food biopreservation
1.7 Molecular methods of screening in food biopreservation
More recently, with the advent of molecular techniques capable of sequencing whole bacterial genomes within 24 hours, molecular screening methods have become feasible as a way of searching for desirable properties in candidate bacterial strains for food preservation. There are now approximately 950 complete bacterial genomes available through the NCBI database, with the elucidation of a further 2100 genomes in progress and additional projects being added continually.
This presents a huge resource for those interested in identifying new bacteriocins, antibiotics and other factors of potential value for the food industry. Each genome of a species can be searched for genes or gene clusters of interest such as those having homology to known bacteriocins, or those encoding for non-ribosomally synthesized peptides (NRSP) such as bacitracin. Once such genes are identified, the gene products can be further characterized for their actual function and strains naturally producing them can then be searched for either in culture collections or in foods of interest with the intention of identifying a strain useful for food production or preservation purposes.
In many genomes however, small open reading frames (ORFs) have not been adequately identified or annotated and often many bacteriocin-encoding genes can be missed. No doubt this issue will be resolved in the future but for now it is important that researchers should not simply rely on the publicly annotated version, but should look closely at small ORFs for their potential to be bacteriocins.
An example of this approach was by Dirix and colleagues (Dirix et al. 2004a;
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Dirix et al. 2004b) who screened for the presence of potential bacteriocins/signal pheromones having double glycine leader sequences in both Gram-positive and negative genomes. They determined that 33% of genomes from Gram-negative bacteria contain one or more transporters carrying a Peptidase C39 domain, compared to 44% of the genomes of Gram-positive bacteria. In addition, more than 40% of the identified peptide genes were either un-annotated or had not yet been recognized as secreted peptides in the genome-sequencing projects.
A second web-based peptide bacteriocin search engine has been developed which has been given the acronym BAGEL (de Jong et al. 2006). BAGEL has been designed to apply a number of ORF prediction tools that take into account genes involved in bacteriocin biosynthesis machinery, regulation, transport function and immunity increasing the likelihood of identifying these loci in genomes. For example, a BAGEL evaluation of the Streptococcus pneumoniae TIGR 4 genome identified 11 significant peptide bacteriocin genes – including all seven originally annotated bacteriocins (Tettelin et al. 2001) and four additional ones. In addition, a further 18 potential bacteriocin genes and 44 ORFs having some homology to bacteriocin genes were identified (Nes et al. 2007).
It is important to note, however, that the mere presence of a bacteriocin structural gene in a genome does not mean that it is produced. For example, in S. pyogenes most M-serotypes encode the salivaricin A structural gene but only M-type 4 S. pyogenes have so far been found to produce the active peptide (Johnson et al. 1979; Simpson et al. 1995). This has been determined to be due to deletions in other genes within the locus such as the transporter and modification genes (Upton et al. 2001; Wescombe et al. 2006a). Similarly, sakacin A structural genes are present within an 8.7 kb sequence on a 60 kb plasmid in L. sakei Lb706 and also in its non-bacteriocinogenic analogue L. sakei Lb706-B (Schillinger and Lücke 1989; Axelsson and Holck 1995). In the case of L. sakei Lb706-B, original plasmid curing attempts using acriflavin caused a mutation in the HPK (sapK) gene region responsible for transport functions resulting in the loss of ability to externalize the active bacteriocin (Axelsson et al. 1993) (pers comm. Urlich Schillinger, May 2007). Additionally, it can be difficult to detect the action of some of the bacteriocins since many are extremely limited in their inhibitory spectrum and this can make the identification of the susceptible species also difficult. Furthermore, bacteriocins can be highly regulated and only expressed in certain circumstances which may not be easily replicated in vitro (Nes and Eijsink 1999; Rawlinson et al. 2002). Such factors mean that phenotypic screening is probably the preferred option for initially identifying strains of interest while molecular methods can be used to further narrow the search down and provide information around the useful aspects of each chosen strain.
Genetic engineering of strains either to disable virulence genes or to increase the efficacy of a particular strain is yet another way to use the power of molecular biology to select or create new strains useful for food preservation. Many bacteriocins are encoded for on plasmids or transposable elements which can be transferred either naturally, or through standard molecular procedures to another
Identifying new protective cultures and culture components 19
12 34 56 78 910 12 34 56 78 920 12 34 56 78 930 12 34 56 78 940 12 4344 45X strain or even species (Jack et al. 1995; Wescombe et al. 2006b). This allows for
the introduction of useful characteristics to species of interest either for the prevention of food spoilage in fermented foods or for other downstream properties such as useful probiotic qualities or survival mechanisms. An example of such an engineered strain is S. mutans BCS3-L1, in which recombinant DNA methods have been used to delete essentially the entire open reading frame (ORF) for lactic acid dehydrogenase (LDH). This mutation created a metabolic blockade that was lethal when exchanged for the wild-type allele, but it was found that replacing the ORF for LDH with the ORF for alcohol dehydrogenase B from Zymomonas mobilis overcame this blockade to yield a viable strain called BCS3-L1 that produced wild-type levels of mutacin 1140 and no lactic acid (Chen et al. 1994;
Hillman et al. 1994; Hillman et al. 2000). Due to the modifications, the strain has significantly reduced cariogenicity, but excellent colonization potential through the production of a natural antibiotic called mutacin 1140. Further modifications were able to be introduced for use in human clinical trials to enable rapid elimination of the strain in case of adverse side effects and to increase genetic stability (Hillman et al. 2007).
The genetic locus for the production of nisin has been used as the basis for a Gram-positive expression system capable of specifically over-expressing genes of interest at controlled time points due to the ability to induce gene expression in response to the addition of exogenous nisin (Kuipers et al. 1995). This system has been named the ‘NIsin Controlled gene Expression system’ or NICE. This powerful system has been able to be transferred into other species including Leuconostoc lactis, Lactobacillus sp., Streptococcus sp., Enterococcus sp. and Bacillus subtilis enabling advances in understanding the pathogenicity of some of the bacteria and helping facilitate dose-response studies for live vaccine work (Kleerebezem et al. 1997; Mierau and Kleerebezem 2005). One of the major problems with this approach is codon usage of the gene to be expressed. Genes from genera closely related to Lactococcus generally have little trouble with expression while those from other organisms depend largely on their use of rare codons for successful expression (Mierau and Kleerebezem 2005). To get around this issue, alternative systems also based on two-component regulatory systems of bacteriocins have been developed such as the SURE system for B. subtilis (Kleerebezem et al. 2004), L. plantarum (Mathiesen et al. 2004), L. sakei (Axelsson et al. 2003) and Enterococcus sp. (Hickey et al. 2003), all of which help to improve the range of Gram-positive organisms that can be genetically manipulated to express proteins of interest. Some of the applications of these systems have included the expression of enzymes for use in food applications such as phage lysins or peptidases and esterases to influence flavour formation in dairy fermentations (de Ruyter et al. 1997; Wegmann et al. 1999; Hickey et al.
2004; Berlec and Strukelj 2009). Given time and a wider acceptance of genetically modified organisms by the public it would be envisioned that the majority of strains used in food preservation will be engineered derivatives combining traits of stability, bacteriocin production and flavour or texture enhancing properties from different species.
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