Functionality of liquid smoke as an all-natural antimicrobial in food preservation

Meat Science 97 (2014) 197–206

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Meat Science journal homepage: www.elsevier.com/locate/meatsci

Review

Functionality of liquid smoke as an all-natural antimicrobial in food preservation Jody M. Lingbeck a, Paola Cordero b, Corliss A. O'Bryan b, Michael G. Johnson b, Steven C. Ricke a,b,c, Philip G. Crandall a,b,⁎ a b c

Sea Star International LLC., 2138 East Revere Place, Fayetteville, AR 72701, USA Department of Food Science and Center for Food Safety, University of Arkansas, 2650 Young Ave., Fayetteville, AR 72704, USA Department of Poultry Science, Division of Agriculture, University of Arkansas, Fayetteville, AR 72704, USA

a r t i c l e

i n f o

Article history: Received 16 October 2013 Received in revised form 28 January 2014 Accepted 2 February 2014 Available online 9 February 2014 Keywords: Liquid smoke Antimicrobial Listeria monocytogenes Salmonella

a b s t r a c t The smoking of foods, especially meats, has been used as a preservation technique for centuries. Today, smoking methods often involve the use of wood smoke condensates, commonly known as liquid smoke. Liquid smoke is produced by condensing wood smoke created by the pyrolysis of sawdust or wood chips followed by removal of the carcinogenic polyaromatic hydrocarbons. The main products of wood pyrolysis are phenols, carbonyls and organic acids which are responsible for the flavor, color and antimicrobial properties of liquid smoke. Several common food-borne pathogens such as Listeria monocytogenes, Salmonella, pathogenic Escherichia coli and Staphylococcus have shown sensitivity to liquid smoke in vitro and in food systems. Therefore liquid smoke has potential for use as an all-natural antimicrobial in commercial applications where smoke flavor is desired. This review will cover the application and effectiveness of liquid smoke and fractions of liquid smoke as an all-natural food preservative. This review will be valuable for the industrial and research communities in the food science and technology areas. © 2014 Published by Elsevier Ltd.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Generation of liquid smoke from wood pyrolysis . . . . . . . . . . . . Antimicrobial activity of liquid smoke . . . . . . . . . . . . . . . . . 3.1. Possible mechanisms of antimicrobial action of liquid smokes . . . 3.2. Activity of phenols . . . . . . . . . . . . . . . . . . . . . . 3.3. Activity of carbonyls . . . . . . . . . . . . . . . . . . . . . . 4. Antimicrobial activity of liquid smoke against Listeria . . . . . . . . . . 4.1. In vitro effects on Listeria . . . . . . . . . . . . . . . . . . . . 4.2. Antilisterial effects in ready-to-eat meats . . . . . . . . . . . . 4.3. Genetic basis of the antimicrobial effects of liquid smoke on Listeria 5. Effects of liquid smoke on Salmonella spp. . . . . . . . . . . . . . . . 6. Effects of liquid smoke on E. coli . . . . . . . . . . . . . . . . . . . . 6.1. In vitro effects of liquid smoke on E. coli . . . . . . . . . . . . . 6.2. Effects of liquid smoke on E. coli in beef . . . . . . . . . . . . . 7. Effect of liquid smoke on Staphylococcus . . . . . . . . . . . . . . . . 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ⁎ Corresponding author at: 2650 Young Ave., Fayetteville, AR 72704, USA.Tel.: +1 479 575 7686. E-mail address: [email protected] (P.G. Crandall).

http://dx.doi.org/10.1016/j.meatsci.2014.02.003 0309-1740/© 2014 Published by Elsevier Ltd.

Traditional smoking of foods, especially meats, has been used as a preservation technique for centuries. Wood smoke, in addition to

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preserving food quality with its antioxidant and antimicrobial properties, also imparts a desirable color, flavor and aroma to smoked foods. Application of liquid smoke requires less time than traditional smoking, is more environmentally friendly, and eliminates potentially toxic compounds while still imparting the desired flavors and aromas of traditional smoking. Use of condensates or “liquid smoke” allows the processor to control the concentration of smoke being applied more readily than generating smoke by burning of wood (Suñen, Fernandez-Galian, & Aristimuño, 2001). Liquid smoke is traditionally applied to meat, fish and poultry and it has also been used to impart flavor to non-meat items such as cheese, tofu and even pet food. Because the smoke flavor is concentrated, application of liquid smoke is best suited for use in marinades, sauces or brines or topically to processed meat items such as hot dogs, sausage, ham and bacon (Rozum, 2009). According to an annual poll conducted by The Center for Food Integrity consumers have less confidence in the safety and quality of the food supply and are demanding more all-natural and minimally processed foods with less synthetic chemical additives (Andrews, 2012). Consumers also have increased interest in organic foods because they believe they are healthier, better tasting, or fresher than conventional products (Wier & Calverley, 2002). However, although free of synthetic chemicals, organic and all-natural foods are not exempt from bacterial contamination and may require the addition of an all-natural antimicrobial to insure their safety. All-natural antimicrobials including those derived from plants, animals and bacteria have been shown to be effective in increasing the safety of food products by destroying or limiting the growth of bacterial pathogens. Several reviews have been written on all-natural antimicrobials from bacterial, plant and animal origin (Davidson, Critzer, & Taylor, 2013; Juneja, Dwivedi, & Yan, 2012; Rai & Chikindas, 2011), as well as their use in organic poultry and meat production (Ricke, Van Loo, Johnson, & O'Bryan, 2012; Sirsat, Muthaiyan, & Ricke, 2009). However, these reviews contain little or no information on the use of liquid smoke as an effective all-natural antimicrobial. The review by Holley and Patel (2005) provides a nice overview on the use of liquid smoke as well as its antimicrobial properties in food systems, especially in fish. This review builds on the information presented in Holley and Patel (2005) as well as provides a more detailed and up to date discussion on the effectiveness of liquid smoke as an all-natural preservative in food products. We will examine the effectiveness of liquid smoke, including ranges of microbial susceptibility and factors

affecting antimicrobial action and discuss currently understood mechanisms of action. 2. Generation of liquid smoke from wood pyrolysis Liquid smoke is produced by condensing wood smoke created by the controlled, minimal oxygen pyrolysis of sawdust or wood chips. The wood is placed in large retorts where intense heat is applied, causing the wood to smolder (not burn), releasing the gases seen in ordinary smoke. These gases are quickly chilled in condensers, which liquefies the smoke. The liquid smoke is then forced through refining vats, and then filtered to remove toxic and carcinogenic impurities. Finally, the liquid is aged for mellowness. Fig. 1 shows a schematic of a typical liquid smoke production facility. Factors influencing the flavor and antimicrobial properties of liquid smoke include the temperature of smoke generation, moisture content of the wood as well as the type of wood used to generate the smoke (Simko, 2005). Common woods include hickory and mesquite, but liquid smoke has also been prepared from rice hulls (Kim et al., 2011, 2012), coconut shells (Zuraida, Sukarno, & Budijanto, 2011) and pecan shells (Van Loo, Babu, Crandall, & Ricke, 2012). In general, woods used to generate liquid smoke are roughly comprised of 25% hemicellulose, 50% cellulose, and 25% lignins (Simko, 2005). See Table 1 for information about composition of specific woods. Pyrolysis occurs in four stages starting with water evaporation, followed by decomposition of hemicelluloses, cellulose decomposition and finally decomposition of lignins. Pyrolysis of hemicellulose and cellulose occurs between 180 °C and 350 °C and produces carboxylic acids and carbonyl compounds while lignins are pyrolyzed between 300 °C and 500 °C and generate phenols (Ramakrishnan & Moeller, 2002; Simko, 2005). Smoke flavor compounds, including phenols, are responsible for the smoke flavor and smoky aroma while carbonyl compounds impart a sweet aroma and color to smoked meat products. In addition to carbonyls, acids, and phenols, pyrolysis of wood often generates unfavorable compounds such as polycyclic aromatic hydrocarbons (PAH). Polycyclic aromatic hydrocarbons are families of compounds, some which are naturally occurring, others are the result of incomplete burning and are typically formed at pyrolysis temperatures between 500 °C and 900 °C (Simko, 2005). The level of PAH formation is also influenced by the wood source (Guillén, Sopelana, & Partearroyo, 2000). Some PAH compounds such as benzo(a)pyrene (B(a)P), have

Fig. 1. Flow diagram of typical liquid smoke production.

J.M. Lingbeck et al. / Meat Science 97 (2014) 197–206 Table 1 Wood carbohydrate composition (%). Wood

Cellulose

Hemicellulose

Lignin

Apple Cherry Chestnut Hard maple Hickory Mesquite Red oak White oak

20.7 20.7 21.4 17.2 41.4 8.0 58.6 21.4

6.9 3.4 3.6 17.2 1.7 8.0 3.4 3.6

37.9 13.8 32.1 55.2 24.1 44.0 24.1 39.3

From Chen and Maga (1993).

been shown to cause birth defects when pregnant mice were exposed to more than 300 ppm in food. Foods containing levels greater than 900 ppm led to liver and blood defects in test animals (EPA, 2008). The European Union (EU) regulations limit the amount of PAH allowed in food while the US Food and Drug Administration has not set an upper limit for PAH exposure (Dolan, Matulka, & Burdock, 2010). Because data has shown that it is possible to reach lower levels of B(a)P in smoked meats, acceptable levels of regulated PAH, specifically B(a)P, will drop from 0.005 to 0.002 ppm in 2014 for these foodstuffs sold in the EU ((EC) No. 835/2011). The 2014 level of PAH is set 150,000 times below the levels known to cause birth defects. Although PAH are extremely toxic, they have low water solubility which allows liquid smoke manufacturers to easily separate out these compounds from their finished products using phase separation and filtration techniques. For more information on PAH in liquid smoke see Guillén and Sopelana (2003) and Simon, de la Calle, Palme, Meier, and Anklam (2005). 3. Antimicrobial activity of liquid smoke Different woods generate different levels of phenols, carbonyls and organic acids upon pyrolysis which affect their antimicrobial properties (see Table 2). Liquid smokes from 20 different types of woods including redwood, black walnut, birch, hickory, aspen, white oak, cherry and chestnut were assessed for their antimicrobial properties against Staphylococcus aureus and Aeromonas hydrophila in broth culture (Boyle, Sofos, & Maga, 1988; Sofos, Maga, & Boyle, 1988). It was found that wood smoke generated from Douglas fir sapwood was inhibitory to both the bacterial strains in that it delayed initiation of growth and growth rates of these organisms while smoke from mesquite or lodge pole pine did little to inhibit these pathogens (Boyle et al., 1988; Sofos et al., 1988). In a separate study, liquid smoke from white mangrove, mahogany and abura were shown to inhibit S. aureus and Escherichia coli. Red mangrove and alstonia were able to inhibit S. aureus, but not E. coli, indicating that not only does the type of wood affect the antimicrobial properties of liquid smoke but also that pathogenic organisms have varying degrees of sensitivity to the ingredients of the liquid smoke (Asita & Campbell, 1990). The susceptibility of major food borne pathogens Listeria monocytogenes, Salmonella, E. coli and Staphylococcus to liquid smoke in laboratory media as well as in model meat systems will be reviewed. Table 3 summarizes the in vitro results of liquid smoke against several food borne pathogens while Table 4 is a summary of the antimicrobial activity of liquid smoke against pathogens in model food systems. 3.1. Possible mechanisms of antimicrobial action of liquid smokes Gram-positive and Gram-negative organisms may behave differently to exposure to liquid smoke or fractions of liquid smoke and there may be varying susceptibility within differing strains of the same organism thus making it difficult to identify the mechanism and compounds responsible for microbial inhibition (Sofos et al., 1988). The amount of phenols present in liquid smoke condensates has been reported to be approximately 9.9–11.1 mg/mL (Ramakrishnan & Moeller, 2002).

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Phenolic compounds are known to disturb the cytoplasmic membranes of bacteria and cause the intracellular fluids to leak (Davidson, 1997). Carbonyls have been reported in liquid smokes in amounts of approximately 2.6 to 4.6% (Milly, Toledo, & Ramakrishnan, 2005). The efficacy of carbonyls as an antimicrobial can be inferred based on the 133 different aldehydes and ketones present in liquid smoke (Montazeri, Oliveira, Himelbloom, Leigh, & Crapo, 2013). Carbonyls inhibit microbial growth by penetrating the cell wall and inactivating enzymes located in the cytoplasm and the cytoplasmic membrane (Milly, 2003). Carbonyls act by condensing with the free, primary amino-groups in the polypeptide chains, primarily in the side-chains of basic amino-acids. These aminogroups may be an essential part of active site of the enzyme, or they may function to bind the substrate by hydrogen-bonding (Painter, 1998). Even if the carbonyls cannot access the interior of a microbial cell, they can still inhibit growth by interfering with the uptake of nutrients. There are 3 proposed mechanisms involved in this interference, termed A, B and C types. Type A inhibition entails the sequestration of amino acids or ammonia by condensation with the carbonyl compounds, thus lowering the effective concentration in the growth medium (Painter, 1998). Some carbonyls, including α-keto-carboxylic acids, enediols, 3-hydroxyketones and 1,3-diones, can also remove essential metal cations by chelation (Painter, 1998). Type B inhibition is active against putrefactive bacteria or molds which excrete exocellular proteases or glycanases to break down proteins or glycans into small fragments which can be taken up by the cells. Carbonyl compounds either inactivate the enzymes as described for type A inhibition, or by immobilizing them in insoluble particles or a three-dimensional polymeric network which physically isolates them from their substrates (Painter, 1998). Type C inhibition involves direct chemical modification of a substrate itself, so that it becomes less accessible or susceptible to the microbial enzymes (Painter, 1998). Antimicrobial activity of phenols and carbonyls is discussed further in the following sections.

3.2. Activity of phenols Early studies on antimicrobial activity of liquid smoke on L. monocytogenes and other pathogens attributed the activity to phenolic compounds. Faith, Yousef, and Luchansky (1992) evaluated several liquid smoke fractions and common smoke phenols for antilisterial activity. A three strain cocktail of L. monocytogenes including isolates Scott A, V7 and 101 M was incubated in hot dog exudate containing 0.2% or 0.6% of the liquid smoke fraction CharSol Supreme (Red Arrow Company, Manitowoc, WI) at 25 °C for up to 114 h. L. monocytogenes levels decreased in liquid smoke treated samples with D-values of 36 h for exudate containing 0.2% liquid smoke and 4.5 h for 0.6% treated samples. When eleven individual smoke phenols were evaluated for their antilisterial activity against L. monocytogenes Scott A in Tryptose Broth, only isoeugenol was able to delay growth. The addition of acetic acid further enhanced inactivation due to its bactericidal activity (Young & Foegeding, 1993). Suñen (1998) measured the antimicrobial activity of seven different smoke fractions used in the Spanish food industry against L. monocytogenes and other pathogenic microorganisms. The MIC values were determined using an agar dilution technique with smoke extracts prepared in buffer, pH adjusted to 7 and used at concentrations up to 2× the maximum level recommended by the manufacturer. Antimicrobial activity was directly related to phenol concentrations in that extracts possessing a high concentration of phenols (94 to 153 mg/kg) corresponded to the fractions with the strongest antimicrobial properties. However, the fraction containing the highest concentration of phenols was not the most active. The most active fraction, in addition to having a high level of phenols (21 mg/kg), also contained the highest concentration of acids (34 mg/kg) which may have contributed to the antimicrobial action of this fraction.

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Table 2 Chemical properties of commercial liquid smokes. Liquid smoke tested

Manufacturer

pH

Titratable acidity as percent acetic acid (wt/wt)

Phenol content (mg/mL)

Carbonyl content (g/100 mL)

References

Charsol Supreme

2.1–2.6

14–16

18–25

20–25

Vitt et al. (2001)

2.2–2.8 No data 2.1–2.6 2.0–2.4 3.0–4.0 7.3–8.1 Not listed 2.5–3.3

14–16 No data 10.5–12 13–15 4.0 maximum No data 10–11 3.5–5.6

15–23 25–30 10–15 9–14 37–42 No data 9–16 mg/g 1.7 maximum

24–30 No data 12–13 16–20 No data No data 12–16 19–22

AM-3

Red Arrow Company (Manitowoc, WI) Red Arrow Company Red Arrow Company Red Arrow Company Red Arrow Company Red Arrow Company Red Arrow Company Red Arrow Company Mastertaste, Inc. (Monterey, TN) Mastertaste, Inc.

4.25–4.85

1.8–2.1

0.3–0.8

16–20

AM-3

Mastertaste, Inc.

4.3

2.2

Not detected

Not listed

List-A-Smoke

Mastertaste Inc.

2.0–2.5

7.0–8.0

1.75–4.25

5–8

Code 10-Poly

Mastertaste Inc.

2.3

10.3

3.22

Not listed

AM-10

Mastertaste Inc.

4.2

2.3

Not detected

Not listed

1291

Mastertaste Inc.

5.7

0.7

0.1

Not listed

Code V

Hickory Specialties (Brentwood, TN) Mastertaste Inc. Mastertaste Inc. Mastertaste Inc. Mastertaste Inc. Mastertaste Inc. Mastertaste Inc. Mastertaste Inc. Mastertaste Inc. Mastertaste Inc. Mastertaste Inc. Mastertaste Inc. Mastertaste Inc. Mastertaste Inc.

2.0

6.8–7.8

1.4–4.0

2.0–7.0

Paranjpye et al. (2004) Faith et al. (1992) Vitt et al. (2001) Vitt et al. (2001) Vitt et al. (2001) Vitt et al. (2001) Paranjpye et al. (2004) Gedela, Escoubas, et al. (2007) and Gedela, Gamble, et al. (2007) Gedela, Escoubas, et al. (2007) and Gedela, Gamble, et al. (2007) Montazeri, Himelbloom, et al. (2013); Montazeri, Oliveira, et al. (2013b) Gedela, Escoubas, et al. (2007) and Gedela, Gamble, et al. (2007) Montazeri, Himelbloom, et al. (2013) and Montazeri, Oliveira, et al. (2013) Montazeri, Himelbloom, et al. (2013) and Montazeri, Oliveira, et al. (2013) Montazeri, Himelbloom, et al. (2013) and Montazeri, Oliveira, et al. (2013) Estrada–Muñoz et al. (1998)

2–3 6.1–7.0 2–3.0 4.1–5.0 2–3.0 2–3.0 5.1–6.0 6.1–7.0 6.1–7.0 2–3 3–3.2 4.1–4.3 6–7.4

4.5–5.9 0–1.4 6.0–7.4 3.0–4.4 6.0–7.4 6.0–7.4 1.5–2.9 0–1.4 0–1.4 4.5–5.9 3.4–3.7 1.5–1.8 0–1.4

0–5 0–5 0–5 20.1–25.0 0–5 0–5 0–5 0–5 0–5 0.3–0.6 0.3–0.6 0.3–0.6 0.3–0.6

151–200.9 101–150.9 101–150.9 0–50.9 101–150.9 51–100.9 51–100.9 101–150.9 51–100.9 151–200 120–132 110–120 100–110

Milly et Milly et Milly et Milly et Milly et Milly et Milly et Milly et Milly et Milly et Milly et Milly et Milly et

Charsol Supreme Charsol Supreme Charsol H-10 Charsol LFB Supreme Poly Aro-Smoke P-50 CharOil C-10 Zesti B

F1 F2 F3 F4 F5 F6 F7 F8 F9 S1 S2 S3 S4

Other studies suggest that the antimicrobial properties of liquid smoke are not attributed to their phenol composition. Smoke fractions from the Spanish food industries (L1, L2, L3 and S) were evaluated for their antimicrobial properties at low temperature against A. hydrophila, Yersinia enterocolitica and L. monocytogenes (Suñen et al., 2001). The pH of the liquid smokes was adjusted to neutrality and used at the maximal concentration recommended by the manufacturer (0.4 to 4%) and were subsequently incubated with 4 to 5 log10 CFU/mL of pathogen for up to 21 days at 4 °C in broth culture. All four extracts were effective at eliminating or suppressing growth of A. hydrophila after 21 days. Fractions L1 and S were bacteriostatic against Y. enterocolitica while fractions L2 and L3 were ineffective at reducing Y. enterocolitica numbers. The most effective fraction against L. monocytogenes was fraction S (3.2 log10 CFU/g reduction after 21 days) while fractions L1 and L2 exhibited slight inhibition by reducing cell counts by 2.1 log10 CFU/mL and 1.9 log 10 CFU/mL, respectively. Fraction L3 did not inhibit L. monocytogenes growth. The most active fraction, fraction S, was lowest in phenol concentration (23 mg/kg), but high in acid concentration (23 mg/kg), while the least effective fraction L3 contained a high level of phenols (99 mg/kg) suggesting that phenol concentration is not indicative of the antimicrobial activity of liquid smoke. 3.3. Activity of carbonyls Carbonyl compounds have also been suggested to contribute to the antimicrobial properties of liquid smoke. Milly et al. (2005) determined minimal inhibitory concentration (MIC) values of low phenolic liquid smoke fractions against Listeria innocua M1 and several Gramnegative bacteria. The MIC values were determined for nine liquid

al. (2005) al. (2005) al. (2005) al. (2005) al. (2005) al. (2005) al. (2005) al. (2005) al. (2005) al. (2008) al. (2008) al. (2008) al. (2008)

smoke fractions (F1–9), eight of which had a phenol concentration of 0 to 5 mg/mL so that the antimicrobial effect of the non-phenolic compounds (i.e. carbonyl compounds and organic acids) could be evaluated. The fraction F1 was found to be the most effective against the Gramnegative cocktail mixture with an MIC value of 1.5%. Fraction F1 had a pH range of 2 to 3 and had the highest carbonyl content at 151 to 200.9 mg/mL. Fractions F3, F5 and F6 had MIC values of 2%, were similar to F1 in pH and phenol level, but were lower in carbonyl content (101 to 150.9 mg/mL for F3 and F5; 51 to 100.9 mg/mL for F6). Fraction F4 which had a phenol content of 20.1 to 25.0 mg/mL, a pH of 4.1 to 5.0 and a carbonyl content of 0 to 50.9 mg/mL produced an MIC value of 3%. The least effective fractions, F7 (MIC 5%) and F9 (MIC 9%) contained the same carbonyl level (51 to 100.9 mg/mL) but differed slightly in pH. Fraction F7 with a lower pH range of 5.1 to 6.0 compared to that of F9, pH 6.1 to 7.0, exhibited significantly improved antimicrobial properties. In assessing the antimicrobial properties of different liquid smoke fractions, these data demonstrate the importance of knowing the concentrations of carbonyl compounds, acids and the pH values of these products. Three refined liquid smoke fractions along with a full strength fraction, Code 10-Poly (Kerry Ingredients and Flavors, Monterey, TN) were tested in vitro for antilisterial properties. The three refined liquid smoke fractions 1291, AM-10 and AM-3 possessed little to no phenols. Using a disk diffusion assay it was determined that all fractions with the exception of 1291 were effective against L. innocua. The largest inhibition zones were seen with Code 10-Poly which had a pH of 2.3, 10.3% titratable acidity and total phenol content of 3.22 mg/mL. Fractions AM10 and AM-3 were also able to reduce the counts of L. innocua in culture media tests, despite their having undetectable phenol levels suggesting

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Table 3 Efficacy of liquid smoke as an antimicrobial against select bacteria in vitro. Fraction

Concentration

Bacteria

Method

Results

Reference

Aro Smoke P-50, CharOil, Charsol H-10, Charsol LFB Supreme Poly, Charsol Supreme Nine commercial LS from Mastertaste F1–F9

0–100%

Listeria monocytogenes ATCC 19115 Listeria innocua ATCC 33090

MIC values determined by a broth dilution method in BHI

Vitt et al. (2001)

10–0.5%

L. innocua M1

MICs determined by a broth dilution method

100% 75% 50% 25% Up to 2× manufacturers recommended concentration 0.4% L1 0.6% L2 4% L3 1% S 1–0.2%

L. innocua ATCC 33090

Disk diffusion

L. monocytogenes CECT 932, L. innocua CECT 4030

Agar dilution

L. monocytogenes CECT 932

Flask containing LS were inoculated and incubated at 4 °C.

MIC values Aro Smoke P-50 = 2.5% CharOil = 5% Charsol H-10 = 1.25% Charsol LFB Supreme Poly = 1.25% Charsol Supreme = 0.5% MIC Values F1 = 1.25% F2 = 2% F3 ≥ 2% F4 = 2% F5 = 2% F6 = 2% F7 = 4% F8 = 2% F9 = 6% Larger zones of inhibition were seen with increasing LS concentration. Code 10-Poly was the most effective fraction at all conditions while 1291 was least effective. Fractions L1, L4, S1 and S3 were not effective. Fraction L2 was inhibitory at 0.6%, L3 at 8% for L. innocua and N8% for L. monocytogenes, S2 at 1% L1 and reduced Listeria by ~2 log CFU/mL S reduced Listeria by 3.2 log CFU/mL and L3 was ineffective at reducing Listeria

Code 10-Poly AM-3 AM-10 1291 Seven fractions from Spanish food industry L1, L2, L3, L4, S1, S2, and S3 (all pH 7) Four fractions from Spanish food industry L1, L2, L3, and S Rice hull smoke extract

Four water based commercial samples and four concentrated extracts from commercial sources Nine commercial LS from Mastertaste F1–F9

96–0.375%

10–0.5%

Lab generated from pyrolysis 1%–0.05% of beachwood chips

96–0.375% Four water based commercial samples and four concentrated extracts from commercial sources Lab generated from pyrolysis 1%–0.05% of beachwood chips

Four water-based commercial samples and four concentrated extracts from commercial sources

96–0.375%

Salmonella Typhimurium MICs were determined in ATCC 14028 nutrient broth with phenol in a microtiter plate Salmonella Enteritidis MICs were determined by a PT 13A microdilution method in microtiter plates at 37 °C.

MIC of rice hull smoke condensate = 0.822%

Milly et al. (2005)

Montazeri, Himelbloom, et al. (2013) and Montazeri, Oliveira, et al. (2013) Suñen (1998)

Suñen et al. (2001)

Kim et al. (2012)

Van Loo et al. (2012) MIC values were 6% for 3 of the 4 water based samples and 12% for the fourth sample. MICs were 0.5% for 3 of the 4 concentrated extracts; the final concentrated extract had an MIC value of 3%. Milly et al. (2005) MIC Values Gram negative cocktail of MIC values were determined F1 = 1.25% by a broth dilution method Salmonella Muenster, F2 ≥ 2% at 37 °C. Salmonella Seftenburg, F3 = 2% Salmonella Typhimurium F4 = 3% and Escherichia coli 8677 F5 = 2% F6 = 2% F7 = 5% F8 = 2% F9 = 9% E. coli ATCC 14948-K12 Smoke condensates were mixed Growth was delayed by 1 day at 0.125%, by Fretheim et al. (1980) with nutrient broth agar followed 1.5 days at 0.166% and no growth was by spread plating of E. coli. Plates observed at concentrations above 0.25% were incubated at 30 °C for 2 weeks. MIC were 6% for the water based samples Van Loo et al. (2012) E. coli O157:H7 MICs were determined by a and 0.5% for 3 of the 4 concentrated ATCC 43888 microdilution method in extracts, the final concentrated extract had microtiter plates an MIC value of 6% Staphylococcus aureus Smoke condensates were mixed No growth was observed at concentrations Fretheim et al. (1980) ATCC 25923 with nutrient broth agar followed above 0.1% by spread plating of E. coli. Plates were incubated at 30 °C for 2 weeks. MIC were 6% for the water based samples Van Loo et al. (2012) MICs were determined by a S. aureus ATCC 25923 and ~0.38% for concentrated extracts. microdilution method in ATCC 6538 microtiter plates Mu50, MRSA Col, MRSA

that organic acids or carbonyls rather than phenols were involved in the antilisterial properties of liquid smoke products they tested (Montazeri, Himelbloom, Oliveira, Leigh, & Crapo, 2013). Milly, Toledo, and Chen (2008) used high-end turkey rolls which were whole parts of turkey breast formed to have no more than 40% binders and broth added, low-end turkey rolls which were minced turkey breast parts with up to 60% binders and broth added before forming and cooking, as well as roast beef cuts to evaluate low phenolic liquid

smoke fractions for antimicrobial properties. Turkey rolls and roast beef cuts were first inoculated with L. innocua M1, subsequently treated with four different liquid smoke fractions (Mastertaste Inc., Brentwood, TN), vacuum packed and refrigerated at 4 °C for up to four weeks of storage. Samples were treated with 2 log10 CFU/25 cm2 of L. innocua M1 and evaluated at 2 and 4 weeks of storage. Samples treated with smoke fractions S1, S2 and S3 had levels below the detection limit for L. innocua at all the sampling times. Samples treated with S4 remained positive after

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Table 4 Efficacy of liquid smoke as an antimicrobial against several bacteria in food systems. Liquid smoke fraction

Liquid smoke concentration

Strain

Processing parameters

Result

Reference

CharSol Supreme

0.6% and 0.2%

Listeria monocytogenes Scott A, V7, 101 M

60%

Listeria innocua

AM-3 AM-10

0.9%

L. innocua ATCC 33090

Listeria counts decreased after 3 days. Estimated D-values are 4.5 h at 0.6% and 36 h for 0.2% A 15 s dip resulted in a 3 log reduction greater reductions were seen with longer dip times Both fractions reduced L. innocua to b2 log CFU/g after 2 weeks.

Faith et al. (1992)

Charsol Supreme

Hot dog exudates containing LS were inoculated with Listeria and incubated at 25 °C for up to 114 h. 100 g chum salmon samples were brined, dipped for 5 min in LS, inoculated and dried in a smoke house Salmon strips were treated with LS, inoculated, vacuum sealed, and stored at 4 °C for up to 49 days.

Fractions from Spanish food industries L1, L2, L3 and S

100%, 1 min dip

L. monocytogenes CECT 932

Filets were brined, inoculated, treated with LS and stored at 4 °C for 21 days

CharSol C-10

100% 50% 25% 10% dipped

L. monocytogenes strains 4–121 and 1455

Salmon filets were brined, treated with LS, inoculated and heat processed.

Zesti-B

100% dipped for 1 or 5 s or sprayed

L. monocytogenes Scott A-2, V7-2, 39-2, 383-2

Zesti-B

100% sprayed

L. monocytogenes Scott A-2, V7-2, 39-2, 383-2

Frankfurters formulated without lactate and diacetate were dipped or sprayed in LS, inoculated, vacuum packed and stored at 1.7 °C for 10 weeks. Frankfurters formulated without lactate and diacetate were sprayed in LS, inoculated, vacuum packed and stored at 6.1 °C for 10 weeks.

Fractions L1 and L2 immediately reduced L. m to below detectable levels. Fraction S slowly reduced Listeria and was below detectable levels by 21 days. Fraction L3 did not show any inhibitory affects. Internal minimum lethality temperatures were N82.8 °C in untreated salmon steaks, 67.2 °C generated smoke was applied throughout the entire smoking process or N 80 °C when smoke was only applied during the last half of the process, 58.9 °C when dipped in 100% CharSol C-10 and 62.8 °C, 68.9 °C and 72.8 °C with 50%, 25% and 10% LS. L. m. was reduced to undetectable numbers after 4 weeks with all treatments

Zesti-B AM-3

100%, 1 s dip

L. monocytogenes Scott A-2, V7-2, 39-2, 383-2

AM-3

100% sprayed to equal 1.8 mL per frank

CharSol-10

100%, dipped

Zesti Smoke

Formulated into franks at 10, 5, and 2.5% (wt/wt)

L. monocytogenes ARS V67, ARS V72, ARS V113, ARS V125, ARS V105, LCDC 81–861 L. monocytogenes LCDC 81–861, M1, M2, M5, C6, serotype 4b derived ATCC 19115 L. monocytogenes

Zesti-B

100%, 1 s dip

L. monocytogenes Scott A-2, V7-2, 39-2, 383-2

Four commercial LS from Mastertaste S1, S2, S3 and S4

100%, 60s dip

L. innocua M1

Four commercial LS from Mastertaste S1, S2, S3 and S4

100%, 60s dip

L. innocua M1

Frankfurters were formulated without lactate or diacetate, dipped in LS, inoculated, vacuum sealed, heat pasteurized for 1 min at 37 °C, chilled and stored at 6.1 °C for 10 weeks Frankfurters were formulated without lactate or diacetate, sprayed with LS, inoculated, sealed and stored at 4 °C for 140 days. Frankfurters were inoculated, dipped in LS, vacuum packed and stored at 4 °C for 72 h. Frankfurters were inoculated, vacuum packed and stored at 4 °C for 12 weeks.

Deli turkey formulated without lactate or diacetate were dipped in LS, inoculated, vacuum packed, heat pasteurized 60s at 93.3 °C, chilled and stored at 6.1 °C for 10 weeks High end turkey rolls (containing less-than 40% binders and broth added), low end turkey rolls (up to 60% binders and broth added) were dipped in LS, inoculated with L. i. in a marked 25 cm2 area, vacuum sealed and stored at 4 °C for 4 weeks. Roast beef cuts were dipped in LS, inoculated with L. i. in a marked 25 cm2 area, vacuum sealed and stored at 4 °C for 4 weeks.

Vitt et al. (2001)

Montazeri, Himelbloom, et al. (2013) and Montazeri, Oliveira, et al. (2013) Suñen et al. (2003)

Poysky et al. (1997)

Gedela, Escoubas et al. (2007)

L. m. was reduced to undetectable levels after 1 week at log 1 inoculum levels, at log 2 the levels declined slowly and were undetectable after 4 weeks. The log 3 inoculum slowly increased over 10 weeks to 0.8 logs higher that inoculum while control increased by 7 logs L. m. levels were undetectable after 3 weeks with both Zesti-B and AM-3 fractions

Gedela, Escoubas et al. (2007)

L. m. was reduced by nearly 3 logs after 30 days and continued to slowly decline for up to 130 days

Martin et al. (2010)

L. m. was reduced initially by at least 1 log and was undetectable after 72 h.

Messina et al. (1988)

Frankfurters formulated with 2.5% LS saw a 0.5 log CFU/mL reduction when inoculated at high L. m. levels and ~2 log CFU/mL when inoculated with lower levels of Listeria. Frankfurters formulated with 5% LS saw greater reductions and were listericidal after 6 weeks. 10% LS was listericidal after 4 weeks. L. m. levels were reduced by 2 logs after 2 weeks and remained low after 10 weeks

Morey et al. (2012)

L. i. levels were reduced to undetectable levels after 2 and 4 weeks of storage on both high and low end turkey rolls with all LS samples tested, with the exception of S4 on low end rolls. Only a small reduction was seen after two weeks (0.53 log CFU) but was not detected after 4 weeks. L. i. levels were reduced to undetectable levels after 2 and 4 weeks of storage with all fractions tested.

Milly et al. (2008)

Gedela, Gamble, et al. (2007)

Gedela, Gamble, et al. (2007)

Milly et al. (2008)

J.M. Lingbeck et al. / Meat Science 97 (2014) 197–206

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Table 4 (continued) Liquid smoke fraction

Liquid smoke concentration

Strain

Processing parameters

Result

Reference

Code V

8%

Escherichia coli O157:H7

Growth was reduced by 2.3 log CFU/g

Estrada-Muñoz et al. (1998)

C10

Staphylococcus aureus

Charsol Supreme

75%

No reduction after 5 days None detected after 5 days None detected after 3 days None detected after 3 days 1.7 log reduction after 3 h compared to control No reduction after 15 h compared to control, however no enterotoxins were present in LS treated samples 0.74 log reduction and no enterotoxins were detected

Paranjpye et al. (2004)

Charsol Supreme

25% 50% 75% 100% 1.25%

Beef trimmings were inoculated and treated with LS after which they were ground and formed into 70–90 g patties. Patties were packaged, and stored at 4 °C for 3 days. Brined strips were dipped for 1 min, inoculated, and processed at 30 °C for 3–5 days

Cocktail mixture of S. aureus ATCC 27664 (enterotoxin E), ATCC 13565 (enterotoxin A), and ATCC 12660 Cocktail mixture of S. aureus ATCC 27664 (enterotoxin E), ATCC 13565 (enterotoxin A), and ATCC 12660

Inoculated, ground pork bellies were treated with LS and heated to 50 °C over 6 h followed by cooling to 7.2 °C for 3–15 h 50 g pieces were inoculated and heated to 50 °C over 6 h. LS was sprayed at hours 4 or 5. Sample were cooled to 7.2 °C for 15 h.

both two and four weeks of cold storage. All liquid smoke fractions tested contained similar phenol concentrations (0.3 to 0.6 mg/mL) while carbonyl concentrations ranged from 110 to 200 mg/mL for fractions S1, S2 and S3 and from 100 to 110 mg/mL for S4. Fraction S4 was also lower in acidity (0 to 1.4%) and higher in pH (6 to 7.4) than the other samples (acidity range1.5 to 5.9% and pH 2 to 4.3). Again, the data suggest that phenols contribute little to the bacteriostatic properties of liquid smoke and that carbonyl compounds and acidity levels are important in assessing the antimicrobial properties of liquid smoke products. The authors also reported that food product composition also plays an important role in bacterial survival in that the lower end turkey rolls were apparently able to support bacterial growth better than the high end turkey rolls or roast beef cuts (Milly, Toledo, & Chen, 2008). Liquid smoke has demonstrated ability at reducing foodborne pathogens including L. monocytogenes, E. coli, S. aureus and Salmonella in RTE foods and other food products. The exact mechanism of action is unknown, and both phenolic compounds and carbonyl compounds are thought to contribute to its antimicrobial properties. Table 4 lists the chemical components of several commercial liquid smokes and other liquid smoke fractions discussed below. 4. Antimicrobial activity of liquid smoke against Listeria L. monocytogenes is a Gram-positive food borne bacterium. Listeria has the ability to grow to infective doses at low temperatures, high salt concentrations, and under acidic and microaerophilic conditions, which make it difficult to control on many ready-to-eat (RTE), refrigerated foods. The infective dose of L. monocytogenes is dependent upon several factors including the bacterial strain and susceptibility of the individual to the bacterium, but it can be as low as 1000 bacteria (SchmidHempel & Frank, 2007). Consumption of an infective dose of Listeria may lead to the food borne infection, listeriosis, in humans especially in older adults or persons who are immunocompromised as well as pregnant women and newborns. Symptoms of listeriosis include fever, muscle aches, nausea or diarrhea and may result in meningitis, premature labor, miscarriage or even death. In the US, 1651 cases of listeriosis and 292 deaths or fetal losses as a result of Listeria infection were reported between 2009 and 2011 (CDC, 2011). 4.1. In vitro effects on Listeria Pittman et al. (2012) studied the effects of individual stressors of cold smoking including freezing, thawing, salt exposure, exposure to liquid smoke and cold storage on virulent and avirulent Listeria. Two

Taormina and Bartholomew (2005)

Taormina and Bartholomew (2005)

strains, virulent HCC7 and avirulent HCC23, both in mid-log growth were examined by transmission electron microscopy (TEM) to identify changes in cell wall integrity due to exposure to a simulated cold smoking process in brain heart infusion (BHI) culture medium. Bacteria were subjected to a sequence of stresses: a freeze thaw cycle (−20 °C for 2 h followed by room temperature thawing for 1 h); exposure to 6% NaCl at 30 °C for 1 h, exposure to 0.6% liquid smoke at 30 °C for 1 h and finally anaerobic storage at 2 °C for 16 h. The authors found that the avirulent strain was more susceptible than the virulent strain to all of the above conditions of the cold-smoking process; the virulent strain was only significantly affected by application of liquid smoke and anaerobic storage. The results suggested that these two different strains utilize different mechanisms to adapt to the above stresses. More importantly, they found that both strains can survive the effects of this simulated cold smoking process, emphasizing the need for additional effective but all-natural antimicrobial interventions to control this pathogen (Pittman et al., 2012). 4.2. Antilisterial effects in ready-to-eat meats Ready-to-eat (RTE) meats such as luncheon meat and frankfurters treated with liquid smoke have shown reduced levels of L. monocytogenes. Beef franks were inoculated by immersion into a 3 log10 CFU/mL cocktail of six strains of L. monocytogenes. After air drying, frankfurters were dipped in full strength CharSol-10, air dried, vacuum packed and stored at 4 °C. After 72 h, frankfurters treated with CharSol-10 exhibited a 3 log10 reduction in L. monocytogenes counts compared to control samples whose bacterial count remained unchanged (Messina, Ahmad, Marchello, Gerba, & Paquette, 1988). Spray application of liquid smoke was also successful in reducing L. monocytogenes levels on frankfurters formulated without the typical Listeria growth inhibitors of sodium lactate and sodium diacetate. Frankfurters were sprayed with the commercial liquid smoke fraction AM-3 followed by inoculation with a six strain cocktail of L. monocytogenes at a concentration of 5 log10 CFU/mL. Frankfurters were stored at 4 °C and bacterial levels were monitored at 2, 24 and 48 h. Liquid smoke treated samples showed a continuous decline of L. monocytogenes with time until no detectable levels were seen at 48 h (Martin et al., 2010). An extended shelf life study in which lactate/ diacetate free frankfurters were sprayed with AM-3 and inoculated with a cocktail of L. monocytogenes, showed that liquid smoke could suppress L. monocytogenes on frankfurters vacuum packed and stored at 4 °C for up to 130 days (Martin et al., 2010). In a similar study lactate/diacetate free frankfurters were inoculated with 3 log10 CFU/mL of a four strain cocktail of L. monocytogenes, sprayed with the liquid

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smoke fraction Zesti B (Mastertaste, Monterey, TN) and stored at the abuse temperature of 6.1 °C for up to 10 weeks. Levels of L. monocytogenes decreased after one week of storage, but thereafter increased by only 0.8 log10 CFU/g above the initial inoculum level by the end of the 10 weeks as compared to a 7 log10 increase in untreated franks (Gedela, Escoubas, & Muriana, 2007). The authors also tested the effectiveness of a dip application of liquid smoke to frankfurters. Retail franks (containing lactate and diacetate) which had previously been shown to permit growth of L. monocytogenes at 1.7 °C after 6 weeks were dipped in Zesti-B liquid smoke for 1 to 90 s followed by inoculation with 1 log10 CFU of a Listeria cocktail. Viable counts were reduced to below the detection level after 4 weeks of storage for all dip times tested whereas untreated controls showed a greater than 4 log10 increase (Gedela, Escoubas, et al., 2007; Gedela, Gamble, et al., 2007). A combination of post-process heat pasteurization and a liquid smoke containing reduced acid levels and low phenolic concentrations was able to reduce L. monocytogenes on frankfurters and deli turkey. Of two smoke extracts tested, the product coded as Zesti-B had 3.5 to 5.6% acidity, a pH 2.5 to 3.3, with a maximum of 1.7 mg/mL of phenols and a carbonyl concentration of 19 to 22 g/100 mL while the product coded as AM-3, a fraction lighter in color and reduced smoke flavor, contained 1.8 to 2.1% acidity, a pH 4.25 to 4.85, with a 0.3 to 0.8 mg/mL phenol concentration and a carbonyl concentration of 16 to 20 g/100 mL. Frankfurters were formulated without lactate/diacetate and dipped for 1 s into Zesti-B or AM-3, inoculated with a four strain mixture of L. monocytogenes at 5 log10 CFU/sample, vacuum packed, pasteurized at 73.9 °C for 1 min and stored at the mild abuse temperature of 6.1 °C for up to 10 weeks. No Listeria was detected immediately after processing and L. monocytogenes levels remained below detectable limits during 10 weeks of storage. Turkey breast chubs formulated without lactate/diacetate or nitrate underwent similar treatment. Samples treated with liquid smoke 60 to 30 s post-process pasteurization resulted in 2 to 3 log 10 reductions of Listeria by week two. L. monocytogenes levels remained consistently low for the remainder of the experiment (Gedela, Gamble, Macwana, Escoubas, & Mariana, 2007). The use of liquid smoke as an ingredient, as opposed to topical application, has also been shown to be effective in reducing L. monocytogenes. Frankfurters, formulated with 2.5%, 5% or 10% (wt/wt) of the liquid smoke fraction Zesti Smoke (Kerry Ingredients and Flavors, TN), were inoculated with either 4 or 8 log10 CFU/mL of L. monocytogenes, vacuum packed and stored at 4 °C for 12 weeks. At select sampling times the frankfurters were rinsed and the rinsates were plated on Modified Oxford agar to detect the presence of Listeria. The liquid smoke incorporated as an ingredient in frankfurters was able to suppress the growth of L. monocytogenes inoculated at both high (8 log10 CFU/mL) and low inoculation levels (4 log10 CFU/mL) at its lowest concentration (2.5%) compared to untreated samples. Inoculated frankfurters not formulated with liquid smoke permitted growth of L. monocytogenes up to more than 8 log10 CFU/mL in 12 weeks for both high and low inoculation levels. The addition of 2.5% liquid smoke to the frankfurters reduced L. monocytogenes levels compared to control levels to 6.2 log10 CFU/mL for the low inoculation level and 7.47 log10 CFU/mL for the high level inoculation. Further suppression was seen with increasing liquid smoke concentrations. However, frankfurters containing 10% liquid smoke were somewhat less acceptable in sensory panel tests as compared to frankfurters containing 2.5% or 5% liquid smoke (Morey, Bratcher, Singh, & McKee, 2012). 4.3. Genetic basis of the antimicrobial effects of liquid smoke on Listeria Liquid smoke has a demonstrated success at limiting the growth of Listeria. Using proteomics Guilbaud et al. (2008) examined the effects of liquid smoke on L. monocytogenes. Listeria was exposed to 30 μg/mL of phenol for 2 h which resulted in the upregulation of ClpP-2, a subunit of the general stress protein ClpP which is required for virulence

expression in L. monocytogenes. Also affected were proteins involved in metabolic pathways including Lmo355 and Lmo2829, proteins involved in membrane bioengineering and lipid metabolism, suggesting that liquid smoke affects the synthesis of the cell membrane. In addition liquid smoke reduced the hemolytic activity of Listeria and may reduce the virulence of Listeria. This may partially explain the extremely limited number of food borne outbreaks involving cold-smoked fish. 5. Effects of liquid smoke on Salmonella spp. Salmonella is a Gram-negative food borne bacterium. Salmonella contamination is especially prevalent in raw poultry and eggs, but the organism can contaminate a wide variety of different foods including dairy, meat and raw vegetables and fruits and animal feeds (Maciorowski, Jones, Pillai, & Ricke, 2004; Foley et al., 2011; Howard, O'Bryan, Crandall, & Ricke, 2012; Finstad, O'Bryan, Marcy, Crandall, & Ricke, 2012). The infectious dose of Salmonella is approximately 5 log10 organisms (Schmid-Hempel & Frank, 2007). Symptoms of Salmonella infection include fever, cramps, vomiting and diarrhea. Generally symptoms only last a few days and leave no lasting effects; however, in some cases life threatening complications may arise (Ricke, Koo, Foley, & Nayak, 2013). A liquid smoke generated from the pyrolysis of rice hulls was shown to be bactericidal against Salmonella Typhimurium in a disk diffusion assay at concentrations ranging from 0.1% to 1%. Larger zones of inhibition were seen at higher rice hull liquid smoke concentrations. The MIC was 0.822% (v/v) as determined by a broth dilution assay. Interestingly, rice hull liquid smoke also enhanced survival of mice infected with a lethal dose of Salmonella. The Balb/c mice were fed a commercial chow diet containing 1% rice hull liquid smoke for 14 days after which they were injected with 5 log10 CFU of Salmonella Typhimurium. Mice fed rice hull liquid smoke survived twice as long (about14 days) compared to mice that were fed the untreated diet who survived approximately 7 days (Kim et al., 2012). Van Loo et al. (2012) determined the MICs of seven commercial liquid smoke samples from three commercial manufacturers against S. Typhimurium using a microdilution method. The liquid smoke samples were separated into two groups. Group I contained water based liquid smokes extracted from woods of hickory, mesquite, apple and pecan. Group II were concentrated liquid smokes extracted from hickory and mesquite. Group II liquid smokes were more effective at reducing Salmonella with MIC values ranging from 0.5% to 4%. Group I was less effective and produced MIC values from 6% to 12%. Similar results were seen with nine commercial liquid smoke samples (Mastertaste, Brentwood, TN) on a cocktail of Gram-negative bacteria consisting of S. Muenster, S. Seftenburg, S. Typhimurium and E. coli. MIC values ranged from 1.5% to 9% (Milly et al., 2005). 6. Effects of liquid smoke on E. coli E. coli is a Gram-negative bacterium found in the intestines of most animals including humans. Although most strains of E. coli are innocuous, some strains such as the Shiga toxin producing strain O157:H7 (STEC) and non O157:H7 Shiga toxin producing strains can cause illness if ingested (Jaeger, 1999). E. coli O157:H7 is infectious at concentrations as low as 10 organisms (Schmid-Hempel & Frank, 2007). Complications arising from STEC strains of E. coli infections include cramps, vomiting, bloody diarrhea, and blood in the urine, with severe infections leading to kidney failure (Belongia et al., 1991). Contamination with STEC strains of E. coli is often found in undercooked meat especially beef, unpasteurized dairy products and raw fruits and vegetables. 6.1. In vitro effects of liquid smoke on E. coli The antimicrobial properties of smoke condensates in concentrations ranging from 0.05% to 1% were evaluated against STEC strains of

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E. coli. These strains were able to grow in up to 0.1% smoke while at 0.125%, growth was delayed by 1 day and at 0.25% no growth was observed (Fretheim, Granum, & Vold, 1980). Values for MICs for several commercial liquid smokes against E. coli O157:H7 were reported by Van Loo et al. (2012). Effective concentrations ranged from 6% to as low as 0.75% depending upon the commercial brand of liquid smoke.

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flavor and also has inhibitory effects on food borne pathogens. The preservative effect of liquid smokes is achieved by antimicrobial and antioxidant compounds such as aldehydes, carboxylic acids and phenols. Liquid smoke also has several advantages over traditional smoking techniques including ease of application, speed of smoking process, good reproducibility of desired characteristics obtained in the final smoked food, and omission of hazardous polycyclic aromatic hydrocarbons.

6.2. Effects of liquid smoke on E. coli in beef The effects of liquid smoke on E. coli in a model meat system have been reported. Beef trimmings were inoculated with 7 log10 CFU/g of the STEC strain E. coli O157:H7 and treated with a final concentration of 8% of the liquid smoke fraction Code V (Hickory Specialties, Brentwood, TN). Trimmings were then ground, formed into patties, heat sealed and stored at 4 °C. Liquid smoke treated trimmings showed a 2.3 log10 CFU/g reduction after three days of refrigerated storage compared to untreated samples (Estrada-Muñoz, Boyle, & Marsden, 1998). 7. Effect of liquid smoke on Staphylococcus Staphylococcus is a Gram-positive spherical-shaped bacterium appearing in grape-like clusters (Betts, 2010). S. aureus is salt tolerant and produces heat-stable enterotoxins which are responsible for staphylococcal foodborne illnesses. Foodborne illness due to S. aureus is a result of eating foods contaminated with toxins produced by the bacteria as opposed to consumption of the bacteria itself (Betts, 2010); it is generally agreed that populations of S. aureus need to reach 5 to 6 log10 organisms for toxin production to occur (SchmidHempel & Frank, 2007). Symptoms of S. aureus infection include nausea, vomiting and diarrhea which generally last 1 to 3 days. Foods commonly contaminated with S. aureus are prepared foods that require no additional cooking such as salads of tuna, egg, chicken, potato and macaroni, sandwiches, bakery products containing a cream filling as well as meat, poultry and eggs (Betts, 2010). Bacon has been shown to also harbor staphylococci (Taormina & Bartholomew, 2005). In order to validate that bacon processing does not permit the growth of S. aureus, cured, raw pork bellies were ground and inoculated with a cocktail of S. aureus followed by application of Charsol Supreme to a final concentration of 1.25%. Samples were slowly heated over the course of 6 h to 50 °C, the peak smoking temperature for bacon, and cooled to 7.2 °C in 3 h to simulate smoking and blast chilling that occurs in commercial plants. S. aureus populations increased by only 0.68 log10 CFU/g in liquid smoke treated samples, when compared to control samples which had an increase of 2.38 log10 CFU/g. When cooling times were increased from 6 to 15 h S. aureus grew by 4 log10 CFU/g in both smoke treated and untreated samples, however no enterotoxins were detected in samples treated with liquid smoke. In a separate experiment, whole pork bellies were sliced into 50 g pieces and inoculated by injection of the bacterial cocktail to a final inoculation concentration of approximately 2 log10 CFU/g. The pieces were then subjected to simulated smoking. A 75% solution of Charsol Supreme was sprayed after 4 and 5 h of the smoking process after which the samples were cooled to 7.2 °C over the course of 15 h and bacterial populations were enumerated. After simulated smoking and extended cooling S. aureus populations were reduced to 0.74 ± 0.53 log10 CFU/g and samples were negative for staphylococcal enterotoxins (Taormina & Bartholomew, 2005). 8. Conclusions Liquid smoke is an effective antimicrobial against an array of bacterial pathogens as demonstrated in both broth culture and food systems. Commercial use of liquid smoke in the food industry may satisfy consumer demand for all-natural foods while still maintaining their safety. Liquid smoke is being used more frequently in preserving protein-based foods, namely meat, fish, and cheese, because it imparts a pleasant

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