Article pp.105-127 du Vol.25 n°2 (2005)

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ARTICLE ORIGINAL ORIGINAL PAPER

An assessment of the bactericidal and fungicidal efficacy of seventeen mineral and organic acids on bacterial and fungal food industry contaminants

H. Martin¹*, P. Maris¹

with technical collaboration of G. Jehannin, A. Rault and R. Fresnel

RÉSUMÉ

Une recherche de l’efficacité bactéricide et fongicide de 17 acides minéraux et organiques sur des bactéries et champignons contami- nants de l’industrie agro-alimentaire

L’efficacité bactéricide et fongicide de dix-sept acides organiques et miné- raux est recherchée pour six souches bactériennes et six moisissures et levures représentatives des contaminants de l’industrie agro-alimentaire.

Une méthode originale inspirée des normes françaises a été employée. Les concentrations minimales bactéricides et fongicides exprimées en concentrations molaires (mol.l^-1) sont comparées, et l’efficacité des acides est dis- cutée selon la valeur de leur pK. Seulement quelques-uns des acides bactéricides testés conservent leur efficacité vis-à-vis des champignons et en particulier vis-à-vis de la souche résistante Absidia corymbifera. C’est le cas des acides nitrique, sulfurique, formique, mandélique, lactique, propioni- que et acétique. De surcroît, une activité intéressante a été notée pour les acides dont le pK se situe entre 3,74 and 3,85 tels que les acides formiques, mandélique et lactique.

L’activité létale sur les bactéries et les champignons des acides faibles se situe dans un intervalle de pH plus compact (2,5 +/– 0,5) par comparaison à celle des acides forts (1,8 +/– 1,3), montrant un effet souche sur l’action toxique des acides forts.

Mots clés

bactéries, champignons, acides, désinfection, microméthode.

1. Agence Française de Sécurité Sanitaire des Aliments – Laboratoire d’Études et de Recherches sur les Médicaments Vétérinaires et les Désinfectants – Unité Produits d’Hygiène Antimicrobiens – La Haute Marche – Javené – BP 90203 – 35302 Fougères cedex – France.

* Correspondance : h.martin@fougeres.afssa.fr

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SUMMARY

The bactericidal and fungicidal efficacy of seventeen organic and mineral acids was investigated on six bacteria and six yeast and fungi representa- tives of food industry contaminants. An original micromethod inspired by French standards was used. The minimal bactericidal or fungicidal concentrations expressed as molar concentrations (mol.l^-1) were compared and efficacy of acids was discussed regarding their pK values. Only few of the bactericidal acids tested kept their efficacy on fungi and in particular on the resistant Absidia corymbifera. This was the case of nitric, sulphuric, formic, mandelic, lactic, propionic and acetic acids. Moreover, an interesting activity for acids with pK values between 3.74 and 3.85 as formic, mandelic and lactic acids was found. Lethal activity of weak acids on bacteria and fungi was in a more compact pH scale (2.5 +/– 0.5) comparing to strong mineral and organic acids (1.8 +/– 1.3) showing that a strain effect does exist on the toxic action of strong acids.

Keywords

bacteria, fungi, acids, disinfection, micromethod.

1 – INTRODUCTION

The effect of low pH against bacteria, yeasts and fungi or viruses has been long recognized. Most of the papers related a preservative or inhibiting effect of acid solutions on microbial activity and growth. The use of washes and sprays containing organic acids has been successful in decontaminating beef, pork and poultry carcasses. The application of 1-1.5 % (v/v) or 2 % (v/v) lactic acid or acetic acid inhibits a weak bacterial contamination of approximately 1.5 log₁₀ (SNIDJERS et al., 1985), low levels of 2-2.4 log₁₀ E. coli 0157:H7, 1.6-1.9 log₁₀ Salmonella typhimurium (CASTILLO et al., 2001) or about 2 log₁₀ of a bovine fae- cal cocktail (DORSA et al., 1998). Organic acids sprays are successfully applied for pathogen reduction in beef carcass processing after the cooler especially when combined with prechill treatments (CASTILLOet al., 2001). Application of organic acid washes to the surface of fruits and vegetables for the purpose of reducing populations of viable micro organisms or for foodstuff preservation also has potential. Some of these agents such as benzoic acid are achieve- ments at the last century decade. Others, such as propionic acid and sorbic acid result from research during the last decade (LÜCK, 1985). So, acidic products as 1% acetic acid limit microbial growth or survival of E. coli 0157 : H7 and Listeria monocytogenes in mustard (RHEE et al., 2003) and inhibitory effects of 1201 ppm sorbic acid, 2435 ppm propionic acid, 3807 ppm acetic acid and 4668 ppm formic acid on spore germination and on mycelial growth of Fusar- ium oxysporum have been noticed (TZATZARAKIS et al., 2000). Then, only few papers talking about the bactericidal and fungicidal effect of acidic solutions on microorganisms were found in the literature (REID, 1932; TANNER and JAMES, 1992; CHERRINGTON et al., 1992; BÖHM, 1987). A number of organic acids as acetic, citric, lactic, formic and propionic acids are used for their bactericidal

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and mild virucidal properties as disinfectants in animal health and food processing. These organic acids have been shown to be less corrosive and toxic than the inorganic sulphuric and hypochloric acids commonly used in animal disease control (KAHRS, 1995). REID (1932) noted a wide difference between the ability of some of these acids to exert a bactericidal effect and to inhibit growth. Then, acids which are strongly bactericidal as oxalic acid exhibit weak inhibitory power in liquid media. On the other hand, acetic, propionic and butyric acids which are weakly bactericidal were the most inhibitory of all the acids tested.

We report in this paper the bactericidal and fungicidal effect of organic and mineral acids on six bacteria and six yeast and fungi representative of food industry contaminants. We compare the sensitivity of these strains to the acids.

We measured the minimal bactericidal or fungicidal concentrations of these acids using a micromethod. This micromethod already developed by MARISet al. (1982) was improved according to Martin et al. (1993) protocol to avoid neu- tralization problems bound to high concentrations acids.

2 – MATERIALS AND METHOD

2.1 Acids

Chemicals selected were 17 organic or mineral acids used in food industry as disinfectants or preservatives. The four mineral acids were: orthophosphoric acid (purity 84%, Prolabo), boric acid (purity 99.8%, Merck), sulphuric acid (purity 95-97%, Merck), nitric acid (purity 65-69%, Prolabo). The 13 organic acids were: formic acid (purity 100%, Prolabo), acetic acid (purity 100%, Pro- labo), citric acid (purity 91.4%, Merck), benzoic acid (purity 91.4%, Merck), sulfamic acid (purity 100%, Sigma), adipic acid (purity 100%, Merck), glutaric acid (purity > 99%, Merck), lactic acid (purity 85%, Sigma), oxalic acid (purity 75%, Prolabo), propionic acid (purity 99%, Merck), succinic acid (purity 99.5%, Merck), tartric acid (purity 99.5%, Merck), mandelic acid (purity 99%, Merck).

Stock solutions were prepared in sterile distilled water at 2% (m/v) for benzoic acid, at 1% (m/v) and 2% (m/v) for adipic and boric acids, at 5% (m/v) for oxalic and succinic acids, at 10% (m/v) for mandelic and sulfamic acids and at 20% (m/v) for citric, glutaric, tartric acids or 20% (v/v) for sulphuric and nitric acids. The stock solutions of the other acids were prepared, when necessary, at 20%, 40% or 80% for formic (v/v), acetic (v/v), orthophosphoric (v/v), propionic (v/v) acids and lactic (m/v) acid.

2.2 Microbial strains

2.2.1 Bacteria

Six bacterial strains which are representative of different species present in the food industry or which are pathogenic for consumers were selected: Entero- coccus hirae CIP 5855, Staphylococcus aureus CIP 53154, Listeria monocy-

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togenes ATCC 19115, Pseudomonas aeruginosa CIP A 22, Salmonella 276 (internal numbering), Escherichia coli CIP 11229.

We prepared bacterial suspensions from bacteria subcultured three times (and incubated during 18-24 h or 42-48 h for Enterococcus hirae) on a tryptic soy agar slope (TSA; Difco) at 37°C. Bacterial suspensions (3.10⁸ CFU.ml^-1) were prepared in saline peptone solution. These bacteria subcultures were realized from stock cultures stored at – 70°C .

2.2.2 Yeast and fungi

One yeast and five fungi were selected according to their importance in food hygiene: Candida albicans ATCC 2091, Absidia corymbifera CIP 1129-75, Geot- richum candidum CIP 285-54, Scopulariopsis brevicaulis CIP 210-53, Aspergil- lus versicolor CIP 1187-79, Penicillium cyclopium CIP 1231-80.

Candida albicans was subcultured on a malt extract agar slope (malt extract 20 g; agar 20 g; freshly distilled water 1000 ml, pH 6.8-7.0; medium sterilized at 121°C +/– 1°C 20 min) during 72 h at 30°C+/– 1°C. Candida vegetative cells suspension (1 or 3.10⁷ CFU.ml^-1) was prepared in an aqueous tryptic casein peptone suspension (tryptic casein peptone 1 g; sodium chloride 8.5 g; freshly distilled water 1000 ml; medium sterilized at 121°C +/– 1°C during 20 min).

Yeast subcultures were realized from stock culture stored at – 70°C .

Fungi were subcultured twice on malt extract agar (see description of medium above) in Roux bottle. Working cultures were prepared in an appropriate volume of diluent (sterile 0.05 % – m/v polysorbate 80 solution in distilled water). The conidiospores were detached from the surface culture with glass beads. The suspension was filtered through a fritted filter (porosity from 40 µm to 100 µm) to separate conidii from mycelium. The spore suspensions so collected were enumerated on malt agar (when necessary after centrifugation at 2000 g for 20 min). The number of spores was adjusted to 1.10⁷ CFU.ml^-1 to 3.10⁷ CFU.ml^-1 in diluent before freezing at – 70°C (stock cultures). The stock cultures were used directly as working cultures for the assays. Incubation times of the subcultures before freezing were the following:

Absidia corymbifera: 15 days at 24°C +/– 1°C and 4 to 6 days at 30°C +/– 1°C.

Aspergillus versicolor: 7 days at 24°C+/– 1°C and 10 days at 24°C+/– 1°C.

Penicillium cyclopium: 15 days at 24°C+/– 1°C and 10-15 days at 24°C+/– 1°C.

Geotrichum candidum: 7 days at 24°C+/– 1°C and 7-15 days at 24°C+/– 1°C.

Scopulariopsis brevicaulis: 7 days at 24°C+/– 1°C and 7-15 days at 24°C+/– 1°C.

2.3 Assay conditions

Acids efficacy was tested without interfering substances.

2.4 Method

A microdilution technique inspired from the ND/1500 method already described by MARTIN and MARIS (1993) was used. Indeed, this study took part in a project of studying the associations of acids with hydrogen peroxide (publica-

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tion in progress). The ND/1500 method was adapted to study the bactericidal efficacy of antiseptics or disinfectants in association. This method was an adaptation of the microdilution technique described by MARIS et al. (1982). This latter had been improved by MARTIN and MARIS (1993) to test high levels of disinfectants in association and to avoid disinfectant neutralization problems. The ND/1500 method was also applied to test fungicidal acid efficacy. This microdilution technique when developed followed the recommendations of the French AFNOR NF T 72-150 standard (1987) and we applied the recommendations of the French AFNOR NF T 72-200 standard (1987) to test disinfectant efficacy on fungi.

2.4.1 Test procedure

The ND/1500 method using the principle of neutralization by dilution included four steps.

2.4.1.1 First step

Serial two-fold dilutions of acids were prepared in sterile distilled water (at least seven dilutions and one control).

2.4.1.2 Second step

Inocula and acid dilutions were left in contact. Each well of a raw of a 96 wells microplate (Falcon) was filled with 75 µl of acid dilution (disinfectant) or distilled water (control), and 75 µl of inoculum. Inoculum was prepared in the microplate with distilled water: 15 µl of 1 or 3.10⁸ CFU.ml^-1 for bacteria (or 15 µl of 1 or 3.10⁷ CFU.ml^-1 for yeast and fungi) were preliminary mixed with 60 µl distilled water or interfering substances. After agitation the mixture was left in contact during 5 min +/– 10 s for bacteria or 15 min +/– 10 s for yeast and fungi at 20°C in temperature controlled room.

2.4.1.3 Third step

The acid activity was neutralized by two transfers in a neutralization solution.

For the first transfer, 10 µl of contact solution were diluted in 90 µl neutralization solution in a microplate. For the second transfer and after agitation, 10 µl of disinfectant in neutralization solution were diluted in 1.5 ml neutralization solution filled in transfer tubes (Dutscher ref. 999602). These two transfer steps must not exceed 15 s for the first and 30 s for the second.

The composition of the neutralization solution was the following: lecithin 3 g;

histidine 1 g; disodium phosphate 34 g; sodium thiosulphate 5 g; polysorbate 80 15 g; distilled water 1000 ml (sterilization at 121°C +/– 1°C during 20 min).

This neutralization solution was diluted (1/10) just before assay.

2.4.1.4 Fourth step

After agitation (vortex) and a minimal 10-min exposure, the transfer tubes contents were poured in Petri dishes and immediately covered with appropriate agar. Bacterial cultures were incubated on PCA agar (Difco) and yeast or fungi cultures on yeast extract agar (yeast extract 5 g; glucose 20 g; Bacto agar 15 g;

freshly distilled water 1000 ml; medium sterilized at 121°C +/– 1°C during 20 min). The agar media were kept at 45-50°C in a water bath before pouring.

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Bacteria were incubated at 37°C during 72 h for Enterococcus hirae and 48 h for the other strains. Incubation was realized at 30°C during 72 h for Candida albicans, at 30°C during 40 h to 70 h for Absidia corymbifera and at 25°C during four days for the other fungal suspensions.

2.4.2 Control

Simultaneously, a control of good neutralization was carried out. For this, 10 µl of each acid dilution were transferred to 190 µl of neutralization solution.

After agitation, 10 µl of acid dilution in neutralization solution were transferred in 1,5 ml neutralization solution in a transfer tube. After agitation and 10 min contact, this acid dilution (1/1500) was filled with 100 µl of pre-diluted inoculum (1 10⁵CFU.ml^-1). So, the inoculum-neutralized acid dilution mixture con- tained 1 10² CFU.ml^-1 to 3 10² CFU.ml^-1. After agitation and at least 15 min contact, the mixture was poured in Petri dishes and covered with appropriate agar. After incubation of the agar cultures as described above for temperature and time, all the agar plates (with acid dilutions and distilled water for control) were enumerated. The neutralization was considered effective for acid dilutions when numeration was higher or equal to the half of the control numeration.

Validation of these results (neutralization validation) was necessary. A varia- tion of one or two dilutions was accepted for the minimal bactericidal concentrations or the minimal fungicidal concentrations. All the results so collected were similar to the results collected with MARIS et al. method (1982), even if microbial reduction detected by this latter method was 10 times more than microbial reduction detected by ND/1500 method (Martin and Maris, 1993).

2.4.3 Results of test procedure

2.4.3.1 Minimal bactericidal concentration and minimal fungicidal concentration Microbial growth (numeration) or total microbial destruction was noted for each agar plate (corresponding to acid dilutions or control). The dilutions without bacterial or fungal growth were considered the minimal bactericidal concentrations (MBC) or the minimal fungicidal concentrations (MFC). At these concentrations at least 1.10⁴ CFU.ml^-1 to 3.10⁴ CFU.ml^-1 (for bacteria) or 1.10³ CFU.ml^-1 to 3.10³ CFU.ml^-1 (for yeast and fungi) were destroyed. Results were expressed in acid percentages (v/v or m/v) and for acid efficacy comparison in molar concentration. We collected so five MBC or five MFC per microbial strain corresponding to acid efficacy without interfering substances.

2.4.3.2 Determination of percentages of dissociated and undissociated acid at bactericidal and fungicidal concentrations

The disinfectant value of strongly dissociated mineral acids depends chiefly upon the number of free hydrogen ions present. For weak organic acids, undissociated molecule and anion may play a role. The amount of undissociated acid is strongly dependent on pH. For each MBC and each MFC, pH in assay conditions was measured and the ratio between undissociated and dissociated acid was calculated. The common equilibrium equation was applied: (A^-)/(HA) = 10^pH-pK (Eklund, 1983) established from the

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common equilibrium equation: ( H⁺)(A^-)/(HA) = K where (H⁺) is the concentration of protons, (A^-) the concentration of anions, (HA) the concentration of undissociated acid and pK the dissociation constant. We applied this equation for both strong and weak acids, and took account the lowest pK value when more than one pK value existed.

3 – RESULTS

3.1 Minimal bactericidal concentrations

The results were dependent on the dissolution capacity of the acids in distilled water, conditioning the concentration of the stock solution. So, no result was collected for adipic and boric acids and only one result for benzoic acid.

Results were collected only for four of the six bacterial strains for tartric, citric, succinic acids. We determined so the molar bactericidal concentrations of 15 acids on one to six bacterial strains (table 1 and table 2).

3.1.1 Pseudomonas aeruginosa

The most sensible strain was Pseudomonas aeruginosa destroyed by acid concentrations varying between 0.001 mol.l^-1 (sulphuric, phosphoric, oxalic, citric, nitric acids) to 0.067-0.104 mol.l^-1 (propionic, glutaric, acetic acids). Propi- onic, glutaric or acetic acids (with pK value between 4.34 and 4.87) had bactericidal molar concentrations 67, 95 or 104 times higher than those of the strong acids cited above. For the other organic acids (tartric, sulfamic, mandelic, formic, lactic and benzoic acids) the bactericidal molar concentrations necessary to kill Pseudomonas aeruginosa were only 2 to 8 times higher than those found for the strong acids. At these molar concentrations, pH medium was near 3 +/– 0.3, excepted for assays with phosphoric acid (pH about 2.2), oxalic acid (pH about 3.6) and benzoic acid (pH about 3.44).

3.1.2 Escherichia coli, Salmonella sp., Listeria monocytogenes

Higher bactericidal molar concentrations were necessary to kill the Gram- negative bacteria Escherichia coli, Salmonella sp., and the Gram-positive bacteria Listeria monocytogenes. For each acid and the three strains cited, molar bactericidal concentrations were close varying from 0.016, 0.009 or 0.004 mol.l^-1 to 0.666 – 0.833 mol.l^-1 (either 42 to 74 or 208 times more) and corresponding to pH values between 2.7 and 2.9 with the exception of some strong acids (pK value < 2) as sulphuric, nitric, sulfamic and phosphoric acids (pH close to 2) and succinic acid (pK value = 4.2) for Escherichia coli (pH about 4.29).

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Table 1

Bactericidal efficacy of acids – Gram-negative bacteria

Tableau 1

Efficacité bactéricide des acides – bactéries Gram négatives

Pseudomonas aeruginosa

acid MW pK MBC % pH mole (AH)/l ISM % AH % H⁺/A^-

sulfuric 98.08 1.92 0.006 3.02 0.001 1 7.36 92.64

phosphoric 98 2.15 0.006 2.22 0.001 1 45.98 54.02

oxalic 90.04 1.27 0.006 3.6 0.001 1 0.47 99.53

citric 192.12 3.14 0.025 3.3 0.001 1 40.89 59.11

nitric 63.02 0.009 3.1 0.001 1 0.08 99.92

tartric 150.09 2.93 0.025 2.72 0.002 2 61.86 38.14

sulfamic 97 1 0.025 2.69 0.003 3 2.00 98.00

mandelic 152.14 3.85 0.05 2.98 0.003 3 88.11 11.89

formic 46.02 3.74 0.025 3.17 0.005 3 78.79 21.21

lactic 90.08 3.83 0.05 3.2 0.006 6 81.01 18.99

benzoic 122.1 4.12 0.1 3.44 0.008 8 82.72 17.28

propionic 74.08 4.87 0.5 3.19 0.067 67 97.95 2.05

glutaric 132.11 4.34 1.25 2.95 0.095 95 96.09 3.91

acetic 60.05 4.79 0.625 3.1 0.104 104 98.00 2.00

E. coli

sulfuric 98.08 1.92 0.156 2.05 0.016 1 42.57 57.43

nitric 63.02 0.156 1.86 0.025 1.56 1.36 98.64

oxalic 90.04 1.27 0.312 1.9 0.035 2.18 19.00 81.00

mandelic 152.14 3.85 0.625 2.62 0.041 2.56 94.44 5.56

sulfamic 97 1 0.625 1.7 0.064 4 16.63 83.37

formic 46.02 3.74 0.312 2.7 0.068 4.25 91.64 8.36

phosphoric 98 2.15 1.25 1.7 0.128 8 73.81 26.19

lactic 90.08 3.83 1.25 2.47 0.139 8.68 95.82 4.18

glutaric 132.11 4.34 2.5 2.61 0.189 11.8 98.17 1.83

succinic 118.09 4.2 2.5 4.29 0.212 13.25 44.84 55.16

propionic 74.08 4.87 2.5 2.89 0.337 21 98.96 1.04

acetic 60.05 4.79 2.5 2.78 0.416 26 99.03 0.97

citric 192.12 3.14 10 1.89 0.521 32.56 94.68 5.32

tartric 150.09 2.93 10 1.82 0.666 41.62 92.80 7.20

Salmonella sp.

oxalic 90.04 1.27 0.08 2.44 0.009 1 6.33 93.67

nitric 63.02 0.078 2.2 0.012 1.33 0.63 99.37

sulfuric 98.08 1.92 0.156 2.05 0.016 1.77 42.57 57.43

sulfamic 97 1 0.312 1.95 0.032 3.55 10.09 89.91

formic 46.02 3.74 0.156 2.86 0.034 3.77 88.35 11.65

lactic 90.08 3.83 0.312 2.75 0.035 3.88 92.32 7.68

mandelic 152.14 3.85 0.625 2.62 0.041 4.55 94.44 5.56

phosphoric 98 2.15 0.625 1.88 0.064 7.11 65.06 34.94

glutaric 132.11 4.34 2.5 2.61 0.189 21 98.17 1.83

citric 192.12 3.14 5 2.06 0.260 28.88 92.32 7.68

propionic 74.08 4.87 2.5 2.89 0.337 37.44 98.96 1.04

acetic 60.05 4.79 2.5 2.78 0.416 46.22 99.03 0.97

succinic 118.09 4.2 5 < 4.29 0.423 47 > 44.83 < 55.16

tartric 150.09 2.93 10 1.82 0.666 74 92.80 7.20

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Table 2

Bactericidal efficacy of acids – Gram-positive bacteria

Tableau 2

Efficacité bactéricide des acides – bactéries Gram positives

MW: Molecular Weight; pK: pK value of acid; pH: pH measure at minimal bactericidal concentration or minimal fungicidal concentration in assay conditions; MBC or MFC: Minimal bactericidal concentration or Minimal fungicidal concentration; ISM: Ratio of acid concentration at MBC or MFC (mole/l) with the lowest MBC or MFC (mole/l) collected for the strain; % AH: percentage of undissociated acid; % H⁺/A^-: percentage of dissociated acid.

PM : poids moléculaire ; pK : pK de l’acide ; pH : mesure dans les conditions de l’essai du pH à la concentration minimale bactéricide ou fongicide ; CMB or CMF : concentration minimale bactéricide ou concentration minimale fongicide ; ISM : Rapport de la concentration molaire (mole/l) d’un acide à la CMB ou à la CMF de cet acide par la plus faible valeur de CMB ou CMF exprimée en concentration molaire obtenue pour la souche ; % AH : pourcentage d’acide non dissocié ; % H⁺/A^- : pourcentage d’acide dissocié.

Listeria monocytogenes

sulfuric 98.08 1.92 0.039 2.35 0.004 1 27.09 72.91

nitric 63.02 0.078 2.2 0.012 3 0.63 99.37

oxalic 90.04 1.27 0.156 2.15 0.017 4.25 11.65 88.35

formic 46.02 3.74 0.156 2.86 0.034 8.5 88.35 11.65

mandelic 152.14 3.85 0.625 2.62 0.041 10.25 94.44 5.56

sulfamic 97 1 0.625 1.7 0.064 16 16.63 83.37

succinic 118.09 4.2 1.25 4.56 0.106 26.5 30.39 69.61

lactic 90.08 3.83 1.25 2.47 0.139 34.75 95.82 4.18

phosphoric 98 2.15 2.5 1.53 0.255 63.75 80.65 19.35

tartric 150.09 2.93 5 2.04 0.333 83.25 88.59 11.41

propionic 74.08 4.87 2.5 2.89 0.337 84.25 98.96 1.04

citric 192.12 3.14 10 1.89 0.521 130.25 94.68 5.32

glutaric 132.11 4.34 10 2.43 0.757 189.25 98.78 1.22

acetic 60.05 4.79 5 2.6 0.833 208.25 99.36 0.64

Staphylococcus aureus

acid MW pK MBC% pH mole (AH)/l ISM % AH % H⁺/A^-

nitric 63.02 0.156 1.86 0.025 1 1.36 98.64

oxalic 90.04 1.27 0.312 1.9 0.035 1.4 19.00 81.00

sulfuric 98.08 1.92 0.625 1.45 0.064 2.56 74.70 25.31

mandelic 152.14 3.85 1.25 2.5 0.082 3.28 95.72 4.28

sulfamic 97 1 2.5 1.3 0.258 10.32 33.39 66.61

formic 46.02 3.74 1.25 2.37 0.272 10.88 95.91 4.09

lactic 90.08 3.83 5 2.17 0.555 22.2 97.86 2.14

propionic 74.08 4.87 5 2.72 0.675 27 99.30 0.70

glutaric 132.11 4.34 10 2.43 0.757 30.28 98.78 1.22

acetic 60.05 4.79 5 2.6 0.833 33.32 99.36 0.64

phosphoric 98 2.15 10 1.08 1.020 40.8 92.16 7.84

E. hirae

nitric 63.02 0.156 1.86 0.025 1 1.36 98.64

sulfuric 98.08 1.92 0.312 1.71 0.032 1.28 61.86 38.14

oxalic 90.04 1.27 0.625 1.7 0.069 2.76 27.09 72.91

mandelic 152.14 3.85 2.5 2.35 0.164 6.56 96.93 3.07

sulfamic 97 1 2.5 1.3 0.258 10.32 33.39 66.61

propionic 74.08 4.87 5 2.72 0.675 27 99.30 0.70

glutaric 132.11 4.34 10 2.43 0.757 30.28 98.78 1.22

formic 46.02 3.74 5 2.02 1.086 43.44 98.13 1.87

lactic 90.08 3.83 10 1.98 1.110 44.4 98.61 1.39

acetic 60.05 4.79 10 2.42 1.665 66.6 99.57 0.43

phosphoric 98 2.15 20 0.8 2.041 81.64 95.72 4.28

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3.1.3 Staphylococcus aureus, Enterococcus hirae

The gram positive bacteria Staphylococcus aureus and Enterococcus hirae, two strains frequently resistant to disinfectants were killed by bactericidal concentrations varying between 0.025 mol.l^-1 and 1.02-2.04 mol.l^-1 (either 41 to 82 times more). Strong acids such nitric, oxalic, sulphuric acids (pK value < 2) were the more active acids. They kill Staphylococcus aureus and Enterococcus hirae with molar bactericidal concentrations either 17 times or 52-104 times more than the molar bactericidal concentrations necessary to kill Pseudomonas aeru- ginosa. At these molar bactericidal concentrations, the pH values of the assays were between 1.45 and 1.9. Surprisingly, the strong phosphoric acid was the less bactericidal of the acids tested. One or two mol.l^-1 of phosphoric acid (either a molar concentration 1600 to 3000 times more than the molar bactericidal concentration found for Pseudomonas aeruginosa) were necessary to kill these two strains. Mortality was then noted for a low pH value (pH = 0.8-1.08).

On the other hand, the weak mandelic acid was found to be the most bactericidal acid among the three acids with similar pK value (pK value between 3.74 and 3.85). So, mandelic acid was bactericidal at molar concentrations of 0.16 or 0.08 mol.l^-1 and formic or lactic acids were bactericidal with 0.3 or 1.08 mol.l^-1 or 0.55 or 1.11 mol.l^-1. Decreasing activity was noted for the other acids with intermediate bactericidal activity for formic and sulfamic acids and lowest bactericidal activity for the following acids: lactic, propionic, glutaric and acetic acids. Concentrations varying between 0.5 and 1.6 mol.l^-1 corresponding to pH value between 2 and 2.72 were necessary.

3.2 Minimal fungicidal concentrations

Molar fungicidal concentrations were obtained for only 5 to 9 acids (table 3).

Fungicidal molar concentrations varied in the range from 5-10 for Absidia corymbifera, Candida albicans, Aspergillus versicolor and Penicillium cyclopium (variations between molar fungicidal concentrations of mandelic, or nitric or formic acids and acetic acid), and from 1 to 31 for Scopulariopsis brevicaulis and 1 to 136 for Geotrichum candidum (variations between molar fungicidal concentrations of formic acid and lactic acid or phosphoric acid).

The acids with pK value < 2, fungicidal on Candida albicans and fungi, were the following: nitric acid (at 0.098-0.397 mol.l^-1), sulphuric acid (at 0.25-1.25 mol.l^-1), oxalic acid (at 0.27 mol.l^-1, fungicidal only on Candida albicans) and phosphoric acid (at 1.02 to 4.08 moles, fungicidal only on Penicillium cyclopium and Geotrichum candidum).

The acids with a pK value between 3.74 and 3.85 fungicidal on Candida albi- cans and fungi were the following: formic (at 0.033-0.54 mol.l^-1), mandelic (at 0.08-0.33 mol.l^-1) and lactic (at 1.1-2.2 mol.l^-1) acids.

The two last acids active on yeast and fungi were propionic acid (at 0.169-2.7 mol.l^-1) or acetic acid (at 0.416-0.83 mol.l^-1). Their pK values are 4.79 and 4.87. The most resistant strain was systematically Absidia corymbi- fera.

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Table 3

Fungicidal efficacy of acids

Tableau 3

Efficacité fongicide des acides

Absidia corymbifera

acid MW pK MFC% pH mole (AH)/l ISM % AH % H⁺/A^-

mandelic 152.14 3.85 5 1.75 0.33 1 99.21 0.79

nitric 63.02 2.5 0.77 0.40 1 14.52 85.48

formic 46.02 3.74 2.5 2.05 0.54 1 98.00 2.00

sulfuric 98.08 1.92 12.5 0.4 1.27 3 97.07 2.93

lactic 90.08 3.83 20 1.9 2.22 6 98.84 1.16

propionic 74.08 4.87 20 2.54 2.70 8 99.53 0.47

acetic 60.05 4.79 20 2.3 3.33 10 99.68 0.32

Candida albicans

mandelic 152.14 3.85 2.5 2.05 0.16 1 98.44 1.56

nitric 63.02 1.25 0.84 0.20 1 12.63 87.37

formic 46.02 3.74 1.25 2.19 0.27 1 97.26 2.74

oxalic 90.04 1.27 2.5 1.17 0.28 1 55.73 44.27

sulfuric 98.08 1.92 5 0.6 0.51 3 95.43 4.57

propionic 74.08 4.87 5 2.7 0.67 4 99.33 0.67

lactic 90.08 3.83 10 2.03 1.11 6 98.44 1.56

acetic 60.05 4.79 10 2.36 1.67 10 99.63 0.37

Scopulariopsis brevicaulis

formic 46.02 3.74 0.31 2.5 0.07 1 94.56 5.44

mandelic 152.14 3.85 1.25 2.05 0.08 1 98.44 1.56

nitric 63.02 1.25 1.02 0.20 2 8.72 91.28

sulfuric 98.08 1.92 2.5 0.85 0.25 3 92.16 7.84

propionic 74.08 4.87 5 2.79 0.67 9 99.18 0.82

acetic 60.05 4.79 5 2.64 0.83 11 99.30 0.70

lactic 90.08 3.83 20 1.9 2.22 31 98.84 1.16

Aspergillus versicolor

nitric 63.02 0.62 1.40 0.10 1.00 3.83 96.17

formic 46.02 3.74 0.62 2.25 0.13 1 96.87 3.13

mandelic 152.14 3.85 5.00 1.75 0.33 3 99.21 0.79

propionic 74.08 4.87 5.00 2.79 0.67 6 99.18 0.82

acetic 60.05 4.79 5.00 2.64 0.83 8 99.30 0.70

Penicillium cyclopium

nitric 63.02 1.25 1.02 0.20 1.00 8.72 91.28

formic 46.02 3.74 2.50 2.05 0.54 2 98.00 2.00

propionic 74.08 4.87 5.00 2.79 0.67 3 99.18 0.82

phosphoric 98 2.15 10 ND 1.02 5 ND ND

acetic 60.05 4.79 6.20 2.64 1.03 5 99.30 0.70

lactic 90.08 3.83 10.00 2.09 1.11 5 98.21 1.79

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© Lavoisier – La photocopie non autorisée est un délit MW: Molecular Weight; pK: pK value of acid; pH: pH measure at minimal bactericidal concentration or minimal fungicidal concentration in assay conditions; MBC or MFC: Minimal bactericidal concentration or Minimal fungicidal concentration; ISM: Ratio of acid concentration at MBC or MFC (mole/l) with the lowest MBC or MFC (mole/l) collected for the strain; % AH: percentage of undissociated acid; % H⁺/A^-: percentage of dissociated acid.

PM : poids moléculaire ; pK : pK de l’acide ; pH : mesure dans les conditions de l’essai du pH à la concentration minimale bactéricide ou fongicide ; CMB or CMF : concentration minimale bactéricide ou concentration minimale fongicide ; ISM : Rapport de la concentration molaire (mole/l) d’un acide à la CMB ou à la CMF de cet acide par la plus faible valeur de CMB ou CMF exprimée en concentration molaire obtenue pour la souche ; % AH : pourcentage d’acide non dissocié ; % H⁺/A^- : pourcentage d’acide dissocié.

3.3 Comparison of fungicidal concentrations with bactericidal concentrations

The ratios between fungicidal concentrations and the bactericidal concentrations of the most sensible bacterial strain (Pseudomonas aeruginosa) were the following:

– 16 to 32 for acetic acid (pH fungicidal assays between 2.3-2.64), – 10 to 40 for propionic acid (pH fungicidal assays between 2.54-2.92), – 13 to 109 for formic acid (pH fungicidal assays between 2.05-2.5), – 25 to 100 for mandelic acid (pH fungicidal assays between 1.75-2.05), – 68 to 278 for nitric acid (pH fungicidal assays between 0.77-1.02), – 198 to 400 for lactic acid (pH fungicidal assays near 2.09), – 397 for oxalic acid (pH fungicidal assays 1.17),

– 425 to 2124 for sulphuric acid (pH fungicidal assays between 0.4-0.85), – 1666-6666 for phosphoric acid (pH non determined).

3.4 Link between acid activity and pK value

3.4.1 Bacteria (figures 1 and 2)

A link between pK value and bactericidal activity was shown. Acid bactericidal activity varied with bacteria and pK values. So, for the sensible strain Pseudomonas aeruginosa, bactericidal molar concentrations followed a pro- gressive elevation parallel to elevation of pK value. Sensibility of Pseudomonas aeruginosa to strong acids (pK < 2) was maximal and superior to sensibility to mandelic acid (pK = 3.85), benzoic acid (pK = 4.12) and mainly to glutaric, ace-

Geotrichum candidum

acid MW pK MFC% pH mole (AH)/l IMS % AH % H⁺/A^-

formic 46.02 3.74 0.15 2.86 0.03 1 88.35 11.65

mandelic 152.14 3.85 1.25 2.05 0.08 2 98.44 1.56

propionic 74.08 4.87 1.25 2.92 0.17 5 98.89 1.11

nitric 63.02 1.25 1.02 0.20 6 8.72 91.28

acetic 60.05 4.79 2.50 2.75 0.42 13 99.10 0.90

sulfuric 98.08 1.92 5.00 0.66 0.51 16 94.79 5.21

lactic 90.08 3.83 10.00 2.09 1.11 37 98.21 1.79

phosphoric 98 2.15 40.00 ND 4.08 136 ND ND

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tic and propionic acids (pK > 4.34). Exception to phosphoric, tartric and citric acids with pK values between 2.15 and 3.14, a similar progression was recorded for Escherichia coli, Salmonella sp. and Listeria monocytogenes. The bactericidal molar concentrations of these last strains were higher than those determined for Pseudomonas aeruginosa and lightly higher than those determined for formic, lactic and mandelic acids with pK values between 3.74 and 3.85.

The Gram-positive Staphylococcus aureus and Enterococcus hirae were killed with bactericidal molar concentrations higher than those necessary for the destruction of the bacteria cited above. We found again, on one hand, the group of phosphoric, tartric and citric acids with weak or without significant bactericidal activity and, on the other hand, the mandelic acid strongly bactericidal among acids of similar pK value.

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70

sulfamic pK = 1 nitric

oxalic pK = 1.27sulfuric pK = 1.92 phosphoric pK = 2.15

tartric pK = 2.93citric pK = 3.14formic pK = 3.74lactic pK = 3.83

mandelic pK = 3.85benzoic pK = 4.12succinic pK = 4.2glutaric pK = 4.34acetic pK = 4.79 propionic pK = 4.87 acids

mol.l–1

Pseudomonas aeruginosa Escherichia coli Salmonella sp

Figure 1

Minimal bactericidal concentrations evolution with pK values of acids (Gram-negative bacteria).

Évolution des concentrations minimales bactéricides en fonction du pK des acides (bactéries Gram négatives).

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3.4.2 Fungi (figure 3)

Acid efficacy link with pK values was more difficult to establish, because numerous fungicidal concentrations were lacking. It was obvious that two strong mineral acids were fungicidal: nitric and sulphuric acids. Formic (pK 3.74) and mandelic (pK 3.85) acids had also a strong fungicidal activity. The third acid of this group (lactic acid with pK 3.83) had a similar fungicidal activity than acetic and propionic acids with superior pK values (4.79 and 4.87). Fungicidal activity of one acid seemed to be more dependent on its total characteristics (structure, MW, pK ) than on one characteristic as pK value.

3.4.3 Action mode of acids

Figures 4 and 5 showing the percentages of dissociated and undissociated acids at bactericidal or fungicidal acid concentrations revealed the different action modes of acids, either by their acidity (activity dependent on concentration of H⁺ions in medium) as strong mineral or organic acids (pK near 1), either by their undissociated form as weak acids (pK near 3). For acids with pK values between 1 and 3, according to the considered strain, combined action of dissociated and undissociated forms were implicated. So, Pseudomonas aeruginosa was more sensible to H+ concentration of acids in medium than resistant bacterial strains and fungi. Surprisingly in figure 5, our results indicated that sulphuric undissociated form was the form active on fungi.

sulfamic pK = 1 nitric

oxalic pK = 1.27sulfuric pK = 1.92 phosphoric pK = 2.15

mandelic pK = 3.85benzoic pK = 4.12succinic pK = 4.2glutaric pK = 4.34acetic pK = 4.79 propionic pK = 4.87 0.00

0.50 1.00 1.50 2.00 2.50

mol.l–1

acids

Listeria monocytogenes Staphylococcus aureus Enterococcus hirae

Figure 2

Minimal bactericidal evolution with pK values of acids (Gram-negative bacteria).

Évolution des concentrations minimales bactéricides en fonction du pK des acides (bactéries Gram négatives).

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0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

nitric

sulfamic pK = 1oxalic pK = 1.27sulfuric pK = 1.92 phosphoric pK = 2.15

mandelic pK = 3.85benzoic pK = 4.12succinic pK = 4.2glutaric pK = 4.34acetic pK = 4.79 propionic pK = 4.87 acids

mol.l–1

Absidia corymbifera Candida albicans Scopulariopsis brevicaulis Aspergillus versicolor Penicillium cyclopium Geotrichum candidum

Figure 3

Minimal fungicidal concentrations evolution with pK values (yeast and fungi).

Évolution des concentrations minimales fongicides en fonction du pK des acides (levure et champignons).

0 10 20 30 40 50 60 70 80 90 100

nitric

sulfamic oxalicsulfuric phosphoric

tartric citric formic lactic

mandélicglutaric acétic propionic

%

Pseudomonas aeruginosa Escherichia coli Salmonella sp.

Listeria monocytogenes Staphylococcus aureus Enterococcus hirae

Figure 4

Percentages of dissociated acids at minimal bactericidal concentrations.

Pourcentages d’acide dissocié aux concentrations minimales bactéricides.

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4 – DISCUSSION

Most of the researches on acid efficacy were related to preservation or inhi- bition of microbial activity and growth. Acetic, lactic, propionic, citric, formic acids alone or in association have an immediate effect on bacterial aerobic flora and pathogens of meat (reviews of DICKSON and ANDERSON, 1992; SMULDERS

and GREER, 1998), and fresh products (review of BEUCHAT, 1998). Generally immersion of meat carcasses and fruit or vegetables in 0.5% to 4% lactic acid or acetic acid solutions during varying contact times (few seconds, 10-15 min or more), produced decreases of 1-1.5 log₁₀ to sometimes 3 log₁₀ of Salmo- nella typhimurium, E coli 0157: H7, Listeria monocytogenes, Listeria innocua, Pseudomonas sp., Yersinia enterolitica or Clostridium sporogenes populations alone or in cocktails (SNIDJERS et al. 1985; DORSA et al., 1998; NASCIOMENTO et al., 2003). A temperature elevation to 40-55°C, an increase of contact time between acid and food (CASTILLO et al., 2001) or complementary treatments as deshydratation (DI PERSIO et al., 2003) or ultra violet (ESCUDERO et al., 2003) improved acid efficacy. Assay conditions are specific and look like to those encountered in the field. Concerning the disinfectant efficacy of acids, more information exists on Gram-negative bacteria and only few data on Gram-posi-

0 10 20 30 40 50 60 70 80 90 100

nitric

sulfamic oxalic sulfuric phosphoric

tartric citric formic lacticmandélic glutaric acétic propionic

%

Candida albicans Absidia corymbifera Scopulariopsis brevicaulis Aspergillus versicolor Penicillium cyclopium Geotrichum candidum

Figure 5

Percentages of dissociated acid at minimal fungicidal concentrations.

Pourcentages d’acide dissocié aux concentrations minimales fongicides.

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tive bacteria and yeast (almost on Candida albicans) (REID, 1932; BÖHM, 1987;

TANNER et al., 1992; CHERRINGTON et al., 1992). In our study, we compare the bactericidal efficacy of acids on Gram-negative contaminants (E. coli and Pseu- domonas aeruginosa) with their bactericidal efficacy on food industry pathogens (Salmonella sp. and Listeria monocytogenes) and with their bactericidal efficacy on Gram-positive resistant strains (Staphylococcus aureus and Enterococcus hirae). Then, our comparison is extended to yeast as Candida albicans, but also to environmental fungi among which the resistant strain Absidia corymbifera.

Our results are collected after 5 min contact time at 20°C for bacteria and after 15 min contact time at 20°C for fungi. These conditions correspond to criteria described in the basic European Standards (NF EN 1040, April 1997; NF EN 1275, June 1997), excepted for the reduction criteria, which are 1 log₁₀ less than standards criteria. In addition, we tested the impact of the four following interfering substances: 30 French degree hard water, 60 French degree hard water, albumin bovine (0.3% w/v) with 30 French degree hard water, albumin bovine (1% w/v) and yeast extract (1% w/v). No or low impact of water hardness and low dirty conditions on acid efficacy was detected (internal results).

For high dirty conditions, concentrations of acids have to be identical or the double (for acid concentrations above 1% - v/v or w/v) or one to 8 times more (for acid concentrations less 1% - v/v or w/v and according to acid bactericidal concentrations) and that for five of the six bacterial strains tested. Pseu- domonas aeruginosa is the exception. This strain is very sensible to low con- centrations of acids and higher acid concentrations (either 1 or 2 times more with hard water or 4 to 64 times more with low dirty or high dirty conditions) were necessary to obtain the same reduction of the population. This observa- tion confirms the relationship between acidity and cell viability already sug- gested by CHERRINGTON et al. (1992) and explains the high impact of organic matter on the low acid bactericidal concentrations collected for Pseudomonas aeruginosa. This last author noted that organic matter as blood, milk and serum but not yeast extract had little effect on the activity of acetic acid and lactic acid on Salmonella sp. Weak impact of water hardness and dirty conditions have to be expected on bactericidal acid concentrations for resistant Gram positive bacteria as Staphylococcus aureus and Enterococcus hirae and resistant fungi as Absidia corymbifera. Our comparative study keeps all its interest in comparing acids efficacy.

Our results are in accordance with usual knowledge of bacterial and fungal behaviours towards pH. Bacterial strains are pH sensitive with minimal growth of Pseudomonas species between pH 4.2 to 4.5 and between pH 1.5 and 3.5 for the majority of bacterial strains, yeasts and fungi (BOUIX and al., 1999).

Results of TANNER and JAMES (1992) using the A.O.A.C. germicidal and deter- gent sanitizer test showed that exposure of Pseudomonas aeruginosa cells (in a presence of 100 ppm hard water) to acidic solutions at pH near 3.0 (succinic acid, sulphuric acid, phosphoric acid) or 2.5 (citric acid, lactic acid, phosphoric acid) destroyed in 60 s 10⁴ viable cells or more per ml. Gram positive bacteria as Listeria monocytogenes, Staphylococcus aureus and yeasts as Candida albicans and Saccharomyces cerevisae were not destructed at pH 3.2 or 3.4. In our study, we detect a 4 log₁₀ reduction in five minutes with all the mineral and organic acids for Pseudomonas aeruginosa and that for pH values near 3 +/– 0.5. For the other bacterial strains and fungi, we can class the acids in three classes ranged as a function of the pK value. On one hand,

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for mineral as well as organic acids with pK value near or less 2 as nitric acid, oxalic acid, sulphuric acid and phosphoric acid, lethal action in five minutes at 20˚C and without interfering substances on bacteria and fungi lies on low and large scale of pH (1.5 +/– 1 pH unity), the more resistant fungus Absidia corymbifera as Scopulariopsis brevicaulis and the yeast Candida albicans being destructed (3 log₁₀ reduction) for the lowest pH values (pH less or equal to 1). On the other hand, for acids with pK value between 4 and 5 as glutaric acid, acetic acid and propionic acid the pH range of lethal action in five min at 20°C and without interfering substances is more compact (2.5 +/– 0.5) and far below the pK values. The third class is the class of acids with pK values between 2 and 4 as tartric acid, citric acid, formic acid, lactic acid and mandelic acid. Some of these acids as tartric and citric acids have in our study an activity limited to few bacterial strains and fungi. Others, as formic, lactic and mandelic acids with pK value between 3.74 and 3.85 have a good bactericidal and fungicidal efficacy in a pH range (between 2 and 3) below their pK values.

Reid in 1932 concluded that the bactericidal activity on Bacillus pyocyanus of the mono basic series of organic acids, acetic, propionic, butyric and valeric increases as the series ascended, either with an increase in molecular weight and a decrease in surface tension. He found the following bactericidal activity range on Bacillus pyocyanus, Bacillus typhosus, Bacillus coli and Staphylococcus aureus: oxalic acid > to lactic acid > to propionic acid > to acetic acid. Oxalic acid was germicidal at normal dilutions varying between N/320 and N/18, so with concentrations 12 to 100 times lower than acetic acid. Succinic acid activity was near the bactericidal activity of propionic and acetic acids. Citric acid activity was not regular, bactericidal for N/18 normal dilution on Bacillus pyocyanus and for normal dilutions varying between N/1.5 and 2 N for the other strains. BÖHM (1987), comparing bactericidal activity of formic acid, citric acid, acetic acid and propionic acid with a suspension test in accordance with the guidelines of the German Society of Veterinary Medicine (DVG) on the bacterial strains laid down in these guidelines: Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Steptococcus faecium, found that formic acid was the more bactericidal of the acids examined. This acid was followed, in the case of Gram-negative bacteria, by acetic acid, and propionic acid. Regarding the effect of acids on Gram- positive bacteria, formic acid was followed in efficiency by propionic acid, acetic acid and citric acid, this last acid activity being irregular. In our study a higher bactericidal and fungicidal effect was found too for oxalic acid in comparison with acetic acid. Acetic acid molar concentrations 24 to 50 times higher, up to 104 times more for Pseudomonas aeruginosa were necessary to kill bacteria. For Candida albicans, the only fungus for which we found oxalic acid efficacy, fungi- cidal acid acetic molar concentration was 6 times more than fungicidal oxalic acid molar concentration. We found a strong bactericidal and fungicidal activity in a narrow pH range for the acids with pK values between 3.74 and 3.85 as formic acid, mandelic acid and lactic acid. Their bactericidal and fungicidal activities were higher than those found for tartric acid and citric acid, but least than bactericidal and fungicidal efficacy of mineral and organic strong acids, exception to phosphoric acid. Comparing to efficacy of the weak acetic acid in terms of molar concentrations (which activity on bacteria and fungi is similar to activity of propionic acid), formic acid was 1.5 times to 24.5 times more active, mandelic acid 2.76 to 34.6 times more active and lactic acid only 1.5 to 3 (or 11.8 for Salmonella sp) times more active. So, among these acids, we found an interesting