Controlling Listeria monocytogenes in Cold Smoked Salmon with the Antimicrobial Peptide Salmine Christopher Cheng, Fletcher Arritt, and Clinton Stevenson
Listeria monocytogenes (LM) is a major safety concern for smoked salmon producers, as it can survive both the brining and smoking process in cold smoked salmon production. Salmine is a cationic antimicrobial peptide derived from the milt of salmon that has been shown to inhibit the growth of LM in vitro. Commercialization of this peptide would add value to a waste product produced when raising salmon. The purpose of this study was to determine the anti-listeria activity of salmine in smoked salmon by measuring the viable counts of LM over time. Cold smoked salmon was treated with a salmine solution or coated with agar or k-carrageenan films incorporating salmine to maintain a high surface concentration of the antimicrobial. Samples were then inoculated with approximately 1.0 × 103 cells of LM. The viable counts were then enumerated throughout 4 wk at 4 °C storage. It was found that 5 mg/g salmine delayed the growth of LM on smoked salmon. These samples had significantly (P < 0.05) lower LM counts than on the untreated samples on days 13 and 22. Edible films did not significantly (P > 0.05) improve the antimicrobial efficacy of salmine. The peptide combined with biopolymers also had lower antimicrobial activity in vitro when compared to salmine alone. These results suggest there is potential for salmine to be used as a natural hurdle to inhibit growth of LM due to post process contamination; however, future investigations for extending this effect throughout the shelf life of smoked salmon products are warranted.
Abstract:
M: Food Microbiology & Safety
Keywords: antimicrobial, Listeria monocytogenes, salmon, smoked
This research indicates that the antimicrobial peptide salmine can potentially be used as a natural antimicrobial compound to inhibit Listeria monocytogenes in cold smoked salmon. Commercialization of this peptide could improve the safety of this product, maintain a clean label, and add value to a waste product from salmon aquaculture.
Practical Application:
Introduction
the smoking process if it is located under the surface of the meat (Eklund and others 1995). There is a zero tolerance policy for LM in ready to eat foods in the United States (Huss and others 1995); thus, there is a need for a technology that eliminates the growth of this pathogen caused by post process contamination. Salmine is a cationic antimicrobial peptide derived from salmon milt tissues, a waste product from the production of salmon with a molecular weight of 5.5 to 13.0 kDa. The peptide may be used to prevent growth of LM because of post-process contamination (Uyttendaele and Debevere 1994). Salmine has many properties similar to the currently used antimicrobial nisin, as both are low molecular weight cationic peptides with hydrophobic properties and they inhibit bacteria by disrupting the cytoplasmic membrane (Bechinger and Lohner 2006). There is an opportunity for it to add value to a traditional waste product of aquaculture. Salmine has been found to inhibit LM in vitro; however, few studies tested the efficacy of this peptide in true food systems (Islam and others 1984; 1985; Uyttendaele and Debevere 1994; Conte and others 2007). Clupeine, a similar antimicrobial peptide derived from herring, caused a 2 to 3 log reduction in LM in milk over 16 d (Potter and others 2005). It was found that higher concentrations of the antimicrobial were needed to achieve the same level of efficacy observed in in vitro conditions. The discrepancy between MS 20142011 Submitted 12/8/2014, Accepted 3/20/2015. Authors are with North Carolina State Univ., Dept. of Food, Bioprocessing and Nutrition Sciences, antimicrobial efficacy in food and in vitro systems is attributed 116A Schaub Hall, Campus Box 7624, Raleigh, NC 27695-7624, U.S.A. Direct to the cationic peptide electrostatically binding to the negatively inquiries to author Stevenson (E-mail: [email protected]). charged food components (Conte and others 2007). Cold smoked salmon is a ready to eat seafood product that lacks a thermal microbial inactivation step. It is processed by brining fish fillets in a salt solution then smoking at approximately 32 to 48 °C for times ranging between 6 and 24 h, giving it a shelf life of approximately 2 to 4 wk (Huss and others 1995). Listeria monocytogenes (LM) is a psychrotrophic and halotolerant pathogen, which is a major safety hazard for smoked salmon producers (Huss and others 1995) because it can survive the cold smoking process. Ingestion of LM can cause serious conditions for vulnerable populations, for example meningitis among immunocompromised persons and stillbirths for pregnant women. It is estimated that there are approximately 1600 illnesses and 260 deaths caused by listeriosis every year in the United States (CDC 2013). The water-phase salt content (calculated as %salt/{%salt + %moisture}) of smoked salmon usually ranges from 3% to 5% and does not have an inhibitory effect on LM growth (Peterson and others 1993). The low temperature of the cold smoking process fails to inactivate the pathogen (Huss and others 1995). Some compounds such as phenolics in smoke can inactivate LM on the surface of the fish, but the bacterium is still able to grow during
M1314
Journal of Food Science r Vol. 80, Nr. 6, 2015
R C 2015 Institute of Food Technologists
doi: 10.1111/1750-3841.12886 Further reproduction without permission is prohibited
Controlling Listeria monocytogenes in . . .
Materials and Methods Preparation of bacterial inoculum L. monocytogenes strains of Scott A., J0161, LW-A46 were used. Scott A and J0161 have been associated with major LM outbreaks (Yildirim and others 2010) and LW-A46 is a strain that has been isolated from salmon (Ratani and others 2012). The 3 strains were grown by incubating individually in brain heart infusion at 37 °C for 18 h. The culture was then centrifuged at 971 × g for 4 min (Intl. Equipment Co. Model CL, Needham Heights, Mass., U.S.A.). The resulting pellet was then suspended in sterile saline solution and the OD600 of each strain solution was adjusted to 0.5. The 3 strains suspended in saline solution were then combined to yield a cocktail of approximately 1.0 × 108 CFU/mL. The cocktail was then diluted to a concentration of 1.0 × 104 CFU/mL for inoculation. Salmine treatment of smoked salmon Preskinned cold smoked salmon purchased from a wholesale grocery outlet was stored at 4 °C on the day of LM inoculation and salmine treatment. Salmon filets were aseptically cut into 2 g samples with an approximate surface area of about 13 cm2 . Protamine sulfate from salmine was purchased from Sigma-Aldrich, St. Louis, Missouri at 81% to 96% purity. Solutions were prepared at a concentration of 0, 1, 5, and 10 mg/mL and filtered by syringe with 22-nm membrane filters. To simulate a spray treatment, salmon samples were then treated with salmine with dropwise addition by pipetting 1 mL of a solution onto the fish to yield final concentrations of 0, 0.5, 2.5, and 5 mg salmine/g salmon sample completely covering the fish sample. When treating salmon samples with antimicrobial films, fish pieces were instead dipped into a 10 mg/mL salmine solution. They were then set out in a biosafety cabinet to dry for 10 min. Each of these treatment combinations was performed in triplicate. Film treatment Four types of films were prepared from negatively charged kcarrageenan (kCGN), neutrally charged agar, a 1:2 blend, and a 2:1 blend of these biopolymers. Film forming solutions were prepared by dissolving 0.5 g of each polymer into 25 mL of deionized water and then autoclaved for 20 min at 123 °C and 17 psi. Salmine solutions were separately prepared by dissolving 0.5 g of salmine into 25 mL water, filter sterilized, then incorporated into the polymer solution. The final solution had a 1% (w/v) biopolymer
concentration and 10 mg/mL of salmine. Smoked salmon samples aseptically cut into 2 g samples with an approximate surface area of 13 cm2 were dipped into the film forming solutions and allowed to air dry in a biosafety cabinet for 10 min. Each of these treatments was performed in triplicate.
L. monocytogenes inoculation After antimicrobial treatment, each salmon sample was then inoculated with approximately 1.0 × 103 cells of LM to simulate LM contamination by pipetting 0.1 mL of culture onto random locations of the meat and allowing it to air dry in the biosafety cabinet. Samples were then vacuum packaged and stored at 4 °C to simulate typical smoked salmon storage during distribution, wholesale and retail storage. Because the typical shelf life of smoked salmon is approximately 2 to 4 wk, LM was enumerated at 1, 4, 7, 13, 22, and 32 d. Microbial analysis LM was enumerated by placing 2 g salmon samples in 18 mL sterile buffered peptone water and homogenized with a 4000 circulator Seward stomacher for 30 s. Appropriate serial dilutions were prepared and 0.1 mL of the final solution was spread on Oxford media (CM 0856 with SR0140 supplement from Fisher Scientific, Waltham, Massachusetts) then incubated for 48 h at 37 °C. The same method was used to enumerate the total viable counts of smoked salmon except plate count agar (Fisher Scientific) was used instead of Oxford media; however, these samples were not inoculated with the LM cocktail. The plates were also incubated for 48 h at 37 °C. In vitro determination of antimicrobial efficacy The antimicrobial efficacy of the edible films was determined using the in vitro method described by Uyttendaele and Debevere (1994). Tubes of tryptic soy broth were combined with filmforming solution. The final 5 mL mixture had 1% (w/v) biopolymer and a salmine concentration of 10 mg/mL. Each tube was then inoculated with 10 μL of LM cocktail with an approximate concentration of 1.0 × 105 CFU/mL. After incubating at 37 °C for 24 h, LM counts were enumerated using Oxford media with selective supplement. Statistical analysis One-way analysis of variance with Tukey-Honest Significant Differences (HSD) means comparison was performed to determine significant difference of salmine concentrations (P < 0.05) using JMP statistical software (developed by SAS).
Results The LM in all salmon samples grew in a sigmoidal fashion (Table 1 and 2). There was an initial lag phase of about 3 d, a log growth phase of over 20 d, and a stationary phase once LM grew to about 8 log CFU/g after 32 d (Table 1). The antimicrobial efficacy of salmine was concentration and time dependent (Table 1). Treating salmon with salmine at 5 mg/g delayed the log phase of LM. The lag phase of these samples was extended by about 6 d. Although significant growth was observed past day 13 in samples treated with 0 and 0.5 mg/g salmine, it was delayed until after 22 d on samples treated with 5 mg/g salmine (Table 1). LM counts on these samples on days 13 and 22 were significantly lower than the counts on the untreated samples by Vol. 80, Nr. 6, 2015 r Journal of Food Science M1315
M: Food Microbiology & Safety
Edible films could minimize unwanted electrostatic interactions between salmine and the food matrix by providing a slow release mechanism of the antimicrobial from the film into the food. The salmine would diffuse throughout the fish until an equilibrium concentration is reached. Edible films could maintain a higher surface concentration where contamination typically occurs. The release rate of antimicrobial compounds can be affected by many extrinsic factors, such as temperature and pH (Teerakarn and others 2002; Choi and others 2005; Min and others 2008). Intrinsic factors of the edible film could also affect the release rate of salmine, for example the dissociation constant may be expected to be lower for anionic biopolymer solutions as compared to those that are relatively more cationic. The objective of this study was to quantify the anti-listeria activity of salmine in smoked salmon and determine the effect of charged and uncharged films on its antimicrobial activity.
Controlling Listeria monocytogenes in . . . Table 1–Listeria monocytogenes counts (log CFU/g) in smoked salmon as affected by salmine. Time (d) Salmine treatment (mg/g salmon) Listeria monocytogenes counts 0 0.5 2.5 5.0 Total viable counts 0 2.5 5
0
1
4
7
13
22
32
3.0 ± 0.1Aa 3.2 ± 0.2Aa 3.0 ± 0.1Aa 3.1 ± 0.1Aa
3.0 ± 0.1Aa 3.0 ± 0.1Aa 3.0 ± 0.1Aa 3.0 ± 0.2Aa
3.0 ± 0.2Aa 3.0 ± 0.1Aab 3.0 ± 0.2Aa 3.0 ± 0.5Aa
3.4 ± 0.4Aa 3.4 ± 0.04Ab 3.1 ± 0.3Aa 3.2 ± 0.2Aa
5.8 ± 1.0Ab 5.2 ± 0.2ABc 4.4 ± 1.3ABb 3.4 ± 0.7Ba
8.2 ± 0.3Ac 8.0 ± 0.2ABd 8.7 ± 0.03Ac 6.5 ± 1.5Bb
8.6 ± 0.4Ac 9.0 ± 0.1Ae 8.5 ± 0.3Ac 8.6 ± 0.2Ac
Listeria monocytogenes (LM) is a major safety concern for smoked salmon producers, as it can survive both the brining and smoking process in cold smoked salmon production. Salmine is a cationic antimicrobial peptide derived from the milt of salmon that has been shown to inhibit the growth of LM in vitro. Commercialization of this peptide would add value to a waste product produced when raising salmon. The purpose of this study was to determine the anti-listeria activity of salmine in smoked salmon by measuring the viable counts of LM over time. Cold smoked salmon was treated with a salmine solution or coated with agar or k-carrageenan films incorporating salmine to maintain a high surface concentration of the antimicrobial. Samples were then inoculated with approximately 1.0 × 103 cells of LM. The viable counts were then enumerated throughout 4 wk at 4 °C storage. It was found that 5 mg/g salmine delayed the growth of LM on smoked salmon. These samples had significantly (P < 0.05) lower LM counts than on the untreated samples on days 13 and 22. Edible films did not significantly (P > 0.05) improve the antimicrobial efficacy of salmine. The peptide combined with biopolymers also had lower antimicrobial activity in vitro when compared to salmine alone. These results suggest there is potential for salmine to be used as a natural hurdle to inhibit growth of LM due to post process contamination; however, future investigations for extending this effect throughout the shelf life of smoked salmon products are warranted.
Abstract:
M: Food Microbiology & Safety
Keywords: antimicrobial, Listeria monocytogenes, salmon, smoked
This research indicates that the antimicrobial peptide salmine can potentially be used as a natural antimicrobial compound to inhibit Listeria monocytogenes in cold smoked salmon. Commercialization of this peptide could improve the safety of this product, maintain a clean label, and add value to a waste product from salmon aquaculture.
Practical Application:
Introduction
the smoking process if it is located under the surface of the meat (Eklund and others 1995). There is a zero tolerance policy for LM in ready to eat foods in the United States (Huss and others 1995); thus, there is a need for a technology that eliminates the growth of this pathogen caused by post process contamination. Salmine is a cationic antimicrobial peptide derived from salmon milt tissues, a waste product from the production of salmon with a molecular weight of 5.5 to 13.0 kDa. The peptide may be used to prevent growth of LM because of post-process contamination (Uyttendaele and Debevere 1994). Salmine has many properties similar to the currently used antimicrobial nisin, as both are low molecular weight cationic peptides with hydrophobic properties and they inhibit bacteria by disrupting the cytoplasmic membrane (Bechinger and Lohner 2006). There is an opportunity for it to add value to a traditional waste product of aquaculture. Salmine has been found to inhibit LM in vitro; however, few studies tested the efficacy of this peptide in true food systems (Islam and others 1984; 1985; Uyttendaele and Debevere 1994; Conte and others 2007). Clupeine, a similar antimicrobial peptide derived from herring, caused a 2 to 3 log reduction in LM in milk over 16 d (Potter and others 2005). It was found that higher concentrations of the antimicrobial were needed to achieve the same level of efficacy observed in in vitro conditions. The discrepancy between MS 20142011 Submitted 12/8/2014, Accepted 3/20/2015. Authors are with North Carolina State Univ., Dept. of Food, Bioprocessing and Nutrition Sciences, antimicrobial efficacy in food and in vitro systems is attributed 116A Schaub Hall, Campus Box 7624, Raleigh, NC 27695-7624, U.S.A. Direct to the cationic peptide electrostatically binding to the negatively inquiries to author Stevenson (E-mail: [email protected]). charged food components (Conte and others 2007). Cold smoked salmon is a ready to eat seafood product that lacks a thermal microbial inactivation step. It is processed by brining fish fillets in a salt solution then smoking at approximately 32 to 48 °C for times ranging between 6 and 24 h, giving it a shelf life of approximately 2 to 4 wk (Huss and others 1995). Listeria monocytogenes (LM) is a psychrotrophic and halotolerant pathogen, which is a major safety hazard for smoked salmon producers (Huss and others 1995) because it can survive the cold smoking process. Ingestion of LM can cause serious conditions for vulnerable populations, for example meningitis among immunocompromised persons and stillbirths for pregnant women. It is estimated that there are approximately 1600 illnesses and 260 deaths caused by listeriosis every year in the United States (CDC 2013). The water-phase salt content (calculated as %salt/{%salt + %moisture}) of smoked salmon usually ranges from 3% to 5% and does not have an inhibitory effect on LM growth (Peterson and others 1993). The low temperature of the cold smoking process fails to inactivate the pathogen (Huss and others 1995). Some compounds such as phenolics in smoke can inactivate LM on the surface of the fish, but the bacterium is still able to grow during
M1314
Journal of Food Science r Vol. 80, Nr. 6, 2015
R C 2015 Institute of Food Technologists
doi: 10.1111/1750-3841.12886 Further reproduction without permission is prohibited
Controlling Listeria monocytogenes in . . .
Materials and Methods Preparation of bacterial inoculum L. monocytogenes strains of Scott A., J0161, LW-A46 were used. Scott A and J0161 have been associated with major LM outbreaks (Yildirim and others 2010) and LW-A46 is a strain that has been isolated from salmon (Ratani and others 2012). The 3 strains were grown by incubating individually in brain heart infusion at 37 °C for 18 h. The culture was then centrifuged at 971 × g for 4 min (Intl. Equipment Co. Model CL, Needham Heights, Mass., U.S.A.). The resulting pellet was then suspended in sterile saline solution and the OD600 of each strain solution was adjusted to 0.5. The 3 strains suspended in saline solution were then combined to yield a cocktail of approximately 1.0 × 108 CFU/mL. The cocktail was then diluted to a concentration of 1.0 × 104 CFU/mL for inoculation. Salmine treatment of smoked salmon Preskinned cold smoked salmon purchased from a wholesale grocery outlet was stored at 4 °C on the day of LM inoculation and salmine treatment. Salmon filets were aseptically cut into 2 g samples with an approximate surface area of about 13 cm2 . Protamine sulfate from salmine was purchased from Sigma-Aldrich, St. Louis, Missouri at 81% to 96% purity. Solutions were prepared at a concentration of 0, 1, 5, and 10 mg/mL and filtered by syringe with 22-nm membrane filters. To simulate a spray treatment, salmon samples were then treated with salmine with dropwise addition by pipetting 1 mL of a solution onto the fish to yield final concentrations of 0, 0.5, 2.5, and 5 mg salmine/g salmon sample completely covering the fish sample. When treating salmon samples with antimicrobial films, fish pieces were instead dipped into a 10 mg/mL salmine solution. They were then set out in a biosafety cabinet to dry for 10 min. Each of these treatment combinations was performed in triplicate. Film treatment Four types of films were prepared from negatively charged kcarrageenan (kCGN), neutrally charged agar, a 1:2 blend, and a 2:1 blend of these biopolymers. Film forming solutions were prepared by dissolving 0.5 g of each polymer into 25 mL of deionized water and then autoclaved for 20 min at 123 °C and 17 psi. Salmine solutions were separately prepared by dissolving 0.5 g of salmine into 25 mL water, filter sterilized, then incorporated into the polymer solution. The final solution had a 1% (w/v) biopolymer
concentration and 10 mg/mL of salmine. Smoked salmon samples aseptically cut into 2 g samples with an approximate surface area of 13 cm2 were dipped into the film forming solutions and allowed to air dry in a biosafety cabinet for 10 min. Each of these treatments was performed in triplicate.
L. monocytogenes inoculation After antimicrobial treatment, each salmon sample was then inoculated with approximately 1.0 × 103 cells of LM to simulate LM contamination by pipetting 0.1 mL of culture onto random locations of the meat and allowing it to air dry in the biosafety cabinet. Samples were then vacuum packaged and stored at 4 °C to simulate typical smoked salmon storage during distribution, wholesale and retail storage. Because the typical shelf life of smoked salmon is approximately 2 to 4 wk, LM was enumerated at 1, 4, 7, 13, 22, and 32 d. Microbial analysis LM was enumerated by placing 2 g salmon samples in 18 mL sterile buffered peptone water and homogenized with a 4000 circulator Seward stomacher for 30 s. Appropriate serial dilutions were prepared and 0.1 mL of the final solution was spread on Oxford media (CM 0856 with SR0140 supplement from Fisher Scientific, Waltham, Massachusetts) then incubated for 48 h at 37 °C. The same method was used to enumerate the total viable counts of smoked salmon except plate count agar (Fisher Scientific) was used instead of Oxford media; however, these samples were not inoculated with the LM cocktail. The plates were also incubated for 48 h at 37 °C. In vitro determination of antimicrobial efficacy The antimicrobial efficacy of the edible films was determined using the in vitro method described by Uyttendaele and Debevere (1994). Tubes of tryptic soy broth were combined with filmforming solution. The final 5 mL mixture had 1% (w/v) biopolymer and a salmine concentration of 10 mg/mL. Each tube was then inoculated with 10 μL of LM cocktail with an approximate concentration of 1.0 × 105 CFU/mL. After incubating at 37 °C for 24 h, LM counts were enumerated using Oxford media with selective supplement. Statistical analysis One-way analysis of variance with Tukey-Honest Significant Differences (HSD) means comparison was performed to determine significant difference of salmine concentrations (P < 0.05) using JMP statistical software (developed by SAS).
Results The LM in all salmon samples grew in a sigmoidal fashion (Table 1 and 2). There was an initial lag phase of about 3 d, a log growth phase of over 20 d, and a stationary phase once LM grew to about 8 log CFU/g after 32 d (Table 1). The antimicrobial efficacy of salmine was concentration and time dependent (Table 1). Treating salmon with salmine at 5 mg/g delayed the log phase of LM. The lag phase of these samples was extended by about 6 d. Although significant growth was observed past day 13 in samples treated with 0 and 0.5 mg/g salmine, it was delayed until after 22 d on samples treated with 5 mg/g salmine (Table 1). LM counts on these samples on days 13 and 22 were significantly lower than the counts on the untreated samples by Vol. 80, Nr. 6, 2015 r Journal of Food Science M1315
M: Food Microbiology & Safety
Edible films could minimize unwanted electrostatic interactions between salmine and the food matrix by providing a slow release mechanism of the antimicrobial from the film into the food. The salmine would diffuse throughout the fish until an equilibrium concentration is reached. Edible films could maintain a higher surface concentration where contamination typically occurs. The release rate of antimicrobial compounds can be affected by many extrinsic factors, such as temperature and pH (Teerakarn and others 2002; Choi and others 2005; Min and others 2008). Intrinsic factors of the edible film could also affect the release rate of salmine, for example the dissociation constant may be expected to be lower for anionic biopolymer solutions as compared to those that are relatively more cationic. The objective of this study was to quantify the anti-listeria activity of salmine in smoked salmon and determine the effect of charged and uncharged films on its antimicrobial activity.
Controlling Listeria monocytogenes in . . . Table 1–Listeria monocytogenes counts (log CFU/g) in smoked salmon as affected by salmine. Time (d) Salmine treatment (mg/g salmon) Listeria monocytogenes counts 0 0.5 2.5 5.0 Total viable counts 0 2.5 5
0
1
4
7
13
22
32
3.0 ± 0.1Aa 3.2 ± 0.2Aa 3.0 ± 0.1Aa 3.1 ± 0.1Aa
3.0 ± 0.1Aa 3.0 ± 0.1Aa 3.0 ± 0.1Aa 3.0 ± 0.2Aa
3.0 ± 0.2Aa 3.0 ± 0.1Aab 3.0 ± 0.2Aa 3.0 ± 0.5Aa
3.4 ± 0.4Aa 3.4 ± 0.04Ab 3.1 ± 0.3Aa 3.2 ± 0.2Aa
5.8 ± 1.0Ab 5.2 ± 0.2ABc 4.4 ± 1.3ABb 3.4 ± 0.7Ba
8.2 ± 0.3Ac 8.0 ± 0.2ABd 8.7 ± 0.03Ac 6.5 ± 1.5Bb
8.6 ± 0.4Ac 9.0 ± 0.1Ae 8.5 ± 0.3Ac 8.6 ± 0.2Ac
