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Effects of treatment with nisin on the microbial flora and sensory properties of a Greek soft acid-curd cheese stored aerobically at 4 °C
ARTICLE IN PRESS
International Dairy Journal 17 (2007) 1254–1258 www.elsevier.com/locate/idairyj
Short communication
Effects of treatment with nisin on the microbial flora and sensory properties of a Greek soft acid-curd cheese stored aerobically at 4 1C Sonia Kykkidoua, Nikolaos Pournisa, Olga K. Kostoulab, Ioannis N. Savvaidisa, a
Laboratory of Food Chemistry and Food Microbiology, Department of Chemistry, University of Ioannina, Ioannina 45110, Greece Laboratory of Molecular Biology, Department of Biological Applications and Technologies, University of Ioannina, Ioannina 45110, Greece
b
Received 22 June 2006; accepted 23 February 2007
Abstract The present study evaluated the use of nisin as an antimicrobial treatment for shelf-life extension of Galotyri, a Greek soft acid-curd cheese, stored aerobically under refrigeration for a period of 42 days. Three different treatments were tested: N0, control sample with no nisin added; N1, 50 IU g1 nisin; and N2, 150 IU g1 nisin, the latter two treatments added post-production to the Galotyri cheese. Of all microorganisms enumerated, lactobacilli, lactococci and yeasts were the groups that prevailed in cheese samples, irrespective of antimicrobial treatment. Based primarily on sensory evaluation (appearance and taste) and a microbiological acceptability limit for yeasts (5 log cfu g1), the use of nisin treatments extended the shelf-life of fresh Galotyri cheese stored at 4 1C by ca. 7 days (N1) and 21 days (N2) with cheese maintaining good sensory characteristics. r 2007 Elsevier Ltd. All rights reserved. Keywords: Microflora; Natural antimicrobials; Nisaplin; Soft acid-curd cheese; Greek cheese; Quality
1. Introduction
2. Materials and methods
Galotyri is a traditional Greek Protected Designation of Origin (PDO) soft acid-curd cheese, with a maximum permitted moisture content of 75% and a minimum fat content in dry matter of 40%. The cheese is white, without a rind and holes, characterized by a pleasant acid taste, a mild aroma and with a creamy texture. Nisin (NISAPLINTM), a bacteriocin produced by strains of Lactococcus lactis subsp. lactis, has been suggested as a natural antimicrobial for use as a biopreservative in foods, including dairy products, and is generally regarded as safe (GRAS) (Davies, Bevis, & Delves-Broughton, 1997). The objectives of the present work were to: (1) monitor the microbiological changes in the natural microbial flora of Galotyri cheese stored aerobically at 4 1C, (2) evaluate the effect of nisin treatments on the natural microbial flora of Galotyri, and (3) determine the shelf-life of Galotyri stored under the aforementioned conditions.
2.1. Sample preparation
Corresponding author. Tel.: +30 2651098343; fax: +30 2651098795.
E-mail address: [email protected] (I.N. Savvaidis). 0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.02.006
Galotyri cheese was produced from ewes’ milk with the addition of commercial starter cultures after the milk was pasteurized and cooled. All samples (1 kg each) were from cheese batches purchased from a local industrial dairy processing plant that were ready for distribution in the market following a short draining (10–18 h at 15 1C) and holding period under refrigeration (4–5 days after preparation). The cheese samples were transported to the laboratory in insulated polystyrene boxes on ice and were used in the experiments within 1 h after transportation. 2.2. Preparation of nisin solutions, addition to the cheese samples, and storage conditions Stock solutions (5000 and 15,000 IU mL1) of nisin (Nisaplinr, activity of 1 106 IU g1 according to the manufacturer, Applin & Barrett Ltd., Beaminster, Dorset, England) were prepared in sterile distilled water immediately
ARTICLE IN PRESS S. Kykkidou et al. / International Dairy Journal 17 (2007) 1254–1258
prior to addition. Nisin treatments were, N0 (no nisin added; control sample), N1 (50 IU g1), and N2 (150 IU g1), the last two treatments added post-production to the cheese. Amounts of nisin added to the Galotyri are typical of those used by cheese manufacturers (Davies et al., 1997). Solutions were added aseptically into the cheese samples (packaged aerobically in plastic containers with lids), the mixtures were then thoroughly mixed with a glass rod, and the cheese samples were stored at 470.5 1C. Three samples were analyzed at predetermined time intervals (0, 4, 7, 14, 21, 28, 35, and 42 days of storage). 2.3. Microbiological, chemical, and sensory analyses Cheese samples (25 g) were transferred aseptically to a stomacher bag with 225 mL of 0.1% buffered peptone water (BPW, Merck, Darmstadt, Germany), and the mixture was homogenized in a stomacher (Lab Blender, Seward, London, UK) for 60 s. For microbial enumeration, 0.1 mL samples of serial dilutions (1:9, BPW, Merck) of cheese homogenates were spread on the surface of dry media. Total mesophilic bacteria and other bacterial species enumerated in the present study were determined by using standard microbiological methods and techniques, previously described (Rogga et al., 2005). Lactobacilli, lactococci, and yeasts enumerated in the present study were not identified. The pH value was recorded using a Metrohm (Herisau, Switzerland), model 691 pH meter equipped with a glass electrode that was immersed directly into the cheese sample. Sensory evaluation was carried out on each day of sampling, according to IDF standards (see IDF, 1995).
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for the pH of control N0 and N1, N2-treated Galotyri samples are in agreement with pH values (3.8–4.3) reported for industrial and artisan Galotyri, also stored aerobically at 4 1C (Rogga et al., 2005). It is noteworthy that such acid/ pH values did not seem to adversely affect the growth of cheese lactic flora (lactobacilli, lactococci, Figs. 1 and 2, respectively) and yeasts (Fig. 3). 3.2. Effect of nisin treatments on natural microbial flora of Galotyri stored aerobically at 4 1C The present study focused on the monitoring of the following groups of microorganisms: lactobacilli (Fig. 1), lactococci (Fig. 2), yeasts (Fig. 3) and pseudomonads, Enterobacteriaceae, enterococci, and pathogenic staphylococci (data not shown), all microbial groups enumerated in earlier studies of Galotyri soft acid-curd cheese (Rogga et al., 2005; Lekkas et al., 2006). The initial Galotyri cheese contained on average (log cfu g1): 8.6 of total mesophilic bacteria, 8.0 of lactobacilli, 8.5 of lactococci, o1.0 of enterobacteria and o2.0 of pseudomonads, enterococci, pathogenic staphylococci, and yeasts. In agreement with our results, Rogga et al. (2005) recently reported high initial counts for total mesophilic bacteria, lactobacilli and lactococci in industrial and artisan Galotyri cheese samples stored aerobically at 4 1C. Our results indicate that both lactobacilli and lactococci comprised the initial (day 0) cheese lactic flora, as shown by colonies (Gram-positive, catalase-negative) grown on trypticase soy agar yeast extract (TSAYE) agar medium (data not shown). Of the remaining bacterial groups tested, pseudomonads, Enterobacteriaceae, enterococci and pathogenic staphylococci (data not shown) did not show any
2.4. Statistical analyses
3. Results and discussion
10
Lactobacilli (log cfu g-1)
Experiments were replicated twice on different occasions with different cheese samples. Analyses were run in triplicate for each replicate (n ¼ 2 3 ¼ 6). Microbiological counts were converted to log cfu g1 and were subjected to Analysis of Variance (ANOVA) using the software Statgraphics (Statistical Graphics Corp., Rockville, MD, USA). Means and standard deviations were calculated. When F-values were significant at the Po0.05 level, individual mean differences were analyzed by the least significant difference (LSD) procedure.
8
6
4
2
3.1. Effect of pH During storage, the pH values of the control N0, N1, and N2-treated Galotyri samples were practically unchanged (P40.05), and increased only incrementally during the first week of storage to reach maximal pH values of 4.3, thereafter attaining mean values in the range of 3.9–4.4 for both control N0 and N1, N2-treated Galotyri samples (data not shown). Our results obtained
0
0
7
14
21 28 Storage (days)
35
42
49
Fig. 1. Changes in the counts of lactobacilli in Galotyri cheese stored aerobically at 4 1C under different nisin treatments: N0, no nisin added (E); N1, nisin 50 IU g1 (m); N2, nisin 150 IU g1 (K). Points represent mean values of two replicate experiments three samples analyzed per replicate (n ¼ 6). Error bars indicate the 95% confidence interval.
ARTICLE IN PRESS S. Kykkidou et al. / International Dairy Journal 17 (2007) 1254–1258
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10
3.3. Effect of nisin treatments on lactic populations of Galotyri stored aerobically at 4 1C
Lactococci (log cfu g-1)
8
6
4
2
0
0
7
14
21 28 Storage (days)
35
42
49
Fig. 2. Changes in the counts of lactococci in Galotyri cheese stored aerobically at 4 1C under different nisin treatments: N0, no nisin added (E); N1, nisin 50 IU g1 (m); N2, nisin 150 IU g1 (K). Points represent mean values of two replicate experiments three samples analyzed per replicate (n ¼ 6). Error bars indicate the 95% confidence interval.
10
Yeasts (log cfu g-1)
8
6
4
2
0
0
7
14
21 28 Storage (days)
35
42
49
Fig. 3. Changes in the counts of yeasts in Galotyri cheese stored aerobically at 4 1C under different nisin treatments: N0, no nisin added (E); N1, nisin 50 IU g1 (m); N2, nisin 150 IU g1 (K). Points represent mean values of two replicate experiments three samples analyzed per replicate (n ¼ 6). Error bars indicate the 95% confidence interval.
significant growth in both control N0 and N1, N2-treated Galotyri samples throughout the entire storage period. The counts were equal to those initially enumerated, probably due to low acid pH values of the cheese samples. In both the control N0, and N1, N2 treated Galotyri cheese samples, the combined effect of acid mean pH values (pH ca. 4) and low temperature (4 1C) could act selectively to permit the growth of acid- and salt-tolerant psychrotrophic bacteria and yeasts.
Lactobacilli counts varied little (Fig. 1), with all counts within 0.5 log range during the first 2 week of storage in the control N0 and N1, N2-treated Galotyri cheese samples. Populations of lactobacilli (ca. 8.0–8.4 log cfu g1) declined after day 4 of storage, in all three Galotyri cheeses up to day 21 of storage, and thereafter increased to reach final populations of ca. 6.7, 5.8 and 5.0 log cfu g1, respectively (Fig. 1). The analysis of variance showed that nisin treatments had a significant effect (Po0.05) on lactobacilli counts for N1 and N2 cheese samples after day 14 and up to the final day 42 of storage at 4 1C (Fig. 1). During this period, by inspection of the lactobacilli bacterial growth curve, the decline in their population was greater for the N2 treatment. The decline in lactobacilli populations for N1 and N2 cheese samples could be attributed to the effect of nisin on this Gram-positive bacterial species. Furthermore, the decrease in lactobacilli counts observed for all three cheeses during this period could be due to the oxygen sensitivity of these anaerobic bacteria that are part of the natural microbial flora of Galotyri cheese, kept under aerobic conditions at 4 1C. Such trend has also been reported for lactic acid bacteria in Mozzarella cheese samples stored at 10 1C for an 8-week period (Eliot, Vuillemard, & Emond, 1998). However, after day 21 of storage, lactobacilli counts in all Galotyri samples increased and stabilized, finally reaching counts of ca. 5.0–6.7 log cfu g1, probably because of oxygen disappearance. The differences recorded in populations of lactobacilli between the control and the treated Galotyri cheeses after 14 and 42 days of storage, in the range of 0.4–0.9 (N1) log cfu g1 and 0.7–1.76 (N2) log cfu g1, could not be attributed to the acid pH, since respective changes in pH values during this storage period were minor. With regard to lactococci in both the control and the treated Galotyri cheese samples, a rather different pattern of growth was observed (Fig. 2). Initial (day 0) lactococci counts were slightly higher compared to respective lactobacilli populations by ca. 0.5 cfu g1, and this trend continued up to 35 days of storage, irrespective of treatment. The lower numbers of lactobacilli populations compared to lactococci throughout the storage period may also be due to the anaerobic incubation of lactobacilli. In the present study, lactococci numbers underwent a significant decrease (Po0.05) beyond day 21 and up to the final day 42 of storage, and counts were significantly lower (Po0.05) for N1 and N2 treatments compared to the control samples. The cause of the sudden drop in lactococci numbers, irrespective of the treatment, between 21 and 42 days of storage is unknown (Fig. 2). It would not seem likely that the drop was a result of reduced nisin treatments over time since the same behaviour was also noted for the control N0 Galotyri cheese samples (Fig. 2). The action of nisin is improved at pH 5.5 or below. As pH values of Galotyri samples remained at pH ca. 4.0,
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nisin activity should have been retained during the entire storage period. Nisin treatments N1 and N2, however, did contribute to lowering of lactobacilli and lactococci populations between days 14–42 and 28–42 of storage, respectively, presumably by causing cell injury. The effectiveness of nisin as an antimicrobial agent against a wide range of Gram-positive bacteria, including lactic acid bacteria, and spore formers is well known (DelvesBroughton, Blackburn, Evans, & Hugenholtz, 1996). Lactic acid bacteria are highly desirable in fermented milk products, including soft acid-curd cheese, and usually exert beneficial and potential probiotic activities. Our results show that although nisin addition at concentrations tested in the present study may alter the biodiversity and the micro-ecological niche of the soft acid-curd cheese, i.e., by reducing population of lactic flora, the shelf-life and sensorial attributes of the product do not seem to be adversely affected (Table 1). One additional benefit of nisin addition is the potential to control the growth of foodborne pathogens, such as Listeria monocytogenes in Galotyri cheese. The pathogen may survive during retail storage despite the low pH of the product (Rogga et al., 2005). In contrast to our study, one other option would be to use a pre-selected L. lactis starter culture to produce nisin in situ under real plant conditions, and then to examine the effect on technological properties (quality, safety, and sensory evaluation) of the product. The measurement of residual nisin activity is required to determine the distribution and stability of nisin during storage of Galotyri cheese to exclude the potential effect of endogenous-produced nisin by ‘‘wild’’ lactococci during fermentation. Such an additional effect cannot be ruled out as nisin activity was not measured. The potential of bacteriocinproducing lactic acid bacteria as a natural method of shelflife extension and improving food safety has been reviewed (Deegan, Cotter, Hill, & Ross, 2006). Such an attempt, however, was not the aim of the present study, and therefore, not investigated.
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3.4. Effect of nisin treatments on yeast populations of Galotyri stored aerobically at 4 1C The yeast populations in the control and treated Galotyri cheese samples stored aerobically at 4 1C are shown in Fig. 3. Analysis of Galotyri cheese samples revealed that the mean initial (day 0) population of yeasts was low (o2 cfu g1) and stayed constant up to days 14 and 28 of storage for the control and treated Galotyri cheese samples, thereafter increasing to reach final counts of ca. 6.8, 5.8 and 5.0 cfu g1, respectively (Fig. 3). After day 14 and up to the final day 42 of storage, yeast populations were significantly lower (Po0.05) for the N1 and N2-treated Galotyri cheese samples compared to the control samples. Yeasts in the control and nisin-treated cheese samples showed a completely different pattern of growth compared to lactic microflora populations. Most unexpectedly and surprisingly, yeast populations were inhibited by both nisin treatments up to day 28 of storage. Certainly, differences in the yeast inhibition between the control and nisin-treated samples cannot be attributed to the low acid pH of the soft acid-curd cheese because changes in their pH values during the 42 days of storage were minor, and both the control and nisin-treated samples resulted in high populations of ca. 5–7 log cfu g1 by the end of the storage period. It appears that an unknown factor (a metabolite produced by the bacteria or yeasts) showing a synergistic effect with nisin against yeasts could be involved, presumably causing cell injury and making cells nisin-sensitive, resulting in a lag phase extension (Fig. 3). Studies describing the effects of nisin in combination with other antimicrobial treatments against yeasts in foods are limited and few. One study has reported on the effectiveness of nisin and CO2 against yeasts and lactic acid bacteria on increasing the shelf-life of fresh pizza (Cabo, Pastoriza, Bernardez, & Herrera, 2001). Although it is unusual for nisin to inhibit yeasts, results of the present study suggest a complementary and synergistic effect of nisin with an unknown antimicrobial factor against yeasts
Table 1 Changesa in appearance and taste scoresb of a Greek soft acid-curd cheese stored aerobically at 4 1C under different nisin treatments: N0, no nisin added; N1, nisin 50 IU g1 and N2, nisin 150 IU g1 added Treatment
0 day
7 days
14 days
21 days
28 days
35 days
42 days
(a) Appearance N0 N1 N2
5.0Aa (0.1) 5.0Aa (0.1) 5.0Aa (0.2)
4.5Bab (0.2) 4.7ABb (0.3) 4.5ABb (0.4)
4.2BCb (0.1) 4.3BCb (0.2) 4.0Bbc (0.2)
3.6Cbc (0.1) 4.0Cbc (0.1) 3.9Bbc (0.1)
3.0Cac (0.1) 4.0Cbc (0.1) 4.0Bbc (0.2)
2.5Dd (0.1) 3.6CDc (0.1) 3.8Bbc (0.3)
2.0Dd (0.1) 2.6CDc (0.3) 3.5BCb (0.4)
(b) Taste N0 N1 N2
5.0Aa (0.1) 5.0Aa (0.1) 5.0Aa (0.2)
4.6Bab (0.2) 4.7ABa (0.3) 4.5ABa (0.4)
4.5BCb (0.1) 4.3BCb (0.2) 4.2Bbc (0.2)
4.0Cbc 4.0Cbc (0.1) 3.9Bbc (0.1)
NA (0.1) 3.8Cbc (0.1) 3.7Bbc (0.2)
NA 3.7CDc (0.1) 3.6Bbc (0.3)
NA NA 3.5Cd (0.1)
a Each value is the mean of two replicate experiments three samples analyzed per replicate (n ¼ 6). Standard deviation values are in parentheses. NA: not analyzed. b Means with different superscript capital letters are significantly different (Po0.05) in the same row; means with different lowercase superscript letters are significantly different (Po0.05) in the same column; a score o3.5 corresponds to quality class III, spoiled sample unfit for human consumption.
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in cheese. The presence of an unknown factor was not investigated in the present study. 3.5. Effects of nisin treatments on sensory properties and shelf-life of Galotyri cheese
imparting a negative impact on the product quality. Prior to recommending the commercial application of the effective nisin treatments described in this study, additional studies under real plant conditions are needed. 4. Conclusions
The extent of growth of certain classes of microorganisms and changes in sensory parameters are widely used to mark the end of the shelf-life of foods (Muir & Banks, 2000). Galotyri resembles other fermented dairy products such as yogurt and labneh, which are initially free of yeasts. This was shown in the present study; however in this case, deterioration after storage is primarily caused by yeasts, reaching high populations (4105 cfu g1) that can result in undesirable flavor defects (Tamime & Robinson, 1999). Microbiological shelf-life was defined as the period in which the yeast population exceeds 5 log cfu g1, and this limit was taken to mark the end of the shelf-life of Galotyri. Results of the present study, based on yeast data, suggest that control samples reached the limit value of yeasts on day 21 of storage. Inspection of control samples on that day showed that the product had developed visible signs of spoilage (mostly whitish and yellowish spots on the surface), with a yeasty taste and judged as an unacceptable product, in agreement with results reported by Rogga et al. (2005). The results of the sensory evaluation (appearance, taste attributes) of the control (N0) and treated (N1, N2) Galotyri cheese samples stored aerobically at 4 1C are presented in Table 1. Individual appearance and taste scores showed a similar pattern of decreasing acceptability for the control and treated samples. During the first 21 days of storage (4 1C), all samples received appearance scores between 3.5 and 5.0. After days 21 and 35 of storage for the N0 and N1 samples, respectively, appearance scores of 3.5 were recorded corresponding to product of acceptable quality (class II), whereas N2 samples reached this score on the final storage day 42. Beyond days 21 and 35 of storage, N0 and N1 samples received a score below 3.5 (class III), whereas N2 samples were acceptable. It is noteworthy that both taste and appearance scores of the control N0 and N1, N2-treated Galotyri samples correlated well with each other (Table 1). Considering a score of 3.5 as corresponding to the end of shelf-life, the control and treated Galotyri had a shelf-life of ca. 21, 35, and 42 days (4 1C). Galotyri cheese was better preserved with the N2 treatment compared to the N1 treatment and the N0 control. Interestingly, both sensorial data (taste, appearance scores) and yeast counts correlated well for cheese samples N0, N1, and N2 between days 21 and 42 of storage. The results of this study further showed that the addition of nisin to Greek soft acid-curd cheese may delay growth of yeasts and thereby extend shelf-life. Although nisin is not currently allowed to be used as a biopreservative in Greek cheeses, our results confirm that nisin as a food grade additive may be used to increase shelf-life without
Based primarily on sensory evaluation and yeast counts, both nisin treatments (N1, N2) resulted in an extension of shelf-life of Galotyri by more than ca. 7 and 21 days, respectively. There is need for a more detailed evaluation of the interactions between lactic flora and yeasts in cheeses, irrespective of the presence or absence of an antimicrobial treatment, identification of the lactic microflora and yeasts in Galotyri. Supplementary experiments should be conducted in order to fully investigate the observed mechanism of yeast inhibition by nisin. Such work could examine the likelihood of antimicrobial substances (e.g., an organic acid such as lactic) being produced, and its synergistic role in the inhibition of yeasts. There appears to be little or no published work on the effect of treatments with nisin on the biopreservation of soft acid-curd cheeses, and this study has demonstrated the effectiveness of such treatment. Acknowledgment The authors express their thanks to Beste Hellas, Athens, Greece, for providing Nisaplin. References Cabo, M. L., Pastoriza, L., Bernardez, M., & Herrera, J. J. R. (2001). Effectiveness of CO2 and Nisaplin on increasing shelf-life of fresh pizza. Food Microbiology, 18, 489–498. Davies, E. A., Bevis, H. E., & Delves-Broughton, J. (1997). The use of bacteriocin nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes. Letters in Applied Microbiology, 24, 343–346. Deegan, L. H., Cotter, P. D., Hill, C., & Ross, P. (2006). Bacteriocins: Biological tools for biopreservation and shelf-life extension. International Dairy Journal, 16, 1058–1071. Delves-Broughton, J., Blackburn, P., Evans, R. J., & Hugenholtz, J. (1996). Applications of the bacteriocin nisin. Antonie van Leeuwenhoek, 69, 193–202. Eliot, S. C., Vuillemard, J. C., & Emond, J. P. (1998). Stability of shredded Mozzarella cheese under modified atmosphere. Journal of Food Science, 63, 1075–1080. IDF. (1995). Guide for the sensory evaluation of cheese. IDF Standard 99A, part IV. Brussels, Belgium: International Dairy Federation. Lekkas, C., Kakouri, A., Paleologos, E., Voutsinas, L. P., Kontominas, M. G., & Samelis, J. (2006). Survival of Escherichia coli O157:H7 in Galotyri cheese stored aerobically at 4 and 12 1C. Food Microbiology, 23, 268–276. Muir, D. D., & Banks, J. M. (2000). Milk and milk products. In D. Kilcast, & P. Subramanian (Eds.), The stability and shelf-life of foods (pp. 197–219). Boca Raton, FL, USA: CRC Press. Rogga, K. J., Samelis, J., Kakouri, A., Katsiari, M. C., Savvaidis, I. N., & Kontominas, M. G. (2005). Survival of Listeria monocytogenes in Galotyri, a traditional soft acid-curd cheese, stored aerobically at 4 and 12 1C. International Dairy Journal, 15, 59–67. Tamime, A. Y., & Robinson, R. K. (1999). Yogurt science and technology. Boca Raton, FL, USA: CRC Press.
International Dairy Journal 17 (2007) 1254–1258 www.elsevier.com/locate/idairyj
Short communication
Effects of treatment with nisin on the microbial flora and sensory properties of a Greek soft acid-curd cheese stored aerobically at 4 1C Sonia Kykkidoua, Nikolaos Pournisa, Olga K. Kostoulab, Ioannis N. Savvaidisa, a
Laboratory of Food Chemistry and Food Microbiology, Department of Chemistry, University of Ioannina, Ioannina 45110, Greece Laboratory of Molecular Biology, Department of Biological Applications and Technologies, University of Ioannina, Ioannina 45110, Greece
b
Received 22 June 2006; accepted 23 February 2007
Abstract The present study evaluated the use of nisin as an antimicrobial treatment for shelf-life extension of Galotyri, a Greek soft acid-curd cheese, stored aerobically under refrigeration for a period of 42 days. Three different treatments were tested: N0, control sample with no nisin added; N1, 50 IU g1 nisin; and N2, 150 IU g1 nisin, the latter two treatments added post-production to the Galotyri cheese. Of all microorganisms enumerated, lactobacilli, lactococci and yeasts were the groups that prevailed in cheese samples, irrespective of antimicrobial treatment. Based primarily on sensory evaluation (appearance and taste) and a microbiological acceptability limit for yeasts (5 log cfu g1), the use of nisin treatments extended the shelf-life of fresh Galotyri cheese stored at 4 1C by ca. 7 days (N1) and 21 days (N2) with cheese maintaining good sensory characteristics. r 2007 Elsevier Ltd. All rights reserved. Keywords: Microflora; Natural antimicrobials; Nisaplin; Soft acid-curd cheese; Greek cheese; Quality
1. Introduction
2. Materials and methods
Galotyri is a traditional Greek Protected Designation of Origin (PDO) soft acid-curd cheese, with a maximum permitted moisture content of 75% and a minimum fat content in dry matter of 40%. The cheese is white, without a rind and holes, characterized by a pleasant acid taste, a mild aroma and with a creamy texture. Nisin (NISAPLINTM), a bacteriocin produced by strains of Lactococcus lactis subsp. lactis, has been suggested as a natural antimicrobial for use as a biopreservative in foods, including dairy products, and is generally regarded as safe (GRAS) (Davies, Bevis, & Delves-Broughton, 1997). The objectives of the present work were to: (1) monitor the microbiological changes in the natural microbial flora of Galotyri cheese stored aerobically at 4 1C, (2) evaluate the effect of nisin treatments on the natural microbial flora of Galotyri, and (3) determine the shelf-life of Galotyri stored under the aforementioned conditions.
2.1. Sample preparation
Corresponding author. Tel.: +30 2651098343; fax: +30 2651098795.
E-mail address: [email protected] (I.N. Savvaidis). 0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.02.006
Galotyri cheese was produced from ewes’ milk with the addition of commercial starter cultures after the milk was pasteurized and cooled. All samples (1 kg each) were from cheese batches purchased from a local industrial dairy processing plant that were ready for distribution in the market following a short draining (10–18 h at 15 1C) and holding period under refrigeration (4–5 days after preparation). The cheese samples were transported to the laboratory in insulated polystyrene boxes on ice and were used in the experiments within 1 h after transportation. 2.2. Preparation of nisin solutions, addition to the cheese samples, and storage conditions Stock solutions (5000 and 15,000 IU mL1) of nisin (Nisaplinr, activity of 1 106 IU g1 according to the manufacturer, Applin & Barrett Ltd., Beaminster, Dorset, England) were prepared in sterile distilled water immediately
ARTICLE IN PRESS S. Kykkidou et al. / International Dairy Journal 17 (2007) 1254–1258
prior to addition. Nisin treatments were, N0 (no nisin added; control sample), N1 (50 IU g1), and N2 (150 IU g1), the last two treatments added post-production to the cheese. Amounts of nisin added to the Galotyri are typical of those used by cheese manufacturers (Davies et al., 1997). Solutions were added aseptically into the cheese samples (packaged aerobically in plastic containers with lids), the mixtures were then thoroughly mixed with a glass rod, and the cheese samples were stored at 470.5 1C. Three samples were analyzed at predetermined time intervals (0, 4, 7, 14, 21, 28, 35, and 42 days of storage). 2.3. Microbiological, chemical, and sensory analyses Cheese samples (25 g) were transferred aseptically to a stomacher bag with 225 mL of 0.1% buffered peptone water (BPW, Merck, Darmstadt, Germany), and the mixture was homogenized in a stomacher (Lab Blender, Seward, London, UK) for 60 s. For microbial enumeration, 0.1 mL samples of serial dilutions (1:9, BPW, Merck) of cheese homogenates were spread on the surface of dry media. Total mesophilic bacteria and other bacterial species enumerated in the present study were determined by using standard microbiological methods and techniques, previously described (Rogga et al., 2005). Lactobacilli, lactococci, and yeasts enumerated in the present study were not identified. The pH value was recorded using a Metrohm (Herisau, Switzerland), model 691 pH meter equipped with a glass electrode that was immersed directly into the cheese sample. Sensory evaluation was carried out on each day of sampling, according to IDF standards (see IDF, 1995).
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for the pH of control N0 and N1, N2-treated Galotyri samples are in agreement with pH values (3.8–4.3) reported for industrial and artisan Galotyri, also stored aerobically at 4 1C (Rogga et al., 2005). It is noteworthy that such acid/ pH values did not seem to adversely affect the growth of cheese lactic flora (lactobacilli, lactococci, Figs. 1 and 2, respectively) and yeasts (Fig. 3). 3.2. Effect of nisin treatments on natural microbial flora of Galotyri stored aerobically at 4 1C The present study focused on the monitoring of the following groups of microorganisms: lactobacilli (Fig. 1), lactococci (Fig. 2), yeasts (Fig. 3) and pseudomonads, Enterobacteriaceae, enterococci, and pathogenic staphylococci (data not shown), all microbial groups enumerated in earlier studies of Galotyri soft acid-curd cheese (Rogga et al., 2005; Lekkas et al., 2006). The initial Galotyri cheese contained on average (log cfu g1): 8.6 of total mesophilic bacteria, 8.0 of lactobacilli, 8.5 of lactococci, o1.0 of enterobacteria and o2.0 of pseudomonads, enterococci, pathogenic staphylococci, and yeasts. In agreement with our results, Rogga et al. (2005) recently reported high initial counts for total mesophilic bacteria, lactobacilli and lactococci in industrial and artisan Galotyri cheese samples stored aerobically at 4 1C. Our results indicate that both lactobacilli and lactococci comprised the initial (day 0) cheese lactic flora, as shown by colonies (Gram-positive, catalase-negative) grown on trypticase soy agar yeast extract (TSAYE) agar medium (data not shown). Of the remaining bacterial groups tested, pseudomonads, Enterobacteriaceae, enterococci and pathogenic staphylococci (data not shown) did not show any
2.4. Statistical analyses
3. Results and discussion
10
Lactobacilli (log cfu g-1)
Experiments were replicated twice on different occasions with different cheese samples. Analyses were run in triplicate for each replicate (n ¼ 2 3 ¼ 6). Microbiological counts were converted to log cfu g1 and were subjected to Analysis of Variance (ANOVA) using the software Statgraphics (Statistical Graphics Corp., Rockville, MD, USA). Means and standard deviations were calculated. When F-values were significant at the Po0.05 level, individual mean differences were analyzed by the least significant difference (LSD) procedure.
8
6
4
2
3.1. Effect of pH During storage, the pH values of the control N0, N1, and N2-treated Galotyri samples were practically unchanged (P40.05), and increased only incrementally during the first week of storage to reach maximal pH values of 4.3, thereafter attaining mean values in the range of 3.9–4.4 for both control N0 and N1, N2-treated Galotyri samples (data not shown). Our results obtained
0
0
7
14
21 28 Storage (days)
35
42
49
Fig. 1. Changes in the counts of lactobacilli in Galotyri cheese stored aerobically at 4 1C under different nisin treatments: N0, no nisin added (E); N1, nisin 50 IU g1 (m); N2, nisin 150 IU g1 (K). Points represent mean values of two replicate experiments three samples analyzed per replicate (n ¼ 6). Error bars indicate the 95% confidence interval.
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10
3.3. Effect of nisin treatments on lactic populations of Galotyri stored aerobically at 4 1C
Lactococci (log cfu g-1)
8
6
4
2
0
0
7
14
21 28 Storage (days)
35
42
49
Fig. 2. Changes in the counts of lactococci in Galotyri cheese stored aerobically at 4 1C under different nisin treatments: N0, no nisin added (E); N1, nisin 50 IU g1 (m); N2, nisin 150 IU g1 (K). Points represent mean values of two replicate experiments three samples analyzed per replicate (n ¼ 6). Error bars indicate the 95% confidence interval.
10
Yeasts (log cfu g-1)
8
6
4
2
0
0
7
14
21 28 Storage (days)
35
42
49
Fig. 3. Changes in the counts of yeasts in Galotyri cheese stored aerobically at 4 1C under different nisin treatments: N0, no nisin added (E); N1, nisin 50 IU g1 (m); N2, nisin 150 IU g1 (K). Points represent mean values of two replicate experiments three samples analyzed per replicate (n ¼ 6). Error bars indicate the 95% confidence interval.
significant growth in both control N0 and N1, N2-treated Galotyri samples throughout the entire storage period. The counts were equal to those initially enumerated, probably due to low acid pH values of the cheese samples. In both the control N0, and N1, N2 treated Galotyri cheese samples, the combined effect of acid mean pH values (pH ca. 4) and low temperature (4 1C) could act selectively to permit the growth of acid- and salt-tolerant psychrotrophic bacteria and yeasts.
Lactobacilli counts varied little (Fig. 1), with all counts within 0.5 log range during the first 2 week of storage in the control N0 and N1, N2-treated Galotyri cheese samples. Populations of lactobacilli (ca. 8.0–8.4 log cfu g1) declined after day 4 of storage, in all three Galotyri cheeses up to day 21 of storage, and thereafter increased to reach final populations of ca. 6.7, 5.8 and 5.0 log cfu g1, respectively (Fig. 1). The analysis of variance showed that nisin treatments had a significant effect (Po0.05) on lactobacilli counts for N1 and N2 cheese samples after day 14 and up to the final day 42 of storage at 4 1C (Fig. 1). During this period, by inspection of the lactobacilli bacterial growth curve, the decline in their population was greater for the N2 treatment. The decline in lactobacilli populations for N1 and N2 cheese samples could be attributed to the effect of nisin on this Gram-positive bacterial species. Furthermore, the decrease in lactobacilli counts observed for all three cheeses during this period could be due to the oxygen sensitivity of these anaerobic bacteria that are part of the natural microbial flora of Galotyri cheese, kept under aerobic conditions at 4 1C. Such trend has also been reported for lactic acid bacteria in Mozzarella cheese samples stored at 10 1C for an 8-week period (Eliot, Vuillemard, & Emond, 1998). However, after day 21 of storage, lactobacilli counts in all Galotyri samples increased and stabilized, finally reaching counts of ca. 5.0–6.7 log cfu g1, probably because of oxygen disappearance. The differences recorded in populations of lactobacilli between the control and the treated Galotyri cheeses after 14 and 42 days of storage, in the range of 0.4–0.9 (N1) log cfu g1 and 0.7–1.76 (N2) log cfu g1, could not be attributed to the acid pH, since respective changes in pH values during this storage period were minor. With regard to lactococci in both the control and the treated Galotyri cheese samples, a rather different pattern of growth was observed (Fig. 2). Initial (day 0) lactococci counts were slightly higher compared to respective lactobacilli populations by ca. 0.5 cfu g1, and this trend continued up to 35 days of storage, irrespective of treatment. The lower numbers of lactobacilli populations compared to lactococci throughout the storage period may also be due to the anaerobic incubation of lactobacilli. In the present study, lactococci numbers underwent a significant decrease (Po0.05) beyond day 21 and up to the final day 42 of storage, and counts were significantly lower (Po0.05) for N1 and N2 treatments compared to the control samples. The cause of the sudden drop in lactococci numbers, irrespective of the treatment, between 21 and 42 days of storage is unknown (Fig. 2). It would not seem likely that the drop was a result of reduced nisin treatments over time since the same behaviour was also noted for the control N0 Galotyri cheese samples (Fig. 2). The action of nisin is improved at pH 5.5 or below. As pH values of Galotyri samples remained at pH ca. 4.0,
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nisin activity should have been retained during the entire storage period. Nisin treatments N1 and N2, however, did contribute to lowering of lactobacilli and lactococci populations between days 14–42 and 28–42 of storage, respectively, presumably by causing cell injury. The effectiveness of nisin as an antimicrobial agent against a wide range of Gram-positive bacteria, including lactic acid bacteria, and spore formers is well known (DelvesBroughton, Blackburn, Evans, & Hugenholtz, 1996). Lactic acid bacteria are highly desirable in fermented milk products, including soft acid-curd cheese, and usually exert beneficial and potential probiotic activities. Our results show that although nisin addition at concentrations tested in the present study may alter the biodiversity and the micro-ecological niche of the soft acid-curd cheese, i.e., by reducing population of lactic flora, the shelf-life and sensorial attributes of the product do not seem to be adversely affected (Table 1). One additional benefit of nisin addition is the potential to control the growth of foodborne pathogens, such as Listeria monocytogenes in Galotyri cheese. The pathogen may survive during retail storage despite the low pH of the product (Rogga et al., 2005). In contrast to our study, one other option would be to use a pre-selected L. lactis starter culture to produce nisin in situ under real plant conditions, and then to examine the effect on technological properties (quality, safety, and sensory evaluation) of the product. The measurement of residual nisin activity is required to determine the distribution and stability of nisin during storage of Galotyri cheese to exclude the potential effect of endogenous-produced nisin by ‘‘wild’’ lactococci during fermentation. Such an additional effect cannot be ruled out as nisin activity was not measured. The potential of bacteriocinproducing lactic acid bacteria as a natural method of shelflife extension and improving food safety has been reviewed (Deegan, Cotter, Hill, & Ross, 2006). Such an attempt, however, was not the aim of the present study, and therefore, not investigated.
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3.4. Effect of nisin treatments on yeast populations of Galotyri stored aerobically at 4 1C The yeast populations in the control and treated Galotyri cheese samples stored aerobically at 4 1C are shown in Fig. 3. Analysis of Galotyri cheese samples revealed that the mean initial (day 0) population of yeasts was low (o2 cfu g1) and stayed constant up to days 14 and 28 of storage for the control and treated Galotyri cheese samples, thereafter increasing to reach final counts of ca. 6.8, 5.8 and 5.0 cfu g1, respectively (Fig. 3). After day 14 and up to the final day 42 of storage, yeast populations were significantly lower (Po0.05) for the N1 and N2-treated Galotyri cheese samples compared to the control samples. Yeasts in the control and nisin-treated cheese samples showed a completely different pattern of growth compared to lactic microflora populations. Most unexpectedly and surprisingly, yeast populations were inhibited by both nisin treatments up to day 28 of storage. Certainly, differences in the yeast inhibition between the control and nisin-treated samples cannot be attributed to the low acid pH of the soft acid-curd cheese because changes in their pH values during the 42 days of storage were minor, and both the control and nisin-treated samples resulted in high populations of ca. 5–7 log cfu g1 by the end of the storage period. It appears that an unknown factor (a metabolite produced by the bacteria or yeasts) showing a synergistic effect with nisin against yeasts could be involved, presumably causing cell injury and making cells nisin-sensitive, resulting in a lag phase extension (Fig. 3). Studies describing the effects of nisin in combination with other antimicrobial treatments against yeasts in foods are limited and few. One study has reported on the effectiveness of nisin and CO2 against yeasts and lactic acid bacteria on increasing the shelf-life of fresh pizza (Cabo, Pastoriza, Bernardez, & Herrera, 2001). Although it is unusual for nisin to inhibit yeasts, results of the present study suggest a complementary and synergistic effect of nisin with an unknown antimicrobial factor against yeasts
Table 1 Changesa in appearance and taste scoresb of a Greek soft acid-curd cheese stored aerobically at 4 1C under different nisin treatments: N0, no nisin added; N1, nisin 50 IU g1 and N2, nisin 150 IU g1 added Treatment
0 day
7 days
14 days
21 days
28 days
35 days
42 days
(a) Appearance N0 N1 N2
5.0Aa (0.1) 5.0Aa (0.1) 5.0Aa (0.2)
4.5Bab (0.2) 4.7ABb (0.3) 4.5ABb (0.4)
4.2BCb (0.1) 4.3BCb (0.2) 4.0Bbc (0.2)
3.6Cbc (0.1) 4.0Cbc (0.1) 3.9Bbc (0.1)
3.0Cac (0.1) 4.0Cbc (0.1) 4.0Bbc (0.2)
2.5Dd (0.1) 3.6CDc (0.1) 3.8Bbc (0.3)
2.0Dd (0.1) 2.6CDc (0.3) 3.5BCb (0.4)
(b) Taste N0 N1 N2
5.0Aa (0.1) 5.0Aa (0.1) 5.0Aa (0.2)
4.6Bab (0.2) 4.7ABa (0.3) 4.5ABa (0.4)
4.5BCb (0.1) 4.3BCb (0.2) 4.2Bbc (0.2)
4.0Cbc 4.0Cbc (0.1) 3.9Bbc (0.1)
NA (0.1) 3.8Cbc (0.1) 3.7Bbc (0.2)
NA 3.7CDc (0.1) 3.6Bbc (0.3)
NA NA 3.5Cd (0.1)
a Each value is the mean of two replicate experiments three samples analyzed per replicate (n ¼ 6). Standard deviation values are in parentheses. NA: not analyzed. b Means with different superscript capital letters are significantly different (Po0.05) in the same row; means with different lowercase superscript letters are significantly different (Po0.05) in the same column; a score o3.5 corresponds to quality class III, spoiled sample unfit for human consumption.
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in cheese. The presence of an unknown factor was not investigated in the present study. 3.5. Effects of nisin treatments on sensory properties and shelf-life of Galotyri cheese
imparting a negative impact on the product quality. Prior to recommending the commercial application of the effective nisin treatments described in this study, additional studies under real plant conditions are needed. 4. Conclusions
The extent of growth of certain classes of microorganisms and changes in sensory parameters are widely used to mark the end of the shelf-life of foods (Muir & Banks, 2000). Galotyri resembles other fermented dairy products such as yogurt and labneh, which are initially free of yeasts. This was shown in the present study; however in this case, deterioration after storage is primarily caused by yeasts, reaching high populations (4105 cfu g1) that can result in undesirable flavor defects (Tamime & Robinson, 1999). Microbiological shelf-life was defined as the period in which the yeast population exceeds 5 log cfu g1, and this limit was taken to mark the end of the shelf-life of Galotyri. Results of the present study, based on yeast data, suggest that control samples reached the limit value of yeasts on day 21 of storage. Inspection of control samples on that day showed that the product had developed visible signs of spoilage (mostly whitish and yellowish spots on the surface), with a yeasty taste and judged as an unacceptable product, in agreement with results reported by Rogga et al. (2005). The results of the sensory evaluation (appearance, taste attributes) of the control (N0) and treated (N1, N2) Galotyri cheese samples stored aerobically at 4 1C are presented in Table 1. Individual appearance and taste scores showed a similar pattern of decreasing acceptability for the control and treated samples. During the first 21 days of storage (4 1C), all samples received appearance scores between 3.5 and 5.0. After days 21 and 35 of storage for the N0 and N1 samples, respectively, appearance scores of 3.5 were recorded corresponding to product of acceptable quality (class II), whereas N2 samples reached this score on the final storage day 42. Beyond days 21 and 35 of storage, N0 and N1 samples received a score below 3.5 (class III), whereas N2 samples were acceptable. It is noteworthy that both taste and appearance scores of the control N0 and N1, N2-treated Galotyri samples correlated well with each other (Table 1). Considering a score of 3.5 as corresponding to the end of shelf-life, the control and treated Galotyri had a shelf-life of ca. 21, 35, and 42 days (4 1C). Galotyri cheese was better preserved with the N2 treatment compared to the N1 treatment and the N0 control. Interestingly, both sensorial data (taste, appearance scores) and yeast counts correlated well for cheese samples N0, N1, and N2 between days 21 and 42 of storage. The results of this study further showed that the addition of nisin to Greek soft acid-curd cheese may delay growth of yeasts and thereby extend shelf-life. Although nisin is not currently allowed to be used as a biopreservative in Greek cheeses, our results confirm that nisin as a food grade additive may be used to increase shelf-life without
Based primarily on sensory evaluation and yeast counts, both nisin treatments (N1, N2) resulted in an extension of shelf-life of Galotyri by more than ca. 7 and 21 days, respectively. There is need for a more detailed evaluation of the interactions between lactic flora and yeasts in cheeses, irrespective of the presence or absence of an antimicrobial treatment, identification of the lactic microflora and yeasts in Galotyri. Supplementary experiments should be conducted in order to fully investigate the observed mechanism of yeast inhibition by nisin. Such work could examine the likelihood of antimicrobial substances (e.g., an organic acid such as lactic) being produced, and its synergistic role in the inhibition of yeasts. There appears to be little or no published work on the effect of treatments with nisin on the biopreservation of soft acid-curd cheeses, and this study has demonstrated the effectiveness of such treatment. Acknowledgment The authors express their thanks to Beste Hellas, Athens, Greece, for providing Nisaplin. References Cabo, M. L., Pastoriza, L., Bernardez, M., & Herrera, J. J. R. (2001). Effectiveness of CO2 and Nisaplin on increasing shelf-life of fresh pizza. Food Microbiology, 18, 489–498. Davies, E. A., Bevis, H. E., & Delves-Broughton, J. (1997). The use of bacteriocin nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes. Letters in Applied Microbiology, 24, 343–346. Deegan, L. H., Cotter, P. D., Hill, C., & Ross, P. (2006). Bacteriocins: Biological tools for biopreservation and shelf-life extension. International Dairy Journal, 16, 1058–1071. Delves-Broughton, J., Blackburn, P., Evans, R. J., & Hugenholtz, J. (1996). Applications of the bacteriocin nisin. Antonie van Leeuwenhoek, 69, 193–202. Eliot, S. C., Vuillemard, J. C., & Emond, J. P. (1998). Stability of shredded Mozzarella cheese under modified atmosphere. Journal of Food Science, 63, 1075–1080. IDF. (1995). Guide for the sensory evaluation of cheese. IDF Standard 99A, part IV. Brussels, Belgium: International Dairy Federation. Lekkas, C., Kakouri, A., Paleologos, E., Voutsinas, L. P., Kontominas, M. G., & Samelis, J. (2006). Survival of Escherichia coli O157:H7 in Galotyri cheese stored aerobically at 4 and 12 1C. Food Microbiology, 23, 268–276. Muir, D. D., & Banks, J. M. (2000). Milk and milk products. In D. Kilcast, & P. Subramanian (Eds.), The stability and shelf-life of foods (pp. 197–219). Boca Raton, FL, USA: CRC Press. Rogga, K. J., Samelis, J., Kakouri, A., Katsiari, M. C., Savvaidis, I. N., & Kontominas, M. G. (2005). Survival of Listeria monocytogenes in Galotyri, a traditional soft acid-curd cheese, stored aerobically at 4 and 12 1C. International Dairy Journal, 15, 59–67. Tamime, A. Y., & Robinson, R. K. (1999). Yogurt science and technology. Boca Raton, FL, USA: CRC Press.