Food Microbiology 39 (2014) 1e6
Contents lists available at ScienceDirect
Food Microbiology journal homepage: www.elsevier.com/locate/fm
Inhibitory effect of combinations of caprylic acid and nisin on Listeria monocytogenes in queso fresco Camila Gadotti, Laura Nelson, Francisco Diez-Gonzalez* Department of Food Science and Nutrition, University of Minnesota, 1354 Eckles Ave., St. Paul, MN 55108, United States
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2013 Received in revised form 19 September 2013 Accepted 16 October 2013 Available online 1 November 2013
Queso fresco (QF), a fresh Hispanic cheese has been linked to outbreaks and recalls caused by Listeria contamination. The use antimicrobial treatments may be a potential solution. The goal of this research was to test the addition of nisin (N), caprylic acid (CA) and trans-cinnamaldehyde (CN) as anti-listerial ingredients in QF. QF batches were inoculated with approx. 104 CFU/g of 5- or 6-strain mixtures of Listeria monocytogenes and treated with antimicrobials. Samples were stored at 4 C for three weeks and Listeria counts were determined by plating on PALCAM agar. The impact on the QF’s natural indicator microorganisms was also assessed during refrigerated storage. All N and CA combinations (0.4 g/kg each) were effective against L. monocytogenes and reduced the final counts by at least 3 log CFU/g after 20 days of storage compared to controls. The levels of most strain mixtures were reduced immediately after treatment and their numbers remained below 103 CFU/g during storage. CN (1.2 g/kg) was bacteriostatic against L. monocytogenes, but it did not reduce initial counts. The addition of CN to the combination of N and CA did not enhance their antimicrobial effect. Results indicated that combinations of N and CA could control L. monocytogenes in QF with little impact on the natural flora of the cheese, providing a solution to control post processing L. monocytogenes contamination of QF. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Nisin Queso fresco trans-Cinnamaldehyde Caprylic acid GRAS Listeria monocytogenes Hispanic cheese
1. Introduction According to the U.S. Census Bureau agency, the Hispanic population increased 43% from 2000 to 2010, making Hispanics the fastest-growing minority group in the U.S. (Census Bureau, 2011). The rapid growth of the Hispanic population in the United States combined with the exposure of people to Latin American culinary culture has resulted in a greater demand for traditional cheeses. There is a wide diversity of Hispanic cheeses, and queso fresco (QF) is one of the most popular soft Hispanic cheeses that is manufactured with little or no starter culture and subsequently does not undergo a fermentation or aging period. Due to the absence of an aging period, the QF cheese is normally consumed within 14 days of manufacture (Renye et al., 2008). Fresh Hispanic cheeses are typically white, have relatively high moisture content (>50%) and have pH values greater than 5.8 (Van Hekken and Farkye, 2003). With near neutral pH, high moisture content and low salt concentration, fresh unripen Hispanic-style cheeses like QF are very susceptible to growth of spoilage organisms and pathogenic bacteria (Lin et al., 2006). The
* Corresponding author. Tel.: þ1 612 624 9756; fax: þ1 612 625 5272. E-mail address: [email protected] (F. Diez-Gonzalez). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.10.007
major food safety concern related to QF stems from the linkage of this cheese to several foodborne disease outbreaks associated with Listeria monocytogenes. Five of 11 cheese associated outbreaks reported to the Centers for Disease Control between 1973 and 1992 were associated with soft cheeses such as queso fresco (Altekruse et al., 1998). The first documented queso fresco outbreak occurred in 1985, in Los Angeles, CA where contaminated QF caused more than 140 listeriosis cases (Linnan et al., 1988). The risk of L. monocytogenes in Hispanic fresh cheeses is based on its ubiquitous occurrence in the environment of dairy plants and its ability to survive and grow on these products. Improvements in the safety of QF need to be effective inhibiting the growth of L. monocytogenes, while maintaining the properties and characteristics of this type of cheese. The use of antimicrobial ingredients may offer a viable solution to this problem. Nisin is a low molecular weight bacteriocin produced by certain strains of Lactococcus lactis subsp, lactis (Delves-Broughton et al., 1996). This antimicrobial is very effective against Gram-positive bacteria such as L. monocytogenes (Boziaris and Adams, 2000). Nisin inhibits target cells by forming pores in the membrane, depleting the trans-membrane potential and the pH gradient, resulting in the leakage of cellular materials (Cleveland et al., 2001). Under U.S. federal regulation, nisin has been affirmed generally-
2
C. Gadotti et al. / Food Microbiology 39 (2014) 1e6
recognized-as-safe (GRAS) status and is allowed in foods at a maximum level of 250 ppm (CFR, 2013). Caprylic acid (CA), or octanoic acid, is an eight-carbon fatty acid naturally present in breast milk, bovine milk and coconut oil (Jensen, 2002). Under federal regulation, CA has GRAS status and is allowed in cheese at levels of 0.04% (CFR, 2013). The exact mechanism of action of CA on bacteria is unknown, however, hypotheses have been suggested to explain the mode of antimicrobial activity of free fatty acids and their monoglycerides (Bergsson et al., 1998). Cinnamaldehyde, or trans-cinnamaldehyde (CN), is the main component of cinnamon flavor (Cinnamomum zeylandicum) and naturally occurs in the bark of cinnamon trees (Burt, 2004). CN has GRAS status, is classified as a flavoring substance, and no usage levels are established by the FDA (CFR, 2013). The mode of action of trans-cinnamaldehyde is still uncertain; studies have shown that the composition of unsaturated fatty acids from the cytoplasmatic membrane of bacterial cells change drastically when exposed to high levels of CN, suggesting that this compound acts on the membrane, altering its lipid profile and structure (Di Pasqua et al., 2007; Di Pasqua et al., 2006). The goal of this research project was to test a combination of GRAS antimicrobial treatments to control a diverse collection of L. monocytogenes strains in queso fresco. GRAS food additives used in this study included nisin (N), caprylic acid (CA) and trans-cinnamaldehyde (CN). 2. Materials and methods 2.1. Bacterial strains The strains used in this research are listed on Table 1. Most of the L. monocytogenes strains are part of the International Life Sciences Institute collection housed at Cornell University provided by Dr. Martin Wiedmann. 2.2. GRAS antimicrobial ingredients The antimicrobial ingredients used in this project included nisin (N; NisaplinÒ, kindly provided by Rex Infanger from Danisco, Inc., Copenhagen, DK), caprylic acid (CA; SigmaeAldrich, Saint Louis, MO), and trans-cinnamaldehyde (CN; SigmaeAldrich).
Table 1 L. monocytogenes strains used in this study and their origin. ID
Ribotype
Serotype
Source
N3-013 J1-158 J1-169 J1-049 C1-056 J1-177 M1-004 J2-020 J1-031 J1-168 J2-031 R2-501
1042B 10142 1052A 1042C 1030A 1051D 1039B 1039C 1059A 116-110-S2 1039E 1042B
4b 4b 3b 3c 1/2a 1/2b NA 1/2a 4a 4a 1/2a 4b
NAb J1-094 NA NA NA NA NA J2-064
ATCC 15313 116-1501-S4 ATCC 51775 H7762 2349 3528 2422 1052
NA 1/2c 1/2a 4a 4a 4a 4a 1/2b
Food, epidemic, UK, pate, 1988 Animal, goat Human, sporadic Human, sporadic Human, sporadic Human, sporadic Human, sporadic Animal, cow Human, sporadic Human, sporadic Animal, cow Human, epidemic, North Carolina, cheese, 2000 Rabbit, Cambridge, England Human, sporadic Cheese, Belgium Franks, Sara Lee outbreak, 1998 Environmental isolate, FSMLa Environmental isolate, FSML Environmental isolate, FSML Animal, cow
a b
Food Safety Microbiology Laboratory, University of Minnesota. NA: Not available.
2.3. Inoculum preparation Stock cultures were stored in glycerol at 55 C and working cultures were grown on tryptic soy agar (TSA) (Neogen Corp., Lansing, MI) at 37 C for 48 h. One colony was picked from each plate, transferred to a tryptic soy broth (TSB) (Neogen Corp.) tube and incubated at 37 C for 18 h. After streaking each culture onto TSA slants and incubating at 37 C for 48 h, working cultures were stored at 4 C for no more than a month. New culture slants were prepared each month. For inoculation of cheese curds, TSB tubes were inoculated individually with working cultures of each strain and incubated at 37 C for 18 h. Aliquots of 2 mL culture samples from each bacterial strain were mixed, serially diluted and applied to 2.3 kg portions of cheese curds in order to obtain an initial count of approximately 104 CFU/g. Groups of five to six strains of L. monocytogenes were used for inoculation. 2.4. Cheese manufacture, inoculation and treatment Approximately 110 L batches of milk were pasteurized for each experimental trial. Non-fat dried milk (2%) was added to the milk before pasteurization. Pasteurized milk was transferred to a sanitized vat and heated to a temperature of 30e33 C in a food processing pilot plant. Calcium chloride (0.02%) was mixed evenly into the milk. The milk was set by adding chymosin (ChymaxÒ, Christian Hansen, Hørsholm, Denmark) (0.01%) and allowing the curd to form for 30 min. Cheese knives that produced 1/2 inches cubes were used to cut the curds. After cutting, the curds were stirred for 1 h and the temperature was increased to 35 C. Whey was drained to about 50% of the original volume and the curds were salted (2%). The curds were bagged and transferred to a biosafety level 2 (BSL2) laboratory. N was weighed to the appropriate amount to obtain a concentration of 0.49 g/kg of curds. CA and CN were measured volumetrically to deliver concentrations between 0.36e0.72 g/kg and 0.3e 1.2 g/kg respectively, before curd mixing. The weighed amounts of these compounds were individually mixed with 100 mL aliquots of wet curds and added separately to a total of 2.3 kg portions of cheese curds before inoculation and mixed for 2 min by hand in sealed bags. Binary combinations of N and CA with or without CN were tested. CN was also tested individually. Curds were molded and allowed to drain for 1 h and the resulting queso fresco portions were bagged and stored at 4 C for 20 days. 2.5. Microbiological analysis During storage time, 11 g composite samples were taken from QF pieces every 2e3 days for microbiological analysis. Samples were homogenized with 99 mL buffered peptone water (BPW) (Neogen Corp.), serially diluted 10-fold and spread plated in duplicate on PALCAM (Neogen Corp.) agar plates. Plates were incubated at 12 C for 10 days, typical colonies were counted and the values were transformed into log CFU/g of cheese sample. The choice of low incubation temperature reduced the interference of natural background bacteria capable of growing on PALCAM producing nontypical Listeria colonies. Non-inoculated control samples were also included in every trial and no presumptive colonies were observed in any of those plates. The pH of samples was also measured directly using a pH electrode (Beckman Instruments, Inc., Fullerton, CA). The lower limit of detection was 100 CFU/g; when no plates had colonies at 101 dilution, a value of 10 CFU/g was assigned. 2.6. Microbiological analysis of natural flora Cheese batches were manufactured following the protocol described above, except that no inocula were used. During storage,
C. Gadotti et al. / Food Microbiology 39 (2014) 1e6
3
11-g composite samples were taken from QF pieces every 3 days for microbiological analysis. Samples were homogenized with 99 mL BPW, diluted and spread plated onto MRS Lactobacilli agar and plate count agar (PCA) (Neogen Corp.) MRS plates were incubated at 37 C for 24 h. All colonies were counted as a lactic acid bacteria count. PCA plates were incubated at 37 C for 24 h for total aerobic plate counts and at 8 C for 10 days for psychrotrophic plate counts. The pH of samples was measured directly on cheese samples using a pH electrode. 2.7. Statistical methods The results for at least two independent experiments of each treatment were used to calculate the mean and standard deviation for each sampling day for each treatment. The average data points at each sampling time were plotted as a time course where the differences among control and treatment groups could be visualized. Bacterial counts in the logarithm form were analyzed using the mixed model analysis of variance (R version 2.11.1; The R foundation for Statistical Computing, Wien, AU). For the analysis, the control was set at zero and the difference between the treatment and control was analyzed to determine if the treatment was significantly different from zero. Tests included whether or not the counts were different at day 11, whether or not the slope was different between the control and treatment and whether or not the line was different in either way. It was assumed that there were no significant differences between trials and that the variation was similar across treatments. 3. Results A series of queso fresco (QF) experiments were conducted to determine the antimicrobial effect of two caprylic acid (CA) concentrations (0.36 and 0.72 g/kg) in combination with nisin (N) (0.49 g/kg). Experiments were conducted using different sets of strains to evaluate the consistency of inhibition among L. monocytogenes (Figs. 1 and 2). All strain mixtures were able to grow relatively rapidly in untreated QF controls. Four of the stains mixture controls reached a count of at least 8 log CFU/g in less than 8 days, and one mixture after 11 days. At both levels of CA, the detectable numbers of an initial group of six strains were less than 100 CFU/g (the detection limit) during the first 17 days, but the counts increased to 3 log CFU/g on the last day of storage at 4 C. Compared to controls, in which active growth was observed, both treatments resulted in more than 5 log CFU/g statistically significant reductions (Fig. 1A, Table 2). The effect of the combination of N and CA was tested with a second set of replicate experiments containing a different mix of five L. monocytogenes strains (Fig. 1B). Initial viable count reductions to undetectable levels were observed with this particular set of strains, but Listeria grew slowly in all treated samples and reached a final count of 4 and 3 log CFU/g at the lowest and highest antimicrobial concentrations, respectively. A third set of identical experiments were conducted with five additional L. monocytogenes ribotypes, but an additional N/CA treatment was included (Fig. 2A). The count of L. monocytogenes in samples treated with 0.36 g/kg CA and 0.49 g/kg N ranged between 100 and 1000 CFU/g during the entire experimental period. Treatments with low CA concentration (0.36 g/kg), and lower concentration of N (0.4 g/kg) also inhibited the growth of Listeria during most of the trial, but in the last week the count was slightly above 3 log CFU/g. Similar results were obtained with a fourth set of L. monocytogenes strains (Fig. 2B). The effect of the combination of N and CA was tested with a fifth set of replicate experiments with a different mix of five
Fig. 1. Effect of nisin (N) and caprylic acid (CA) addition to queso fresco on the Listeria monocytogenes count during storage at 4 C. Strain mixtures: (A): R2-500, N1-227, N3031, J1-110, J1-119, and R2-502. (B): ATCC 15313, H7762, 2349, 3528, 2422. Symbols e Circles: no addition; closed triangles: treatment with 0.49 g/kg N and 0.36 g/kg CA; open triangles: treatment with 0.49 g/kg N and 0.72 g/kg CA.
L. monocytogenes strains (Fig. 2C). The count of L. monocytogenes in treated samples grew from undetectable levels to approximately 4 log CFU/g during the experimental period. In comparison to the previous experiments, the initial count reached undetectable levels after 2 days in some of the treatments. Despite the differences observed among the five groups of strains, in all cases the reductions of L. monocytogenes counts by combinations of N and CA were more than 3 log CFU/g and statistically significant (Table 2). The effects of CN alone and in combination with N and CA were tested against a mixture of five L. monocytogenes strains (Fig. 3A and B). All CN treatments (0.3, 0.6, 1.2 g/kg) inhibited growth, but only at a concentration of 1.2 g/kg CN had comparable effects to the N/CA combination. With 0.6 g/kg of CN, the microbial counts were 2 log CFU/g lower than the control at the end of the storage period. CN was also tested in combination with N and CA, and little differences in bacterial counts of L. monocytogenes in QF were observed compared with the treatment with only N and CA (Fig. 3A and B). When CN was used at 0.6 g/kg in addition to N and CA (0.4 g/ kg) the counts of the pathogen mixtures in treated samples were at least 4 log CFU/g smaller than the control and at least 1 log CFU/g less than the N/CA only combination. Combinations of N, CA and CN were further tested to determine their impact on naturally present bacteria in QF and to assess their relevance in shelf life. None of the treatments completely inhibited the growth of lactic acid bacteria, aerobic organisms and psychrotrophic bacteria throughout the 32 days of storage at 4 C (Fig. 4AeC), and only the combination with higher concentration of CN had any significant effect in delaying growth of natural flora (p < 0.05).
4
C. Gadotti et al. / Food Microbiology 39 (2014) 1e6 Table 2 List of combination antimicrobial treatments used to inhibit L. monocytogenes in queso fresco and the statistical significance when the differences of L. monocytogenes counts were compared to untreated samples. Antimicrobial added per kg of curds
Fig. 2. Effect of nisin (N) and caprylic acid (CA) addition to queso fresco on the Listeria monocytogenes count during storage at 4 C. Strain mixtures (A): N3-013, J1-158, J1169, J1-049, and C1-056. (B): DUP-1051D, DUP-1039B, 116-1501-S-4, DUP-1039C, DUP-1059A, and (C): 116-110-S-2, DUP-1039E, DUP-1052, ATCC 51775, DUP-1042B. Symbols e Circles: no addition; closed triangles: 0.49 g/kg N and 0.36 g/kg CA; open triangles: 0.49 g/kg N and 0.72 g/kg CA; closed squares: 0.40 g/kg N and 0.36 g/kg CA.
4. Discussion The combination of N and CA applied to cheese at concentrations within regulatory levels was effective against all strains of Listeria tested in this project. However, there was a distinction in the way different sets of strains behaved throughout the 20 days of storage time. Some strains were more resistant to N as the treatment did not significantly reduce numbers initially. Three different sets of strains that were not initially resistant to the antimicrobial combination, at time zero the counts were reduced by at least 2 log CFU/g (compared to the controls). In Fig. 2A, the same effect
Nisin (g/kg)
Caprylic acid (g/kg)
trans-cinnamaldehyde (g/kg)
e e e 0.4 0.49 0.49 0.49 0.49
e e e 0.36 0.36 0.36 0.36 0.72
0.3 0.6 1.2 e e 0.3 0.6 e
# Independent trials
p-value
2 2 2 3 6 2 2 5
0.0799 0.009
Contents lists available at ScienceDirect
Food Microbiology journal homepage: www.elsevier.com/locate/fm
Inhibitory effect of combinations of caprylic acid and nisin on Listeria monocytogenes in queso fresco Camila Gadotti, Laura Nelson, Francisco Diez-Gonzalez* Department of Food Science and Nutrition, University of Minnesota, 1354 Eckles Ave., St. Paul, MN 55108, United States
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2013 Received in revised form 19 September 2013 Accepted 16 October 2013 Available online 1 November 2013
Queso fresco (QF), a fresh Hispanic cheese has been linked to outbreaks and recalls caused by Listeria contamination. The use antimicrobial treatments may be a potential solution. The goal of this research was to test the addition of nisin (N), caprylic acid (CA) and trans-cinnamaldehyde (CN) as anti-listerial ingredients in QF. QF batches were inoculated with approx. 104 CFU/g of 5- or 6-strain mixtures of Listeria monocytogenes and treated with antimicrobials. Samples were stored at 4 C for three weeks and Listeria counts were determined by plating on PALCAM agar. The impact on the QF’s natural indicator microorganisms was also assessed during refrigerated storage. All N and CA combinations (0.4 g/kg each) were effective against L. monocytogenes and reduced the final counts by at least 3 log CFU/g after 20 days of storage compared to controls. The levels of most strain mixtures were reduced immediately after treatment and their numbers remained below 103 CFU/g during storage. CN (1.2 g/kg) was bacteriostatic against L. monocytogenes, but it did not reduce initial counts. The addition of CN to the combination of N and CA did not enhance their antimicrobial effect. Results indicated that combinations of N and CA could control L. monocytogenes in QF with little impact on the natural flora of the cheese, providing a solution to control post processing L. monocytogenes contamination of QF. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Nisin Queso fresco trans-Cinnamaldehyde Caprylic acid GRAS Listeria monocytogenes Hispanic cheese
1. Introduction According to the U.S. Census Bureau agency, the Hispanic population increased 43% from 2000 to 2010, making Hispanics the fastest-growing minority group in the U.S. (Census Bureau, 2011). The rapid growth of the Hispanic population in the United States combined with the exposure of people to Latin American culinary culture has resulted in a greater demand for traditional cheeses. There is a wide diversity of Hispanic cheeses, and queso fresco (QF) is one of the most popular soft Hispanic cheeses that is manufactured with little or no starter culture and subsequently does not undergo a fermentation or aging period. Due to the absence of an aging period, the QF cheese is normally consumed within 14 days of manufacture (Renye et al., 2008). Fresh Hispanic cheeses are typically white, have relatively high moisture content (>50%) and have pH values greater than 5.8 (Van Hekken and Farkye, 2003). With near neutral pH, high moisture content and low salt concentration, fresh unripen Hispanic-style cheeses like QF are very susceptible to growth of spoilage organisms and pathogenic bacteria (Lin et al., 2006). The
* Corresponding author. Tel.: þ1 612 624 9756; fax: þ1 612 625 5272. E-mail address: [email protected] (F. Diez-Gonzalez). 0740-0020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2013.10.007
major food safety concern related to QF stems from the linkage of this cheese to several foodborne disease outbreaks associated with Listeria monocytogenes. Five of 11 cheese associated outbreaks reported to the Centers for Disease Control between 1973 and 1992 were associated with soft cheeses such as queso fresco (Altekruse et al., 1998). The first documented queso fresco outbreak occurred in 1985, in Los Angeles, CA where contaminated QF caused more than 140 listeriosis cases (Linnan et al., 1988). The risk of L. monocytogenes in Hispanic fresh cheeses is based on its ubiquitous occurrence in the environment of dairy plants and its ability to survive and grow on these products. Improvements in the safety of QF need to be effective inhibiting the growth of L. monocytogenes, while maintaining the properties and characteristics of this type of cheese. The use of antimicrobial ingredients may offer a viable solution to this problem. Nisin is a low molecular weight bacteriocin produced by certain strains of Lactococcus lactis subsp, lactis (Delves-Broughton et al., 1996). This antimicrobial is very effective against Gram-positive bacteria such as L. monocytogenes (Boziaris and Adams, 2000). Nisin inhibits target cells by forming pores in the membrane, depleting the trans-membrane potential and the pH gradient, resulting in the leakage of cellular materials (Cleveland et al., 2001). Under U.S. federal regulation, nisin has been affirmed generally-
2
C. Gadotti et al. / Food Microbiology 39 (2014) 1e6
recognized-as-safe (GRAS) status and is allowed in foods at a maximum level of 250 ppm (CFR, 2013). Caprylic acid (CA), or octanoic acid, is an eight-carbon fatty acid naturally present in breast milk, bovine milk and coconut oil (Jensen, 2002). Under federal regulation, CA has GRAS status and is allowed in cheese at levels of 0.04% (CFR, 2013). The exact mechanism of action of CA on bacteria is unknown, however, hypotheses have been suggested to explain the mode of antimicrobial activity of free fatty acids and their monoglycerides (Bergsson et al., 1998). Cinnamaldehyde, or trans-cinnamaldehyde (CN), is the main component of cinnamon flavor (Cinnamomum zeylandicum) and naturally occurs in the bark of cinnamon trees (Burt, 2004). CN has GRAS status, is classified as a flavoring substance, and no usage levels are established by the FDA (CFR, 2013). The mode of action of trans-cinnamaldehyde is still uncertain; studies have shown that the composition of unsaturated fatty acids from the cytoplasmatic membrane of bacterial cells change drastically when exposed to high levels of CN, suggesting that this compound acts on the membrane, altering its lipid profile and structure (Di Pasqua et al., 2007; Di Pasqua et al., 2006). The goal of this research project was to test a combination of GRAS antimicrobial treatments to control a diverse collection of L. monocytogenes strains in queso fresco. GRAS food additives used in this study included nisin (N), caprylic acid (CA) and trans-cinnamaldehyde (CN). 2. Materials and methods 2.1. Bacterial strains The strains used in this research are listed on Table 1. Most of the L. monocytogenes strains are part of the International Life Sciences Institute collection housed at Cornell University provided by Dr. Martin Wiedmann. 2.2. GRAS antimicrobial ingredients The antimicrobial ingredients used in this project included nisin (N; NisaplinÒ, kindly provided by Rex Infanger from Danisco, Inc., Copenhagen, DK), caprylic acid (CA; SigmaeAldrich, Saint Louis, MO), and trans-cinnamaldehyde (CN; SigmaeAldrich).
Table 1 L. monocytogenes strains used in this study and their origin. ID
Ribotype
Serotype
Source
N3-013 J1-158 J1-169 J1-049 C1-056 J1-177 M1-004 J2-020 J1-031 J1-168 J2-031 R2-501
1042B 10142 1052A 1042C 1030A 1051D 1039B 1039C 1059A 116-110-S2 1039E 1042B
4b 4b 3b 3c 1/2a 1/2b NA 1/2a 4a 4a 1/2a 4b
NAb J1-094 NA NA NA NA NA J2-064
ATCC 15313 116-1501-S4 ATCC 51775 H7762 2349 3528 2422 1052
NA 1/2c 1/2a 4a 4a 4a 4a 1/2b
Food, epidemic, UK, pate, 1988 Animal, goat Human, sporadic Human, sporadic Human, sporadic Human, sporadic Human, sporadic Animal, cow Human, sporadic Human, sporadic Animal, cow Human, epidemic, North Carolina, cheese, 2000 Rabbit, Cambridge, England Human, sporadic Cheese, Belgium Franks, Sara Lee outbreak, 1998 Environmental isolate, FSMLa Environmental isolate, FSML Environmental isolate, FSML Animal, cow
a b
Food Safety Microbiology Laboratory, University of Minnesota. NA: Not available.
2.3. Inoculum preparation Stock cultures were stored in glycerol at 55 C and working cultures were grown on tryptic soy agar (TSA) (Neogen Corp., Lansing, MI) at 37 C for 48 h. One colony was picked from each plate, transferred to a tryptic soy broth (TSB) (Neogen Corp.) tube and incubated at 37 C for 18 h. After streaking each culture onto TSA slants and incubating at 37 C for 48 h, working cultures were stored at 4 C for no more than a month. New culture slants were prepared each month. For inoculation of cheese curds, TSB tubes were inoculated individually with working cultures of each strain and incubated at 37 C for 18 h. Aliquots of 2 mL culture samples from each bacterial strain were mixed, serially diluted and applied to 2.3 kg portions of cheese curds in order to obtain an initial count of approximately 104 CFU/g. Groups of five to six strains of L. monocytogenes were used for inoculation. 2.4. Cheese manufacture, inoculation and treatment Approximately 110 L batches of milk were pasteurized for each experimental trial. Non-fat dried milk (2%) was added to the milk before pasteurization. Pasteurized milk was transferred to a sanitized vat and heated to a temperature of 30e33 C in a food processing pilot plant. Calcium chloride (0.02%) was mixed evenly into the milk. The milk was set by adding chymosin (ChymaxÒ, Christian Hansen, Hørsholm, Denmark) (0.01%) and allowing the curd to form for 30 min. Cheese knives that produced 1/2 inches cubes were used to cut the curds. After cutting, the curds were stirred for 1 h and the temperature was increased to 35 C. Whey was drained to about 50% of the original volume and the curds were salted (2%). The curds were bagged and transferred to a biosafety level 2 (BSL2) laboratory. N was weighed to the appropriate amount to obtain a concentration of 0.49 g/kg of curds. CA and CN were measured volumetrically to deliver concentrations between 0.36e0.72 g/kg and 0.3e 1.2 g/kg respectively, before curd mixing. The weighed amounts of these compounds were individually mixed with 100 mL aliquots of wet curds and added separately to a total of 2.3 kg portions of cheese curds before inoculation and mixed for 2 min by hand in sealed bags. Binary combinations of N and CA with or without CN were tested. CN was also tested individually. Curds were molded and allowed to drain for 1 h and the resulting queso fresco portions were bagged and stored at 4 C for 20 days. 2.5. Microbiological analysis During storage time, 11 g composite samples were taken from QF pieces every 2e3 days for microbiological analysis. Samples were homogenized with 99 mL buffered peptone water (BPW) (Neogen Corp.), serially diluted 10-fold and spread plated in duplicate on PALCAM (Neogen Corp.) agar plates. Plates were incubated at 12 C for 10 days, typical colonies were counted and the values were transformed into log CFU/g of cheese sample. The choice of low incubation temperature reduced the interference of natural background bacteria capable of growing on PALCAM producing nontypical Listeria colonies. Non-inoculated control samples were also included in every trial and no presumptive colonies were observed in any of those plates. The pH of samples was also measured directly using a pH electrode (Beckman Instruments, Inc., Fullerton, CA). The lower limit of detection was 100 CFU/g; when no plates had colonies at 101 dilution, a value of 10 CFU/g was assigned. 2.6. Microbiological analysis of natural flora Cheese batches were manufactured following the protocol described above, except that no inocula were used. During storage,
C. Gadotti et al. / Food Microbiology 39 (2014) 1e6
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11-g composite samples were taken from QF pieces every 3 days for microbiological analysis. Samples were homogenized with 99 mL BPW, diluted and spread plated onto MRS Lactobacilli agar and plate count agar (PCA) (Neogen Corp.) MRS plates were incubated at 37 C for 24 h. All colonies were counted as a lactic acid bacteria count. PCA plates were incubated at 37 C for 24 h for total aerobic plate counts and at 8 C for 10 days for psychrotrophic plate counts. The pH of samples was measured directly on cheese samples using a pH electrode. 2.7. Statistical methods The results for at least two independent experiments of each treatment were used to calculate the mean and standard deviation for each sampling day for each treatment. The average data points at each sampling time were plotted as a time course where the differences among control and treatment groups could be visualized. Bacterial counts in the logarithm form were analyzed using the mixed model analysis of variance (R version 2.11.1; The R foundation for Statistical Computing, Wien, AU). For the analysis, the control was set at zero and the difference between the treatment and control was analyzed to determine if the treatment was significantly different from zero. Tests included whether or not the counts were different at day 11, whether or not the slope was different between the control and treatment and whether or not the line was different in either way. It was assumed that there were no significant differences between trials and that the variation was similar across treatments. 3. Results A series of queso fresco (QF) experiments were conducted to determine the antimicrobial effect of two caprylic acid (CA) concentrations (0.36 and 0.72 g/kg) in combination with nisin (N) (0.49 g/kg). Experiments were conducted using different sets of strains to evaluate the consistency of inhibition among L. monocytogenes (Figs. 1 and 2). All strain mixtures were able to grow relatively rapidly in untreated QF controls. Four of the stains mixture controls reached a count of at least 8 log CFU/g in less than 8 days, and one mixture after 11 days. At both levels of CA, the detectable numbers of an initial group of six strains were less than 100 CFU/g (the detection limit) during the first 17 days, but the counts increased to 3 log CFU/g on the last day of storage at 4 C. Compared to controls, in which active growth was observed, both treatments resulted in more than 5 log CFU/g statistically significant reductions (Fig. 1A, Table 2). The effect of the combination of N and CA was tested with a second set of replicate experiments containing a different mix of five L. monocytogenes strains (Fig. 1B). Initial viable count reductions to undetectable levels were observed with this particular set of strains, but Listeria grew slowly in all treated samples and reached a final count of 4 and 3 log CFU/g at the lowest and highest antimicrobial concentrations, respectively. A third set of identical experiments were conducted with five additional L. monocytogenes ribotypes, but an additional N/CA treatment was included (Fig. 2A). The count of L. monocytogenes in samples treated with 0.36 g/kg CA and 0.49 g/kg N ranged between 100 and 1000 CFU/g during the entire experimental period. Treatments with low CA concentration (0.36 g/kg), and lower concentration of N (0.4 g/kg) also inhibited the growth of Listeria during most of the trial, but in the last week the count was slightly above 3 log CFU/g. Similar results were obtained with a fourth set of L. monocytogenes strains (Fig. 2B). The effect of the combination of N and CA was tested with a fifth set of replicate experiments with a different mix of five
Fig. 1. Effect of nisin (N) and caprylic acid (CA) addition to queso fresco on the Listeria monocytogenes count during storage at 4 C. Strain mixtures: (A): R2-500, N1-227, N3031, J1-110, J1-119, and R2-502. (B): ATCC 15313, H7762, 2349, 3528, 2422. Symbols e Circles: no addition; closed triangles: treatment with 0.49 g/kg N and 0.36 g/kg CA; open triangles: treatment with 0.49 g/kg N and 0.72 g/kg CA.
L. monocytogenes strains (Fig. 2C). The count of L. monocytogenes in treated samples grew from undetectable levels to approximately 4 log CFU/g during the experimental period. In comparison to the previous experiments, the initial count reached undetectable levels after 2 days in some of the treatments. Despite the differences observed among the five groups of strains, in all cases the reductions of L. monocytogenes counts by combinations of N and CA were more than 3 log CFU/g and statistically significant (Table 2). The effects of CN alone and in combination with N and CA were tested against a mixture of five L. monocytogenes strains (Fig. 3A and B). All CN treatments (0.3, 0.6, 1.2 g/kg) inhibited growth, but only at a concentration of 1.2 g/kg CN had comparable effects to the N/CA combination. With 0.6 g/kg of CN, the microbial counts were 2 log CFU/g lower than the control at the end of the storage period. CN was also tested in combination with N and CA, and little differences in bacterial counts of L. monocytogenes in QF were observed compared with the treatment with only N and CA (Fig. 3A and B). When CN was used at 0.6 g/kg in addition to N and CA (0.4 g/ kg) the counts of the pathogen mixtures in treated samples were at least 4 log CFU/g smaller than the control and at least 1 log CFU/g less than the N/CA only combination. Combinations of N, CA and CN were further tested to determine their impact on naturally present bacteria in QF and to assess their relevance in shelf life. None of the treatments completely inhibited the growth of lactic acid bacteria, aerobic organisms and psychrotrophic bacteria throughout the 32 days of storage at 4 C (Fig. 4AeC), and only the combination with higher concentration of CN had any significant effect in delaying growth of natural flora (p < 0.05).
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C. Gadotti et al. / Food Microbiology 39 (2014) 1e6 Table 2 List of combination antimicrobial treatments used to inhibit L. monocytogenes in queso fresco and the statistical significance when the differences of L. monocytogenes counts were compared to untreated samples. Antimicrobial added per kg of curds
Fig. 2. Effect of nisin (N) and caprylic acid (CA) addition to queso fresco on the Listeria monocytogenes count during storage at 4 C. Strain mixtures (A): N3-013, J1-158, J1169, J1-049, and C1-056. (B): DUP-1051D, DUP-1039B, 116-1501-S-4, DUP-1039C, DUP-1059A, and (C): 116-110-S-2, DUP-1039E, DUP-1052, ATCC 51775, DUP-1042B. Symbols e Circles: no addition; closed triangles: 0.49 g/kg N and 0.36 g/kg CA; open triangles: 0.49 g/kg N and 0.72 g/kg CA; closed squares: 0.40 g/kg N and 0.36 g/kg CA.
4. Discussion The combination of N and CA applied to cheese at concentrations within regulatory levels was effective against all strains of Listeria tested in this project. However, there was a distinction in the way different sets of strains behaved throughout the 20 days of storage time. Some strains were more resistant to N as the treatment did not significantly reduce numbers initially. Three different sets of strains that were not initially resistant to the antimicrobial combination, at time zero the counts were reduced by at least 2 log CFU/g (compared to the controls). In Fig. 2A, the same effect
Nisin (g/kg)
Caprylic acid (g/kg)
trans-cinnamaldehyde (g/kg)
e e e 0.4 0.49 0.49 0.49 0.49
e e e 0.36 0.36 0.36 0.36 0.72
0.3 0.6 1.2 e e 0.3 0.6 e
# Independent trials
p-value
2 2 2 3 6 2 2 5
0.0799 0.009
