Antibacterial Activity of Dextran-Conjugated Lysozyme against Escherichia coli and Staphylococcus aureus in Cheese Curd

Transcription

1 411 Journal of Food Protection, Vol. 71, No. 2, 2008, Pages Copyright, International Association for Food Protection Research Note Antibacterial Activity of Dextran-Conjugated Lysozyme against Escherichia coli and Staphylococcus aureus in Cheese Curd S. AMIRI, 1 R. RAMEZANI, 1 AND M. AMINLARI 2 * 1 Department of Food Science and Technology, School of Agriculture, and 2 Department of Biochemistry, School of Veterinary Medicine, Shiraz University, Shiraz, 71345, Iran MS : Received 5 December 2006/Accepted 18 June 2007 ABSTRACT The purposes of this research were to glycosylate lysozyme with dextran under Maillard reaction conditions and assess the antimicrobial characteristics of the lysozyme-dextran conjugate in a culture medium and cheese curd. Solutions containing lysozyme and dextran were incubated at 60 C and at 79% relative humidity. Gel permeation chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis were used to follow the glycosylation process. Under optimum conditions 3.7 mol of dextran were coupled to 1 mol of lysozyme. Lytic activity of the conjugate against the cell wall of Micrococcus luteus was about 62% of that of native lysozyme. Evaluation of the lysozyme-dextran conjugate against test microorganisms (Staphylococcus aureus and Escherichia coli) in culture media indicated a progressive increase in antimicrobial activity, with an increase in enzyme-conjugate concentration. The lysozyme-dextran conjugate was also effective against E. coli in a natural food system, as it reduced the bacterial count by 3 log in cheese curd after 40 days of storage. Unlike E. coli, the antimicrobial action of lysozyme against S. aureus was not improved by conjugation with dextran in both in vitro and in vivo tests. Antimicrobial activity of the lysozyme-dextran conjugate against gram-negative bacteria is probably related to the remaining lytic activity as well as the excellent surfactant properties of the lysozyme-dextran conjugate. These results might increase the applicability of lysozyme as a natural antimicrobial ingredient in different food products. A widespread trend in many countries is the movement toward natural food products. In an effort to meet this demand, the food industry has expressed increased interest in using antimicrobial preservatives that are perceived as more natural (2). Lysozyme is well-known enzyme that has the ability to lyse bacterial cells (17, 19). Lysozyme is found abundantly in nature and is produced by bacteria, fungi, plants, birds, and mammals. Some viruses even contain the genetic code for lysozyme (3). Chicken egg-white lysozyme has a molecular weight of 14,600 (129 amino acid residues) and a high isoelectric point (pi 11.0). Because of the presence of four disulfide bonds, flexibility of the main chain is very restricted, even in solution, resulting in high structural stability (18). Lysozyme is a single-peptide protein that possesses enzymatic activity against the - 1,4-glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine found in peptidoglycan, the major component of the cell wall of both gram-positive and gramnegative bacteria (5). Hydrolysis of the cell wall by lysozyme can damage structural integrity, leading to bacterial cell lysis (4). When modified with fatty acids or hydrophobic peptides, lysozyme can be converted into a potent bactericidal agent against Escherichia coli K-12 (7, 11). The lysozyme-dextran conjugate may act on the outer membrane of gram-negative bacteria, and the conjugate is ex- * Author for correspondence. aminlari@shirazu.ac.ir. pected to show a lytic action spectrum different from that of native lysozyme. The purpose of this investigation was to assess the antimicrobial characteristics of the lysozyme-dextran conjugate and the possibility of its application as a new preservative in the cheese industry. MATERIALS AND METHODS Materials. Dextran (molecular weight 10,000), Sephadex G-100, trinitrobenzene sulfonate, and Micrococcus luteus cells were obtained from Sigma (St. Louis, Mo.). Lysozyme was provided by Canadian Inovatech (Abbotsford, British Columbia). Protein molecular-weight markers were obtained from Fermentas (Molndal, Sweden). Plate count agar, nutrient broth, MacConkey agar, and Baird Parker medium were obtained from Merck (Darmstadt, Germany). E. coli IFO 1399 and Staphylococcus aureus IFO 1112 were purchased from the Persian Type Culture Collection (Tehran, Iran). All other chemicals were reagent grade and were commercially available. Preparation of the lysozyme-dextran conjugate. The lysozyme-dextran conjugate was prepared as described by Scaman et al. (18). Four hundred milligrams of lysozyme and 2 g of dextran were mixed in 5 ml of 0.1 M sodium phosphate buffer (ph 7.0) and lyophilized for 7 h. The mixture was incubated at 60 C and 78.9% relative humidity for 1 week in a container containing a saturated KBr solution. A control (without dextran) was treated under the same conditions. The lysozyme-dextran conjugate thus prepared was separated from the unreacted lysozyme by gel permeation chromatography, using a Sephadex G-100 column. The

2 412 AMIRI ET AL. J. Food Prot., Vol. 71, No. 2 column was equilibrated and eluted with 0.05 M sodium phosphate (ph 7.4). Fifty milligrams of conjugated lysozyme was dissolved in 1.0 ml of 0.05 M phosphate buffer (ph 7.4). The solution was gently mixed and centrifuged at 2,500 g for 10 min. The supernatant was applied to a Sephadex G-100 column (90 by 1.5 cm), and the column was connected to the buffer reservoir. The protein content in each fraction was detected by measuring absorbance at 280 nm. All fractions containing the lysozyme dextran conjugate were pooled, concentrated by ultrafiltration, and lyophilized. Fifty milligrams of unconjugated lysozyme was treated similarly. Measurement of protein content. The soluble protein content of all samples was determined by the method of Lowry et al. (14). Electrophoresis. Sodium dodecyl sulfate (SDS) slab gel electrophoresis was conducted according to the method of Laemmli using a 5 to 20% gradient acrylamide separating gel (13). Protein samples were added to the loading buffer to give a final concentration of 1 mg/ml protein, 0.01 mol/ml Tris-HCl (ph 6.8), 0.4% SDS, 100 g/liter glycerol, and 0.04 g/liter bromophenol blue. The running gel (140 by 140 by 1 mm) was a 50- to 200-g/liter gradient polyacrylamide gel in 1.2 mol/liter Tris-HCl (ph 8.8) and 3 g/liter SDS. The stacking gel contained 30 g/liter acrylamide in 0.25 mol/liter Tris-HCl (ph 6.8) and 2 g/liter SDS. The electrode buffer contained mol/liter Tris-HCl, mol/liter glycine, and 1.5 g/liter SDS at ph Electrophoresis was performed at a constant 25-mA current, and the gel was stained with 2.5 g/liter Coomassie brilliant blue R-250 in 500 g/liter acetic acid 250 g/liter methanol and de-stained with 100 g/liter acetic acid 70 g/liter methanol. Measurement of the amino groups of the lysozyme-dextran conjugate. The content of free amino groups in the lysozyme-dextran conjugate was determined by the trinitrobenzene sulfonate method (6). To 1 ml of protein solution (1 mg/ml) were added 1 ml of 4% NaHCO 3 (ph 8.5) and 1 ml of 0.1% trinitrobenzene sulfonate in water. The solution was incubated at 40 C for 2 h. Then, 1 ml of 10% SDS was added, followed by 0.5 ml of 1 N HCl. Absorbance of the solution, diluted with 0.01 N HCl if necessary, was read at 344 nm against a blank treated as above, but containing 1 ml of water instead of the protein solution. Lysozyme activity. Lysozyme activity was measured by the lysis of M. luteus cell walls (12). Nine milligrams of dried M. luteus cell walls was dissolved in 25 ml of 0.1 M potassium phosphate buffer (ph 7.0) and diluted to a final volume of 30 ml with the same buffer. Lysozyme or modified lysozymes at a concentration of 1 mg/ml were dissolved in cold distilled water. The M. luteus cell wall suspension (2.9 ml) was poured into a cuvette and incubated for 4 to 5 min to achieve temperature equilibration. The enzyme solution (0.1 ml) was added to the cuvette, and the change in absorbance at 450 nm was then recorded. The activity is presented as the rate of decrease in absorbance per min of the initial velocity of reaction. FIGURE 1. Elution profiles of native lysozyme, heated lysozyme without dextran, and the lysozyme-dextran conjugate on a Sephadex G-100 column (90 by 1.5 cm). Glycosylated lysozyme was prepared under optimum conditions (60 C, ph 7.0 for 1 week). The column was equilibrated and eluted at a flow rate of 0.5 mil/ min, with 0.05 M sodium phosphate (ph 7.4). Detection of antimicrobial activity. The antimicrobial assay for lysozyme derivatives used E. coli (as a representative gramnegative bacterium) and S. aureus (as a gram-positive bacterium). The bacteria were cultivated in nutrient broth at 37 C for 16 h and then decimally diluted to give a concentration of 10 6 CFU/ ml. To 4.5 ml of the bacterial suspension in 50 mm potassium buffer (ph 7.0) was added 0.5 ml of lysozyme or modified lysozyme solutions in 50 mm potassium buffer to give final lysozyme concentrations of 100, 250, and 400 g/ml. The mixture was incubated at 37 C for 16 h. Decimal dilutions were carried out in sterile physiological saline solution adjusted to ph 7.2. A 100- l aliquot was spread plated on plate count agar for both E. coli and S. aureus. Manufacture of cheese curd. Ten liters of whole milk (2.7% fat) was pasteurized at 65 C for 30 min. Rennet coagulation was done at 35 C. The extracted curds were placed in plastic molds at 30 C for about 2.5 h and were not salted. One milliliter of the bacterial suspension (10 6 CFU/ml in 50 mm potassium buffer [ph 7.0]) and 0.5 ml of lysozyme or modified lysozyme solution in the same medium (to give a final lysozyme concentration of 400 g/ml) were added to 40 g of cheese curd. The curds were mixed completely and allowed to ripen at 4 C for 40 days. The microbial counts were obtained every 3 days on the cheese curds. Sampling plan. Samples were removed every 3 days for bacterial counts. Briefly, 1 g of cheese curd was diluted in 9 ml of sterile physiological saline solution. The diluted samples were shaken for 2 min. Decimal dilutions (0.1 ml) were spread plated on Baird Parker agar (with 0.05% potassium tellurite) for S. aureus and pour plated in MacConkey agar for E. coli. All colonies were counted after 24 h of incubation at 37 C. Statistical analysis. All data were analyzed by the ANOVA procedure of COSTAT, and comparison of means was performed using the Duncan multiple range test (P 0.05). RESULTS AND DISCUSSION Incubation of chicken egg lysozyme at 60 C and 79% relative humidity for 7 days resulted in pale browning of the protein powder, indicative of the Maillard reaction. Figure 1 shows the elution profiles of lysozyme-dextran conjugates from gel permeation chromatography on a Sephadex G-100 column. Compared with the unmodified lysozyme, the glycosylated lysozyme was eluted in the void volume of the G-100 gel permeation chromatography column, suggesting an increase in the size and molecular weight of lysozyme due to the covalent attachment of dextran to lysozyme. Evidence for covalent attachment of dextran to lysozyme was obtained from SDS slab polyacrylamide gel electrophoresis of the conjugate, as shown in Figure 2. The electrophoretic patterns of the lysozyme-dextran conjugate

3 J. Food Prot., Vol. 71, No. 2 ANTIBACTERIAL ACTIVITY OF DEXTRAN-CONJUGATED LYSOZYME IN CHEESE CURD 413 TABLE 1. Enzymatic activity and free amino groups in the lysozyme-dextran conjugate a Enzyme Free amino groups Enzymatic activity (U/mg) Lysozyme 7 A A Lysozyme-dextran 3.3 B B Heat-lysozyme 7 A C a Each point is the average of three replicates. For each property, values with different letters are significantly different (P 0.05). FIGURE 2. SDS polyacrylamide gel electrophoresis of dextranconjugated lysozyme before gel filtration chromatography (5 to 20% polyacrylamide gel, 20 g of protein per well). M, molecular weight markers; 1, unmodified lysozyme; 2, lysozyme (no dextran, 1 week at 60 C); 3, lysozyme (5 mg of dextran per mg of lysozyme, 1 week at 60 C). before gel permeation chromatography showed a broad, diffused band, the molecular weight of which was higher than the band corresponding to the native lysozyme, suggesting covalent attachment of dextran to lysozyme. Similar results have been reported by other investigators using different molecules binding to lysozyme, including perillaldehyde (7), dextran (1), glucose-stearic monoester (19), and galactomannan (18). The extensively diffused band is indicative of the multiplicity of the conjugated derivatives obtained during the reaction of lysozyme with dextran. These multiple forms probably originate from the formation of molecules with different numbers of polysaccharides attached to each protein. Lysozyme has seven free amino groups (1). It is therefore conceivable that these multiple derivatives belong to lysozyme with one to seven dextrans covalently attachment to amino groups. The 3.7 mol of dextran shown in Table 1 is therefore the average number. Measurement of free amino groups of the conjugate showed that when lysozyme was heated at 60 C for 1 week, 3.7 mol of dextran was attached to 1 mol of lysozyme (Table 1). Lysozyme activity of the conjugate was measured by lytic activity against Micrococcus luteus cell wall material. Enzymatic activity of the conjugate decreased to 62.37% (Table 1). Both steric hindrance and blocking of the positive charges might be responsible for the decrease in lytic activity of the conjugated enzyme observed in this study. This result is in agreement with those of Ibrahim et al. (7, 11), who reported a 44% reduction in enzyme activity of lysozyme derivatives incorporating four palmitic acid residues with respect to unmodified lysozyme. These researchers also observed a decrease in lysozyme activity as the degree of modification increased up to four residues of perillaldehyde. They observed a 27% loss in activity when four residues of perillaldehyde were attached. Ibrahim et al. showed that the progressive loss in lysozyme activity in the absence of modifying agents, occurred with increasing temperature (to 80 C) for 20 min at ph 6.0, whereas a complete loss of activity was reached by heating at ph 7.0 (8). From the results of the present study and those of Ibrahim et al. (11), one can conclude that chemical modification can protect lysozyme against heat-induced loss of lytic activity. Antimicrobial activity of modified lysozyme. Antibacterial action of lysozyme derivatives on E. coli and S. aureus was also studied. Figure 3 shows the activity of lysozyme and modified lysozymes on E. coli as a function of enzyme concentration. The highest antimicrobial activity was observed when the lysozyme-dextran conjugate was used. Bactericidal activity against E. coli at 37 C increased as the concentration of the conjugate increased. Moreover, lysozyme and the heated enzyme without dextran showed some antimicrobial effect against E. coli only when used at a concentration of 400 g/ml. Antimicrobial effects of the conjugate against gram-positive bacteria were also investigated using one typical strain (Fig. 4). It is well known that lysozyme has antimicrobial effects against gram-positive bacteria. As indicated in Figure 4, lysozyme showed bactericidal activity against S. aureus at 37 C, which increased with enzyme concentration. As expected, the antimicrobial effects of the lysozyme-dextran conjugate were almost the same as those of lysozyme for S. aureus. Lysozyme attacks only glycosidic bonds between N-acetylhexosamines of the peptidoglycan layer in bacterial cell walls. However, since the cell envelope of these bacteria contains a significant amount of hydrophobic material FIGURE 3. Antimicrobial activity of lysozyme derivatives against E. coli. The enzyme-bacterial suspensions were incubated at 37 C for 16 h. Each point is the average of three replicates. Columns with different letters are significantly different (P 0.05).

4 414 AMIRI ET AL. J. Food Prot., Vol. 71, No. 2 FIGURE 4. Antimicrobial activity of lysozyme derivatives against S. aureus. The enzyme-bacterial suspensions were incubated at 37 C for 16 h. Each point is the average of three replicates. Columns with different letters are significantly different (P 0.05). FIGURE 6. Antimicrobial activity of lysozyme derivatives against S. aureus in cheese curd at 4 C. Each point is the average of three replicates. such as lipopolysaccharide associated with the thin peptidoglycan layer, native lysozyme fails to lyse gram-negative bacteria when it is simply added to the cell suspension. Synergistic factors such as detergents and heat destabilize and consequently solubilize the outer membranes that consist mainly of lipopolysaccharide (15). For example, 1% SDS solubilizes the purified outer membrane isolated from E. coli K-12 without any sediment (16). However, addition of detergents or some surface-active compounds (such as SDS) is not always desirable nor applicable in the food industry. However, various hybrid proteins with polysaccharide (such as dextran or galactomannan) exhibited excellent functional properties (1, 15). Therefore, to improve surface properties of the protein, conjugation with a polysaccharide is desirable for industrial applications. In addition, these compounds are capable of solubilizing the outer membranes of gram-negative bacteria (16). Strong surface activity of the lysozyme-dextran conjugate leads to destruction of the outer membrane of gram-negative bacteria (9, FIGURE 5. Antimicrobial activity of lysozyme derivatives against E. coli in cheese curd at 4 C. Each point is the average of three replicates. 16), which might enhance the lytic activity of lysozyme toward the peptidoglycan layer in the inner membrane. Antimicrobial effects of the lysozyme-dextran conjugate in cheese curd. The antimicrobial activity of various forms of lysozyme against E. coli and S. aureus in the cheese curd as a natural food environment was investigated. Cheese curds, made from pasteurized cow milk, were inoculated with E. coli or S. aureus (10 6 CFU/40 g) with these bacterial populations measured every three days. As shown in Figure 5, lysozyme and modified enzymes were effective against E. coli at a concentration of 400 g/ml in the cheese curd. However, this reduction was significantly higher for the glycosylated enzyme compared with native lysozyme. The lysozyme-dextran conjugate was bactericidal, decreasing the populations of E. coli by 3 log (Fig. 5) as was also shown in in vitro tests. The differences between control (without enzyme), unmodified lysozyme and the lysozyme-dextran conjugate were significant (P 0.05). Figure 6 shows the antimicrobial effect of the enzyme and its derivatives against S. aureus. In cheese curd, lysozyme or modified enzymes were effective against S. aureus at 400 g/ml (P 0.05). This reduction was not as profound as it was with E. coli. There were no significant differences between the bactericidal effect of lysozyme and the lysozyme-dextran conjugate (P 0.05). However, these results were in good agreement with those from the in vitro tests. Taken together, the results of this study show that the lysozyme-dextran conjugate is a suitable candidate as a protein ingredient with antimicrobial activity (in addition to its improved solubility at different temperature and ph values, and better emulsion and foam stability (1)) in different food systems, as compared with the native enzyme or the heated lysozyme without dextran. ACKNOWLEDGMENT This research was financially supported by grants 85-GR-AGR-29 and 85-GR-VT-11 from Shiraz University Research Council.

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