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Use of active compounds for prolonging the shelf life of mozzarella cheese
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International Dairy Journal 18 (2008) 624–630 www.elsevier.com/locate/idairyj
Use of active compounds for prolonging the shelf life of mozzarella cheese Milena Sinigaglia, Antonio Bevilacqua, Maria Rosaria Corbo, Sandra Pati, Matteo Alessandro Del Nobile Faculty of Agricultural Science, Department of Food Science, University of Foggia, Via Napoli, 25-71100 Foggia, Italy Received 6 May 2007; accepted 29 November 2007
Abstract The effectiveness of lysozyme and ethylenediaminetetraacetic disodium salt (Na2-EDTA) against the spoilage microorganisms of mozzarella cheese was studied. Mozzarella cheeses were packaged in a conditioning solution (diluted brine), which contained lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (10, 20 and 50 mmol L1), and stored at 4 1C for 8 days. The population of spoilage microorganisms (total coliforms and Pseudomonadaceae), along with the functional microbiota of mozzarella cheese (lactic acid bacteria) was enumerated. Lysozyme and Na2-EDTA significantly inhibited the growth of coliforms and Pseudomonadaceae during the first 7 days of storage, whereas the functional microbiota (or lactic acid bacteria) were not affected. The results of this study showed that it is possible to extend the shelf life of mozzarella cheese through the use of lysozyme and Na2-EDTA in the conditioning brine. r 2008 Elsevier Ltd. All rights reserved.
1. Introduction Traditional fresh mozzarella cheese can be made using either a mesophilic or thermophilic starter, which have the role to produce enough lactic acid during cheese making to demineralize and transform the curd into a state that will stretch in hot water at the target pH. The most commonly used acidification methods are (1) milk ferment inoculation (IDC), (2) chemical acidification (CA). These methods reduce the acidification time (1.5–2.5 h), and hence operation times can be considerably reduced in comparison with traditional processes. The IDC method produces larger yields of approximately 14–18%, compared with approximately 10–12% with the CA method (Ferrari et al., 2003). Traditional mozzarella is packaged in a dilute solution of salts (NaCl and/or CaCl2) (called conditioning brine) and has a short shelf life (5–7 days). Contrary to the traditional way of packaging, mozzarella cheese in some part of the world is packaged without the conditioning solution. The shelf life of this cheese is longer (14–28 weeks) than that Corresponding author. Tel.: +39 881 589 242; fax: +39 881 589 231.
E-mail address: [email protected] (M.A. Del Nobile). 0958-6946/$ - see front matter r 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.11.022
reported for the Italian product (Aly, 1996; Kuo & Gunaserakan, 2003; Lilbaek et al., 2006); The short shelf life of traditional mozzarella cheese has been attributed to microbiological spoilage. This spoilage is often caused by the growth of coliforms, Pseudomonas spp. and/or by psychrotrophic bacteria that grow on the cheese surface, mostly coming from water used in the manufacture (Cabrini & Neviani, 1983; Cantoni, Iacumin, & Comi, 2003; Cantoni, Stella, Cozzi, Iacumin, & Comi, 2003; Parisi, 2003; Rondinini & Garzaroli, 1990). According to the Italian law (Anonymous, 1997), coliforms are the test microorganisms for evaluating shelf life of mozzarella cheese. Presence of coliforms in cheese is an indication of poor sanitation (Farkye, 2000). Coliforms grow rapidly during the first few days of storage, producing lactic acid, acetic acid, formic acid, succinic acid, ethanol, 2,3-butyleneglycol, H2 and CO2 and causing the gassy defect and the swelling of the plastic bags (Farkye, 2000). One approach for extending the microbiological shelf life of mozzarella cheese is to introduce antimicrobials, preferably naturally occurring antimicrobials (Gill & Holley, 2000a). Altieri, Scrocco, Sinigaglia, and Del Nobile (2005), for example, added a lactic acid/chitosan solution
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directly to the whey starter used for the traditional mozzarella cheese manufacturing. They reported that chitosan inhibited the growth of some spoilage microorganisms (such as coliforms), whereas it did not influence the growth of the functional microbiota. Alternatively, we hypothesize that addition of lysozyme and ethylenediaminetetraacetic acid (EDTA) in the conditioning brine could improve the shelf life of mozzarella cheese. Lysozyme is a lytic enzyme found in foods such as milk and eggs, which can hydrolyze b-1,4 linkages between N-acetylmuramic acid and N-acetylglucosamine (Cunningham, Proctor, & Goetsch, 1991). Commercially, lysozyme has been used primarily to prevent late blowing in semihard cheeses, caused by Clostridium tyrobutyricum (Bester & Lombard, 1990; Cunningham et al., 1991). However, EDTA, a chelator used in a wide variety of food products to prevent oxidation and other deteriorative reactions catalyzed by metal ions, is known to potentiate the activity of antimicrobials (such as lysozyme), especially against Gram-negative microorganisms (Branen & Davidson, 2004; Gill & Holley, 2000a; Razavi-Rohani & Griffiths, 1994; Stevens, Sheldon, Klapes, & Klaenhammer, 1991). In the USA, several EDTA salts have a history of food use; Na2-EDTA, in fact, is approved for use in canned carbonated soft drinks, canned cooked vegetables, potato salad and other foods and in aqueous multivitamins preparation (21 CFR 72) (Code of Federal Regulations, 1998) at concentrations ranging from 35 to 800 ppm (Heimbach et al., 2000). In addition, it is approved for indirect food additives uses as components of adhesive (21 CFR 175.105) (Code of Federal Regulations, 1998) and as a component of an aqueous sanitizing solution used in food-processing equipments (21 CFR 178.1010) (Code of Federal Regulations, 1998); its NOAEL value (no observed adverse lethal effect level) is 500 mg kg1 day1. Moreover, it is not carcinogenic and does not show a bioaccumulation potential (SIAM, 2001). To our knowledge, only few studies have been conducted on food systems in regard to the synergistic efficacy as an antimicrobial of lysozyme and EDTA (Gill & Holley, 2000a, b, 2003), while no studies to our knowledge have been conducted on mozzarella cheese. Therefore, this study aimed to determine if the microbiological shelf life of mozzarella cheese could be improved by using lysozyme and EDTA. 2. Materials and methods 2.1. Sample preparation The research focused on mozzarella cheese, made from cows’ milk, and was divided in two different experimental phases. In the 1st phase, mozzarella cheeses (50 g of weight and 5–7 cm of diameter; moisture, 55%; fat, 24%; fat/ proteins, 1.2), produced through the biological acidification (whey starters), were purchased from a cheese factory located in Puglia (a region of Southern Italy) and brought
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to our laboratory under refrigeration (4 1C). In Italy, mozzarella is sold in small size pieces, individually packaged in bags containing diluted saline solution (conditioning solution). Cheeses were removed from their packages and placed in individual polyethylene pouches containing 200 mL of the conditioning solution (12% NaCl brine), which contained 0.25 mg mL1 of lysozyme (Sigma-Aldrich, Milan, Italy) and different concentrations of Na2-EDTA (J.T. Baker, Milan, Italy), i.e., 50, 20 and 10 mmol L1. High barrier 170 mm 250 mm plastic bags, made of 102 mm nylon/polyethylene (Tecnovac, San Paolo D’Argon, Bergamo, Italy), were used. The bags had a CO2 and O2 permeability of 3.26 1019 mol mm2 s1 Pa1 and 9.23 1019 mol mm2 s1 Pa1, respectively, and water vapor transmission rate of 1.62 1010 kg m2 s1. Mozzarella cheeses, packaged in conditioning brine without lysozyme and Na2-EDTA, were used as controls. As the addition of Na2-EDTA produced a decrease in pH to 4.5, a second control was prepared by packaging mozzarella balls in 200 mL of the conditioning brine, acidified to pH 4.5 with 0.1 M HCl, to observe the influence of pH on microflora (Table 1). In the 2nd experimental phase, in order to prevent the decrease of pH due to the addition of Na2-EDTA, 50-g samples of cow mozzarella cheese, purchased from the same cheese factory, were packaged with 200 mL of a dilute salt solution to which a phosphate buffer (50 mmol L1; pH 6.5; K2HPO4/KH2PO4: J.T. Baker) was added previously. The conditioning solution contained lysozyme (0.25 mg mL1) and Na2-EDTA (50 and 20 mmol L1). Mozzarella cheeses, packaged in a buffered diluted brine, were used as controls (Table 1). The samples of 1st and 2nd experimental phases were analyzed immediately after the
Table 1 Samples of mozzarella cheese of 1st and 2nd experimental phases Sample
Conditioning solution
1st experimental phase Control A Diluted salt solution Active B Diluted salt solution+lysozymea+Na2-EDTA 50 mmol L1 samples C Diluted salt solution+lysozyme+Na2-EDTA 20 mmol L1 D Diluted salt solution+lysozyme+Na2-EDTA 10 mmol L1 Acidified E Diluted salt solution, acidified to pH 4.5 control 2nd experimental phase Bufferedb diluted salt solution Control Ab Active Bb Buffered diluted salt solution+lysozymea+Na2samples EDTA 50 mmol L1 Cb Buffered diluted salt solution+lysozyme+Na2EDTA 20 mmol L1 a
0.25 mg mL1. The diluted salt solution was buffered to pH 6.5 using a phosphate buffer 50 mmol L1. b
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packaging and after 1, 3, 6 and 8 days of storage under refrigerated conditions (4 1C).
2.2. Microbiological analyses Samples of 10 g of mozzarella cheese were diluted with 90 mL of a sterile saline solution (0.9%, w/v, NaCl) in a Stomacher bag and blended for 1 min with a Stomacher Lab Blender 400 (PBI International, Milan, Italy). Decimal dilutions of cheese homogenates were performed and microbiological counts carried out. The media and the conditions used for the enumerations were as follows: MRS Agar, modified by adding 0.17 g L1 of cycloheximide (Sigma-Aldrich) after autoclaving at 121 1C for 15 min, incubated at 30 1C for 4 days under anaerobic conditions for lactic acid bacteria; Violet Red Bile Agar (VRBA), incubated at 37 1C for 18–24 h for total coliforms; Slanetz Bartley Medium, incubated at 37 1C for
Sinigaglia (2006), was adopted: logðcfu g1 Þ ¼ logðcfu g1 Þmax A exp exp ðmmax 2:71Þ l S:L: þ1 þ A exp exp A lt ðmmax 2:71Þ þ1 A
where A (log(cfu g1)), mmax (log(cfu g1) day1) and l (days) are the Gompertz parameters (maximum increase of cell load attained in the stationary phase; maximal growth rate and lag phase, respectively), log(cfu g1)max is the threshold value for total coliforms and Pseudomonadaceae, S.L. is the shelf life and t is the time (days). When a decrease of microbial concentration followed by a cell load increase was detected, the re-parameterized Gompertz equation was cast in the following form:
n nh i oo 8 l t > < logðcfu g1 Þ ¼ K 1 þ A1 exp exp ðm1max 2:7182Þ A1 1 þ 1 n nh i oo > : logðcfu g1 Þ ¼ K 2 þ A2 exp exp ðm2max 2:7182Þ l2 ½tt þ 1 A2
48 h for enterococci; Pseudomonas Agar Base, modified by adding Pseudomonas CFC selective supplement after autoclaving at 121 1C for 15 min, incubated at 30 1C for 48 h for Pseudomonas spp. All the media and the supplements used were from Oxoid (Milan, Italy). 2.3. Evaluation of pH The pH was evaluated on mozzarella cheese homogenates and on the conditioning solution by a pH meter (Crison, mod Micro-pH 2001, Barcelona, Spain). 2.4. Statistical analysis and shelf life calculation In each experimental phase, the analyses were performed in duplicate and cheeses were obtained at two independent time points. The experimental data were submitted to oneway ANOVA and Tukey test (po0.05) through the software Statistica for Windows (Statsoft, Tulsa, OK, USA). The shelf life of the samples was evaluated through the population numbers of coliforms and Pseudomonadaceae. The critical values were set to 1 105 cfu g1 and 5 105 cfu g1 for coliforms and Pseudomonadaceae, respectively, as reported by the Italian law (Anonymous, 1997) and Bishop and White (1986). The re-parameterized Gompertz equation, proposed by Corbo, Del Nobile, and
(1)
tot t4t
(2)
where t is the time at which there is the switch from the decreasing to the increasing trend of cell load data; K1 (initial cell load), A1, m1max and l1 are the fitting parameters of the first Gompertz equation (decreasing trend of cell load data) and K2, A2, m2max and l2 the Gompertz values of the increasing length of the curve. The values of K1 and K2 were calculated through the following equations: K 2 ¼ ½logðcfu g1 Þmax A2 exp exp ðm2max 2:7182Þ l2 ½S:L: t þ1 (3) A2 l1 t 1 K 1 ¼ A1 exp exp ðmmax 2:7182Þ þ1 A1 þ K 2 þ A2 exp exp ðm2max 2:7182Þ l2 þ1 (4) A2 The confidence intervals of model parameters were evaluated as follows: first, a fit was run with the original data; then, using the data points standard deviation, 100 additional fits were run on artificial data sets, which were generated by randomly varying the data around the fitted function. From these additional fits, a distribution of values for each parameter was obtained. The sets of data
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obtained for each parameter were statistically treated to obtain the 95% confidence interval. 3. Results and discussion As reported in Section 2, the research was divided in two phases and focused on the possibility of prolonging the shelf life of mozzarella cheese through the addition of an antimicrobial mix (lysozyme and Na2-EDTA at 10, 20 and 50 mmol L1) to the conditioning brine. In the 1st phase, a dilute salt solution was used as the conditioning brine (with or without the antimicrobial mix), whereas in the 2nd part of the research, the mozzarella cheeses were packaged in a diluted salt solution buffered to pH 6.5 through a mixture of K2HPO4/KH2PO4. The analyses were performed for 8 days, in order to simulate a commercial storage time. Fig. 1 shows the evolution of total coliforms over storage time in the packed mozzarella cheese samples of the 1st experimental phase. As the addition of Na2-EDTA decreased the pH of the conditioning solution to 4.5, a second control sample was prepared (mozzarella cheese packaged in a conditioning brine acidified to pH 4.5), in order to evaluate if the antimicrobial activity was exerted by the active compounds or the low pH. As expected, total coliforms were able to proliferate in the control sample (A) and in the acidified control (E). Lysozyme+Na2-EDTA 50 mmol L1 (sample B) decreased the coliforms number from 4 log cfu g1 to the undetectable levels at 6 days; the cell load, however, increased up to 3.51 log cfu g1 after 8 days of storage. A different trend was observed in sample C (lysozyme+Na2-EDTA 20 mmol L1): in fact, a slight decrease of coliforms number (from 4.1 to 3.4 log cfu g1) within the storage time was observed. In the 2nd phase, the
Fig. 1. Evolution of coliforms population of traditional mozzarella cheese, packaged in a conditioning solution (12% NaCl), with lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (1st phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
Fig. 2. Evolution of coliforms population of traditional mozzarella cheese, packaged in a buffered conditioning solution (12% NaCl, pH 6.5), with lysozyme (0.25 mg mL1) and different amounts of Na2EDTA (2nd phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
conditioning solution was buffered to pH 6.5 to delete the effect of the acidification of the brine on the spoiling microorganisms. Moreover, the experiments were performed on higher amounts of Na2-EDTA (50 and 20 mmol L1), as they were more effective in inhibiting the spoiling microflora. Fig. 2 shows the evolution of total coliforms on mozzarella samples of the 2nd phase. As described previously, the addition of lysozyme and Na2-EDTA inhibited significantly the growth of coliforms, as it could be inferred by the decrease of coliforms population up to the undetectable level after 6 days of storage. The significance of Pseudomonadaceae and other psychrotrophic bacteria on cheese is well known (Cantoni, Iacumin et al., 2003; Cantoni, Stella et al., 2003); moreover, the contamination of mozzarella cheese by Pseudomonas spp. is a major concern for the Italian product, as they could affect the odor within the storage time (Bevilacqua, Corbo, & Sinigaglia, 2007). Fig. 3 shows the evolution of Pseudomonas spp. in mozzarella cheese samples of the 1st phase. The effect of lysozyme and Na2-EDTA was similar to that observed for total coliforms: in fact, an initial viability loss, followed by an increase of cell number after 3 days of storage, was observed in sample B. Otherwise, Pseudomonas spp. was able to proliferate in the controls (A and E) and in the samples packaged in the conditioning brine which contained lower amounts of Na2-EDTA (samples C and D). A similar result (decrease of cell number within 3 days, followed by an increase of the population) was recovered for the samples of the 2nd phase (Fig. 4). The initial decrease of pseudomonads, followed by the increase of cell load in the later days of storage, along with the total coliforms, could be due to a reversible stress on the cells, as reported by Johnston and Brown (2002) and Richards and Cavill (1976). The use of Na2-EDTA could cause a reversible damage to the outer membrane,
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Fig. 3. Evolution of Pseudomonas spp. population of traditional mozzarella cheese, packaged in a conditioning solution (12% NaCl), with lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (1st phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
Fig. 4. Evolution of Pseudomonas spp. population of traditional mozzarella cheese, packaged in a buffered conditioning solution (12% NaCl, pH 6.5), with lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (2nd phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
inhibiting the ability of making colonies on plates. Within a prolonged stress condition, the cell would repair the damages to the membrane acquiring the culturable ability. As the discussed figures are ‘‘qualitative results’’, the experimental data were modeled through a re-parameterized Gompertz equation, proposed by Corbo et al. (2006), and the shelf life was evaluated, in order to quantify the antimicrobial action of lysozyme and Na2-EDTA. The proposed function contains the shelf life value as a fitting parameter; therefore, it was possible to evaluate this parameter and its confidence interval and point out significant differences. Shelf life values were evaluated both through coliforms and Pseudomonas spp. cell number and the break-points were set to 1 105 (D.P.R. 54/97, Anonymous, 1997) for total coliforms and 5 105 for Pseudomonas spp. (Bishop & White, 1986).The results are shown in Table 2. The addition of the active compounds to the conditioning solution prolonged the shelf life evaluated through the coliforms. In fact, it was approximately 3.7 days for sample A, whereas in samples B (Na2-EDTA 50 mmol L1) and C (Na2-EDTA 20 mmol L1) it was 48 days (Table 2). A similar result was obtained for the pseudomonads which showed a shelf life value of 3.62 days in the control sample (A) and 5.64–6.60 days in samples B and C. It was not possible to point out the effect of Na2-EDTA concentration, as a superposition of shelf life values was observed. The acidified control (sample E) showed both for coliforms and pseudomonads a shelf life value, similar (p40.05) to those observed for sample A (control), but significantly lower (po0.05) than the shelf life of samples B, C and D. Therefore, we could suppose that the active compounds exerted an ‘‘intrinsic antimicrobial action’’, different from the acidification of the conditioning solution. As the shelf life value was evaluated through the cell number of two different targets (coliforms and Pseudomonas spp.), the actual shelf life (Table 2) of mozzarella cheese was assumed as the lower value of the calculated ones. In the conditions applied in this research, Pseudomonas spp. showed a shelf life value lower than that observed for total coliforms and the differences were significant (po0.05) for the sample with higher amounts of Na2-EDTA (samples B and C);
Table 2 Shelf life (days) of mozzarella cheese samples (1st experimental phase), packaged in diluted saline solutiona Samples
Coliforms
Pseudomonas spp.
Actual shelf lifeb
A B C D E
3.7380 (3.0888, 5.0763)c –d – 7.2493 (6.6185, 8.7916 1027) 4.3480 (4.0858, 5.8653)
3.6252 6.5911 5.6448 6.6067 4.2442
3.6252 6.5911 5.6448 6.6067 4.2442
a
(3.1357, (6.2746, (5.1263, (6.1579, (3.5879,
4.6041) 6.7918) 6.2457) 7.1196) 4.9651)
(3.1357, (6.2746, (5.1263, (6.1579, (3.5879,
4.6041) 6.7918) 6.2457) 7.1196) 4.9651)
Shelf life values were evaluated on the basis of coliforms and pseudomonads cell number. The critical values were set to 1 105 and 1 105 log cfu g1, respectively. b The actual shelf life value was assumed as the lowest one of the calculated shelf life values using coliforms and Pseudomonas spp. data. c 95% confidence interval. d It was not possible to evaluate shelf life values, because within 8 days the cell load of coliforms did not reach the break-point (5 log cfu g1).
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therefore, they were assumed as the actual shelf life values of the analyzed samples of mozzarella cheese, confirming that in some conditions the Pseudomonadaceae are the critical microorganisms for the shelf life of Italian mozzarella cheese, as reported by Corbo, Bevilacqua, and Sinigaglia (2007). As expected by the growth/death kinetics of coliforms and Pseudomonadaceae spp., the addition of Na2-EDTA and lysozyme increased the actual shelf life values (6.69 days) in the samples of the 2nd phase and no significant differences were recorded amongst the two amounts of Na2-EDTA or the microbial targets (coliforms and Pseudomonadaceae) (data not shown).
The active compounds did not inhibit lactic acid bacteria and enterococci. As an example, Figs. 5 and 6 show the evolution of lactic acid bacteria which increased from 5–6 to 7–8 log cfu g1, both in the samples of 1st and 2nd phase. Similar results were obtained for enterococci (data not shown). Within the complex microbiota of mozzarella cheese, lactic acid bacteria are the functional microorganisms, responsible for the acidification of the curd through the production of lactic acid from lactose and able to exert a probiotic action on the human health (Nousiainen & Seta¨la¨, 1998); therefore, the mix lysozyme–Na2-EDTA did not affect the functional characteristics of mozzarella cheese. As reported earlier, the addition of lysozyme and Na2-EDTA decreased significantly the pH of the conditioning brine (from 6.2–6.5 to 4.5), whereas the pH of mozzarella cheese (6.5) was less affected in the first days; it decreased, however, after 6 days of storage (5.8 in the control sample and 5.0 in the active ones—B, C and D) (data not shown). The addition of the phosphate buffer prevented the decrease, both in the conditioning brine and in mozzarella cheese. After 6 days, a drop, however, was observed in the conditioning solution, probably due to the loss of the buffering ability of KH2PO4–K2HPO4 mix (data not shown). Sensory analyses need to be investigated; odor, in fact, seemed to be not affected by the antimicrobial compounds or by the acidification of the conditioning brine, but we would assume that the acidification of the brine, due to the addition of Na2-EDTA, could result in differences in the texture of mozzarella cheese.
Fig. 5. Evolution of lactic acid bacteria population of traditional mozzarella cheese, packaged in a conditioning solution (12% NaCl), with lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (1st phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
4. Conclusions Na2-EDTA and lysozyme were effective in inhibiting the growth of spoilage microorganisms such as coliforms and Pseudomonas spp., without affecting the lactic acid bacteria. The studied method could be advantageously used to prolong the shelf life of mozzarella cheese, allowing the distribution of this product beyond the market borders. Further investigations are proposed to determine the minimum inhibitory concentration of EDTA, because of the toxicity of this compound for human health, and to identify new compounds, able to prolong the shelf life of traditional mozzarella cheese, without affecting its functional microbiota and rheological properties. Acknowledgment This work was supported by the Apulian region under Grant no. PS_031 ‘‘Miglioramento della qualita` dieteticonutrizionale e sicurezza di produzioni casearie tipiche della Capitanata’’.
Fig. 6. Evolution of lactic acid bacteria population of traditional mozzarella cheese, packaged in a buffered conditioning solution (12% NaCl, pH 6.5), with lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (2nd phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
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Gill, A. O., & Holley, R. A. (2000b). Surface application of lysozyme, nisin and EDTA to inhibit spoilage and pathogenic bacteia on ham and bologna. Journal of Food Protection, 63, 1338–1346. Gill, A. O., & Holley, R. A. (2003). Interactive inhibition of meat spoilage and pathogenic bacteria by lysozyme, nisin and EDTA in the presence of nitrite and sodium chloride at 24 degrees C. International Journal of Food Microbiology, 80, 251–259. Heimbach, J., Rieth, S., Mohamedshah, F., Slesinski, R., SamuelFernando, P., Sheenan, T., et al. (2000). Safety assessment of iron EDTA [sodium iron (Fe3+) ethylene diamine tetraacetic acid]: Summary of toxicological, fortification and exposure data. Food and Chemical Toxicology, 38, 99–111. Johnston, M. D., & Brown, M. H. (2002). An investigation into the changed physiological state of Vibrio bacteria as a survival mechanism in response to cold temperatures and studies on their sensitivity to heating and freezing. Journal of Applied Microbiology, 92, 1066–1077. Kuo, M. I., & Gunaserakan, S. (2003). Effect of frozen storage on physical properties of pasta filata and nonpasta filata mozzarella cheeses. Journal of Dairy Science, 86, 1108–1117. Lilbaek, H. M., Broe, M. L., Hoier, E., Fatum, T. M., Ipsen, R., & Sorensen, N. K. (2006). Improving the yield of mozzarella cheese by phospholipase treatment of milk. Journal of Dairy Science, 89, 4114–4125. Nousiainen, J., & Seta¨la¨, J. (1998). Lactic acid bacteria as animal probiotics. In S. Salminen, & A. von Wright (Eds.), Lactic acid bacteria. Microbiology and functional aspects (2nd ed., pp. 437–473). New York, NY, USA: Marcel Dekker Inc. Parisi, S. (2003). Curve predittive per la crescita dei batteri coliformi in prodotti lattiero-caseari. Industrie Alimentari, 421, 29–37. Razavi-Rohani, S. M., & Griffiths, S. M. (1994). The effect of mono and polyglycerol laurate on spoilage and pathogenic bacteria associated with foods. Journal of Food Safety, 14, 131–151. Richards, R. M. E., & Cavill, R. H. (1976). Electron microscope study of the effect of benzalkonium chloride and edetate disodium on cell envelope of Pseudomonas aeruginosa. Journal of Pharmaceutical Science, 65, 76–80. Rondinini, G., & Garzaroli, C. (1990). Mozzarelle prodotte per acidificazione chimica: Aspetti microbiologici e alterativi. Industrie Alimentari, 29, 329–333. SIAM. (2001). SIDS initial assessment profile. CAS No. 60-00-4. EDTA. /http://www.jetoc.or.sp/HP-SIDSpdffiles/60-00-4.pdfwww.jetoc.or.sp/ HP-SIDSpdffiles/60-00-4.pdfS; accessed 19.07.07. Stevens, K. A., Sheldon, B. W., Klapes, N. A., & Klaenhammer, T. R. (1991). Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria. Applied and Environmental Microbiology, 57, 3613–3615.
International Dairy Journal 18 (2008) 624–630 www.elsevier.com/locate/idairyj
Use of active compounds for prolonging the shelf life of mozzarella cheese Milena Sinigaglia, Antonio Bevilacqua, Maria Rosaria Corbo, Sandra Pati, Matteo Alessandro Del Nobile Faculty of Agricultural Science, Department of Food Science, University of Foggia, Via Napoli, 25-71100 Foggia, Italy Received 6 May 2007; accepted 29 November 2007
Abstract The effectiveness of lysozyme and ethylenediaminetetraacetic disodium salt (Na2-EDTA) against the spoilage microorganisms of mozzarella cheese was studied. Mozzarella cheeses were packaged in a conditioning solution (diluted brine), which contained lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (10, 20 and 50 mmol L1), and stored at 4 1C for 8 days. The population of spoilage microorganisms (total coliforms and Pseudomonadaceae), along with the functional microbiota of mozzarella cheese (lactic acid bacteria) was enumerated. Lysozyme and Na2-EDTA significantly inhibited the growth of coliforms and Pseudomonadaceae during the first 7 days of storage, whereas the functional microbiota (or lactic acid bacteria) were not affected. The results of this study showed that it is possible to extend the shelf life of mozzarella cheese through the use of lysozyme and Na2-EDTA in the conditioning brine. r 2008 Elsevier Ltd. All rights reserved.
1. Introduction Traditional fresh mozzarella cheese can be made using either a mesophilic or thermophilic starter, which have the role to produce enough lactic acid during cheese making to demineralize and transform the curd into a state that will stretch in hot water at the target pH. The most commonly used acidification methods are (1) milk ferment inoculation (IDC), (2) chemical acidification (CA). These methods reduce the acidification time (1.5–2.5 h), and hence operation times can be considerably reduced in comparison with traditional processes. The IDC method produces larger yields of approximately 14–18%, compared with approximately 10–12% with the CA method (Ferrari et al., 2003). Traditional mozzarella is packaged in a dilute solution of salts (NaCl and/or CaCl2) (called conditioning brine) and has a short shelf life (5–7 days). Contrary to the traditional way of packaging, mozzarella cheese in some part of the world is packaged without the conditioning solution. The shelf life of this cheese is longer (14–28 weeks) than that Corresponding author. Tel.: +39 881 589 242; fax: +39 881 589 231.
E-mail address: [email protected] (M.A. Del Nobile). 0958-6946/$ - see front matter r 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.11.022
reported for the Italian product (Aly, 1996; Kuo & Gunaserakan, 2003; Lilbaek et al., 2006); The short shelf life of traditional mozzarella cheese has been attributed to microbiological spoilage. This spoilage is often caused by the growth of coliforms, Pseudomonas spp. and/or by psychrotrophic bacteria that grow on the cheese surface, mostly coming from water used in the manufacture (Cabrini & Neviani, 1983; Cantoni, Iacumin, & Comi, 2003; Cantoni, Stella, Cozzi, Iacumin, & Comi, 2003; Parisi, 2003; Rondinini & Garzaroli, 1990). According to the Italian law (Anonymous, 1997), coliforms are the test microorganisms for evaluating shelf life of mozzarella cheese. Presence of coliforms in cheese is an indication of poor sanitation (Farkye, 2000). Coliforms grow rapidly during the first few days of storage, producing lactic acid, acetic acid, formic acid, succinic acid, ethanol, 2,3-butyleneglycol, H2 and CO2 and causing the gassy defect and the swelling of the plastic bags (Farkye, 2000). One approach for extending the microbiological shelf life of mozzarella cheese is to introduce antimicrobials, preferably naturally occurring antimicrobials (Gill & Holley, 2000a). Altieri, Scrocco, Sinigaglia, and Del Nobile (2005), for example, added a lactic acid/chitosan solution
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directly to the whey starter used for the traditional mozzarella cheese manufacturing. They reported that chitosan inhibited the growth of some spoilage microorganisms (such as coliforms), whereas it did not influence the growth of the functional microbiota. Alternatively, we hypothesize that addition of lysozyme and ethylenediaminetetraacetic acid (EDTA) in the conditioning brine could improve the shelf life of mozzarella cheese. Lysozyme is a lytic enzyme found in foods such as milk and eggs, which can hydrolyze b-1,4 linkages between N-acetylmuramic acid and N-acetylglucosamine (Cunningham, Proctor, & Goetsch, 1991). Commercially, lysozyme has been used primarily to prevent late blowing in semihard cheeses, caused by Clostridium tyrobutyricum (Bester & Lombard, 1990; Cunningham et al., 1991). However, EDTA, a chelator used in a wide variety of food products to prevent oxidation and other deteriorative reactions catalyzed by metal ions, is known to potentiate the activity of antimicrobials (such as lysozyme), especially against Gram-negative microorganisms (Branen & Davidson, 2004; Gill & Holley, 2000a; Razavi-Rohani & Griffiths, 1994; Stevens, Sheldon, Klapes, & Klaenhammer, 1991). In the USA, several EDTA salts have a history of food use; Na2-EDTA, in fact, is approved for use in canned carbonated soft drinks, canned cooked vegetables, potato salad and other foods and in aqueous multivitamins preparation (21 CFR 72) (Code of Federal Regulations, 1998) at concentrations ranging from 35 to 800 ppm (Heimbach et al., 2000). In addition, it is approved for indirect food additives uses as components of adhesive (21 CFR 175.105) (Code of Federal Regulations, 1998) and as a component of an aqueous sanitizing solution used in food-processing equipments (21 CFR 178.1010) (Code of Federal Regulations, 1998); its NOAEL value (no observed adverse lethal effect level) is 500 mg kg1 day1. Moreover, it is not carcinogenic and does not show a bioaccumulation potential (SIAM, 2001). To our knowledge, only few studies have been conducted on food systems in regard to the synergistic efficacy as an antimicrobial of lysozyme and EDTA (Gill & Holley, 2000a, b, 2003), while no studies to our knowledge have been conducted on mozzarella cheese. Therefore, this study aimed to determine if the microbiological shelf life of mozzarella cheese could be improved by using lysozyme and EDTA. 2. Materials and methods 2.1. Sample preparation The research focused on mozzarella cheese, made from cows’ milk, and was divided in two different experimental phases. In the 1st phase, mozzarella cheeses (50 g of weight and 5–7 cm of diameter; moisture, 55%; fat, 24%; fat/ proteins, 1.2), produced through the biological acidification (whey starters), were purchased from a cheese factory located in Puglia (a region of Southern Italy) and brought
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to our laboratory under refrigeration (4 1C). In Italy, mozzarella is sold in small size pieces, individually packaged in bags containing diluted saline solution (conditioning solution). Cheeses were removed from their packages and placed in individual polyethylene pouches containing 200 mL of the conditioning solution (12% NaCl brine), which contained 0.25 mg mL1 of lysozyme (Sigma-Aldrich, Milan, Italy) and different concentrations of Na2-EDTA (J.T. Baker, Milan, Italy), i.e., 50, 20 and 10 mmol L1. High barrier 170 mm 250 mm plastic bags, made of 102 mm nylon/polyethylene (Tecnovac, San Paolo D’Argon, Bergamo, Italy), were used. The bags had a CO2 and O2 permeability of 3.26 1019 mol mm2 s1 Pa1 and 9.23 1019 mol mm2 s1 Pa1, respectively, and water vapor transmission rate of 1.62 1010 kg m2 s1. Mozzarella cheeses, packaged in conditioning brine without lysozyme and Na2-EDTA, were used as controls. As the addition of Na2-EDTA produced a decrease in pH to 4.5, a second control was prepared by packaging mozzarella balls in 200 mL of the conditioning brine, acidified to pH 4.5 with 0.1 M HCl, to observe the influence of pH on microflora (Table 1). In the 2nd experimental phase, in order to prevent the decrease of pH due to the addition of Na2-EDTA, 50-g samples of cow mozzarella cheese, purchased from the same cheese factory, were packaged with 200 mL of a dilute salt solution to which a phosphate buffer (50 mmol L1; pH 6.5; K2HPO4/KH2PO4: J.T. Baker) was added previously. The conditioning solution contained lysozyme (0.25 mg mL1) and Na2-EDTA (50 and 20 mmol L1). Mozzarella cheeses, packaged in a buffered diluted brine, were used as controls (Table 1). The samples of 1st and 2nd experimental phases were analyzed immediately after the
Table 1 Samples of mozzarella cheese of 1st and 2nd experimental phases Sample
Conditioning solution
1st experimental phase Control A Diluted salt solution Active B Diluted salt solution+lysozymea+Na2-EDTA 50 mmol L1 samples C Diluted salt solution+lysozyme+Na2-EDTA 20 mmol L1 D Diluted salt solution+lysozyme+Na2-EDTA 10 mmol L1 Acidified E Diluted salt solution, acidified to pH 4.5 control 2nd experimental phase Bufferedb diluted salt solution Control Ab Active Bb Buffered diluted salt solution+lysozymea+Na2samples EDTA 50 mmol L1 Cb Buffered diluted salt solution+lysozyme+Na2EDTA 20 mmol L1 a
0.25 mg mL1. The diluted salt solution was buffered to pH 6.5 using a phosphate buffer 50 mmol L1. b
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packaging and after 1, 3, 6 and 8 days of storage under refrigerated conditions (4 1C).
2.2. Microbiological analyses Samples of 10 g of mozzarella cheese were diluted with 90 mL of a sterile saline solution (0.9%, w/v, NaCl) in a Stomacher bag and blended for 1 min with a Stomacher Lab Blender 400 (PBI International, Milan, Italy). Decimal dilutions of cheese homogenates were performed and microbiological counts carried out. The media and the conditions used for the enumerations were as follows: MRS Agar, modified by adding 0.17 g L1 of cycloheximide (Sigma-Aldrich) after autoclaving at 121 1C for 15 min, incubated at 30 1C for 4 days under anaerobic conditions for lactic acid bacteria; Violet Red Bile Agar (VRBA), incubated at 37 1C for 18–24 h for total coliforms; Slanetz Bartley Medium, incubated at 37 1C for
Sinigaglia (2006), was adopted: logðcfu g1 Þ ¼ logðcfu g1 Þmax A exp exp ðmmax 2:71Þ l S:L: þ1 þ A exp exp A lt ðmmax 2:71Þ þ1 A
where A (log(cfu g1)), mmax (log(cfu g1) day1) and l (days) are the Gompertz parameters (maximum increase of cell load attained in the stationary phase; maximal growth rate and lag phase, respectively), log(cfu g1)max is the threshold value for total coliforms and Pseudomonadaceae, S.L. is the shelf life and t is the time (days). When a decrease of microbial concentration followed by a cell load increase was detected, the re-parameterized Gompertz equation was cast in the following form:
n nh i oo 8 l t > < logðcfu g1 Þ ¼ K 1 þ A1 exp exp ðm1max 2:7182Þ A1 1 þ 1 n nh i oo > : logðcfu g1 Þ ¼ K 2 þ A2 exp exp ðm2max 2:7182Þ l2 ½tt þ 1 A2
48 h for enterococci; Pseudomonas Agar Base, modified by adding Pseudomonas CFC selective supplement after autoclaving at 121 1C for 15 min, incubated at 30 1C for 48 h for Pseudomonas spp. All the media and the supplements used were from Oxoid (Milan, Italy). 2.3. Evaluation of pH The pH was evaluated on mozzarella cheese homogenates and on the conditioning solution by a pH meter (Crison, mod Micro-pH 2001, Barcelona, Spain). 2.4. Statistical analysis and shelf life calculation In each experimental phase, the analyses were performed in duplicate and cheeses were obtained at two independent time points. The experimental data were submitted to oneway ANOVA and Tukey test (po0.05) through the software Statistica for Windows (Statsoft, Tulsa, OK, USA). The shelf life of the samples was evaluated through the population numbers of coliforms and Pseudomonadaceae. The critical values were set to 1 105 cfu g1 and 5 105 cfu g1 for coliforms and Pseudomonadaceae, respectively, as reported by the Italian law (Anonymous, 1997) and Bishop and White (1986). The re-parameterized Gompertz equation, proposed by Corbo, Del Nobile, and
(1)
tot t4t
(2)
where t is the time at which there is the switch from the decreasing to the increasing trend of cell load data; K1 (initial cell load), A1, m1max and l1 are the fitting parameters of the first Gompertz equation (decreasing trend of cell load data) and K2, A2, m2max and l2 the Gompertz values of the increasing length of the curve. The values of K1 and K2 were calculated through the following equations: K 2 ¼ ½logðcfu g1 Þmax A2 exp exp ðm2max 2:7182Þ l2 ½S:L: t þ1 (3) A2 l1 t 1 K 1 ¼ A1 exp exp ðmmax 2:7182Þ þ1 A1 þ K 2 þ A2 exp exp ðm2max 2:7182Þ l2 þ1 (4) A2 The confidence intervals of model parameters were evaluated as follows: first, a fit was run with the original data; then, using the data points standard deviation, 100 additional fits were run on artificial data sets, which were generated by randomly varying the data around the fitted function. From these additional fits, a distribution of values for each parameter was obtained. The sets of data
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obtained for each parameter were statistically treated to obtain the 95% confidence interval. 3. Results and discussion As reported in Section 2, the research was divided in two phases and focused on the possibility of prolonging the shelf life of mozzarella cheese through the addition of an antimicrobial mix (lysozyme and Na2-EDTA at 10, 20 and 50 mmol L1) to the conditioning brine. In the 1st phase, a dilute salt solution was used as the conditioning brine (with or without the antimicrobial mix), whereas in the 2nd part of the research, the mozzarella cheeses were packaged in a diluted salt solution buffered to pH 6.5 through a mixture of K2HPO4/KH2PO4. The analyses were performed for 8 days, in order to simulate a commercial storage time. Fig. 1 shows the evolution of total coliforms over storage time in the packed mozzarella cheese samples of the 1st experimental phase. As the addition of Na2-EDTA decreased the pH of the conditioning solution to 4.5, a second control sample was prepared (mozzarella cheese packaged in a conditioning brine acidified to pH 4.5), in order to evaluate if the antimicrobial activity was exerted by the active compounds or the low pH. As expected, total coliforms were able to proliferate in the control sample (A) and in the acidified control (E). Lysozyme+Na2-EDTA 50 mmol L1 (sample B) decreased the coliforms number from 4 log cfu g1 to the undetectable levels at 6 days; the cell load, however, increased up to 3.51 log cfu g1 after 8 days of storage. A different trend was observed in sample C (lysozyme+Na2-EDTA 20 mmol L1): in fact, a slight decrease of coliforms number (from 4.1 to 3.4 log cfu g1) within the storage time was observed. In the 2nd phase, the
Fig. 1. Evolution of coliforms population of traditional mozzarella cheese, packaged in a conditioning solution (12% NaCl), with lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (1st phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
Fig. 2. Evolution of coliforms population of traditional mozzarella cheese, packaged in a buffered conditioning solution (12% NaCl, pH 6.5), with lysozyme (0.25 mg mL1) and different amounts of Na2EDTA (2nd phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
conditioning solution was buffered to pH 6.5 to delete the effect of the acidification of the brine on the spoiling microorganisms. Moreover, the experiments were performed on higher amounts of Na2-EDTA (50 and 20 mmol L1), as they were more effective in inhibiting the spoiling microflora. Fig. 2 shows the evolution of total coliforms on mozzarella samples of the 2nd phase. As described previously, the addition of lysozyme and Na2-EDTA inhibited significantly the growth of coliforms, as it could be inferred by the decrease of coliforms population up to the undetectable level after 6 days of storage. The significance of Pseudomonadaceae and other psychrotrophic bacteria on cheese is well known (Cantoni, Iacumin et al., 2003; Cantoni, Stella et al., 2003); moreover, the contamination of mozzarella cheese by Pseudomonas spp. is a major concern for the Italian product, as they could affect the odor within the storage time (Bevilacqua, Corbo, & Sinigaglia, 2007). Fig. 3 shows the evolution of Pseudomonas spp. in mozzarella cheese samples of the 1st phase. The effect of lysozyme and Na2-EDTA was similar to that observed for total coliforms: in fact, an initial viability loss, followed by an increase of cell number after 3 days of storage, was observed in sample B. Otherwise, Pseudomonas spp. was able to proliferate in the controls (A and E) and in the samples packaged in the conditioning brine which contained lower amounts of Na2-EDTA (samples C and D). A similar result (decrease of cell number within 3 days, followed by an increase of the population) was recovered for the samples of the 2nd phase (Fig. 4). The initial decrease of pseudomonads, followed by the increase of cell load in the later days of storage, along with the total coliforms, could be due to a reversible stress on the cells, as reported by Johnston and Brown (2002) and Richards and Cavill (1976). The use of Na2-EDTA could cause a reversible damage to the outer membrane,
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Fig. 3. Evolution of Pseudomonas spp. population of traditional mozzarella cheese, packaged in a conditioning solution (12% NaCl), with lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (1st phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
Fig. 4. Evolution of Pseudomonas spp. population of traditional mozzarella cheese, packaged in a buffered conditioning solution (12% NaCl, pH 6.5), with lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (2nd phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
inhibiting the ability of making colonies on plates. Within a prolonged stress condition, the cell would repair the damages to the membrane acquiring the culturable ability. As the discussed figures are ‘‘qualitative results’’, the experimental data were modeled through a re-parameterized Gompertz equation, proposed by Corbo et al. (2006), and the shelf life was evaluated, in order to quantify the antimicrobial action of lysozyme and Na2-EDTA. The proposed function contains the shelf life value as a fitting parameter; therefore, it was possible to evaluate this parameter and its confidence interval and point out significant differences. Shelf life values were evaluated both through coliforms and Pseudomonas spp. cell number and the break-points were set to 1 105 (D.P.R. 54/97, Anonymous, 1997) for total coliforms and 5 105 for Pseudomonas spp. (Bishop & White, 1986).The results are shown in Table 2. The addition of the active compounds to the conditioning solution prolonged the shelf life evaluated through the coliforms. In fact, it was approximately 3.7 days for sample A, whereas in samples B (Na2-EDTA 50 mmol L1) and C (Na2-EDTA 20 mmol L1) it was 48 days (Table 2). A similar result was obtained for the pseudomonads which showed a shelf life value of 3.62 days in the control sample (A) and 5.64–6.60 days in samples B and C. It was not possible to point out the effect of Na2-EDTA concentration, as a superposition of shelf life values was observed. The acidified control (sample E) showed both for coliforms and pseudomonads a shelf life value, similar (p40.05) to those observed for sample A (control), but significantly lower (po0.05) than the shelf life of samples B, C and D. Therefore, we could suppose that the active compounds exerted an ‘‘intrinsic antimicrobial action’’, different from the acidification of the conditioning solution. As the shelf life value was evaluated through the cell number of two different targets (coliforms and Pseudomonas spp.), the actual shelf life (Table 2) of mozzarella cheese was assumed as the lower value of the calculated ones. In the conditions applied in this research, Pseudomonas spp. showed a shelf life value lower than that observed for total coliforms and the differences were significant (po0.05) for the sample with higher amounts of Na2-EDTA (samples B and C);
Table 2 Shelf life (days) of mozzarella cheese samples (1st experimental phase), packaged in diluted saline solutiona Samples
Coliforms
Pseudomonas spp.
Actual shelf lifeb
A B C D E
3.7380 (3.0888, 5.0763)c –d – 7.2493 (6.6185, 8.7916 1027) 4.3480 (4.0858, 5.8653)
3.6252 6.5911 5.6448 6.6067 4.2442
3.6252 6.5911 5.6448 6.6067 4.2442
a
(3.1357, (6.2746, (5.1263, (6.1579, (3.5879,
4.6041) 6.7918) 6.2457) 7.1196) 4.9651)
(3.1357, (6.2746, (5.1263, (6.1579, (3.5879,
4.6041) 6.7918) 6.2457) 7.1196) 4.9651)
Shelf life values were evaluated on the basis of coliforms and pseudomonads cell number. The critical values were set to 1 105 and 1 105 log cfu g1, respectively. b The actual shelf life value was assumed as the lowest one of the calculated shelf life values using coliforms and Pseudomonas spp. data. c 95% confidence interval. d It was not possible to evaluate shelf life values, because within 8 days the cell load of coliforms did not reach the break-point (5 log cfu g1).
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therefore, they were assumed as the actual shelf life values of the analyzed samples of mozzarella cheese, confirming that in some conditions the Pseudomonadaceae are the critical microorganisms for the shelf life of Italian mozzarella cheese, as reported by Corbo, Bevilacqua, and Sinigaglia (2007). As expected by the growth/death kinetics of coliforms and Pseudomonadaceae spp., the addition of Na2-EDTA and lysozyme increased the actual shelf life values (6.69 days) in the samples of the 2nd phase and no significant differences were recorded amongst the two amounts of Na2-EDTA or the microbial targets (coliforms and Pseudomonadaceae) (data not shown).
The active compounds did not inhibit lactic acid bacteria and enterococci. As an example, Figs. 5 and 6 show the evolution of lactic acid bacteria which increased from 5–6 to 7–8 log cfu g1, both in the samples of 1st and 2nd phase. Similar results were obtained for enterococci (data not shown). Within the complex microbiota of mozzarella cheese, lactic acid bacteria are the functional microorganisms, responsible for the acidification of the curd through the production of lactic acid from lactose and able to exert a probiotic action on the human health (Nousiainen & Seta¨la¨, 1998); therefore, the mix lysozyme–Na2-EDTA did not affect the functional characteristics of mozzarella cheese. As reported earlier, the addition of lysozyme and Na2-EDTA decreased significantly the pH of the conditioning brine (from 6.2–6.5 to 4.5), whereas the pH of mozzarella cheese (6.5) was less affected in the first days; it decreased, however, after 6 days of storage (5.8 in the control sample and 5.0 in the active ones—B, C and D) (data not shown). The addition of the phosphate buffer prevented the decrease, both in the conditioning brine and in mozzarella cheese. After 6 days, a drop, however, was observed in the conditioning solution, probably due to the loss of the buffering ability of KH2PO4–K2HPO4 mix (data not shown). Sensory analyses need to be investigated; odor, in fact, seemed to be not affected by the antimicrobial compounds or by the acidification of the conditioning brine, but we would assume that the acidification of the brine, due to the addition of Na2-EDTA, could result in differences in the texture of mozzarella cheese.
Fig. 5. Evolution of lactic acid bacteria population of traditional mozzarella cheese, packaged in a conditioning solution (12% NaCl), with lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (1st phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
4. Conclusions Na2-EDTA and lysozyme were effective in inhibiting the growth of spoilage microorganisms such as coliforms and Pseudomonas spp., without affecting the lactic acid bacteria. The studied method could be advantageously used to prolong the shelf life of mozzarella cheese, allowing the distribution of this product beyond the market borders. Further investigations are proposed to determine the minimum inhibitory concentration of EDTA, because of the toxicity of this compound for human health, and to identify new compounds, able to prolong the shelf life of traditional mozzarella cheese, without affecting its functional microbiota and rheological properties. Acknowledgment This work was supported by the Apulian region under Grant no. PS_031 ‘‘Miglioramento della qualita` dieteticonutrizionale e sicurezza di produzioni casearie tipiche della Capitanata’’.
Fig. 6. Evolution of lactic acid bacteria population of traditional mozzarella cheese, packaged in a buffered conditioning solution (12% NaCl, pH 6.5), with lysozyme (0.25 mg mL1) and different amounts of Na2-EDTA (2nd phase). The data represent the average of two repetitions7S.D. The lines are the best fit to the experimental points through the modified Gompertz equation.
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