CHEMICAL CHARACTERISTICS AND ANTILISTERIAL TESTING OF CHEMICAL CHARACTERISTICS AND ANTILISTERIAL TESTING OF LOW COLOR AND FLAVOR LIQUID SMOKES LOW COLOR AND FLAVOR LIQUID SMOKES

Naim Montazeri, Brian Himelbloom, Alexandra Oliveira & Charles CrapoFishery Industrial Technology Center, School of Fisheries and Ocean Sciences

University of Alaska Fairbanks, Kodiak AK 99615

Chemical characteristics and antilisterial tests of low color and flavor liquid smoke fractions were conducted as a potential application for cold-smoked salmon. Recent studies showed the effectiveness of these fractions (low in phenol and carbonyl) against Listeria monocytogenes. We selected four commercial smokes Code 10-Poly having strong color and flavor, and refined fractions AM-3, AM-10 and 1291 (Kerry Ingredients and Flavors, Monterey, TN). The Gardner Delta Color Comparator showed color values of 15, 10, 5 and 2, respectively. Lowest pH and highest titratable acidity were for Code 10-Poly (2.3 and 10.3%) and reverse for 1291 (5.7 and 0.7%) followed by AM-3 (4.3 and 2.2%) and AM-10 (4.2 and 2.3%). Two spectrophotometric methods based on the Gibbs reaction were used to measure total phenolic compounds. Both showed good linearity (R2= 0.99) for standard phenol solutions. However, only Code 10-Poly showed color formation. Volatile compounds of the liquid smokes were identified using a gas chromatograph-mass spectrometer. Code 10-Poly had detectable levels of phenolic compounds which verified the negligible phenol concentrations in other fractions. Various carbonyl and aldehyde compounds were identified in the fractions. Disc diffusion assay on plate count agar was used to evaluate antilisterial properties of the liquid smokes (at 0, 25, 50, 75 and 100% concentrations) at 25 and 35oC against L. innocua (as a model for L. monocytogenes). Significant inhibition (P<0.05) was in all fractions tested except for 1291 which had slight inhibition zones at high concentrations (mean = 8 mm). Code 10-Poly resulted in the largest inhibition zones (mean = 36 mm) followed by AM-10 (mean = 30 mm) and AM-3 (mean = 28 mm) with no differences (P>0.05) in the inhibitory zones of each fraction for incubation temperatures. This study forms the basis for assessing of liquid smokes as antilisterial supplements to cold-smoked salmon.

Liquid smokes Four water soluble liquid smokes (LSs) namely Code 10-Poly, AM-3, AM-10 and 1291 (Kerry Ingredients and Flavors, Monterey, TN) were analyzed.Chemical and antibacterial properties of LSsColor: Gardner Delta Color Comparator (BYK, Additives and Instruments, Columbia, MD).Titratable acidity (TA%) and pH: Titration of diluted liquid smoke (1:4, w/v deionized water) to pH = 8.3 with 0.1N NaOH (Bratzler et al., 1969).Total phenol: Two spectrophotometric methods based on the reaction of Gibbs reagent (2,6-Dichloroquinone-4-chloroimide) to phenolic compounds were used to measure total phenol in diluted LSs (at 40, 400 and 4000 levels): Nicholson (1984) as method A; and Vitt et al. (2001) as method B.Aromatic compounds: LSs were extracted using dichlormethane (1:2, v/v, LS to solvent) at 45 °C. The solvent was removed using a separatory funnel, evaporated under flow of N2. Compostion of the LSs extracts were analyzed using gas chromatography coupled to mass spectroscopy (Guillen et al., 1995)Antilisterial properties of liquid smokes: Inhibitory zone was measured with paper disk-agar diffusion test of liquid smokes against L. innocua in plate count agar media after incubation at 4, 25 and 35 °C.Cold-smoking process: Wood smoking process was conducted as illustrated in Figure 2.Quality Characteristics of cold-smoked sockeye salmon:Muscle pH (Ruiz-Capillas and Moral, 2001)Water Phase Salt (WPS%) (Hilderbrand, 2000)Total phenol in acetonitrile extract of fish tissue (Nicholson, 1984)

Bratzler LJ, et al., 1969. Journal of Food Science. 34, 146-148.Centers for Disease Control and Prevention (CDC). 2011. Listeriosis. URL: http://www.cdc.gov/nczved/divisions/dfbmd/diseases/listeriosis/ (Accessed on 03/07/2011).FDA, 2001. Processing Parameters Needed to Control Pathogens in Cold Smoked Fish. Supplement to vol. 66, No. 7, 2001—Journal of Food Science.FDA, 2003. Listeria monocytogenes Risk Assessment: I. Introduction. URL: http://www.fda.gov/Food/ScienceResearch/ResearchAreas/RiskAssessmentSafetyAssessment/ucm184052.htm (Accessed on 03/07/2011).Gibbs HD, 1927. Journal of Biological Chemistry. 72, 649-664.Guillen MD, et al., 1995. Journal of Agricultural and Food Chemistry. 43, 463-468.Hilderbrand KS, 2000. Quick determination of water phase salt content of smoked fish. Oregon State University. Extension Sea Grant, Newport, OR.Milly PJ, et al., 2005. Journal of Food Science. 70, M12-M17.Nicholson MD, 1984. Tar-depleted liquid smoke treatment of food casings. In: United States Patent, Vol. 4431032, Union Carbide Corporation, US.Ruiz-Capillas C, and Moral A, 2001. Food Research International. 34, 441-447.Vitt SM, et al., 2001. Journal of Food Safety. 21, 111-125.

Gibbs reaction is pH dependent and only occurs in alkaline media. Different buffering capacities of the methods shifted pH values to different values (9.4 and 8.8, respectively)Method A was more reliable because pH is adjusted to the optimum suggested value of 9.4 (Gibbs, 1927) Phenol was not measurable in three refined LSs High acidity of LSs might prevent the reaction to occur at lower dilutionsVery low or undetectable concentration of phenolic compounds in the fraction which was later confirmed by GCMS resultsLiquid smokes AM-3 and AM-10 were the refined fractions with the highest antilisterial inhibitory properties. Curing step resulted relatively high amount of salt to the fish. Shorter curing time will be required to keep WPS near 3.5% to meet recommended level while keeping the salt at a level desired by consumers

Kodiak Area Marine Science Symposium, April 9–12, 2011

Kodiak Area Marine Science Symposium, April 9–12, 2011

Abstract

Listeria monocytogenes, the causative agent of foodborne listeriosis, are widely spread in the environment. They are facultative anaerobic rods and capable of growth at temperatures ranging from 1 to 45 °C and in media with salt content (NaCl) up to 10% (w/v). Several cases of illnesses and outbreaks occur throughout the world as the result of cross-contamination and lack of a thermal process for killing bacteria. In the United States, listeriosis causes about 1,600 cases of illness and 260 deaths each year (CDC, 2011) and FDA maintains a "zero-tolerance" policy for the ready-to-eat foods (FDA, 2003).

Among high-risk foods, cold-smoked salmon, though not linked to epidemic listeriosis, has a relatively high level of contamination potential to Listeria (36%). Thermal treatment in cold-smoking process does not provide enough heat to eliminate vegetative cells so it favors bacterial growth. Overexposing the product to smoke may increase the antibacterial properties due to elevated antibacterial smoke components but it may result in unpleasant smoky flavor or appearance. Refined liquid smokes (reduced in smoky color and flavor) have been shown to have antilisterial properties (Milly et al., 2005), may be applied as a post-process lethality step (just before packaging) to inhibit the growth of Listeria without sensory alteration in the product. The object of this study is to provide basic information on the quality characteristics of liquid smokes and cold-smoked sockeye salmon.

Introduction

Material and methods

Code 10-Poly was the darkest LS with strong smoky flavor, highest acidity and lowest TA% (Figure 1; Table 1)Two methods for determination of total phenol showed good linearity with standard solutions (R2= 0.99; Figure 3)Total phenol was measurable only in Code 10-Poly diluted 400 times. Two methods resulted in different values; 3.22 ± 0.03 and 1.33 ± 0.50 mg/ml for method A and B, respectivelyMajor components of LS volatiles are presented in Table 2.Phenol were detected in Code 10-Poly and in small amounts in 1291Syringol and guaiacol were the most abundant phenols in Code 10-Poly Three refined fractions were more abundant in carbonyls (esp. in 1-Hydroxy-2-butanone)2(5H)-Furanone and its derivatives were less abundant in AM-10 than AM-3 AM-10, a refined fraction of AM-10 was higher in organic acids but lower in furan and pyran containing compounds Paper disk-agar diffusion test showed the most inhibition for Code 10-Poly, followed by AM-10, AM-3 and 1291 (Figure 4 & 5)Cold-smoking process deposited 2.7 ± 0.8 mg of phenolic compounds per 100 g of cold-smoked fish and reduced pH to to 6.0 ± 0.1 Salt content was 4.9 ± 0.6 % resulted in 7.79 ± 0.8 % water-phase salt (WPS), which is higher than minimum level of 3.5% WPS set by FDA (2001)

Results

Discussion

References

Figure 1 Liquid smokes with their Gardner Color Value (From top to bottom: Code 10-Poly, AM-3, AM-10 and 1291)

Figure 2 Traditional cold-smoking process of Sockeye salmon

Figure 3 Standard curve for phenol standard solutions according to the method A and B (top and bottom)

Standard curve for determination of total phenolic content (Method A)

0.0000.002

0.0040.006

0.0080.010

0.0120.014

0.016

Concentration (mg/ml)

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Abso

rban

ce

Fit: y = 0.01 + 76.65*xR2= 0.996

Standard curve for determination of total phenolic content (Method B)

0.000 0.002 0.004 0.006 0.008 0.010 0.012

Concentration (mg/ml)

-0.20.00.20.40.60.81.01.21.41.61.82.0

Abso

rban

ce

Fit: y = 0.07 + 172.50xR2 = 0.993

Code 10 - Poly AM-3 AM-10 1291Gardner Color Value 16 11 5 2

Acidity (pH) 2.3 ± 0.0 4.3 ± 0.0 4.2 ± 0.0 5.7 ± 0.0Titratable Acidity % 10.3 ± 0.0 2.2 ± 0.0 2.3 ± 0.0 0.7 ± 0.0

Table 1 Color, pH and TA% of liquid smokes

Main Compounds Code 10-Poly AM-3 AM-10 1291Furan and pyran containing compounds 14.3 44.7 25.8 14.62-methoxytetrahydrofuran 6.4Furfural 5.6 2H-Pyran-3(4H)-one, dihydro- 1.3 1.1 2(5H)-Furanone 2.7 23.9 22.0 2.22(5H)-Furanone, 5-methyl- 0.2 2.2 0.9 4-Methyl-5H-furan-2-one 0.7 3.2 2-Furancarboxaldehyde, 5-(hydroxymethyl)- 1.0 10.6 Butyrolactone 0.5 4.0Phenolic compounds 37.0 0.0 0.0 0.1Phenol 0.5 Phenol, 4-methyl- (p-Cresol) 0.6 Phenol, 2-methoxy- (Guiacol) 3.9 1,2-Benzenediol 3.3 1,2- Benzenediol, 3-methyl- 0.8 0.1Phenol, 2,6-dimethoxy- (Syringol) 12.3 Eugenol 0.2 Vanillin 1.0 Benzaldehyde, 4-hydroxy- 3,5-dimethoxy- 2.2 Ethanone,1-4(hydroxy-3,5-dimethoxyphenyl)- 2.6 Aldehyde and ketones 17.6 29.3 28.2 55.7Propanal 1.9 5.7 8.2 6.31-Hydroxy-2-butanone 2.7 7.6 11.2 46.02-Cyclopenten-1-one 2.8 1.0 0.91,2- Cyclopentanedione 2.4 2-Cyclopenten, 1-one, 3-methyl- 1.1 Organic acids 6.7 1.0 7.4 2.3Butanoic acid, 2-Propenyl ester 4.6 Benzeneacetic acid, 4-hydroxy-3-methoxy- 0.4 Propanoic acid 0.6 2.4 1.63-Hydroxy-4-methoxybenzoic acid 4.9 Miscellaneous 2.0 5.0 13.1 0.01,4:3,6-Dianhydro-.alpha.-d-glucopyranose 0.5 0.5 7.3 2,3-Anhydro-d-mannosan 0.5 4.6 4.2 Unknowns with peak are >2 % 9.5 2.1 8.9 11.6

Table 2 Aromatic compounds in volatile fraction of liquid smoke with their relative proportions in % TIC (total ion count)

AM-10 was chosen to apply to smoked salmon for the next experiment for its higher antilisterial properties while having lower flavor and colorThe potential of post-process elimination of L. innocua cold-smoked salmon by applying liquid smoke (as a hurdle technology) is the next goal of the project

Future research

Figure 4 Growth inhibition of L. innocua in PCA plate by LS AM-10 at different concentrations

Figure 5 Paper disk-agar diffusion test of LSs against L. innocua at different incubation conditions. Letters (a to c) and (x to z) indicate significant difference b/w LSs at each concentration and b/w concentrations, respectively (p<0.05).

Paper disk-agar diffusion test of LSs against L. innocua (48 h incubation at 35 °C)

Concentration (%)

Inhi

bitio

n zo

ne (c

m)

LS: Poly-10LS: AM-3LS: AM-10LS: 1291

25 50 75 100-505

1015202530354045

a, z

a, ya, xy

a, xy

a, xy a, x

a, zb, y

a, x

a, za, y

a, x

b, y c, y

b, x b, x

Paper disk-agar diffusion test of LSs against L. innocua (72 h incubation at 25 °C)

Concentration (%)

Inhi

bitio

n zo

ne (c

m)

LS: Poly-10LS: AM-3LS: AM-10LS: 1291

25 50 75 100-505

10152025303540

a, z

a, ya, xy

a, x

a, z

a, zb, yz

b, yb, xy

b, x

b, x

b, x

c, y c, y c, xy

c, x

Paper disk-agar diffusion test of LSs against L. innocua(14 days incubation at 4 °C followed by 24 h at 35 °C )

Concentration (%)

Inhi

bitio

n zo

ne (c

m)

LS: Poly-10LS: AM-3LS: AM-10LS: 1291

25 50 75 100-10

0

10

20

30

40

50

a, x a, xa, xa, x

b, yb, xy b, xy

b, x

b, x

b, xb, x

b, x bc, xb, x

c, xc, x

Receiving fish and filletingReceiving fish and filleting

Dry-salting (curing) in salt and brown sugar mixture (1:1) for 7 hDry-salting (curing) in salt and brown sugar mixture (1:1) for 7 h

Rinsing cured fish with cold tap water to remove excess of cure, putting the fillets on the racks

Rinsing cured fish with cold tap water to remove excess of cure, putting the fillets on the racks

Traditional cold-smoking process using Alder woodchips:1) Drying for 30 min at 18 °C 2) Applying smoke for 8 h at 18 °C

Traditional cold-smoking process using Alder woodchips:1) Drying for 30 min at 18 °C 2) Applying smoke for 8 h at 18 °C

Vacuum-packagingVacuum-packaging

Funding for this project was provided by USDA-CSREES and Alaska Sea Grant College ProgramFunding for this project was provided by USDA-CSREES and Alaska Sea Grant College Program