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Journal of Food Protection, Vol. 74, No. 7, 2011, Pages 1119–1125 doi:10.4315/0362-028X.JFP-11-052 Copyright G, International Association for Food Protection

Effect of Antimicrobial Spice and Herb Extract Combinations on Listeria monocytogenes, Staphylococcus aureus, and Spoilage Microflora Growth on Cooked Ready-to-Eat Vacuum-Packaged Shrimp Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/7/1119/1685434/0362-028x_jfp-11-052.pdf by guest on 24 September 2021 NIMSHA S. WEERAKKODY,1 NOLA CAFFIN,1 GARY A. DYKES,2 AND MARK S. TURNER1*

1School of Agriculture and Food Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia; and 2School of Science, Monash University, Bandar Sunway, Selangor, Malaysia

MS 11-052: Received 3 February 2011/Accepted 24 March 2011

ABSTRACT Two spice and herb extract combinations from galangal (Alpinia galanga), rosemary (Rosmarinus officinalis), and lemon iron bark (Eucalyptus staigerana) were evaluated for their ability to inhibit the growth of inoculated Listeria monocytogenes and Staphylococcus aureus and naturally present spoilage microflora on cooked ready-to-eat shrimp stored for 16 days at 4 or 8uC. A combination of galangal, rosemary, and lemon iron bark significantly reduced (P , 0.05) levels of aerobic bacteria and lactic acid bacteria at 4uC on day 12 by 1.6 and 1.59 log CFU/g, respectively. By day 16, levels of these bacteria were equivalent to those of controls. The shrimp treated with this spice and herb extract combination had significantly lower (P , 0.05) lipid oxidation from day 4 to day 16. Similarly, a combination of galangal and rosemary extract significantly reduced (P , 0.05) levels of aerobic bacteria and lactic acid bacteria at 8uC on day 8 by 2.82 and 2.61 log CFU/g, respectively. By days 12 and 16, levels of these bacteria were equivalent to those of controls. The shrimp treated with this spice and herb combination had significantly lower (P , 0.05) lipid oxidation on days 4 and 16. None of the spice and herb extract combinations had an effect on levels of L. monocytogenes or S. aureus or changed the color or pH of the shrimp during storage. The results of this study indicate that combinations of galangal, rosemary, and lemon iron bark extracts can be used to control the growth of spoilage microflora on ready-to-eat shrimp.

Spices and herbs have been used for centuries as food processing (20), and low temperature (25). A combination additives to enhance food flavor and as preservatives to of physical hurdles rather than a single hurdle extends the extend shelf life and may act as natural preservatives to meet shelf life of food products by controlling microbial consumer demand for healthy and minimally processed population and retaining freshness and quality without ready-to-eat (RTE) foods (15). The antimicrobial properties altering the sensory and nutritive value of the product (17). of various spices and herbs have been studied extensively in The consumption of raw and RTE shrimp has increased vitro, and their major chemical compounds have been steadily over the years. In the United States, the per capita identified (24, 27, 30, 31). In addition to their antimicrobial consumption of shrimp was 1.3 kg in 1998 and 1.9 kg in 2004 properties, some spices and herbs have antioxidant activity (11). Shrimp has a short shelf life because of its high protein and may therefore reduce oxidative rancidity in foods. content and high pH (,7.5) (12). Cooked shrimp is retailed as Although spices and herbs have strong antimicrobial and either a chilled or a frozen product. Frozen cooked shrimp are antioxidant effects in vitro, some do not have these same thawed prior to retail display or sale. During thawing, pre- and properties when incorporated into a food matrix possibly postprocessing microflora populations can increase, leading to because of insufficient concentrations of active components spoilage, especially under abusive storage conditions. Ac- and/or reduced activity due to reactions with food cording to Food Standards Australia New Zealand (FSANZ) ingredients (2). These inconsistent effects have limited the (10), the average temperatures recorded for chilled cooked practical use of spices and herbs as natural preservatives. prawn samples collected from retail outlets in Perth, Brisbane, Thus, spice and herb extracts may be best considered as part Melbourne, and Sydney in a 4-week period during July and of a microbial growth hurdle approach in combination with August 2002 ranged from 25.5 to 12.8uC. Temperatures other preservation technologies such as modified atmo- higher than 5uC also were found in displayed cooked shrimp sphere packaging (25), irradiation (16), high-pressure in retail outlets. Therefore, additional measures are needed to control the growth of naturally occurring microflora and * Author for correspondence. Tel: z61-7 33657364; Fax: z61-7 pathogenic microorganisms under abusive storage conditions 33651177; E-mail: [email protected]. to ensure quality and safety. 1120 WEERAKKODY ET AL. J. Food Prot., Vol. 74, No. 7

Microbial contamination of shrimp can occur at various Test microorganisms. L. monocytogenes Scott A (serotype stages of production: on the ship, in the processing plant 4b) and L. monocytogenes V7 (serotype 1/2a) were obtained from environment, during handling, during transportation, and the U.S. Food and Drug Administration (Cincinnati, OH). S. during storage under fluctuating temperature conditions aureus ATCC 25925, S. aureus ATCC 29213, and L. monocyto- (10). In addition to spoilage microbes, shrimp may be genes ATCC 7644 were obtained from the American Type Culture Collection (Manassas, VA). S. aureus SA113 was supplied by the contaminated with pathogenic microorganisms such as Institute for Biomedical Innovation (Queensland University of Listeria monocytogenes, Staphylococcus aureus, Salmonel- Technology, Brisbane, Australia). All bacterial growth media used la Typhimurium, and Clostridium botulinum type E (20, in this study were supplied by Oxoid (Basingstoke, UK). All 23). L. monocytogenes is a psychrotolerant bacterium that bacteria were maintained as frozen stocks in 40% glycerin at can grow at close to 0uC and is an important pathogen in 280uC. Working cultures were grown and maintained on tryptic chilled RTE products such as cooked and peeled shrimp soy agar. stored in modified atmosphere packaging (MAP) (3). RTE Preparation and inoculation of shrimp. Frozen RTE

cooked shrimp and shrimp salad have been contaminated Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/7/1119/1685434/0362-028x_jfp-11-052.pdf by guest on 24 September 2021 with L. monocytogenes (7, 14). Cooked, peeled shrimp can cooked, peeled, and deveined shrimps (Penaeus vannamei), which be vacuum packed to limit the passage of water vapor and had the last abdominal shell (plus telson and uropods) still present, were obtained from a Brisbane supermarket 1 day before the oxygen and minimize dehydration and oxidation. Vacuum experiment. Shrimps were transported on ice to the laboratory and packaging also controls the growth of L. monocytogenes stored at 4uC to thaw overnight. The next day, the last abdominal (Scott A) (13). S. aureus is a common foodborne pathogenic shell with the telson and uropods was removed from each shrimp, postprocessing contaminant of vacuum-packaged and re- and the shell-less shrimp abdomen was weighed. Shrimps that were duced-moisture products (1). S. aureus can grow under 10 ¡ 1 g were selected for the experiments. temperature abuse conditions and produce enterotoxins All six bacterial strains were grown separately in 2 ml of without spoiling foods. tryptic soy broth at 37uC for 18 h to a population of ,9.0 log CFU/ In our previous work, we identified combinations of ml. Cells were harvested by centrifugation at 9,000 | g for 10 min three spice and herb extracts that had synergistic antimicro- at room temperature. The supernatant was decanted, and the pellet bial activity against foodborne pathogens (30). The combi- was resuspended in sterile 0.85% saline by vortexing. A 100-ml nation of galangal (Alpinia galanga), rosemary (Rosmarinus aliquot of each S. aureus strain or 50 ml of each L. monocytogenes strain was added to 500 ml of 0.85% saline solution to provide a officinalis), and lemon iron bark (Eucalyptus staigerana) three-strain mixture containing ,5.0 to 6.0 log CFU/ml, which extract had optimal synergistic activity against L. monocyto- resulted in an attachment level of ,4.0 to 5.0 log CFU/g of shrimp. genes, and the combination of galangal and rosemary extract Shrimps were inoculated with a three-strain mixture of L. had optimal synergistic antimicrobial activity against S. monocytogenes or a three-strain mixture of S. aureus or were not aureus. The objectives of this study were to evaluate the inoculated. The noninoculated shrimps were included to determine effectiveness of combined spice and herb extracts to control the growth of natural microflora and pH, color, and oxidation inoculated L. monocytogenes and S. aureus and naturally changes. Shrimps were inoculated by immersion for 5 min at 25uC occurring spoilage microflora on vacuum-packed cooked in the bacterial suspensions and agitation with a glass rod to ensure RTE shrimp stored at 4 and 8uC. The effects of the spice and an even distribution of the organisms. After inoculation, shrimps herb extract combinations on lipid oxidation, color, and pH of were removed and air dried for 15 min in a biosafety cabinet to the shrimp also were examined. allow bacterial attachment. Treatment of shrimps. In a previous study, we investigated MATERIALS AND METHODS various combinations of three herb and spice extracts (galangal, Extraction of spices and herbs. Fresh galangal was rosemary, and lemon iron bark) for synergistic activity against purchased from a local shop (Brisbane, Australia). Fresh leaves foodborne pathogens in laboratory growth media (30). Galangal of lemon iron bark were donated by Australian Rainforest Products and rosemary extracts had synergistic antimicrobial activity against (Lismore, New South Wales, Australia). A dried commercial S. aureus, and galangal, rosemary, and lemon iron bark extracts preparation of rosemary (Masterfoods, Wyong, Australia) was had synergistic antimicrobial activity against L. monocytogenes purchased from a Brisbane supermarket. Fresh A. galanga rhizome (30). was washed under running water and the outer skin removed. The The stock solutions (500 mg/ml) of galangal, rosemary, and rhizome was sliced and dried at 40uC overnight and subjected to lemon iron bark extracts were diluted in 80% glycerin to prepare cryogrinding with a freezer mill (SPEX Sample Prep, Metuchen, lower concentrations. Food grade glycerin was used as a solvent NJ) for 5 min to make a fine powder. Fresh leaves of E. staigerana instead of ethanol because glycerin is a generally recognized as were dried at 40uC for 24 h and cryoground for 3 min. R. officinalis safe (GRAS) substance. The S. aureus–inoculated and some also was cryoground for 3 min to a fine powder. noninoculated shrimps were treated with a mixture of 5 mg/ml Hexane was used to extract A. galanga, and ethanol was used galangal and 10 mg/ml rosemary extracts, and the L. monocyto- to extract R. officinalis and E. staigerana as described in genes–inoculated shrimps and some noninoculated shrimps were Weerakkody et al. (31). These extracts produced with these treated with a mixture of 5 mg/ml galangal, 10 mg/ml rosemary, solvents possessed the highest levels of antimicrobial activity and 2.5 mg/ml lemon iron bark extracts. compared with extracts obtained with other solvents or water (30). Aliquots of 500 ml of the mixtures were spread on each side of Dried ethanol extracts were dissolved in 90% ethanol, and the single shrimp surfaces and left for 10 min. Controls for this dried galangal hexane extract was dissolved in glycerin (Mel- experiment included sets of shrimps that were inoculated with bourne Food Ingredient Depot, Melbourne, Australia) to make equivalent volumes of 80% glycerin only or with water containing stock solutions (500 mg/ml), which were stored at 4uC until use. 0.25 ml/ml ethanol (ethanol was added at 0.025%, similar to the J. Food Prot., Vol. 74, No. 7 EFFECT OF ANTIMICROBIAL SPICE AND HERB EXTRACTS ON RTE SHRIMP 1121

TABLE 1. Antimicrobial activity of spice and herb extracts against L. monocytogenes and native microflora on shrimp stored at 4uC Populaton (log CFU/g) on storage dayb:

Bacteria Treatmenta 04 81216

L. monocytogenes No spice control 5.34 ¡ 0.30 6.55 ¡ 0.24 7.40 ¡ 0.74 8.19 ¡ 0.36 8.77 ¡ 0.18 Glycerin control 5.28 ¡ 0.33 5.82 ¡ 0.37 6.57 ¡ 0.58 7.90 ¡ 0.73 8.45 ¡ 0.84 X3 spice mix 4.99 ¡ 0.24 5.61 ¡ 0.54 5.89 ¡ 0.56 7.43 ¡ 1.08 8.21 ¡ 0.89 Aerobic bacteria No spice control ,3.48 A a ,3.48 A a6.89¡ 0.64 B b8.07¡ 0.68 B c8.82¡ 0.36 A d Glycerin control ,3.48 A a ,3.48 A a5.04¡ 0.26 A b6.71¡ 0.76 A c8.06¡ 0.86 A d X3 spice mix ,3.48 A a ,3.48 A a3.94¡ 0.64 A a5.11¡ 0.53 C b7.57¡ 0.35 A c Lactic acid bacteria No spice control ,3.48 A a4.28¡ 0.27 A a6.23¡ 0.84 B b7.81¡ 0.83 B c8.57¡ 0.80 A d Glycerin control ,3.48 A a3.69¡ 0.29 A a4.73¡ 0.96 A a6.32¡ 1.05 A b7.61¡ 0.45 A c X3 spice mix ,3.48 A a ,3.48 A a4.06¡ 0.32 A a4.73¡ 0.37 C a6.97¡ 0.64 A b Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/7/1119/1685434/0362-028x_jfp-11-052.pdf by guest on 24 September 2021 a No spice control shrimp were treated with water (plus ethanol) only. Glycerin control shrimp were treated with glycerin only. X3 spice mix shrimp were treated with extracts of galangal, rosemary, and lemon iron bark. b Within a column, mean values followed by the same uppercase letter are not significantly different (P . 0.05). Within a row, mean values with the same lowercase letter are not significantly different (P . 0.05). concentration present in the spice combination mixtures) and left for described by Nirmal and Benjakul (21). A 1-g sample of ground 10 min. These controls allowed determination of the antimicrobial shrimp was mixed with 9 ml of a solution containing 0.375% activity due to glycerol and allowed comparison of bacterial levels thiobarbituric acid (Sigma-Aldrich, Steinheim, Germany), 15% found in shrimps that were not treated with extracts or glycerol. trichloroacetic acid (Ajax Finchem, Cheltenham, Victoria, Aus- Each shrimp was placed in a separate vacuum bag (Micris Pty. tralia), and 0.25 N HCl. The mixture was heated in a boiling water Ltd., Cleveland, Queensland, Australia) to which an additional 1 ml bath for 10 min, cooled with running water, and then centrifuged at of the corresponding spice and herb extract mixture or control liquid 4,000 | g for 20 min. The supernatant was collected, and the (80% glycerin or water plus 0.025% ethanol) was added. The absorbance was read at 532 nm with a spectrophotometer vacuum bags have an oxygen transmission rate of 80 cc/m2/24 h/atm (Lovibond, Dortmund, Germany). The TBARS value was at 23uC and a water vapor transmission rate of 7 g/m2/24 h at 38uC. calculated from a standard curve of malonaldehyde (1,1,3,3- The bags were vacuum sealed (Vacumatic 282, Vacumatic tetramethoxypropane; Sigma-Aldrich) (0 to 2 ppm) and expressed Australia, Narre Warren, Victoria, Australia) at 80 mm Hg. After as milligrams of malonaldehyde per kilogram of shrimp meat. vacuum packaging, L. monocytogenes–inoculated shrimps were stored at 4uC for 16 days, and S. aureus–inoculated shrimps were pH measurement. Ten grams of shrimp sample was stored at 8uC for 16 days. We selected two temperatures, 4 and 8uC, transferred to a stomacher bag with 90 ml of 0.85% saline solution for the two pathogenic bacteria, which would allow growth during and homogenized for 60 s with a stomacher. The pH of this storage. Glycerol (80%)–treated and water (plus 0.025% ethanol)– homogenized sample was measured with a pH meter (TPS Pty. treated control samples were stored separately at both temperatures. Ltd., Brisbane, Australia). Shrimps were analyzed for bacterial populations, pH, color, and lipid oxidation after 0, 4, 8, 12, and 16 days of storage. This entire shrimp Color measurement. Color changes occurring on the surface trial was repeated independently two more times. of shrimp samples were quantified with a chromameter (Konica Minolta, Sensing, Japan) based on CIE L, a, and b values (L, Microbial enumeration. Each shrimp was transferred to an lightness; a, redness and greenness; b, yellowness and blueness). individual sterile stomacher bag, 0.85% saline was added to Color measurements were conducted at predetermined storage produce a 1:10 (wt/vol) dilution, and the mixture was homogenized intervals. Measurements were taken perpendicular to the sample in a stomacher (240 rpm per paddle; BA6021, Colworth Stomacher surface at three places on each shrimp specimen. 400, Seward, London, UK) for 60 s. Serial dilutions of the bacterial suspensions were prepared with 0.85% saline, and 0.1 ml of the Statistical analysis. Shrimp samples were analyzed in dilutions was spread onto appropriate media. PALCAM agar was triplicate on different days. Microbial counts were log transformed used to enumerate L. monocytogenes from inoculated samples, and before the analysis of variance was conducted in MINITAB plates were incubated at 37uC for 48 h. Baird-Parker agar was used (Minitab, Inc., State College, PA). Tukey’s test for comparison of to enumerate S. aureus from inoculated samples, and plates were means was performed with the same program. The significance incubated 37uC for 24 h. From the noninoculated shrimp samples, level was defined at P , 0.05. native microflora including aerobic bacteria and lactic acid bacteria were enumerated. Aerobic bacteria were enumerated by plating RESULTS 100 ml on plate count agar, which was incubated at 30uC for 2 days. Effect of spice and herb extract mixture on L. Lactic acid bacteria were enumerated by plating 100 ml on de Man monocytogenes and spoilage microflora on shrimps Rogosa Sharpe agar, which was incubated at 25 C for 3 days u stored at 4'C. Counts of naturally occurring spoilage anaerobically in a GasPak Anaerobic System (Oxoid). For each bacteria and inoculated L. monocytogenes on shrimps stored sample, at least two appropriate dilutions were enumerated. Bacteria were enumerated on plates with 30 or more colonies. at 4uC are given in Table 1. The galangal, rosemary, and lemon iron bark extract mixture did not significantly inhibit Determination of TBARS. Thiobarbituric acid reactive (P . 0.05) the growth of inoculated L. monocytogenes over substances (TBARS) in the shrimp samples were determined as the 16 days of storage as compared with glycerin-treated or 1122 WEERAKKODY ET AL. J. Food Prot., Vol. 74, No. 7

TABLE 2. TBARS values for shrimp treated with spice and herb extracts, vacuum packaged, and stored at 4uC TBARS (mg malonaldehyde/kg shrimp) on storage dayb:

Treatmenta 0481216

No spice control 4.92 ¡ 0.80 A a 4.87 ¡ 0.61 A a 4.68 ¡ 0.37 B a 4.84 ¡ 0.12 A a 5.80 ¡ 0.23 A a Glycerin control 4.49 ¡ 0.40 A a 5.69 ¡ 0.42 A b 5.94 ¡ 0.48 A c 5.99 ¡ 0.10 A d 6.01 ¡ 0.10 A e X3 spice mix 4.74 ¡ 0.55 A a 4.54 ¡ 0.23 B a 4.04 ¡ 0.36 C a 3.95 ¡ 0.14 B a 3.64 ¡ 0.30 B a a No spice control shrimp were treated with water (plus ethanol) only. Glycerin control shrimp were treated with glycerin only. X3 spice mix shrimp were treated with extracts of galangal, rosemary, and lemon iron bark. b Within a column, mean values followed by the same uppercase letter are not significantly different (P . 0.05). Within a row, mean values with the same lowercase letter are not significantly different (P . 0.05). untreated shrimps. On day 12 of storage, aerobic bacteria and rosemary extract mixtures did not significantly inhibit Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/7/1119/1685434/0362-028x_jfp-11-052.pdf by guest on 24 September 2021 and lactic acid bacteria counts on extract-treated shrimps (P . 0.05) the growth of inoculated S. aureus during were significant lower (P , 0.05) that those on glycerin- 16 days of storage as compared with glycerin-treated or treated shrimps. The difference in these counts were 1.60 untreated shrimps. The levels of aerobic bacteria and lactic and 1.59 CFU/g, respectively. However, by day 16 levels acid bacteria were significantly lower (2.82 and 2.61 log of aerobic and lactic acid bacteria in extract-treated CFU/g, respectively) in shrimps treated with the spice and shrimps were equivalent to those in glycerin-treated herb extract mixture only on day 8 of storage compared with shrimps. The glycerin-treated shrimp did have significant- the glycerin-treated control. However, aerobic and lactic ly lower aerobic and lactic acid bacteria counts (P , acid bacterial growth increased by days 12 and 16, and no 0.05) on days 8 and 12 compared with these counts on difference in counts between extract-treated shrimp and untreated shrimps. However, glycerin did not inhibit glycerin-treated shrimps was found on those days. Glycerin growth of L. monocytogenes. did not have an effect on spoilage microflora except on day 4, when significant inhibition (P , 0.05) was noted for Lipid peroxidation in shrimp stored at 4'C. The aerobic and lactic acid bacteria. TBARS values of treated and untreated shrimps stored at 4uC for 16 days are listed in Table 2. The TBARS values for shrimp Lipid peroxidation in shrimp stored at 8'C. The treated with the galangal, rosemary, and lemon iron bark extract TBARS values of shrimps stored at 8uC for 16 days are listed in mixture were significantly lower (P , 0.05) than those for Table 4. Significantly lower TBARS values (P , 0.05) were glycerin-treated and untreated shrimps on all days except day 0. obtained on days 4 and 16 of storage for extract-treated shrimp The glycerin treatment did not have an effect on TBARS values compared with glycerin-treated and untreated shrimp controls. (compared with the untreated control) throughout the storage The glycerin treatment did not have an effect on TBARS values period, except on day 8 when significant higher values were (compared with the untreated control) throughout the storage obtained for glycerin-treated shrimp. period. The TBARS values did not significantly change over time except when day 16 values were compared with day 0 Effect of spice and herb extract mixture on S. values for glycerin-treated and untreated shrimp. aureus and spoilage microflora on shrimps stored at 8'C. Counts of spoilage bacteria and inoculated S. aureus pH of shrimp stored at 4 and 8'C. Neither of the two on shrimps stored at 8uC are shown in Table 3. Galangal spice and herb extract mixtures caused a significant change

TABLE 3. Antimicrobial activity of spice and herb extracts against S. aureus and native microflora on shrimp stored at 8uC Populaton (log CFU/g) on storage dayb:

Bacteria Treatmenta 04 81216

S. aureus No spice control 5.08 ¡ 0.29 5.52 ¡ 0.42 5.89 ¡ 0.60 5.60 ¡ 0.45 5.98 ¡ 0.36 Glycerin control 5.18 ¡ 0.16 5.38 ¡ 0.31 5.91 ¡ 0.44 6.22 ¡ 0.34 6.28 ¡ 0.43 X2 spice mix 4.99 ¡ 0.25 5.22 ¡ 0.27 5.35 ¡ 0.44 5.24 ¡ 0.61 5.76 ¡ 0.30 Aerobic bacteria No spice control ,3.48 A a7.59¡ 1.57 B b8.89¡ 0.25 A c9.10¡ 0.10 A d9.13¡ 0.18 A e Glycerin control ,3.48 A a4.65¡ 0.71 A a8.06¡ 0.57 A b9.09¡ 0.03 A b8.96¡ 0.04 A c X2 spice mix ,3.48 A a ,3.48 A a5.24¡ 0.65 B b7.88¡ 0.72 A c7.96¡ 0.83 A d Lactic acid bacteria No spice control ,3.48 A a6.95¡ 0.99 B b8.41¡ 0.81 A c8.56¡ 0.38 A d9.00¡ 0.23 A e Glycerin control ,3.48 A a4.69¡ 0.66 A a7.59¡ 0.56 A b8.63¡ 0.65 A c9.15¡ 0.03 A d X2 spice mix ,3.48 A a4.68¡ 0.00 A a4.98¡ 1.24 B a7.34¡ 0.19 A b8.36¡ 0.81 A c a No spice control shrimp were treated with water (plus ethanol) only. Glycerin control shrimp were treated with glycerin only. X2 spice mix shrimp were treated with extracts of galangal and rosemary. b Within a column, mean values followed by the same uppercase letter are not significantly different (P . 0.05). Within a row, mean values with the same lowercase letter are not significantly different (P . 0.05). J. Food Prot., Vol. 74, No. 7 EFFECT OF ANTIMICROBIAL SPICE AND HERB EXTRACTS ON RTE SHRIMP 1123

TABLE 4. TBARS values for shrimp treated with spice and herb extracts, vacuum packaged, and stored at 8uC TBARS (mg malonaldehyde/kg shrimp) on storage dayb:

Treatmenta 0481216

No spice control 5.06 ¡ 0.66 A a 4.87 ¡ 0.32 A a 5.27 ¡ 0.48 A a 5.79 ¡ 0.15 A a 6.98 ¡ 0.75 A b Glycerin control 4.72 ¡ 0.30 A a 5.68 ¡ 0.27 A a 5.90 ¡ 0.69 A a 5.48 ¡ 0.27 A a 6.82 ¡ 1.12 A b X2 spice mix 5.16 ¡ 0.14 A a 3.79 ¡ 0.53 B a 4.82 ¡ 0.87 A a 4.18 ¡ 0.07 A a 3.90 ¡ 0.04 B a a No spice control shrimp were treated with water (plus ethanol) only. Glycerin control shrimp were treated with glycerin only. X2 spice mix shrimp were treated with extracts of galangal and rosemary. b Within a column, mean values followed by the same uppercase letter are not significantly different (P . 0.05). Within a row, mean values with the same lowercase letter are not significantly different (P . 0.05).

(P . 0.05) in the pH of the shrimp immediately after flora (initial levels of ,3.48 log CFU/g) and therefore Downloaded from http://meridian.allenpress.com/jfp/article-pdf/74/7/1119/1685434/0362-028x_jfp-11-052.pdf by guest on 24 September 2021 application or during storage (data not shown). allowed the higher initial levels (,4 to 5 log CFU/ml) of inoculated S. aureus and L. monocytogenes to grow on the Color changes of shrimp stored at 4 and 8'C. No shrimp. The low growth of S. aureus in untreated shrimp significant differences were found in L, a, and b values of also may be due to its well known poor ability to compete treated and untreated shrimps stored at 4 and 8uC (data not with other bacteria (3). The total aerobic bacterial counts in shown). shrimp were ,100 times greater than the S. aureus counts after 4 days of storage. Application of the spice and herb DISCUSSION extract mixtures may have been patchy due to poor contact Spice and herb extracts with antimicrobial properties on some places of the shrimp surface, thereby allowing can be used to control the growth of foodborne pathogenic enhanced bacterial growth. The naturally occurring micro- bacteria and spoilage bacteria, which can in turn lead to an flora on shrimp also may be have been sublethally injured or extension of the shelf life of processed food products. stressed during frozen storage before the assays and According to FSANZ guidelines (9), RTE food products are therefore may have been more sensitive than the inoculated considered satisfactory when total aerobic bacterial counts pathogens to the herb and spice extract mixtures. are #6 log CFU/g and pathogenic bacteria are undetectable. L. monocytogenes populations increased over the 16- According to this guideline, when stored at 4uC untreated day storage period in shrimp stored at 4uC. These results are shrimp would be unfit for consumption between days 5 and in agreement with those of Farber (7), who found that 8, glycerin-treated shrimp would be unfit between days 9 Listeria increased by 2 to 3 log CFU/g in cooked shrimp and 12, and shrimp treated with the galangal, rosemary, and stored at 4uC for 7 days. MAP with CO2 was more effective lemon iron bark extract mixture would be unfit between against L. monocytogenes than vacuum packaging and air days 13 and 16. A similar pattern was seen for shrimp stored packaging (25). In addition to the spice and herb extract at 8uC; untreated shrimp were unfit for consumption treatment, the combination of different physical hurdles between days 1 and 4, glycerin-treated shrimp were unfit such as vacuum packaging and refrigerated storage used in between days 5 and 8, and shrimp treated with galangal and our study extended the shelf life of RTE shrimp by rosemary extract mixture were unfit between days 9 and 12. controlling the populations of spoilage microorganisms Generally, chilled cooked crustaceans have a 10-day shelf and the physiological quality of the shrimp during the life at 21uC (6, 10). This shelf life was achieved at higher storage period. Other researchers also have used various temperatures (4 and 8uC) when treatment with spice and hurdle technologies in food preservation. Mastromatteo et herb extracts was combined with vacuum packaging. al. (19) found that a combination of MAP and thymol We found an inhibitory effect of spice and herb extract coating (1,000 ppm) on shrimp improved the shelf life by combinations on tested spoilage microorganisms, including more than 9 days at 4uC compared with samples packaged aerobic bacteria and lactic acid bacteria. Galangal and in air and stored at 4uC. In this same study, MAP alone did rosemary extracts have produced antibacterial activity not improve the shelf life of uncoated shrimp. In another against the lactic acid bacterium Lactococcus lactis (8, 24, study, L. monocytogenes growth in RTE shrimp under MAP 26), which agrees with the results of our study. However, was inhibited by higher numbers of inoculated lactic acid inoculated S. aureus and L. monocytogenes were not bacteria at 2uC but only for low initial inoculum levels (2 inhibited by treatment with spice and herb extracts during log CFU/g) of L. monocytogenes (20). the 16 days of storage, even though the extracts previously In the present study we used glycerin, which is a GRAS had strong synergistic antimicrobial activity (30). The food additive (28), as a solvent for the extracts. Glycerin reason for this difference in findings is not clear, but this absorbs moisture from the environment, causing a reduction situation highlights the fact that results obtained with in the water activity of the product (28). The 80% glycerin laboratory media can be different from those obtained in used in our study did not have a significant inhibitory effect food matrices. One possible explanation for the lack of on S. aureus and L. monocytogenes. According to Litsky et bacterial inhibition in shrimp is that the spice and herb al. (18), a higher concentration of glycerin (88.8%) had a extracts more actively inhibited competing spoilage micro- bacteriostatic effect on S. aureus, and Escherichia coli, 1124 WEERAKKODY ET AL. J. Food Prot., Vol. 74, No. 7

Salmonella Typhimurium, and Pseudomonas aeruginosa these herb and spice extracts might be useful as a shrimp were unable to grow in solutions of 58.6, 48.1, and 40.5% preservative. Further studies are required to determine the glycerin, respectively. effect of these spice and herb extracts at different None of the spice and herb extract combinations used in concentrations and to identify other suitable solvents that this study changed the color or pH of shrimp during storage. diffuse the antimicrobial compounds efficiently. Shrimp tissue has a natural buffering capacity, and the lactic acid generated by the growth of natural lactic acid bacteria REFERENCES on shrimp during storage can be neutralized by ammonia 1. Borneman, D. L., S. C. Ingham, and C. Ane. 2009. Predicting produced from the concurrent proteolysis of shrimp protein growth–no growth of Staphylococcus aureus on vacuum-packaged (19, 22). This buffering effect may explain the lack of ready-to-eat meats. J. Food Prot. 72:539–548. significant changes in pH of shrimps throughout the storage 2. Burt, S. 2004. Essential oils: their antibacterial properties and potential applications in foods—a review. Int. J. Food Microbiol. 94: period despite significant increases in the levels of lactic 223–253. acid bacteria.

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