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Control of Listeria monocytogenes in model sausages by enterocin AS-48
International Journal of Food Microbiology 103 (2005) 179 – 190 www.elsevier.com/locate/ijfoodmicro
Control of Listeria monocytogenes in model sausages by enterocin AS-48 Samir Ananoua, Margarita Garrigab, Marta Hugasb, Mercedes Maquedaa, Manuel Martı´nez-Buenoa, Antonio Ga´lvezc, Eva Valdiviaa,d,T a
Departamento de Microbiologı´a, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 18071-Granada, Spain b IRTA-Centro de Tecnologı´a de la Carne, 17121-Monells, Girona, Spain c´ Area de Microbiologı´a, Facultad de Ciencias Experimentales, Universidad de Jae´n, Paraje Las Lagunillas, Jae´n, Spain d Instituto de Biotecnologı´a, Universidad de Granada, 18071-Granada, Spain Received 18 May 2004; received in revised form 18 December 2004; accepted 29 December 2004
Abstract In this work we describe the control of Listeria monocytogenes CECT 4032 in sausage by adding the enterocin AS-48 producer strains Enterococcus faecalis A-48-32 and Enterococcus faecium S-32-81, and also by adding a semi-purified preparation of the bacteriocin. Addition of preformed AS-48 caused a significant decrease ( Pb0.01) in the number of viable listeria even at the lowest bacteriocin concentration tested (112 AU/g). At a higher concentration (225 AU/g) listeria were below the detection level (1.99 log units/g) in meat at 3 days of incubation but growth of listeria was observed again after 9 days. For an AS-48 concentration of 450 AU/g, no viable listeria were detected after 6 and 9 days of incubation. When E. faecalis A-48-32 was used as inoculum at approximately 107 cfu/g, listeria counts decreased progressively from start of experiment, being below detection level at day 9. The best results were obtained with E. faecium S-32-81, since listeria were undetectable at 6 days of incubation. Bacteriocin concentrations in samples reached concentrations of 60 and 80 AU/g for strains A-48-32 and S-32-81, respectively. These results clearly indicate that AS-48 can be used in the control of L. monocytogenes in sausages. D 2005 Elsevier B.V. All rights reserved. Keywords: Listeria monocytogenes; Biopreservation; Sausages; Enterocins; Bacteriocins; AS-48
1. Introduction
T Corresponding author. Departamento de Microbiologı´a, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 18071Granada, Spain. Tel.: +34 958 243244; fax: +34 958 249486. E-mail address: [email protected] (E. Valdivia). 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.12.024
Raw meat is highly sensitive to microbial spoilage because of its ecological properties (a w, pH and nutrients). In addition, meat and meat products are responsible for many food-borne illnesses outbreaks. Listeria monocytogenes is an ubiquitous bacterial pathogen with a high prevalence in many foods,
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mainly milk and cheeses, meat and meat products, fish, shellfish and vegetables (Farber and Peterkin, 1991; Samelis and Metaxopoulos, 1999). The ubiquity and also the psychrotrophy of this bacterium make its control to be extremely difficult and it can be considered an in house bacterium in processing plants of meat products. Thus, a study has reported that as many as 68% of the environmental samples in meat processing plants were positive for Listeria (Salvat et al., 1995). Several investigations from European institutions responsible for public health have shown that Listeria occurs in 12–16% of industrial fermented food products (European Report, 1999; AFSSA, 2000). In fact, it is known that L. monocytogenes can survive the commercial dry sausage manufacturing process despite the various hurdles such as low pH, salt and nitrites (Hugas et al., 1995; Varabioff, 1992; Incze, 1998). Some bacteriocins like nisin, enterocins A and B, sakacin, or pediocin AcH, have been tested, alone or in combination with several physico-chemical treatments (modified atmosphere packaging, high hydrostatic pressure, heat, pH and chemical preservatives) as an additional hurdle to control proliferation of listeria in meat products (Nielsen et al., 1990; Cutter and Siragusa, 1996; Aymerich et al., 2000; Cleveland et al., 2001; Garriga et al., 2002). Also, several bacteriocinogenic lactic acid bacteria, especially pediococci strains, have been used as bioprotective cultures for food manufacturing processes in attempts to control this bacterium (Foegeding et al., 1992; Hugas et al., 1995; Hugas, 1998; Mataragas et al., 2003; Budde et al., 2003). Enterocin AS-48 is a cationic cyclic bacteriocin produced by Enterococcus faecalis S-48 with broad bactericidal activity against most of the Gram-positive bacteria, including several pathogens like L. monocytogenes (Mendoza et al., 1999), Staphylococcus aureus, Mycobacterium sp., Bacillus cereus, and some Gram-negative bacteria (Ga´lvez et al., 1989; Abriouel et al., 1998, 2002, 2003). The features of AS-48 (broad spectrum of antimicrobial activity, stability in a wide range of temperature and pH, and sensitivity to digestive proteases; Ga´lvez et al., 1986; Samyn et al., 1994) point to it as a promising alternative to chemical preservatives to be used in a future as biopreservative in foods. Currently, we are performing a series of studies intended to know how AS-48 acts on the main food-borne pathogenic
bacteria as well as the influence of environmental factors concurring in foods on its antimicrobial effectiveness. In a previous study Mendoza et al. (1999) demonstrated the in vitro inhibitory effect of AS-48 on L. monocytogenes CECT 4032 in Brain Hearth Infusion broth. In this study we have investigated the ability of enterocin AS-48 to control L. monocytogenes CECT 4032 in model sausages, by adding the bacteriocinogenic strains E. faecalis A-4832 and Enterococcus faecium S-32-81 or a semipurified preparation of the enterocin.
2. Materials and methods 2.1. Bacterial strains and culture conditions Two enterococcal strains were used as bacteriocin AS-48 producers (AS-48+): E. faecalis A-48-32 and E. faecium S-32-81. The last is a strain isolated from cheese, lacking virulence determinants, and harbouring plasmid pAM401-81 that encodes for AS-48 production and immunity (Maqueda et al., 2003). As negative controls two Bac- strains E. faecalis S-47 and E. faecium S-32 were used, respectively. E. faecalis S-47 was used as indicator strain. The strain L. monocytogenes CECT 4032, associated with a case of meningitis, was used as challenge in sausages. Bacterial strains were grown on brain heart infusion (BHI) (Scharlab, Barcelona, Spain) at 30 8C. Solid media were prepared by adding 1.5% agar (Scharlab) to the broths. Enterococci and listeria cultures were maintained at 4 8C. 2.2. Preparation of bacteriocin AS-48 E. faecalis A-48-32 was grown for 8 h at 30 8C in a modified complex medium (MCM broth) (Abriouel et al., 2003): 0.2% Casaminoacids (Difco, Detroit, MI, USA), 0.2% BHI, 1% Glucose, 0.1% yeast nitrogen base (YNB; Difco), and 0.001% MgSO 4d 7H20 (PanReac, Barcelona, Spain) dissolved in 0.1 M sodium phosphate buffer (pH 7.2). YNB solution was sterilized by filtration (0.45 Am, Millipore Ibe´rica, Madrid, Spain) before being added to the heatsterilized medium. Bacteriocin recovery was done by cation exchange chromatography as described by Abriouel et al. (2003). Eluted fractions were sterilized
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by filtration (0.45 Am, Millipore) and tested for bacteriocin activity against the indicator strain S-47 by the agar well diffusion method using steel cylinders of 8 mm (outer) diameter (Ga´lvez et al., 1986). The titre of bacteriocin was defined as the reciprocal of the highest dilution showing inhibition of the indicator lawn and was expressed in arbitrary units (AU) per milliliter. 2.3. Inoculum preparation To prepare inocula, Enterococcus and Listeria cultures were grown overnight at 30 8C in BHI broth. The cells were washed, harvested by centrifugation, resuspended in 0.85% NaCl (ca. 1109 cfu/ml) and diluted to appropriate concentration in the same diluent before adding directly to sausage batter as described below. 2.4. Model sausage manufacture Meat was tempered at 1/0 8C, ground through a 6 mm plate at 1 8C, and mixed together with the ingredients. The sausage mixture contained lean pork and backfat pork (3:1) (in g/kg) sodium chloride, 25; sodium nitrite 0.1; potassium nitrate 0.3; sodium ascorbate, 0.5; sodium pyrophosphate (pH 5.0), 1.5; dextrose, 7; lactose, 10; skimmed milk powder, 10; sodium caseinate, 10; Ponceau 4R, 0.02; black pepper, 3; water, 50. The cultures, dissolved in the water of the formulation, or the bacteriocins were applied to the meat mixture when indicated. The prepared sausage mixture (90–100 g) was placed in Petri dishes completely filled to avoid air bubbles. The incubation was performed at 20 8C for 9 days in a laboratory incubator Heraeus BK-600 (Kendro Laboratory Products, D-63450 Hanau, Deustchland). 2.4.1. Trials – Trial A was carried out to investigate the effectiveness of the addition of bacteriocin AS-48 produced ex situ in controlling listeria in model sausages. This trial consisted of four treatments challenged with L. monocytogenes (103 cfu/g): a control batch, without AS-48, and three batches containing 112, 225 or 450 AU/g AS-48 respectively.
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– Trial B was intended to assess the biocontrol of L. monocytogenes by a strain producing enterocin AS-48. This trial consisted of three treatments contaminated with L. monocytogenes (103 cfu/g): a control batch without added enterococci, one batch inoculated with E. faecalis S-47 (AS-48 ) (106 cfu/g) and one batch with E. faecalis A-48-32 (AS48+) (106 cfu/g). – Trial C was carried out to investigate the influence of bacteriocinogenic inoculum size on the biocontrol of L. monocytogenes. This trial consisted of three treatments: a control batch contaminated with L. monocytogenes (103 cfu/g) and two batches contaminated with Listeria and inoculated with AS-48 strain E. faecalis S-47 (approximately 107 cfu/g) or AS-48+ strain E. faecalis A-48-32 (approximately 107 cfu/g), respectively. – Trial D was performed to assay the ability of strain E. faecium S-32-81 (AS-48+) to control listeria in model sausages. This trial consisted of three treatments: a control batch contaminated with L. monocytogenes (103 cfu/g) and two batches challenged with Listeria and inoculated with approximately 107 cfu/g E. faecium S-32 (AS-48 ) or E. faecium S-32-81 (AS-48+), respectively. 2.5. Sausage sampling Triplicates from each treatment were sampled at selected times to determine Listeria, enterococci, lactic acid bacteria, pH values and bacteriocin concentration. For the microbiological determinations, 10 g were aseptically removed and mixed (1:10) with dilution medium (0.1% peptone, 0.85% NaCl). The homogenisation was done in a Stomacher Lab-blender (model 400, Cooke Laboratory products, Alexandria, Virginia) for 1 min, serially 10-fold diluted and plated on the respective selective media: MRS agar (Scharlab) for lactic acid bacteria incubated inside anaerobic jars (Gaspak System, BBL, Becton Dickinson, Cockeysville, MD) using anaerobic atmosphere generators (Biome´rieux, Mercy L’Etoile, France) at 30 8C for 72 h; Kanamycin Sodium Azide agar (KAA, Oxoid, Basingstoke, England) for enterococci, incubated at 37 8C for 48 h; PALCAM agar base added of PALCAM Listeria Selective Supplement (Merck, Darmstadt, Germany) for L. monocytogenes incubated at 30 8C for 72 h.
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The pH of sausages was measured at selected times by inserting a pH electrode (Mod. 5202, Crison, Barcelona, Spain) connected to a pH meter GLP21 (Crison). 2.6. Bacteriocin production To assess the in situ AS-48 production and recovery, bacteriocin was extracted at selected times from model sausages according to Garriga et al. (2002). Briefly, sausage samples (10 g) were homogeneised in 90 ml of the following solution: 50 mM sodium acetate, 100 mM EDTA and 0.2% Triton X100 at pH 5 with an a Stomacher Lab-blender for 1 min, boiled for 10 min, cooled and filtrated in order to obtain the liquid phase. The bacteriocin in the liquid phase was precipitated with ammonium sulphate 300 g/L and the pellet was dissolved in 2 ml of 50 mM phosphate buffer, pH 7.2 to obtain a 5-fold concentrated bacteriocin solution. The sample was heated at 80 8C for 10 min and stored at 20 8C. The bacteriocin titre of the extracts was determined by the agar spot test method (Tagg and McGiven, 1971) using E. faecalis S-47 as indicator strain and expressed in arbitrary units (AU) per milliliter. 2.7. Statistical analyses Statistical analyses were performed using the SPSS-PC 11.0 software (SPSS, Chicago, Ill., USA). Data relating to microbiological counts and pH along the incubation period of model sausages were subjected to ANOVA. The presence of enterocin AS-48 or bacteriocinogenic E. faecalis A-48-32 or E. faecalis S-32-81 strains was used as factor with three categories: sausages manufactured with enterocin AS-48 or with A-48-32 or S-32-81 strains (AS48+) and sausages without AS-48 or A-48-32+ strains.
3. Results 3.1. Inhibition of L. monocytogenes by addition of enterocin AS-48 Trial A was carried out to test the control of L. monocytogenes by semipurified enterocin AS-
48, produced by conventional fermentation and added at different concentrations to the meat mixtures. Growth of L. monocytogenes, lactic acid bacteria and enterococci, pH values and titers of extracted bacteriocin along the incubation period are illustrated in Fig. 1. L. monocytogenes decreased significantly in sausages added with enterocin AS-48 compared to control sausages in proportion to the AS-48 concentration. Remarkably, even at the lowest bacteriocin concentration tested (112 AU/g) listeria counts after 3 days at 20 8C were 2.4 log units lower compared with the control ( Pb0.01) (Fig. 1A and B). As expected, higher concentrations of AS-48 (225 AU/ g) achieved a more drastic effect on viability of listeria, which were below the detection limit in meat mixtures after 3 days (Fig. 1C). However, at sampling time 9 days listeria counts were 2 log cfu/g. Treatment with the highest AS-48 concentration (450 AU/g) showed a progressive bactericidal effect on listeria with no further recovery after 6 days (Fig. 1D). Lactic acid bacteria (LAB) in every single batch reached similar counts at 3, 6 and 9 days of incubation. Although the differences were not significant, there was a general delay in growth of LAB in bacteriocin-added samples, which reached highest cell densities at day 6 versus day 3 in the controls. In general, highest counts were ca. one log unit lower in bacteriocin added samples compared with the controls. Counts of enterococci were very similar in all batches (Fig. 1A–C). Recovery of bacteriocin from meat mixtures only was possible in the batch added with the highest AS-48 concentration (450 AU/g). In this case, the percentage of bacteriocin adsorption to meat mixtures was 80% at time 0 (right after mixing) and increased with further incubation. After 3, 6 and 9 days of incubation, the percentages of activity recovered were 6.7%, 7.7% and 9% respectively, of the total activity added to meat mixture (Fig. 1D). The pH of the control batch decreased to 5.62, 5.1 and 4.96 after 3, 6 and 9 days respectively. The evolution of pH in sausages added with AS-48 was very similar for the different bacteriocin concentrations treatments, and values were always higher compared to the control batch (Fig. 2).
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Fig. 1. Effect of exogenous added bacteriocin AS-48 on the viability of L. monocytogenes in a model meat sausage system. Trial A. A: Control batch; B: batch added with AS-48 at 112 AU/g; C: batch added with AS-48 at 225 AU/g; D: batch added with AS-48 at 450 AU/g. Growth of enterococci (rhombus), total lactic acid bacteria (triangle), L. monocytogenes (square). Bars: extracted bacteriocin (AU/g). Values are the average of triplicates.
3.2. Inhibition of L. monocytogenes by an enterococcal strain producing AS-48
Fig. 2. Influence of bacteriocin addition on the evolution of pH values in a model meat sausage system, trial A. Control batch (rhombus); batches treated with 112 AU/g (square), 225 AU/g (triangle) and 450 AU/g (circle) of AS-48. Values are the average of triplicates.
The biocontrol exerted by the bacteriocinogenic strain E. faecalis A-48-32 on the growth of L. monocytogenes was followed in trial B. Listeria, lactic acid bacteria, enterococci and pH values are illustrated in Fig. 3. In the control batch not inoculated with enterococci, Listeria counts increased from an initial value of 7.8102 cfu/g to 1.16105 cfu/g at day 3, and 3.12104 cfu/g at day 9 of incubation at 20 8C (Fig. 3A). In the batch inoculated with E. faecalis S-47 (AS-48 ), growth of Listeria was slightly slower, reaching counts of 2.13104 cfu/g and 1.47104 cfu/ g after 3 days and 9 days respectively (Fig. 3B). In batch inoculated with bacteriocinogenic strain A-4832 listeria did not growth throughout the whole incubation period (Fig. 3C).
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Fig. 3. Biocontrol of L. monocytogenes by strain E. faecalis A-48-32 in a model meat sausage system. Trial B. A: Control batch. B: Batch inoculated with approximately 106 cfu/g of E. faecalis S-47 (AS-48 ). C: Batch inoculated with approximately 106 cfu/g E. faecalis A-48-32 (AS-48+). Growth of enterococci (rhombus), total lactic acid bacteria (triangle), L. monocytogenes (square). Bars, extracted bacteriocin (AU/g). D: Evolution of pH along the incubation time. Control batch (rhombus); batch AS-48 lote (triangle); batch AS-48+ (square). Values are the average of triplicates.
The bacteriocinogenic strain A-48-32 was well adapted in meat mixtures. In samples inoculated with the AS-48+ strain, enterococci represented approximately 80% of the total lactic acid bacteria versus 5.9% in the AS-48 batch. The AS-48+ strain was able to produce AS-48 in meat mixtures and a residual bacteriocin activity of 40 AU/g was detected (Fig. 3C). The pH curves for control (AS-48 ) and (AS-48+) batches were almost identical (Fig. 3D), and pH values decreased from ca. 6.0 to 5.03 after 9 days. In order to assess the effect of inoculum size on control of listeria, a higher concentration of enterococci (approximately 107 cfu/g) was tested in trial C (Fig. 4). In the control batch, the evolution of bacterial counts (Fig. 4A) was similar in comparison with trial B. In the batch inoculated with E. faecalis S-47 (AS-48 ), growth of Listeria was inhibited to a great extent, (1.38, 1.16 and 1.53 log compared to control after 3, 6
and 9 days respectively) but viable counts were still of 7.7102 cfu/g after 9 days (Fig. 4B). In batch inoculated with bacteriocinogenic strain A-48-32 Listeria decreased progressively throughout the incubation period, down to a concentration below the detection limit (1102 cfu/g) after 9 days of incubation (Fig. 4C). The concentrations of residual bacteriocin activity in meat sausages reached highest values after 6 days of incubation (60 AU/g), decreasing to 30 AU/g after 9 days. For trial C, acidification of meat samples inoculated with enterococci occurred much more rapidly compared with the control (Fig. 4D) and also with results from trial B. Trial D (Fig. 5) consisted of model sausages inoculated with the non-bacteriocinogenic E. faecium S-32 (AS-48 ) and E. faecium S-32-81 (AS48+). The inoculum of enterococci was approxi-
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Fig. 4. Biocontrol of L. monocytogenes by strain E. faecalis A-48-32 in a model meat sausage system. Trial C. A: Control batch. B: Batch inoculated with approximately 107 cfu/g of E. faecalis S-47 (AS-48 ). C: Batch inoculated with approximately 107 cfu/g E. faecalis A-48-32 (AS-48+). Growth of enterococci (rhombus), total lactic acid bacteria (triangle), L. monocytogenes (square). Bars, extracted bacteriocin (AU/g). Evolution of pH along the incubation time. Control batch (rhombus); batch AS-48 (triangle); batch AS-48+ (square). Values are the average of triplicates.
mately 107 cfu/g. Results obtained for the control batch were similar to controls from trials B and C. In the batch inoculated with strain E. faecium S-32, growth of listeria was inhibited, but the concentration of viable listeria after 9 days of incubation was 1.1103 cfu/g (Fig. 5B). In the batch inoculated with strain E. faecium S-32-81 counts of listeria decreased markedly after day 3 of incubation, and were below the detection level (1.99 log cfu/g) after days 6 and 9 (Fig. 5C). The concentration of bacteriocin AS-48 in samples increased progressively during incubation, up to 80 AU/g at day 9 (Fig. 5C). The pH of samples inoculated with E. faecium S-32 and E. faecium S-32-81 was quite similar, with no significant differences (Fig. 5D). The pH of batches inoculated with enterococci decreased more
rapidly compared with controls, reaching values of 5.03 and 5.2, respectively, at day 3. After 9 days of incubation, the pH of batches inoculated with enterococci were slightly higher (4.9) compared to the control (4.7).
4. Discussion L. monocytogenes has emerged as an important foodborne pathogen in the later part of 20th century causing various clinical syndromes. Listeria species are commonly found in raw and unprocessed foods (milk, leafy vegetables, fish and meats). Major outbreaks of listeriosis with high morbidity and mortality have been associated with consumption of a variety of foods, including soft cheeses, meat and
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Fig. 5. Biocontrol of L. monocytogenes by strain E. faecium S-32-81 in a model meat sausage system. Trial D. A: Control batch. B: Batch inoculated with approximately 107 cfu/g of E. faecium S-32 (AS-48 ). C: Batch inoculated with approximately 107 cfu/g E. faecium S-32-81 (AS-48+). Growth of enterococci (rhombus), total lactic acid bacteria (triangle), L. monocytogenes (square). Bars, extracted bacteriocin (AU/g). D: Evolution of pH along the incubation time. Control batch (rhombus); batch AS-48 (triangle); batch AS-48+ (circle).Values are the average of triplicates.
vegetable products (Schlech, 2000). In spite of listeriosis remaining an uncommon infection with only sporadic cases and outbreaks of illness (Anonymous, 2001), it is still of great concern for several reasons: the high mortality of L. monocytogenes infections (20–30%, Rocourt and Cossart, 1997), its ubiquity in raw foods, its capacity to overcome different hurdles used in processed foods (like, for example, low storage temperature, mild heat treatments, chemical preservatives, and acidic pH) and especially an increase in the numbers of susceptible individuals (YOPIS: young, old, pregnant and immunocompromised people). Therefore, it seems necessary to find new methods to control listeria in foods. Bacteriocins produced by lactic acid bacteria are interesting tools for biocontrol of listeria, either alone or in combination with classical hurdles. Nisin is the most widely used bacteriocin in food preservation.
However, its application on meat is clearly limited, especially if pH is above 5.0 (El-Khateib et al., 1993; Fang and Lin, 1994). In fact, it is currently applied only on a few meat products, and a high concentration (250 Ag/g) is recommended in these cases (Thomas et al., 2000). Therefore other bacteriocin-producing lactic acid bacteria are being searched for antimicrobial substances alternatives to nisin. Bacteriocins can be applied in meat systems by two basic methods: by adding crude, purified or semipurified bacteriocin preparations or by inoculation with pure cultures of the bacteriocinogenic strains. Both approaches offer advantages and disadvantages, and the choice of either one will depend on the bacteriocin, the producer strain, the food system and the target organism. Therefore, before using a given bacteriocin for biopreservation, it is necessary to study its efficacy for each particular food system, to
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determine the concentrations of added bacteriocin required to achieve an efficient control of foodborne pathogens, or the capacity of bacteriocinogenic strains for growth and bacteriocin production in the food system. The results presented in this work are the first contribution on the antilisterial activity of bacteriocin AS-48 in a meat system such as sausages, be it by in situ production by strains E. faecalis A-48-32 and E. faecium S-32-81 or by addition of partially purified bacteriocin preparations. Addition of different AS-48 concentrations (112, 225 and 450 AU/g) had a pronounced inhibitory effect on growth of Listeria, even at the lowest concentration tested. However, the bacteriocin concentrations required for complete inhibition of Listeria during prolonged incubation (between 225 and 450 AU/ml, corresponding to approximately 20 and 40 Ag/ml respectively) were markedly higher compared to the value of 0.1 Ag/ml obtained in BHI broth (Mendoza et al., 1999). It is known that activity of bacteriocins can be influenced by the chemical composition and the physical conditions of food (Cleveland et al., 2001). The apparently lower effectiveness of AS-48 in sausages compared to BHI broth, in spite of the higher bacteriocin concentration added, could be attributed to a higher retention of the bacteriocin molecules by meat and fat components, to a slower diffusion, and also to the irregular distribution of the bacteriocin molecules in the meat matrix with a higher dry matter content compared to liquid media. In fact, it was not possible to detect bacteriocin activity in sausages manufactured with 112 or 225 AU/ml even at time 0. Nevertheless, AS-48 had a marked antilisterial effect in both cases, suggesting that the matrixbound bacteriocin remains active and is released slowly. It is also interesting that, for the highest concentration tested, even though 80% of the activity was retained by the meat matrix at time 0, the concentrations of viable listeria decreased gradually over incubation time until they were not detected, suggesting once more a slow release of bacteriocin molecules from the meat matrix. These results also indicate that the remaining listeria detected at lower bacteriocin concentrations can be efficiently eliminated by higher concentrations, avoiding adaptation to AS-48 as was suggested in our previous work (Mendoza et al., 1999).
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Another result of interest is the higher pH of meat mixtures added of AS-48. This can be attributed to a slight inhibition of the lactic acid bacteria by the continued bacteriocin activity during the whole incubation period. On the contrary, in biocontrol experiments using a bacteriocinogenic strain, the population of lactic acid bacteria was not affected noticeably and the evolution of pH values was almost identical for all samples in spite of the fact that the bacteriocin concentrations detected in meat mixtures after day 3 were similar to those found in previous experiments with exogenous added bacteriocin. Therefore, it seems that the effect of AS-48 on lactic acid bacteria depends greatly on the initial bacteriocin concentration, which was much higher when exogenous bacteriocin was added as compared to in situ production. Biocontrol experiments of L. monocytogenes using an inoculum of the producer strain E. faecalis A-4832 at 106 cfu/g was less efficient since it only was able to suppress growth of the listeria. Nevertheless, viable counts were significantly lower compared with the control batch (by 1.69, 1.67 and 1.21 log cfu/g at 3, 6 and 9 days respectively). Such inhibition was attributed to bacteriocin production, since no such effect was observed in the batch inoculated with the AS-48 strain. Furthermore, inhibition of listeria by the AS48+ strain occurred at pH close to 6.0. This, together with the fact that the evolution of pH values was similar for the different batches, clearly indicate that inhibition of the listeria was independent of meat acidification. Previous studies (Nielsen et al., 1990; Hugas et al., 1995; Mendoza et al., 1999) have shown that the effectiveness of bacteriocins appears to be dependent upon concentration of both target bacteria and bacteriocin molecules which in turn may depend on the concentration of producer cells. Because it is conceivable that a higher bacteriocin concentration should be achieved in a shorter incubation time by using a higher inoculum of the bacteriocinogenic strain, a tenfold higher inoculum has also been tested. Previous results carried out in BHI broth indicate that a threshold concentration of the bacteriocin-producing strain of at least 107 cfu/g is required for an efficient inhibition of Listeria, irrespectively of the incubation temperature (37 or 15 8C) and the initial ratio of bacteriocinogenic strain/listeria (100:1, 10:1 and 1:1)
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(Mendoza et al., 1999). In meat environments, a higher concentration of bacteriocin-producing cells may be necessary to compensate adsorption of bacteriocin molecules to the meat matrix. Our results have confirmed this hypothesis, since an inoculum of 107 cfu/g of the bacteriocinogenic strains (E. faecalis A-48-32 and especially E. faecium S-3281) was much more effective in lowering the viable counts of listeria. For strain S-32-81, the results were similar to those obtained for an exogenous added bacteriocin concentration of 40 Ag/g, although the decrease in listeria viability was delayed. Noticeably, the concentrations of bacteriocin extracted from meat samples were also very high, even if compared with samples with exogenous added bacteriocin. Interestingly, the optimum inoculum concentration of the bacteriocinogenic strain that achieved an efficient inhibition of Listeria had no significant adverse effects on the LAB population, probably because the main population of LAB was conformed by bacteriocinogenic enterococci. The general conclusion of this study is that bacteriocin AS-48 can control L. monocytogenes in this food system be it either by exogenous addition of an acceptably low bacteriocin concentration or by in situ production, being the first time that AS-48 production in a meat model system is reported. Although a large fraction of bacteriocin activity becomes undetectable at the early stages, probably due to adsorption to meat components, there is still sufficient activity to control L. monocytogenes efficiently. It is also possible that gradual bacteriocin desorption takes place during incubation time, but initial bacteriocin concentration seems to be a key factor to control L. monocytogenes. Ways to achieve this purpose are dual: addition of sufficient exogenous bacteriocin seems adequate for biopreservation of nonfermented meat products, but it may interfere with lactic acid bacteria in fermented products unless selected starters resistant to AS-48 are used. Inoculation with a sufficient inoculum of an AS-48 producing strain seems an adequate procedure to control L. monocytogenes in fermented meat. The use of enterococci in fermented meat is considered by some authors as technologically unacceptable (Holley et al., 1988) and there is controversy over considering them as GRAS (Generally Recognized As Safe) microorganisms (Giraffa
et al., 1997). Nevertheless, some strains, especially those belonging to E. faecium, which have much lower pathogenicity potential, have been used in many different applications as dairy starter cultures (Giraffa et al., 1997) and probiotics (Fuller, 1989). In meat fermented products, enterococci, especially E. faecium, represent one of the LAB species that can be found in relatively high numbers during fermentation and they may contribute, together with lactobacilli, to the fermentation, conferring flavor to products by their glycolytic, proteolytic and lipolytic activities (Hugas et al., 2003). Among the features of technological relevance that bacteriocinogenic strains should meet are a good capacity for food colonisation and for bacteriocin production in the food matrix, where other preservative agents such as nitrites, sodium chloride, organic acids, pepper and thermal treatments may also occur. Strain E. faecium S-32-81 exhibits many features that make it suitable for development of starter or adjunct cultures: it lacks intrinsic virulence factors, has a good capacity for colonisation of meat and produces sufficient amounts of bacteriocin in meat mixtures. Therefore, further studies should be carried out in order to eliminate antibiotic resistance traits carried by the plasmid pAM401 in which the AS-48 gene cluster was cloned for transformation of E. faecium S-32. Alternatively, integration of the AS48 encoding region into the bacterial chromosome should also be attempted. The corresponding derivative strains may find practical application in food preservation against L. monocytogenes.
Acknowledgements This work was supported by grant (AGL20013315-C02-01) from Spanish Ministry of Science and Technology. Samir Ananou was beneficiary of a fellowship from AECI (Spanish Ministry of Foreign Affairs) and also received several grants from the University of Granada Research Programme.
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Ga´lvez, A., Maqueda, M., Valdivia, E., Quesada, A., Montoya, E., 1986. Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis. Can. J. Microbiol. 32, 765 – 771. Ga´lvez, A., Maqueda, M., Martı´nez-Bueno, M., Valdivia, E., 1989. Bactericidal and bacteriolytic action of peptide antibiotic AS-48 against Gram-positive and Gram-negative bacteria and other organisms. Res. Microbiol. 140, 57 – 68. Garriga, M., Aymerich, M.T., Costa, S., Monfort, J.M., Hugas, M., 2002. Bactericidal synergism through bacteriocins and high pressure in meat model system during storage. Food Microbiol. 19, 509 – 518. Giraffa, G., Carminati, D., Neviani, E., 1997. Enterococci isolated from dairy products: a review of risks and potential technological use. J. Food Prot. 60, 732 – 738. Holley, R.A., Lammerding, A.M., Tittiger, F., 1988. Occurrence and significance of streptococci in fermented Italian type dry sausage. Int. J. Food Microbiol. 7, 63 – 79. Hugas, M., 1998. Bacteriocinogenic lactic acid bacteria for the biopreservation of meat and meat products. Meat Sci. 49, 139 – 150. Hugas, M., Garriga, M., Aymerich, M.T., Monfort, J.M., 1995. Inhibition of Listeria in dry fermented sausage by the bacteriocinogenic Lactobacillus sake CTC494. J. Appl. Microbiol. 79, 322 – 330. Hugas, M., Garriga, M., Aymerich, M.T., 2003. Functionality of enterococci in meat products. Int. J. Food Microbiol. 88, 223 – 233. Incze, K., 1998. Dry fermented sausages. Meat Sci. 49, 169 – 177. Maqueda, M., Ferna´ndez, M., Martı´n, M.C., Valdivia, E., Martı´nezBueno, M., 2003. Expresio´n del cara´cter AS-48 en diversas bacterias la´cticas. Proceed. XIX Congreso Nacional de Microbiologı´a, Santiago de Compostela. Mataragas, M., Drosinos, E.H., Metaxopoulos, J., 2003. Antagonistic activity of lactic acid bacteria against Listeria monocytogenes in sliced cooked cured pork shoulder stored under vacuum or modified atmosphere at 4F2 8C. Food Microbiol. 20, 259 – 265. Mendoza, F., Maqueda, M., Ga´lvez, A., Martı´nez-Bueno, M., Valdivia, E., 1999. Antilisterial activity of peptide AS-48 and study of changes induced in the cell envelope properties of an AS-48-adapted strain of Listeria monocytogenes. Appl. Environ. Microbiol. 65, 618 – 625. Nielsen, J.W., Dickson, J.S., Crouse, J.D., 1990. Use of a bacteriocin produced by Pediococcus acidilactici to inhibit Listeria monocytogenes associated with fresh meat. Appl. Environ. Microbiol. 56, 2142 – 2145. Rocourt, J., Cossart, P., 1997. Listeria monocytogenes. In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology, Fundamentals and Frontiers. ASM Press, Washington DC, pp. 337 – 352. Salvat, G., Toquin, M.T., Michel, Y., Colin, P., 1995. Control of Listeria monocytogenes in the delicatessen industries: the lessons of a listeriosis outbreak in France. Int. J. Food Microbiol. 25, 75 – 81. Samelis, J., Metaxopoulos, J., 1999. Incidence and principal sources of Listeria spp. and Listeria monocytogenes contamination in
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Control of Listeria monocytogenes in model sausages by enterocin AS-48 Samir Ananoua, Margarita Garrigab, Marta Hugasb, Mercedes Maquedaa, Manuel Martı´nez-Buenoa, Antonio Ga´lvezc, Eva Valdiviaa,d,T a
Departamento de Microbiologı´a, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 18071-Granada, Spain b IRTA-Centro de Tecnologı´a de la Carne, 17121-Monells, Girona, Spain c´ Area de Microbiologı´a, Facultad de Ciencias Experimentales, Universidad de Jae´n, Paraje Las Lagunillas, Jae´n, Spain d Instituto de Biotecnologı´a, Universidad de Granada, 18071-Granada, Spain Received 18 May 2004; received in revised form 18 December 2004; accepted 29 December 2004
Abstract In this work we describe the control of Listeria monocytogenes CECT 4032 in sausage by adding the enterocin AS-48 producer strains Enterococcus faecalis A-48-32 and Enterococcus faecium S-32-81, and also by adding a semi-purified preparation of the bacteriocin. Addition of preformed AS-48 caused a significant decrease ( Pb0.01) in the number of viable listeria even at the lowest bacteriocin concentration tested (112 AU/g). At a higher concentration (225 AU/g) listeria were below the detection level (1.99 log units/g) in meat at 3 days of incubation but growth of listeria was observed again after 9 days. For an AS-48 concentration of 450 AU/g, no viable listeria were detected after 6 and 9 days of incubation. When E. faecalis A-48-32 was used as inoculum at approximately 107 cfu/g, listeria counts decreased progressively from start of experiment, being below detection level at day 9. The best results were obtained with E. faecium S-32-81, since listeria were undetectable at 6 days of incubation. Bacteriocin concentrations in samples reached concentrations of 60 and 80 AU/g for strains A-48-32 and S-32-81, respectively. These results clearly indicate that AS-48 can be used in the control of L. monocytogenes in sausages. D 2005 Elsevier B.V. All rights reserved. Keywords: Listeria monocytogenes; Biopreservation; Sausages; Enterocins; Bacteriocins; AS-48
1. Introduction
T Corresponding author. Departamento de Microbiologı´a, Facultad de Ciencias, Universidad de Granada, Fuentenueva s/n, 18071Granada, Spain. Tel.: +34 958 243244; fax: +34 958 249486. E-mail address: [email protected] (E. Valdivia). 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.12.024
Raw meat is highly sensitive to microbial spoilage because of its ecological properties (a w, pH and nutrients). In addition, meat and meat products are responsible for many food-borne illnesses outbreaks. Listeria monocytogenes is an ubiquitous bacterial pathogen with a high prevalence in many foods,
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mainly milk and cheeses, meat and meat products, fish, shellfish and vegetables (Farber and Peterkin, 1991; Samelis and Metaxopoulos, 1999). The ubiquity and also the psychrotrophy of this bacterium make its control to be extremely difficult and it can be considered an in house bacterium in processing plants of meat products. Thus, a study has reported that as many as 68% of the environmental samples in meat processing plants were positive for Listeria (Salvat et al., 1995). Several investigations from European institutions responsible for public health have shown that Listeria occurs in 12–16% of industrial fermented food products (European Report, 1999; AFSSA, 2000). In fact, it is known that L. monocytogenes can survive the commercial dry sausage manufacturing process despite the various hurdles such as low pH, salt and nitrites (Hugas et al., 1995; Varabioff, 1992; Incze, 1998). Some bacteriocins like nisin, enterocins A and B, sakacin, or pediocin AcH, have been tested, alone or in combination with several physico-chemical treatments (modified atmosphere packaging, high hydrostatic pressure, heat, pH and chemical preservatives) as an additional hurdle to control proliferation of listeria in meat products (Nielsen et al., 1990; Cutter and Siragusa, 1996; Aymerich et al., 2000; Cleveland et al., 2001; Garriga et al., 2002). Also, several bacteriocinogenic lactic acid bacteria, especially pediococci strains, have been used as bioprotective cultures for food manufacturing processes in attempts to control this bacterium (Foegeding et al., 1992; Hugas et al., 1995; Hugas, 1998; Mataragas et al., 2003; Budde et al., 2003). Enterocin AS-48 is a cationic cyclic bacteriocin produced by Enterococcus faecalis S-48 with broad bactericidal activity against most of the Gram-positive bacteria, including several pathogens like L. monocytogenes (Mendoza et al., 1999), Staphylococcus aureus, Mycobacterium sp., Bacillus cereus, and some Gram-negative bacteria (Ga´lvez et al., 1989; Abriouel et al., 1998, 2002, 2003). The features of AS-48 (broad spectrum of antimicrobial activity, stability in a wide range of temperature and pH, and sensitivity to digestive proteases; Ga´lvez et al., 1986; Samyn et al., 1994) point to it as a promising alternative to chemical preservatives to be used in a future as biopreservative in foods. Currently, we are performing a series of studies intended to know how AS-48 acts on the main food-borne pathogenic
bacteria as well as the influence of environmental factors concurring in foods on its antimicrobial effectiveness. In a previous study Mendoza et al. (1999) demonstrated the in vitro inhibitory effect of AS-48 on L. monocytogenes CECT 4032 in Brain Hearth Infusion broth. In this study we have investigated the ability of enterocin AS-48 to control L. monocytogenes CECT 4032 in model sausages, by adding the bacteriocinogenic strains E. faecalis A-4832 and Enterococcus faecium S-32-81 or a semipurified preparation of the enterocin.
2. Materials and methods 2.1. Bacterial strains and culture conditions Two enterococcal strains were used as bacteriocin AS-48 producers (AS-48+): E. faecalis A-48-32 and E. faecium S-32-81. The last is a strain isolated from cheese, lacking virulence determinants, and harbouring plasmid pAM401-81 that encodes for AS-48 production and immunity (Maqueda et al., 2003). As negative controls two Bac- strains E. faecalis S-47 and E. faecium S-32 were used, respectively. E. faecalis S-47 was used as indicator strain. The strain L. monocytogenes CECT 4032, associated with a case of meningitis, was used as challenge in sausages. Bacterial strains were grown on brain heart infusion (BHI) (Scharlab, Barcelona, Spain) at 30 8C. Solid media were prepared by adding 1.5% agar (Scharlab) to the broths. Enterococci and listeria cultures were maintained at 4 8C. 2.2. Preparation of bacteriocin AS-48 E. faecalis A-48-32 was grown for 8 h at 30 8C in a modified complex medium (MCM broth) (Abriouel et al., 2003): 0.2% Casaminoacids (Difco, Detroit, MI, USA), 0.2% BHI, 1% Glucose, 0.1% yeast nitrogen base (YNB; Difco), and 0.001% MgSO 4d 7H20 (PanReac, Barcelona, Spain) dissolved in 0.1 M sodium phosphate buffer (pH 7.2). YNB solution was sterilized by filtration (0.45 Am, Millipore Ibe´rica, Madrid, Spain) before being added to the heatsterilized medium. Bacteriocin recovery was done by cation exchange chromatography as described by Abriouel et al. (2003). Eluted fractions were sterilized
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by filtration (0.45 Am, Millipore) and tested for bacteriocin activity against the indicator strain S-47 by the agar well diffusion method using steel cylinders of 8 mm (outer) diameter (Ga´lvez et al., 1986). The titre of bacteriocin was defined as the reciprocal of the highest dilution showing inhibition of the indicator lawn and was expressed in arbitrary units (AU) per milliliter. 2.3. Inoculum preparation To prepare inocula, Enterococcus and Listeria cultures were grown overnight at 30 8C in BHI broth. The cells were washed, harvested by centrifugation, resuspended in 0.85% NaCl (ca. 1109 cfu/ml) and diluted to appropriate concentration in the same diluent before adding directly to sausage batter as described below. 2.4. Model sausage manufacture Meat was tempered at 1/0 8C, ground through a 6 mm plate at 1 8C, and mixed together with the ingredients. The sausage mixture contained lean pork and backfat pork (3:1) (in g/kg) sodium chloride, 25; sodium nitrite 0.1; potassium nitrate 0.3; sodium ascorbate, 0.5; sodium pyrophosphate (pH 5.0), 1.5; dextrose, 7; lactose, 10; skimmed milk powder, 10; sodium caseinate, 10; Ponceau 4R, 0.02; black pepper, 3; water, 50. The cultures, dissolved in the water of the formulation, or the bacteriocins were applied to the meat mixture when indicated. The prepared sausage mixture (90–100 g) was placed in Petri dishes completely filled to avoid air bubbles. The incubation was performed at 20 8C for 9 days in a laboratory incubator Heraeus BK-600 (Kendro Laboratory Products, D-63450 Hanau, Deustchland). 2.4.1. Trials – Trial A was carried out to investigate the effectiveness of the addition of bacteriocin AS-48 produced ex situ in controlling listeria in model sausages. This trial consisted of four treatments challenged with L. monocytogenes (103 cfu/g): a control batch, without AS-48, and three batches containing 112, 225 or 450 AU/g AS-48 respectively.
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– Trial B was intended to assess the biocontrol of L. monocytogenes by a strain producing enterocin AS-48. This trial consisted of three treatments contaminated with L. monocytogenes (103 cfu/g): a control batch without added enterococci, one batch inoculated with E. faecalis S-47 (AS-48 ) (106 cfu/g) and one batch with E. faecalis A-48-32 (AS48+) (106 cfu/g). – Trial C was carried out to investigate the influence of bacteriocinogenic inoculum size on the biocontrol of L. monocytogenes. This trial consisted of three treatments: a control batch contaminated with L. monocytogenes (103 cfu/g) and two batches contaminated with Listeria and inoculated with AS-48 strain E. faecalis S-47 (approximately 107 cfu/g) or AS-48+ strain E. faecalis A-48-32 (approximately 107 cfu/g), respectively. – Trial D was performed to assay the ability of strain E. faecium S-32-81 (AS-48+) to control listeria in model sausages. This trial consisted of three treatments: a control batch contaminated with L. monocytogenes (103 cfu/g) and two batches challenged with Listeria and inoculated with approximately 107 cfu/g E. faecium S-32 (AS-48 ) or E. faecium S-32-81 (AS-48+), respectively. 2.5. Sausage sampling Triplicates from each treatment were sampled at selected times to determine Listeria, enterococci, lactic acid bacteria, pH values and bacteriocin concentration. For the microbiological determinations, 10 g were aseptically removed and mixed (1:10) with dilution medium (0.1% peptone, 0.85% NaCl). The homogenisation was done in a Stomacher Lab-blender (model 400, Cooke Laboratory products, Alexandria, Virginia) for 1 min, serially 10-fold diluted and plated on the respective selective media: MRS agar (Scharlab) for lactic acid bacteria incubated inside anaerobic jars (Gaspak System, BBL, Becton Dickinson, Cockeysville, MD) using anaerobic atmosphere generators (Biome´rieux, Mercy L’Etoile, France) at 30 8C for 72 h; Kanamycin Sodium Azide agar (KAA, Oxoid, Basingstoke, England) for enterococci, incubated at 37 8C for 48 h; PALCAM agar base added of PALCAM Listeria Selective Supplement (Merck, Darmstadt, Germany) for L. monocytogenes incubated at 30 8C for 72 h.
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The pH of sausages was measured at selected times by inserting a pH electrode (Mod. 5202, Crison, Barcelona, Spain) connected to a pH meter GLP21 (Crison). 2.6. Bacteriocin production To assess the in situ AS-48 production and recovery, bacteriocin was extracted at selected times from model sausages according to Garriga et al. (2002). Briefly, sausage samples (10 g) were homogeneised in 90 ml of the following solution: 50 mM sodium acetate, 100 mM EDTA and 0.2% Triton X100 at pH 5 with an a Stomacher Lab-blender for 1 min, boiled for 10 min, cooled and filtrated in order to obtain the liquid phase. The bacteriocin in the liquid phase was precipitated with ammonium sulphate 300 g/L and the pellet was dissolved in 2 ml of 50 mM phosphate buffer, pH 7.2 to obtain a 5-fold concentrated bacteriocin solution. The sample was heated at 80 8C for 10 min and stored at 20 8C. The bacteriocin titre of the extracts was determined by the agar spot test method (Tagg and McGiven, 1971) using E. faecalis S-47 as indicator strain and expressed in arbitrary units (AU) per milliliter. 2.7. Statistical analyses Statistical analyses were performed using the SPSS-PC 11.0 software (SPSS, Chicago, Ill., USA). Data relating to microbiological counts and pH along the incubation period of model sausages were subjected to ANOVA. The presence of enterocin AS-48 or bacteriocinogenic E. faecalis A-48-32 or E. faecalis S-32-81 strains was used as factor with three categories: sausages manufactured with enterocin AS-48 or with A-48-32 or S-32-81 strains (AS48+) and sausages without AS-48 or A-48-32+ strains.
3. Results 3.1. Inhibition of L. monocytogenes by addition of enterocin AS-48 Trial A was carried out to test the control of L. monocytogenes by semipurified enterocin AS-
48, produced by conventional fermentation and added at different concentrations to the meat mixtures. Growth of L. monocytogenes, lactic acid bacteria and enterococci, pH values and titers of extracted bacteriocin along the incubation period are illustrated in Fig. 1. L. monocytogenes decreased significantly in sausages added with enterocin AS-48 compared to control sausages in proportion to the AS-48 concentration. Remarkably, even at the lowest bacteriocin concentration tested (112 AU/g) listeria counts after 3 days at 20 8C were 2.4 log units lower compared with the control ( Pb0.01) (Fig. 1A and B). As expected, higher concentrations of AS-48 (225 AU/ g) achieved a more drastic effect on viability of listeria, which were below the detection limit in meat mixtures after 3 days (Fig. 1C). However, at sampling time 9 days listeria counts were 2 log cfu/g. Treatment with the highest AS-48 concentration (450 AU/g) showed a progressive bactericidal effect on listeria with no further recovery after 6 days (Fig. 1D). Lactic acid bacteria (LAB) in every single batch reached similar counts at 3, 6 and 9 days of incubation. Although the differences were not significant, there was a general delay in growth of LAB in bacteriocin-added samples, which reached highest cell densities at day 6 versus day 3 in the controls. In general, highest counts were ca. one log unit lower in bacteriocin added samples compared with the controls. Counts of enterococci were very similar in all batches (Fig. 1A–C). Recovery of bacteriocin from meat mixtures only was possible in the batch added with the highest AS-48 concentration (450 AU/g). In this case, the percentage of bacteriocin adsorption to meat mixtures was 80% at time 0 (right after mixing) and increased with further incubation. After 3, 6 and 9 days of incubation, the percentages of activity recovered were 6.7%, 7.7% and 9% respectively, of the total activity added to meat mixture (Fig. 1D). The pH of the control batch decreased to 5.62, 5.1 and 4.96 after 3, 6 and 9 days respectively. The evolution of pH in sausages added with AS-48 was very similar for the different bacteriocin concentrations treatments, and values were always higher compared to the control batch (Fig. 2).
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Fig. 1. Effect of exogenous added bacteriocin AS-48 on the viability of L. monocytogenes in a model meat sausage system. Trial A. A: Control batch; B: batch added with AS-48 at 112 AU/g; C: batch added with AS-48 at 225 AU/g; D: batch added with AS-48 at 450 AU/g. Growth of enterococci (rhombus), total lactic acid bacteria (triangle), L. monocytogenes (square). Bars: extracted bacteriocin (AU/g). Values are the average of triplicates.
3.2. Inhibition of L. monocytogenes by an enterococcal strain producing AS-48
Fig. 2. Influence of bacteriocin addition on the evolution of pH values in a model meat sausage system, trial A. Control batch (rhombus); batches treated with 112 AU/g (square), 225 AU/g (triangle) and 450 AU/g (circle) of AS-48. Values are the average of triplicates.
The biocontrol exerted by the bacteriocinogenic strain E. faecalis A-48-32 on the growth of L. monocytogenes was followed in trial B. Listeria, lactic acid bacteria, enterococci and pH values are illustrated in Fig. 3. In the control batch not inoculated with enterococci, Listeria counts increased from an initial value of 7.8102 cfu/g to 1.16105 cfu/g at day 3, and 3.12104 cfu/g at day 9 of incubation at 20 8C (Fig. 3A). In the batch inoculated with E. faecalis S-47 (AS-48 ), growth of Listeria was slightly slower, reaching counts of 2.13104 cfu/g and 1.47104 cfu/ g after 3 days and 9 days respectively (Fig. 3B). In batch inoculated with bacteriocinogenic strain A-4832 listeria did not growth throughout the whole incubation period (Fig. 3C).
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Fig. 3. Biocontrol of L. monocytogenes by strain E. faecalis A-48-32 in a model meat sausage system. Trial B. A: Control batch. B: Batch inoculated with approximately 106 cfu/g of E. faecalis S-47 (AS-48 ). C: Batch inoculated with approximately 106 cfu/g E. faecalis A-48-32 (AS-48+). Growth of enterococci (rhombus), total lactic acid bacteria (triangle), L. monocytogenes (square). Bars, extracted bacteriocin (AU/g). D: Evolution of pH along the incubation time. Control batch (rhombus); batch AS-48 lote (triangle); batch AS-48+ (square). Values are the average of triplicates.
The bacteriocinogenic strain A-48-32 was well adapted in meat mixtures. In samples inoculated with the AS-48+ strain, enterococci represented approximately 80% of the total lactic acid bacteria versus 5.9% in the AS-48 batch. The AS-48+ strain was able to produce AS-48 in meat mixtures and a residual bacteriocin activity of 40 AU/g was detected (Fig. 3C). The pH curves for control (AS-48 ) and (AS-48+) batches were almost identical (Fig. 3D), and pH values decreased from ca. 6.0 to 5.03 after 9 days. In order to assess the effect of inoculum size on control of listeria, a higher concentration of enterococci (approximately 107 cfu/g) was tested in trial C (Fig. 4). In the control batch, the evolution of bacterial counts (Fig. 4A) was similar in comparison with trial B. In the batch inoculated with E. faecalis S-47 (AS-48 ), growth of Listeria was inhibited to a great extent, (1.38, 1.16 and 1.53 log compared to control after 3, 6
and 9 days respectively) but viable counts were still of 7.7102 cfu/g after 9 days (Fig. 4B). In batch inoculated with bacteriocinogenic strain A-48-32 Listeria decreased progressively throughout the incubation period, down to a concentration below the detection limit (1102 cfu/g) after 9 days of incubation (Fig. 4C). The concentrations of residual bacteriocin activity in meat sausages reached highest values after 6 days of incubation (60 AU/g), decreasing to 30 AU/g after 9 days. For trial C, acidification of meat samples inoculated with enterococci occurred much more rapidly compared with the control (Fig. 4D) and also with results from trial B. Trial D (Fig. 5) consisted of model sausages inoculated with the non-bacteriocinogenic E. faecium S-32 (AS-48 ) and E. faecium S-32-81 (AS48+). The inoculum of enterococci was approxi-
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Fig. 4. Biocontrol of L. monocytogenes by strain E. faecalis A-48-32 in a model meat sausage system. Trial C. A: Control batch. B: Batch inoculated with approximately 107 cfu/g of E. faecalis S-47 (AS-48 ). C: Batch inoculated with approximately 107 cfu/g E. faecalis A-48-32 (AS-48+). Growth of enterococci (rhombus), total lactic acid bacteria (triangle), L. monocytogenes (square). Bars, extracted bacteriocin (AU/g). Evolution of pH along the incubation time. Control batch (rhombus); batch AS-48 (triangle); batch AS-48+ (square). Values are the average of triplicates.
mately 107 cfu/g. Results obtained for the control batch were similar to controls from trials B and C. In the batch inoculated with strain E. faecium S-32, growth of listeria was inhibited, but the concentration of viable listeria after 9 days of incubation was 1.1103 cfu/g (Fig. 5B). In the batch inoculated with strain E. faecium S-32-81 counts of listeria decreased markedly after day 3 of incubation, and were below the detection level (1.99 log cfu/g) after days 6 and 9 (Fig. 5C). The concentration of bacteriocin AS-48 in samples increased progressively during incubation, up to 80 AU/g at day 9 (Fig. 5C). The pH of samples inoculated with E. faecium S-32 and E. faecium S-32-81 was quite similar, with no significant differences (Fig. 5D). The pH of batches inoculated with enterococci decreased more
rapidly compared with controls, reaching values of 5.03 and 5.2, respectively, at day 3. After 9 days of incubation, the pH of batches inoculated with enterococci were slightly higher (4.9) compared to the control (4.7).
4. Discussion L. monocytogenes has emerged as an important foodborne pathogen in the later part of 20th century causing various clinical syndromes. Listeria species are commonly found in raw and unprocessed foods (milk, leafy vegetables, fish and meats). Major outbreaks of listeriosis with high morbidity and mortality have been associated with consumption of a variety of foods, including soft cheeses, meat and
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Fig. 5. Biocontrol of L. monocytogenes by strain E. faecium S-32-81 in a model meat sausage system. Trial D. A: Control batch. B: Batch inoculated with approximately 107 cfu/g of E. faecium S-32 (AS-48 ). C: Batch inoculated with approximately 107 cfu/g E. faecium S-32-81 (AS-48+). Growth of enterococci (rhombus), total lactic acid bacteria (triangle), L. monocytogenes (square). Bars, extracted bacteriocin (AU/g). D: Evolution of pH along the incubation time. Control batch (rhombus); batch AS-48 (triangle); batch AS-48+ (circle).Values are the average of triplicates.
vegetable products (Schlech, 2000). In spite of listeriosis remaining an uncommon infection with only sporadic cases and outbreaks of illness (Anonymous, 2001), it is still of great concern for several reasons: the high mortality of L. monocytogenes infections (20–30%, Rocourt and Cossart, 1997), its ubiquity in raw foods, its capacity to overcome different hurdles used in processed foods (like, for example, low storage temperature, mild heat treatments, chemical preservatives, and acidic pH) and especially an increase in the numbers of susceptible individuals (YOPIS: young, old, pregnant and immunocompromised people). Therefore, it seems necessary to find new methods to control listeria in foods. Bacteriocins produced by lactic acid bacteria are interesting tools for biocontrol of listeria, either alone or in combination with classical hurdles. Nisin is the most widely used bacteriocin in food preservation.
However, its application on meat is clearly limited, especially if pH is above 5.0 (El-Khateib et al., 1993; Fang and Lin, 1994). In fact, it is currently applied only on a few meat products, and a high concentration (250 Ag/g) is recommended in these cases (Thomas et al., 2000). Therefore other bacteriocin-producing lactic acid bacteria are being searched for antimicrobial substances alternatives to nisin. Bacteriocins can be applied in meat systems by two basic methods: by adding crude, purified or semipurified bacteriocin preparations or by inoculation with pure cultures of the bacteriocinogenic strains. Both approaches offer advantages and disadvantages, and the choice of either one will depend on the bacteriocin, the producer strain, the food system and the target organism. Therefore, before using a given bacteriocin for biopreservation, it is necessary to study its efficacy for each particular food system, to
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determine the concentrations of added bacteriocin required to achieve an efficient control of foodborne pathogens, or the capacity of bacteriocinogenic strains for growth and bacteriocin production in the food system. The results presented in this work are the first contribution on the antilisterial activity of bacteriocin AS-48 in a meat system such as sausages, be it by in situ production by strains E. faecalis A-48-32 and E. faecium S-32-81 or by addition of partially purified bacteriocin preparations. Addition of different AS-48 concentrations (112, 225 and 450 AU/g) had a pronounced inhibitory effect on growth of Listeria, even at the lowest concentration tested. However, the bacteriocin concentrations required for complete inhibition of Listeria during prolonged incubation (between 225 and 450 AU/ml, corresponding to approximately 20 and 40 Ag/ml respectively) were markedly higher compared to the value of 0.1 Ag/ml obtained in BHI broth (Mendoza et al., 1999). It is known that activity of bacteriocins can be influenced by the chemical composition and the physical conditions of food (Cleveland et al., 2001). The apparently lower effectiveness of AS-48 in sausages compared to BHI broth, in spite of the higher bacteriocin concentration added, could be attributed to a higher retention of the bacteriocin molecules by meat and fat components, to a slower diffusion, and also to the irregular distribution of the bacteriocin molecules in the meat matrix with a higher dry matter content compared to liquid media. In fact, it was not possible to detect bacteriocin activity in sausages manufactured with 112 or 225 AU/ml even at time 0. Nevertheless, AS-48 had a marked antilisterial effect in both cases, suggesting that the matrixbound bacteriocin remains active and is released slowly. It is also interesting that, for the highest concentration tested, even though 80% of the activity was retained by the meat matrix at time 0, the concentrations of viable listeria decreased gradually over incubation time until they were not detected, suggesting once more a slow release of bacteriocin molecules from the meat matrix. These results also indicate that the remaining listeria detected at lower bacteriocin concentrations can be efficiently eliminated by higher concentrations, avoiding adaptation to AS-48 as was suggested in our previous work (Mendoza et al., 1999).
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Another result of interest is the higher pH of meat mixtures added of AS-48. This can be attributed to a slight inhibition of the lactic acid bacteria by the continued bacteriocin activity during the whole incubation period. On the contrary, in biocontrol experiments using a bacteriocinogenic strain, the population of lactic acid bacteria was not affected noticeably and the evolution of pH values was almost identical for all samples in spite of the fact that the bacteriocin concentrations detected in meat mixtures after day 3 were similar to those found in previous experiments with exogenous added bacteriocin. Therefore, it seems that the effect of AS-48 on lactic acid bacteria depends greatly on the initial bacteriocin concentration, which was much higher when exogenous bacteriocin was added as compared to in situ production. Biocontrol experiments of L. monocytogenes using an inoculum of the producer strain E. faecalis A-4832 at 106 cfu/g was less efficient since it only was able to suppress growth of the listeria. Nevertheless, viable counts were significantly lower compared with the control batch (by 1.69, 1.67 and 1.21 log cfu/g at 3, 6 and 9 days respectively). Such inhibition was attributed to bacteriocin production, since no such effect was observed in the batch inoculated with the AS-48 strain. Furthermore, inhibition of listeria by the AS48+ strain occurred at pH close to 6.0. This, together with the fact that the evolution of pH values was similar for the different batches, clearly indicate that inhibition of the listeria was independent of meat acidification. Previous studies (Nielsen et al., 1990; Hugas et al., 1995; Mendoza et al., 1999) have shown that the effectiveness of bacteriocins appears to be dependent upon concentration of both target bacteria and bacteriocin molecules which in turn may depend on the concentration of producer cells. Because it is conceivable that a higher bacteriocin concentration should be achieved in a shorter incubation time by using a higher inoculum of the bacteriocinogenic strain, a tenfold higher inoculum has also been tested. Previous results carried out in BHI broth indicate that a threshold concentration of the bacteriocin-producing strain of at least 107 cfu/g is required for an efficient inhibition of Listeria, irrespectively of the incubation temperature (37 or 15 8C) and the initial ratio of bacteriocinogenic strain/listeria (100:1, 10:1 and 1:1)
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(Mendoza et al., 1999). In meat environments, a higher concentration of bacteriocin-producing cells may be necessary to compensate adsorption of bacteriocin molecules to the meat matrix. Our results have confirmed this hypothesis, since an inoculum of 107 cfu/g of the bacteriocinogenic strains (E. faecalis A-48-32 and especially E. faecium S-3281) was much more effective in lowering the viable counts of listeria. For strain S-32-81, the results were similar to those obtained for an exogenous added bacteriocin concentration of 40 Ag/g, although the decrease in listeria viability was delayed. Noticeably, the concentrations of bacteriocin extracted from meat samples were also very high, even if compared with samples with exogenous added bacteriocin. Interestingly, the optimum inoculum concentration of the bacteriocinogenic strain that achieved an efficient inhibition of Listeria had no significant adverse effects on the LAB population, probably because the main population of LAB was conformed by bacteriocinogenic enterococci. The general conclusion of this study is that bacteriocin AS-48 can control L. monocytogenes in this food system be it either by exogenous addition of an acceptably low bacteriocin concentration or by in situ production, being the first time that AS-48 production in a meat model system is reported. Although a large fraction of bacteriocin activity becomes undetectable at the early stages, probably due to adsorption to meat components, there is still sufficient activity to control L. monocytogenes efficiently. It is also possible that gradual bacteriocin desorption takes place during incubation time, but initial bacteriocin concentration seems to be a key factor to control L. monocytogenes. Ways to achieve this purpose are dual: addition of sufficient exogenous bacteriocin seems adequate for biopreservation of nonfermented meat products, but it may interfere with lactic acid bacteria in fermented products unless selected starters resistant to AS-48 are used. Inoculation with a sufficient inoculum of an AS-48 producing strain seems an adequate procedure to control L. monocytogenes in fermented meat. The use of enterococci in fermented meat is considered by some authors as technologically unacceptable (Holley et al., 1988) and there is controversy over considering them as GRAS (Generally Recognized As Safe) microorganisms (Giraffa
et al., 1997). Nevertheless, some strains, especially those belonging to E. faecium, which have much lower pathogenicity potential, have been used in many different applications as dairy starter cultures (Giraffa et al., 1997) and probiotics (Fuller, 1989). In meat fermented products, enterococci, especially E. faecium, represent one of the LAB species that can be found in relatively high numbers during fermentation and they may contribute, together with lactobacilli, to the fermentation, conferring flavor to products by their glycolytic, proteolytic and lipolytic activities (Hugas et al., 2003). Among the features of technological relevance that bacteriocinogenic strains should meet are a good capacity for food colonisation and for bacteriocin production in the food matrix, where other preservative agents such as nitrites, sodium chloride, organic acids, pepper and thermal treatments may also occur. Strain E. faecium S-32-81 exhibits many features that make it suitable for development of starter or adjunct cultures: it lacks intrinsic virulence factors, has a good capacity for colonisation of meat and produces sufficient amounts of bacteriocin in meat mixtures. Therefore, further studies should be carried out in order to eliminate antibiotic resistance traits carried by the plasmid pAM401 in which the AS-48 gene cluster was cloned for transformation of E. faecium S-32. Alternatively, integration of the AS48 encoding region into the bacterial chromosome should also be attempted. The corresponding derivative strains may find practical application in food preservation against L. monocytogenes.
Acknowledgements This work was supported by grant (AGL20013315-C02-01) from Spanish Ministry of Science and Technology. Samir Ananou was beneficiary of a fellowship from AECI (Spanish Ministry of Foreign Affairs) and also received several grants from the University of Granada Research Programme.
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