Michael E. Stiles

Lactic acid bacteria of foods and their current taxonomy

Application of molecular genetic techniques to determine the relatedness of food-associated lactic acid bacteria has resulted in significant changes in their taxonomic classification. During the 1980s the genus Streptococcus was separated into the three genera Enterococcus, Lactococcus and Streptococcus. The lactic acid bacteria associated with foods now include species of the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. The genus Lactobacillus remains heterogeneous with over 60 species (ymol% G+C content ranging from 33 to 55), of which about one-third are strictly heterofermentative. However, many changes have been made and reorganization of the genus along lines that do not follow previous morphological or phenotypic differentiation from Leuconostoc and Pediococcus is being studied. Phylogenetically belonging to the Actinomyces branch of the bacteria, Lactobacillus bifidus has been moved to the genus Bifidobacterium also on account of its greater than 50 mol% G+C content. It is therefore no longer considered one of the lactic acid bacteria senso strictu, which form part of the Clostridium branch of the bacteria. The new genus Weissella has been established to include one member of the genus Leuconostoc (Leuc, paramesenteroides) and heterofermentative lactobacilli with unusual interpeptide bridges in the peptidoglycan. Contrary to the clear-cut division of the streptococci, morphological and physiological features of Weissella do not directly support this grouping which now incorporates species that produce D(-)- as well as DL-lactate. The new genus Carnobacterium is morphologically similar to the lactobacilli, but it shares some physiological similarities (e.g. growth at pH 9.5) and a common phylogenetic branch with the genus Enterococcus. The review includes information on the taxonomic changes and the relationship of the bacteria of food fermentation and spoilage.

Enterococci at the crossroads of food safety

Enterococci are gram-positive bacteria and fit within the general definition of lactic acid bacteria. Modern classification techniques resulted in the transfer of some members of the genus Streptococcus, notably some of the Lancefields group D streptococci, to the new genus Enterococcus. Enterococci can be used as indicators of faecal contamination. They have been implicated in outbreaks of foodborne illness, and they have been ascribed a beneficial or detrimental role in foods. In processed meats, enterococci may survive heat processing and cause spoilage, though in certain cheeses the growth of enterococci contributes to ripening and development of product flavour. Some enterococci of food origin produce bacteriocins that exert anti-Listeria activity. Enterococci are used as probiotics to improve the microbial balance of the intestine, or as a treatment for gastroenteritis in humans and animals. On the other hand, enterococci have become recognised as serious nosocomial pathogens causing bacteraemia, endocarditis, urinary tract and other infections. This is in part explained by the resistance of some of these bacteria to most antibiotics that are currently in use. Resistance is acquired by gene transfer systems, such as conjugative or nonconjugative plasmids or transposons. Virulence of enterococci is not well understood but adhesins, haemolysin, hyaluronidase, aggregation substance and gelatinase are putative virulence factors. It appears that foods could be a source of vancomycin-resistant enterococci. This review addresses the issue of the health risk of foods containing enterococci.

Biopreservation by lactic acid bacteria.

Biopreservation refers to extended storage life and enhanced safety of foods using the natural microflora and (or) their antibacterial products. Lactic acid bacteria have a major potential for use in biopreservation because they are safe to consume and during storage they naturally dominate the microflora of many foods. In milk, brined vegetables, many cereal products and meats with added carbohydrate, the growth of lactic acid bacteria produces a new food product. In raw meats and fish that are chill stored under vacuum or in an environment with elevated carbon dioxide concentration, the lactic acid bacteria become the dominant population and preserve the meat with a ‘hidden’ fermentation. The same applies to processed meats provided that the lactic acid bacteria survive the heat treatment or they are inoculated onto the product after heat treatment. This paper reviews the current status and potential for controlled biopreservation of foods.

Enterococci in foods—a conundrum for food safety

Enterococci form part of the lactic acid bacteria (LAB) of importance in foods. They can spoil processed meats but they are on the other hand important for ripening and aroma development of certain traditional cheeses and sausages, especially those produced in the Mediterranean area. Enterococci are also used as human probiotics. However, they are important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics, but antibiotic resistance alone cannot explain the virulence of some of these bacteria. Virulence factors such as adhesins, invasins and haemolysin have been described. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some harbour virulence traits and generally, Enterococcus faecalis harbours more of them than Enterococcus faecium. Regulations in Europe stipulate that safety of probiotic or starter strains is the responsibility of the producer; therefore, each strain intended for such use should be carefully evaluated. For numerous questions, immediate answers are not fully available. It is therefore suggested that when considering an Enterococcus strain for use as a starter or probiotic culture, it is imperative that each particular strain should be carefully evaluated for the presence of all known virulence factors. Ideally, such strains should harbour no virulence determinants and should be sensitive to clinically relevant antibiotics. In general, E. faecium appears to pose a lower risk for use in foods, because these strains generally harbour fewer recognised virulence determinants than E. faecalis. Generally, the incidence of such virulence determinants among E. faecium strains is low, as compared to E. faecalis strains, probably as a result of the presence of pheromone-responsive plasmids.

Double‐glycine‐type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis

Many non‐lantibiotic bacteriocins of lactic acid bacteria are produced as precursors which have N‐terminal leader peptides that share similarities in amino acid sequence and contain a conserved processing site of two glycine residues in positions ‐1 and ‐2. A dedicated ATP‐binding cassette (ABC) transporter is responsible for the proteolytic cleavage of the leader peptides and subsequent translocation of the bacteriocins across the cytoplasmic membrane. To investigate the role that these leader peptides play in the recognition of the precursor by the ABC transporters, the leader peptides of leucocin A, lactococcin A or colicin V were fused to divergicin A, a bacteriocin from Carnobacterlum divergens that is secreted via the cells general secretion pathway. Production of divergicin was monitored when these fusion constructs were introduced into Leuconostoc gelidum, Lactococcus lactis and Escherichia coli, which carry the secretion apparatus for leucocin A, lactococcins A and B, and colicin V, respectively. The different leader peptides directed the production of divergicin in the homologous hosts. In some cases production of divergicin was also observed when the leader peptides were used in heterologous hosts. For ABC‐transporter‐dependent secretion in E. coli the outer membrane protein TolC was required. Using this strategy, colicin V was produced in L. lactis by fusing this bacteriocin behind the leader peptide of leucocin A.

Characteristics and genetic determinant of a hydrophobic peptide bacteriocin, carnobacteriocin A, produced by Carnobacterium piscicola LV17A.

Carnobacteriocin A is a hydrophobic nonlantibiotic bacteriocin that is detected early in the growth cycle of Carnobacterium piscicola LV17A and encoded by a 49 MDa plasmid. The bacteriocin was purified using hydrophobic interaction and gel filtration chromatography, and reversed-phase HPLC. Three different active peaks (A1, A2 and A3) were detected, but the purified samples had identical N-terminal amino acid sequences for the first 15 amino acids as determined by Edman degradation analysis. Only a 2.4 kb fragment of the EcoRI digest of the plasmid pCP49 hybridized with a 23-mer oligonucleotide probe derived from amino acids 5 to 13 of the amino acid sequence. The structural gene for carnobacteriocin A is located 600 base pairs into the 2.4 kb EcoRI fragment, but no other genetic information was detected on this unit. The structural gene includes an 18 amino acid N-terminal extension of the bacteriocin, ending with Gly-Gly residues in the -2, -1 positions with respect to the cleavage site. The bacteriocin consists of 53 amino acids that differ markedly from the majority of hydrophobic peptide bacteriocins characterized to date. Based on the amino acid sequence derived from the nucleotide sequence a molecular mass of 5052.85 Da was calculated. Mass spectrometric analysis showed that the molecular mass of the major component (A3) was 2 Da lower, thereby indicating the presence of a disulphide bridge between Cys 22 and Cys 51. Carnobacteriocin A2 has a similar structure except that Met 52 is oxidized to a sulphoxide, whereas A1 appears to be a mixture of peptides derived proteolytically from A3 or A2.

Effect of growth of selected lactic acid bacteria on storage life of beef stored under vacuum and in air.

The effect of growth of different types of lactic acid bacteria (LAB) on the storage life of normal pH beef was determined anaerobically (under vacuum) and aerobically. Four LAB from meat were inoculated separately onto sterile slices of lean beef. Inoculated samples were stored anaerobically at 2 degrees C for 10 weeks or stored aerobically in an oxygen permeable film at 7 degrees C for 10 days, with and without previous storage under vacuum at 2 degrees C. The LAB strains used were Carnobacterium maltaromicus (previously C. piscicola) LV17 and UAL26, Leuconostoc gelidum UAL187-22 and Lactobacillus sake Lb706. Storage life was determined by sensory panel evaluation of colour and odour. Under anaerobic conditions Lb. sake Lb706, inoculated at log 2 CFU/cm2, grew rapidly to reach maximum population within three weeks of storage. L. gelidum UAL187-22 also grew on the meat but at a slower rate. In contrast, growth of C. maltaromicus LV17 and UAL26 was unpredictable, achieving maximum population after 2 to 8 weeks. None of the test strains caused spoilage of the meat within the 10-week storage period under vacuum. When the test organisms were inoculated at an initial level of log 4 CFU/cm2, C. maltaromicus LV17 and UAL26 produced off-odours after 8 weeks of storage under vacuum at 2 degrees C. Under aerobic conditions at 7 degrees C, all four of the strains grew well on the beef samples. C. maltaromicus LV17 and UAL26 and Lb. sake Lb706 all caused off-odours and discoloration. The rate of aerobic deterioration in meat quality was faster with increased time of storage under vacuum. L. gelidum UAL187-22 could be a suitable antagonistic strain with the potential to extend the storage life of beef, stored anaerobically and packaged aerobically for retail sale, without producing undesirable sensory changes.

Effect of Amino Acid Substitutions on the Activity of Carnobacteriocin B2 OVERPRODUCTION OF THE ANTIMICROBIAL PEPTIDE, ITS ENGINEERED VARIANTS, AND ITS PRECURSOR IN ESCHERICHIA COLI

Carnobacteriocin B2, a 48-amino acid antimicrobial peptide containing a YGNGV motif that is produced by the lactic acid bacterium Carnobacterium piscicola LV17B, was overexpressed as fusion with maltose-binding protein in Escherichia coli. This fusion protein was cleaved with Factor Xa to allow isolation of the mature bacteriocin that was identical in all respects to that obtained from C. piscicola Similar methodology permitted production of the precursor precarnobacteriocin B2 (CbnB2P), which has an 18-amino acid leader, as well as six mutants of the mature peptide: CbnF3 (Tyr3 → Phe), CbnS33 (Phe33 → Ser), CbnI34 (Val34 → Ile), CbnI37 (Val37 → Ile), CbnG46 (Arg46 → Gly), and Cbn28 (truncated frameshift mutation: (carnobacteriocin B2 1-28) + ELTHL). Examination of these compounds for antimicrobial activity showed that although CbnI34, CbnI37, and CbnG46 were fully active, CbnB2P, CbnF3, CbnS33, Cbn28, and all of the fusion proteins had greatly reduced or no antimicrobial activity. Expression of the immunity protein that protects against the action of the parent carnobacteriocin B2 in a previously sensitive organism also protects against the active mutants. Because carnobacteriocin B2 also acts as an inducer of bacteriocin production in C. piscicola, the ability of the precursor CbnB2P and the mutants to exert this effect was examined. All were able to induce Bac− cultures and reestablish the Bac+ phenotype except for the truncated Cbn28. The results demonstrate that very minor changes in the peptide sequence may drastically alter antimicrobial activity but that the induction of bacteriocin production is much more tolerant of structural modification, especially at the N terminus.

Bacteriocin production by Carnobacterium piscicola LV 61

Carnobacterium piscicola LV 61 produces a bacteriocin designated piscicolin 61, that is heat resistant, active over a wide pH range and inactivated by alpha-chymotrypsin, pepsin, trypsin and papain. It is effective against strains of the genera Carnobacterium, Enterococcus and Listeria. Other lactic acid bacteria tested were less sensitive or resistant to piscicolin. It is produced at temperatures from 1 to 30 degrees C. Maximum bacteriocin activity was detected after the culture had entered the stationary phase of growth and when the culture was grown in a medium with an initial pH between 8.0 and 9.0. The same high amounts of bacteriocin could be obtained in a culture at a constant pH of 6.5. No bacteriocin was produced at pH 5.0. Peptone in the growth medium promotes bacteriocin production, whereas meat and yeast extract did not influence the amounts of bacteriocin produced. Bacteriocin production and immunity are probably encoded by a 22 kb plasmid.

Microbial ecology of fresh pork stored under modified atmosphere at −1, 4.4 and 10°C

Abstract The prevalent bacteria on fresh pork packaged in modified atmosphere with elevated CO2 were determined by selection of representative colonies from the greatest dilution of meat samples. The pork samples were stored in two packaging films of different oxygen permeability at three storage temperatures. Strains were classified and those identified as lactic acid bacteria were screened for production of inhibitory substances. The types of bacteria isolated from samples stored in the two packaging films were similar. Storage temperature influenced the type of bacteria that dominated the microbial population. At 10°C the prevalent microflora consisted of aeromonads, Enterobacteriaceae and lactic acid bacteria but at 4.4 and −1°C, aeromonads, Brochothrix thermosphacta and lactic acid bacteria dominated. Listeriae were detected as part of the prevalent microflora on samples stored at −1°C, but not on samples stored at 4.4 or 10°C. Species of lactic acid bacteria dominating the microflora were influenced by growth medium. The majority of isolates taken from Plate Count agar were carnobacteria whereas those from Lactobacilli MRS agar were homofermentative lactic acid bacteria. Of the 538 lactic acid bacteria isolates screened for production of inhibitory substances, 162 strains showed deferred inhibition toward a range of lactic acid bacteria and nonlactic acid bacteria indicator strains.