Purification of lysozyme from shell liquor of eastern oysters (crassostrea virginica) and its use in antimicrobial films to preserve smoked fish

quor of a bivalve mollusk on a laboratory and large scale levels. The modification of protocol consisted of omitting the gel filtration step and directly applying the sample to the first ion exchange chromatography (Xue et al., 2004). Large volumes of oyster shell liquor were purified using two-step ion exchange chromatography. The protein purified from the shell liquor showed high lytic activity against M. lysodeikticus. SDS-PAGE showed a single band at about 18 KDa which was of similar size to plasma lysozyme (Xue et al., 2004). From the results of Minimum Inhibitory Concentration assays we found that purified oyster lysozyme from the shell liquor had antimicrobial activity against both gram-positive and gram negative bacteria. There have been few studies which dealt with the use of bivalve lysozyme for antimicrobial activities against Gram-positive and Gram-negative bacteria (Nilsen et al., 1999; Xue et al., 2004). Lysozymes are known to be active against Gram 38 positive bacteria due to thick peptidoglycan layer whereas Gram- negative bacteria have a single layer of peptidoglycan and a thick outer membrane which acts as a barrier preventing the access of lysozyme (Masschalck and Michiels, 2003). However, some investigators (During et al., 1999; Pellegrini et al., 1992; Ibrahim et al., 1996) have reported lysozyme to be active against gram negative bacteria. Our study also show that lysozyme from oyster shell liquor has activities against some Gram negative bacteria and Gram positive bacteria (Table 3.8). Oyster lysozyme showed highest inhibition towards Clostridium perfringens. Adam (1974) showed that ultrahigh temperature treated Clostridium perfringens spores with EDTA sensitized the spores to lysozyme (Adams, 1974). However Hughey and Johnson (1987) reported Clostridium perfringens to be resistant to the action of hen egg white lysozyme. Hen egg white lysozyme is used in food industry against C. botulinum (Hughey et al., 1989; Johnson, 1994). It has been shown in studies that lysozyme may have different inhibitory activities against the same bacteria but different species (Johnson, 1994; Losso et al, 2000). Hen egg white lysozyme is known to be effective against certain gram negative bacteria when the cells are pre-treated with EDTA (Wooley et al., 1974). Pellegrini et al., 1992 showed that lysozyme could be effective against certain Gram negative bacteria even without the EDTA pretreatment. The bactericidal activity of lysozyme was attributed to the cationic and hydrophobic properties of lysozyme (Pellegrini et al., 1992). Our results also show that there is a marked decrease in absorbance of Gram negative C. coli, C. jejuni without any addition of EDTA. C. jejuni was shown to be weakly inhibited by lysozyme when the bacteria were inoculated into complex broth media containing 20-200 mg/liter of lysozyme (Hughey and Johnson, 1987). 39 Lysozyme is effective against L. monocytogenes and several studies have been carried out using lysozyme against L .monocytogenes (Carminati and Carini, 1989; Hughey et al., 1989; Wang and Shelef, 1991). Our study shows that at highest concentration tested i.e. 160 µg/ml, oyster lysozyme inhibited L. monocytogenes. The susceptibility of L. monocytogenes to lysozyme inactivation was found to be dependent on the physiological status of the bacterium and also the medium of the food in which lysozyme was suspended (Johnson, 1994). Staphylococcus aureus, a gram positive bacterium is not inhibited by action of lysozyme (Salton and Pavlik, 1960; Wooley et al., 1974). Our study show that oyster lysozyme at 160 µg/ml did not inhibit the growth of S. aureus. Similarly, Yersina enterocolitica and E.coli O157:H7 were not inhibited by 160 µg/ml of oyster lysozyme. Hughey and Johnson (1987) reported Yersina enterocolitica and E.coli O157:H7 to be resistant to the action of hen egg white lysozyme. Thus, from the minimum inhibitory concentration assays we conclude that oyster lysozyme and hen egg white lysozyme have antimicrobial activities against both Gram- positive and Gram-negative bacteria. 40 CHAPTER 4 CONTROL OF LISTERIA MONOCYTOGENES AND SALMONELLA ANATUM ON THE SURFACE OF SMOKED SALMON COATED WITH EDIBLE COATINGS CONTAINING OYSTER LYSOZYME AND NISIN 4.1 Introduction Cold-smoked salmon is a ready-to-eat (RTE) food product which is normally purchased vacuum-packed and has a shelf life of 3 to 5 weeks at refrigerated temperatures (Rovik, 2000; Leroi et al., 2001). Regulatory agencies in the United States have adopted zero- tolerance policy towards contamination of RTE products with Listeria monocytogenes which can grow easily on the surface of these food products. A major concern of the smoked fish industry is contamination of their products with L. monocytogenes and Salmonella species (Heintz and Johnson, 1998). The highest incidence of L. monocytogenes is associated with cold-smoked fish rather than hot smoked fish because this pathogen does not survive the hot smoke process (Eklund et al., 1994). Contamination of smoked fish with L. monocytogenes ranges from 17.9% to 22.3% (Heintz and Johnson, 1998). Studies have shown that cold-smoked salmon is a good substrate for the growth of L. monocytogenes even under vacuum conditions (Rorvik et al., 1991). Salmonella species have also been associated with smoked fish outbreaks (Heintz et al., 2000). The incidence of Salmonella in smoked fish is 3.9% with Salmonella newport or Salmonella anatum as the most prominent spp isolated from smoked fish in United States (Heintz et al., 2000). Antimicrobial films act as a protective barrier which retards the food deterioration and when the film is incorporated with antimicrobial agents can extend the food shelf life (Cha and Chinnan, 2004). Antimicrobial agents incorporated into edible films are released slowly onto the surface of food products and these coatings also serve as a 41 barrier to moisture and oxygen which are cost effective and can protect the food material even when the package of the food material is opened (Cha and Chinnan, 2004). Edible film coatings can be polysaccharide-based films, protein-based films or lipid-based films. One of the widely used polysaccharide film is alginate based films. Alginates are extracted from the brown seaweeds that belong to the Phaephyceae class, They are salts of alginic acid, which is a linear polymer of D-mannuronic and L- guluronic acid monomers. Alginates have the ability to react with di-valent and tri- valent cations and this property is being utilized in alginate film formation. Calcium ions are widely used as effective gelling agents (Cha and Chinnan, 2004). Antimicrobial agents have been incorporated into calcium alginate gels and used to preserve a variety of food products (Wan et al., 1997; Cutter and Siragudsa, 1996; Williams et al., 1978; Lazarus et al., 1976). The objective of this study was to determine the feasibility of using oyster lysozyme in antimicrobial films coatings to enhance the preservation of cold-smoked salmon. 4.2 Materials and Methods 4.2.1 Culture Growth Conditions Listeria monocytogenes strain V7 (Serotype ½ a) and Salmonella anatum were obtained from the Centers for Disease Control, (Atlanta, Ga.). The bacterial cultures were grown for 24 h at 37◦C in brain heart infusion (BHI) broth and decimally diluted with 0.1 M potassium phosphate buffer (pH 7.0) (PBS buffer). The pure cultures were stored at -70◦ C and subcultured twice in BHI broth at 37°C for 24 h before use. 42 4.2.2 Preparation of Edible Coatings The agar gel coating was prepared by dissolving 0.75 g of Bacto agar (Difco) into 100 ml of sterile distilled water. Zein propylene glycol liquid solution was obtained from Freeman Industries (Tuckahoe, N.Y.). Calcium alginate coating was made by the combination of 1g of calcium carbonate (Sigma) and 1g of sodium alginate (ISP Alginates, Inc.) into 100 ml of sterile distilled water. All the solutions were autoclaved at 121°C for 20 min and stored until use. 4.2.3 Preparation of Antimicrobials and Fish Samples Purified oyster lysozyme was diluted to a concentration of 160 µg/ml because this is the minimum inhibitory concentration of oyster lysozyme needed against L. monocytogenes and S. anatum (Chapter 3). Hen egg white lysozyme crystals were obtained from Sigma. The concentration of hen egg white lysozyme used during this study was also160 µg/ml for the same reasons as for oyster lysozyme. Nisaplin, a commercial source of nisin was obtained from Aplin & Barrett Ltd ( Trowbridge, Wilts, England). Nisaplin stock solution was made by dissolving 1g of Nisaplin into 1 ml of 0.02 N HCl to yield a final concentration of 1000 IU/g. The Nisaplin stock was filter sterilized through a 0.22µm low protein binding filter. The stock solution was autoclaved at 121°C for 20 min and stored until used. Smoked salmon samples were purchased from a local supermarket. They were cut into 1g pieces and kept in sterile Whirl-Pack bags that were frozen at -20°C until used. 4.2.4 Determination of the Most Effective Film Coating for Enhancing the Antimicrobial Activity of Oyster Lsyozyme (Appendix 1) Fish samples (1g) were immersed into 24 h broth cultures of L. monocytogenes V7 (Serotype ½ a) or Salmonella anatum for 1 min, allowed to drip free of excess inoculum 43 and dried for 20 min. The control samples were dipped into the broth cultures. Smoked salmon samples with and without L. monocytogenes or S. anatum were dipped into various treatments. The following six treatments were examined for bacterial counts during this study: 1) Control smoked salmon with no treatment; 2) Inoculated control smoked salmon; 3) inoculated smoked salmon dipped into 1000IU/g of nisin (N), Hen egg white lysozyme at 160 µg/ml (HEWL), or Oyster lysozyme at 160 µg/ml (LYO); 4) Inoculated smoked salmon dipped into edible coatings without antimicrobial treatments; 5) Inoculated smoked salmon dipped into edible coatings containing either N, HEWL or LYO; 6) Inoculated smoked salmon with N and HEWL or LYO incorporated inside edible coatings. The samples were allowed to air dry for 20 min before putting them into sterile bags and refrigerated at 4◦ C for 24 h. Bacterial counts were determined the next day by adding PBS to make a 1/10 dilution in each bag, stomaching for 2 min then plating serial dilutions onto XLD agar for Salmonella anatum and Oxford medium base with Oxford supplement for L. monocytogenes. The plates were then incubated at 37°C for 24 h and CFU/g was determined. The effect of three different edible coatings; calcium alginate, 0.75% agar edible coating and zein coatings containing antimicrobial agents against Listeria monocytogenes and Salmonella anatum on the surface of the smoked fish at 4◦ C after 24 h were determined. 4.2.5 Effect of Oyster Lysozyme Incorporated into Calcium Alginate Coatings against Listeria monocytogens and Salmonella anatum during 35 days Storage (Appendix 2) Smoked salmon samples (1g) were immersed into 24 h broth cultures of L. monocytogenes V7 (Serotype ½ a) or S. anatum for 1 min, allowed to drip free of excess inoculum and dried for 20 min. The following six treatments were examined for bacterial 44 counts during this study 1) Control smoked salmon with no treatment; 2) Inoculated control smoked salmon; 3) Inoculated smoked salmon dipped into1000IU/g of nisin (N), Hen egg white lysozyme at160µg/ml (HEWL) or Oyster lysozyme at 160µg/ml (LYO); 4) Inoculated smoked salmon dipped into edible coatings without antimicrobial treatments; 5) Inoculated smoked salmon dipped into edible coatings containing either N, HEWL or LYO; 6) Inoculated smoked salmon with N and HEWL or LYO incorporated inside edible coating. The samples were allowed to air dry for 20 min before putting them into sterile bags and refrigerated at 4° C. Bacterial counts were determined at 0, 7, 14, 21, 28, and 35 d by adding PBS to make a 1/10 dilution in each bag, stomaching for 2 min, and.then plating serial dilutions onto XLD agar for S. anatum or Oxford medium base with Oxford supplement for L. monocytogenes. The plates were incubated at 37°C for 24 h and CFU/g was determined. 4.2.6 Statistical Analysis The inhibitory effects of the edible coatings with or without the antimicrobial agents against growth of L. monocytogenes or S. anatum on the ready-to-eat cold-smoked salmon samples at refrigerated temperatures were analyzed by statistical comparisons of all pairs using Student’s t-test following one-way analysis of the variance (ANOVA) (SAS Institute Inc., Cary, N.C., U.S.A.). Statistical significance occurs for P > 0.05. 4.3 Results After 24 h, L. monocyotogenes counts were reduced by 0.52 log CFU/g when oyster lysozyme was incorporated into 1% calcium alginate coatings on the surface of the smoked salmon (Figure 4.1). When nisin was added along with oyster lysozyme into the coating, it caused further reduction of bacterial counts by 1.63 log CFU/g. There was no 45 significant difference between oyster lysozyme and hen egg white lysozyme in reducing the bacterial counts, either when used alone or when used in combination with nisin inside the calcium alginate coating. ee e bbccd f dd a 0 1 2 3 4 5 6 7 8 C H ew l Ly o N C aA lg C aA lg H ew l C aA lg Ly o C aA lg N C aA lg N H ew l C aA lg N Ly o Treatments Lo g C FU /g Figure 4.1: Effect of Calcium Alginate Edible Coating containing Antimicrobial Agents against Listeria monocytogenes on the Surface of Smoked Salmon at 4◦ C after 24 h. a All analysis were based on two separate experiments. Means followed by the same letter are not significantly different (P = 0.05) from each other. Statistical comparisons of all pairs were analysed using Student’s t-test following one-way analysis of the variance (ANOVA) (SAS Institute, Inc., Cary, NC). b Abbreviations of treatments are as follows: C, Control; CaAlg, Calcium alginate coating; Hewl, Hen Egg White Lysozyme; N, Nisin; Lyo, Oyster lysozyme; CaAlgN, Calcium alginate coating with Nisin; CaAlgHewl, Calcium alginate coating with Hen Egg White Lysozyme; CaAlgLyo, Calcium alginate coating with Lysozyme; CaAlgNHewl, Calcium alginate coating with Nisin and Hen egg white lysozyme; CaAlgNLyo, Calcium alginate coating with nisin and Lysozyme L. monocyotogenes counts were reduced by 0.82 log CFU/g when oyster lysozyme was incorporated into 0.75% agar coatings on the surface of the smoked salmon after 24 h (Figure 4.2). Nisin along with oyster lysozyme inside the coating caused further reduction of bacterial counts by 1.82 log CFU/g. There was no significant 46 difference between oyster lysozyme and hen egg white lysozyme in reducing the bacterial counts, either when used alone or when used in combination with nisin inside the 0.75% agar coating. ccc bb a d bb a 0 1 2 3 4 5 6 7 8 C H ew l Ly o N Ag Ag H ew l Ag Ly o Ag N Ag N H ew l Ag N Ly o Treatments Lo g C FU /g Figure 4.2: Effect of 0.75% Agar Edible Coating containing Antimicrobial Agents against Listeria monocytogenes on the Surface of Smoked Salmon at 4◦ C after 24 h. a All analysis were based on two separate experiments. Means followed by the same letter are not significantly different (P = 0.05) from each other. Statistical comparisons of all pairs were analysed using Student’s t-test following one-way analysis of the variance (ANOVA) (SAS Institute, Inc., Cary, NC). b Abbreviations of treatments are as follows: C, Control; Ag, Agar coating; Hewl, Hen Egg White Lysozyme; N, Nisin; Lyo, Oyster lysozyme; AgN, Agar coating with Nisin; AgHewl, Agar coating with Hen Egg White Lysozyme; AgLyo, Agar coating with Lysozyme; AgNHewl, Agar coating with Nisin and Hen Egg White Lysozyme and; AgNLyo, Agar coating with Nisin and Lysozyme After 24 h, L. monocyotogenes counts were reduced by 0.61 log CFU/g when oyster lysozyme was incorporated into zein coatings on the surface of the smoked salmon (Figure 4.3). When nisin was added along with oyster lysozyme inside the coating it caused further reduction of bacterial counts by 1.79 log CFU/g. There was no significant difference between oyster lysozyme and hen egg white lysozyme in reducing the bacterial 47 counts, either when used alone or when used in combination with nisin inside the zein coating. ccc bbb d bb a 0 1 2 3 4 5 6 7 8 C H ew l Ly o N Z ZH ew l ZL yo ZN ZN H ew l ZN Ly o Treatment Lo g C FU /g Figure 4.3: Effect of Zein Coating containing Antimicrobial Agents against Listeria monocytogens on the Surface of Smoked Salmon at 4◦ C after 24 h a All analysis were based on two separate experiments. Means followed by the same letter are not significantly different (P = 0.05) from each other. Statistical comparisons of all pairs were analysed using Student’s t-test following one-way analysis of the variance (ANOVA) (SAS Institute, Inc., Cary, NC). b Abbreviations of treatments are as follows: C, Control; Z, Zein coating; Hewl, Hen Egg White Lysozyme; N, Nisin; Lyo, Oyster lysozyme; ZN, Zein coating with Nisin; ZHewl, Zein coating with Hen Egg White Lysozyme; ZLyo, Zein coating with Lysozyme; ZNHewl, Zeincoating with Nisin and Hen Egg White Lysozyme ; ZNLyo, Zein coating with Nisin and Lysozyme After 24 h, S. anatum counts were reduced by 1.82 log CFU/g when oyster lysozyme was incorporated into 1% calcium alginate coating on the surface of the smoked salmon Figure (4.4). Oyster lysozyme when incorporated into the calcium alginate coatings showed the most significant difference from all other treatment and was found to be the most effective treatment. Oyster lysozyme along with nisin when incorporated inside the calcium alginate coatings also showed significant reduction in bacterial counts. 48 edb f dcd abbc 0 1 2 3 4 5 6 7 8 C H ew l Ly o N C aA lg C aA lg H ew l C aA lg Ly o C aA lg N C aA lg N H ew l C aA lg N Ly o Treatments Lo g C FU /g a Figure 4.4: Effect of Calcium Alginate Coating containing Antimicrobial Agents against Salmonella anatum on the Surface of Smoked Salmon at 4◦ C after 24 h aAll analysis were based on two separate experiments. Means followed by the same letter are not significantly different (P = 0.05) from each other. Statistical comparisons of all pairs were analysed using Student’s t-test following one-way analysis of the variance (ANOVA) (SAS Institute, Inc., Cary, NC). b Abbreviations of treatments are as follows: C, Control; CaAlg, Calcium alginate coating; Hewl, Hen Egg White Lysozyme; N-Nisin; Lyo, Oyster lysozyme; CaAlgN, Calcium alginate coating with Nisin; CaAlgHewl, Calcium alginate coating with Hen Egg White Lysozyme; CaAlgLyo, Calcium alginate coating with Lysozyme; CaAlgNHewl, Calcium alginate coating with Nisin and Hen Egg White Lysozyme; CaAlgNLyo, Calcium alginate coating with nisin and Lysozyme After 24 h, S. anatum counts were reduced by 0.4 log CFU/g when oyster lysozyme was incorporated into 0.75% agar coatings on the surface of the smoked salmon (Figure 4.5). When nisin was added along with oyster lysozyme inside the coating it caused further reduction of bacterial counts by 0.54 log CFU/g. The most 49 effective treatment was when nisin and oyster lysozyme were incorporated inside the calcium alginate film. db abcdabcabccdc a 0 1 2 3 4 5 6 7 8 C H ew l Ly o N A g A gH ew l A gL yo A gN A gN H ew l A gN Ly o Treatments Lo g C FU /g ab Figure 4.5: Effect of 0.75 % Agar Coating containing Antimicrobial Agents against Salmonella anatum on the Surface of Smoked Salmon at 4◦ C after 24 h. a All analysis are based on two separate experiment. Means followed by the same letter are not significantly different (P = 0.05) from each other. Statistical comparisons of all pairs were analysed using Student’s t-test following one-way analysis of the variance (ANOVA) (SAS Institute, Inc., Cary, NC). b Abbreviations of treatments are as follows: C, Control; Ag,Agar coating; Hewl, Hen Egg White Lysozyme; N, Nisin; Lyo, Oyster lysozyme; AgN, Agar coating with Nisin; AgHewl, Agar coating with Hen Egg White Lysozyme; AgLyo, Agar coating with Lysozyme; AgNHewl, Agar coating with Nisin and Hen Egg White Lysozyme ; AgNLyo, Agar coating with Nisin and Lysozyme After 24 h, S. anatum counts were reduced by 0.97 log CFU/g when oyster lysozyme was incorporated into zein coatings on the surface of the smoked salmon (Figure 4.6) after 24 h. When nisin was added along with oyster lysozyme inside the coating it caused further reduction of bacterial counts by 1.14 log CFU/g. Hence, it was seen that for all the treatments, edible coating along with antimicrobials and nisin showed 50 the most reduction in bacterial counts compared to the control. The best film among the three films was then chosen for the shelf life study. e dde cbcabca 0 1 2 3 4 5 6 7 8 C H ew l Ly o N Z ZH ew l ZL yo ZN ZN H ew l ZN Ly o Treatments Lo g C FU /g Figure 4.6: Effect of Zein Coating containing Antimicrobial Agents against Salmonella anatum on the Surface of Smoked Salmon at 4◦ C after 24 h. a All analysis are based on two separate experiment. Means followed by the same letter are not significantly different (P = 0.05) from each other. Statistical comparisons of all pairs were analysed using Student’s t-test following one-way analysis of the variance (ANOVA) (SAS Institute, Inc., Cary, NC). b Abbreviations of treatments are as follows: C, Control; Z, Zein coating; Hewl, Hen Egg White Lysozyme; N, Nisin; Lyo, Oyster lysozyme; ZN, Zein coating with Nisin; ZHewl, Zein coating with Hen Egg White Lysozyme; ZLyo, Zein coating with Lysozyme; ZNHewl, Zeincoating with Nisin and Hen Egg White Lysozyme; ZNLyo, Zein coating with Nisin and Lysozyme From the 35 days shelf life study, we observed that at 4◦ C an initial inoculation of Listeria monocytogenes increased from 6.57 log CFU/g to 9.86 log CFU/g on day 35 in the control samples (Table 4.1). Bacterial counts reached the control level when the surface of smoked salmon was treated with oyster lysozyme by the end of shelf life period. After 35 days of storage there was a 1.05 log CFU/g reduction of L. monocyotogenes counts on the surface of smoked fish coated with calcium alginate coating containing 160µ/ml oyster lysozyme as compared to the control. With the 51 addition of nisin along with oyster lysozyme in the calcium alginate coatings, L. monocyotogenes counts were further reduced by 2.75 log CFU/g on the surface of the smoked salmon by day 35. The most significant treatment was oyster lysozyme and hen egg white lysozyme when incorporated into the calcium alginate coating. There was no significant difference between oyster lysozyme and hen egg white lysozyme. At 4◦ C, an initial inoculation of Salmonella anatum increased from 5.78 log CFU/g to 6.12 log CFU/g on day 35 in the control samples (Table 4.2). Bacterial counts were reduced by 0.73 log CFU/g when the surface of smoked salmon was treated with oyster lysozyme at the end of the shelf life period. After 35 days of storage there was a 1.52 log reduction of S. anatum counts on the surface of the smoked salmon coated with calcium alginate containing 160 µg/ml oyster lysozyme as compared to the control. With the addition of nisin along with oyster lysozyme in the calcium alginate coatings S. anatum counts were reduced by 2.25 log CFU/g on the surface of the smoked fish by day 35. The most effective treatment was obtained when oyster lysozyme and hen egg white lysozyme was incorporated into the calcium alginate coating. 4.4 Discussion Smoked fish and shellfish products can be contaminated with foodborne pathogens like Listeria monocytogenes and Salmonella species (Heintz and Johnson, 1998). Studies carried out in cold-smoked salmon processing plant showed that the primary source of L. monocytogenes contamination was the surface of the frozen or raw fish coming into the plant (Eklund et al., 2004). Listeria can survive the cold-smoking process and has been isolated from cold smoked salmon (Guyer and Jemmi, 1991; Dillon et al., 1994; Jemmi, 52 1993). Salmonella species have also been associated with smoked seafood contamination with an incidence of 3.9% in smoked fish (Heinitz et al., 2000). Listeria monocyotogenes and Salmonella anatum counts were reduced by 0.80 CFU/g on the surface of smoked salmon when the samples were coated with calcium alginate or zein coatings compared to the control non-treated samples. These findings are in agreement with other studies that have shown that calcium alginate edible films coated on lamb or beef cuts reduced total bacterial counts by about 1 log cycle compared to the non-coated samples (Lazarus et al., 1976; Williams et al., 1978). Our study has shown that adding oyster lysozyme with or without nisin into the edible coatings significantly reduced L. monocytogenes and S. anatum counts compared to the control nontreated samples on the surface of smoked salmon. Furthermore studies have shown that zein edible coatings are more effective with the addition of food grade antimicrobial agents against foodborne pathogens on the surface of ready-to-eat chicken (Janes et al., 2002). Zein films containing a high concentration of nisin reduced L. monocytogenes counts by 8 log cycles compared to zein films without nisin (Hoffman et al., 2001). We chose calcium alginate edible coatings because it showed more promise for the control of S. anatum on the surface of smoked salmon with oyster lysozyme. The S. anatum counts were reduced to 1.82 log CFU/g when oyster lysozyme was incorporated into the calcium alginate film compared to 0.97 and 0.4 log CFU/g reduction in bacterial numbers when smoked salmon samples were coated with zein or agar coatings respectively. 53 Table 4.1 Effect of Different Antimicrobials with and without Calcium Alginate Coating on the Growth of Listeria monocytogenes at 4◦ C for 35 Days. Log CFU/g* Treatment* Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 C 6.57± 0.03 a 6.81± 0.21 a 7.86 ± 0.25 a 8.88 ± 0.12 a 9.39 ± 0.14 a 9.86 ± 0.17 a CaAlg 6.45 ± 0.01 a 6.19 ± 0.14 b 7.01 ± 0.12 b 8.24 ± 0.07 b 9.44 ± 0.13 a 9.93 ± 0.19 a N 5.09 ± 0.16 d 4.58 ± 0.36 g 5.96 ± 0.23 c 7.68 ± 0.11 d 8.77 ± 0.43 b 9.39 ± 0.13 b LYO 5.40 ± 0.07 cd 5.22 ± 0.12 de 6.95± 0.15 b 7.91 ± 0.02 cd 8.98 ± 0.18 abc 9.61 ± 0.07 a HEWL 5.53 ± 0.09 b 5.17± 0.14 d 6.99 ± 0.02 b 7.9 ± 0.07 c 9.10 ± 0.19 ab 9.61 ± 0.14 ab CaAlgN 5.22 ± 0.11 cd 4.78 ± 0.02 fg 5.83 ± 0.33 d 6.9 ± 0.15 e 7.99 ± 0.19 d 8.30± 0.10 d CaAlgLYO 5.82 ± 0.27 b 5.64 ± 0.18 c 6.37± 0.77 c 7.16 ± 0.20 e 8.47 ± 0.26 cd 8.81 ± 0.27 c CaAlgHEWL 5.61 ± 0.11 b 5.43 ± 0.09 cd 6.19 ± 0.12 cd 7.05 ± 0.10 e 8.57 ± 0.38 b 8.83 ± 0.21 c CaAlNHEWL 5.71± 0.36 b 4.97 ± 0.12 ef 6.01 ± 0.23 c 6.11 ± 0.10 f 7.04 ± 0.16 e 7.15 ± 0.17 e CaAlgNLYO 5.63 ± 0.19 b 4.90± 0.02 e 6.06 ± 0.21 c 6.35 ± 0.13 f 7.15 ± 0.15 e 7.11 ± 0.16 e * All analyses were based on two separate experiments with each mean + standard deviation being average of three determinations. Means within each vertical column followed by the same letter are not significantly different (P = 0.05) from each other. Statistical comparisons of all pairs were analyzed using Student’s t-test following one-way analysis of variance (ANOVA) (SAS Institute Inc., Cary, NC). 54 Table 4.2 Effect of Different Antimicrobials with and without Calcium Alginate Coating on the Growth of Salmonella anatum at 4◦ C for 35 Days. Log CFU/g* Treatment* Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 C 5.78 ± 0.10 a 5.90 ± 0.34 a 5.91 ± 0.14 a 6.07± 0.11 a 6.05 ± 0.06 a 6.12 ± 0.01 a CaAlg 5.70 ± 0.14 ab 5.68 ± 0.29 a 5.74 ± 0.24 a 5.89 ± 0.17 ab 5.78 ± 0.30 a 6.16 ± 0.15 a N 5.59 ± 0.30 a 5.81 ± 0.07 a 5.98 ± 0.12 a 5.79 ± 0.02 a 6.06 ± 0.12 a 5.63 ± 0.72 a LYO 5.21± 0.10 cd 5.58 ± 0.04 a 5.54 ± 0.07 b 5.81 ± 0.21 abc 5.60 ± 0.26 b 5.39 ± 0.08 b HEWL 5.35 ± 0.07 abc 5.75 ± 0.36 a 5.61± 0.02 a 5.66 ± 0.14 b 5.58 ± 0.21 b 5.43 ± 0.13 b CaAlgN 5.77 ± 0.16 a 5.70 ± 0.01 a 5.93 ± 0.20 a 5.52 ± 0.19 c 5.90 ± 0.10 ab 5.94 ± 0.02 ab CaAlgLYO 4.88 ± 0.15 d 4.84 ± 0.31 b 4.79 ± 0.21 c 4.93 ± 0.02 d 4.83 ± 0.17 c 4.60 ± 0.36 c CaAlgHEWL 4.88 ± 0.21 d 4.99 ± 0.22 b 5.03 ± 0.16 c 4.89 ± 0.14 d 4.93 ± 0.10 c 4.64 ± 0.20 c CaAlgNHEWL 4.97 ± 0.09 d 4.88 ± 0.11 b 4.86 ± 0.18 c 4.71± 0.13 d 3.88 ± 0.12 d 3.91 ± 0.11 d CaAlgNLYO 4.88 ± 0.07 d 4.64 ± 0.20 b 4.85 ±0.22 c 4.64 ± 0.20 d 3.82 ± 0.16 d 3.87 ± 0.14 d * All analyses were based on two separate experiments with each mean + standard deviation being average of three determinations. Means within each vertical column followed by the same letter are not significantly different (P = 0.05) from each other. Statistical comparisons of all pairs were analyzed using Student’s t test following one-way analysis of variance (ANOVA) (SAS Institute Inc., Cary, NC. 55 At 4°C, an initial inoculum of Listeria monocytogenes increased from 6.57 log CFU/g to 6.81 log CFU/g on day 7 in the control samples. Conversely, studies have shown that L. monocytogenes can grow rapidly on cold-smoked salmon increasing up to 5 log within 7 days at 4°C (Nilsson et al., 1997). The slow growth of L. monocyotgenes in our case could be due to the presence of different microflora on the surface of the smoked salmon samples which can interfere with the growth of Listeria species on the surface of the smoked fish. Studies have shown that interaction between Listeria species and other microflora on the surface of raw food material is complex. Depending on the presence of the microflora, the growth of Listeria can either be inhibited or enhanced (Guyer and Jemmi, 1991). The low growth of Listeria could be due to the competition with the natural microflora on the surface of the fish (Ben Embarek, 1994). The bacterial flora of stored smoked fish consists of Lactobacillus spp and if it is predominantly present on the fish it could inhibit the growth of Listeria as a result of competition (Guyer and Jemmi, 1991; Ben Embarek, 1994; Nilsson et al., 1997). On the other hand, at an initial inoculation level of 5.78 log CFU/g, S. anatum numbers on control samples remained almost constant throughout the experiment (Gill and Holley, 2000). The Salmonella anatum strain used in this study did not grow at refrigerated temperatures because this pathogen is a mesophilic organism and does not proliferate at refrigerated temperatures. However, Salmonella spp. can retain their biochemical and serological characters at low temperatures thus maintaining their pathogenic potential (Wilson et al., 1974). Nisin is a food grade antimicrobial effective mainly against Gram-positive bacteria. In our study, Nisin significantly reduced L. monocytogenes counts on day 0 56 (Nattress et al., 2001; Cutter and Siragusa, 1997) but nisin did not reduce S. anatum counts. However, when S. anatum was treated with nisin and lysozyme a significant reduction in bacterial counts occurred. Nisin combined with a chelating agent was bactericidal against Salmonella spp (Steven et al., 1991). Our study has shown that nisin did not retain its antimicrobial activity throughout the 35 day period. It has been reported that nisin has significant antimicrobial activity at the starting of the storage during the refrigerated storage but its effectiveness decreases as the storage period increases (Rose et al., 1999; Janes et al., 2002) Although lysozyme is known to be mainly effective against Gram-positive organism, our study shows that lysozyme was able to inhibit the growth of the Gram- negative S. anatum. Studies have shown that lysozyme acts on Gram-negative bacteria by a mechanism independent of its enzymatic action (Ibrahim et al., 2001; Pellegrini et al., 1992). Nattress et al.,(2001) reported that lysozyme in combination with nisin improves the efficiency of the mixture, show greater antimicrobial activity and extend the time during which nisin could be effective. Synergy between nisin and lysozyme against Carnobacterium was also observed and a combination of nisin and lysozyme was more effective against Carnobacterium than the individual component (Chung and Hancock, 2000).Electron microscopy results showed an increased surface disruption of the bacterial membrane and proposed that nisin could be inhibiting the energy dependent processes that repair lysozyme damage (Chung and Hancock, 2000). This synergy between the mixtures of nisin and lysozyme could be used to extend the shelf life of a variety of food products in the food industry when samples need to be stored for longer periods of time. 57 Calcium alginate edible coatings increased the effectiveness of nisin, hen egg white lysozyme, and oyster lysozyme when incorporated inside the coatings. Nisin and oyster lysozyme in the calcium alginate coatings reduced L. monocyotogenes and S. anatum counts by 2.75 log CFU/g and 2.25 log CFU/g, respectively by day 35. Wan et al., (1997) showed that the effectiveness of nisin increased when incorporated in calcium alginate micro-particles. We found that there was no significant difference between Hen egg white lysozyme and oyster lysozyme on the bacterial growth when incorporated into the coating. Our results agree with Cutter and Siragusa (1996) who immobilized nisin in a calcium alginate film and found that bacterial reduction was greater when the bacteriocin was incorporated into the film rather than when applied alone. Our results show that incorporation of antimicrobial agents into the calcium alginate edible film was able to retain the effectiveness of the antimicrobial agents throughout the 35 days storage making it more effective than when they were applied alone on the samples. These results are in agreement with studies using calcium alginate gel containing antimicrobial agents that effectively reduced foodborne pathogens on a wide variety of food products (Cutter and Siragudsa, 1996; Wan et al., 1997; Lazarus et al., 1976; Williams et al., 1978). Our results indicated that the effectiveness of oyster lysozyme was enhanced when combined with nisin incorporated into the calcium alginate coating, which could be used to preserve ready-to-eat smoked salmon at refrigerated temperatures. 58 CHAPTER 5 SUMMARY AND CONCLUSION The present study aimed at purifying lysozyme from oyster shell liquor, and evaluating its use against major foodborne pathogens, and determining the feasibility of using oyster lysozyme in antimicrobial films to enhance the preservation of smoked salmon. Shell liquor lysozyme was purified using a series of ion exchange chromatographies. There was a seasonal variation in lysozyme activity and the spring season showed the highest lysozyme activity. Three liters of shell liquor were collected during this season and scale up purification protocol was carried which yielded 205 mg of protein. The purified shell liquor lysozyme was then tested for its antibacterial activities against major food borne pathogens. From the Minimum Inhibitory Concentration assay we found that purified oyster lysozyme from the shell liquor had antimicrobial activity against a number of both gram-positive and gram negative bacteria. To control Listeria monocytogenes and Salmonella anatum on the surface of smoked salmon with edible film coatings, oyster lysozyme was incorporated into edible film coatings. Calcium alginate coating showed promise with the control of S. anatum and hence was chosen for 35 days shelf life study. 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Mol Biol. 139:11-25. 64 APPENDIX 1: DETERMINATION OF THE MOST EFFECTIVE FILM COATING FOR ENHANCING THE ANTIMICROBIAL ACTIVITY OF OYSTER LYSOZYME The following abbreviations stand for: Z, Zein; CaAlg, Calcium alginate; Ag, Agar; EC, Edible coating; HEWL, Hen egg white lysozyme; LYO; Oyster lysozyme; N, Nisin Edible coating + Antimicrobials + Nisin EC + LYO + N EC + HEWL + N Edible coatings AgZ CaAlg N LYO HEWL Antimicrobials Control: No treatment 1 g smoked salmon Control: Inoculated Control Edible coating + Antimicrobials EC + N EC + HEWL EC + LYO Immersed 24 h Bacterial suspension 65 APPENDIX 2: EFFECT OF OYSTER LYSOZYME INCORPORATED INTO CALCIUM ALGINATE COATINGS AGAINST LISTERIA MONOCYTOGENES AND SALMONELLA ANATUM DURING 35 DAYS STORAGE 24 h Broth culture Treatments Air dry for 20 mins Determination of bacterial counts 0 day 7 days 14 days 21 days 28 days 35 days Bacterial counts Log CFU/g 66 VITA Shreya Datta was born on February 4, 1978, in Bareilly, India. She earned her Bachelor of Science degree in microbiology in 1999 from Sardar Patel University, Gujarat, India. She then got her Master of Science degree in microbiology in 2001 from Department of Biosciences, Sardar Patel University, Gujarat, India. She worked as a lecturer in the Microbiology Department, V.P. Science College, Sardar Patel University, V.V.Nagar, India from 2001-2002. She joined as a master’s student in the Department of Food Science at Louisiana State University, Baton Rouge, Louisiana, in 2003.