Life of Fresh Crab Meat

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Role of Package Type on Shelf- life of Fresh Crab Meat Carla Gutierrez Tyler,1 Michael Jahncke,*1,3 Susan Sumner,1 Renee Boyer,1 Cameron Hackney2 and Tom Rippen4 1Virginia Polytechnic Institute and State University, Food Science and Technology, 22 Duck Pond Road, Blacksburg, VA 24061, USA; 2The Davis College of Agriculture, Forestry Natural Resources and Design, West Virginia University, Morgantown, WV 26506, USA; 3Virginia Seafood AREC, 102 S. King St., Hampton, VA 23669, USA; 4University of Maryland Eastern Shore, 2122 Richard A. Henson Center, Princess Anne, MD 21853, USA

INTRODUCTION ABSTRACT In the United States, competition Microbiological quality of fresh (not pasteurized) crab from crab meat imports has adversely meat stored at 4°C in a 340 g (12 oz) food grade polyethylene impacted the fresh crab meat industry. traditional snap-lid container and equally fresh crab meat The Virginia Marine Resources Com- stored at 4°C in a 227 g (8 oz) SimpleStep® tray sealed with mission estimates that Virginia’s yearly Cryovac™ film (oxygen transmission rate of 10,000 cc/m2/24 h) crab harvest has been decreasing since 1995 (21). Several contributing factors was evaluated over a 12-day period. Aerobic plate counts were have influenced the decline of the blue conducted on storage days 0, 4, 6, 8, 10 and 12. Anaerobic plate crab in the Chesapeake Bay area, includ- counts were conducted on storage days 0, 5 and 12. Aerobic ing a decrease in the number of blue plate counts of crab meat from the two containers did not differ crabs available in the Chesapeake Bay, (P > 0.05). Analysis of anaerobic microbial growth indicates a decline in the ecological health of the that sampling days were significant (P < 0.05), but container type Chesapeake Bay, and a dramatic rise in the importation of crab meat from Asia or style was not significant (P > 0.05). Oxygen and CO2 in the package headspace was significantly different between container (15). Imported crab meat directly com- types (P < 0.05). Gas concentration between sampling days was petes with domestic crab meat and is not significant P( > 0.05). Results of this study demonstrate that sold to local restaurants at cheaper and there were no significant differences in refrigerated shelf life of more predictable prices (21). The com- crab meat packaged in SimpleStep® trays with Cryovac™ film bination of abundant supply, low labor versus the traditional polyethylene snap-lid container packaging cost, and growing demand for crab meat (P > 0.05). have all contributed to the popularity of imports, forcing a number of large domestic producers out of business (11). New packaging could boost U.S. sales and give the domestic sellers an edge. Prior to the introduction of a new package style, research studies should be conducted to evaluate container head-space gases, microbial growth and shelf life, A peer-reviewed article chemical decomposition, sensory quality and possibility of toxin production by *Author for correspondence: +1 757.727.4861; Fax: +1 757.727.4871 [email protected] Clostridium botulinum (8).

796 FOOD PROTECTION TRENDS | DECEMBER 2010 The bacterial flora on crabs reflect is an oxygen permeable film and com- commercially packaged snap-lid tubs the environment from which they were plies with the FDA’s Fish and Fisheries and 21 SimpleStep® trays were packed in harvested; the flora may change from Products Hazard and Control Guidance styrofoam ice chests with ice packs and season to season depending on the water (Third Edition) (4). Cryovac™’s film is shipped overnight to Blacksburg, Vir- quality, water temperature and harvest designed to maintain freshness and color ginia. Upon arrival, the crab meat con- o location (5). The flora are also influenced of food products without employing CO2 tainers were stored at 4 C. Three Sim- by environmental factors such as temper- treatments (4). The benefits of packag- pleStep® trays and three snap-lid tubs ature, packaging and duration of storage ing crab meat in a polypropylene Simp- were evaluated for aerobic organisms on ® ™ (5). Fresh (unpasteurized) crab meat is leStep tray with Cryovac 10,000 OTR days 0, 4, 6, 8, 10, and 12. Three simple usually hand picked, with no further film include innovative convenience fea- step trays and 3 snap lid tubs were evalu- processing, which contributes to higher tures, such as being microwavable, easy ated for anaerobic organisms on days 0, bacterial numbers than pasteurized crab opening, reusable and resealable (18). 5, and 12. Testing day 0 was designated meat (22). Furthermore, fresh crab meat The new packaging also has the po- as the time the crab meat arrived at the is a perishable product that will undergo tential to maintain quality and safety of Virginia Tech Food Science & Technol- spoilage and flavor loss within 10–14 crab meat while providing smaller por- ogy building. days or less during storage (22). Under tion sizes for a broader consumer base At each sampling time, an 11 g sam- refrigeration, spoilage of seafood occurs (9). Smaller, thinner packages or pouch- ple of crab meat was aseptically removed because of growth of psychrotrophic es, boil-in-bag packages and molded from each container with a sterile spatula bacteria such as Pseudomononas spp. trays and cups can significantly increase and placed in a separate sterile stomacher (20) and Achromobacter (2). A storage the heating and cooling rates of their bag (Nasco, Ft. Atkinson, WI) with 99 temperature of 4.4°C or lower is recom- contents, saving the processor money ml of 0.1% peptone (Oxoid, Basing- mended for refrigerated, microbiologi- and energy (9, 19). stoke, Hampshire, England). The sam- cally sensitive products (6). The shelf life This study evaluates the shelf life ples were blended in a Stomacher Lab of crab meat depends on several contrib- of fresh crab meat packaged in tradi- Blender 400 (Tekmar Co., Cincinnati, uting factors, including initial microbial tional polyethylene snap-lid container OH) for 30 seconds. ® counts and container integrity (18). and a new SimpleStep tray sealed with a 10,000 cc/m2/24 h OTR, Cryovac™ New food packaging technologies Enumeration of aerobes and can improve the quality and safety of food film. Identification of aerobic and- an aerobic bacteria over the shelf life of the anaerobes from fresh crab commodities. Packaging not only acts as meat a barrier for food products, but also can meat was also completed. control the growth of microorganisms To enumerate aerobes from the MATERIALS AND METHODS already present in the food when it is crab meat, the homogenate was diluted packaged. Polyethylene and polypropy- The shelf life of fresh crab meat using 9 ml peptone blanks, and dilutions lene are rigid or semi rigid acrylic plas- stored in two different package types were spread plated onto trypticase soy tics approved for food contact. Fresh (traditional polyethylene snap-lid con- agar (TSA; BBL, Sparks, MD and MP blue crab meat in the Chesapeake Bay tainer, and SimpleStep® tray sealed with Biomedicals, LLC, Solon, Ohio). Plates area and the Virginia coast area is sold a 10,000 cc/m2/24 h OTR, Cryovac™ were incubated at 35oC for 48 h and in traditional plastic (polyethylene) film) and incubated at 4°C was evalu- colonies were counted. snap-lid containers of 8 oz., 12 oz. or ated over a period of 12 days. Oxygen Anaerobic testing was performed ac- and CO analyses were also conducted. 16 oz. Pasteurized crab meat is sold 2 cording to methods outlined by Holde- in metal cans of 8 oz., 12 oz. or 16 oz. The entire study was conducted in tripli- man and Moore (10). One ml aliquots plastic snap-lid containers sealed with cate. The first two replications were per- from homogenate dilutions were placed formed with crabs harvested in Fall 2007; metal pop top lids. Plastic and alumi- in a glass anaerobe roll tube containing the third replication was performed with num, commonly used to package crab Brain Heart Infusion agar (BHI; BBL, crabs harvested in Fall 2008. meat, give longer shelf lives and bet- Sparks, MD). After the tubes were inoc- ter sensory and microbial qualities than Fresh crab meat sample ulated, they were placed on a horizontal crab meat packaged in steel cans (9). It preparation spinner (Bellco, Houston, TX) until the has been found that vacuum skinned medium solidified. Roll tubes were in- packaging can improve sensory qualities Fresh, handpicked crab meat was cubated at 30oC for five days. After five of freshly cooked and picked crab meat obtained from a commercial processor days, the colonies were examined under (9). in Cambridge, MD. The crab meat was a dissecting microscope and counted The FDA has a minimum standard purchased in the morning after picking through the glass roll tube. of 10,000 cc/m2/24 h oxygen transfer was complete. On the day of purchase, rate (OTR) for fresh seafood (7). The 8 oz. of crab meat was transferred from Cellular fatty acid identification transmission rate allows the transfer of several commercially packaged 12 oz. preparation for aerobes gas generated from food and the outside (340 g) snap-lid tubs into the polypro- environment, preventing the genera- pylene based SimpleStep® trays (8 oz., After colonies were counted, well- tion of potentially harmful bacteria (4). 227 g), and vacuum sealed with Cryo- isolated colonies were picked and sepa- Cryovac™ produces an OTR film that vac™ 10,000 OTR film. Twenty-one rately streaked onto TSA plates and

DECEMBER 2010 | FOOD PROTECTION TRENDS 797 FIGURE 1. aerobic (A) and anaerobic (B) plate count (log CFU/g) of (MIS, Microbial ID Inc., Newark, DE), microorganisms isolated from fresh crab meat packaged in SimpleStep® trays which uses the cellular fatty acid profile and traditional snap-lid tubs stored at 4oC for 12 days. to identify microorganisms. The procedure for cell sample preparation uses four reagents to saponify, esterify, extract and base wash the fatty acid extract, follow- ing MIS protocol (12). After base washing the fatty acid extract, approximately 100 µl of the washed extract was placed into 100 µl glass inserts (Agilent, Newark, DE). The individual glass inserts are housed in phenyl methyl silicone glass vials (25 mm × 0.2 mm ID × 0.3 µm film thick- ness) (Hewlett-Packard Co., Palo Alto, CA). Eleven mm crimp tops (Agilent, Newark, DE) were securely fastened to the top of the vials to prevent evapora- tion of the bacterial cellular fatty acid. Standards and blanks were analyzed in the HP 5890A gas chromatograph (Hewlett-Packard Co., Palo Alto, CA) to standardize the equipment prior to the injection of the unknown samples. The chromatograph is equipped with a model HP 6763 autosampler (Hewlett- Packard), a flame ionization detector and a model HP-3392A integrator (Hewlett- Packard). The air gas flow rate through the chromatograph was 400 ml/min, 30 ml/min for hydrogen, and 30 ml/min for nitrogen. The temperature used was 250oC in the injection port and 300oC for the detector. After injection, the oven temperature of the apparatus was ramped from 170oC to 270oC at a rate of 5oC/ min, followed by an additional increase o o o incubated for 24 h at 35oC. For mixed ies demonstrated that no strict anaerobic from 270 C to 310 C at a rate of 30 C/ cultures, the microorganisms were re- microorganisms were present. min. This end temperature was held for o peatedly streaked until a pure culture After 24 h, the broth tubes were 2 min before returning to 170 C prior to was obtained. When a pure culture was checked and viewed for gas produc- the injection of the subsequent sample. obtained, the colonies were transferred tion and microbial growth. Under the The MIS software was used to calcu- into a clean (12 × 100), Teflon-lined constant stream of anaerobe grade CO2 late the percentage of area for each com- screw capped tube, labeled and placed gas, a Pasteur pipette (FisherScientific, pound in its library, comparing it with in a commercial freezer (-18oC, up to 15 Pittsburg, PA) was used to dispense 6 the total area of the compound detected. days) until cellular fatty acid identifica- drops of the cooked meat broth into a Compounds were identified by using the tion. rubber stoppered 12 × 100 mm glass Aerobic TSBA Version 4.0 Library and tube peptone-yeast extract basal medium the 3.9 version for anaerobes. Cellular fatty acid identification broth (PYG), a custom-made solution (10). The inoculated PYG solution was preparation for anaerobes incubated for 24 h at 30oC. After 24 h, Gas analyzer After colonies were counted, well- the PYG was centrifuged (Sorvall, GLC- The ratio of gas present in the Sim- isolated colonies were selected for iden- 1, Newtown, CT) at 3000 RPM for 10 pleStep® trays and the snap-lid tubs was tification. Under a constant stream of minutes. The supernatant was removed analyzed using the CheckPoint O /CO anaerobe grade CO , the colonies were and the remaining pellet subjected to cel- 2 2 2 (PBI Dansensor America, Glenrock, pierced with a sterile needle and a sample lular fatty acid identification. NJ). Testing was conducted on days 0, of the colony was placed in a small an- 4, 6, 8, 10, 12, using 25 gauge 1 ½" ster- aerobic roll tube of cooked meat broth Cellular fatty acid identification ile needles (Becton Dickinson, Franklin (CM, Difco, Sparks, MD) and placed in for aerobes and anaerobes a 30oC incubator for 24 h. All cultures Lakes, NJ) and 13 mm filters (FisherSci- in the anaerobic CM were then grown on All aerobic and anaerobic identifica- entific, Pittsburgh, PA). Tabs of weather TSA plates for identification purposes, tions were performed using the Sherlock stripping were placed on the Cryovac™ because results from preliminary stud- Microbial Identification System software film and the snap-lid tops to protect the

798 FOOD PROTECTION TRENDS | DECEMBER 2010 Table 1. Bacteria isolated from fresh crab meat stored at 4°C in SimpleStep® trays with Cryo- vac™ 10,000 cc/m2/24 h oxygen transmission rate (OTR) film and traditional polyethylene snap-lid tubs. Microbial colonies were evaluated at each sampling time. This list includes the collective microorganisms isolated from each package type throughout the study.

Microorganism SimpleStep® trays Polyethylene snap-lid tubs

Aerococcus viridans + + Aeromonas caviae + Acinetobacter calcoace + Actinetobacter johnsonii + + Alcaligene + Bacillus cereus + Bacillus marinus + Bacillus sphaericus + Carnobacterium piscicola + + Cellulomonas fimi + Chromobacterium + Corynebacterium ammoniage + Enterococcus faecalis + Erwinia carotovora + Exiguobacterium acetylicum + + Kocuria varians + + Kurthia gibsonii + Lactococcus plantarum + Micrococcus luteus + Myroides odoratus + + Neisseria + Pseudomonas nautica + Pseudomonas putida + Shewanella putrefaciens + + Staphylococcus arlettae + Staphylococcus caseolyticus + Staphylococcus chromogenes + Staphylococcus cohnii + + Staphylococcus gallinarum + Staphylococcus hominis + Staphylococcus kloosii + + Staphylococcus sanguis + Staphylococcus warneri + Staphylococcus xylosus + + Streptococcus bovis + + Streptococcus mutans + + indicates that bacterium was found

DECEMBER 2010 | FOOD PROTECTION TRENDS 799 Table 2. Headspace gas composition (%) of SimpleStep® trays with Cryovac™ 10,000 cc/m2/24 h oxygen transmission rate (OTR) film and the traditional polyethylene snap-lid tubs for fresh crab meat stored at 4°C. (Packaging procedure for trial 3 was performed at a different location than trials 1 and 2.)

Simple Step® trays with Cryovac film Traditional polyethylene snap lids Days of Storage Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3

CO2 O2 CO2 O2 CO2 O2 CO2 O2 CO2 O2 CO2 O2 0 0.1 21.0 0.2 20.7 0.0 20.7 0.0 20.9 0.2 20.7 0.0 20.8 4 0.1 20.8 0.0 21.1 0.1 20.7 0.1 20.6 0.0 21.0 1.1 3.9 6 0.1 20.9 0.0 20.8 0.1 20.6 0.1 20.9 0.0 20.7 8.6 0.8 8 0.1 20.9 0.0 20.9 0.0 20.8 0.1 20.9 0.0 21.1 12.5 2.7 10 0.1 21.1 0.0 20.7 0.1 20.6 0.1 0.9 0.0 20.6 12.8 0.5 12 0.1 20.8 0.0 20.7 0.1 20.6 1.1 20.8 0.0 20.8 0.1 16.5

integrity of the package prior to the in- CFU/g (considered spoiled) in both in the snap-lid tubs, but O2 increased sertion of the needle. package types (Fig. 1 A). By day 12, to normal levels. Both CO2 and O2 re- aerobic plate count for the SimpleStep® mained constant throughout the second

Statistical analysis trays was 7.50 log CFU/g (+0.62) and repetition. In the third repetition, CO2 7.53 log CFU/g (+0.15) for the tradi- increased on day 6 and remained elevat- Data were analyzed by use of a com- tional snap-lids, respectively. There was ed until day 12. The O levels dropped pletely randomized design. All statistical 2 no significant difference in the aerobic on days 4–10, but had recovered on day analyses were conducted using SAS, ver- counts between package types on each 12. There were differences in O levels sion 9.1 (SAS Institute, Cary, NC). The 2 sampling day (P > 0.05). within replications (P < 0.05). Carbon mean log survival of aerobic and anaero- The anaerobic microbial counts on dioxide concentrations were higher in bic bacterial growth from standard plate ® day 0 for the SimpleStep trays was 4.57 the snap-lid containers than in the Sim- counts on TSA and BHI agar (respective- log CFU/g (+0.32) and 4.23 log CFU/g pleStep® trays (P < 0.05). Overall, the ly) were analyzed using the general linear (+0.40) for traditional snap-lids, respec- gas concentrations were not significantly model (GLM), and a model mean of the tively. The anaerobic plate count on day different between sample days (P > 0.05) data was compared with the method of ® 12 for the SimpleStep trays was 7.13 (Table 2). least squares means (LSD) for effect. The log CFU/g (+0.42) and 7.33 log CFU/g data readings from the O2 and CO2 gas (+0.55) for the traditional snap-lids. analysis output and coliform MPN were Anaerobic plate counts after the twelve DISCUSSION also analyzed using the GLM, and the day incubation period showed that There were no differences in shelf model means of the data were compared anaerobic growth between the two pack- life of crab meat packaged in traditional using LSD. age types was not statistically significant polyethylene snap-lid cups versus the (P > 0.05). An evaluation of the bacterial SimpleStep® trays with Cryovac™ 10,000 organisms identified in the SimpleStep® RESULTS OTR film (P > 0.05). The crab meat in trays and polyethylene snap-lid tubs is the SimpleStep® tray with the Cryovac™ Microbial spoilage in the shelf- shown in Table 1. 10,000 OTR film showed aerobic bacte- life study rial growth similar to that of the meat in For this study, spoilage was Gas analysis the polyethylene snap-lid cups. Gates et defined as microbial counts at or above al. tested oxygen barrier pouch packag- The concentration of O2 and CO2 7.0 log CFU/g (14). The aerobic plate gas remained consistent in both types of ing, non-barrier pouches and vacuum- count of the crab meat on day 0 for the packaging during the first 8 days of sam- skin packaging on fresh crab meat and SimpleStep® trays was 5.12 log CFU/g pling in the first two trials. On day 10 for concluded that no packaging material (+0.32) and 4.97 log CFU/g (+0.40) for improved the microbiological shelf life trial 1, the concentration of CO2 in the the traditional snap-lids, respectively. By polyethylene snap-lid tubs had increased (9). The results from this study support

10 days of storage, the aerobic counts slightly, and O2 levels had decreased. By Gates et al., indicating that there is no of the fresh crab meat reached 7.0 log day 12, the CO2 was still slightly higher difference in shelf life of the crab meat

800 FOOD PROTECTION TRENDS | DECEMBER 2010 in either of the package types that were Regardless of package type, if CO2 4. anonymous. 1997−2009. Cryo- tested (9). is allowed to build up, the crab meat vac 10K OTR bag for fresh fish. Environmental conditions (e.g., can deteriorate quickly. The difference Duncan, SC. Available at: http:// picking and packing room temperature, in the concentration of CO2 in the Sim- www.sealedair.com/products/food/ Chesapeake Bay water temperature etc.) pleStep® trays compared with the poly- fishsf/10k_otr_bag.html. Accessed and handling practices (e.g., picking ethylene snap-lid tubs on days 10 and October 2009. table cleanliness, use of bare hands or 12 (first repetition) and day 4 through 5. Cockey, R. R., and T. Chai. 1991. clean gloved hands, etc.) at the crab 12 of the third replication may be due Microbiology of crustacea process- to the production of CO gas from fer- ing: Crabs. p. 41 63. D.R. Ward, and meat processor and the purchaser’s estab- 2 − lishment all contribute to the level of mented lactose or the consumption of C.R. Hackney (ed.), Microbiology of O by aerobic microorganisms . Marine Food Products, Van Nos- contaminants in the fresh crab meat and 2 (13, 16) thereby affect the data. Spoiled meat Carbon dioxide can inhibit the growth trand Reinhold, New York. was determined as meat with bacterial of spoilage microorganisms, increasing 6. FDA. 2001. Fish and fisheries pro- counts at or above 107 CFU/g (14). No the shelf life of certain food products (1, ducts, hazards and controls guid- . No consistent trends in CO levels ance. Pathogen growth and toxin strict anaerobes were detected in any of 3) 2 the replications, which is similar to the were observed in any repetition, mak- formation (other than Clostridium results from a fresh crab meat study per- ing it difficult to identify a cause for the botulinum) as a result of time/tem- CO gas fluctuation. Both CO increases perature abuse. Chapter 12. 3rd formed by Suklim et al. (20). Additionally, 2 2 Ward et al. noted that when anaerobic in replications one and three occurred in ed. Available at: http://www.fda. colonies were examined, the organisms the polyethylene snap-lid tubs, suggest- gov/Food/GuidanceCompliance- isolated anaerobically were identified as ing that the tubs may be less efficient RegulatoryInformation/Guidance- in releasing CO into the outside envi- Documents/Seafood/FishandFish- facultative lactobacilli (22), further in- 2 ™ dicating that no strict anaerobes were ronment compared with the Cryovac eriesProductsHazardsandControls- present in the sampled crab meat. Both 10,000 cc/m2/24 h OTR film. Guide/ucm092162.htm. Accessed packaging types were found to have a August 2007. variety of Staphylococcus species, likely a ACKNOWLEDGMENTS 7. FDA. 2001. Fish and Fisheries result of the handling conditions. Also products hazards and controls isolated were different spoilage bacteria, The authors wish to thank Heng- guidance. Clostridium botulinum including Schewanella, Carnobacterium, jian Wang, Dianne W. Bourne and Brian toxin formation. Chapter13. 3rd ed. and Pseudomonas species. Pseudomonas Smith for their assistance in this project. Available at: http://www.cfsan.fda. species have been previously reported as This research was funded through the gov/~comm/haccp4.html. Accessed major spoilage organisms in seafood (22). Virginia Sea Grant Project “Develop, August 2007. The processor from whom the crab meat Evaluate and Characterize Different 8. Gates, K., Y. Huang, A. H. Parker, was purchased advertises Chesapeake Package Types on the Quality, Shelf Life and D. P. Green. 1996. Quality char- Blue Crab meat and Indonesian pasteur- and Market Acceptability of Pasteurized acteristics of fresh blue crab meat ized crab meat. 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