoclaving, whereas larger bacteriocins such as helveticin J (Joerger and Klaenhammer 1986) and zoocin A (Simmonds et al. 1996) are inactivated by 10–30 min at temperatures ranging between 60 and 100 °C.
A landmark observation in the field of LAB bacteriocin research was the confirmation in 1947 that the inhibitory activity of some lactococci (then referred to as group N streptococci) toward other LAB was at least in part attributable to an antimicrobial substance called nisin (for group N inhibitory substance) (Mattick et al. 1947). Nisin, now approved for use as a food additive in more than 50 countries, is regarded as the prototype of the bacteriocins of Gram-positive bacteria and more specifically of those belonging to the lantibiotic class.
Interestingly, the original discovery of nisin (Rogers 1928), the progenitor of the commercially-applicable peptide antibiotics, was one year earlier than the much more celebrated discovery of penicillin (Fleming 1929), still the benchmark of the clinically-significant non-proteinaceous antibiotics.
The success of nisin as a food preservative adjunct stimulated frenetic prospecting for alternative inhibitory agents that might find comparable application to food preservation. In 1976, the first review of the then burgeoning studies of the bacteriocinogenicity of Gram-positive bacteria (Tagg et al. 1976) predicted that this field would continue to flourish and that it would be largely motivated by the perceived potential for applications of these bacteriocins to bacterial interference and food preservation. Indeed, several groups of enthusiasts continued to explore the potential application of bacterial interference through the early days of the antibiotic era, mostly targeting Staphlyococcus aureus, due to its predilection for
Identifying new protective cultures and culture components 9
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X antibiotic resistance development. More recently however, bacterial interference
research has become more focused on modulation of the microflora of the human oral cavity in an attempt to control a variety of ailments ranging from halitosis to dental caries and streptococcal pharyngitis (Tagg and Dierksen 2003). Bacteriocin-producing probiotic strains to gain commercial traction for the control of oral infections are S. salivarius K12 (producer of the lantibiotics salivaricin A and salivaricin B) (Power et al. 2008) and the genetically-modified S. mutans JH1140 (producer of mutacin 1140 and mutacin IV) (Hillman et al. 2007).
Studies of the bacteriocins of LAB now dominate the literature in this field, most of the reports however containing only relatively superficial descriptions of bacteriocin activity spectra against randomly-selected collections of indicator bacteria and ending with optimistic predictions of the potential of these bacteriocins for commercial application. Few bacteriocins have actually lived up to these aspirations, among the more successful being nisin and the pediocins (class 2 bacteriocins of various Pediococcus species) (Schillinger et al. 1996; Paul Ross et al. 2002; Parada et al. 2007). An account of bacteriocinogenic LAB viewed as useful to the food industry has been prepared by Schillinger et al. (1996) and some examples that have progressed into commercial applications are presented in Table 1.3. More recent examples include the Lactococcus lactis producer of the two-component lantibiotic lacticin 3147, which has been used to control Listeria on the surface of smear-ripened cheese (O’Sullivan et al. 2006) and a leucocin-producing strain of Leuconostoc carnosum (Budde et al. 2003) which has been
Table 1.3 Examples of bacteriocins that may potentially be useful in the food industry Bacteriocin Producer Active against Food
applications Example Reference Nisin A L. lactis Variety of
Gram-positive strains
Dairy, bakery, vegetable products
Nisaplin® Paul Ross et al. (2002) Pediocin
PA-1 P.
acidilactici Listeria Processed
meats Alta 2341® Rodríguez et al. (2002) Leucocin
A/B L. carnosum
4010 Listeria, LAB Processed
meats SafePro®
B-SF-43 Budde et al.
(2003) Lacticin
3147 L. lactis Broad range of Gram-positive strains
Dairy, fermented meats, biomedical applications in humans and animals
No commercial product
O’Sullivan et al. (2006)
Lacticin 481 L. lactis LAB,
clostridia Cheese, meat products, vegetables
No commercial product
O’Sullivan et al. (2003)
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incorporated into a commercial bio-preservation product for the control of Listeria in stored meat.
One of the more recent strains to enter the commercial arena is Streptococcus macedonicus ACA-DC 198, a producer of the SA-FF22 look-alike lantibiotic macedocin (Georgalaki et al. 2002). It has been suggested that highly-competitive strains such as this that can be found as part of the natural microbiota of foodstuffs can perhaps be considered to mediate a rudimentary form of ‘immunity’ in food (Cotter et al. 2005). As such, the directed modification of the natural food microbiota by supplementation with safe but highly-competitive LAB is perhaps the equivalent of the microbiota-modification strategies adopted for the implementation of microbial interference/colonization resistance to prevent infections of the human host.