International Journal of Food Microbiology 191 (2014) 53–59

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Control of Listeria monocytogenes in fresh cheese using protective lactic acid bacteria M.C. Coelho, C.C.G. Silva ⁎, S.C. Ribeiro, M.L.N.E. Dapkevicius, H.J.D. Rosa CITA-A, Centro de Investigação e Tecnologias Agrárias dos Açores, Universidade dos Açores, Angra do Heroísmo, Açores, Portugal

a r t i c l e

i n f o

Article history: Received 20 February 2014 Received in revised form 4 July 2014 Accepted 23 August 2014 Available online 29 August 2014 Keywords: Bacteriocin Lactic acid bacteria Listeria monocytogenes Fresh cheese Enterococcus faecalis Lactococcus lactis

a b s t r a c t In the past years, there has been a particular focus on the application of bacteriocins produced by lactic acid bacteria (LAB) in controlling the growth of pathogenic bacteria in foods. The aim of this study was to select LAB strains with antimicrobial activity, previously isolated from a traditional Azorean artisanal cheese (Pico cheese), in order to identify those with the greatest potential in reducing Listeria monocytogenes in fresh cheese. Eight bacteriocin producer strains identified as Lactococcus lactis (1) and Enterococcus faecalis (7) were tested. In general, the bacteriocin-producing strains presented a moderate growth in fresh cheese at refrigeration temperatures (4 °C), increasing one log count in three days. They exhibited slow acidification capacity, despite the increased production of lactic acid displayed by some strains after 24 h. Bacteriocin activity was only detected in the whey of fresh cheese inoculated with two Enterococcus strains, but all cheeses made with bacteriocin-producing strains inhibited L. monocytogenes growth in the agar diffusion bioassay. No significant differences were found in overall sensory evaluation made by a non-trained panel of 50–52 tasters using the isolates as adjunct culture in fresh cheese, with the exception of one Enterococcus strain. To test the effect of in situ bacteriocin production against L. monocytogenes, fresh cheese was made from pasteurized cows' milk inoculated with bacteriocin-producing LAB and artificially contaminated with approximately 106 CFU/mL of L. monocytogenes. The numbers of L. monocytogenes were monitored during storage of fresh cheese at refrigeration temperature (4 °C) for up to 15 days. All strains controlled the growth of L. monocytogenes, although some Enterococcus were more effective in reducing the pathogen counts. After 7 days, this reduction was of approximately 4 log units compared to the positive control. In comparison, an increase of 4 log CFU/mL in pathogen numbers was detected over the same period, in the absence of bacteriocin-producing LAB. The combination of two bacteriocin producing Enterococcus sp. optimized the reduction of L. monocytogenes counts in fresh cheese, reducing by approximately 5 log units after 7 days. The present work demonstrates that using bacteriocin-producing strains in the manufacture of fresh cheese might contribute to preventing the growth of undesirable pathogenic bacteria such as L. monocytogenes. A blend of two strains demonstrated great potential as a protective culture for the cheese making process. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Listeria monocytogenes is the causative agent of listeriosis, a severe disease in humans and one of the most significant foodborne diseases in industrialized countries (Schlech, 2000). L. monocytogenes is a psychrotrophic microorganism widely distributed in the environment that can grow at refrigerated temperatures and is highly acidic and salttolerant (review by McLauchlin et al., 2004). These characteristics make L. monocytogenes difficult to control in dairy foods and in particular in soft cheeses. As a result, contaminated cheese has been implicated in some of the major listeriosis outbreaks reported worldwide (Almeida et al., 2013; Castro et al., 2012; Farber and Peterkin, 1991; Hitchins, 1996). ⁎ Corresponding author at: Centro de Investigação de Tecnologias Agrárias dos Açores (CITA-A), Departamento de Ciências Agrárias, Universidade dos Açores, Rua Capitão João D'Avila, 9700-042 Angra do Heroísmo, Terceira, Portugal. E-mail address: [email protected] (C.C.G. Silva).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.08.029 0168-1605/© 2014 Elsevier B.V. All rights reserved.

Latin-style fresh cheese is a popular dairy product in Portugal and Spain (Evert-Arriagada et al., 2013). It is a non-ripened cheese, produced by enzymatic coagulation of milk with rennet, without adding starter cultures. It exhibits a soft texture, a slightly acidic flavor and has low salt and high moisture content. Due to the absence of starters, fresh cheese presents high pH values (above 5.0) and should be consumed shortly after production. Fresh cheese is also typically made without preservatives, requiring refrigerated temperatures for conservation. Therefore, fresh cheeses are particularly sensitive to colonization by L. monocytogenes through post-process contamination (Kabuki et al., 2004). Due to the relatively high pH and water activity allowing the growth of this microorganism during cheese storage at 4 °C, fresh cheese deserves particular attention from a hygienic/safety perspective (Castro et al., 2012). Consequently, the use of additional strategies to control the growth and survival of L. monocytogenes is imperative. One of the approaches used to prevent the growth of undesirable microorganisms in food is the use of bacteriocins or bacteriocin-

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M.C. Coelho et al. / International Journal of Food Microbiology 191 (2014) 53–59

producing lactic acid bacteria (LAB). Bacteriocins provide a more natural means of preserving food by reducing the requirement for chemical preservatives. A large number of bacteriocins possess anti-listerial activity and many of them have been applied to the control of L. monocytogenes in cheeses (Dal Bello et al., 2012; Liu et al., 2008; Ross et al., 1999). Although underutilized in the majority of cases, some enterocins produced by enterococci are among the most active bacteriocins in combating L. monocytogenes and may offer potential benefits in controlling this microorganism in foods. However, direct application of bacteriocins/enterocins may result in decrease or the complete loss of antimicrobial activity due to problems related to interaction with food components (Aasen et al., 2003; Chollet et al., 2008). Alternatively, the incorporation of live bacteriocin-producing strain(s), either through direct addition to the food or in an immobilized form on packaging, may present a potential benefit in controlling L. monocytogenes in dairy products. In this study, eight LAB bacteriocin producers isolated from an artisanal cheese (Pico cheese), and identified as Enterococcus faecalis (7) and Lactococcus lactis (1) were tested. The growth and in situ bacteriocin production of these LAB strains were investigated during the manufacture and storage of fresh cheese. Moreover, the effect of adding these LAB, on the physicochemical parameters and sensory acceptance of fresh cheese was evaluated. In addition, these bacteriocin-producing strains were tested with regard to controlling L. monocytogenes growth during the manufacture and storage of fresh cheese. The capacity for several blends of two Enterococcus strains to control L. monocytogenes growth during the manufacture and storage of fresh cheese was also assessed. To our knowledge, this represents the first report involving the use of bacteriocin producing LAB in fresh cheese with a view to controlling L. monocytogenes. 2. Material and methods 2.1. Microorganisms and culture conditions A strain of L. lactis producer of lacticin 481 (L3A21M1) and 7 strains of E. faecalis previously isolated from an Azorean artisanal cheese (Ribeiro et al., 2014) were included in this study. Before each experiment, the cultures were incubated for 24 h at 30 °C in MRS broth (AES, Bruz, France). Prior to fresh cheese production, all revitalized strains were grown in ultra-pasteurized (UHT) skim milk and incubated for 48 h at 30 °C reaching a cell concentration of about 108 cells/mL. The indicator strain L. monocytogenes ATCC 7466 was propagated in Nutrient Broth (AES) and incubated for 18 h at 37 °C. For inoculation in cheese, the revitalized cells of L. monocytogenes were pelleted by centrifugation (Centrifuge 5415D, Eppendorf, Germany), for 10 min at 5900 g, washed twice and resuspended in buffered peptone water (AES) and subsequently diluted to give the desired cell number (108 CFU/mL). 2.2. Manufacture of fresh cheese Cow's raw milk (3.5% fat, wt/wt) obtained from the Azores University farm (Chegalvorada, Angra do Heroismo, Portugal) was pasteurized at 73 °C for 16 s in a shaking water bath (Julabo, Model SW22, Germany) and then cooled in wet ice. Calcium chloride (0.2 g/L; Merck) and NaCl (10 g/L) were then added to the milk. In each trial, milk warmed at 32 °C was distributed in five 0.5 L vats and individually inoculated with each LAB culture (1%). Rennet (LMF 1/15,000, 0.2 g/L) was then added to the milk and incubated at 32 °C for aprox. 40 min. Control cheese was made without any inoculum. Once the coagulum was sufficiently firm, it was cut into 1–2 cm cubes and heated at 37 °C for 25 min. Whey was drained off and curds were distributed into perforated sterile stain steel circular cheese containers (6.5 cm in diameter). Cheeses were stored under refrigeration (4 °C) inside appropriated plastic boxes with a mesh covered with sterilized cheese cloth allowing whey drainage.

Cheeses were made in three trials carried out on six different days. In each day, five types of cheeses were made: one control (2 cheeses) and four inoculated with four different LAB strains (2 cheeses for each LAB). 2.3. Analysis of the experimental fresh cheeses Cheeses were sampled in duplicate (two different cheeses of the same trial) for pH, titratable acidity, LAB counts and bacteriocin activity in the beginning of storage (time = 0) and after 0, 6, 24, 48 and 72 h of storage at 4 °C. Cheese pH was measured directly with a pH meter (WTW Inolab pH Level 1, Germany). Titratable acidity was determined by direct titration of 4 g of fresh cheese dissolved in warm water, according to AOAC method # 920.124 (AOAC, 1995). For microbiological analyses, 25 g of cheese was transferred into a stomacher (400 Circulator, Seward, United Kingdom) containing 225 mL of sterile 0.1% (wt/vol) buffered peptone water (AES). Further dilutions were made from this original dilution and the quantification of microbial counts was carried out using the pour plate technique. The lactococci were enumerated on M17 agar (Biokar) and incubated under aerobic conditions at 30 °C for 72 h, whereas the enterococci were enumerated on KF Streptococcus agar (Biokar) at 37 °C for 48 h under aerobic conditions. Each experiment was conducted in duplicate. The antimicrobial activity of cheese samples was detected by the agar disk diffusion assay (Ribeiro et al., 2014). Briefly, cheese samples (5 g) were centrifuged at 4500 g for 10 min. Supernatants were neutralized with phosphate buffer (0.5 M, pH 7.0), filtered through a 0.22 μm membrane filter (Sartorius Stedim Biotech, Germany) and placed in duplicate into wells (6 mm diameter) made in pour plates of Plate Count Agar (AES) containing cultures of L. monocytogenes as indicator microorganism. After anaerobic incubation at 37 °C for 12 h, the diameter of the zone of growth inhibition was measured and bacteriocin activity expressed in mm. In addition, uniform cheese pieces were cut using a sterile cork borer (5 mm) and transferred into wells (6 mm diameter) of Plate Count Agar (AES) inoculated with indicator organism as previously indicated. The dishes were incubated at 37 °C for 12 h and checked for a clear halo around the cheese samples. All determinations were done using three independent samples (cheeses made on different days). 2.4. Sensory analysis To evaluate the influence of the eight bacteriocin producers in the final sensorial characteristics of fresh cheese, the different cheese productions were subjected to a panel evaluation. Sensory analysis was performed on fresh (2 days old) cheese by a non-trained panel of tasters comprising 50 to 52 participants from both genders, with ages ranging from 19 to 60 years old. The attributes judged were acidity, salty taste, firmness, flavor and general acceptability. Cheese scoring was conducted on a one to five scale (in which 1 stands for absence and 5 for presence at a strong level). Prior to assessment, each cheese was divided into various portions, and equilibrated at room temperature. Panelists were exposed to each sample on an individual petri plastic dish, and were asked to assess the specific attributes. Two evaluation sessions were performed and, in each session, four samples with LAB inoculate and one control (without any inoculums) were tested. 2.5. Evaluation of L. monocytogenes growth in fresh cheese Prior to cheese production, all revitalized LAB isolates were grown in UHT skim milk and incubated for 48 h at 30 °C reaching a cell concentration of about 108 cells/mL. L. monocytogenes (1%) was added to the milk in a suspension of 105–107 CFU/mL, by the time of LAB inoculation (Section 2.2) and left for 20 min before rennet addition. Fresh cheeses were then prepared as indicated in Section 2.2. A control cheese was made containing only L. monocytogenes inoculate. To enumerate

M.C. Coelho et al. / International Journal of Food Microbiology 191 (2014) 53–59

L. monocytogenes levels in the cheeses, 25 g samples were taken at different time intervals (0, 6, 24 h, 2, 3 and 7 days) and diluted in 225 ml of buffered peptone water (AES). After stomaching (2 min at 230 rpm), the mixture was serially diluted, plated onto PALCAM Listeria agar with added supplement (AES) and incubated at 37 °C for 48 h. Cheeses were made in three trials carried out on different days.

55

3.2. Bacteriocin production in fresh cheese Bacteriocin activity in cheeses made with the different lactic cultures is registered in Table 2. Bacteriocin production was determined by two methods, either by detection in cheese whey by the well-diffusion method, or by placing a piece of cheese on agar plates containing L. monocytogenes as indicator strain. When the cheese samples were placed on agar plates containing the indicator strain, clear inhibition zones were seen around the cheese pieces with the bacteriocinproducing strains. Thus, all cheeses made with bacteriocin-producing LAB exhibited bacteriocin activity from the introduction of curds into molds (time = 0) throughout the whole storage period (72 h). However, bacteriocin activity in cheese whey was only detected in cheese inoculated with two Enterococcus strains: L2B21K3 detected throughout the storage time and L3A21K6 detected after 48 h of storage at 4 °C (Table 2).

2.6. Statistical analysis Cheese pH and titratable acidity were analyzed using ANOVA repeated measures, with strain between subject factor and time as the within subject variable. When differences were statistically significant (P b 0.05), post hoc multiple comparisons were determined by the PLSD test. Values from each trial were determined from the means of duplicate data. Each cheese trial was repeated three times and the final results represent the average of each set of experiments. Sensory evaluations were statistically analyzed using one-way ANOVA. Post hoc multiple comparisons were determined by Tukey's test. Differences were considered statistically significant at P b 0.05. Statistical tests were performed using IBM SPSS Statistics, version 20.0 (IBM Corporation, New York, USA).

3.3. Sensory analysis The sensory profiles of the experimental fresh cheeses showed that only two attributes (acidity and firmness) were significantly different among cheeses (Table 3). All cheeses made with LAB cultures were found to have flavor, salty taste and general acceptability comparable to the control cheese made without any protective culture. Tasters recognized low cheese scores for acidity (1.4 to 2.3), salt content (2.0 to 2.7) and flavor (1.7 to 2.3), as well as average scores for firmness (2.4 to 3.4) and general acceptability (3.0 to 3.5). The most notable and significant (P b 0.05) differences between cheeses were evidenced through perceived firmness and acidity. Nevertheless, cheeses inoculated with bacteriocin-producing LAB showed no significant differences from control cheeses in the sensory attributes tested.

3. Results 3.1. Fresh cheeses inoculated with bacteriocin producer LAB Results obtained for pH, titratable acidity in fresh cheeses made with the different lactic cultures are indicated in Table 1. Cheese pH and titratable acidity were significantly influenced (P b 0.05) by the lactic culture (Table 1). The results of the pH readings show that the tested LAB cultures were poor acidifiers. Values of pH were initially between 6.4 and 6.5, and reduced slightly to 6.0 and 6.4 after 72 h. Conversely, the control cheese increased pH levels to 6.6. The most significant reduction of these was observed for cheeses produced with two enterococcus strains (L3A21M3 and L3A21M8). These strains also presented the highest values of titratable acidity. However, there was no apparent relationship between pH and titratable acidity for cheeses inoculated with other strains (e.g. strain L2B21K3). The titratable acidity remained unchanged (0.04 g/100 g) in the control cheese and cheeses produced with three enterococcus strains (L3A21K6, L3A21K7 and L3B1K3). Cell counts of the starter culture bacteria were also recorded during storage of fresh cheese at 4 °C (Fig. 1). All LAB grew well in fresh cheese samples, increasing at least one log unit to reach counts around 109 CFU/ mL after 72 h of refrigeration. By this time, all the strains were in the stationary phase.

3.4. Control of L. monocytogenes in fresh cheese The effect of in situ bacteriocinogenic producers on the behavior of L. monocytogenes in fresh cheese was determined during the storage period at 4 °C (Fig. 2). During storage at refrigeration temperatures, in the absence of a protective culture, L. monocytogenes grew through a seven-day period, reaching large numbers (about 108 CFU/g). However, L. monocytogenes counts were reduced albeit variable extents in all the samples of fresh cheese that had been inoculated with LAB, regardless of the strain used. Nonetheless, such reduction was more pronounced in E. faecalis strains than in L. lactis (L3A21M1), where a bacteriostatic effect was observed (Fig. 2A). After seven days of refrigeration, a reduction of L. monocytogenes by two log units was observed in cheese inoculated with L. lactis. In contrast, all E. faecalis strains decreased L. monocytogenes counts by 3 to 4 log units compared to the control (Fig. 2A, B). Strains L3A21M3 and L3A21M8 were the most effective,

Table 1 Values of pH and titratable acidity of fresh cheeses inoculated with bacteriocinogenic LAB. Values are presented as mean ± SEM of two experiments. Time (h)

pH

Titratable acidity (g/100 g)

0 Control L. lactis L3A21M1 E. faecalis L3A21M3 L3A1M6 L3A21M8 L2B21K3 L3B1K3 L3A21K6 L3A21K7 a–g

6

24

48

72

0

6

24

48

72

a

0.04 ± 0.00

0.04 ± 0.00

0.04 ± 0.00

0.04 ± 0.00

0.04 ± 0.00a

0.05 ± 0.01

0.05 ± 0.00

0.07 ± 0.00

0.07 ± 0.00

0.08 ± 0.01b

0.04 0.04 0.05 0.04 0.05 0.05 0.05

0.05 0.05 0.07 0.05 0.05 0.05 0.05

0.07 0.08 0.12 0.08 0.05 0.05 0.05

0.12 0.09 0.16 0.09 0.05 0.05 0.05

0.13 0.11 0.18 0.10 0.07 0.05 0.05

6.5 ± 0.1

6.5 ± 0.1

6.6 ± 0.0

6.6 ± 0.0

6.6 ± 0.0

6.5 ± 0.0

6.7 ± 0.1

6.7 ± 0.1

6.6 ± 0.0

6.4 ± 0.0a

6.5 6.5 6.5 6.4 6.4 6.4 6.4

6.4 6.4 6.4 6.6 6.5 6.5 6.6

6.3 6.4 6.3 6.6 6.5 6.6 6.5

6.1 6.3 6.1 6.3 6.4 6.4 6.5

6.0 6.2 6.1 6.2 6.2 6.2 6.3

± ± ± ± ± ± ±

0.1 0.1 0.0 0.0 0.0 0.0 0.0

± ± ± ± ± ± ±

0.0 0.0 0.0 0.1 0.0 0.1 0.1

± ± ± ± ± ± ±

0.0 0.1 0.1 0.1 0.1 0.1 0.0

± ± ± ± ± ± ±

0.0 0.1 0.0 0.1 0.1 0.1 0.0

± ± ± ± ± ± ±

0.0b 0.1bc 0.1b 0.1cd 0.1c 0.0cd 0.0d

± ± ± ± ± ± ±

0.00 0.00 0.00 0.00 0.00 0.00 0.00

Mean values in the same column not followed by the same letter are significantly different (P b 0.05).

± ± ± ± ± ± ±

0.00 0.00 0.00 0.00 0.00 0.00 0.00

± ± ± ± ± ± ±

0.00 0.01 0.01 0.01 0.00 0.00 0.00

± ± ± ± ± ± ±

0.01 0.00 0.00 0.00 0.00 0.00 0.00

± ± ± ± ± ± ±

0.00c 0.00d 0.00e 0.01d 0.00f 0.00g 0.00g

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M.C. Coelho et al. / International Journal of Food Microbiology 191 (2014) 53–59

B

10

9

Log (CFU/g)

Log (CFU/g)

A

8

7

10

9

8

7

6

6 0

12

24

36

48

60

72

0

12

24

36

48

60

72

Time (h)

Time (h)

Fig. 1. Growth of bacteriocinogenic LAB in fresh cheese stored at 4 °C. A) Viable counts were monitored in cheeses containing L. lactis L3A21M1 (◊) and E. faecalis L3A21M3 (●), L3A1M6 (■), and L3A21M8 (□). B) Viable counts were monitored in cheeses containing E. faecalis L2B21K3 (◊), L3B1K3 (●), L3A21K6 (■), and L3A21K7 (□).

reducing cell counts of L. monocytogenes at the beginning (6 h) and throughout the storage time (Fig. 2A). The most effective strains were combined on the basis of coexistence tests and used in different preparations (L3A21M3 + L3A21M8, L3B1K3 + L3A21M3 and L3A21K6 + L3A21K7) to control L. monocytogenes in fresh cheese (Fig. 2C, D). The mixtures L3A21M3 + L3A21M8 and L3B1K3 + L3A21M3 were found to have a considerable inhibitory effect on L. monocytogenes, reducing this pathogen by ~ 4 log CFU/g in the first three days of storage and by ~ 5 log CFU/g on day seven (Fig. 2C). In a separate experiment, a lower inoculum of L. monocytogenes was tested with similar results (Fig. 2D).

not fall as with common cheeses. Thus, the strains tested may not be suitable as starter organisms, but they may still be useful as adjunct cultures to improve safety in this type of fresh cheese. In the present study, all strains apparently produced bacteriocin-like substances throughout the cheese making and storage. This was detected by the direct application of cheese samples to L. monocytogenes. However, the tested cheese samples also contain the producer strains, which can produce bacteriocins during the 12 hour incubation of the bacteriocin production test. Nevertheless, the high antimicrobial activity in cheeses detected since the beginning of storage at 4 °C is probably mainly due to bacteriocin production during cheese making and cannot be attributed to a reduction in pH or production of lactic acid as demonstrated by the low values of titratable acidity, since in most strains these values are identical to the control. Furthermore, a slight increase in the inhibition halos was observed in the first 6 h, which corresponds to the exponential growth of strains. In later growth phases, where an increase in lactic acid concentration and the concomitant decrease in pH were observed, no increase in the diameter of the inhibition halos occurred. In addition, the hypothesis that inhibition could be the result of organic acids produced by LAB was not consistent with the absence of inhibition halos detected in cheese whey from six strains, some of them being the highest producers of acid (e.g. L3A21M8 and L3A21M3, Table 1). In opposition, inhibition halos were detected with cheese whey produced by strains L3B1K3 and L3A21K6 that were poor acid producers (Table 1). Although the kinetics of bacteriocin production was not evaluated in the present study, several authors report that bacteriocin production increases during the exponential phase and stabilizes during

4. Discussion LAB comprises a heterogeneous collection of microorganisms having in common the metabolic property of lactic acid production (Mayo et al., 2010). The strains tested in the present work were isolated from a traditional cheese and all produced lactic acid in different amounts. However, the values detected for titratable acidity were low and none of the strains reduced pH below 6.0. These results are in agreement with other studies reporting poor acid production by L. lactis and enterococci in milk (Ayad et al., 2004). In a systematic study, Sarantinopoulos et al. (2001) examined the acidifying ability of Enterococcus strains grown in skimmed milk for 6 h at 37 °C and observed that only three out of the 56 E. faecalis strains reduced the pH below 5.9. Latin-style fresh cheeses are traditionally produced without any starter and consequently, pH does

Table 2 Bacteriocin activity in fresh cheese presented as diameter of inhibition zones (mm). Values are presented as mean ± SEM of two experiments. Bacteriocin activity from cheese whey was only detected with two strains (L3B1K3 and L3A21K6). Inhibition test

Cheese

Cheese whey

ND: No inhibition observed.

Strain

Control L. lactis L3A21M1 E. faecalis L3A21M3 L3A1M6 L3A21M8 L2B21K3 L3B1K3 L3A21K6 L3A21K7 E. faecalis L3B1K3 L3A21K6

Storage time (hours) 0

6

12

24

72

ND

ND

ND

ND

ND

14.00 ± 0.13

13.71 ± 0.06

14.31 ± 0.01

14.72 ± 0.02

14.75 ± 0.01

14.43 15.28 14.45 15.11 16.23 15.85 16.73

14.00 15.32 14.71 15.42 16.90 16.99 17.51

14.48 15.74 16.22 15.74 17.61 17.01 17.67

15.65 16.65 16.26 16.65 17.99 17.57 17.90

14.75 15.97 16.13 15.31 17.66 16.49 16.20

± ± ± ± ± ± ±

0.06 0.08 0.08 0.15 0.05 0.24 0.01

10.77 ± 0.00 ND

± ± ± ± ± ± ±

0.01 0.16 0.52 0.01 0.23 0.08 0.11

11.65 ± 0.18 ND

± ± ± ± ± ± ±

0.04 0.02 0.16 0.02 0.16 0.20 0.23

10.89 ± 0.41 ND

± ± ± ± ± ± ±

0.01 0.23 0.08 0.23 0.01 0.04 0.03

12.24 ± 0.04 11.64 ± 0.08

± ± ± ± ± ± ±

0.21 0.35 0.06 0.69 0.42 0.11 0.31

12.24 ± 0.04 11.92 ± 0.21

M.C. Coelho et al. / International Journal of Food Microbiology 191 (2014) 53–59

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Table 3 Sensory evaluation of fresh cheeses inoculated with bacteriocinogenic LAB. Trial

Cheeses

Acidity

1

Control L3A21M1 L3A1M6 L3A21M3 L3A21M8 Control L2B21K3 L3B1K3 L3A21K6 L3A21K7

1.83 1.44 2.33 2.31 1.81 1.78 1.70 1.80 1.94 1.90

2

± ± ± ± ± ± ± ± ± ±

0.10ab 0.09a 0.16b 0.17b 0.13ab 0.13 0.12 0.12 0.14 0.12

Salty taste

Firmness

2.52 2.29 2.67 2.62 2.46 2.00 2.06 2.34 2.26 2.48

2.90 2.46 3.06 2.94 2.37 2.82 2.46 3.42 2.80 3.14

± ± ± ± ± ± ± ± ± ±

0.13 0.13 0.13 0.15 0.12 0.13 0.15 0.14 0.13 0.15

± ± ± ± ± ± ± ± ± ±

Flavor

0.13ab 0.12ac 0.13b 0.15ab 0.12c 0.12ab 0.13a 0.11b 0.13ab 0.15b

1.92 1.96 2.29 2.27 1.85 1.88 1.74 2.12 1.90 2.06

± ± ± ± ± ± ± ± ± ±

General acceptability 0.13 0.14 0.15 0.15 0.14 0.13 0.13 0.15 0.12 0.14

3.37 3.44 3.17 3.02 3.37 3.22 3.22 3.48 3.34 3.28

± ± ± ± ± ± ± ± ± ±

0.12 0.15 0.17 0.16 0.16 0.13 0.15 0.12 0.13 0.15

a–c

Mean values in the same trial and column not followed by the same letter are significantly different (P b 0.05). Mean data ± SEM from 52 consumers in trial 1 and 50 consumers in trial 2, and based on a 5-point scale (1 stands for absence and 5 for presence at a strong level).

the stationary phase (Benkerroum et al., 2012; Han et al., 2013; Martínez et al., 2013). The results obtained by the two strains that displayed anti-listeria activity in cheese whey confirmed the production of bacteriocin like activity by the Enterococcus strain L2B21K3 from storage time onwards. In cheese inoculated with this strain a slight increase in the diameter of the inhibition halos was observed over time, whereas in the case of the strain L3A21K6, the anti-listeria activity of cheese whey was only detected after 48 h. The absence of anti-listeria activity observed in the whey cheese of other strains tested may indicate the occurrence of adsorption of bacteriocins on the surface of producer cells or milk proteins. Similar results were obtained by various authors who failed to detect the presence of bacteriocin activity on cheese whey, while it was detected when cheese samples were placed directly into wells of agar

B 9

9

8

8

7

7

Log ufc/g

Log ufc/g

A

containing indicator bacteria (Foulquié-Moreno et al., 2003; Rodríguez et al., 2005; Sarantinopoulos et al., 2002). In the present study, only two Enterococcus strains produce bacteriocins detectable in the cheese whey. The bacteriocins produced by these strains have different chemical properties, in particular an overall positive charge (results not presented), unlike most bacteriocins, which are anionic and can bind strongly to phosphate groups of casein. The application of new strains to the cheese making process requires a positive sensory acceptance by the consumers. In the present study, cheeses made with the bacteriocin-producer strains resulted in an overall positive appreciation similar to the control cheeses (made without any culture). In general, tasters have assigned a low score to the acidity, salt content and flavor of experimental cheeses. With regard to fresh cheeses, satisfactory sensory outcomes require the use of strains with

6 5

6 5

4

4

3

3 2

2 0

1

2

3

4

5

6

0

7

1

2

D

9

4

5

6

7

5

6

7

9

8

8

7

7

Log ufc/g

Log ufc/g

C

3

Time (days)

Time (days)

6 5

6 5

4

4

3

3 2

2 0

1

2

3

4

Time (days)

5

6

7

0

1

2

3

4

Time (days)

Fig. 2. Growth of L. monocytogenes in fresh cheese at 4 °C in the presence of bacteriocin-producer LAB cultures: A) L. monocytogenes alone (●) and L. monocytogenes plus L. Lactis L3A21M1 (■) and Enterococcus strains L3A21M3 (▲), L3A1M6 (□), and L3A21M8 (□); B) L. monocytogenes alone (●) and L. monocytogenes plus Enterococcus strains L2B21K3 (■), L3B1K3 (▲), L3A21K6 (□), and L3A21K7 (□); C) L. monocytogenes alone (●) and L. monocytogenes plus two Enterococcus strains: L3A21M3 + L3A21M8 (▲), L3B1K3 + L3A21M3 (♦) and L3A21K6 + L3A21K7 (■); D) Lower initial inoculum of L. monocytogenes (●) plus Enterococcus strains: L3A21M3 + L3A21M8 (▲), L3B1K3 + L3A21M3 (♦) and L3A21K6 + L3A21K7 (■).

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mild acidifying capacities to prevent an excessive formation of organic acids (Gomes et al., 2011). Mild flavor and texture of the final product are also demanded by the consumers of fresh cheeses (Gammariello et al., 2009). In relation to salt content, it is usual to add salt at the time of cheese consumption, so the quantity will be adjusted to satisfy the tastes of individual consumers. Therefore, no negative sensory attributes were indicated by the non-trained tasters when LAB strains were used in the fresh cheese making process. According to Pereira et al. (2011) microorganisms inoculated in cheeses made with cows' milk do not exert any influence on the flavor of the cheese in the early days of ripening, which confirms the results obtained in the present study. Bioavailable bacteriocin activity will most probably be the determining factor for an in situ antibacterial action. Of major importance to any application of bacteriocin-producing LAB to cheese making is the finding that bacteriocin-like activity is produced at sufficient levels and is active enough during cheese manufacture and storage. The results obtained from in situ bacteriocin producers clearly show a high listeria killing activity in fresh cheese made with the Enterococcus strains. The L. lactis strain tested produced lacticin 481 (data not show), which proved to be less effective in reducing L. monocytogenes (reduction of two log units after seven days). These results are in agreement with other studies concerning L lactis carried out in cottage cheese. Reductions of 2–3 log units of listeria counts in cheese were observed after a week of maturation using bacteriocinogenic L. lactis (Rodríguez et al., 2005; Ross et al., 2000). In cottage cheese, Dal Bello et al. (2012) also observed a reduction of L. monocytogenes by three log units after incubation with a lacticin-481 producer strain of L. lactis. However, after this initial period, L. monocytogenes counts increased to values close to the control. This was justified by the fact that cheese presented little acidification and thus did not inhibit growth of the pathogen. In contrast, the bacteriocinogenic Enterococcus strains tested in the present study were more efficient in reducing L. monocytogenes counts at the beginning and throughout the storage time. The highest levels of antilisterial activity in cheese (~5 log reduction) were observed by the application of two E. faecalis combinations (L3A21M3 + L3A21M8 and L3B1K3 + L3A21M3). The enterocins produced by these strains have not yet been identified, as none of them match enterocins described in literature (results not shown). The potential benefits of applying enterocins to control listeria in cheeses have also been well documented (Foulquié-Moreno et al., 2006; Giraffa, 1995; Nuñez et al., 1997). However, none of the studies published to date present a reduction of L. monocytogenes as high as in the current study. Pingitore et al. (2012) also tested various bacteriocinogenic Enterococcus strains in fresh cheese, having observed a reduction in L. monocytogenes to three log units after 12 days. Nevertheless, the enterococci exerted a bacteriostatic effect and did not reduce in the initial counts of L. monocytogenes. Thus, some of the strains tested in the present study have a great potential for being applied to fresh cheese production with the aim of reducing possible contamination from the L. monocytogenes. In particular, the use of L3B1K3 and L3A21M3 strains in combination could be advantageous in controlling L. monocytogenes in cheese. Although these two strains have been identified as E. faecalis, raising some safety issues regarding their application in foods, they have a reduced number of virulence factors (Ribeiro et al., 2014). The genus Enterococcus is the most controversial group of lactic acid bacteria present in food products. Even so, the enterococci are a component of the natural microbiota of dairy products, and may have beneficial effects (Rivas et al., 2012). Studies on the microbiota of traditional cheeses produced mainly from raw milk from ewes, goats or cows, indicate that enterococci are a relevant component of the natural cultures and play an important role in cheese ripening, typical taste and flavor (Foulquié-Moreno et al., 2006). Some of the enterococci can grow and produce bacteriocins in milk and cheeses, which make them good candidates for use as protective cultures against pathogens. In this way, these strains may be of great technological importance in cheese manufacture in controlling L. monocytogenes.

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