Inhibiting Factors Produced by Lactic Acid Bacteria. 1.0Xygen Metabolites and Catabolism End-Products

Lait (1991) 71, 525-541 525 © Elsevier/INRA Review

Inhibiting factors produced by lactic acid bacteria. 1.0xygen metabolites and catabolism end-products

Je Piard *, M Desmazeaud

INRA, Station de Recherches Laitières, Unité de Microbiologie, 78350 Jouy-en-Josas, France

(Received 29 April 1991; accepted 7 June 1991)

Summary - Lactic acid bacteria can produce a variety of antimicrobial compounds which may affect both the lactic acid bacteria themselves as weil as undesirable or pathogenic strains. In this first section, we describe the biosynthesis, mode of action and activity spectra of these inhibitors. Metabolites of oxygen (hydrogen peroxide and free radicals) exhibit a bacteriostatic or bactericidal activity against lactic or non-Iactic flora. When associated with the lactoperoxidase/thiocyanate system, hydrogen peroxide leads to the formation of inhibitory compounds which are bacteriostatic for lactic acid bacteria and bactericidal for Gram-negative bacteria. The antimicrobial activity of organic acids (Iactic, acetic and formic) and of pH are closely linked. It appears that the non-dissociated fraction of these acids is the major inhibitory form. Thus, acetic acid whose pKa is higher than that of lactic acid exhibits the highest inhibitory activity. The antimicrobial activities of diacetyl, acetaldehyde and of the D isomers of amino acids are also described, although their effects are slight in usual lactic fermentations. 8acteriocins are the last type of inhibitory substance produced by some lactie acid bacteria. They will be described in a second section. lactic acid bacteria / oxygen 1 acidity 1diacetyll acetaldehyde

Résumé - Facteurs inhibiteurs produits par les bactéries lactiques. 1. Métabolites de l'oxy- gène et produits du catabolisme. Les bactéries lactiques sont capables de produire une variété de produits inhibiteurs dont les effets peuvent se répercuter sur la flore lactique el/e-même mais aussi sur les flores indésirables ou pathogènes. Dans cette première partie, nous décrivons les méca- nismes d'apparition, les modes d'action et les spectres d'activité de ces inhibiteurs. Les métabolites de l'oxygène (le peroxyde d'hydrogène et les radicaux libres) peuvent avoir des effets biologiques de nature bactériostatique ou bactéricide sur la flore lactique ou non lactique. Le peroxyde d'hydrogène associé au système lactoperoxydase/thiocyanate, catalyse la formation de produits inhibiteurs, bac- tériostatiques pour la flore lactique et bactéricides pour les bactéries Gram-négative. Les pouvoirs antibactériens des acides organiques et du pH sont intimement liés. 1/ semble que la fraction non dissociée de ces acides soit la forme inhibitrice majeure. De ce fait l'acide acétique de pKa supérieur au pKa de l'acide lactique, a un pouvoir inhibiteur supérieur. Les activités inhibitrices du diacétyle, de l'acétaldéhyde et des acides aminés de forme isomérique D sont décrites bien que leurs effets soient mineurs dans les fermentations lactiques habituel/es. Les bactériocines produites par cer- taines bactéries lactiques seront décrites dans une seconde revue. bactérie lactique / oxygène / acidité / diacétyle / acétaldéhyde

* Correspondence and reprillts 526 Je Piard,M Desmazeaud

INTRODUCTION pH, diacetyl, acetaldehyde, 0 isomers of amino acids). In the second section, we will examine inhibitions due to small antimi- Aside from their fundamental roles in the crobial molecules or to bacteriocins. Inhibi- formation of organoleptic and rheological tion resulting from inter-strain competition characters during the transformation of (Juillard et al, 1987) is not within the scope milk to cheese and yogurt, lactic acid bac- of this literature review and so will not be teria are used and studied for their capaci- discussed. ty to inhibit unwanted bacteria, and thus For convenience, Lactococcus lactis for increasing the shelf-life of the products. subsp lactis, L lactis subsp lactis biovar di- It is known empirically that the decrease acetylactis and L lactis subsp cremoris will of pH in itself is a factor of fundamental im- be designated L tectis, L diacetylactis and portance in food safety, since it leads to L cremoris respectively. the elimination of unwanted bacteria and even pathogens. This is important since the ecological niches favorable to lactic OXYGEN METABOLITES acid bacteria have high water activity and are rich in nutrients, thus being very favorable for bacteria proliferation. However, Mechanism of appearance the capacity of lactic acid bacteria to effec- tively inhibit other species not only results Lactic acid bacteria are called facultative from their ability to lower the pH, but de- anaerobes, or aerotolerant, and they cata- pends on the nature of the organic acids bolize sugars via fermentation. This means that they produce. Also, other antimicrobi- that electron transport chains do not inter- ais such as oxygen metabolites (eg hydre- vene, but in subsequent oxidoreduction gen peroxide), bacteriocins, diacetyl, acet- steps nicotinamide adenine dinucleotide aldehyde and 0 isomers of amine acids (NAD) participates, undergoing cycling oxi- are Iikely to enhance the inhibitory activity dation and reduction. In aerobic conditions, of lactic acid bacteria. these NAD molecules react with oxygen to form hydrogen peroxide (H202) or water as Renewed interest in these aspects of a result of the action of various enzymes the metabolism of lactic acid bacteria has (table 1).The dismutation of endogenous occurred. Industrial accidents leading to superoxide anions (02-) (table 1)can also food poisoning, notably by Listeria mono- add a slight contribution to the accumula- cytogenes have provided the impetus for tion of H202 by the action of a superoxide research on the potential use of lactic acid dismutase (SaD) present in most lactic bacteria to improve food safety and even acid bacteria (Britton et al, 1978) or as a to create new fermented products. result of manganese, present in high con- The present review will deal with cur- centrations (20-25 mmol/I) in the cyto- rent knowledge on the inhibitory potential plasm of bacteria lacking SaD (Archibald of lactic acid bacteria. We will also discuss and Fridovitch, 1981). the possible benefit to food safety that With the exception of several isolated could result from better control of these cases (Whittenbury, 1964; Kono and Frido- various inhibitory systems. vitch, 1983), lactic acid bacteria lack cata- This section deals with inhibition phe- lase because they cannot synthesize he- nomena due to oxygen metabolites and moporphyrins. Thus, lactic acid bacteria end-products of catabolism (organic acids, can rid themselves of hydrogen peroxide Inhibition by lactic acid bacteria 527

Table 1. Reactions between lactic acid bacteria and oxygen or oxygen metabolites (atter Archibald and Fridovich, 1981; Condon, 1987). Reactions des bactéries lactiques avec l'oxygène ou ses métabolites (d'après Archibald et Fridovich, 1981; Condon, 1987).

Reactions and catalysing enzymes Lactic acid bacteria in which the enzyme has been identified

NADH :H,O, oxidase NADH + H+ + 02 > NAD+ + H202 Most lactic acid bacteria

NADH:H,O oxidase 2NADH + 2H+ + 02 > NAD+ + 2H20 Most lactic acid bacteria

pyruvate oxidase Pyruvate + phosphate + 02 ------:> acetylphosphate L delbrueckii, L plantarum, TPP, FAD + CO2 + H202 P halophilus, S mutans

oxidase a-GP Lactic acid bacteria utilizing a-Glycerophosphate + 02 > dihydroxyacetone phosphate glycerol in aerobiosis + H202 (enterococci,lactobacilli, P pentosaceus)

SOD 202- + 2H+ > H202 + 02 Most lactic acid bacteria

NADH peroxidase NADH + H+ + H202 > 2H2O Most lactic acid bacteria

Mn2+ 202-+ 2 H+ > H202 + 02 Bacteria lacking SOD

TPP: thiamine pyrophosphate; FAD: flavine adenine dinucleotide; GP: glycerophosphate; SOD: superoxide dismutase.

formed, only by their NADH peroxidase. It OH- + OH" + O2 (Gregory and Fridovltch, follows that the concentration of H202 1974). Aerobic growth of lactic acid bacte- formed in aerobiosis is variable and de- ria thus leads to the formation of 3 princi- pends on the activities of NADH oxidase pal derivatives of oxygen: H202• 02- and and NADH peroxidase, catalyzing the for- OH" which are vectors of "oxygen toxicity". mation and degradation of H202 (Anders et al, 1970). Mode of action The simultaneous presence of hydrogen peroxide and superoxide anions can be Although the toxicity of oxygen has been the origin of hydroxyl radical formation ac- dernonstrated, its modes of action have cording to the reaction: 02- + H202 -> not been as c1earlyelucidated. Two types 528 Je Piard, M Desmazeaud may be distinguished, one leading to a presence of molecular oxygen may rein- bacteriostatic effect, the other being bacte- force the damage, perhaps by a direct ef- ricidal. fect at the level of the affected membrane sites. Bacteriostatic effect Bactericidal effect Homolactic bacteria catabolize glucose via the Embden-Meyerhof pathway, while het- Free radicals and hydrogen peroxide can erolactic bacteria follow the pentose phos- damage bacterial nucleic acids, leading phate shunt. In the case of homo/actic fer- to reversible or irreversible alterations. mentation, the 2 molecules of NAD Ananthaswamy and Eisenstark (1977) reduced during the formation of glyceral- demonstrated the modification of DNA by dehyde-3-phosphate (G3P) are reoxidized hydrogen peroxide by showing that E coli to favor the reduction of pyruvate to lactic mutants lacking DNA repair systems were acid by a lactic dehydrogenase (LDH). more sensitive to H202 than strains with in- When the cells are in aerobic conditions, tact repair systems. Hydrogen peroxide ap- their NADH oxidase and NADH peroxi- parently causes breaks in the carbon- dase systems are induced (Murphy and phosphate backbone of DNA, releasing nu- Condon, 1984), which compete with LDH c1eotidesand preventing chromosome rep- for available NADH. This results in a de- Iication (Freese et al, 1967). crease in the acidifying activity of the bac- Hydroxyl radicals can attack the methyl teria. The same reasoning applies to het- group of thymine, thereby damaging DNA erofermenting bacteria using the pentose (Byczkowski and Gessner, 1988). Active phosphate pathway. molecular oxygen can also react with gua- The oxidation of sulfhydryl compounds nine and cause breaks in one strand of may have a bacteriostatic or bactericidal DNA. This was shown in E coli, where effect, depending on whether or not the re- treatment of plasmid DNA prevented trans- action is reversible when the cells return to formation (Di Mascio et al, 1989). anaerobic conditions. The denaturing ef- Gram-negative bacteria are better pro- fect of oxygen and its metabolites on G3P tected from the toxic effect of oxygen me- dehydrogenase has been shown (Kong tabolites because of the presence of an and Davison, 1980) and it is probable that outer Iipopolysaccharide layer. This enve- oxidation of sulfhydryl to disulfide affects a lope traps active molecular oxygen (Dahl number of enzymes (lactate dehydroge- et al, 1989). Thus, the kinetics of cell death nase, atcohol dehydrogenase) or coen- is a multistep process in Salmonella sp zymes with sulfhydryl groups (coenzyme (multihit killing), while it is linear in the case A) (Haugaard, 1968). of L lactis. Lactic acid bacteria have an ad- The toxicity of oxygen may result from ditional handicap in the resistance to mo- the peroxidation of membrane lipids lecular oxygen because they lack mem- (Haugaard, 1968), which would explain the brane carotenoids. Active molecular increased membrane permeability caused oxygen thus diffuses through the peptido- glycan layer rapidly and reacts with sensi- by H202, O2- and OH" (Kong and Davi- son, 1980). This hypothesis was support- tive membranes sites (Dahl et al, 1989). ed by the finding that the degree of membrane lipid peroxidation was related to a Effect on lactic flora 1055 of viability by E coli (Harley et al, 1978). Once cell envelopes are damaged The relative toxicity of H202• O2- and OH" by the above-mentioned metabolites, the towards bacteria is somewhat controver- Inhibitionbylacticacidbacteria 529 sial. Concerning lactococci, there is an ap- sensitive to hydrogen peroxide and -ex- parent consensus that hydrogen peroxide posed to a sublethal concentration of the is the major inhibitor (Condon, 1987). compound became capable of growth in Anders et al (1970) reported that 0.2 the presence of a lethal concentration of mmol/l H202 was the concentration which hydrogen peroxide (Condon, 1987). The inhibited the growth of these bacteria by author observed the simultaneous induc- 50% and that 1.5 mmol/l induced cell tion of NADH peroxidase and, to a lesser death. Autoinhibition by H202, however, is extent, that of NADH oxidase. The same not universal among lactococci. Thus, induction was shown when cells were sub- Smart and Thomas (1987) showed that in jected to a sublethal temperature, leading oxygen-insensitive strains, there is an ade- the author to suggest that it may be a quateness between the activities of question of stress proteins. These induc- NADH:H202 oxidase and NADH peroxi- tions may partiaily explain the variability of (jase,' preventing the accumulation of hy- results presented above. drogen peroxide. In some strains, reduc- Similar phenomena have been de- tion of oxygen to water by an NADH:H202 scribed in other bacteria. Thus, 30 proteins oxidase enables these bacteria to remain are induced in Salmonella typhimurium unaffected by aerobiosis. when cells are treated with sublethal doses Although inhibition is occasionally ob- of hydrogen peroxide (Morgan et al, 1986). served in lactobacilli in aerobic conditions, Five of these proteins are also induced af- the modes of action remain to be estab- ter a heat shock. Other stresses, such as Iished. It is generally admitted that hydro- treatment with nalidixic acid or ethanol, gen peroxide is not responsible for this in- lead to the synthesis of the same 30 pro- hibition (Gregory and Fridovitch, 1974; teins, in addition to others. On the other Yousten et al, 1975; Condon, 1983). Some hand, the proteins induced by hydrogen authors have advanced the notion that the peroxide are not the same as those in- absence of SOD in Lactobacillus could en- duced by aerobiosis. able the formation of hydroxyl radicals via a reaction between the superoxide anion Effect on non-Iactic flora and hydrogen peroxide (Gregory and Fridovitch, 1974). The authors attributed Among the non-Iactic acid bacteria that the toxicity of oxygen to these OH" radi- may be present in rnilk, it is of interest to cals. Subsequently, however, Archibald note that the catalase + character of un- and Fridovitch (1981) reported that lactic wanted Gram-negative bacteria (Pseudo- acid bacteria lacking SOD had a dismuta- mones, E coli) does not render them par- tion activity resulting from their high Mn2+ ticularly tolerant to oxygen metabolites levels (see table 1).This system protects (Rolfe et al, 1978). Price and Lee (1970) them from the superoxide anion and pre- concluded that hydrogen peroxide was re- vents the formation of hydroxyl radicals. sponsible for the inhibition of Pseudomo- The authors concluded that inhibition re- nas, Bacillus and Proteus by Lb plantarum. sulted from the combination of several fac- Dahiya and Speck (1968) reported that hy- tors (Condon, 1983) or from the shunting drogen peroxide produced by Lactobacillus of carbohydrate metabolism towards other inhibited Staphylococcus aureus and the metabolic pathways where energy produc- optimal H202 production was obtained at tion is slower (Murphy and Condon, 1984). 5 -c. Recent data suggest that lactic acid ln conclusion, oxygen metabolites may bacteria can adapt to oxygen. Lactococci affect both lactic acid flora itself as weil as 530 JC Piard, M Desmazeaud

unwanted species. Various avenues of in- mechanism of inhibition of the system but vestigation are promising for obtaining op- also stabilizes lactoperoxidase at high dilu- timal benefit from the inhibition of these tions (Steele and Morrison, 1969). metabolites. The construction of strains Native milk does not contain hydrogen containing amplified NADH oxidase sys- peroxide (Reiter and Hârnulv, 1984), al- tems will lead to excess production of though it has been observed that milk lac- H202. Detailed studies on the mechanisms tic acid flora (Iactococci and lactobacilli) at- of resistance to these compounds (notably ter milking can produce H202 in aerobic via stress proteins) should be undertaken conditions, even when the milk is stored at in parallel to avoid phenomena of autoinhi- low temperature (Dahiya and Speck, bition. 1968). Saliva taken aseptically from human salivary glands contains hydrogen peroxide (Pruitt et al, 1982). THE LACTOPEROXIDASE- THIOCYANATE-HYDROGEN Mode of action and biological effects PEROXIDE SYSTEM OF MILK

ln the presence of H202, lactoperoxidase The inhibition observed is based on the in- catalyzes the oxidation of thiocyanate to teraction between three compounds: lac- non-inhibitory end-products, such as 2 toperoxidase, thiocyanate and hydrogen S04 -, CO2 and NH3 (Oram and Reiter, peroxide. Lactoperoxidase is the name 1966). In the course of this oxidation, how- given to milk peroxidase, of which cow's ever, more or less inhibitory intermediates milk contains 10-30 j1g/ml (Reiter, 1985). form, eg hypothiocyanate (OSCN-) (Aune This activity is more than sufficient to acti- and Thomas, 1977) or higher oxyacids vate the lactoperoxidase system (LPS), 02SCN- and 03SCN- (Pruitt et al, 1982). since Pruitt and Reiter (1985) showed that The major effect of these metabolites the reactions of the LPS could be cata- occurs at the level of the oxidation of sulf- Iyzed by a lactoperoxidase concentration hydryl . groups of metabolic enzymes as low as 0.5 ng/ml. (Thomas and Aune, 1978). Thus, hexoki- Lactoperoxidase can tolerate an acidity nases are totally inhibited, and aldolases equivalent to pH 3 in vitro (Wright, 1958) and 6-phosphogluconate dehydrogenase and resists human gastric juice (Gothefors are partially inactivated by the LPS. Law and Marklund, 1975). It is relatively heat- and John (1981), however, showed that stable, since it is only partially inactivated energy-transducing o-lactate dehydroge- by a short pasteurization at 74°C (Wright nase was also inhibited by this system, and Tramer, 1958), after which there sub- even though its active site does not con- sists enough residual activity for the LPS tain SH groups. to remain effective. The LPS also causes lesions in the The thiocyanate anion (SCN-) is widely cytoplasmic membrane, causing the leak- distributed in animal secretions and tis- age of potassium ions, amino acids and sues and its concentration is a reflection of polypeptides from the cell. Sugar and ami- eating habits and lifestyle of the individuals no acid transport systems are also inhibit- (Reiter and Hârnulv, 1984). The thiocya- ed, as are the synthesis of DNA, RNA and nate concentration in milk varies from 1 to proteins (Reiter and Hârnulv, 1984). 10 ppm and the compound has a dual role As in the case of the direct toxicity of in the LPS: it is directly involved in the oxygen metabolites, the catalase + charac- Inhibition by lactic acid bacteria 531

ter of Gram-negative bacteria confers no Among undesirable Gram-positive species, advantage on them, and is even detrimen- Siragusa and Johnson (1989) showed that tai. Marshall and Reiter (1980) showed the LPS delayed the development of that the LPS is bacteriostatic for L lactis L monocytogenes. and bactericidal for E coli (fig 1), which may be due to structural differences in the Field of application cell walls. Oram and Reiter (1966) showed that LPS-resistant strains of lactic acid ln raw milk, the LPS is a powerful inhibitor bacteria contained an enzyme which cata- from hydrogen peroxide producing strains. Iyzed the oxidation of NADH2 in the pres- ln addition, this factor augments the toxici- ence of an intermediate product of thiocya- ty of H202 by a factor of about 100: active nate oxidation. lt is th us possible that lactic against lactococci at a concentration of 1 acid bacteria can neutralize the oxidation mmol/l (Anders et al, 1970), hydrogen per- products of thiocyanate or else can repair oxide present at 10 nmol/I is sufficient to the damage caused (Reiter and Hàrnulv, activate the LPS (Thomas, 1985). 1984). The acid resistance of the LPS and the The LPS has a broad spectrum of activi- fact that SCN- arises from raw milk or from ty, including most Gram-negative bacteria the digestion of certain plants led research- found in milk, eg Pseudomonas and E coli ers to examine the antibacterial activity of (Bjërck et al, 1975), Salmonella (Purdy et the LPS in vivo. This was shawn in calves al, 1983) and Shigella (table Il). Pruitt and having absorbed raw milk enriched with E Reiter (1985) published a Iist of the biologi- coli: only 1 to 10% of the strain was found cal effects caused by the LPS in bacteria. in the abomasum, the fourth (true) stom-

8 CV E. coli 8 ® L. loctis 7 7 E ::> 6 6 u, u a 5 5 0; a -l 4 4

3 3 2 4 6 2 4 6 Time (h) Time (h)

Fig 1. Effect of various concentrations of OSCN- on the viability of E coli (a) and L lactis (b) in synthe- tic medium pH 5.5. OSCN- concentrations: (0), 0; (.), 5 urnol/l: (0), 10 Jlmol/l; (.), 20 Jlmolll; (L'i), 25 Jlmol/l; (Â), 35 Ilmol/l (after Marshall and Reiter, 1980; reprinted by permission of the Society for Gen- erai Microbiology). Effet de concentrations variables de OSCfIr sur la viabilité de E coli (a) et de L lactis (b) dans du milieu synthétique à pH 5,5. Concentrations de OSCfIr: (0), 0; (e), 5 j.lmoll/; (0), 10 j.lmoll/; (_), 20 umou'; (.1), 25 umolâ; (~), 35 j.lmol/I (d'après Marshall et Reiter, 1980; reproduit avec la permission de la Society for General Microbiology). 532 Je Piard, M Desmazeaud

Table Il. Antibacteriai activity of the lactoperoxi- ppm) (Reiter and Hârnulv, 1984). This dase system against various bacteria (afler practice led to a reduction in the undesira- Asperger, 1986; Siragusa and Johnson, 1989). ble flora of raw milk, especially Pseudomo- Activité antibactérienne du système lactoperoxydase contre diverses bactéries (d'après nas sp, whose proteolytic and Iipolytic ac- Asperger, 1986; Siragusa et Johnson, 1989). tivities pose an important problem when processing milk. The LPS has been used in pharmacolo- Target strain Biological activity gy by incorporating the ingredients neces- Bacterio- Bacteri- sary to activate the system in toothpastes static cidal or contact lens solutions. This system will undoubtedly be developed in the future in the food and agriculture industry. The LPS Lactic acid bacteria is a very interesting system in this field, Lactococcus sp x Thermophilic lactobacilli x since it is bactericidal for Gram-negative bacteria and only bacteriostatic for Gram- Spoilage bacteria positive species. Whenever we speak of a Pseudomonas f1uorescens x bacteriostatic effect, it is almost certainly a Gram-negative bacteria x saturable system. lts use to improve the Bacil/us cereus x storage of raw milk thus should not have major effects on the subsequent growth of Pathogenic bacteria lactic starters. It is also to be noted that if Staphylococcus aureus x the milk is pasteurized before processing, Escherichia coli x a part of the lactoperoxidase is inactivated. Salmonella sp x Pseudomonas aeruginosa x Listeria monocytogenes x END-PRODUCTS OF CATABOLISM

End-products of carbohydrate catabolism, effect of pH ach of ruminants, whereas if one of the components of the LPS system was neu- The production of acids by lactic acid bac- tralized, the number of viable coliform bacteria contributes to energy production in teria was maintained (Reiter and Hârnulv, this bacterial group, as we will see below. 1984). The acidity which forms is detrimental for The safety of this system towards eu- the development of unwanted bacteria and karyotic cells remained to be shown. thus greatly improves the hygienic quality Reiter and Hârnulv (1984) reported that of dairy products. However, this aéidity can the LPS did not change the respiration of also affect the lactic flora itself. It is to be liver cells in vitro and did not cause Iysis of remembered that the dairy industry most erythrocytes. Hanstrôrn et al (1983) even often uses mixed cultures of lactic acid showed that the LPS protected human bacteria whose composition is more or cells from hydrogen peroxide toxicity. less known with certainty. A different acidi- The LPS is thus a valuable system for fying activity and a different acid tolerance some authors, who suggested improving of the strains composing the starter may the storage properties of raw milk by add- lead to the dominance of the more acid- ing traces of SCN- (12 ppm) and H202 (8 resistant strains. Inhibition by lactic acid bacteria 533

Bioenergetics in /actic acid bacteria Osmoregu/ation and ragu/ation of intracellu/ar pH in /actic acid bacteria Bacteria have 2 mechanisms for generating ATP: Lactic acid, the final major product of car- - phosphorylation of ADP to ATP via gly- bohydrate catabolism by lactic acid bacte- colysis intermediates; ria is in equilibrium according to the reaction: - chemosmotic energy characterized by a proton gradient. CHs-CHOH-COOH + H20 <-> The latter pathway functions by the CHs-CHOH-COo- + HsO+ presence of the following secondary transport systems: - coupling of lactate efflux and protons (fig ACID 2) in the ratio of 2 H+/lactate (Konings, 1985); - coupling between the entry of solutes and the influx of protons at 1 or 2 Hr/solute molecule. Finally, the ATPase system leads to the expulsion of protons with energy consump- tion (1 mol ATP/2H+) or the entry of protons with energy production. During glycolysis, the coupling of lactate and protons efflux creates an electric potential (negative in the intracellular com- partment) and a pH gradient (alkaline in the cytoplasm), whose resultant is the pro- Fig 2. Scheme of metabolic energy-generating ton motive force (pmf) which generates and energy-consuming processes in lactococci ATP via the ATPase system (fig 2). This (after Konings and Otto, 1983; reprinted by per- -system offers a positive energy balance to mission of Kluwer Academic Publishers). Four lactic acid bacteria during a part of their pathways related to bioenergetics of the cell are shown: 1) ATP synthesis via glycolysis; 2) the growth phase. Each molecule of glucose coupling of lactate and protons efflux; 3) the consumed leads to the formation of 2 coupling of protons influx or efflux with respecti- molecules of lactate. During their excre- vely concomitant ATP synthesis or consump- tion, they enable the bacterium to extrude tion; 4) the coupling of solutes and protons in- 4 protons. When we subtract the 2 protons flux. Schéma du processus de synthèse et d'utilisa- resulting from glucose catabolism, the ef- tion de l'énergie chez les lactocoques (d'après flux of lactate leads to the translocation of Konings et Otto, 1983; reproduit avec la permis- 2 protons per glucose molecule. If they are sion de Kluwer Academie Publishers). Quatre utilized by the ATPase system, they ena- voies liées à la bioénergie cellulaire sont repré- ble the synthesis of 1 molecule of ATP, sentées : 1) la synthèse d'ATP par /'intermé- which is an energy gain of 50% in compari- diaire de la glycolyse; 2) le couplage des efflux de lactate et de protons; 3) le couplage des in- son to the 2 molecules of ATP produced flux ou des efflux de protons avec respective- by phosphorylation during glycolysis in ho- ment, synthèse d'ATP ou consommation d'ATP; mofermenting bacteria (Ten Brink and 4) le couplage des influx de solutés et de pro- Konings, 1982). tons. 534 Je Piard, M Desmazeaud

Since its pKa is 3.86 and the initial intra- -Effects of end-products cellular pH is close to neutrality, most lac- of sugar catabolism on different tlora tic acid is ionized. The bacteria must th us Effect on lactic acid bacteria face 2 situations: eliminate the proton sup- ply in order to maintain intracellular pH At uncontrolled pH, acidification and the (pHc) at a value consistent with vital meta- decrease of intracellular pH can lead to de- bolic processes, and eliminate excess lac- creased activity of metabolic enzymes, tate in order to restore osmotic balance. even their denaturation, as reported eg by Accolas et al (1980) for the hexokinase of Konings and Otto (1983) determined the factors governing these regulations in cultures grown at uncontrolled pH. 8acteri- al growth is characterized by the presence of a lactate gradient (àlact) which causes a pH gradient (~pH) as a result of H+ et- flux/lactate coupling "(fig 3). This intervenes 200 at the level of the pmf, which simultane- 120 ously increases. As cell growth continues, T the external lactate concentration increas- 160 L es, ~Iact and thus ~pH and ~p decrease. s E É <, This drop in the pmf leads to decreases in ~ C' 80 20 "- both the absorption of nutrients and H+/ '"u .S Ci c: lactate stoichiometry. The cell is th us LL -0ë" forced to gradually tap its energy reserves 80 Ci to eliminate H+ with the ATPase system. 40 .. u Cell death occurs when the cell no longer 40 has energy available to control its pHc. ln cultures run at controlled pH, there is no pH gradient and the pmf reflects only 120 240 360 480 the difference in membrane potential, ie it Time (min) is constant during growth and decreases when the sugar in the medium is consumed. Note than in a controlled pH cul- Fig 3. Evolution of proton motive force and of ture, there is no cell death, and so a pmf the lactate gradient in L cremoris during growth will reappear if the bacteria once again in batch cultures on complex medium with lactose (2 g/I) as sole energy source. (e), proton have an available carbon source (Konings motive force; (0), membrane potential; (to), lac- and Otto, 1983). tate gradient; (A) pH gradient; (---), cell protein The absence of a proton gradient in concentration (after Konings and Otto, 1983; re- controlled pH cultures means that the pmf printed by permission of Kluwer Academie Pub- Iishers). will have a lower value during the expo- Évolution de la force proton motrice et du gra- nential phase of growth. As a result, the dient de lactate au cours de la croissance de L growth rate will be lower than in the case cremoris en culture discontinue dans un milieu of a culture at uncontrolled pH. This phe- complexe avec le lactose (2 g/I) comme seule nomenon has apparently not been report- source d'énergie. (e), force proton motrice; (0), ed, probably because it is transitory and potentiel membranaire; (,1), gradient de lactate; ("'), gradient de pH; (---) concentration de pro- growth rate calculations are generally téines cellulaires (d'après Konings et Otto, 1983; made over the entire exponential phase of reproduit avec la permission de Kluwer Acade- growth. mic Publishers). Inhibition by lactic acid bacteria 535

S thermophilus. The tolerance of metabolic Effect on non-Iactic flora enzymes and of transport systems to acid There are very few non-Iactic bacteria ca- pH is variable depending on the strain, pable of growing at pH values lower than which could explain the variable acid toler- the threshold pH of lactic acid bacteria ance in lactic acid bacteria which in mixed (table III). Good lactic acidification should cultures leads to phenomena of domi- thus inhibit the growth of E coli, Pseudo- nance. Thus, Accolas et al (1977) reported monas, Salmonella and Clostridium. that at uncontrol/ed pH, L bulgaricus con- Although it is very easy to determine the tinued to grow after S thermophilus stopped growing, while at control/ed pH, pH, this parameter is not a reliable indica- tor for presuming whether or not microor- both strains stopped at the same time (Juil- lard et al, 1987). ganisms wil/ develop. The pH is only a par- tial illustration of the concentration of weak Homofermenting lactic acid bacteria acids in a medium, since a large part of produce lactic acid from glucose in almost stoichiometric quantities. When the bacteria are nutritional/y deprived, however, Table III. Limit pH values allowing the initiation of growth of various microorganisms (after their metabolism also yields formic and Gray and Killinger, 1966; Stadhouders and acetic acids and ethanol (Cogan et al, Langeveld, 1966; Asperger, 1986). 1989). Heterofermenting bacteria degrade Valeurs de pH limites permettant l'initiation de la croissance de divers microorganismes glucose to lactic acid, ethanol, CO2 and in some cases acetic acid. The cel/ mem- (d'après Gray et Kil/inger, 1966; Stadhouders et Langeveld, 1966; Asperger, 1986). brane is impermeable to ionized hydrophil- ic acid, while non-ionized hydrophobie acids diffuse passively (Kashket, 1987). Gram-negative Limit Weak organic acids, such as acetic acid bacteria pH (pKa = 4.74) will thus be present at the same concentration on both sides of the Escherichia coli 4.4 membrane. The intracellular medium is ba- Klebsiella pneumoniae 4.4 sic in comparison to the external medium Proteus vulgaris 4.4 and will favor the ionization of these mole- Pseudomonas aeruginosa 5.6 cules and thus an increase in their intracel- Salmonella paratyphi 4.5 lular concentration. This causes not only Salmonella typhi 4.0-4.5 Vibrioparahaemolyticus 4.8 an acidification of the cytoplasm, but also the creation of inhibitions, especial/y of en- Gram-positive zymes, by salt excesses. Final/y, Kashket (1987) reported that the organic acids and Bacillus cereus 4.9 alcohols produced could act as protono- Clostridium botulinum 4.7 phores in the membrane and accentuate Enterococcus sp 4.8 Lactobacil/us sp 3.8-4.4 the anarchie entry of protons. There are Micrococcus sp 5.6 considerable knowledge gaps in this field: Staphylococcus aureus 4.0 although lactic acid bacteria have been ex- Lactococcus lactis 4.3-4.8 tensively studied during their growth, rela- Brevibacterium linens 5.6 tively little is known about their behavior in Listeria monocytogenes 5.5 stationary or declining phase. 536 Je Piard, M Desmazeaud these compounds may be non-dissociated 6.0 and 5.6 in the case of acetate, formate as a function of pH. We saw above, how- and lactate, respectively, and attributed the ever, that in the case of weak organic ac- major inhibitory power to acetate. In terms ids, it is precisely this non-dissociated frac- of spore germination, the same authors cit- tion that diffuses across the membrane ed, in order, formate, lactate and acetate and is more or less ionized depending on which inhibited spore germination by 50% intracellular pH. This type of molecule ex- at 0.1 molli and at pH 4.4, 4.3 and 4.2, re- erts a shadowy effect which cannot be de- spectively. The cumulated effects of the 3 termined by only measuring the pH of the acids were not studied, however, even medium. In this context, the study by though they may be produced simultane- Chung and Goepfert (1970) is interesting, ously in certain conditions of the growth of since it showed that salmonellas are inhib- lactococci (Cogan et al, 1989). ited at pH values < 4.4 for lactic acid and Concerning other sporulated bacteria, 5.4 for acetic acid. In light of the respective especially Clostridium tyrobutyricum which pKa values of 3.86 and 4.75 for the 2 acids is responsible for the late blowing of and of their dissociation constant, inhibi- cheeses, a review by Blocher and Busta tion apparently occurs when the non- (1983) indicated that vegetative cells were ionized fraction reaches about 20%. Daly inhibited at pH 4.65, while spore germina- et al (1972) showed that L diacetylactis tion was affected at pH 4.6. couId inhibit a wide range of unwanted Finally, it is ta be noted that the sensitiv- bacteria in dairy products. Using S aureus ity of bacteria to acids depends on varia- as the model strain, the authors estab- tions which in turn depends on the simulta- Iished that the inhibitory activities of acetic neous action of other factors (salt levels, and lactic acids increased as the pH de- activity of water, redox potential, heat treat- creased. Adams and Hall (1988) investi- ment, etc). For example, Kleter et al (1984) gated the individual and cumulated effects showed the synergy between acidity and of lactic and acetic acids towards E coli the NaCI concentration in the inhibition of and Salmonella enteritidis. They confirmed C tyrobutyricum. that the molecular form of these 2 acids is ln conclusion, it is important to note the the toxic factor for the bacteria (fig 4). high inhibitory capacity of organic acids in They attributed the higher toxicity of acetic the molecular (non-ionized) form. lt is con- acid to the fact that its pK is higher than a sistent to imagine several improvements that of lactic acid. The authors also for the more efficient utilizatiôn of this sys- showed that the 2 acids acted synergisti- tem. Improved acid and salt tolerance of cally in weakly-buffered media: lactic acid the enzymes of lactic acid bacteria would decreased the pH of the medium, thereby enable the lactic flora to more efficiently increasing the toxicity of acetic acid. compete with unwanted bacteria. As in the Several research groups have exam- case of the induced resistance to hydrogen ined the inhibitory capacity of lactic acid peroxide, a more thorough understanding bacteria in terms not only of the growth of of the stress proteins of lactic acid bacteria sporulated bacteria, but also towards the could indicate interesting prospects in this spores of these bacteria. Thus, Wong and field. It would also be of interest to design Chen (1988) showed that Bacillus cereus lactic acid bacteria which at the proper is first progressively inhibited and then time would switch their catabolism to killed when cocultured with various lactic heterofermenting pathways in order to acid bacteria. They showed that growth profit from the high inhibitory power of ace- completely stopped at pH values of 6.1, tic acid at low pH. Inhibition by lactic acid bacteria 537

120 100

Q) '§ 50 J:: 25 i ~

u'" ;;: 120 'u Q) 100 a. ~ C/) 75 0 50

a 2 4 8 16 32 63 126251500 a 2 4 8 16 32 63 126251500 Total ocid (mmol/I)

Fig 4. Effect of total acid concentration on microbial specific growth rate (r:I) and % dissociation of acid ("'). (a) E coli and acetic acid, (b) E coli and lactic acid, (c) Salmonella enteritidis and acetic acld, (d) Salmonella enteritidis and lactic acid. Specifie growth rate units are expressed on an arbitrary scale (atter Adams and Hall, 1988; reprinted by permission of International Journal of Food Science and Technology). Effet de la concentration totale d'acide sur le taux de croissance bactérien spécifique (0) et sur le % de dissociation de l'acide ("). (a) E coli et acide acétique, (b) E coli et acide lactique, (c) Salmonella enteritidis et acide acétique, (d) Salmonella enteritidis et acide lactique. Les unités de taux de croissance spécifique sont exprimées sur une échelle arbitraire (d'après Adams et Hall, 1988; reproduit avec la permission de International Journal of Food Science and Technology).

Diacetyl diacetyl up to the dose of 350 llg/ml. Gram-negative bacteria, on the other L diacetylactis and Leuconostoc sp can hand, begin to be inhibited at 100 llg/ml use citrate present in milk (== 8.3 mmol/l). and are almost totally inhibited at 200 1191 Among the products formed during this ml. The author indicated that diacetyl is le- metabolism (Cogan, 1980), diacetyl is im- thal for Gram-negative bacteria and only portant since it is the major aromatic con- bacteriostatic for Gram-positive bacteria. stituent of butter and fresh cheeses. The mean diacetyl concentration pro- The antibacterial properties of diacetyl duced by L diacetylactis in pure culture or have been shown in a large number of in coculture with Leuconostoc is 4 llg/ml studies involving different microorganisms. (Cogan, 1980) and so there is little chance Jay (1982) published a thorough table of that the compound is antibacterial in lactic the inhibitory capacity of diacetyl, showing fermentations, but it could act synergisti- that lactic acid bacteria are insensitive to cally with other factors (Gilliland, 1985). In 538 Je Piard. M Desmazeaud light of the value of the broad spectrum of acetaldehyde produced in yogurt, suggest- antibacterial action of diacetyl, especially ing that the compound could play a role, against Gram-negative bacteria, Jay undoubtedly minor but not negligible, in an- (1982) suggested its use in manufacturing tagonism among different flora. processes in the food and agriculture industry, as an aseptic agent for surfaces D-isomers of amino acids and utensils. Its volatility would avoid con- taminating the flavor of the manufactured Gilliland and Speck (1968) reported that a products. In addition, work is currently un- compound present in the supernatant of a der way to construct hyperproducing dia- lactic starter culture inhibited the same cetyi bacteria. The use of these strains starter. The compound was identified as D- could reinforce the antagonist role of dia- leucine, inhibitory at the concentration of 1 cetyI. Uncertainties remain, however, con- mg/ml. Teeri (1954) showed that several D- cerning the mutagenic potential of diacetyl amino acids inhibited lactobacilli. (Jay, 1982). The mechanism of these inhibitions has not been elucidated. The initial idea was Acetaldehyde that D-amino acids compete with L-forms, essential for the growth of lactic acid bac- Acetaldehyde production in certain dairy teria. Another hypothesis is that D-amino products and notably in yogurt, where it is acids are accidentally incorporated in pro- the principal aromatic constituent, is attrib- teins, rendering them inactive (Gilliland uted principally to L bulgaricus. This spe- and Speck, 1968). cies contains a threonine aldolase which The conditions for the appearance of D- c1eaves threonine into acetaldehyde and amino acids are unknown. Even though glycine (Accolas et al, 1980). The flora of sorne bacterial antibiotics contain D-amino yogurt (L bulgaricus and S thermophilus) acids, there is to our knowledge no proof cannot rnetabolize acetaldehyde and so it of the presence of a racemase in lactic accumulates in the product at a concentra- acid bacteria. This subject requires further tion of", 25 ppm (Accolas et al, 1980). study, ail the more so since unwanted The possible inhibition of lactic acid bacteria such as Pseudomonas are also bacteria by acetaldehyde is relatively apparently sensitive to D-amino acids undocumented, limited to the work of (Daniels, 1966). Kulshrestha and Marth (1975), who showed that the lactic acid bacteria tested CONCLUSION (L lactis, L citrovorum, S thermophilus) were very slightly inhibited at acetaldehyde doses starting at 10 ppm, reaching The first part of this review has examined 3-10% inhibition at 100 ppm. The growth the wide range of inhibitory substances in of undesirable bacteria in dairy products, lactic acid bacteria resulting from the me- eg Staphylococcus aureus, Salmonella ty- tabolism of oxygen and from other catabol- phimurium or E coli decreased at acetalde- ic pathways. These inhibitors are only very hyde concentrations of 10-100 ppm. It is partially used in the fight against undesira- also to be noted that cell division of E ble flora. Future applications are neverthe- coli is inhibited by 10-3 mol/I (44 ppm) ac- less promising. etaldehyde (Egyud, 1967). These data are We have seen that the lactoperoxidase- to be compared with the level of 25 ppm of thiocyanate-hydrogen peroxide system is a Inhibition by lactic acid bacteria 539 powerful inhibitor, capable of exerting a Adams MR, Hall CJ (1988) Growth inhibition of bactericidal effect on a number of patho- food borne pathogens by lactic and acetic ac- gens. This system ls understood to a suffi- ids and their mixtures. Int J Food Sei Technol 23,287-292 cient extent to be used and optimized at the pilot scale for the preservation of milk. Ananthaswamy HN, Eisenstark A (1977) Repair of hydrogen peroxide-induced single-strand More detailed studies on the mechanism of breaks in Escherichia coli deoxyribonucleic resistance to this system by lactic acid acid. J Bacterio/130, 187-191 bacteria are necessary to envision its use Anders RF, Hogg DM, Jago GR (1970) Forma- in lactic fermentations. tion of hydrogen peroxide by group N strepto- Inhibitions resulting from oxygen metab- cocci and its effect on their growth and me- olism remain poorly elucidated. There ex- tabolism. Appl Microbio/19, 608-612 ists a wide variability of sensitivity to these Archibald FS, Fridovich 1(1981) Manganese, su- compounds among lactic acid bacteria and peroxide dismutase and oxygen tolerance in some lactic acid bacteria. J Bacteriol 146, this variability is of value wh en studying 928-936 the mechanisms involved in the resistance Asperger H (1986) Wirkungen von to these metabolites. Stress proteins are Milchsaurebakterien auf andere mikroorga- apparently involved and more detailed nismen. Oesterr Milchwirts 41, suppl 1 to is- knowledge of these phenomena could sue 4,1-22 have repercussions on other aspects, eg Aune TM, Thomas EL (1977) Accumulation of the heat resistance of lactic acid bacteria. hypothiocyanite ion during peroxidase- Organic acids are the major products of catalyzed oxidation of thiocyanate ion. Eur J sugar catabolism. lt is important to note Biochem 80, 209-214 that it is the molecular, or non-ionized, Bjërck L, Rosen CG, Marshall VM, Reiter B (1975) Antibacterial activity of the lactoperox- form of these acids which exerts the great- idase system in milk against pseudomonads est inhibition. Technological advantages and other Gram-negative bacteria. Appl Mi- could arise from these observations. crobio/30, 199-204 Blocher JC, Busta FF (1983) Bacterial spore resistance to acids. 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