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Effects of free fatty acids on P Boyaval, C Corre, C Dupuis, E Roussel

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P Boyaval, C Corre, C Dupuis, E Roussel. Effects of free fatty acids on propionic acid bacteria. Le Lait, INRA Editions, 1995, 75 (1), pp.17-29. ￿hal-00929416￿

HAL Id: hal-00929416 https://hal.archives-ouvertes.fr/hal-00929416 Submitted on 1 Jan 1995

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lait (1995) 75, 17-29 17 © Elsevier/INRA

Original article

Effects of free fatty.acids on propionic acid bacteria

P Boyaval 1, C Corre 1, C Dupuis 1, E Roussel 2

1 Laboratoire de Recherches de Technologie Laitière, INRA, 65, rue de St Brieuc, 35042 Rennes Cedex; 2 Standa-Industrie, 184, rue Maréchal-Galliéni, 14050 Caen, France

(Received 10 May 1994; accepted 21 November 1994)

Summary - The seasonal variations in composition, especially du ring the grazing period, often lead to poor eye formation in Swiss-type cheese. The influence of free fatty acids on the grow1h and

of the dairy propionibacteria has been studied in this work. Linoleic (C1B:2), laurie (C12:0), myristic (C14:0) and oleic acids (C1B:1) inhibited the growth and acid production of P treudenreichii subsp shermanii in the reference medium. The antibacterial activity of Iinoleic acid can be overcome by additions of cholesterol and soya lecithin to the medium. The four species and !WO subspecies of dairy could be divided into !WO groups according to their high susceptibility to unsaturated fatty acids (P freudenreichii subsp shermanii and subsp freudenreichit) or low susceptibility (P acidipro- pionici, P jensenii and P thoeniJ). Nevertheless, this inhibitory action of free fatty acids was not observed in milk, retentate media or lactic curd. A total extraction of the milk led to the recovery of this inhibitory effect on Propionibacterium. The possible modes of action of these molecules are discussed on the basis of the observed effluxes and disturbances in the cell membranes.

free 1 Propionibacterium 1inhibition llinoleic acid Ilauric acid

Résumé - Effet des acides gras libres sur les bactéries propioniques. Les variations saisonnières de composition du lait peuvent avoir des répercussions importantes sur la qualité des fromages. Les laits de printemps conduisent souvent à de graves défauts d'ouverture en fabrication de fromage à pâte pressée cuite (PPC) et notamment d'emmental. Ce travail montre l'action fortement inhibitrice de 1 faibles quantités d'acides gras libres (10 à 100 mg/t-I) sur le développement, dans leur milieu de cul- ture de référence, des bactéries propioniques, agents de l'ouverture de ces fromages. Les acides linoléique (CIB:2), laurique (CI2:0), myristique (CI4:0) et oléique (CIB:I) inhibent la croissance et la pro- duction d'acides propionique et acétique de Propionibacterium freudenreichii subsp shermanii. La 1 présence de cholestérol ou de lécithine de soja dans le milieu de culture restaure l'activité cellulaire. Deux grands groupes de susceptibilités différentes à ces actions inhibitrices peuvent être formés 1 parmi les souches des 4 espèces de bactéries propioniques laitières: les 2 sous-espèces de P freu- denreichii (grande susceptibilité) d'une part et P acidipropionici, P jensenii et P thoenii (faible sus- ceptibilité) d'autre part. Néanmoins, l'action inhibitrice de l'acide linoléique ne se retrouve ni lors de cul- 1 ture sur lait ou sur rétentat (facteur de concentration volumique = 5), ni dans des caillés préparés selon la technologie des fromages PPC. Une délipidation totale du lait permet à nouveau l'expression

1

1

1 18 P Boyaval et al du caractère inhibiteur de cet acide gras libre sur les bactéries propioniques. Les modes d'action pos- sibles de ces acides gras sont discutés au regard des résultats de pertes de potassium et des per- turbations engendrées par ces acides gras libres au niveau des membranes cellulaires. acide gras libre /Propionibacterium / inhibition / acide linoléique / acide laurique

INTRODUCTION fatty acids in milk fat, especially the feed- ing, the lactation stage, the animal species or the breed of cow. The seasonal varia- The inhibitory action of rancid milk on the tions in the composition of milk fat influence growth of numerous bacteria has been dairy processing. This assertion is particu- shown (Tarassuk and Smith, 1940) and con- larly true in cooked hard cheese process- firmed later (Costilow and Speck, 1951). ing, where the milk fat represents 45 to 55% The inhibitory effects and also the growth of the dry matter. This fat influences the promoting properties of fatty acids were rheological properties (Creamer and Oison, reviewed by Nieman (1954). The most pro- 1982), the flavour (Adda et al, 1982) and nounced antibacterial effects of low con- centrations of fatty acids have been noted the microbial transformation of the cheese with Gram-positive bacteria. (Chen et al, 1979 ). The antibacterial activity of free fatty acids As the natural lipase of milk is almost was shown at the same time, especially on completely destroyed by the cooked hard bacteria (Humfeld, 1947; Poz- cheese processing (Driessen, 1983), the nanski et al, 1968). This activity is some- main Iipolytic activities in this type of cheese times bactericidal but, with most microor- are of microbial origin: psychrotrophic bac- ganisms, the antibacterial action of fatty teria (Law et al, 1976), lactic and propionic acids is bacteriostatic (Nieman, 1954). starters and surface flora for Comté and Kodicek and Worden (1945) have shown Beaufort. that oleic and linolenic acids inhibit the Lactic acid bacteria exhibit a low lipolytic growth of Lactobacillus helveticus. Lauric activity (Stadhouders and Veringa, 1973), and myristic acids also proved to be but propionic acid bacteria, which are the inhibitory for this microorganism (Williams main ripening agents in Swiss-type cheeses, and Fieger, 1946). Later, Anders and Jago are lipolytic (Knaut and Mazurek, 1974; (1964a, b) showed that , accumu- Dupuis and Boyaval, 1993; Dupuis et al, lated early during the cheese ripening pro- 1993). Free fatty acid concentrations as high cess, inhibited the viability of lactic acid as 14 9 kg-1 of hard cheese were reported streptococci. This oleic acid alters the pyru- after ripening (Woo et al, 1984). After pro- vate metabolism of group N streptococci pionic and acetic acids, which are the direct (Anders and Jago, 1970). The cis-form of result of propionic acid bacteria activity, the unsaturated fatty acids exhibited a greater major fatty acids are oleic (C18:1), palmitic antibacterial activity than the correspond- (C16:0), stearic (C18:0) and myristic acids ing trans isomers on L helveticus, M tuber- (C14:0), as in milk fat (Langler and Day, culosis and B subtilis (Nieman, 1954; Gal- 1966; Masson et al, 1978; Deeth et al, 1983; braith and Miller, 1973; Kabara, 1979). de Jong and Badings, 1990). Milk fat mainly consists of The seasonal variations which change of fatty acids (97 to 98%) (Kurtz, 1965). the proportions of these acids, especially in Many factors determine the proportion of spring (Decaen and Journet, 1966; Gray, Fatty acids and propionibacteria 19

1973; Masson et al, 1978), affect the Swiss- labo, France). Ali media were sterilized by heat type cheese ripening. Poor eye formation treatment (120°C, 15 min). and lower levels of propionic and acetic Cultures on milk were carried out with a low acids in the curd suggest a poor propionic heat milk powder (milk 'G') recommended by acid bacteria activity. This problem has a Chamba and Prost (1989) for the evaluation of acidifying activity of thermophilic lactic acid bac- considerable economic impact for the manu- te ria. facturers who need to know the exact factors Cheese curds were prepared according to leading to these depreciated cheeses. Buisson et al (1987) with microfiltrated milk ln this study, we have examined the (Trouvé et al, 1991). The phase of pressing was influence of free fatty acids on growth, acid not carried out. production (propionic and acetic acids) and lactate consumption of Propionibacterium freudenreichii subsp shermsnii, the most Analysis frequently found propionic acid bacteria in Swiss-type cheese and of a type-strain of Bacterial growth was followed by optical each dairy species of this genus. Moreover, measurements (650 nm; Beckman spectropho- potassium efflux rates, consump- tometer DU 7400, Gagny, France) correlated with tion and ~\jI (transmembrane electrical dry weight measurements. Lactic, propionic and potential) alterations generated by Iinoleic concentrations were measured using an HPLC system (Beckman, Gagny, France) molecules on Propionibacterium have been equipped with an UV detector (214 nm; Scan- measured in order to understand the nature ning detector module 167). Separation of sam- of this influence. pie of 20 JlI took place in a 7.5 x 300 mm (Aminex A6, exchange, Biorad) stainless steel column,

operated at ambient temperature with H2S04 MATERIALS AND METHODS 5 mmoll-1 (1 ml rnirr'") as eluent.

Strains and growth media Chemicals reagents

Five type-strains (Cummins and Johnson, 1986), Acetic acid (C2, Merck, Darmstadt, Germany); of each species or subspecies of dairy Propioni- propionic acid (Cs, Sigma, St Quentin Fallavier, bacterium were used during this study: P freuden- France); n- (C4, Sigma); reichii subsp freudenreichii CIP 103026 (Collec- (Ca, Sigma); (Cs' Merck); tion de l'Institut Pasteur, Paris, France), (C1Q, Merck); laurie acid (C12, Prolabo, Paris, P freudenreichii subsp shermanii CIP 103027, France); oleic acid (C1S:1' Prolabo) and Iinoleic P jenseniiCIP 103028, P thoeniiCIP 103029 and acid (C1S:2' Prolabo) were used in this study, dis- P acidipropionici DSM 4900 (Deutsche Samm- solved in ethyl alcohol of a high chemically pure lung von Mikroorganismen und Zellkulturen, form (Prolabo). Cholesterol was purchased from Braunschweig, Germany). Moreover, one strain Prolabo, soya lecithin from Lucas-Meyer (France, frequently used in cheese factories, Propioni- 'Emulfluid E') and bovine albumin (BSA, essen- bacterium freudenreichii subsp shermanii LRTL tially globulin free) from Sigma. 30 (INRA, Rennes, France), was also used throughout this study. Strains were propagated in a lactate-yeast extract based medium (YEL) Growth and activity in the presence (Malik et al, 1968) at 30°C without shaking. Via- of free fatty acids bility was evaluated by plating 1 ml of cell sus- pension on YEL agar and incubated at 30°C for 4 days. Cultures were carried out at 30°C in 100 ml glass Stock cultures were maintained at -70°C in bottles with 10 and 100 mg 1-1 of each fatty acid YEL medium containing 15% (vlv) glycerol (Pro- in the YEL medium. Controls were made for each 20 P Boyaval et al

() concentration employed. The ditions, except that the ce Ils were pretreated with pH was not monitored. the protonophore TCS (3, 3', 4', 5 - tetrachloro- salicylanilide) (10 j.lmoll-1, final concentration).

Oxygen consumption measurements RESULTS

Oxygen consumption was measured polaro- graphically using a Clarke-type electrode con- nected to a Gilson oxygraph (Model K-ITC-C, Inhibitoryaction of free fattyacids (FFA) Middleton, Wl, USA). Measurements were car- ried out at 30°C on cells incubated in phosphate butter (50 mmol 1-1, pH 6.50) at a final concen- Even at very low concentrations « 1a mg 1 tration of 0.3 mg ml-1 (dry weight: DW). 1- ), the palmitic (C16:0) and stearic acids (C18:0) were not soluble in ethanol. They were soluble in propanol-1 and chloroform Potassium efflux measurements but formed a precipitate in the YEL medium. Consequently, they were not tested further

The variations in the potassium content of the in this study. cells (K+in) were determined by measuring the At 10 mg 1-1, growth of P freudenreichii changes of the potassium concentration in the subsp shermanii LRTL 30 on YEL medium external mediurn (K+out) with a potassium-vali- was clearly inhibited by the and nomycin-selective electrode (Radiometer) asso- ciated with a calomel reference electrode con- slightly by the lauric (C12:0), myristic (C14:0) taining a secondary salt bridge filled with a and oleic (C18:1) acids. These bacteriostatic solution of 100 mmol 1-1 NaCI (Boulanger and effects were clearly evidenced at 100 mg Letellier, 1988). The cation electrode potential 1 1- , particularly for the lauric (C12:0) and was calibrated with KCI solutions of known con- Iinoleic (C18:2) acids (table 1). A restoration centrations, which were prepared in the experi- of growth after 25 h was observed for the mental butter. To estimate the total potassium content of the bacteria, the cation was released by oleic acid (C18:1). The lactate consumption treatment of the cells with 0.4 mmoll-1 Iinoleate was affected in the same way with no or 2 mmol 1-1 laurate. The amount of released decrease for laurie (C12:0) and linoleic acids cation was th en estimated with the potassium (C18:2) and a slight decrease for myristic electrode. K+in was calculated from the value of (C14:0) and oleic acids (C18:1). The propi- K+ and expressed in nmol mg-1 ml-1 (cell DW) out onate and acetate productions reflected assuming that 4 x 109 cells correspond to 1 mg ml-1 (cell DW). Whatever the product nature exactly the lactate evolution. Moreover, a added to the cells, the ethanol concentration was low positive (or no) effect of the fatty acids always less than 1% (v/v). from acetic (C2:0) to capric acids (ClO:0) on the initial (24 h) lactate consumption and propionic and acetic acids production was Measurement of the transmembrane noted. electrical potential (.6.0/) The effects of caproic acid (C6:0) and linoleic acid (C18:2) on the type-strains of tl.\jI was determined from the accumulation of the four species and two subspecies of dairy [14C] TPP+ (tetraphenylphosphonium ion) (10 propionibacteria are shown in table II. A 1 umol lr", final concentration, 2.17 GBq mmol- ) slight positive effect of the caproic acid on (Ghazi et al, 1986). The cells were filtered on the growth of the two P freudenreichii strains glass fibre filters (GF/F Whatman), washed with 4 ml of butter and counted for radioactivity. TPP+ was observed. This effect was not signifi- uptake was corrected for unspecific binding by cant for the three other species. Linoleic subtracting a blank obtained under identical con- acid drastically affected the growth of Fatty acids and propionibacteria 21

Table 1. Effect 01 caproic acid (Cs:o) and linoleic acid (C1S:2) at 100 mg 1-1 on the growth 01 the live type-strains 01 dairy Propionibacterium. 1 Effets des acides caproïque (C6:0) et linoléique (CI8:2) à 100 mg 1- sur la croissance des 5 souches-type de bactéries propioniques laitières.

FFA Strain

103026 103027 103028 103029 4900

C6:0 o o o

C18:2

Inhibitory action: --- high: .- medium: - low; 0, no activity. Action inhibitrice: --- forte; -- moyenne; • faible; 0 : pas d'activité.

Table Il. Inhibiting action 01 Iree latty acids on P freudenreichii subsp freudenreichii and, the growth, propionate production and lactate to a lesser extent, the growth of P freuden- consumption 01 P freudenreichii subsp sher- reichii subsp shermanii, P theonii. P jensenii manii LRTL 30 compared ta a control without and P acidipropionici growth was affected lattyacid. during the first 30 h, but the number of cells Action des acides gras libres sur la croissance, la production de propionate et la consommation de sharply increased afterwards to reach a level lactate de Propionibacterium Ireudenreichii subsp higher than the control cultures (28% more shermanii LRTL 30 (comparée à un témoin sans for P jensenii and 33% for P acidipropionicl). addition d'acide gras libre). Globally, these results were confirmed by the lactate consumption and the propionic and acetic acid production. Fattyacid Growth Propionate Lactate production consumption As the natural medium of Propionibac- terium multiplication in Swiss-type cheese technology is milk, we have tried to repeat C2 0 + + our observations on milk. The main results C3 0 + 0 are summarized in figure 1. Even in a milk C4 0 + 0 containing a low concentration (500 mg Cs 0 + 0 1-1), linoleic acid, at concentrations up to 5 9 Cs 0 + 1-1, had no effect on cell multiplication or on C1Q + acid production. The inhibitory effect of C12 Iinoleic acid was recovered after a previous C14 extraction of the lipids from the milk pow- C1S:1 der with before milk rehydration. C1S:2 Moreover, the inhibition of Propionibacterium freudenreichii subsp shermanii LRTL 30 was also not evidenced in milk retentate Inhibition: -", high; -', medium; " low. Activation: +++, high; ++, medium; +, low; 0, no effect. (volume concentration factor of 5, obtained tnhibition: --', forte; --, moyenne; " faible. Activation: by ultrafiltration; not shown). In order to bet- +++, forte; ++ moyenne; + faible; 0 sans effet. ter master the possible influence of free fatty 22 P Boyaval et al

acids in Swiss-type cheese technology, we caproic acid (C6:0), nor by the presence of have examined the development of this biotin at 10-5 or 10-4 9 1-1. However, soya strain in a cheese curd made from microfil- lecithin (100 mg 1-1) and cholesterol (100 trated milk (which contained only lactic mg 1-1) greatly reduced this negative effect starters before addition of propionic acid (fig 2). bacteria). The cell multiplication, during the 340 h of the trial, was not affected by the presence of 5 9 1-1 of linoleic acid (not .~ 3.5

ai" 2.5 .2a Alterations of the FFA inhibition activity 0

byaddition of compounds 1.5

No growth stimulating effect of Tween 40, 0.5 Tween 60, Tween 80 (at 100 mg 1-1) and o ~=~:!!:=:==~!:::::;:::~----< biotin (at 10-5 or 1Q-4 9 1-1)was observed. o 20 40 60 80 On the other hand, Tween 80 at 30 9 1-1 Timelh) counteracted the inhibitory effect of linoleic 4 acid for the P jensenii CIP 103028 culture 3.5 (not shown). The inhibitory effect of linoleic . 1 acid (C18:2) at 100 mg 1- was reversed nei- ther by the presence of 10 or 100 mg 1-1 of

_ 1.8 1.5

o 0 0.5 ot:=~~~~~~~~-----i o 20 40 60 80 Timelh)

Fig 2. Effect of linoleic acid (C18:2) (100 mg 1-1) 60 80 100 120 (.); linoleic (100 mg 1-1) + cholesterol (100 mg 1-1) Time(h) (0); linoleic (100 mg 1-') + biotin (10-4 g 1-1) (+); Fig 1. Effect of linoleic acid (5 g 1-1) on propio- linoleic (100 mg 1-1) + soya lecithin (100 mg 1-1) nic acid production by P freudenreichii subsp (e); linoleic (100 mg 1-1) + caproic acid (C6:0) 10 shermanii LRTL 30 in milk G (.) and in milk G mg 1-1 (.À.) and 100 mg 1-1 (1:\) on the growth of P previously treated with hexane (.À.). Control freudenreichii subsp shermanii LRTL 30. Contrais without addition of linoleic acid (0). Contrais are are carried out with the same solve nt concentra- carried out with the same solve nt concentrations tions as in trials. as in trials. Effets des acides linoléique (CI8:2)(100 mg rI) Effet de J'acide linoléique (5 g rI) sur la pro- (.); linoléique (100 mg rI) + cholesterol (100 mg duction d'acide propionique par des cellules de rI) (0); linoléique (100 mg rI) + biotine (1D-4 g P freudenreichii subsp shermanii LRTL 30 cul- rI) (+); linoléique (100 mg rI) + lécithine de soja tivées sur lait G (.) et sur lait G après traite- (100 mg t:'} (e); linoléique (100 mg rI) + acide l ment à J'hexane (~). (0) représente la produc- caproïque (C6:0) à 10 mg.r (~) et à 100 mg r! tion d'acide propionique sans addition d'acide (1:\) sur la croissance de P freudenreichii subsp linoléique. Les témoins sont réalisés avec les shermanii LRTL 30. Les témoins sont réalisés mêmes concentrations de solvant que dans les avec les mêmes concentrations de solvant que essais. dans les essais. Fatty acids and propionibacteria 23

Alterations of the cell membrane 200 integrity: potassium efflux

Total cellular K+ initially present in the Pro- pionibacterium cells was evaluated at 1650 nmol mg-1 dry weight, a value close to the 1800 nmol mg-1 determined by Duperray et al (1992) for the closely related species of Corynebacterium glutamicum. Figure 3 re- presents the effect of increasing concen- trations of linoleic acid on the initial rate of K+ efflux. The minimum concentration of linoleic 50 100 150 200 acid necessary to detect an efflux of K+ in Linoleic acid (/Lmol-1) cells incubated at pH 6.50 is 6 Ilmoll-1 (20 Ilmoll-1 for laurie acid). Addition of 1251lmol Fig 3. Effect of Iinoleic acid on inhibition of O 1-1of linoleic acid induced a complete and 2 consumption (e), K+ efflux rate (0) and L'l'V (.) in 1 rapid efflux of K+ (1600 urnol min- Propionibacterium freudenreichii subsp sherma- mg-1 (DW)). also induced an niiLRTL30. efflux of cytoplasmic K+. This efflux was Effet de l'acide linoléique sur la consommation maximal only for a laurie acid concentration d'02 des cellules de Propionibacterium freuden- reichii subsp shermanii LRTL 30 (e), la vitesse de of 900 Ilmoll-1, more than 7 times the con- sortie du potassium intracellulaire (0) et le L'l'V centration of linoleic acid for the same rate (.). (fig 4). Addition of 50 umol t' of BSA to the cell suspension after addition of the free

fatty acid totally counteracted the action of 200 251lmoll-1 of linoleic acid on K+ efflux (the same effect was observed for 200 Ilmoll-1 BSA after addition of 51 umol r' of linoleic acid). Moreover, a 1 min incubation with 50 umol r' BSA totally protected the internai K+ cellular pool against the action of 51 ~011-1 of linoleic acid. The addition of MgS04 at 1 mmol 1-1 completely counteracted the K+ efflux induced by the previous adjunction of 52 urnol 1-1of linoleic acid to the bacterial cells. Morever, in the presence of 20 mmol 1-1 MgS0 in the medium, 50 urnol 1-1 of 4 800 1000 linoleic acid have no effect on the K+ efflux Laurie acid (JUllol-1) from the P freudenreichii subsp shermanii cells. A previous incubation of the Propio- Fig 4. Effect of laurie acid on inhibition of O2 nibacterium cells with caproic acid (780 umol consumption (e), K+ efflux rate (0) and L'l'V (.) in 1-1) had no protective effect on K+ efflux Propionibacterium freudenreichii subsp sherma- induced later by linoleic acid (25 urnol 1-1) niiLRTL30. (not shown). Effet de l'acide laurique sur la consommation d'02 des cellules de Propionibacterium freu- The presence of 15 mg of Tween 80 in denreichii subsp shermanii LRTL 30 (e), la the cell suspension had a total protective vitesse de sortie du potassium intracellulaire (0) effect against linoleic acid action (on K+ et le L'l'V (.). 24 P Boyaval et al efflux and on oxygen consumption), at least growth. Addition of 60 Ilmoll-1 of B8A had up to 200 urnol 1-1 ! Moreover, addition of no action on the cell oxygen consumption, Tween 80 (15 mg) after addition of linoleic but protected the cells against the action of acid (50 Ilmoll-1) immediately stopped the 100 Ilmoll-1 linoleic acid. But even with this K+ efflux. A 2 min incubation of the cells protective action, 180 urnol 1-1 of Iinoleic with 200 Ilmoll-1 of cholesterol reduced by acid induced a 90% decrease in oxygen 74% the K+ efflux induced by 1281lmoll-1 of consumption. Lauric acid at 440 urnol 1-1 linoleic acid (not shown). But the addition achieved the same inhibition. This is the of 100 urnol 1-1 or 200 urnol 1-1 of choles- same ratio observed as without B8A pro- terol after addition of 50 urnol t' of linoleic tection (2.4 more laurie acid than Iinoleic acid had no effect on that K+ efflux. acid).

Action of FFA on the transmembrane DISCUSSION electrical potential Inhibitoryaction ofFFA At pH 6.50, the Ô\j1 was 158 mV, a value close to the Ô\j1 determined for other Gram- The main effects of the fatty acids on Pro- positive bacteria (162 mV for S aureus and pionibacterium freudenreiehii subsp sher- 113 mV for S Jaetis (Kashket, 1981) and manii grawth on YEL medium enable a sep- 190 mV for Corynebacterium glutamicum aration into three classes: c1ass l, from C2:0 (Duperray et al, 1992)). The cell membrane to ClO:0 no effect on growth and a low pos- also became more permeable to H+ after itive effect on the fermentative capacities; addition of linoleic acid since a decrease of class Il, C14:0 and C18:1 negative effect on the Ô\j1 was observed (fig 3). This decreas- growth and acid production, intermediate ing Ô\j1 fram 158 mV to a mV was evidenced effect between classes 1 and III; class III, in the same range of linoleate concentra- C18:2 and C12:0 complete inhibition of the tion than K+ efflux. At a concentration of 900 growth and metabolism of these bacteria. urnol l"" of laurie acid, the ô\j1 was a (fig 4). To our knowledge, no information has been published on the action of free fatty acids on propionic acid bacteria. Lactobacilli, Inhibition of oxygen consumption although generally not sensitive to saturated fatty acids, can be sometimes inhibited by them if the compounds have a chain length The inhibition of oxygen consumption, ini- of around C12, these being the most active tially evaluated at 15 ± 2 nmol O min-1 2 (Hassinen et al, 1951). In this study, we mg-1 (DW) was shown within the same have observed the same effects on propio- range as Ô\j1 decrease (fig 3). Lauric acid nic acid bacteria. inhibitory effect towards oxygen consump- A few explanations on the exact influ- tion was roughly the same as observed for ence of the inhibitory fatty acids on microbial linoleic acid except that total inhibition cells have been proposed but not confirmed: occurred at 250 Ilmoll-1 for laurie acid and i) they decrease the bacterial respiration at 100 Ilmoll-1 for linoleic acid (fig 4). (Galbraith and Miller, 1973); ii) they build Caproic acid at concentrations up to 800 an adsorption layer around the cell which Ilmoll-1 showed no action on oxygen con- changes the cell permeability, leading to an sumption and K+ intracellular level (not outward diffusion of vital cellular cornpo- shown), confirming the above results on cell nents or bloc king the adsorption of essential Fatty acids and propionibacteria 25

nutrients (Nieman, 1954); iii) they are able, teins or by other means. These indications in the form of , to lower the surface lead to an investigation into the role of these tension of the media (Nieman, 1954) and compounds, at the cell membrane level, they inhibit the active transport of amino with and without fatty acids in the medium. acids (Galbraith and Miller, 1973). ln this study we were unable to detect As numerous works have shown that sev- any positive effect of the polyoxyethylene eral surface active compounds do not inhibit derivative of fatty acid monoesters of sor- the growth of microorganisms, the first and bitan ( of oleic acid = Tween 80; ester second hypotheses must be controlled of = Tween 60 and ester of experimentally. The fact that Gram-positive = Tween 40) at 100 mg 1-1 on bacteria are more sensitive than Gram-neg- the growth of P freudenreichii subsp sher- ative bacteria supports the intervention of manii LRTL 30. This growth stimulation was the cell membrane structures in this phe- frequently evidenced in the past (Dubos, nomenon. Moreover, the work of Moss et al 1947; Ledeoma et al, 1977; Cummins and (1969) on the fatty acid composition of pro- Johnson, 1986). The neutralization of the pionibacteria leads to the separation of the antimicrobial properties of Iinoleic acid by species into two groups: P freudenreichii Tween 80 at 30 9 1-1 was also evidenced subsp freudenreichii and subsp shermanii by Baker et al (1983) for laurie acid. The on the one hand and the three other species exact mechanism of the Tween in neutral- on the other hand. This separation is in close izing the activity of this acid has not been agreement with the two groups of Propioni- explained in his paper. bacterium species reactivity towards fatty The use of saturated fatty acids to inhibit acids found in this study. the negative action of unsaturated fatty acids observed by Hassinen et al (1950) on L bifidus was not confirmed using our strain. Alterations of the FFA inhibition activity If the mode of action of these acids is an byaddition of compounds interaction with the membrane layers of the cells, the affinity of the membranes for the The presence of protein can counteract the fatty acid considered and a concentration inhibition of fatty acids as observed by Dubos dependence of the phenomena are proba- (1947) for the serum albumin, which has high bly involved. The ratios under investigation affinity binding sites for FFA (Frapin et al, here may be out of the correct range to 1993). Many other components act similarly: observe a detoxification. saponin, lecithin, charcoal, starch, choles- Biotin is required for the growth of most terol (Kodicek and Worden, 1945). The anta- strains of dairy propionibacteria (Delwich, gonistic effect of cholesterol and lecithin on an 1949). No positive effect on growth of dairy antibacterial fatty acid, as observed in this propionibacteria by additions of biotin was study, was already noticed by Wynne and observed in this study. The high level of Foster (1950), working with Micrococcus biotin of our medium had probably hidden pyogenes var aureus and oleic acid. No clear this potential effect. Williams et al (1947) interpretation of this type of results has been supposed that biotin promotes, in some way, proposed to our knowledge. the formation of unsaturated fatty acids ln 1990, Somkuti and Johnson showed essential for the bacteria. The relationships that P freudenreichii cells were able to bind between fatty acids and biotin in the nutrition the cholesterol present in the medium by of microorganisms is still far from being polysaccharide or membrane bound pro- clearly defined. 26 P Boyaval et al

Alteration of the cell membrane activity chain free fatty acids were evidenced as far more pote nt uncouplers than the saturated acids (Borst et al, 1962). But we cannot If ~'" = 0, at 100 urnol 1-1 of linoleic acid, exclude the possibility of some cofactor leak- ~'" remained at 60% of its initial value after age from the cells. The protective effect of addition of 100 Ilmoll-1 of laurie acid. More- BSA against the inhibitory effect of an over, 250 umol 1-1 of lauric acid induced uncoupler, as we observed in this study, only a very slow K+ efflux in the cells of P has already been observed on mitochon- treudenreichii subsp shermanii. It was then dria since 1956 (Pullman and Racker, 1956). evidenced that linoleic acid is a much more It has been related to the presence of an active compound than laurie acid as a Pro- hydrophobic site in that protein, which has a pionibacterium cell membrane disturber. great affinity for fatty acids. The addition of linoleic or laurie acids in As underlined by Borst et al (1962) the Propionibacterium cell suspensions results membrane disturbing activity of free fatty in an immediate perturbation of the permea- acids may probably be distinguished from bility properties of the cytoplasmic mem- that brought about by agents breaking up brane and of the bacterial energetic state: mitochondrial or bacterial envelope struc- cells lose cytoplasmic potassium, they ture in that it is readily reversible by the sub- become partially or totally depolarized and sequent addition of serum albumin. How- their respiration is inhibited. Ali these ever, we have no c1ear interpretation of the changes indicate that the target of the free protective action of MgS04 on Propioni- fatty acids is the cytoplasmic membrane. bacterium cells. To our knowledge, no information is avail- The fatty acid inhibitory effects observed able about the K+ transport system(s) in on the growth and metabolism of dairy Pro- Propionibacterium cells. However, we have pionibacterium are of great industrial impor- calculated in our experiments that one cell tance. Indeed, the interest in propionic acid reached by fatty acid lost potassium at a production by is increasing rate of 4.8 106 K+ S-1. This rate is many (Boyaval and Corre, 1987; Boyaval, 1992). orders of magnitude higher than one expects This revival for biological propionic acid pro- for passive efflux through the lipid bilayer duction is based on the highly increased estimated at 20 ions s-1/cell by Gomperts productivities allowed by the technology of 14 (1976) (if a permeability coefficient of 10- membrane bioreactors. As yeast extracts, m S-1 is assumed) . This suggests that the which are very frequently used in that type inhibitory action of free fatty acids occurred of fermentation, contain unsaturated fatty by some globallipid bilayer disturbances. acids (Eddy, 1958) the producer must take The K+ efflux is not the consequence of into account the susceptibility to these membrane depolarization since the cells inhibitory effect in the selection of the pro- incubated with the protonophore TCS at 10 pionic acid strains. Ilmoll-1, do not show K+ efflux (not shown). But the most important point is the pos- Even if the time necessary to reach the com- sible involvement of these fatty acids in the plete loss of K+ by the cells increased when inhibition of dairy Propionibacterium in hard the free fatty acid concentrations decreased, cheese manufacturing. These cheeses re- the level "0" is always attained. present more than 212 000 tons per year Free fatty acids are known to uncouple in France, only for Emmental (CNIEL, 1994). oxidative phosphorylation in mitochondria, The highly predominant species employed chloroplasts and bacterial cells (Rottenberg in hard cheese technology is P treudenreichii and Hashimoto, 1986). Unsaturated long- subsp shermanii. If the response of most of Fatty acids and propionibacteria 27

the strains of this species is similar to the spring. The cycle of cheese manufacturing response of the strain LRTL 30, it will prob- must be completed in the initial presence ably beinteresfinq to mix them with strains of Iinoleic acid in arder to discard this hypoth- of other species in spring, when the problem esis or not. of eye formation occurs. Unfortunately, we have been unable to find any information on the level of free fatty acids in the cheese ACKNOWLEDGMENTS curd at the end of the cold ripening, just before the development of the propionibac- The authors gratefully acknowledge the region teria. These analyses are currently under of Brittany for the financial support. E Faivre for investigation. This finding underlines once the preparation of the cheese curd, A Ghazy and again that the lipid content of the medium L Letellier for their expert help during K+ efflux has a drastic protective effect on the cells, measurements and paper elaboration and C probably by integration of the free fatty acid Guinard and E Rees for the English improvement in the lipid globules. But scanning electron of the tex!. microscopie examinations of Saint Paulin cheeses suggest that the globule membrane could be disorganised by change in pH, REFERENCES enzymatic action and by the mechanical action of pressing (Rousseau. 1988). Mem- Adda J, Gripon JC, Vassal L (1982) The chemistry of brane debris and fractured fat globule were flavour and texture generation in cheese. Food Chem 9,115-125 observed. Moreover, some water was pref- Anders RF, Jago GR (1964a) The effect of fatty acids on erentially localized around the globules, the metabolism of lactic acid streptococci. 1. Inhibi- leading to a kind of 'barrier' between the tion of bacterial growth and proteolysis. J Dairy Res globules and the hydrophobie fatty acids. 31,81-89 These observations were completed by a Anders RF, Jago GR (1964b) The effect of fatty acids on study on Emmental cheese where fat glob- the metabolism of lactic acid streptococci. II. Resis- tance of a variant of Streptococcus cremoris strain ules had lost their initial structure to give C13. J Dairy Res 31,91-94 large masses with diverse forms (Rousseau Anders RF, Jago GR (1970) The effect of fatty acids on and Le Gallo, 1990). These points under- the metabolism of pyruvate in Iactic acid strepto- line that the fat globules, with such modifi- cocci. J Dairy Res 37, 445-456 cations and reduced surface have a Baker RC, Vadehra DV, Gravani RB (1983) Neutraliza- decreased ability to trap free fatty acids in tion of antimicrobial properties of Lauricidin by Tweens. J Food Safety6, 1-12 the cheese body. Our test with a curd which Borst P, Loos JA, Christ EJ, Slater EC (1962) Uncoupling has not followed the complete cheese tech- activity of long-chain fatty acids. Biochim Biophys nology process (not pressed), was proba- Acta 62, 509-518 bly not sufficient to answer the question Boulanger P, Letellier L (1988) Characterization of ion asked. Moreover, the alterations of fat glob- channels involved in the penetration of phage T 4 ules, during processing and ripening of these DNA into cells. J Biol Chem 263, 9767-9775 cheeses, could overcome their trapping Boyaval P, Corre C (1987) Continuous fermentation of effect on free fatty acids as observed in milk sweet whey permeate for propionic acid production and fresh cheese curd. Even if we have no in a CSTR with UF recycle. Biotechnol Lett9, 801- clear biochemical data on the evolutionof 806 the lipid phase in a cheese curd from the Boyaval P (1992) Production d'acides organiques en beginning of the technology to the end of bioréacteur à membrane. Ind Aliment Agric 109, 953- 958 the ripening period, these results cast doubt Buisson V, Kerjean JR, Courroye M (1987) Miniaturisa- on the major role of free fatty acids in the tion de la fabrication fromagère. Tech Lait Market alteration of the eye formation during the 1024,17-23 28 P Boyaval et al

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