Growth inhibition of Listeria monocytogenes by bacteriocin-producing Staphylococcus equorum SE3 in cheese models

Food Control 71 (2017) 50e56

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Growth inhibition of Listeria monocytogenes by bacteriocin-producing Staphylococcus equorum SE3 in cheese models Wilhelm Bockelmann a, *, Margarita Koslowsky a, Stefanie Goerges b, Siegfried Scherer b, Charles M.A.P. Franz a, Knut J. Heller a a

Max Rubner-Institut, Department of Microbiology and Biotechnology, Hermann-Weigmann-Str. 1, 24103 Kiel, Germany €hrungs- und Lebensmittelforschung (ZIEL), Wissenschaftszentrum Weihenstephan, Technische Universita €t Abteilung Mikrobiologie, Zentralinstitut für Erna München, Weihenstephaner Berg 3, 85350 Freising, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 March 2016 Received in revised form 1 June 2016 Accepted 18 June 2016 Available online 21 June 2016

The anti-listerial activity of Staphylococcus equorum SE3 isolated from cheese brine was tested in two different model cheese systems to ascertain its potential for use as a protective culture for smear cheese ripening. Co-cultivation of Listeria monocytogenes L129 and the antilisterial S. equorum SE3 was performed on “milk agar” or “modified milk agar” (model cheese surface systems). S. equorum inoculated at concentrations of 106 cfu/cm2 completely inhibited growth of L. monocytogenes inoculated at 10e500 cfu/ cm2 on modified milk agar within 24 h of incubation, in the negative controls L129 grew to >107 cfu/cm2. At a higher inoculation level, growth inhibition was still more than 7 log units after 24 h. L. monocytogenes strains of different serotypes were also inhibited. Co-cultivation of S. equorum SE3 with other smear bacteria or yeasts, however, showed no growth inhibition of these important ripening microorganisms. The antilisterial effect was not diminished on the modified milk agar when cocultivation was performed with the added smear cheese microbiota. However, on milk agar with no adjuncts (“green cheese model”), only a slight (<1 log unit) growth inhibition of L. monocytogenes was observed. Addition of peptides or amino acids to milk agar could restore growth inhibition of listeriae at different levels. © 2016 Published by Elsevier Ltd.

Keywords: Listeria monocytogenes Staphylococcus equorum Growth inhibition Cheese model Smear cheeses Smear bacteria

1. Introduction Surface-ripened cheeses such as Tilsit, Limburg, Chaumes, Appenzell and many other varieties possess a surface microbiota called red smear (Bockelmann, 2002; Brennan et al., 2002; Cogan, 2011; Mounier et al., 2005). Apart from coryneform bacteria, such as Brevibacterium (B.) linens, Microbacterium (M.) gubbeenense and Corynebacterium (C.) casei, yeasts [(e.g. Debaryomyces (D.) hansenii)] and staphylococci [(e.g. Staphylococcus (S.) equorum)] make up the surface microbiota of smear cheeses (Rea et al., 2007). A natural reservoir for yeasts and staphylococci occurring on smear cheese are the cheese brines, which contain average concentrations of 100 cfu/mL or more (Bockelmann, Koslowsky, Hammer, & Heller, 2006; Jaeger, Hoppe-Seyler, Bockelmann, & Heller, 2002). A certain degree (<100
>105 cfu*cm
2) of contamination of smear cheeses with enterobacteria, enterococci, pseudomonads and

* Corresponding author. E-mail address: [email protected] (W. Bockelmann). http://dx.doi.org/10.1016/j.foodcont.2016.06.019 0956-7135/© 2016 Published by Elsevier Ltd.

moulds is also quite common (Bockelmann, 2003; Bockelmann & Hoppe-Seyler, 2001; Bockelmann et al., 1997). In spite of modern hygiene concepts and improved technology, contamination with Listeria monocytogenes still occurs sporadically (Pintado, Oliveira, Pampulha, & Ferreira, 2005; Rudolf & Scherer, 2001). According to Goulet, Hedberg, LeMonnier, and deValk (2008), the incidence of food-borne listeriosis in European countries is even increasing. Brevibacterium linens was the first commercial smear culture and is today sold by all major starter culture suppliers. Therefore, early research interest was directed towards the selection of antilisterial B. linens strains for cheese-ripening applications (MaisnierPatin & Richard, 1995; Stewart, Tompkin, & Cole, 2002; ValdesStauber & Scherer, 1996). A disadvantage of this approach, is that B. linens is acid-sensitive and can only grow when the cheese pH is above pH 6.0, thus it cannot be used as a biocontrol agent to protect young cheeses from growth of Listeria monocytogenes, which can grow in a broad pH range of pH 4 to pH 9.6 (Lado & Yousef, 2007; Chap. 6). The isolation of an anti-listerial, acid- and salt-tolerant Staphylococcus equorum strain WS2733 was reported by Carnio

W. Bockelmann et al. / Food Control 71 (2017) 50e56

et al. (2000). This strain synthesizes a peptide antibiotic termed micrococcin P1, which was shown to efficiently inhibit the growth of L. monocytogenes in cheese model systems. However, growth of many other Gram-positive bacteria, including the technologically important cheese coryneforms of the genera Brevibacterium, Corynebacterium, Arthrobacter, Microbacterium and Micrococcus, were also inhibited. The acid sensitivity of B. linens and the broad spectrum of inhibition of Gram-positive bacteria by S. equorum WS2733 were probably the reasons why no commercial applications of these anti-listerial cultures were established. Since yeasts and staphylococci are acid- and salt-tolerant and grow fast during the first days of cheese ripening, they were the most promising candidates for development of biocontrol strains that protect young smear cheeses from listerial growth. Goerges et al. (2011) isolated and characterized a yeast strain with antilisterial properties. The present study aimed to characterize a previously isolated smear-cheese associated S. equorum strain that was able to inhibit L. monocytogenes without affecting the technologically important coryneforms and yeasts. Such cultures could be applied either to the smear liquid or directly to the cheese brines (Jaeger et al., 2002). The bactericidal activity of S. equorum against several L. monocytogenes strains, as well as its inactivity against technologically important coryneforms and yeasts, were studied in two different model smear cheese systems. These represented a young or ‘green’ cheese using “milk agar”, as well as a ripened cheese type, using milk agar with added peptides and vitamins (“modified milk agar”). 2. Materials and methods 2.1. Microorganisms and culture conditions Twenty cheese brines from 11 northern German cheese producers were (previously) screened for antilisterial staphylococci (Bockelmann et al., 2006). Nearly all 2000 isolates from smear cheese- and non-smear cheese-producing environments were S. equorum, showing the importance of this species in cheese manufacture. Only three isolates possessed clear antilisterial activity (Bockelmann et al., 2006). The most active strain, i.e. Staphylococcus equorum SE3, was used in this study. The typical smear microorganisms S. equorum SE3, B. linens Br5, Corynebacterium casei CA3, Microbacterium gubbeenense CA12, Debaryomyces hansenii 6004, Kluyveromyces marxianus 5675, Candida krusei 60132 and Geotrichum candidum 6215 were kept in the culture collection of the Department of Microbiology and Biotechnology (Max RubnerInstitut), Kiel. Listeria monocytogenes strains (L93, L100, L129, L395 and L400) were kindly provided by the Department of Quality and Safety of Milk and Fish (Max Rubner-Institut), Kiel. The latter strains belonged to several serotypes and had all been isolated from food poisoning incidents (Table 1). L. monocytogenes strains were incubated on blood agar (Merck, Darmstadt, Germany) over night at 37  C and maintained at 4  C. For co-cultivation experiments, 10 mL of brain heart infusion (BHI) was inoculated with fresh L. monocytogenes grown on blood agar

Table 1 Listeria monocytogenes strains isolated from food poisoning incidents used in the study. Collection no.

Source (cheese)

year

Serotype

L93 L100 L129 (SLCC 8683) L395 L400

Limburg cheese Steinbuscher Vacherin Mont d’Or acid curd cheese Tilsit

1987 1987 1983 2006 2007

1/2a 1/2b 4b 4b 3a

51

and incubated at 30  C for 16e18 h. If not stated otherwise, media were purchased from Merck (Darmstadt, Germany). Smear bacteria and staphylococci were cultivated on modified Plate-Count (mPC) agar according to HoppeSeyler, Jaeger, Bockelmann, and Heller (2000). One litre of medium contained 22.5 g Plate-Count agar, 1 g skim milk powder, 10 g casein hydrolysate, 10 g brain heart infusion and 30 g sodium chloride. The pH was adjusted to pH 7.0 with NaOH. Yeasts and moulds were cultivated on YGC agar. 2.2. Co-cultivation of L. monocytogenes and S. equorum For co-cultivation of listeria and smear microorganisms (yeasts, coryneforms, staphylococci), a “modified milk agar” (mMA) consisting of the mPC agar mentioned above, with additional skim milk powder (10 g/Litre), was used as a model for a ripened smear cheese surface environment. This medium contains a high amount of proteolysis products, vitamins and minerals, which are supplied by the brain heart infusion and casein hydrolysate of the mPC medium and by the addition of a 100x BME vitamins solution (Sigma-Aldrich, Taufkirchen, Germany), described in detail by Hoppe-Seyler et al. (2000). Milk Agar (MA), used as a model for a green cheese surface, was prepared with commercial UHT skim milk containing 1.4% agar-agar. The pH was adjusted to 7.0 with NaOH. In contrast to the “modified milk agar”, this milk agar medium did not contain additional peptides and vitamins and salt. In both cheese models, the effect of salt was tested at 0%, 1%, 2% and 3% NaCl. Pre-cultures for agar surface co-cultivation (staphylococci, listeria, coryneforms, yeasts as mentioned above) were grown over night in liquid media. Yeasts were grown in malt extract broth, staphylococci and coryneforms smear bacteria were grown in modified Plate-Count broth (same recipe as mPC, without agar) and listeria, which were grown in brain heart infusion broth. For co-cultivation of staphylococci and listeria, the method of Goerges, Aigner, Silakowski, and Scherer (2006) developed for yeasts and listeria was modified. The concentration of staphylococci, coryneforms or yeasts was adjusted to 105 - 106 cfu/cm2 agar surface, while the concentration of listeria was adjusted to approximately 101 cfu/cm2. This value was considered to be a realistic contamination level for smear cheeses (Goerges et al., 2006). Higher concentrations of listeria were used in some experiments to test the efficiency of inhibition. For co-cultivation, listeriae were serially diluted in a tenfold dilution series to the required cell density. 300 mL of a 24 h BHI culture of S. equorum SE3 was centrifuged for 3 min at 2  C and 14,000 rpm, and the pellet was resuspended in 100 mL of quarter-strength Ringer’s solution. After this, 100 mL of diluted Listeria culture was added to the Staphylococcus suspension and plated onto mMA or MA. To determine the surface counts after incubation at 30  C for 24 h, a disk was stamped out of the agar with the rim of an open sterile, disposable plastic tube (surface 5.7 cm2) and was placed upside down in a 100 mL Erlenmeyer flask containing 8 mL of quarter strength Ringer’s solution. Cell lawns on top of the agar pieces were then suspended by shaking flasks at 200 rpm for 30 min. The resulting bacterial suspensions were transferred to sterile tubes. The remaining agar disks in the flasks were washed a second time with an additional 2 mL of Ringer’s solution to obtain a total volume of 10 ml. This 10 mL suspension was defined as the first dilution step (10
1) and was then serially diluted in ten-fold dilution steps. Aliquots of 0.1 mL of appropriate ten-fold dilutions were spread plated and incubated on Palcam agar (24 h at 37  C) and SK agar (48 h at 30  C), for selective enumeration of the listeria and staphylococci, respectively. Samples were analysed in duplicate with 2 dilutions per sample (quadruplicate counts). For determination of Listeria concentrations below the detection limit of

W. Bockelmann et al. / Food Control 71 (2017) 50e56

1.7 cfu/cm2, a second agar sample was incubated in 20 mL of ½ Fraser bouillon at 37  C for 24 h, shaken at 100 rpm for enrichment and plated on Palcam agar for detection of L. monocytogenes. Plates were incubated for 24 h at 37  C. The same method was used when listeria were co-cultivated with coryneforms or yeasts (Goerges et al., 2011). mPC bouillon was used for coryneforms and lactose-lactate- yeast extractpeptone (LLYP) broth for yeasts mentioned above. LLYP contained per litre of medium: lactose 10 g, 10 mL of a 90% lactic acid solution, soy peptone 5 g, casein peptone 5 g, yeast extract 5 g; the pH was adjusted to pH 5.6 with concentrated NaOH. Yeasts and coryneforms were grown in Erlenmeyer flasks over night at 25  C while shaking at 220 rpm. Plating of coryneforms after cocultivation was done on mPC agar, plating of yeasts on YGC agar.

1010 109 counts after 24 h [cfu/cm 2]

52

L.m. in coculture

L.m. control

SE3 in coculture

108 107 106 105 104 103 102 101 100

0 L93

0

0

0

0

L100

L129

L395

L400

L. monocytogenes strain

2.3. Characterisation of the bacteriocin activity

3. Results and discussion 3.1. Co-cultivation of L. monocytogenes and S. equorum in the mMA cheese model For surface ripened cheeses, low contamination levels of L. monocytogenes are frequently observed (Rudolf & Scherer, 2001). For reasons unknown, listeriae sporadically grow to high titres especially on mould cheeses and smear cheeses (Koch et al., 2010; Rudolf & Scherer, 2001). In the present study, high concentrations (ca. 106 cfu/cm2) of the listeriolytic S. equorum SE3 cells were cocultivated with 10e100 cfu/cm2 of L. monocytogenes L129 for 24 h. This L. monocytogenes/S. equorum ratio was considered to be realistic, simulating a probable situation in a smear cheese plant (Goerges et al., 2006). A complete inhibition of 5 L. monocytogenes strains of different serotypes by S. equorum SE3 was observed when growing these bacteria in co-culture on modified milk agar (Fig. 1) for 24 h. At higher initial concentrations of listeria (3.5  104 cfu/cm2 or 1:10 and 1:100 dilutions, i.e. ca. 3.5  103 and 3.5  102 cfu/cm2, respectively), the reduction of listerial counts was more than 5 log units compared to negative controls (pure L.m. cultures without S. equorum, Fig. 2). Comparable results were obtained when the experiment was repeated with a mixture of equal parts of the 5 L. monocytogenes strains (initial L.m. concentrations: 4.4  104 cfu/ cm2 or 1:10, 1:100 and 1:1000 dilutions, data not shown). Complete inhibition of listeria was observed at 4.4  102 and 4.4  101 cfu/ cm2. In all experiments, the L. monocytogenes counts were between 107-108 cfu/cm2 in the negative controls. The final concentration of S. equorum SE3 after co-cultivation was ca. 109 cfu/cm2 in all experiments. 3.2. Co-cultivation of L. monocytogenes with S. equorum and smear microorganisms in the mMA cheese model Single strains of typical smear yeasts showed no inhibition of

Fig. 1. Co-cultivation of five L. monocytogenes (L.m.) strains of different serotypes (initial concentration 1.1  101e1.9  101 cfu/cm2) and S. equorum SE3 (initial concentration 3.1  106cfu/cm2) on modified milk agar. The detection limit for listeria was 1.7 cfu/cm2, after enrichment in Fraser broth 0.17 cfu/cm2. The zero value represents no growth in enrichment broth. As negative controls, L. monocytogenes strains were grown on mMA without S. equorum SE3. Co-cultivation was performed in duplicate, error bars show the values of the single experiments.

1010 109 counts after 24 h [cfu/cm 2]

Bacteriocin production was tested by the spot on lawn assay (Ahn and Stiles, 1996) and for this, tryptose soft agar was prepared containing 2.6% tryptose broth and 0.8% agar agar. S. equorum SE3 was spotted on the surface of a tryptose soy agar plate and grown overnight at 30  C. The next day, 3 mL of a-chymotrypsin (5 mg/mL in distilled water) was spotted 20 mm next to the colony spot and incubated at 37  C for 3 h. After this, 100 mL of an overnight culture of the indicator strain L. monocytogenes L129 grown in brain heart infusion broth was inoculated into tryptose soft agar and poured over the S. equorum colony. The agar plates were incubated at 30  C for 24 h and evaluated for zones of inhibition.

L.m. in coculture

SE3 in coculture

L.m. control

108 107 106 105 104 103 102 101 100

0

0 > 10

> 100

> 1000

> 10000

initial L.m. concentration [cfu/cm2] Fig. 2. Co-cultivation of L. monocytogenes L129 and S. equorum SE3 on mMA. The initial concentration of S. equorum was around 1.8  106cfu/cm2. The initial L.m. concentrations in the 3 experiments were 1.3  101, 3.5  101 and 4.4  101 cfu/cm2 in the highest dilution. The detection limit for listeria was 1.7 cfu/cm2, after enrichment in Fraser broth 0.17 cfu/cm2. The zero value represents no growth in enrichment broth. The bars show mean values of 3 independent experiments performed over 3 weeks, error bars show the standard deviation of measurements.

Table 2 Co-cultivation of L. monocytogenes (L.m.) L129 (initial concentration 10 cfu/cm2) with typical smear microorganisms on modified milk agar. The detection limit for listeria was 1.7 cfu/cm2. Cultures

Initial concentration Counts after 24 h cocultivation L.m.

Kluyveromyces marxianus Candida krusei Pichia norvegensis Debaryomyces hansenii Brevibacterium linens Corynebacterium casei Staphylococcus equorum Microbacterium gubbeenense

2.5 1.4 5.3 1.0 5.5 5.1 4.9 3.2

L.m. control (pure)

10

       

5

10 105 106 105 106 106 106 106

1.7  2.6  5.2  3.7  2.9  7.3  <1,7 6.5 

Cheese culture 7

10 107 106 107 105 103 105

5.9 4.5 1.7 2.8 2.5 4.7 2.9 3.1

5.1  106 e

       

105 106 107 106 108 108 108 108

W. Bockelmann et al. / Food Control 71 (2017) 50e56

1010

L.m. S. equorum

109 108 107 106 105

102

0% 10

MA

90

%

MA

75

%

MA

% 50

MA

25

%

MA

10

%

< 17.5

103

< 17.5

104

< 17.5

counts after 24h [cfu/cm2 ]

L. monocytogenes when grown in co-culture on mMA; single strains of typical smear bacteria growing to high cell densities could also not prevent growth of L. monocytogenes (Table 2). The visible growth reduction of L. monocytogenes (1e2 log units) by cocultivation with single strains of coryneforms is probably not a specific antilisterial effect as seen for S. equorum SE3, which was able to prevent Listeria growth completely (Table 2). Co-cultivation of L. monocytogenes L129 and S. equorum SE3 on mMA resulted in lowered pH values of the mMA surface between pH 5.8 and pH 6.5 after 24 h of incubation. However, as L. monocytogenes is considered to be quite acid tolerant and has a minimal growth pH of ca. 4.5 (Ryser & Marth, 1987), this lowered pH was not likely to be the cause of inhibition by S. equorum. A typical smear cheese has a surface pH > 7 as a result of lactate degradation by yeasts and liberation of alkaline metabolites, e.g. ammonia, by coryneforms and other bacteria of the surface microbiota (Bockelmann, 2002). Since the growth optimum of L. monocytogenes, on the other hand, is between pH 6.5 and pH 7.5 (Ryser & Marth, 1987), co-cultivation of listeria and staphylococci was repeated in the presence of various smear consortia, typical of semi-hard, soft, or acid curd cheeses, to test the antilisterial properties under optimum growth conditions for L. monocytogenes (Fig. 3). In the presence of microorganisms of the smear consortia, as added to the cheese model, the strong listerial inhibition by S. equorum SE3 was similar as observed for the L. monocytogenes pure culture control (Fig. 3). Typical growth of the smear consortia was observed after 24 h incubation. Yeasts reached counts of >107 cfu/cm2, coryneforms and staphylococci counts were >108 cfu/cm2. In all co-cultivation batches, high pH values of the smear cheese model surface (pH > 7.0) were observed after 24 h (data not shown). This showed that S. equorum SE3 possessed strong antilisterial activity even under optimum growth conditions, which are also suited for growth of listeria. In contrast to S. equorum WS2733 (Carnio et al., 2000), which was reported to inhibit other smear microorganisms, growth of these microorganisms was not affected by S. equorum SE3, indicating that S. equorum SE3 would be better suited as an adjunct culture for surface ripening of smear cheeses. To determine whether the listerial inhibition was presumptively caused by a bacteriocin and not by some other

53

MA

A mM

0% 10

Fig. 4. Co-cultivation of S. equorum SE3 (initial conc. 1.8  107 cfu/cm2) and L. monocytogenes L129 (initial concentration 30 cfu/cm2) on milk agar (MA) and serial dilutions of milk agar with modified milk agar (mMA). The pH was measured after 24 h incubation at 30  C. The detection limit for listeria shown in the figure was 17.5 cfu/ cm2.

unknown factor, the proteolytic sensitivity of the inhibiting compound was tested in a spot-on-the-lawn assay with a-chymotrypsin. S. equorum SE3 spots showed antimicrobial activity towards the Listeria monocytogenes indicator strain by a clear zone of inhibition around the S. equorum colony, except for the side of the colony where the a-chymotrypsin was applied and where an inhibition zone did not develop (data not shown). 3.3. Co-cultivation of L. monocytogenes with S. equorum SE3 in the milk agar (MA) cheese model Since S. equorum is a natural component of smear cheese consortia and of cheese brines in general, results appeared to be quite promising for a future application of antilisterial staphylococci in cheese plants (Bockelmann et al., 2006). Milk agar, resembling a green cheese surface with no or a low degree of proteolysis, was used as a second cheese model for inhibition studies. This model would simulate the case in which a young cheese was recently contaminated by L. monocytogenes. Surprisingly, no listerial inhibition was observed using MA for co-cultivation (Fig. 4, left bar). Mixing of MA with different amounts of mMA, which increased peptide and vitamin levels could, however, restore the antilisterial activity of S. equorum SE3. A fully restored inhibition of L. monocytogenes growth was achieved at a ratio of 25:75% MA:mMA (Fig. 4). This indicated that a nutrient-rich environment, as present in mMA (amino acids, peptides, vitamins etc.) and which resembles a matured smear cheese surface, is necessary for the antilisterial activity of S. equorum SE3. 3.4. Modification of milk agar and mMA

Fig. 3. Co-cultivation of S. equorum SE3, B. linens Br5 and L. monocytogenes L129 (13 cfu/cm2) with combinations of smear microorganisms resulting in typical smear consortia; Debaryomyces hansenii (DH), Geotrichum candidum (GC), Kluyveromyces marxianus (KM), Candida krusei (CK), Corynebacterium casei (CC), Microbacterium gubbeenense (MG). The initial concentrations of all smear microorganisms were approximately 106 cfu/cm2, initial concentrations of L. monocytogenes L129 were ca. 13 cfu/cm2. Cultures were plated on modified milk agar and incubated for 24 h at 30  C. The detection limit for listeria was ca. 1.7 cfu/cm2 (shown as “0”).

Since milk agar did not contain salt while modified milk agar (mMA) contains 3% salt, co-cultivation was repeated in both model systems using either 0%, 1%, 2% and 3% NaCl for both models (data not shown). The previously observed complete listerial inhibition on mMA with 3% NaCl (>7 log units) was reduced by 1e3 log units at 2%, 1% and 0% NaCl. On milk agar containing 1e3% NaCl, listeriae were slightly inhibited (<1 log unit), which was not the case for milk agar without salt. More co-cultivation experiments were performed to identify the factor(s) playing a role in L. monocytogenes inhibition. The removal

54

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Table 3a Co-cultivation of L. monocytogenes (L.m.) L129 and S. equorum SE3 on modified milk agar with different compositions. Modifications of mod. milk agar (mMA)

L.m. counts

S. equorum counts

mMA mMA mMA mMA mMA mMA mMA mMA mMA mMA mMA mMA

<1.7a 8.7  101 <1.7 4.7  101 <1.7 5.1  101 <1.7 <17.5 <1.7 1.8  101 <17.5 <17.5

4.6  108 6.0  108 9.5  108 1.8  109 9.4  108 6.9  108 7.6  108 >5.3  108 1.2  109 1.1  109 >5.3  108 4.0  108

positive control e vitamins b e skim milk e NaCl e brain heart infusion e casein peptone & yeast extr. þ sodium lactate þ lactate - glucose e casein hydrolysate prepared with wheyc, not water e glucose þ galactose (3 g/L)

a Depending on the dilution used, the detection limit was either 1.7 or 17.5 cfu/ cm2. b Removal of a component (
), addition of a component (þ). c The nearly protein-free whey was prepared from raw skim milk ultrafiltrated through a membrane (10,000 kDa cut-off).

of single components of mMA did not diminish the excellent antilisterial properties of S. equorum SE3 (Table 3a). The addition of whey, lactate and galactose had no negative effect on inhibition (Table 3a). The addition of single mMA constituents to the milk agar model could partially restore Listeria inhibition (Table 3b). L. monocytogenes inhibition was fully restored by the addition of 2% casein peptone, consisting mainly of oligopeptides with chain lengths <4 mer (51.80%), 7 mer (38.19%), 37 mer (0.07%) and >74 mer (0.04%) according to product data provided by the supplier Merck. Addition of an equal concentration of casamino acids (Merck) instead of casein peptone restored inhibition considerably less (Table 3b). Since the concentrations of some amino acids are lower in casamino acids compared to same amounts of casein peptone (data from Merck), a mix of 18 amino acids was prepared at

a higher concentration (4.5%, final concentration). Under these conditions, Listeria inhibition was better compared to MA with 2% casamino acids, but not as good when using an agar containing 2% casein peptone (Table 3b). The addition of single amino acids, including phosphorylated serine, which is present in casein and perhaps in casein peptone (no data available), and 2 mixes of 10 amino acids each, had no restoring effect on listerial inhibition in the milk agar model (Table 3b). In another co-cultivation experiment on MA, a typical old-young smear was simulated: “old-young” smearing is the traditional, low (no) cost procedure of cheese producers, who treat (brush, spray) mature cheeses before young cheeses in the same machine (Bockelmann, 2002). This so-called “old-young smear” contains high amounts of recycled bacteria as well as degradation products from the action of the highly proteolytic mature smear microbiota (Bockelmann et al., 1997) To mimick “old-young” smearing, concentrations of casein peptone (5 mg, 10 mg, 20 mg, 40 mg per plated volume each) were added to the plating suspension together with staphylococci and listeria, which resulted in an increasing L. monocytogenes inhibition by 0.5, 1, 2 and 3 log units after 24 h of incubation, only (data not shown).

3.5. Reproducibility of co-cultivation The adapted co-cultivation assay, originally developed for yeasts by Goerges et al. (2006), also gave reproducible results for growth of mixed cultures of L. monocytogenes and S. equorum (Fig. 5). Pure cultures of L. monocytogenes as positive control were included in order to show uninhibited growth of the pathogen in the cheese models mMA or MA. The degree of inhibition was calculated by comparing L. monocytogenes pure culture counts on mMA with counts in co-culture. When models were modified, mMA and MA were included as positive and negative control (Table 3a, b). The experiments were performed over more than 12 months. Mean

Table 3b Co-cultivation of L. monocytogenes (L.m.) L129 and S. equorum SE3 on milk agar with various additional ingredients taken from the mMA recipe. Modifications of milk agar (MA)

L.m. counts

S. equorum counts

mMA positive control MA negative control MA (no fat) MA þ plate count (22.5 g/L) MA þ casein hydrolysate (10 g/L) MA þ brain heart infusion (10 g/L) MA þ yeast extract MA þ glucose

<17 8.1  5.4  8.8  1.3  5.3  8.6  8.4 

3.9 2.6 2.1 3.7 9.6 4.9 3.0 1.7

       

108 108 107 108 108 108 108 107

mMA positive control MA negative control MA þ 0.75% casein pepton MA þ 1% casein pepton MA þ 2% casein pepton MA þ 0.75% casamino acids MA þ 1% casamino acids MA þ 2% casamino acids

4.7 6.5  1.9  6.7  <1.7 2.8  2.8  2.8 

105 104 105

3.5 1.4 4.6 5.6 4.2 6.3 3.9 3.9

       

108 107 108 108 108 108 108 108

101 106e9.5  107 106 102 107 107

n.d.a n.d. 2.1  5.4  4.4  9.3 

107 108 105 106

MA MA MA MA MA MA a

þ þ þ þ þ þ

2% casein pepton positive control 11 single amino acidsb phoshoserine (0.1%) 18 amino acids (total 4.5%) 10 amino acids (set A)c 10 amino acids (set B)d

5.4 4.6 8.9 4.0 4.6 7.7

     

106 106 101 103 102 102 106 107 102 101

n.d. ¼ not determined. 11 separate experiments were performed by adding a single amino acid to Milk Agar for each assay; they were selected according to the composition of casein peptone given by the manufacturer (Merck, Darmstadt, Germany), the final concentration in milk agar was 0.25% (Ala, Arg, Cys, Gly, His, Ile, Leu, Lys, Met, Pro, Val). c Mix A consisted of Ala, Arg, Asp, Cys, Glu, Gly, His, Ile, Leu, Ser-P (each amino acid: 0.25%, Ser-P: 0.1%). d Mix B contained Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Ser-P (each amino acid: 0.25%, Ser-P: 0.1%). b

W. Bockelmann et al. / Food Control 71 (2017) 50e56

109

L.m. pure culture

L.m. in coculture

SE3 in coculture

counts after 24h [cfu/cm2 ]

108 107 106

55

antilisterial culture in smear cheese manufacture. Until now there have been no reports on the use of antilisterial S. equorum in commercial cheese ripening. Further studies should concentrate on identifying the factors affecting inhibition of L. monocytogenes. On the basis of the results of this study, one may speculate that inhibition might be related to peptide transport or metabolism, as the inhibition could be restored by addition of casein peptides (Fig. 4).

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Acknowledgements

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This research project was supported by the German Ministry of Economics and Technology (via AiF) and the FEI (Forschungskreis €hrungsindustrie e.V., Bonn). Project AiF14486N. der Erna

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References

100 A mM

MA am

% +2 MA

s ca

in

s cid oa % +2 MA

in se ca

ne pto pe

Fig. 5. Reproducibility of results of co-cultivated S. equorum SE3 and L. monocytogenes L129. Pure cultures of L. monocytogenes were plated on a parallel plate as uninhibited controls. The error bars show the standard deviation of bacterial counts after 24 h of incubation on modified milk agar (mMA, n ¼ 15), milk agar (MA, n ¼ 11), milk agar with added casamino acids (n ¼ 3) and milk agar with added casein peptone (n ¼ 6). Initial concentrations on agar were 20 cfu/cm2 (std. dev. 13) for L. monocytogenes and 1.7  106 cfu/cm2 (std. dev. 1.1  106) for S. equorum SE3.

values and standard deviation of counts during this period are shown in Fig. 5. Data of the most interesting co-culture assays (addition of casein peptone and casamino acids) are also shown. 4. Conclusions Functional antilisterial S. equorum cultures would be an ideal adjunct for cheese factories, not only for smear cheese producers, because S. equorum was found in all analysed industrial cheese brines, independent of the cheese type produced. This underlines the general importance of this species for the cheese environment (foil-ripened, waxed, smear-ripened, mould-ripened) (Bockelmann et al., 2006). The presence of antilisterial S. equorum in cheese brines, smearing machines and smear cheese surfaces could help to reduce trace amounts of L. monocytogenes, which seem to be present in many cheese factories (Rudolf & Scherer, 2001). S. equorum SE3 would be an ideal candidate as adjunct for smear cultures, because growth of other smear microorganism was not inhibited in these mixed cultures, as was shown to be the case for S. equorum WS2733 (Carnio et al. 2000). However, it is unclear, whether the antilisterial activity of S. equorum SE3 would actually lead to inhibition of L. monocytogenes in cheese brines or on smear cheeses. The anti listerial effect would be most important at the beginning of cheese ripening. The use of milk agar as a model for green cheese, however, indicated that green cheese surfaces at the beginning of smear ripening might not have the required nutrient composition for L. monocytogenes inhibition by S. equorum SE3. On the other hand, the use of modified milk agar, representing a mature smear cheese surface, demonstrated strong inhibition of listerial growth by S. equorum SE3 in contrast to the milk agar model. Since the development of the proteolytic smear on cheese proceeds fast within days (Bockelmann, Willems, Neve, & Heller, 2005), the question remains, whether the inhibition of L. monocytogenes in real cheese environment will be as excellent as found on mMA or as poor as on MA. Current studies analysing the ripening of Tilsit, Limburg and acid curd cheese artificially contaminated with L. monocytogenes will show whether S. equorum SE3 can be used as

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