International Journal of Food Microbiology 211 (2015) 38–43
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Stability, antimicrobial activity, and effect of nisin on the physico-chemical properties of fruit juices Adelson Alves de Oliveira Junior a, Hyrla Grazielle Silva de Araújo Couto b, Ana Andréa Teixeira Barbosa c,⁎, Marcelo Augusto Guitierrez Carnelossi b, Tatiana Rodrigues de Moura c a b c
Departamento de Nutrição,Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brazil Departamento de Tecnologia de Alimentos, Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brazil Departamento de Morfologia, Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brazil
a r t i c l e
i n f o
Article history: Received 29 January 2015 Received in revised form 23 June 2015 Accepted 26 June 2015 Available online 2 July 2015 Keywords: Bacteriocin Food preservation Microbial inactivation Listeria monocytogenes Alicyclobacillus acidoterrestris
a b s t r a c t Heat processing is the most commonly used hurdle for inactivating microorganisms in fruit juices. However, this preservation method could interfere with the organoleptic characteristics of the product. Alternative methods have been proposed and bacteriocins such as nisin are potential candidates. However, the approval of bacteriocins as food additives is limited, especially in foods from vegetal origin. We aimed to verify the stability, the effect on physico-chemical properties, and the antimicrobial activity of nisin in different fruit juices. Nisin remained stable in fruit juices (cashew, soursop, peach, mango, passion fruit, orange, guava, and cupuassu) for at least 30 days at room or refrigerated temperature and did not cause any significant alterations in the physico-chemical characteristics of the juices. Besides, nisin favored the preservation of vitamin C content in juices. The antimicrobial activity of nisin was tested against Alicyclobacillus acidoterrestris, Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes in cashew, soursop, peach, and mango juices. Nisin caused a 4-log reduction in viable cells of A. acidoterrestris in soursop, peach, and mango juices after 8 h of incubation, and no viable cells were detected in cashew juices. After 24 h of incubation in the presence of nisin, no viable cells were detected, independently of the juices. To S. aureus, at 24 h of incubation in the presence of nisin, viable cells were only detected in mango juices, representing a 4-log decrease as compared with the control treatment. The number of viable cells of B. cereus at 24 h of incubation in the presence of nisin represented at least a 4-log decrease compared to the control treatment. When the antimicrobial activity of nisin was tested against L. monocytogenes in cashew and soursop juices, no reduction in the viable cell number was observed compared to the control treatment after 24 h of incubation. Viable cells were four and six times less than in the control treatment, in peach and mango juices respectively. The most sensitive microorganism to nisin was A. acidoterrestris and the least sensitive was L. monocytogenes. Still, a reduction of up to 90% of viable cells was observed in peach and mango juices inoculated with L. monocytogenes. These results indicate that the use of nisin could be an alternative in fruit juice processing. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The most commonly used hurdle for inactivating microorganisms and enzymes in fruit juices, and thereby extending the product shelf life, is heat processing (Braddock, 1999; Jiménez-Sánchez et al., 2015). However, it is well known that the organoleptic characteristics of the product may be altered by this processing method. This leads to an increased interest in innovative non-thermal processing technologies, which can enhance the shelf life of high quality foods, with reduced or no application of heat, and without affecting the quality and safety of ⁎ Corresponding author at: Departamento de Morfologia Universidade Federal de Sergipe Cidade Universitária Prof. José Aloísio de Campos Jardim Rosa Elze São Cristóvão, SE 49100–000, Brazil. E-mail address: [email protected] (A.A.T. Barbosa).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.06.029 0168-1605/© 2015 Elsevier B.V. All rights reserved.
the product (Bi et al., 2013; Jiménez-Sánchez et al., 2015; Timmermans et al., 2014). A considerable number of studies have been conducted showing the application of pulsed electric field (Bi et al., 2013; Timmermans et al., 2014), high hydrostatic pressures (Espina et al., 2013), essential oils (Espina et al., 2012), ozone (Torlak, 2014), and bacteriocins from lactic acid bacteria (Carvalho et al., 2008; Grande et al., 2005; Komitopoulou et al., 1999; Pei et al., 2013) in the preservation of fruit juices. Amongst these, the use of bacteriocins from lactic acid bacteria is a promising alternative. The application of bacteriocins in food conservation is widespread; however, little is known about its use in fruit products (Grande et al., 2005; Pei et al., 2013). Bacteriocins previously tested in fruit juices are nisin (Komitopoulou et al., 1999), enterocin AS48 (Grande et al., 2005), bificin C6564 (Pei et al., 2013), and bovicin HC5 (Carvalho
A.A. de Oliveira Junior et al. / International Journal of Food Microbiology 211 (2015) 38–43
et al., 2008). Amongst these, only nisin is approved as food additive, but its approval in fruit products is limited. Nisin is an antimicrobial peptide that has an antimicrobial activity against several food-borne spoilage and pathogenic microorganisms, including Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, and Alicyclobacillus acidoterrestris (Cleveland et al., 2001; Cotter et al., 2005; Deegan et al., 2006; Komitopoulou et al., 1999). Microorganisms such as L. monocytogenes have been reported to be capable of surviving in raw fruit and vegetable juices (Barbosa et al., 2013; Burnett and Beuchat, 2001; Mutaku et al., 2005), and A. acidoterrestris is increasingly being reported as a cause of spoilage of fruit juices (Oteiza et al., 2014; Zhang et al., 2013). B. cereus and S. aureus have also been isolated from fruit products (Carvalho et al., 2007; Vantarakis et al., 2011). Nisin could therefore be a promising bacteriocin for use in controlling the growth of these microorganisms in fruit products. The knowledge on nisin application in fruit juices is still limited. Some studies only showed the effect of nisin against A. acidoterrestris in apple and orange juices (Pathanibul et al., 2009; Walker and Phillips, 2006), and against indigenous microorganisms in litchi juice (Li et al., 2012; Yu et al., 2013). In these studies, nisin stability and its effects on the physico-chemical properties of these juices were not reported. In this study, we aimed to verify the stability of nisin and its effect on the physico-chemical properties of different fruit juices, and evaluate its antimicrobial activity against A. acidoterrestris, L. monocytogenes, B. cereus and S. aureus in cashew, soursop, peach, and mango juices to confirm the potential of nisin in the conservation of fruit juices.
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2.4. Antimicrobial activity of nisin in fruit juices The antimicrobial activity of nisin was tested against L. monocytogenes, S. aureus, B. cereus, and A. acidoterrestris in cashew, soursop, peach, and mango juices. Approximately 106 CFU/mL (100 μL of a 12 h culture) of the microorganisms were inoculated in fruit juices that were supplemented with 5000 IU/mL nisin. The fruit juices were incubated at microorganism growth temperature, therefore to S. aureus ATCC 8095, B. cereus ATCC 4504, and L. monocytogenes ATCC 7644 the juices were incubated at 37 °C and to A. acidoterrestris DSMZ 2498 at 40 °C. After 0, 8, and 24 h of incubation, samples were withdrawn, diluted (in tenfold increments) and viable cell numbers were determined by plate counting. The cell number was calculated considering the following: CFU/mL = CFU × DF/aliquot volume, where CFU = colony-forming units, DF = dilution factor (reciprocal of the dilution), and aliquot volume = sample volume plated (in mL). Controls without the bacteriocin nisin were also performed. 2.5. Statistical analysis All treatments were repeated three times and each repetition was conducted in triplicate. For physico-chemical analysis, the results obtained were submitted to analysis of variance (ANOVA) at 5% of probability, and means were compared with Tukey's test using the SAS statistical software (Statistical Analysis Systems, 2004). In the figures, data and error bars represent arithmetic mean values and standard deviation, respectively.
2. Material and methods
3. Results
2.1. Microorganisms and growth conditions
3.1. Stability of nisin in different fruit juices
S. aureus ATCC 8095, B. cereus ATCC 4504, and L. monocytogenes ATCC 7644 were grown in BHI media (HIMDEDIA, Mumbai, India) at 37 °C. A. acidoterrestris DSMZ 2498 was grown at 40 °C in AAM (A. acidoterrestris media) according to methods described by Yamazaki et al. (2000). The indicator strain Lactococcus lactis ATCC 19435 was cultivated in MRS media (HIMDEDIA, Mumbai, India) at 30 °C.
The antimicrobial activity of nisin was detected in all fruit juices for at least 30 days of storage, independently of the incubation temperature (Table 1). If the storage time was extended to 70 days, nisin remained stable in fruit juices at 4 °C, independently of the juice (Table 1). If the storage temperature was increased to 30 °C, the antimicrobial activity of nisin was detected in passion fruit, orange, soursop, and cupuassu juices for at least 70 days. In cashew, mango, peach, and guava juices, no residual activity of nisin was detected at 70 days of storage at this temperature (Table 1). Fruit juices that have not been supplemented with nisin (control treatment) did not show any antimicrobial activity (data not shown).
2.2. Stability of nisin in different fruit juices The commercial fruit juices used in this study were purchased from local stores in Aracaju city, Sergipe state, Brazil. The juices tested for nisin stability were cashew, soursop, peach, mango, passion fruit, orange, guava, and cupuassu. The stability of nisin in fruit juices was tested by adding bacteriocin at a final concentration of 5000 IU/mL. After 0, 4, 8, 12, 30, and 70 days of incubation at room temperature (30 °C ± 2) or at 4 °C, samples were withdrawn and tested for antimicrobial activity by the well diffusion assay described by Tagg et al. (1976). The assay was performed using L. lactis ATCC 19435 as the indicator strain. Approximately, 106 CFU/mL of the microorganisms was inoculated in solid media and dispensed into Petri dishes. A well of approximately 5 mm diameter was created in the center of the dish and 25 μL of the sample was added in the well. The dishes were incubated at 4 °C for 12 h for sample diffusion and at 30 °C for microorganism growth. After 24 h, the inhibition zones formed were analyzed. A control treatment of juice without nisin was also included. 2.3. Effect of nisin on the physico-chemical properties of fruit juices Peach, mango, cashew, and soursop juices were supplemented with nisin at 5000 IU/mL and stored at 4 °C. After 0, 5, 15, and 45 days of storage, samples were withdrawn and the soluble solids (expressed in °Brix), total titratable acidity, pH level, vitamin C content, and browning index were evaluated (Carnelossi, 2000). Controls without nisin were also performed.
3.2. Effect of nisin on the physico-chemical properties of fruit juices When fruit juices were stored at 4 °C with or without nisin, no significant differences (at 5% by Tukey's test) in pH or total titratable acidity were observed after 45 days of storage (Tables 2, 3, 4, and 5). Regarding the vitamin C content, a reduction was observed in all the juices, with or without nisin, after 45 days of storage. However, the reduction was always higher in control juice samples without nisin (Tables 2, 3, 4, and 5). For peach juice, the reduction in vitamin C content after this storage time was 70% and 73%, respectively, with and without nisin (Table 2). For mango juice, the reduction was 75% in control samples and 53% in samples with added nisin (Table 3). In cashew juices, the samples with added nisin showed a reduction of 70%, and in control samples, the reduction was 82% (Table 3). When the soursop juice was tested, the decrease in vitamin C content was found to be 70% in the control sample and 56% in samples with added nisin, after 45 days of storage (Table 5). When the browning index was analyzed after 45 days of storage, the index was higher in peach and cashew juices stored without nisin (Tables 2 and 4). In mango and soursop juices, no significant differences were observed in the browning index for samples with or without nisin at this same storage day (Tables 3 and 5). In soursop juice, the browning
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A.A. de Oliveira Junior et al. / International Journal of Food Microbiology 211 (2015) 38–43
Table 1 Nisin stability in different fruit juices at 4 °C and at 30 °C. Juice
Incubation days 0a
4a
4 °C Cashew Passion fruit Mango Orange Soursop Peach Guava Cupuassu a b
20 ± 0.01 20 ± 0.08 20 ± 0.01 20 ± 0.03 20 ± 0.05 20 ± 0.1 20 ± 0.07 20 ± 0.14
b
8a
12a
30a
70a
30 °C
4 °C
30 °C
4 °C
30 °C
4 °C
30 °C
4 °C
30 °C
4 °C
30 °C
20 ± 0.18 20 ± 0.012 20 ± 0.23 20 ± 0.01 20 ± 0.03 20 ± 0.09 20 ± 0.13 20 ± 0.19
20 ± 0.09 20 ± 0.06 20 ± 0.19 20 ± 0.24 20 ± 0.27 20 ± 0.01 20 ± 0.20 20 ± 0.09
20 ± 0.01 20 ± 0.05 20 ± 0.20 20 ± 0.18 19 ± 0.22 18 ± 0.01 18 ± 0.05 18 ± 0.03
20 ± 0.23 20 ± 0.04 20 ± 0.01 20 ± 0.36 20 ± 0.01 20 ± 0.09 20 ± 0.05 20 ± 0.01
20 ± 0.05 20 ± 0.01 20 ± 0.03 20 ± 0.18 18 ± 0.09 16 ± 0.17 16 ± 0.03 15 ± 0.09
20 ± 0.12 20 ± 0.00 20 ± 0.08 20 ± 0.07 20 ± 0.10 10 ± 0.00 10 ± 0.04 15 ± 0.28
20 ± 0.01 20 ± 0.09 20 ± 0.00 17 ± 0.00 20 ± 0.25 8 ± 0.01 11 ± 0.00 14 ± 0.00
19 ± 0.01 16 ± 0.04 10 ± 0.04 15 ± 0.07 20 ± 0.00 10 ± 0.00 10 ± 0.06 15 ± 0.05
10 ± 0.01 10 ± 0.00 10 ± 0.00 16 ± 0.05 20 ± 0.06 12 ± 0.00 11 ± 0.06 14 ± 0.00
22 ± 0.03 16 ± 0.06 12 ± 0.05 22 ± 0.01 21 ± 0.03 20 ± 0.06 19 ± 0.05 22 ± 0.18
0 ± 0.00 10 ± 0.03 0 ± 0.00 10 ± 0.03 20 ± 0.01 0 ± 0.00 0 ± 0.00 12 ± 0.00
Inhibition zone (mm). Standard deviation.
Table 2 Effect of nisin on the physicochemical characteristics of peach juice. Control treatments without nisin are also shown. Parameter
Treatment
pH
Control Nisin Control Nisin Control Nisin Control Nisin Control Nisin
Acidity (% citric acid) Vitamin C (% ascorbic acid) Browning index Solids soluble (°Brix) a
Storage daya 0
5
15
45
3.79 ± 0.02 aA 3.79 ± 0.02 aA 0.34 ± 0.01 aA 0.35 ± 0.00 aA 149.65 ± 0.80 aA 136.05 ± 0.66 aA 44.71 ± 0.60 bC 254.03 ± 22.0 aB 11.91 ± 0.10 bC 17.91 ± 0.10 aB
3.80 ± 0.04 aA 3.77 ± 0.02 aA 0.29 ± 0.00 bB 0.33 ± 0.00 aA 111.11 ± 0.01 aA 125.00 ± 0.46 aA 465.95 ± 0.15 aAB 465.95 ± 0.10 aAB 13.16 ± 0.10 bB 17.91 ± 0.27 aB
3.67 ± 0.06 aB 3.66 ± 0.02 aB 0.28 ± 0.01 bB 0.35 ± 0.01 aA 98.76 ± 0.12 aA 111.11 ± 0.18 aA 355.16 ± 0.10 bBC 676.40 ± 0.40 aA 13.25 ± 0.00 bB 19.66 ± 0.83 aA
3.66 ± 0.03 aB 3.63 ± 0.00 aB 0.30 ± 0.01 aB 0.32 ± 0.00 aA 39.68 ± 0.07 aB 39.68 ± 0.00 aB 736.44 ± 0.80 aA 451.46 ± 0.10 bAB 14.41 ± 0.20 bA 19.25 ± 0.00 aA
Means for the same parameter followed by the same letter, uppercase in line and lowercase in column, did not differ significantly by Tukey test at 5% probability.
index was lower at time 0 for samples with added nisin. However, there was an increase in this index during the first 5 days of storage and, from that point onwards, there was no statistically significant difference observed between samples, with or without added nisin (Table 5). After 45 days of storage, the soluble solids (°Brix) were consistently higher in samples supplemented with nisin (Tables 2, 3, 4, and 5).
3.3. Antimicrobial activity of nisin in fruit juices When A. acidoterrestris was inoculated in fruit juices, control treatment remained at 106 CFU/mL after 24 h of incubation in cashew and peach juices (Fig. 1a and c). In soursop and mango juices, 1-log increase in cell number was observed at this incubation time (Fig. 1b and d). In samples supplemented with nisin after 8 h of incubation, no viable cells were detected in cashew juices (Fig. 1a) and a 4-log reduction in viable cells was observed for soursop (Fig. 1b), peach (Fig. 1c), and mango
(Fig. 1d) juices. After 24 h of incubation in the presence of nisin, no viable cells were detected, independently of the juices (Fig. 1). With S. aureus, the concentration of viable cells in control treatments remained at least at 106 CFU/mL after 24 h of incubation in all tested juices (Fig. 2). For cashew (Fig. 2a), peach (Fig. 2c), and mango (Fig. 2d) juices, a 2-log reduction was observed after 8 h of incubation in samples supplemented with nisin. At 24 h of incubation in the presence of nisin, viable cells were only detected in mango juices (Fig. 2), representing a 4-log decrease as compared with the control treatment (Fig. 2d). Control treatments of B. cereus remained at least 105 CFU/mL after 24 h of incubation in fruit juices (Fig. 3). After 8 h of incubation in the presence of nisin, viable cell numbers showed a 4-log decrease in cashew and soursop juices (Fig. 3a and b). In peach and mango juices, the reduction was not greater than 1-log at this incubation time (Fig. 3c and d). The number of viable cells of B. cereus at 24 h of incubation in the presence of nisin represented at least a 4-log decrease compared to the control treatment (Fig. 3).
Table 3 Effect of nisin on the physicochemical characteristics of mango juice. Control treatments without nisin are also shown. Parameter
Treatment
Storage daysa 0
5
15
45
pH
Control Nisin Control Nisin Control Nisin Control Nisin Control Nisin
3.66 ± 0.07 aA 3.66 ± 0.07 aA 0.39 ± 0.00 aA 0.39 ± 0.00 aA 136.05 ± 0.66 bA 190.47 ± 0.66 aA 506.14 ± 0.90 aA 941.87 ± 0.40 aA 10.91 ± 0.10 bB 12.91 ± 0.10 aB
3.66 ± 0.01 aA 3.79 ± 0.05 aA 0.34 ± 0.02 aB 0.34 ± 0.01 aB 125.00 ± 0.46 aA 188.11 ± 0.01 bA 536.43 ± 0.80 aA 602.21 ± 0.60 aA 11.58 ± 0.10 bA 16.58 ± 0.20 aA
3.70 ± 0.05 aA 3.68 ± 0.01 aA 0.31 ± 0.00 bB 0.36 ± 0.01 aAB 123.45 ± 0.12 bA 188.14 ± 0.18 bA 339.97 ± 0.15 bA 656.00 ± 0.30 aA 11.75 ± 0.00 bA 16.25 ± 0.00 aA
3.63 ± 0.04 aA 3.60 ± 0.02 aA 0.33 ± 0.00 bB 0.36 ± 0.02 bAB 47.61 ± 0.83 aB 89.68 ± 0.72 bB 628.14 ± 0.90 aA 675.65 ± 0.30 aA 11.08 ± 0.10 bB 16.58 ± 0.10 aA
Acidity (% citric acid) Vitamin C (% ascorbic acid) Browning index Solids soluble (°Brix) a
Means for the same parameter followed by the same letter, uppercase in line and lowercase in column, did not differ significantly by Tukey test at 5% probability.
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Table 4 Effect of nisin on the physicochemical characteristics of cashew juice. Control treatments without nisin are also shown. Parameter
Treatment
pH
Control Nisin Control Nisin Control Nisin Control Nisin Control Nisin
Acidity (% citric acid) Vitamin C (% ascorbic acid) Browning index Solids soluble (°Brix) a
Storage daysa 0
5
15
45
3.72 ± 0.01 aB 3.72 ± 0.01 aA 0.31 ± 0.01 aA 0.30 ± 0.00 aA 408.16 ± 0.86 aA 421.76 ± 0.66 aA 201.01 ± 0.40 aAB 126.51 ± 0.30 aA 11.83 ± 0.10 bA 16.66 ± 0.10 aA
3.73 ± 0.03 aB 3.68 ± 0.06 aA 0.27 ± 0.00 aB 0.2842 ± 0.00 aAB 402.77 ± 0.10 aAB 375.00 ± 0.46 bAB 122.05 ± 0.10 aBC 98.94 ± 0.30 aA 11.83 ± 0.10 bA 16.83 ± 0.44 aA
3.89 ± 0.01 aA 3.73 ± 0.01 bA 0.23 ± 0.01 bC 0.2662 ± 0.00 aB 358.02 ± 0.12 aB 333.33 ± 0.18 bB 72.70 ± 0.20 aC 102.73 ± 0.80 aA 12.00 ± 0.10 bA 16.75 ± 0.63 aA
3.58 ± 0.01 aC 3.61 ± 0.00 aA 0.25 ± 0.00 aBC 0.27 ± 0,00 aB 71.42 ± 0.00 aC 125.23 ± 0.00 bC 296.54 ± 0.70 aA 126.68 ± 0.30 bA 11.91 ± 0.27 bA 15.91 ± 0.10 aB
Means for the same parameter followed by the same letter, uppercase in line and lowercase in column, did not differ significantly by Tukey test at 5% probability.
Table 5 Effect of nisin on the physicochemical characteristics of soursop juice. Control treatments without nisin are also shown. Parameter
Treatment
Storage daysa 0
5
15
45
pH
Control Nisin Control Nisin Control Nisin Control Nisin Control Nisin
3.45 ± 0.02 aB 3.45 ± 0.02 aAB 0.45 ± 0.01 bA 0.49 ± 0.00 aA 163.26 ± 0.86 aA 108.84 ± 0.66 bA 206.42 ± 0.10 aAB 77.42 ± 0.10 bB 12.41 ± 0.10 bC 19.16 ± 0.10 aA
3.52 ± 0.01 aA 3.49 ± 0.04 aA 0.34 ± 0.00 bB 0.39 ± 0.00 aB 138.88 ± 0.10 aAB 138.88 ± 0.01 aAB 209.34 ± 0.30 aA 210.34 ± 0.20 aA 12.83 ± 0.20 bB 18.00 ± 0.17 aBC
3.49 ± 0.02 aAB 3.42 ± 0.03 bB 0.36 ± 0.02 bB 0.39 ± 0.00 aB 123.45 ± 0.12 aB 111.11 ± 0.00 aAB 99.80 ± 0.30 aC 129.54 ± 0.60 aAB 13.25 ± 0.00 bA 18.25 ± 0.00 aB
3.44 ± 0.02 aB 3.44 ± 0.00 aAB 0.36 ± 0.00 aB 0.36 ± 0.03 aB 47.61 ± 0.00 aC 47.61 ± 0.00 aC 104.14 ± 0.10 aBC 121.00 ± 0.80 aAB 12.50 ± 0.00 bBC 17.83 ± 0.10 aC
Acidity (% citric acid) Vitamin C (% ascorbic acid) Browning index Solids soluble (°Brix)
Means for the same parameter followed by the same letter, uppercase in line and lowercase in column, did not differ significantly by Tukey test at 5% probability.
4. Discussion To consolidate the approval and/or the use of nisin as a preservative in fruit juices, more studies are needed to show its effectiveness in different fruit juices against pathogenic and spoilage microorganisms. To date, only four studies have shown the effects of this bacteriocin on the preservation of fruit products. Barbosa et al. (2013) studied the incorporation of nisin in acetate films to package minimally processed mangoes. Walker and Phillips (2006) and Pathanibul et al. (2009) demonstrated the ability of nisin to control Listeria innocua and A. acidoterrestris, respectively, in apple juice. Yu et al. (2013) verified the effect of nisin on the inactivation of indigenous microorganisms in litchi juice. In this study, we showed that nisin could inhibit S. aureus, A. acidoterrestris, L. monocytogenes, and B. cereus strains in fruit juices. The application of bacteriocins as preservatives in fruit juices can be limited by their properties such as the solubility of the peptide, the interaction of bacteriocins with juices components, juice pH, and inactivation by proteases (Ganzle et al., 1999; Pei et al., 2013). Therefore, the stability of its antimicrobial activity is an essential characteristic for a bacteriocin to be used in the conservation of fruit juices. Our results showed that nisin remained stable in different fruit juices for at least 30 days of storage at room or refrigerated temperature, and that 24 h of incubation with nisin was enough to eliminate up to 107 CFU/mL of inoculated microorganisms. These results indicated that nisin could remain stable in fruit juices to ensure the microbiological safety of the product.
However, this study showed that the effect of the antimicrobial activity of nisin was heterogeneous and depended on both the type of juice and microorganism. The most sensitive microorganism to nisin
Viable cell number (Log CFU/mL)
When the antimicrobial activity of nisin was tested against L. monocytogenes in cashew and soursop juices, no reduction in the viable cell number was observed compared to the control treatment after 24 h of incubation (Fig. 4a and b). Viable cells were four and six times less than in the control treatment, in peach and mango juices respectively (Fig. 4c and d). Control treatment remained at least 106 CFU/mL after 24 h of incubation (Fig. 4).
10
a
10
8
8
6
6
4
4
2
2
b
0
0 0
Viable cell number (Log CFU/mL)
a
8
0
24
10
c
10
8
8
6
6
4
4
2
2
0
8
24
d
0 0
8 24 Incubation time (h)
0
8 24 Incubation time (h)
Fig. 1. Antimicrobial activity of nisin against A. acidoterrestris DSMZ2498 in cashew (a), soursop (b), peach (c), and mango (d) juices. Nisin was added at 5000 IU/mL (black bars) or not added in control treatments (white bars). A. acidoterrestris was inoculated at approximately 106 CFU/mL and viable cells were counted after 0, 8, and 24 h of incubation.
10
a
10
b
8
8
6
6
4
4
2
2
0 10
0 0
8
24
10
0
8
8
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0 0
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8 24 Incubation time (h)
8 24 Incubation time (h)
a
10
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2
2
Viable cell number (Log CFU/mL)
Viable cell number (Log CFU/mL)
was A. acidoterrestris and the least sensitive was L. monocytogenes. Still, a reduction of up to 90% of viable cells was observed in peach and mango juices inoculated with L. monocytogenes after 24 h of incubation. Moreover, the microbial load used in our experiment was higher than would normally occur in real situations of contamination. The maximum microbial load expected to be found in real-life situations would be 102 or 103 viable cells per mL of juice or food (Burnett and Beuchat, 2001; Rosenquist et al., 2005; Vantarakis et al., 2011).
10
10
a
b
8
8
6
6
4
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2
2
0 10
0
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24
0 10
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c
d
8
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24
0
0
8 24 Incubation time (h)
0 8 Incubation time (h)
24
Fig. 3. Antimicrobial activity of nisin against B. cereus ATCC4504 in cashew (a), soursop (b), peach (c), and mango (d) juices. Nisin was added at 5000 IU/mL (black bars) or not added in control treatments (white bars). B. cereus was inoculated at approximately 106 CFU/mL and viable cells were counted after 0, 8, and 24 h of incubation.
b
0
0 0
8
0
24
c
10
10
8
8
6
6
4
4
2
2
8
24
d
0
0 0
Fig. 2. Antimicrobial activity of nisin against S. aureus ATCC8095 in cashew (a), soursop (b), peach (c), and mango (d) juices. Nisin was added at 5000 IU/mL (black bars) or not added in control treatments (white bars). S. aureus was inoculated at approximately 106 CFU/mL and viable cells were counted after 0, 8, and 24 h of incubation.
10
8
24
d
c 8
Viable cell number (log CFU/mL)
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Viable cell number (log CFU/mL)
Viable cell number (Log CFU/mL)
Viable cell number (Log CFU/mL)
42
8 24 Incubation time (h)
0
8 24 Incubation time (h)
Fig. 4. Antimicrobial activity of nisin against L. monocytogenes ATCC7644 in cashew (a), soursop (b), peach (c) and mango (d) juices. Nisin was added at 5000 IU/mL (black bars) or not added in control treatments (white bars). B. cereus was inoculated at approximately 106 CFU/mL and viable cells were counted after 0, 8, and 24 h of incubation.
Cashew juice with added nisin caused a rapid (in the first 8 h) decrease in the viability of A. acidoterrestris, S. aureus, and B. cereus. After 24 h of incubation, the antimicrobial activity of nisin for B. cereus and S. aureus was lower in mango juice, whereas the reverse was observed for L. monocytogenes. Pei et al. (2013) and Yamazaki et al. (2000) also reported heterogeneous effects of the antimicrobial activity of bacteriocins in different fruit juices and attributed these to the combination of bacteriocins and polyphenols found in the juices, although the exact factors were not completely defined. It is well known that fruits such as cashew, soursop, peach, and mango contain divalent ions, such as calcium and magnesium (Soares et al., 2004). These divalent cations have also been reported to decrease bacteriocin activity (Davies et al., 1996; Houlihan and Russel, 2006; Kumar and Srivastava, 2011). For L. monocytogenes, Davies et al. (1996) reported that nisin resistance was associated with binding of divalent metals by ribitol and glycerol phosphates of lipoteichoic acids on the cell surface. Therefore, the heterogeneous activity of nisin in fruit juices could be explained by the intrinsic properties of the juices and also by features of the microorganisms. Bacteriocins are well known to not cause any changes in the organoleptic characteristics of foods (Cleveland et al., 2001; Deegan et al., 2006), but to date, the studies showing the effect of nisin in physicochemical properties of foods are limited. The physico-chemical analyses performed in this study also showed that nisin did not cause any significant alterations in the physico-chemical characteristics of the fruit juices tested. The properties of the juices supplemented or not with nisin were unchanged, except the solid soluble content that was always higher with nisin. This result was expected as a solute (nisin) was added to these samples. Moreover, the addition of nisin favored the preservation of the vitamin C content in all the juices tested, which is a desirable feature. Thus, nisin can ensure the microbiological safety of juices without causing significant negative changes in the product. Despite these results, further studies are needed to see consumer acceptability of juices with added nisin. The initial concentration of nisin could be adjusted according to its antimicrobial effects and its sensorial properties that affect consumer acceptability.
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Considering the risk of pathogenic microorganisms to contaminate fruit juices, the United States Food and Drug Administration (FDA) imposed Hazard Analysis and Critical Control Point (HACCP) requirements on juice processors. The HACCP require that processors of fruit juices achieve a 5-log reduction of a target pathogen (FDA, 2001). Our results showed that nisin achieved a 5-log reduction of A. acidoterrestris, S. aureus, and B. cereus after 24 h of incubation in all juices tested. A similar result was observed for L. monocytogenes in peach and mango juices only. These results show that nisin reaches the requirements established by the FDA. In conclusion, in this study we state that nisin remained stable in different fruit juices without causing changes in organoleptic characteristics and showed antimicrobial activity against spoilage and pathogenic microorganisms. The application of this bacteriocin together with a mild heat treatment or other antimicrobial agent can be an alternative in the processing of fruit juices. Acknowledgments The authors would like to thank the Conselho de Desenvolvimento Científico e Tecnológico (CNPq), Brasília, Brazil and the Fundação de Apoio à Pesquisa e à inovação Tecnológica do Estado de Sergipe (FAPITEC), Sergipe, Brazil, (FAPITEC/SE /FUNTEC/CNPq N° 04/2011), for providing fellow ships and financial support for this research. References Barbosa, A.A.T., Silva de Araújo, H.G., Matos, P.N., Carnelossi, M.A.G., Castro, A.A., 2013. Effects of nisin-incorporated films on the microbiological and physicochemical quality of minimally processed mangoes. Int. J. Food Microbiol. 164, 135–140. Bi, X., Liu, F., Rao, L., Li, J., Liu, B., Liao, X., Wu, J., 2013. Effects of electric field strength and pulse rise time on physicochemical and sensory properties of apple juice by pulsed electric field. Innovative Food Sci. Emerg. Technol. 17, 85–92. Braddock, R.J., 1999. Handbook of Citrus By-Products and Processing Technology. John Wiley and Sons, Inc., New York, NY, USA. Burnett, S.L., Beuchat, L.R., 2001. Human pathogens associated with raw produce and unpasteurized juices, and difficulties in decontamination. J. Ind. Microbiol. Biotechnol. 27, 104–110. Carnelossi, M.A.G., 2000. Fisiologia pós-colheita de folhas de couve (Brassicaoleracea cv. Acephala) minimamente processadas Doctoral Thesis, Universidade Federal de Viçosa, Brazil, Departamento de Microbiologia (83 pp.). Carvalho, A.A.T., Costa, E.D., Mantovani, H.C., Vanetti, M.C.D., 2007. Effect of bovicin HC5 on growth and spore germination of Bacillus cereus and Bacillus thuringiensis isolated from spoiled mango pulp. J. Appl. Microbiol. 102, 1000–1009. Carvalho, A.A.T., Vanetti, M.C.D., Mantovani, H.C., 2008. Bovicin HC5 reduces thermal resistance of Alicyclobacillusacidoterrestris in acidic mango pulp. J. Appl. Microbiol. 104, 1685–1691. Cleveland, J., Montville, T.J., Nes, I.F., Chikinda, M.L., 2001. Bacteriocins: safe, natural antimicrobials for food preservation. Int. J. Food Microbiol. 71, 1–20. Cotter, P.D., Hill, C., Ross, P., 2005. Bacteriocins: developing innate immunity for food. Nat. Rev. 3, 777–788. Davies, E.A., Falahee, M.B., Adams, M.R., 1996. Involvement of the cell envelope in Listeria monocytogenes in the acquisition of nisin resistance. J. Appl. Bacteriol. 81, 139–146. Deegan, L.H., Cotter, P.D., Hill, C., Ross, P., 2006. Bacteriocins: biological tools for biopreservation and shelf-life extension. Int. Dairy J. 16, 1058–1071. Espina, L., Somolinos, M., Ouazzou, A.A., Condón, S., García-Gonzalo, D., Pagán, R., 2012. Inactivation of Escherichia coli O157:H7 in fruit juices by combined treatments of citrus fruit essential oils and heat. Int. J. Food Microbiol. 159, 9–16. Espina, L., García-Gonzalo, D., Laglaoui, A., Mackey, B.M., Pagán, R., 2013. Synergistic combinations of high hydrostatic pressure and essential oils or their constituents and their use in preservation of fruit juices. Int. J. Food Microbiol. 161, 23–30.
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FDA, 2001. Hazard analysis and critical control point (HACCP); procedures for the safe and sanitary processing and importing of juice: final rule (21 CFR Part 120). Fed. Regist. vol. 66. US Food and Drug Administration, Washington, D.C., pp. 6137–6202 Ganzle, M.G., Weber, S., Hammes, W.P., 1999. Effect of ecological factors on the inhibitory spectrum and activity of bacteriocins. Int. J. Food Microbiol. 46, 207–217. Grande, M.J., Lucas, R., Abriouel, H., Ben Omar, N., Maqueda, M., Martínez-Bueno, M., Martínez-Cañamero, M., Valdivia, E., Gálvez, A., 2005. Control of Alicyclobacillusacidoterrestris in fruit juices by enterocin AS-48. Int. J. Food Microbiol. 104, 289–297. Houlihan, A.J., Russel, J.B., 2006. The effect of calcium and magnesium on the activity of bovicin HC5 and nisin. Curr. Microbiol. 365–369. Jiménez-Sánchez, C., Lozano-Sánchez, J., Segura-Carretero, A., Fernández-Gutiérrez, A., 2015. Alternatives to conventional thermal treatments in fruit-juice processing. Part 2: effect on composition, phytochemical content, and physicochemical, rheological, and organoleptic properties of fruit juices. Crit. Rev. Food Sci. Nutr. http://dx.doi. org/10.1080/10408398.2014.914019 (in press). Komitopoulou, E., Boziaris, I.S., Davies, E.A., Delves-Broughton, J., Adam, M.R., 1999. Alicyclobacillusacidoterrestris in fruit juices and its control by nisin. Int. J. Food Sci. Technol. 34, 81–85. Kumar, M., Srivastava, S., 2011. Effect of calcium and magnesium on the antimicrobial action of enterocin LR/6 produced by Enterococcus faecium LR/6. Int. J. Antimicrob. Agents 572–575. Li, H., Zhao, L., Wu, J., Zhang, Y., Liao, X., 2012. Inactivation of natural microorganisms in litchi juice by high-pressure carbon dioxide combined with mild heat and nisin. Food Microbiol. 30, 139–145. Mutaku, I., Erku, W., Ashenafi, M., 2005. Growth and survival of Escherichia coli O157:H7 in fresh tropical fruit juices at ambient and cold temperatures. Int. J. Food Sci. 56, 133–139. Oteiza, J.M., Soto, S., Alvarenga, V.O., Sant'Ana, A.S., Giannuzzi, L., 2014. Flavorings as new sources of contamination by deteriogenic Alicyclobacillus of fruit juices and beverages. Int. J. Food Microbiol. 172, 119–124. Pathanibul, P., Taylor, T.M., Davidson, P.M., Harte, F., 2009. Inactivation of Escherichia coli and Listeria innocua in apple and carrot juices using high-pressure homogenization and nisin. Int. J. Food Microbiol. 28, 316–320. Pei, J., Yuan, Y., Yue, T., 2013. Control of Alicyclobacillus acidoterrestris in fruit juices by a newly discovered bacteriocin. World J. Microbiol. Biotechnol. 30, 855–863. Rosenquist, H., Smidr, L., Andersen, S.R., Jensen, G.B., Wilcks, A., 2005. Occurrence and significance of Bacillus cereus and Bacillus thuringiensis in ready-to-eat food. FEMS Microbiol. Lett. 250, 129–136. Soares, L.M.V., Shishido, K., Moraes, A.M.M., Moreira, V.A., 2004. Composição mineral de sucos concentrados de frutas brasileiras. Cienc. Tecnol. Aliment. Campinas 24, 202–206. Statistical Analysis Systems, 2004. User's Guide: Stat. Version 9. SAS Institute, Cary. Tagg, J.R., Dajani, A.S., Wannamaker, L.W., 1976. Bacteriocins of gram-positive bacteria. Bacteriol. Rev. 40, 722–756. Timmermans, R.A.H., NieropGroot, M.N., Nederhoff, A.L., van Boekel, M.A.J.S., Matser, A.M., Mastwijk, H.C., 2014. Pulsed electric field processing of different fruit juices: impact of pH and temperature on inactivation of spoilage and pathogenic micro-organisms. Int. J. Food Microbiol. 173, 105–111. Torlak, E., 2014. Efficacy of ozone against Alicyclobacillus acidoterrestris spores in apple juice. Int. J. Food Microbiol. 172, 1–4. Vantarakis, A., Affifi, M., Kokkinos, P., Tsibouxi, M., Papapetropoulou, M., 2011. Occurrence of microorganisms of public health and spoilage significance in fruit juices sold in retail markets in Greece. Anaerobe 17, 288–291. Walker, M., Phillips, C.A., 2006. The effect of preservatives on Alicyclobacillusacidoterrestris and Propionibacteriumcyclohexanicum in fruit juice. Food Control 19, 974–981. Yamazaki, K., Murakami, M., Kawai, Y., Inoue, N., Matsuda, T., 2000. Use of nisin for inhibition of Alicyclobacillus acidoterrestris in acidic drinks. Int. J. Food Microbiol. 17, 315–320. Yu, Y., Wu, J., Xiao, G., Xu, Y., Tang, D., Chen, Y., Zhang, Y., 2013. Combined effect of dimethyl dicarbonate (DMDC) and nisin on indigenous microorganisms of litchi juice and its microbial shelf life. J. Food Sci. 78, 1236–1241. Zhang, J., Yue, T., Yuan, Y., 2013. Alicyclobacillus contamination in the production line of kiwi products in China. PLoS One 8 (7), e67704.
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Stability, antimicrobial activity, and effect of nisin on the physico-chemical properties of fruit juices Adelson Alves de Oliveira Junior a, Hyrla Grazielle Silva de Araújo Couto b, Ana Andréa Teixeira Barbosa c,⁎, Marcelo Augusto Guitierrez Carnelossi b, Tatiana Rodrigues de Moura c a b c
Departamento de Nutrição,Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brazil Departamento de Tecnologia de Alimentos, Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brazil Departamento de Morfologia, Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brazil
a r t i c l e
i n f o
Article history: Received 29 January 2015 Received in revised form 23 June 2015 Accepted 26 June 2015 Available online 2 July 2015 Keywords: Bacteriocin Food preservation Microbial inactivation Listeria monocytogenes Alicyclobacillus acidoterrestris
a b s t r a c t Heat processing is the most commonly used hurdle for inactivating microorganisms in fruit juices. However, this preservation method could interfere with the organoleptic characteristics of the product. Alternative methods have been proposed and bacteriocins such as nisin are potential candidates. However, the approval of bacteriocins as food additives is limited, especially in foods from vegetal origin. We aimed to verify the stability, the effect on physico-chemical properties, and the antimicrobial activity of nisin in different fruit juices. Nisin remained stable in fruit juices (cashew, soursop, peach, mango, passion fruit, orange, guava, and cupuassu) for at least 30 days at room or refrigerated temperature and did not cause any significant alterations in the physico-chemical characteristics of the juices. Besides, nisin favored the preservation of vitamin C content in juices. The antimicrobial activity of nisin was tested against Alicyclobacillus acidoterrestris, Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes in cashew, soursop, peach, and mango juices. Nisin caused a 4-log reduction in viable cells of A. acidoterrestris in soursop, peach, and mango juices after 8 h of incubation, and no viable cells were detected in cashew juices. After 24 h of incubation in the presence of nisin, no viable cells were detected, independently of the juices. To S. aureus, at 24 h of incubation in the presence of nisin, viable cells were only detected in mango juices, representing a 4-log decrease as compared with the control treatment. The number of viable cells of B. cereus at 24 h of incubation in the presence of nisin represented at least a 4-log decrease compared to the control treatment. When the antimicrobial activity of nisin was tested against L. monocytogenes in cashew and soursop juices, no reduction in the viable cell number was observed compared to the control treatment after 24 h of incubation. Viable cells were four and six times less than in the control treatment, in peach and mango juices respectively. The most sensitive microorganism to nisin was A. acidoterrestris and the least sensitive was L. monocytogenes. Still, a reduction of up to 90% of viable cells was observed in peach and mango juices inoculated with L. monocytogenes. These results indicate that the use of nisin could be an alternative in fruit juice processing. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The most commonly used hurdle for inactivating microorganisms and enzymes in fruit juices, and thereby extending the product shelf life, is heat processing (Braddock, 1999; Jiménez-Sánchez et al., 2015). However, it is well known that the organoleptic characteristics of the product may be altered by this processing method. This leads to an increased interest in innovative non-thermal processing technologies, which can enhance the shelf life of high quality foods, with reduced or no application of heat, and without affecting the quality and safety of ⁎ Corresponding author at: Departamento de Morfologia Universidade Federal de Sergipe Cidade Universitária Prof. José Aloísio de Campos Jardim Rosa Elze São Cristóvão, SE 49100–000, Brazil. E-mail address: [email protected] (A.A.T. Barbosa).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.06.029 0168-1605/© 2015 Elsevier B.V. All rights reserved.
the product (Bi et al., 2013; Jiménez-Sánchez et al., 2015; Timmermans et al., 2014). A considerable number of studies have been conducted showing the application of pulsed electric field (Bi et al., 2013; Timmermans et al., 2014), high hydrostatic pressures (Espina et al., 2013), essential oils (Espina et al., 2012), ozone (Torlak, 2014), and bacteriocins from lactic acid bacteria (Carvalho et al., 2008; Grande et al., 2005; Komitopoulou et al., 1999; Pei et al., 2013) in the preservation of fruit juices. Amongst these, the use of bacteriocins from lactic acid bacteria is a promising alternative. The application of bacteriocins in food conservation is widespread; however, little is known about its use in fruit products (Grande et al., 2005; Pei et al., 2013). Bacteriocins previously tested in fruit juices are nisin (Komitopoulou et al., 1999), enterocin AS48 (Grande et al., 2005), bificin C6564 (Pei et al., 2013), and bovicin HC5 (Carvalho
A.A. de Oliveira Junior et al. / International Journal of Food Microbiology 211 (2015) 38–43
et al., 2008). Amongst these, only nisin is approved as food additive, but its approval in fruit products is limited. Nisin is an antimicrobial peptide that has an antimicrobial activity against several food-borne spoilage and pathogenic microorganisms, including Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, and Alicyclobacillus acidoterrestris (Cleveland et al., 2001; Cotter et al., 2005; Deegan et al., 2006; Komitopoulou et al., 1999). Microorganisms such as L. monocytogenes have been reported to be capable of surviving in raw fruit and vegetable juices (Barbosa et al., 2013; Burnett and Beuchat, 2001; Mutaku et al., 2005), and A. acidoterrestris is increasingly being reported as a cause of spoilage of fruit juices (Oteiza et al., 2014; Zhang et al., 2013). B. cereus and S. aureus have also been isolated from fruit products (Carvalho et al., 2007; Vantarakis et al., 2011). Nisin could therefore be a promising bacteriocin for use in controlling the growth of these microorganisms in fruit products. The knowledge on nisin application in fruit juices is still limited. Some studies only showed the effect of nisin against A. acidoterrestris in apple and orange juices (Pathanibul et al., 2009; Walker and Phillips, 2006), and against indigenous microorganisms in litchi juice (Li et al., 2012; Yu et al., 2013). In these studies, nisin stability and its effects on the physico-chemical properties of these juices were not reported. In this study, we aimed to verify the stability of nisin and its effect on the physico-chemical properties of different fruit juices, and evaluate its antimicrobial activity against A. acidoterrestris, L. monocytogenes, B. cereus and S. aureus in cashew, soursop, peach, and mango juices to confirm the potential of nisin in the conservation of fruit juices.
39
2.4. Antimicrobial activity of nisin in fruit juices The antimicrobial activity of nisin was tested against L. monocytogenes, S. aureus, B. cereus, and A. acidoterrestris in cashew, soursop, peach, and mango juices. Approximately 106 CFU/mL (100 μL of a 12 h culture) of the microorganisms were inoculated in fruit juices that were supplemented with 5000 IU/mL nisin. The fruit juices were incubated at microorganism growth temperature, therefore to S. aureus ATCC 8095, B. cereus ATCC 4504, and L. monocytogenes ATCC 7644 the juices were incubated at 37 °C and to A. acidoterrestris DSMZ 2498 at 40 °C. After 0, 8, and 24 h of incubation, samples were withdrawn, diluted (in tenfold increments) and viable cell numbers were determined by plate counting. The cell number was calculated considering the following: CFU/mL = CFU × DF/aliquot volume, where CFU = colony-forming units, DF = dilution factor (reciprocal of the dilution), and aliquot volume = sample volume plated (in mL). Controls without the bacteriocin nisin were also performed. 2.5. Statistical analysis All treatments were repeated three times and each repetition was conducted in triplicate. For physico-chemical analysis, the results obtained were submitted to analysis of variance (ANOVA) at 5% of probability, and means were compared with Tukey's test using the SAS statistical software (Statistical Analysis Systems, 2004). In the figures, data and error bars represent arithmetic mean values and standard deviation, respectively.
2. Material and methods
3. Results
2.1. Microorganisms and growth conditions
3.1. Stability of nisin in different fruit juices
S. aureus ATCC 8095, B. cereus ATCC 4504, and L. monocytogenes ATCC 7644 were grown in BHI media (HIMDEDIA, Mumbai, India) at 37 °C. A. acidoterrestris DSMZ 2498 was grown at 40 °C in AAM (A. acidoterrestris media) according to methods described by Yamazaki et al. (2000). The indicator strain Lactococcus lactis ATCC 19435 was cultivated in MRS media (HIMDEDIA, Mumbai, India) at 30 °C.
The antimicrobial activity of nisin was detected in all fruit juices for at least 30 days of storage, independently of the incubation temperature (Table 1). If the storage time was extended to 70 days, nisin remained stable in fruit juices at 4 °C, independently of the juice (Table 1). If the storage temperature was increased to 30 °C, the antimicrobial activity of nisin was detected in passion fruit, orange, soursop, and cupuassu juices for at least 70 days. In cashew, mango, peach, and guava juices, no residual activity of nisin was detected at 70 days of storage at this temperature (Table 1). Fruit juices that have not been supplemented with nisin (control treatment) did not show any antimicrobial activity (data not shown).
2.2. Stability of nisin in different fruit juices The commercial fruit juices used in this study were purchased from local stores in Aracaju city, Sergipe state, Brazil. The juices tested for nisin stability were cashew, soursop, peach, mango, passion fruit, orange, guava, and cupuassu. The stability of nisin in fruit juices was tested by adding bacteriocin at a final concentration of 5000 IU/mL. After 0, 4, 8, 12, 30, and 70 days of incubation at room temperature (30 °C ± 2) or at 4 °C, samples were withdrawn and tested for antimicrobial activity by the well diffusion assay described by Tagg et al. (1976). The assay was performed using L. lactis ATCC 19435 as the indicator strain. Approximately, 106 CFU/mL of the microorganisms was inoculated in solid media and dispensed into Petri dishes. A well of approximately 5 mm diameter was created in the center of the dish and 25 μL of the sample was added in the well. The dishes were incubated at 4 °C for 12 h for sample diffusion and at 30 °C for microorganism growth. After 24 h, the inhibition zones formed were analyzed. A control treatment of juice without nisin was also included. 2.3. Effect of nisin on the physico-chemical properties of fruit juices Peach, mango, cashew, and soursop juices were supplemented with nisin at 5000 IU/mL and stored at 4 °C. After 0, 5, 15, and 45 days of storage, samples were withdrawn and the soluble solids (expressed in °Brix), total titratable acidity, pH level, vitamin C content, and browning index were evaluated (Carnelossi, 2000). Controls without nisin were also performed.
3.2. Effect of nisin on the physico-chemical properties of fruit juices When fruit juices were stored at 4 °C with or without nisin, no significant differences (at 5% by Tukey's test) in pH or total titratable acidity were observed after 45 days of storage (Tables 2, 3, 4, and 5). Regarding the vitamin C content, a reduction was observed in all the juices, with or without nisin, after 45 days of storage. However, the reduction was always higher in control juice samples without nisin (Tables 2, 3, 4, and 5). For peach juice, the reduction in vitamin C content after this storage time was 70% and 73%, respectively, with and without nisin (Table 2). For mango juice, the reduction was 75% in control samples and 53% in samples with added nisin (Table 3). In cashew juices, the samples with added nisin showed a reduction of 70%, and in control samples, the reduction was 82% (Table 3). When the soursop juice was tested, the decrease in vitamin C content was found to be 70% in the control sample and 56% in samples with added nisin, after 45 days of storage (Table 5). When the browning index was analyzed after 45 days of storage, the index was higher in peach and cashew juices stored without nisin (Tables 2 and 4). In mango and soursop juices, no significant differences were observed in the browning index for samples with or without nisin at this same storage day (Tables 3 and 5). In soursop juice, the browning
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Table 1 Nisin stability in different fruit juices at 4 °C and at 30 °C. Juice
Incubation days 0a
4a
4 °C Cashew Passion fruit Mango Orange Soursop Peach Guava Cupuassu a b
20 ± 0.01 20 ± 0.08 20 ± 0.01 20 ± 0.03 20 ± 0.05 20 ± 0.1 20 ± 0.07 20 ± 0.14
b
8a
12a
30a
70a
30 °C
4 °C
30 °C
4 °C
30 °C
4 °C
30 °C
4 °C
30 °C
4 °C
30 °C
20 ± 0.18 20 ± 0.012 20 ± 0.23 20 ± 0.01 20 ± 0.03 20 ± 0.09 20 ± 0.13 20 ± 0.19
20 ± 0.09 20 ± 0.06 20 ± 0.19 20 ± 0.24 20 ± 0.27 20 ± 0.01 20 ± 0.20 20 ± 0.09
20 ± 0.01 20 ± 0.05 20 ± 0.20 20 ± 0.18 19 ± 0.22 18 ± 0.01 18 ± 0.05 18 ± 0.03
20 ± 0.23 20 ± 0.04 20 ± 0.01 20 ± 0.36 20 ± 0.01 20 ± 0.09 20 ± 0.05 20 ± 0.01
20 ± 0.05 20 ± 0.01 20 ± 0.03 20 ± 0.18 18 ± 0.09 16 ± 0.17 16 ± 0.03 15 ± 0.09
20 ± 0.12 20 ± 0.00 20 ± 0.08 20 ± 0.07 20 ± 0.10 10 ± 0.00 10 ± 0.04 15 ± 0.28
20 ± 0.01 20 ± 0.09 20 ± 0.00 17 ± 0.00 20 ± 0.25 8 ± 0.01 11 ± 0.00 14 ± 0.00
19 ± 0.01 16 ± 0.04 10 ± 0.04 15 ± 0.07 20 ± 0.00 10 ± 0.00 10 ± 0.06 15 ± 0.05
10 ± 0.01 10 ± 0.00 10 ± 0.00 16 ± 0.05 20 ± 0.06 12 ± 0.00 11 ± 0.06 14 ± 0.00
22 ± 0.03 16 ± 0.06 12 ± 0.05 22 ± 0.01 21 ± 0.03 20 ± 0.06 19 ± 0.05 22 ± 0.18
0 ± 0.00 10 ± 0.03 0 ± 0.00 10 ± 0.03 20 ± 0.01 0 ± 0.00 0 ± 0.00 12 ± 0.00
Inhibition zone (mm). Standard deviation.
Table 2 Effect of nisin on the physicochemical characteristics of peach juice. Control treatments without nisin are also shown. Parameter
Treatment
pH
Control Nisin Control Nisin Control Nisin Control Nisin Control Nisin
Acidity (% citric acid) Vitamin C (% ascorbic acid) Browning index Solids soluble (°Brix) a
Storage daya 0
5
15
45
3.79 ± 0.02 aA 3.79 ± 0.02 aA 0.34 ± 0.01 aA 0.35 ± 0.00 aA 149.65 ± 0.80 aA 136.05 ± 0.66 aA 44.71 ± 0.60 bC 254.03 ± 22.0 aB 11.91 ± 0.10 bC 17.91 ± 0.10 aB
3.80 ± 0.04 aA 3.77 ± 0.02 aA 0.29 ± 0.00 bB 0.33 ± 0.00 aA 111.11 ± 0.01 aA 125.00 ± 0.46 aA 465.95 ± 0.15 aAB 465.95 ± 0.10 aAB 13.16 ± 0.10 bB 17.91 ± 0.27 aB
3.67 ± 0.06 aB 3.66 ± 0.02 aB 0.28 ± 0.01 bB 0.35 ± 0.01 aA 98.76 ± 0.12 aA 111.11 ± 0.18 aA 355.16 ± 0.10 bBC 676.40 ± 0.40 aA 13.25 ± 0.00 bB 19.66 ± 0.83 aA
3.66 ± 0.03 aB 3.63 ± 0.00 aB 0.30 ± 0.01 aB 0.32 ± 0.00 aA 39.68 ± 0.07 aB 39.68 ± 0.00 aB 736.44 ± 0.80 aA 451.46 ± 0.10 bAB 14.41 ± 0.20 bA 19.25 ± 0.00 aA
Means for the same parameter followed by the same letter, uppercase in line and lowercase in column, did not differ significantly by Tukey test at 5% probability.
index was lower at time 0 for samples with added nisin. However, there was an increase in this index during the first 5 days of storage and, from that point onwards, there was no statistically significant difference observed between samples, with or without added nisin (Table 5). After 45 days of storage, the soluble solids (°Brix) were consistently higher in samples supplemented with nisin (Tables 2, 3, 4, and 5).
3.3. Antimicrobial activity of nisin in fruit juices When A. acidoterrestris was inoculated in fruit juices, control treatment remained at 106 CFU/mL after 24 h of incubation in cashew and peach juices (Fig. 1a and c). In soursop and mango juices, 1-log increase in cell number was observed at this incubation time (Fig. 1b and d). In samples supplemented with nisin after 8 h of incubation, no viable cells were detected in cashew juices (Fig. 1a) and a 4-log reduction in viable cells was observed for soursop (Fig. 1b), peach (Fig. 1c), and mango
(Fig. 1d) juices. After 24 h of incubation in the presence of nisin, no viable cells were detected, independently of the juices (Fig. 1). With S. aureus, the concentration of viable cells in control treatments remained at least at 106 CFU/mL after 24 h of incubation in all tested juices (Fig. 2). For cashew (Fig. 2a), peach (Fig. 2c), and mango (Fig. 2d) juices, a 2-log reduction was observed after 8 h of incubation in samples supplemented with nisin. At 24 h of incubation in the presence of nisin, viable cells were only detected in mango juices (Fig. 2), representing a 4-log decrease as compared with the control treatment (Fig. 2d). Control treatments of B. cereus remained at least 105 CFU/mL after 24 h of incubation in fruit juices (Fig. 3). After 8 h of incubation in the presence of nisin, viable cell numbers showed a 4-log decrease in cashew and soursop juices (Fig. 3a and b). In peach and mango juices, the reduction was not greater than 1-log at this incubation time (Fig. 3c and d). The number of viable cells of B. cereus at 24 h of incubation in the presence of nisin represented at least a 4-log decrease compared to the control treatment (Fig. 3).
Table 3 Effect of nisin on the physicochemical characteristics of mango juice. Control treatments without nisin are also shown. Parameter
Treatment
Storage daysa 0
5
15
45
pH
Control Nisin Control Nisin Control Nisin Control Nisin Control Nisin
3.66 ± 0.07 aA 3.66 ± 0.07 aA 0.39 ± 0.00 aA 0.39 ± 0.00 aA 136.05 ± 0.66 bA 190.47 ± 0.66 aA 506.14 ± 0.90 aA 941.87 ± 0.40 aA 10.91 ± 0.10 bB 12.91 ± 0.10 aB
3.66 ± 0.01 aA 3.79 ± 0.05 aA 0.34 ± 0.02 aB 0.34 ± 0.01 aB 125.00 ± 0.46 aA 188.11 ± 0.01 bA 536.43 ± 0.80 aA 602.21 ± 0.60 aA 11.58 ± 0.10 bA 16.58 ± 0.20 aA
3.70 ± 0.05 aA 3.68 ± 0.01 aA 0.31 ± 0.00 bB 0.36 ± 0.01 aAB 123.45 ± 0.12 bA 188.14 ± 0.18 bA 339.97 ± 0.15 bA 656.00 ± 0.30 aA 11.75 ± 0.00 bA 16.25 ± 0.00 aA
3.63 ± 0.04 aA 3.60 ± 0.02 aA 0.33 ± 0.00 bB 0.36 ± 0.02 bAB 47.61 ± 0.83 aB 89.68 ± 0.72 bB 628.14 ± 0.90 aA 675.65 ± 0.30 aA 11.08 ± 0.10 bB 16.58 ± 0.10 aA
Acidity (% citric acid) Vitamin C (% ascorbic acid) Browning index Solids soluble (°Brix) a
Means for the same parameter followed by the same letter, uppercase in line and lowercase in column, did not differ significantly by Tukey test at 5% probability.
A.A. de Oliveira Junior et al. / International Journal of Food Microbiology 211 (2015) 38–43
41
Table 4 Effect of nisin on the physicochemical characteristics of cashew juice. Control treatments without nisin are also shown. Parameter
Treatment
pH
Control Nisin Control Nisin Control Nisin Control Nisin Control Nisin
Acidity (% citric acid) Vitamin C (% ascorbic acid) Browning index Solids soluble (°Brix) a
Storage daysa 0
5
15
45
3.72 ± 0.01 aB 3.72 ± 0.01 aA 0.31 ± 0.01 aA 0.30 ± 0.00 aA 408.16 ± 0.86 aA 421.76 ± 0.66 aA 201.01 ± 0.40 aAB 126.51 ± 0.30 aA 11.83 ± 0.10 bA 16.66 ± 0.10 aA
3.73 ± 0.03 aB 3.68 ± 0.06 aA 0.27 ± 0.00 aB 0.2842 ± 0.00 aAB 402.77 ± 0.10 aAB 375.00 ± 0.46 bAB 122.05 ± 0.10 aBC 98.94 ± 0.30 aA 11.83 ± 0.10 bA 16.83 ± 0.44 aA
3.89 ± 0.01 aA 3.73 ± 0.01 bA 0.23 ± 0.01 bC 0.2662 ± 0.00 aB 358.02 ± 0.12 aB 333.33 ± 0.18 bB 72.70 ± 0.20 aC 102.73 ± 0.80 aA 12.00 ± 0.10 bA 16.75 ± 0.63 aA
3.58 ± 0.01 aC 3.61 ± 0.00 aA 0.25 ± 0.00 aBC 0.27 ± 0,00 aB 71.42 ± 0.00 aC 125.23 ± 0.00 bC 296.54 ± 0.70 aA 126.68 ± 0.30 bA 11.91 ± 0.27 bA 15.91 ± 0.10 aB
Means for the same parameter followed by the same letter, uppercase in line and lowercase in column, did not differ significantly by Tukey test at 5% probability.
Table 5 Effect of nisin on the physicochemical characteristics of soursop juice. Control treatments without nisin are also shown. Parameter
Treatment
Storage daysa 0
5
15
45
pH
Control Nisin Control Nisin Control Nisin Control Nisin Control Nisin
3.45 ± 0.02 aB 3.45 ± 0.02 aAB 0.45 ± 0.01 bA 0.49 ± 0.00 aA 163.26 ± 0.86 aA 108.84 ± 0.66 bA 206.42 ± 0.10 aAB 77.42 ± 0.10 bB 12.41 ± 0.10 bC 19.16 ± 0.10 aA
3.52 ± 0.01 aA 3.49 ± 0.04 aA 0.34 ± 0.00 bB 0.39 ± 0.00 aB 138.88 ± 0.10 aAB 138.88 ± 0.01 aAB 209.34 ± 0.30 aA 210.34 ± 0.20 aA 12.83 ± 0.20 bB 18.00 ± 0.17 aBC
3.49 ± 0.02 aAB 3.42 ± 0.03 bB 0.36 ± 0.02 bB 0.39 ± 0.00 aB 123.45 ± 0.12 aB 111.11 ± 0.00 aAB 99.80 ± 0.30 aC 129.54 ± 0.60 aAB 13.25 ± 0.00 bA 18.25 ± 0.00 aB
3.44 ± 0.02 aB 3.44 ± 0.00 aAB 0.36 ± 0.00 aB 0.36 ± 0.03 aB 47.61 ± 0.00 aC 47.61 ± 0.00 aC 104.14 ± 0.10 aBC 121.00 ± 0.80 aAB 12.50 ± 0.00 bBC 17.83 ± 0.10 aC
Acidity (% citric acid) Vitamin C (% ascorbic acid) Browning index Solids soluble (°Brix)
Means for the same parameter followed by the same letter, uppercase in line and lowercase in column, did not differ significantly by Tukey test at 5% probability.
4. Discussion To consolidate the approval and/or the use of nisin as a preservative in fruit juices, more studies are needed to show its effectiveness in different fruit juices against pathogenic and spoilage microorganisms. To date, only four studies have shown the effects of this bacteriocin on the preservation of fruit products. Barbosa et al. (2013) studied the incorporation of nisin in acetate films to package minimally processed mangoes. Walker and Phillips (2006) and Pathanibul et al. (2009) demonstrated the ability of nisin to control Listeria innocua and A. acidoterrestris, respectively, in apple juice. Yu et al. (2013) verified the effect of nisin on the inactivation of indigenous microorganisms in litchi juice. In this study, we showed that nisin could inhibit S. aureus, A. acidoterrestris, L. monocytogenes, and B. cereus strains in fruit juices. The application of bacteriocins as preservatives in fruit juices can be limited by their properties such as the solubility of the peptide, the interaction of bacteriocins with juices components, juice pH, and inactivation by proteases (Ganzle et al., 1999; Pei et al., 2013). Therefore, the stability of its antimicrobial activity is an essential characteristic for a bacteriocin to be used in the conservation of fruit juices. Our results showed that nisin remained stable in different fruit juices for at least 30 days of storage at room or refrigerated temperature, and that 24 h of incubation with nisin was enough to eliminate up to 107 CFU/mL of inoculated microorganisms. These results indicated that nisin could remain stable in fruit juices to ensure the microbiological safety of the product.
However, this study showed that the effect of the antimicrobial activity of nisin was heterogeneous and depended on both the type of juice and microorganism. The most sensitive microorganism to nisin
Viable cell number (Log CFU/mL)
When the antimicrobial activity of nisin was tested against L. monocytogenes in cashew and soursop juices, no reduction in the viable cell number was observed compared to the control treatment after 24 h of incubation (Fig. 4a and b). Viable cells were four and six times less than in the control treatment, in peach and mango juices respectively (Fig. 4c and d). Control treatment remained at least 106 CFU/mL after 24 h of incubation (Fig. 4).
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Fig. 1. Antimicrobial activity of nisin against A. acidoterrestris DSMZ2498 in cashew (a), soursop (b), peach (c), and mango (d) juices. Nisin was added at 5000 IU/mL (black bars) or not added in control treatments (white bars). A. acidoterrestris was inoculated at approximately 106 CFU/mL and viable cells were counted after 0, 8, and 24 h of incubation.
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Viable cell number (Log CFU/mL)
was A. acidoterrestris and the least sensitive was L. monocytogenes. Still, a reduction of up to 90% of viable cells was observed in peach and mango juices inoculated with L. monocytogenes after 24 h of incubation. Moreover, the microbial load used in our experiment was higher than would normally occur in real situations of contamination. The maximum microbial load expected to be found in real-life situations would be 102 or 103 viable cells per mL of juice or food (Burnett and Beuchat, 2001; Rosenquist et al., 2005; Vantarakis et al., 2011).
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Fig. 3. Antimicrobial activity of nisin against B. cereus ATCC4504 in cashew (a), soursop (b), peach (c), and mango (d) juices. Nisin was added at 5000 IU/mL (black bars) or not added in control treatments (white bars). B. cereus was inoculated at approximately 106 CFU/mL and viable cells were counted after 0, 8, and 24 h of incubation.
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Fig. 2. Antimicrobial activity of nisin against S. aureus ATCC8095 in cashew (a), soursop (b), peach (c), and mango (d) juices. Nisin was added at 5000 IU/mL (black bars) or not added in control treatments (white bars). S. aureus was inoculated at approximately 106 CFU/mL and viable cells were counted after 0, 8, and 24 h of incubation.
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A.A. de Oliveira Junior et al. / International Journal of Food Microbiology 211 (2015) 38–43
Viable cell number (log CFU/mL)
Viable cell number (Log CFU/mL)
Viable cell number (Log CFU/mL)
42
8 24 Incubation time (h)
0
8 24 Incubation time (h)
Fig. 4. Antimicrobial activity of nisin against L. monocytogenes ATCC7644 in cashew (a), soursop (b), peach (c) and mango (d) juices. Nisin was added at 5000 IU/mL (black bars) or not added in control treatments (white bars). B. cereus was inoculated at approximately 106 CFU/mL and viable cells were counted after 0, 8, and 24 h of incubation.
Cashew juice with added nisin caused a rapid (in the first 8 h) decrease in the viability of A. acidoterrestris, S. aureus, and B. cereus. After 24 h of incubation, the antimicrobial activity of nisin for B. cereus and S. aureus was lower in mango juice, whereas the reverse was observed for L. monocytogenes. Pei et al. (2013) and Yamazaki et al. (2000) also reported heterogeneous effects of the antimicrobial activity of bacteriocins in different fruit juices and attributed these to the combination of bacteriocins and polyphenols found in the juices, although the exact factors were not completely defined. It is well known that fruits such as cashew, soursop, peach, and mango contain divalent ions, such as calcium and magnesium (Soares et al., 2004). These divalent cations have also been reported to decrease bacteriocin activity (Davies et al., 1996; Houlihan and Russel, 2006; Kumar and Srivastava, 2011). For L. monocytogenes, Davies et al. (1996) reported that nisin resistance was associated with binding of divalent metals by ribitol and glycerol phosphates of lipoteichoic acids on the cell surface. Therefore, the heterogeneous activity of nisin in fruit juices could be explained by the intrinsic properties of the juices and also by features of the microorganisms. Bacteriocins are well known to not cause any changes in the organoleptic characteristics of foods (Cleveland et al., 2001; Deegan et al., 2006), but to date, the studies showing the effect of nisin in physicochemical properties of foods are limited. The physico-chemical analyses performed in this study also showed that nisin did not cause any significant alterations in the physico-chemical characteristics of the fruit juices tested. The properties of the juices supplemented or not with nisin were unchanged, except the solid soluble content that was always higher with nisin. This result was expected as a solute (nisin) was added to these samples. Moreover, the addition of nisin favored the preservation of the vitamin C content in all the juices tested, which is a desirable feature. Thus, nisin can ensure the microbiological safety of juices without causing significant negative changes in the product. Despite these results, further studies are needed to see consumer acceptability of juices with added nisin. The initial concentration of nisin could be adjusted according to its antimicrobial effects and its sensorial properties that affect consumer acceptability.
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Considering the risk of pathogenic microorganisms to contaminate fruit juices, the United States Food and Drug Administration (FDA) imposed Hazard Analysis and Critical Control Point (HACCP) requirements on juice processors. The HACCP require that processors of fruit juices achieve a 5-log reduction of a target pathogen (FDA, 2001). Our results showed that nisin achieved a 5-log reduction of A. acidoterrestris, S. aureus, and B. cereus after 24 h of incubation in all juices tested. A similar result was observed for L. monocytogenes in peach and mango juices only. These results show that nisin reaches the requirements established by the FDA. In conclusion, in this study we state that nisin remained stable in different fruit juices without causing changes in organoleptic characteristics and showed antimicrobial activity against spoilage and pathogenic microorganisms. The application of this bacteriocin together with a mild heat treatment or other antimicrobial agent can be an alternative in the processing of fruit juices. Acknowledgments The authors would like to thank the Conselho de Desenvolvimento Científico e Tecnológico (CNPq), Brasília, Brazil and the Fundação de Apoio à Pesquisa e à inovação Tecnológica do Estado de Sergipe (FAPITEC), Sergipe, Brazil, (FAPITEC/SE /FUNTEC/CNPq N° 04/2011), for providing fellow ships and financial support for this research. References Barbosa, A.A.T., Silva de Araújo, H.G., Matos, P.N., Carnelossi, M.A.G., Castro, A.A., 2013. Effects of nisin-incorporated films on the microbiological and physicochemical quality of minimally processed mangoes. Int. J. Food Microbiol. 164, 135–140. Bi, X., Liu, F., Rao, L., Li, J., Liu, B., Liao, X., Wu, J., 2013. Effects of electric field strength and pulse rise time on physicochemical and sensory properties of apple juice by pulsed electric field. Innovative Food Sci. Emerg. Technol. 17, 85–92. Braddock, R.J., 1999. Handbook of Citrus By-Products and Processing Technology. John Wiley and Sons, Inc., New York, NY, USA. Burnett, S.L., Beuchat, L.R., 2001. Human pathogens associated with raw produce and unpasteurized juices, and difficulties in decontamination. J. Ind. Microbiol. Biotechnol. 27, 104–110. Carnelossi, M.A.G., 2000. Fisiologia pós-colheita de folhas de couve (Brassicaoleracea cv. Acephala) minimamente processadas Doctoral Thesis, Universidade Federal de Viçosa, Brazil, Departamento de Microbiologia (83 pp.). Carvalho, A.A.T., Costa, E.D., Mantovani, H.C., Vanetti, M.C.D., 2007. Effect of bovicin HC5 on growth and spore germination of Bacillus cereus and Bacillus thuringiensis isolated from spoiled mango pulp. J. Appl. Microbiol. 102, 1000–1009. Carvalho, A.A.T., Vanetti, M.C.D., Mantovani, H.C., 2008. Bovicin HC5 reduces thermal resistance of Alicyclobacillusacidoterrestris in acidic mango pulp. J. Appl. Microbiol. 104, 1685–1691. Cleveland, J., Montville, T.J., Nes, I.F., Chikinda, M.L., 2001. Bacteriocins: safe, natural antimicrobials for food preservation. Int. J. Food Microbiol. 71, 1–20. Cotter, P.D., Hill, C., Ross, P., 2005. Bacteriocins: developing innate immunity for food. Nat. Rev. 3, 777–788. Davies, E.A., Falahee, M.B., Adams, M.R., 1996. Involvement of the cell envelope in Listeria monocytogenes in the acquisition of nisin resistance. J. Appl. Bacteriol. 81, 139–146. Deegan, L.H., Cotter, P.D., Hill, C., Ross, P., 2006. Bacteriocins: biological tools for biopreservation and shelf-life extension. Int. Dairy J. 16, 1058–1071. Espina, L., Somolinos, M., Ouazzou, A.A., Condón, S., García-Gonzalo, D., Pagán, R., 2012. Inactivation of Escherichia coli O157:H7 in fruit juices by combined treatments of citrus fruit essential oils and heat. Int. J. Food Microbiol. 159, 9–16. Espina, L., García-Gonzalo, D., Laglaoui, A., Mackey, B.M., Pagán, R., 2013. Synergistic combinations of high hydrostatic pressure and essential oils or their constituents and their use in preservation of fruit juices. Int. J. Food Microbiol. 161, 23–30.
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