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Biochemical Society Transactions 694 12. Okui, S., Uchiyama, M., Mizugaki, M. & Sugawara, A. (1063) Hiochim. Hiophys. Acta 70, 348-35 1 13. Farbood. M. I., Morris, J. A., Sprecker, M. A,. Hienkowski, I,. J., Miller, K. lā.? Vock, M. 11. & Hagedorn, M. I,. ( 1 990) US. Pat. 4,960,597 14. Gregory, 1ā. & Eilerman, K.G. (1989) PCT Int. Appl. WO 8912-134A 15. Hilton, M. D. & Cain, W. J. (1990) Appl. Environ. Microbiol. 56.623-627 16. Farbood, M. I., Willis, H. J. & Christenson, 1ā. A. (1086) S. African ZA 8.5 04,306 17. Farbood. M. I., Morris, J. A. & Lhwney, A. 1. (1000) U S . Pat. 4,970,163 18. Huckholz, I,., Farbood, M. I., Kossiakoff, N. & Scharpf, I,. G. (1990) 1J.S.Pat. 4,000,603 Keceived 12 April 1091 Anti-microbial substances produced by food associated micro-organisms Hans Horn and Christina Msrtvedt MATFORSK, Norwegian Food Research Institute, Osloveien I , N- I430 As, Norway Introduction Fermentative breakdown of carbohydrates results in a range of small molecular mass organic molecules which exhibit anti-microbial action, including lactate, propionate, acetate and ethanol. Conditions favouring the growth of bacteria producing these metabolic end products were discovered and used thousands of years ago and are still exploited in the production of a variety of products. They are employed in different manners, either as food additives after production in industrial fermentors, or produced in situ during fermentation of meat, vegetables and milk. Today there is considerable renewed interest in the use of naturally produced anti-microbial substances for food preservation and protection owing to the low energy demand of the fermentation process and the discovery of an abundance of additional anti-microbial activities associated with the fermentative microbes. Recombinant DNA technology has made it possible to identify and clone genes encoding these anti-microbials and electrotransformation has enabled the transfer of the genes to other bacteria, thus opening the era of tailormade starter cultures. The present paper will survey the antimicrobials produced by food associated microorganisms with the main emphasis on the lactic acid bacteria and related organisms. Metabolic end products Present knowledge suggests that the commonest form of anti-microbial activity expressed in foods is that associated with lactic, acetic and propionic acids. Lactic acid results from the metabolism of many different types of bacteria, primarily LactoAbbreviation used: 3-HPA, 3-hydroxypropanal. Volume 19 bacillus, Lactococcus, Leuconostoc and Pediococcus. Lactic acid is produced from breakdown of hexoses via the Embden-Meyerhof-Parnas pathway. Ideally, in homofermentative breakdown one mole of hexose gives rise to two moles of lactic acid and two moles of ATP. The anti-microbial action of lactic acid is only moderate. The acid produced lowers the pH and the growth of most spoilage and pathogenic organisms is inhibited to some extent. Lactic acid causes the leakage of hydrogen ions across the cell membrane. This results in acidification of the cell interior and inhibition of nutrient transport. The energy-yielding metabolism is not influenced [ 11. Lactic acid is the primary acid produced during fermentation of sausage, sauerkraut, olives, yoghurt, etc. In heterofermentative breakdown of sugars, the dissimilation proceeds via the pentosephosphate shunt. One mole of hexose gives rise to one mole of lactic acid, ethanol, CO, and ATP, respectively. However, oxidation of NADH + H + using alternative hydrogen acceptors (see below) sometimes leads to the formation of acetic acid and ATP instead of ethanol. Acetic acid is one of the most used anti-microbials from micro-organisms. Industrially it is produced through aerobic oxidation of ethanol by members of the genus Acetobacter. Acetic acid is mainly used as a food additive, in the form of vinegar. It is added as a preservative substance and a flavouring agent to many different foods including mayonnaise, dressings, pickles and mustard. Acetic acid has a wide range of inhibitory activity, inhibiting yeasts and moulds as well as bacteria. Its action cannot be explained by pH reduction alone. The undissociated form penetrates the cell and hereby exerts its inhibitory action, which is consistent with its anti-microbial activity increasing with decreasing pH values. Food Biotechnology Propionibacterium produces propionic acid from lactic acid. In short, 3 moles of lactic acid give rise to 2 moles of propionic acid, one mole of acetic acid, C 0 2 and ATP. In ripe Swiss and Jarlsberg cheese up to 1% of propionic acid may be detected. The inhibitory action of propionic acid is partially caused by inhibition of nutrient transport [2], and to accumulation in the cell resulting in inhibition of cell metabolism [3]. Propionates are mainly used as niould inhibitors, but the organism which causes rope in bread, Bacillus mesentericus is also inhibited. Consequently, the main application of propionates is in the baking industry. The three organic acids described here have an optimum anti-microbial function located close to and below their pK, values. At these pH values a large fraction of the acids are undissociated, they solubilize in the cell membrane, block transport of necessary growth substances, acidify the cell interior by dissociation and exert other inhibitory activities on cell growth. The Food and Agriculture Organization (FAO) and World Health Organization (WHO) have set no limit for the acceptable daily intake of these acids. [8] indicate that active transport across the membrane is affected. A more delayed reaction is also described. After 2 hours exposure to the lactoperoxidase system cell death can be demonstrated. Cytoplasm is extruded from a polar position, possibly when the last cell division envelope is formed PI. When iodine substitutes for thiocyanate, direct halogenation of bacterial proteins seems to be the inhibitory mechanism. Hypochlorous acid, which exhibits multiple effects on bacterial cells, apparently is formed from chloride [9]. The lactoperoxidase system is not much used as an antimicrobial food additive. In vivo mainly the H,O,-producing organisms are influenced. The spoilage psychrotrophs do not activate the system. By addition of glucose and glucose oxidase or hydrogen peroxide (at non toxic levels) the lactoperoxidase system is activated and it is possible to extend the lag period of psychotrophs in refrigerated milk from 2 to 5 days. Residual H20, and the inhibitory thiocyanate product are both inactivated by pasteurization. This process has been patented by Alfa Laval. The lactoperoxidase system Reuterin: 3-hydroxypropionaldehyde In 1924, Hanssen [4] observed that freshly drawn milk exhibited bactericidal properties. The inhibitory activity was lost after heat treatment (75"C, 15 min) and the activity was mistakenly attributed to peroxidative enzymes transferred from plants. Today, it is known that the enzyme responsible, lactoperoxidase, is present in high concentrations in milk and other body fluids and that the lactoperoxidase system is dependent on two additional factors to exhibit its bactericidal effect [ 51. Together with thiocyanate and/or halides, also present in milk, and hydrogen peroxide, produced by aerobically growing micro-organisms, an enzyme reaction product of bactericidal nature is formed. When thiocyanate is utilized by the lactoperoxidase-HzO, system, it is converted from a weak to a strong bactericidal agent. The instability of the actual compound formed has made it difficult to isolate the active substance and to establish its exact chemical composition. According to Bjorck et a.! 1975 [6] the bactericide produced is a low molecular mass compound, easily destroyed by heat, which exerts the most pronounced activity against Lactococci and Gram-negative bacteria. The nature of the bactericidal action is not well defined. An immediate reaction has been described [7] where hexokinase or other glycolytic enzymes are involved. Other results Recently, research on Lactobacillus reuteri has revealed the nature of an extremely broad-spectrum anti-microbial, called reuterin [lo]. When grown on a mixture of glucose and glycerol, L. reuteri produces a catabolite from glycerol which can act as an acceptor for the NADH + H + formed from glucose. The catabolite, 3-hydroxypropanal (3-HPA), is formed by an enzymatic dehydration of glycerol and is subsequently reduced to 1,3-propanediol by an NADH + H+-specific dehydrogenase (Fig. 1). The N A D H + H + needed for this reaction to proceed, is gained from the main heterofermentative pathway by the shifi from ethanol to acetic acid production [ 111. The aldehyde has been shown to be the inhibitory compound. Because it is readily reduced to the diol, 3-HPA cannot be detected during the active phase of growth when reducing power is plentiful, but begins to accumulate in the growth medium when the cells enter the stationary phase of growth [12]. Interestingly, 3-HPA is formed throughout growth when L. reuteri is cocultured with any one of a number of different Gram positive and Gram negative species, including Eschm'chia coli [13]. This is probably of great ecological significance, giving L. reuteri an advantage in competition with other members of the gut bacterial flora. The enzymes involved in glycerol dissimilation appear to be constitutive in L. reuteri 1991 695 Biochemical Society Transactions Bacteriocins Fig. I The proposed reactions taking place in the utilization of glycerol as hydrogen acceptor 696 Framed substance is reuterin. Hz-q-OH H-?-OH Hz - C - O H Glycerol Glycerol dehydratase 1 H-T=O H-q-H HzC-OH FNr:I +3 3-hydroxypropionaldehyde(3-HPA) Main pathway reductase 3-HPA Hz?-OH H-C-H I HzC-OH I ,3-propanediol [ 141 in contrast with the situation in Klebsiella pneumoniae, for example, where they are inducible [ 111. Dissimilation of glycerol is reported to take place by the same pathway, in Clostridiurn spp. [15], in K. pneumoniae [ 161 and in other Lactobacillus spp. [ 171. The actual accumulation and excretion of 3-HPA is however, a trait confined to L. reuteri alone. The mechanism underlying lack of accumulation and excretion in other glycerol dissimilating bacteria is not yet understood but dismutation and/or isomerization of 3-HPA have been proposed by several authors [ l l , 12, 171. Isolation and purification of reuterin led to the discovery of an equilibrium mixture of 3-HPA consisting of the monomer, hydrated monomer and a cyclic dimeric form [ 121. Reuterin is produced and exhibits its bactericidal function at pH 4-9, with an optimum around pH 6-8. Reuterin has a very broad activity spectrum. All micro-organisms tested so far are sensitive, although to different degrees. Experiments show that it acts as an anti-bacterial, anti-fungal, anti-protozoal and anti-viral compound [121. Even the producer strain itself is influenced. Lactic acid bacteria are in general more resistant than other micro-organisms [ 131. These findings are supported by the demonstration of inhibitory effect on sulphydryl enzymes [18], essential in DNA synthesis and other vital cell functions. Volume 19 Bacteriocins are anti-microbial substances which are produced and excreted by micro-organisms and act on the same or closely related species. Bacteriocins contain an essential protein moiety and are supposed to show a bactericidal mode of action. A wealth of basic information concerning bacteriocins is available. Most of today's knowledge is deduced from research on E. coli bacteriocins (colicins), and the reader is referred to Pugsley and Oudega, 1987 [ 191 for a detailed review. Nisin, produced by Lactococcus lactis, is the best known bacteriocin produced by a food associated micro-organism. Nisin has a well documented activity, a broad inhibitory spectrum and is affirmed for use in certain foods [201. Many other bacteriocins have been described, of which those produced by food associated bacteria have the greatest potential value. So far more than 20 different bacteriocins produced by lactic acid bacteria have been reported [ 19, 201. Although only a few of these have been purified to homogeneity, they appear to constitute a heterogenous group of anti-microbials. Estimation of size based on molecular sieve procedures often indicates a molecular mass considerably higher than the actual size determined after purification. During purification, activity is often reduced severely [2 1, 221. The genetic determinants involved in bacteriocin production encode at least two different single traits, i.e. production and immunity. Starter cultures producing bacteriocins may prove to be useful in improving the safety of the food fermentation processes. Genes encoding production of and immunity to bacteriocins may be transferred to strains used as starter cultures by means of electrotransformation. The genes represent easily selectable genetic markers, which may be used to enhance the stability of additional genes, when used in edible vectors and will give the transformed starter culture an advantage compared with nonbacteriocin-producing sensitive strains. Screening for bacteriocin producing organisms is usually performed with the deferred method [23] where fresh mature colonies are overlaid with soft agar containing a sensitive strain. After further incubation to allow the indicator strain to grow, bacteriocin producing colonies are seen with clear inhibition zones (Fig. 2). T o quantify bacteriocin activity, a microtitre plate assay is used. Twofold dilutions of sterile bacteriocin extracts are inoculated with a freshly prepared indicator culture. Bac- Food Biotechnology teriocin units (BUIml) are usually presented as the reciprocal of the highest dilution exhibiting growth inhibition of the indicator organism [24]. The demonstration of bacteriocin production in synthetic media does not necessarily reflect production and activity in fermented foods. Nutrient availability, temperature, pH, inoculum size and other factors will all influence production [ZO]. We have developed an easy and safe procedure to demonstrate plasmid encoded bacteriocin activity in fermented food. Lactobacillus sake L45, isolated from naturally fermented dry sausage, produced a bacteriocin Fig. 2 Result from the deferred assay, demonstrating the production of the bacteriocin, lactocin S, by L. sake L45, as clear inhibition zones surrounding the individual colonies Fig. 3 Effect of bacteriocin production in fermented sausage (*) denotes producing strain (Bac', Imm') and (0) the sensitive (Bac-, Irnm-, E$) strain. *I * * 108 - ;t( 4.. \ .-n L \ Y n n L 0 n 107- 5 z 0 4 8 12 Time (days) 16 20 24 designated Lactocin S [25]. The genes encoding bacteriocin production and self-resistance are located on a 50 kb plasmid. A cured strain was obtained through segregation experiments and shown to be both bacteriocin negative and sensitive (Bac-, Imm-) [25]. Electro-transformation was performed [26] to construct a Bac-, Imm-, Ery' variant of L. sake L45, with growth characteristics identical with the parental strain. Dry sausage was made using a mixture of L. sake L45 Bat+, Imm' and Bac-, Imm-, Ery' isolates as the starter culture. After four days the number of Ery' cells dropped in contrast with the Bac+, Imm+ count (Fig. 3). This method may prove useful in demonstrating bacteriocin production in fermented food, since all additional anti-microbial activity can be neglected. 1. Busta, F. F. & Foegeding, P. M. (1983) in Disinfection, Sterilization and Preservations (Block, S.S., ed.), pp. 657-664, Lea & Febinger, Philadelphia, USA. 2. Eklund, T. (1980)J. Appl. Bact. 48,423-432 3. Luck, E. (1980) Antimicrobial Food Additives Characteristics, Uses, Effects, pp. 186-21 1, SpringerVerlag, Berlin, Heidelberg & New York 4. Hanssen, F. W. (1924) B. J. Exp. Pathol. 5,271-280 5. Bjorck, L. P. (1978)J. Dairy Res. 45, 109-1 17 6. Bjorck, L. P., Rosen, C. G., Marshall, V. & Reiter, B. (1975) Appl. Microbiol. 3, 199-204 7. Reiter, B. & Oram, J. D. (1967) Nature, (London) 216, 328-330 8. Reiter, B. (1976) in Inhibition and Inactivation of Vegetative Microbes (Skinner, F. A. & Hugo, W. B., eds.), pp. 42-48, Academic Press Ltd, London 9. Davidson, P. M., Post, L. S., Branen, A. L. & McCurdy, A. R. (1983) in Antimicrobials in Foods (Branen, A. L. & Davidson, P. M., eds.), pp. 371-385, Marcel Dekker, Inc., New York and Base1 10. Axelsson, L. T., Chung, T. C., Dobrogosz, W. J. & Lindgren, S. E. (1989) Microb. Ecol. Health Disease 2, 131-136 11. Forage, R. G. & Lin, E. C. C. (1982) J. Bacteriol. 151, 591-599 12. Axelsson, I,. T. (1990) Dissertation Report 44, Uppsala, Sweden 13. Chung, T. C., Axelsson, L. T., Lindgren, S. E. & Dobrogosz, W. J. (1989) Microb. Ecol. Health Disease 2, 137- 144 14. Talarico, T. L. & Dobrogosz, W. J. (1990) Appl. Environ. Microbiol. 56, 1195-1 197 15. Forsberg, C. V. (1987) Appl. Environ. Microbiol. 53, 639-643 16. Forage, R. G. & Foster, M. A. (1983)J. Bacteriol. 149, 413-419 17. Sobolov, M. & Smiley, K. L. (1960) J. Bacteriol. 79, 26 1-266 1991 697 Biochemical Society Transactions 698 18. Dobrogosz, W. J., Casas, I. A., Pagano, G. A., Talarico, T. I,., Sjoberg, B.-M. & Karlsson, M. (1989) in The Regulatory and Protective Role of the Normal Microflora (Gruff, R., Midtvedt, T. & Norin, E., eds.), Wenner-Gren Cent. Int. Symp. Ser., vol. 52, pp. 283-292, Macmillan Press, London 19. Pugsley, A. P. & Oudega, B. (1987) in Plasmids (Hardy, M. G. ed.), pp. 105-161, IRL Press, Oxford, Washington DC 20. Daeschel, M. A. (1990) in Biotechnology and Food Safety (Bills, D. D. & Kung, S-D., eds.), pp. 91-104, Butterworth-Heinemann,Stoneham,MA, U S A . 21. Schillinger, U. & Lucke, F. K. (1989) Appl. Environ. Microbiol. 55, 1901-1906 22. Barefoot, S. E. & Klaenhammer, T. R. (1984) Antimicrob. Agents Chemother. 26,328-333 23. Joerger, M. C. & Klaenhammer, T. R. (1986) J. Bacteriol. 167,439-446 24. Mayr-Harting, A., Hedges, A. J. & Berkeley, R. C. W. (1972)Methods Microbiol. 7,315-422 25. Msrtvedt, C. I. & Nes, I. F. (1990) J. Gen. Microbiol. 136,1601-1607 26. Aukrust, T. W. & Nes, I. F. (1988) FEMS Lett. 52, 127-132 Received 12 April 1991 DNA probes and the detection of food-borne pathogens using the polymerase chain reaction Raymond A. McKee, Christopher M. Gooding, Stephen D. Garrett, Hilary A. Powell, Barbara M. Lund and Margaret Knox A.F.R.C., Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, U.K. Introduction In recent years the increased incidence of foodborne microbial pathogens [ 1, 2, 31 has highlighted the need for more rapid and reliable tests than those currently available. In the case of short shelf-life products this would enable manufacturers to quickly recognize contaminated products. Standard microbiological tests are available for the vast majority of microbial contaminants which may be found in foods. Each identification procedure will vary with the organism and food concerned, but would normally involve pre-enrichment, and enrichment incubations. These allow an increase in cell number, sufficient for detection and identification using a battery of microbiological and biochemical tests [4]. These standard microbiological techniques have been supplemented with recently developed methods to ease the labour intensive nature of the process, such as immunological detection [51. Determination of the presence of living cells within the sample by ATP monitoring, flow cytometry and electrical conductance measurement can give an indication of the need to proceed with microbiological tests. The use of nucleic acid probes for the detection of microbial pathogens is based on the unique ability of a molecule of DNA or RNA to hybridize Abbreviations used: PCR, polymerase chain reaction; CFU, colony-formingunit, Volume 19 specifically with a complementary sequence. A major advantage of nucleic acid probes over other detection methods is their information content. An antibody for example may recognize a unique stretch of six amino-acid residues, but owing to the redundancy in the genetic code these six amino acids may be coded for by up to several thousand different DNA molecules. There are a number of choices as to which nucleic acid sequence should be used as the target. Genomic DNA sequences from known cloned genes such as those encoding toxins have been used and offer high specificity. However, even if the target gene is present on a plasmid it is unlikely to be present at greater than one hundred copies per cell. When the number of target organisms per sample is low [see later, polymerase chain reaction (PCR) methodologies] this concentration would not be detected using standard hybridization protocols. With this in mind a number of commercially available probes target ribosomal RNA (rRNA), as this can be present at from 1000 to 10000 copies per cell. One of the drawbacks to this approach is the high degree of conservation of primary sequence in rRNA from different species, particularly closelyrelated species. At the Institute of Food Research we have an interest in developing DNA probes for the detection of List& species and in particular List& monocytogenes. W e have used an approach developed by Ward and his co-workers [6] which allows the