Novel bacteriocins from lactic acid bacteria (LAB): various structures and applications
© Perez et al.; licensee BioMed Central Ltd. 2014
Published: 29 August 2014
Bacteriocins are heat-stable ribosomally synthesized antimicrobial peptides produced by various bacteria, including food-grade lactic acid bacteria (LAB). These antimicrobial peptides have huge potential as both food preservatives, and as next-generation antibiotics targeting the multiple-drug resistant pathogens. The increasing number of reports of new bacteriocins with unique properties indicates that there is still a lot to learn about this family of peptide antibiotics. In this review, we highlight our system of fast tracking the discovery of novel bacteriocins, belonging to different classes, and isolated from various sources. This system employs molecular mass analysis of supernatant from the candidate strain, coupled with a statistical analysis of their antimicrobial spectra that can even discriminate novel variants of known bacteriocins. This review also discusses current updates regarding the structural characterization, mode of antimicrobial action, and biosynthetic mechanisms of various novel bacteriocins. Future perspectives and potential applications of these novel bacteriocins are also discussed.
Main differences between (LAB) bacteriocins and conventional antibiotics.
Intensity of bioactivity
Active at nano-to-micro molar range
Active at micro-to-milli molar range
Proteolytic enzyme degradability
Active pH range
Amenability to bioengineering
Possible mechanism of target cell developing resistance
Adaptation through changes in cell membrane composition
Genetically transferable determinant that inactivates the active compound
Mode of action
Pore formation, inhibition of cell wall biosynthesis
Cell membrane or intercellular targets
Toxicity towards eukaryotic cells
Bacteriocin classification with representative bacteriocins isolated in our laboratory.
Lantibiotics, small (<5 kDa) peptides containing lanthionine and β-methyllanthionine
Nukacin ISK-1 
Small (<10 kDa), heat-stable, non-lanthionine-containing peptides
Small heat-stable peptides, synthesized in a form of precursor which is processed after two glycine residues, active against Listeria, have a consensus sequence of YGNGVXC in the N-terminal
Leucocin A 
Two component systems: two different peptides required to form an active poration complex
Lactococcin Q ,
Enterocin NKR-5-3AZ ,
Enterocin X 
N- and C- termini are covalently linked, resulting in a circular structure
Other class II bacteriocins, including sec-dependent bacteriocins and leaderless bacteriocins
Large molecules heat sensitive peptides
Class IIa bacteriocins have a distinct conserved sequence (YGNGVXC) in the N-terminal region that is responsible for their high potency against the food pathogen Listeria monocytogenes [15, 16]. The class IIb bacteriocins are two-peptide bacteriocins that require both peptides to work synergistically to be fully active [17, 18]. Class IIc bacteriocins, arguably the most poorly understood of the bacteriocins, are grouped based on their circular structural configuration. The N- and C-termini of class IIc bacteriocins are covalently linked giving the peptide an extremely stable structure [19, 20]. On the other hand, class IId bacteriocins comprise the remaining bacteriocins combined as miscellaneous, or as a one-peptide non-pediocin linear group . Sec- dependent bacteriocins  and leaderless bacteriocins  belong to this class.
Despite the huge potential of bacteriocins in a number of applications, nisin remains the only commercially available bacteriocin used for food applications. However, the use of bacteriocins in clinical settings has been limited to animal, rather than human, health . Nevertheless, the approval of nisin for application in food by the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives, as well as the approval by the US Food and Drug Agency (FDA) for its use in pasteurized, processed cheese spreads, should establish a legal precedent for the use of other bacteriocins as food preservatives. In clinical settings, the increasing number of researchers developing bioengineered bacteriocins with enhanced properties targeting clinical pathogens should fast track the widespread use of bacteriocins. Thus, it is important to discover more novel bacteriocins with unique properties in order to enhance the arsenal of antimicrobials we have for controlling both undesirable bacteria in food and clinical pathogens. In this review we describe an efficient screening system employed in our lab that fast tracks the discovery of novel LAB bacteriocins. We also discuss a few examples of novel bacteriocins that are studied in our lab, describing their structures and characteristics as well as highlighting their exemplary properties that could be deemed useful for their future applications.
Biology of bacteriocins
Bacteriocin production is widespread among bacteria. It has been suggested that the majority of bacterial species synthesize bacteriocins [6, 12]. This is because their biosynthetic machineries are relatively simple and are often associated with transferable elements such as conjugative transposons or plasmids . As highlighted earlier, bacteriocins are ribosomally synthesized peptides. Genes related to bacteriocin biosynthesis are generally clustered, and are encoded on plasmids, chromosome and/or transposons with minimum genetic machinery consisting of structural cognate immunity genes . Bacteriocins are usually synthesized as biologically inactive prepeptides that include an N-terminal leader peptide attached to the C-terminal propeptide [5, 10, 12]. The leader peptide: (i) serves as a recognition site which directs the prepeptide towards maturation and transport proteins, (ii) protects the producer strain by keeping the bacteriocin in an inactive state while it is inside the producer strain, and (iii) interacts with the propeptide domain to ensure it is in a suitable conformation for enzyme-substrate interaction of the modification machinery [10, 23–25].
Like lantibiotics, class II bacteriocins are synthesized as an inactive prepeptide that usually contain a characteristic double-glycine proteolytic processing site [15, 16]. However, some class II bacteriocins are synthesized with a typical N-terminal signal sequence of a sec-dependent type, and secreted through the general secretory pathway . Unlike lantibiotics, class II bacteriocins do not undergo extensive post-translational modification. Following the translation of the prepeptide, it is processed by specific enzymes to cleave off the leader peptide concomitant with its translocation to the extracellular space through a dedicated ABC-transporter that sometimes require an accessory protein [15, 16]. However, there are a growing number of newly reported bacteriocins that lack leader sequences; these are of interest as they are active immediately after translation [21, 22, 28].
Fast tracking the screening of novel bacteriocins
Bacteriocins must be obtained in their purified form in order to be studied and characterized. However, it is well known that establishing a purification system for bacteriocins is expensive, time-consuming, and tedious. Moreover, in many cases, these systems often identify previously reported bacteriocins. This has led us to develop a rapid system that can screen for novel bacteriocins from various sources during the early stage of the screening and isolation of bacteriocin-producing strains. This has enabled us to save time, effort, and money in fast tracking the discovery of more novel bacteriocins.
When the molecular mass of the bacteriocin is determined, supernatants with or without pretreatment of overnight culture with LAB strains are injected to the ESI-LC/MS system for analysis. In cases where bacteriocin production is low, in which direct LC/MS analysis of the supernatant is unable to visualize possible bacteriocin signals, sample pretreatment of the culture supernatants is essential to remove any media borne impurities, and to concentrate the bacteriocin in the supernatant. The mass detector is manually set at the mass range of m/z 500-3000, which is the range of molecular mass for most bacteriocins . This enabled us to determine the molecular mass of the active compound in the supernatant, which is compared to the molecular mass of reported bacteriocins. When molecular weights of the bacteriocins do not resemble any previously reported strains, we consider them novel. Moreover, in cases where no molecular masses are detected from the ESI-LC/MS analyses, we still classify them as potentially novel to minimize the possibility of false negative results.
For statistical analysis, antimicrobial spectra of the LAB strain are determined using the spot-on-lawn method  against a set of indicator strains (usually about 10 strains), and are analysed using PCA with the data sets from reported bacteriocins as the correlating variable. In most cases, bacteriocins are clustered into three groups, nisin variants, class IIa (anti-Listeria) bacteriocins, and narrow-spectrum bacteriocins. Based on our experience, most novel bacteriocins do not fall within any of these clusters. Nevertheless, novel bacteriocins falling within a distinct cluster are sometimes observed, especially novel variants of known bacteriocins. Thus, data obtained from the PCA should be combined with that from the molecular mass analysis .
Structural determination and characterization of novel LAB bacteriocins
Novel nisin variant
To illustrate the effectiveness of the screening system described above, one could refer to the paper that reported the discovery of the nisin variant, nisin Q . Moreover, Zendo et al. (2008) clearly described the capacity of the system to discriminate between nisin variants A, Z, and Q . Nisin Q, the third nisin variant to be reported, was isolated from Lactococcus lactis 61-14, an LAB isolated from a Japanese river . Although nisin Q appears to have similar structure to that of nisin A and Z, which were analysed by nuclear magnetic resonance (NMR) spectroscopy , gene annotation of the nisin Q locus revealed significantly lower similarity (only 82%) to other nisin variants, whereas nisin A and Z have almost identical gene loci. This indicates that, from an evolutionary perspective, the locus of nisin Q is at a greater genetic distance from the loci of both nisin A and Z. Nevertheless, nisin Q still shares the same biosynthetic pathway with other nisin variants as it was successfully produced when its structural gene, nisQ, was introduced to the nisin Z-producing strain together with nisin Z. Furthermore, nisin Q can stimulate pheromone activity in the regulation system of nisin A (NisRK), albeit to a much lower degree than that by nisin A .
Novel class IIa: anti-listerial bacteriocins
The high specificity of class IIa bacteriocins against the psychrophilic food pathogen L. monocytogenes has brought this group to prominence in its potential use against this lethal pathogen. Enterocin NKR-5-3C (Ent53C) is one of the bacteriocins produced by Enterococcus faecium NKR-5-3, an LAB isolated from the Thai fermented fish Pla-ra [35, 36]. Ent53C showed very strong microbial activity (in nanomolar range) against Listeria spp. and other Gram-positive species, a typical characteristic of class IIa bacteriocins .
Novel class IIb: two-peptide bacteriocins
While most bacteriocins exist as a single active peptide molecule, two-peptide (class IIb) bacteriocins, as the name suggests, consist of two different individual peptide molecules that require equal amounts of each peptide to exert its optimal antimicrobial activity [17, 42]. In our laboratory, we have three novel bacteriocins belonging to this class: lactococcin Q (Figure 4B) , enterocin X , and enterocin NKR-5-3AZ .
Lactococcin Q, a novel two-peptide bacteriocin isolated from L. lactis QU 4, an LAB isolated from corn, is comprised of two peptides: Qα and Qβ . These two peptides show high similarity to lactococcin Gα and Gβ, peptides that comprise the two-peptide bacteriocin lactococcin G . Both lactococcins G and Q have very narrow and specific antimicrobial spectra, with antimicrobial activity only against strains derived from L. lactis [43, 45]. Structural analysis of lactococcin Q peptides showed the presence of α-helical structures in the same positions as in lactococcin G, thus suggesting that these bacteriocins have similar modes of action . Furthermore, hybrid combinations of these homologous lactococcins have identical activity spectra to the original combinations, although some lead to activities distinct from the original. When mixed with an equimolar concentration of Gβ, Lactococcin Qα had 32 times lower specific activity than the original combination, whereas Gα and Qβ in combination had specific activity comparable to the original lactococcin Q combination .
Enterocin X is a novel two-peptide bacteriocin from E. faecium KU-B5 that has different activity against target microorganisms than to its component peptides, enterocins Xα and Xβ. When equimolar concentrations of these peptides were tested against a panel of indicator strains, the combined antimicrobial activity was not uniformly enhanced, with activities of 0.13-130-fold or 1.0-1020-fold than that of Xα and Xβ alone, respectively .
Conversely, enterocin NKR-5-3AZ (Ent53AZ) is one of the multiple bacteriocins produced by the bacterial strain E. faecium NKR-5-3 . This bacteriocin showed very high homology to another two-peptide bacteriocin, brochocin-C, which is comprised of brochocins A and B . Although purification of Ent53Z from the culture supernatant of strain NKR-5-3 was unsuccessful , as occurred with its homologue, brochocin B , its putative structural gene, ent53Z, was found directly downstream of the putative structural gene of Ent53A in the genome of NKR-5-3 .
Novel class IIc: circular bacteriocins
Circular bacteriocins (class IIc) are characterized by their unique structural feature of a head-to-tail cyclization of their backbones [19, 20]. The circular nature of their structure provides greater structural stability, higher thermal stress resistance, and superior stability against proteolytic digestion, compared to their linear counterparts [47, 48]. Owing to their exceptional sturdiness, the use of circular bacteriocins as food preservatives and stable antimicrobial agents for clinical setting is seriously being considered. However, in order for this full potential to be recognized, it is important that their biosynthetic mechanisms are understood, which currently remains unknown.
In our laboratory, we have isolated two novel circular bacteriocins that share high identity with each other despite their producer strains belonging to distinct genera. Lactocyclicin Q (LycQ), isolated from Lactococcus sp. QU 12 , and leucocyclicin Q (LcyQ), isolated from Leuconostoc mesenteroides TK41401 , are both circular bacteriocins composed of 61 amino acid residues (Figure 4C). DNA sequence analysis of structural genes revealed their identical nature. LycQ and LcyQ share 72% DNA sequence identity. Moreover, both their precursor peptides contain a leader sequence of two amino acid residues in which cyclization (to yield the mature bacteriocin) occurs between L3 and W63 . Secondary structure prediction analyses of both bacteriocins revealed four identical α-helices, all having subtle amphiphilic characteristics that are thought to play an important role in their antimicrobial action.
Like any typical circular bacteriocin, LycQ and LcyQ both exhibit high stability against thermal, pH, and proteolytic enzyme stresses [49, 50]. For instance, LycQ maintains full activity after exposure to 121°C for 15 min under acidic condition, as well as exposure to a pH range of 3.0-9.0 . Other bacteriocins, such as nisin Z, lose the majority of their antimicrobial activity when exposed to similar conditions .
As with other circular bacteriocins, the biosynthetic mechanisms of LycQ and LcyQ remain unclear. Although attempts to heterologously express LcyQ failed, identification of its putative biosynthetic gene cluster was recently reported by Mu et al. , who also identified the biological function of LcyD in the maturation of LcyQ. LcyD is a protein belonging to a large family of proteins of unknown functions - these are the DUF95 superfamily membrane proteins that are widespread in the biosynthetic gene clusters of circular bacteriocins [19, 20].
Novel leaderless bacteriocins (class IId)
While most bacteriocins are synthesized as inactive prepeptides, which contain an N-terminal leader peptide attached to the C-terminal propeptide moiety , some bacteriocins are atypical in the sense that they are synthesized without an N-terminal leader sequence. Since the leader peptides of typical bacteriocins have essential roles in recognition sites for secretion and maturation during the bacteriocin synthesis, as well as to protect the producer strain by keeping them in an inactive state while still inside the producer bacterial cells, leaderless bacteriocins are presumed to have unique biosynthetic mechanisms [10, 53]. From an application perspective, leaderless bacteriocins have promising commercial potential since they can be readily synthesized without the need to cleave the leader peptide. This makes scale-up production considerably easier, even in heterologous eukaryotic production systems.
In our laboratory, we have isolated and studied a number of novel leaderless bacteriocins. For example, lacticin Q and its homologue lacticin Z were produced by L. lactis QU 5 and L. lactis QU 14 respectively. Strain QU 5 is an LAB isolated from corn  while strain QU 14 was isolated from horse intestine . In addition, Weissella hellenica QU 13, an LAB isolated from Japanese pickles, Takana-zuke, was recently found to produce two novel leaderless bacteriocins, weissellicins Y and M .
Lacticin Q and Z share 94% identity, varying only in three amino acid residues at positions 10, 33, and 44. Furthermore, they both have a formylated-methionine at the initiation residue (Figure 4D). They are both 53-amino-acid highly cationic peptides that show very strong antimicrobial activity (at nanomolar concentrations) as well as high stability against various stresses [22, 54]. Lacticin Q contains two amphiphilic helices that play an important role in its antimicrobial activity [56–58]. Owing to the very high homology and comparable activity spectra of these two leaderless bacteriocins, it was inferred that they have the same mode of antimicrobial action .
The genes involved in the biosynthesis of lacticin Q and Z were recently elucidated . The biosynthetic gene clusters lnqBCDEF and lnzBCDEF are involved in the secretion and self-immunity of lacticin Q and Z, respectively. Overexpression of lnqQ in L. lactis NZ9000 resulted in the intracellular accumulation of lacticin Q. This led to growth suppression as a result of the intracellular toxicity of the bacteriocin, but the growth suppression was circumvented when lnqBCDEF was co-expressed . Further analyses on the functions of the gene products of lnqBCDEF revealed that secretion of lacticin Q to the extracellular space is strictly controlled by the gene products of this gene cluster while the self-immunity system is more flexible. The ABC transporter-type immunity LnqEF is sufficient to confer minimum immunity, whereas LnqBCD are thought to be accessory proteins that support the activity of LnqEF .
Applications of LAB bacteriocins
LAB have been associated with food fermentations dating all the way back to ancient times due to their beneficial influence on nutrition, organoleptic, and shelf-life of foods . However, it is their ability to produce antimicrobial peptides, bacteriocins, that have made them particularly promising in both food and pharmaceutical industries. In the food industry, bacteriocins have huge potential in the biopreservation of various foods, either alone, or in combination with other methods of preservation, which is known as hurdle technology . The antimicrobial activity of many bacteriocins, especially the class IIa bacteriocins, against the highly pathogenic and food-borne L. monocytogenes offers an ideal solution to the problem caused by this sturdy pathogen, which is commonly reported to contaminate ready-to-eat refrigerated food products . There are different approaches by which bacteriocins may be applied to food systems. While the use of bacteriocin as a food preservative in its purified form is the most commonly used approach, direct inoculation of bacteriocin-producing LAB into the food system is also effective. The pediocin PA-1/AcH-producing Pediococcus pentosaceus BCC 3772 when used as starter culture for Nham, a traditional Thai fermented pork sausage, effectively controlled the growth of L. monocytogenes without compromising the quality of Nham . The utilization of the product that is fermented by a bacteriocin-producing LAB as a raw material for food processing has also been shown to be effective . In addition, the incorporation of bacteriocins into the food packaging film or surfaces has also been explored [64, 65]. In our recent study, when pediocin PA-1/AcH was incorporated into a biocomposite packaging film, the initial load of L. monocytogenes on the meat surface was significantly reduced .
The clinical potential of bacteriocins has been the subject of on-going investigation by many scientists all over the world due to the activity of some bacteriocins against Gram-positive human and animal pathogens, including MDR pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) strain and vancomycin-resistant Enterococcus faecalis (VRE) strain [12, 66, 67]. In addition, biofilm associated infections among patients with indwelling medical devices have remained a challenge for medical researchers. In our recent study, we showed that nisin A and lacticin Q exert strong bactericidal activities against MRSA both in its planktonic and biofilm cells, whereas vancomycin, the commonly used antibiotic to treat MRSA infections, showed bactericidal activity against planktonic cells only .
Furthermore, because bacteriocins are ribosomally synthesized, they have relatively simpler biosynthetic mechanisms compared to those of conventional antibiotics. The gene-encoded nature of bacteriocins makes them easily amenable through bioengineering to either increase their activity or specify target microorganism. Owing to this feature of bacteriocins, antibiotic therapy would become less damaging to the natural gut microflora, which is a common drawback of conventional antibiotic use.
In Japan, as well as the utilization of nisin as a food preservative, we succeeded in developing a nisin A-containing hand wash and oral hygiene gel [68, 69]. This is anchored on the food-grade distinction of nisin A, an important prerequisite of the government regulation for the application of bacteriocin in Japan . This nisin A-containing hand wash is better in terms of stability and bioactivity compared to general hand wash. Although antimicrobial activities of most bacteriocins are limited against Gram-positive bacteria, it is well known that nisin and other bacteriocins become potent against Gram-negative pathogens when they are exposed to agents, such as surfactants, that could compromise the integrity of their outer membranes [71, 72]. Whereas, Oralpeace™, was shown to be effective in controlling tooth cavities (caused by Streptococcus mutans) and bacterial gingivitis (caused by Porphyromonas gingivalis) [68, 69, 73]. Oralpeace™ contains only natural edible materials and compounds, making it a better option for individuals who have difficulty gargling, such as the elderly and physically challenged individuals. Furthermore, in order to prolong the effectiveness of nisin in application systems, we successfully developed a liposome-encapsulated nisin that showed significantly prolonged activity than "naked" nisin in inhibiting the synthesis of S. mutans glucan-biofilm .
On the other hand, the use of nisin in the veterinary industry as a preventive drug and as a cure for bovine mastitis has also been explored. Bovine mastitis is a disease that has a major economic impact, not only in Japan, but also in the worldwide dairy industry, as it is the major source of economic loss among cattle farmers. However, there is a rising concern regarding the rampant use of conventional antibiotics to control bovine mastitis, which has resulted in antibiotic contamination in milk. We have shown that our nisin-based injectable drug can control almost 99.9% of bacteria causing mastitis such as Staphylococcus aureus and Streptococcus agalactiae 60 s after drug administration .
Furthermore, in order to gain insights and preempt any possible mechanism of the emergence of bacteriocin-resistant strains, we studied the two-component systems (TCSs) of various commensal strains to bacteriocin-producing LAB strains such as S. aureus and S. mutans. Two novel TCSs NsrRS (nisin A-resistant TCS) and LcrRS (lacticin 481-resistant TCS) in S. mutans were found to be associated with resistance against the lantibiotics nisin A, nukacin ISK-1, and lacticin 481. NsrRS regulates expression of the protein NsrX that functions like the nisin immunity protein NisI, which binds to the nisin molecule. LcrRS on the other hand, was found to regulate the expression of the ABC transporter LctFEG, which also functions as an immunity protein complex for lacticin 481 . Additionally, in the case of S. aureus, we demonstrated that there are several TCS associated with its resistance to antimicrobial compounds produced by other bacteria. BraRS (previously reported to be associated with bacitracin resistance) system functions specifically to negate the antimicrobial action of nisin A and nukacin ISK-1, whereas GraRS (reportedly involved in susceptibility to cationic peptides such as defensins, gentamicin, and vancomycin) and VraRS (vancomycin resistance-associated TCS) confer broad-spectrum resistance against general cationic peptides and cell-wall synthesis inhibiting compounds, respectively .
Bacteriocins from Generally Recognized as Safe (GRAS) LAB have continued to gain great interest among an increasing number of research groups due to their huge application potential both in food, and in pharmaceutical industries. In the food industry, bacteriocins have long been proposed as a solution to the problems of food spoilage and food-borne infections. However, up to now, nisin remains the only commercially available and industrially utilized bacteriocin despite a vast array of bacteriocins being discovered in the past two decades. It was suggested that the combined lack of awareness of what bacteriocins can achieve in food systems, and the lack of enthusiasm to move away from existing food-preservation techniques, is the reason for the under-utilization of bacteriocin in the food industry . Fortunately, there is an upward trend of both consumer preference and regulatory demand toward minimally processed food products without the use of chemical preservatives. This provides a genuine opportunity that could jump-start the widespread application of bacteriocins in the food industry. However, the use of bioengineered bacteriocins for food applications could face consumer resistance as in the case Genetically Modified Organisms (GMO's). As anti-GMO advocates have always argued, safety of their use must first be thoroughly evaluated.
On the other hand, perhaps one of the greatest concerns that human kind is facing in the 21st century is the growing problem of MDR pathogens and the rapidly decreasing antibiotic arsenal to combat them. Researchers around the world are scrambling for possible alternatives to address this problem. The high specific activity of some bacteriocins against clinical pathogens, even against MDR strains, offers a possible solution to this growing problem. But perhaps it is their amenability to bioengineering that makes them exceptionally promising. There is an increasing number of reported bioengineered bacteriocins with narrow spectrum of activity (which minimize damage to the natural gut flora), enhanced bioactivity, and higher stability. Furthermore, the cyclization mechanism of circular bacteriocins could open up new ways to engineer circular therapeutic compounds to enhance their stability.
The advancement of science and technology has led to an increase in bacteriocin research, in terms of both quality and quantity, and consequently more novel bacteriocins with unique properties have been reported. It is apparent that there is still more to learn about this family of peptide antibiotics. Thus, in order to further enhance the bacteriocin arsenal against these undesirable microorganisms (spoilage and pathogens) it is important, not only to advance the study (mode of antimicrobial action and their biosynthetic mechanisms) of known bacteriocins, but also to continue the search for more novel bacteriocins with promising properties.
The studies described here were supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Grant number 24380051) and Novozymes Japan Research Fund. We are also grateful to the Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho), Japan for providing a scholarship grant to R.H. Perez.
This article has been published as part of Microbial Cell Factories Volume 13 Supplement 1, 2014: Proceedings of the 11th International Symposium on Lactic Acid Bacteria. The full contents of the supplement are available online at http://www.microbialcellfactories.com/supplements/13/S1. Publication charges for this supplement were funded by the Stichting Symposium on Lactic Acid Bacteria. The articles have undergone the journal's standard peer review process for supplements. EJ is employed by Chr Hansen A/S, is a shareholder in Chr Hansen A/S; and has applied for patents relating to the content of an article which he did not edit but which forms part of this supplement. MK is an employee of Nizo. The other Supplement Editors declare that they have no competing interests.
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