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Investigation on ropiness development by isolates of the genera Lactobacillus, Alcaligenes and Feta cheese starter cultures
ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 20 (2003) 503–509
www.elsevier.nl/locate/jnlabr/yfmic
Investigation on ropiness development by isolates of the genera Lactobacillus, Alcaligenes and Feta cheese starter cultures F.I. Samarasa,*, C. Kehagiasb, J.S. Arkoudelosa, M.I. Bocarisa b
a Institute of Technology of Agricultural Products, National Agricultural Research Foundation, S. Venizelou 1, Lycovrysi, 14123 Athens, Greece Technological Educational Institution of Athens, Faculty of Food Technology and Nutrition, Agiou Spyridonos Str., 12210, Egaleo, Athens, Greece
Received 5 April 2002; received in revised form 5 December 2002; accepted 5 December 2002
Abstract Lactobacillus pseudoplantarum and Alcaligenes spp. were isolated from ropy brine and milk, respectively, and their ability to develop ropiness was investigated. Vigorous ropiness was developed by L. pseudoplantarum in whey with 7% NaCl, after 21 days of incubation at 151C. Alcaligenes spp. developed ropy colonies on PCA, containing up to 2% NaCl at incubation temperature of 41C and 151C. Also reconstituted skim milk inoculated with Alcaligenes spp. became ropy after 5–13 days of incubation only at 41C. Ropiness was not noticed when NaCl was added to the skim milk. In order to investigate the ropiness development in Feta cheese brine, a commercial yoghurt culture was used to make Feta cheese in combination with the two bacteria isolates and a ropy yoghurt culture. The results revealed that only the ropy yoghurt culture and not the normal one, developed very high ropiness in brine during ripening at 151C. The results also showed that Alcaligenes spp. and L. pseudoplantarum intensified ropiness of brine. To avoid ropiness, it is recommended that yoghurt starter cultures used for Feta should be selected and bacterial contaminants that could cause ropiness in brine should be eliminated. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cheese; Cultures; Ropiness; Lactobacillus; Alcaligenes
1. Introduction Certain bacteria growing on food products produce exopolysaccharides, which might increase viscosity of liquid products or cause sticky appearance of the solid ones. The alteration in the textural properties by exopolysaccharides is known as ropiness. This is considered as a defect in some products like milk, cream, cider, wine, while in some fermented milks like stirred yoghurt the production of exopolysaccharides under certain conditions can improve texture (DuenasChasco et al., 1998). Ropy cultures of lactic acid bacteria are used by manufacturers of stirred yoghurt for improving body, mouthfeel, and preventing wheying off (Driessen and Loones, 1992). Feta is a soft, white cheese, ripened and kept in brine until it is sold to consumers. Occasionally, the viscosity of brine increases and becomes ropy. Ropy appearance *Corresponding author. Tel.: +30-1-28-45-940-12; fax: +30-1-2840-740. E-mail address: [email protected] (F.I. Samaras).
of brine is not normally associated with any undesirable organoleptic characteristics but affects the appearance of the cheese. This has a negative effect on the consumer perceptibility, which is, nowadays, of more concern since most Feta cheese is sold to the consumers not from the barrels (without having a view on the brine) but in retail packages. Studies concerning the ropy appearance of Feta cheese brine have not been reported in a systematic manner. Glossiotis (1984) working in a Feta cheese factory, observed the appearance of ropy brine mainly during the winter period. Samaras (1994) reported that ropiness of brine is a phenomenon that appears suddenly and it does not remain stable during storage after manifestation. Chomakov (1967) studied various factors that influence the ropy appearance of white cheese brine. He found a strain of Lactobacillus plantarum to be responsible for the exopolysaccharides reformation. Cerning (1990) reviewed the production and the properties of exopolysaccharides by lactic acid bacteria. The instability of the slime producing trait in
0740-0020/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0740-0020(02)00175-2
ARTICLE IN PRESS 504
F.I. Samaras et al. / Food Microbiology 20 (2003) 503–509
thermophilic and mesophilic lactic acid bacteria has been reported (Cerning, 1990). Certain lactic acid cultures increase viscosity of whey but might lose this property when frequently transferred or at high temperatures. The majority of the dairy companies produce Feta cheese with the addition of a yoghurt culture containing L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus (Kehagias et al., 1995). During recent years there has been a tendency to combine these thermophilic bacteria with mesophilic ones. The purpose of this study was to investigate the conditions under which micro-organisms that can contaminate Feta cheese brine as well as cultures used for the preparation of the cheese, can cause ropiness.
2. Materials and methods 2.1. Isolation and identification of the micro-organisms A rope-inducing micro-organism was isolated analyzing 15 samples of ropy Feta cheese brine taken randomly (Samaras, 1994). Another strain was accidentally taken from extreme viscous retail milk, stored at 41C for a long period of time. Both strains were purified by multiple propagation. The schematic key for tentative generic identification of foodborne bacteria introduced by Jay (2000) was followed for the strain identification. The former strain was identified as L. pseudoplantarum (ID 82%) by API 50 CHL. The latter one was estimated to belong to the genus Alcaligenes by performing the biochemical tests according to the Kersters and De Ley (1984). Both cultures (Lactobacillus and Alcaligenes) were grown overnight on MRS (Oxoid) agar and PCA (Oxoid) at 301C, respectively, before use. 2.2. Growth and development of ropiness in various media Whey, reconstituted skim milk and plate count agar (PCA) were used for ropiness development and determination. Nutrient broth was also used for growth measurement. The whey was obtained after Feta cheese curd making in our laboratory. It was sterilized by tyndallization at 621C, NaCl was added to obtain concentrations of 3%, 5%, 7%, 9%, and 11% (w/v) and transferred into 50 ml flasks. After L. pseudoplantarum was inoculated into the medium and the flasks were stored at 151C and 321C. Three replicated experiments were conducted and two samples were taken from each treatment after 0, 5, 10, 15, 20, 25, and 30 days of storage and analyzed. The pH of the whey was measured by using an ORION expandable ion Analyzer (EA 940). The viscosity was also measured by using a viscosimeter as described
elsewhere. The pH and the viscosity were used as indexes of growth and ropiness, respectively. In order to study the salt tolerance and the influence of temperature on Alcaligenes spp. the micro-organism was loop streaked on PCA containing 2%, 4%, 6%, and 8% NaCl. Plate dishes were incubated at 41C, 151C, and 321C for 72 h. The growth of the micro-organism was subjectively evaluated by using the scale according to the colony size: 0––no growth, 1––start growth, 2–– medium growth, 3––high growth, 4––very high growth and 5––extremely high growth. The ropiness development was also subjectively evaluated from colony sliminess as it is described in another paragraph below. In addition, Alcaligenes spp. was inoculated into approximately 50 ml of nutrient broth (Oxoid) and reconstituted skim milk with various NaCl concentrations (0%, 2%, 4%, 6%, and 8% w/v) in flasks. Incubation took place at 41C, 151C, and 321C, respectively. The growth in the nutrient broth was estimated by measuring transmittance of the inoculated medium by Spectronic 20 (Bausch & Lomb) at 550 nm. The ropiness development in skim milk was measured by a viscosimeter, as it is described in another paragraph below. 2.3. Development of ropiness in Feta cheese brine Feta cheese was prepared from pasteurized ewe’s milk in the laboratory according to the method of Glossiotis (1984). Four batches of Feta cheese were made in two trials. Yoghurt cultures, which are composed of L. delbrueckii subsp. bulgaricus and S. thermophilus are commonly used as starters for the manufacture of Feta. For the experiment needs the commercial yoghurt culture CH1 (Hansen, Copenhagen, Denmark) was used alone or in combination with another yoghurt culture V2 (Wiesby, Niebull, Germany) and the two bacteria isolates. The experimental design comprised four treatments as follows: (a) starter culture CH1 alone, (b) combination of two starter cultures CH1 and V2, (c) combination of starter culture CH1 and L. pseudoplantarum and (d) combination of starter culture CH1 and Alcaligenes spp. Approximately 1 kg of Feta cheese was placed in cans and the containers were filled with brine. The cans were closed using lids with an opening covered by a plastic stopper. Cheese was kept at 15–181C. During storage the pH of the brine was measured and ropiness was evaluated subjectively, as it is described in the next paragraph. 2.4. Evaluation of ropiness The evaluation and the measurement of ropiness are very hard tasks to perform. Ropiness system is unstable, any treatment could cause a collapse. Even more is not able to use an instrument in some instances, small
ARTICLE IN PRESS F.I. Samaras et al. / Food Microbiology 20 (2003) 503–509
particles in the system could interfere and give biased readings. It was a challenge to find out a method of ropiness measurement and the empirical one was developed. The ropiness of the whey and the reconstituted skim milk media was estimated by measuring the viscosity with a Brookfield LV viscosimeter (probe 1, revolution speed 60, measuring unit m Pa/s—milli Pascal per second). The viscosimeter could not be used for viscosity measurement of Feta brine because it contained small particles, which could interfere and give biased readings. Thus the ropiness development in Feta brine was evaluated subjectively by a method which was developed in our laboratory. A loop was inserted into the medium and was drawn out slowly. The formation of a thread and its length before breaking were taken into consideration for ropiness evaluation according to the following scale: 0––zero, 1––low, 2––moderate, 3–– high, 4––very high, and 5––extremely high. The method for evaluating ropiness of the colonies on PCA was based on the same principle and the same scale was also used. A loop was brought into contact with the colony and the formation of a thread was evaluated.
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spp. The morphological and physiological characteristics of the micro-organisms are presented in Table 1. 3.2. Growth and development of ropiness by L. pseudoplantarum in whey Generally, Feta cheese is manufactured under various conditions by quite a large number of firms. The cheese curd occurs at 341C, while the ripening process takes place at 16–181C. Since the ropiness occurs in the Feta brine, which mainly consists of whey, whey was used as a medium. The changes of pH and viscosity during incubation of L. pseudoplantarum in whey at 151C and 321C are shown in Fig. 1. The viscosity of both samples, the control and the inoculated, changed very little within the first 10 days of incubation at 151C and 321C, respectively. At the same time a pH decrease was observed in the inoculated sample indicating growth. This decrease of pH was even more pronounced in samples incubated at 321C. After 21 days of incubation a strong increase in viscosity was observed, indicating development of ropiness, only in the sample inoculated
Table 1 Morphological and physiological characteristics of L. pseudoplantarum and Alcaligenes spp. Characteristics
3.1. Morphological and physiological characteristics of the isolated micro-organisms Several experiments were carried out in various media under different conditions in order to collect information about the behavior and the properties of the two micro-organisms grown in various media under different conditions. Since L. pseudoplantarum was isolated from Feta cheese brine, whey was chosen as medium. Following the methodology described in Materials and Methods, the micro-organism isolated from ropy Feta brine was identified as L. pseudoplantarum (ID 82%) and the one isolated from ropy milk as Alcaligenes
6
Motility Size Gram Catalase Anaerobic Spores Glucose fermentation Lactose fermentation Growth at 12–151C Litmus milk Gelatine hydrolysis
L. pseudoplantarum
Alcaligenes spp.
Small +
Medium +
+( ) +
Nd
a
, +b Slimy colonies Acidic Nd
Ropiness in milk Alkaline
Nd: not determined. a Aerobic conditions. b Anaerobic conditions.
32o C
15 o C
14 12
5 pH
10 8
4
6 4
3
Viscosity mPas -1
3. Results and discussion
2 0
5
10 15 20 25 30 Time (days)
0
5
10 15 20 25 30 Time (days)
Fig. 1. Changes of pH and viscosity in whey during growth of L. pseudoplantarum at 151C and 321C: pH without inoculation (n), pH with inoculation (m), viscosity without inoculation (&), viscosity with inoculation (’).
ARTICLE IN PRESS F.I. Samaras et al. / Food Microbiology 20 (2003) 503–509
with L. pseudoplantarum at 151C. Contrarily, the viscosity did not increase in the samples incubated at 321C. On the other hand, ropiness development takes place the first days during ripening of Feta cheese at 15–181C, which was confirmed by the findings of this experiment. Usually, the finished product of Feta cheese contains approximately 3% NaCl. During processing dry salt is added to the cheese curd or the curd is put in brine of various concentrations, depending upon the producer. The NaCl concentration in the brine and in the curd varies until equilibrium has been achieved. This variation may favors ropiness development at a specific concentration. For this purpose six NaCl concentrations (0%, 3%, 5%, 7%, 9%, and 11%) were chosen, in order to find out which one is the optimum for ropiness development. The whey with 0% NaCl was not inoculated and used as control. In Fig. 2 the effect of NaCl concentration on pH and viscosity during growth
6
of L. pseudoplantarum in whey at 151C is presented. The results clearly showed that this micro-organism developed ropiness by increasing the viscosity of the whey containing 7% NaCl. There was only a slight increase of viscosity in samples containing 5% and 3% NaCl and no increase in the samples with 9%, 11% salt and the control. The difference in the pH values with 3%, 5%, and 7% NaCl between inoculated and not inoculated samples is an indication of the growth of the microorganism, which occurred before the 20th day and maintained up to 40th day. On the other hand the pH value of the whey after taking Feta cheese curd is slightly below 5. Preliminary experiments have also showed development of ropiness in whey after the 20th day. At 9% and 11% NaCl concentrations the growth of the micro-organism was inhibited, since the pH remained constant and the viscosity did not change. The results of this study indicated that ropiness development in whey was affected by both temperature (151C) and
16
(B)
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10 8 4
6
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4 2
3
16
(D)
(C)
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(F)
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5 pH
10 8 4
6
Viscosity mPa.s-1
14
4 3
2 20
25 30 35 Time (days)
40
20
25 30 35 Time (days)
40
Fig. 2. Effect of the NaCl concentration on pH and viscosity during growth of L. pseudoplantarum in whey at 151C: pH without inoculation (n), pH with inoculation (m), viscosity without inoculation (&), viscosity with inoculation (’). (A) 0% NaCl; (B) 3% NaCl; (C) 5% NaCl; (D) 7% NaCl; (E) 9% NaCl; and (F) 11% NaCl.
ARTICLE IN PRESS F.I. Samaras et al. / Food Microbiology 20 (2003) 503–509
the salt concentration (7% NaCl). Also, it occurred at a pH range of 4–4.5 (Figs. 2B–D). 3.3. Growth and development of ropiness by Alcaligenes spp. in solid and liquid media The effect of NaCl on the growth of Alcaligenes spp. on PCA and the ropy appearance of colonies is shown in Fig. 3. The micro-organism proliferated at all incubation temperatures (41C, 151C, and 321C) and only at NaCl concentration of 0% and 2%. However, ropiness was detected only at 41C and 151C. Feta is ripened initially at 16–181C, then it is kept at 41C until consumption. Therefore, during ripening and storage of Feta, any contamination of cheese or brine by Alcaligenes might lead to ropiness when NaCl concentration is not higher than 2%. The effect of NaCl concentration on the growth of Alcaligenes spp. in nutrient broth, expressed as transmittance, at various temperatures of 41C, 151C, and 321C is given in Fig. 4. This micro-organism proliferated at all three temperatures used and at a NaCl concentration of up to 4%. However, growth was also observed at 6% NaCl only at 151C which is an indication that at this temperature the conditions were more favorable for growth of the micro-organism. Preliminary studies indicated that ropiness was not developed at 151C and 321C (Fig. 3). Thereby the influence of NaCl concentration on viscosity during growth of Alcaligenes spp. in skim milk at 41C was studied. From Fig. 5, it can be seen that highest viscosity was measured when no NaCl was added to the milk. This finding comes in agreement with the data presented in Fig. 3 during growth of Alcaligenes spp. on PCA where ropiness was developed mainly at 0% NaCl and at 2%. The results of this study indicated that Alcaligenes spp. did not grow at NaCl concentrations higher than 6% (Fig. 4) and developed ropy colonies (Fig. 3) or ropiness in milk (Fig. 5) only when the NaCl
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concentration was up to 2% or 0%, respectively. However, since the micro-organism can grow at relatively low temperatures (41C), it will be interesting to investigate whether contamination of milk, cheese or brine by Alcaligenes spp. could influence the appearance of ropiness in brine solution.
3.4. Development of ropiness in Feta cheese brine prepared under various conditions In order to put in practice the findings described above, the two bacteria isolates were used in combination with commercial lactic acid starter cultures in order to make Feta cheese and study the development of ropiness. The changes in pH and the appearance of ropiness of Feta cheese brine are presented in Fig. 6. In the first five days, there was no increase in ropiness in brine in all samples. After eight days, ropiness increased gradually in the samples where CH1 culture was combined in cheese milk with each one of L. pseudoplantarum, Alcaligenes spp. and V2 culture. Ropiness increased further after the 8th day and was constantly high after the second week (Fig. 6). The sample in which CH1 culture was combined with V2 culture gave the highest ropiness (Fig. 6D), followed by the sample with CH1 culture+Alcaligenes spp. (Fig. 6A). In contrary, the sample of brine with CH1 culture alone showed no or very little increase of ropiness (Fig. 6C). From Fig. 6, it can be concluded, that the drop of pH followed a different pattern for the two pairs of samples: CH1 culture with V2 culture (C) and CH1 culture with Alcaligenes spp. (A) on the one hand, CH1 culture alone (C) and CH1 culture with L. pseudoplantarum (B) on the other. Under our experimental conditions, the pH drop alone did not seem to be the only factor in the development of ropiness (experiments C and B). The presence of the proper micro-organisms in the milk samples is essential too (experiments D and A).
Growth - Ropiness Intensity
5 4 oC 15 oC
4
32 oC o
o
15 C
32 C
3 o 4 C
2
No growth and no ropiness
1 0 0
2
4 NaCl %
6
8
Fig. 3. Effect of NaCl concentration on growth and ropy appearance of colonies of Alcaligenes spp. on PCA at various temperatures; 41C, 141C, 321C: (&) growth, (’) ropiness.
ARTICLE IN PRESS F.I. Samaras et al. / Food Microbiology 20 (2003) 503–509
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(A)
Transmittance (%)
Transmittance (%)
120 100 80 60 40 20 0
120 100 80
1
2 3 4 Time (days)
5
0
(C)
60 40 20 0 1
2 3 4 Time (days)
5
120 100 80 60 40 20 0
(E)
Transmittance (%)
0
0
6
1
(B)
60 40 20 0
6
Transmittance (%)
Transmittance (%)
0
120 100 80
120 100 80 60 40 20 0
1
2 3 4 Time (days)
5
6
2 3 4 Time (days)
5
6
(D)
0
2 3 4 Time (days)
1
5
6
Fig. 4. Effect of the NaCl concentration on the growth, expressed as Transmittance (% at 550 nm) of Alcaligenes spp. in nutrient broth at 41C(n), 151C(&) and 321C (J): (A) 0% NaCl; (B) 2% NaCl; (C) 4% NaCl; (D) 6% NaCl and (E) 8% NaCl.
21 Viscosity mPa.s -1
18 15 12 9 6 3 0 4
5
6
7 8 9 Time (days)
10
11
Fig. 5. Effect of the NaCl concentration: 0% (n), 2% (&), 4% (J), 6% ( * ), 8% (+) on viscosity during growth of Alcaligenes spp. in skim milk at 41C.
During recent years, there has been intensive interest in the bacteria producing ropiness in food, not only because of their effect on texture but also for the potential bioactive role of exopolysaccharides (Vemura
et al., 1998). However, information on the development of ropiness of cheeses in brine is limited (Chomakov, 1967; Samaras, 1994). This study revealed that a ropy yoghurt culture (V2) could produce ropiness in brine of Feta cheese under certain conditions (151C) which are quite different from those used for the manufacture of stirred yoghurt. The other one (CH1) did not cause ropiness in brine. Since the yoghurt bacteria (S. thermophilus and L. delbrueckii subsp. bulgaricus) are used as starters in Feta cheesemaking, it should be taken into consideration the selection of the no ropy strains, keeping always in mind that the ropy appearance of Feta cheese is not desirable. It is well known that ropy and not ropy strains exist in yoghurt starter cultures (Rawson and Marshall, 1997). The results of this study also showed that bacteria, which are inhabitants in dairy plants, like Alcaligenes and Lactobacillus can intensify ropiness when contaminating milk, brine or cheese. Alcaligenes spp. increased ropy appearance of brine at 151C but did not develop ropiness at the same temperature in salted milk. Our
ARTICLE IN PRESS F.I. Samaras et al. / Food Microbiology 20 (2003) 503–509
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5
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(B)
4
2
4
5
3
pH
pH
3
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4 5
2
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1
1 3 6
(C)
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4
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(D)
5
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Ropiness
pH
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1 3
0 0
5
10 15 Time (days)
Ropiness
(A)
20
3
Ropiness
6
509
0 0
5
10 15 Time (days)
20
Fig. 6. Changes of pH (’) and viscosity (&) in the brine of Feta cheese by different cheese cultures and micro-organisms Alcaligenes spp. and L. pseudoplantarum at different periods of time: (A) CH1+Alcaligenes spp.; (B) CH1+L. pseudoplantarum; (C) CH1 culture and (D) CH1+V2 cultures).
results clearly showed that NaCl composition of the substrate as well as the temperature are both factors associated with the development of ropiness. It is essential to eliminate contaminants, which can cause ropiness by cleaning and disinfecting of the facilities of cheese plants.
References Cerning, J., 1990. Exocellular polysaccharides produced by lactic acid bacteria. FEMS Microbiol. Rev. 87, 113–130. Chomakov, C., 1967. Isolation of lactic acid bacteria causing ropiness of white cheese brine. Milchwissenschaft 22, 569–573. Driessen, F.M., Loones, A., 1992. Developments in the fermentation process (liquid, stirred and set fermented milks). Bull. Int. Dairy Fed. 277, 28–40. Duenas-Chasco, M.T., Rodriguez-Carvajal, M.A., Tejero-Mateo, P., Espartero, J.L., Irastorza-Iribas, A., Gil-Serrano, A.M., 1998. Structural analysis of the exopolysaccharides produced by Lactobacillus spp. G-77. Carbohydr. Res. 307, 125–133.
Glossiotis, C., 1984. Products from ewe milk. Proceedings of the Seminar in Dairy Science, Greek National Dairy Committee, pp. 233–256. Jay, J.M., 2000. Modern Food Microbiology. D. Van Nostrand, New York, USA, pp. 313–317. Kehagias, C., Koulouris, S., Samona, A., Malliou, S., Koumoutsos, G., 1995. Effect of various starters on the quality of cheese in brine. Food Microbiol. 12, 413–419. Kersters, K., De Ley, J., 1984. Genus Alcaligenes Castellani and Chalmers 1919, 936AL. In: Krieg, N.R., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, MD, USA, pp. 361–366. Rawson, H., Marshall, V.M., 1997. Effect of ropy strains of Lactobacillus delbrueckii spp. bulgaricus and Streptococcus thermophilus on rheology of stirred yogurt. Int. J. Food Sci. and Technol. 32, 213–220. Samaras, F., 1994. Factors affecting the ropiness development in Feta cheese brine. Proceedings of the Seminar in Dairy Science, Greek National Dairy Committee, pp. 145–155. Vemura, J., Itoh, T., Kaneko, T., Noda, K., 1998. Chemical characterization of exocellular polysaccharide from Lactobacillus delbrueckii subsp. Bulgaricu OLL1073R-1. Milchwissenschaft 53, 443–446.
www.elsevier.nl/locate/jnlabr/yfmic
Investigation on ropiness development by isolates of the genera Lactobacillus, Alcaligenes and Feta cheese starter cultures F.I. Samarasa,*, C. Kehagiasb, J.S. Arkoudelosa, M.I. Bocarisa b
a Institute of Technology of Agricultural Products, National Agricultural Research Foundation, S. Venizelou 1, Lycovrysi, 14123 Athens, Greece Technological Educational Institution of Athens, Faculty of Food Technology and Nutrition, Agiou Spyridonos Str., 12210, Egaleo, Athens, Greece
Received 5 April 2002; received in revised form 5 December 2002; accepted 5 December 2002
Abstract Lactobacillus pseudoplantarum and Alcaligenes spp. were isolated from ropy brine and milk, respectively, and their ability to develop ropiness was investigated. Vigorous ropiness was developed by L. pseudoplantarum in whey with 7% NaCl, after 21 days of incubation at 151C. Alcaligenes spp. developed ropy colonies on PCA, containing up to 2% NaCl at incubation temperature of 41C and 151C. Also reconstituted skim milk inoculated with Alcaligenes spp. became ropy after 5–13 days of incubation only at 41C. Ropiness was not noticed when NaCl was added to the skim milk. In order to investigate the ropiness development in Feta cheese brine, a commercial yoghurt culture was used to make Feta cheese in combination with the two bacteria isolates and a ropy yoghurt culture. The results revealed that only the ropy yoghurt culture and not the normal one, developed very high ropiness in brine during ripening at 151C. The results also showed that Alcaligenes spp. and L. pseudoplantarum intensified ropiness of brine. To avoid ropiness, it is recommended that yoghurt starter cultures used for Feta should be selected and bacterial contaminants that could cause ropiness in brine should be eliminated. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cheese; Cultures; Ropiness; Lactobacillus; Alcaligenes
1. Introduction Certain bacteria growing on food products produce exopolysaccharides, which might increase viscosity of liquid products or cause sticky appearance of the solid ones. The alteration in the textural properties by exopolysaccharides is known as ropiness. This is considered as a defect in some products like milk, cream, cider, wine, while in some fermented milks like stirred yoghurt the production of exopolysaccharides under certain conditions can improve texture (DuenasChasco et al., 1998). Ropy cultures of lactic acid bacteria are used by manufacturers of stirred yoghurt for improving body, mouthfeel, and preventing wheying off (Driessen and Loones, 1992). Feta is a soft, white cheese, ripened and kept in brine until it is sold to consumers. Occasionally, the viscosity of brine increases and becomes ropy. Ropy appearance *Corresponding author. Tel.: +30-1-28-45-940-12; fax: +30-1-2840-740. E-mail address: [email protected] (F.I. Samaras).
of brine is not normally associated with any undesirable organoleptic characteristics but affects the appearance of the cheese. This has a negative effect on the consumer perceptibility, which is, nowadays, of more concern since most Feta cheese is sold to the consumers not from the barrels (without having a view on the brine) but in retail packages. Studies concerning the ropy appearance of Feta cheese brine have not been reported in a systematic manner. Glossiotis (1984) working in a Feta cheese factory, observed the appearance of ropy brine mainly during the winter period. Samaras (1994) reported that ropiness of brine is a phenomenon that appears suddenly and it does not remain stable during storage after manifestation. Chomakov (1967) studied various factors that influence the ropy appearance of white cheese brine. He found a strain of Lactobacillus plantarum to be responsible for the exopolysaccharides reformation. Cerning (1990) reviewed the production and the properties of exopolysaccharides by lactic acid bacteria. The instability of the slime producing trait in
0740-0020/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0740-0020(02)00175-2
ARTICLE IN PRESS 504
F.I. Samaras et al. / Food Microbiology 20 (2003) 503–509
thermophilic and mesophilic lactic acid bacteria has been reported (Cerning, 1990). Certain lactic acid cultures increase viscosity of whey but might lose this property when frequently transferred or at high temperatures. The majority of the dairy companies produce Feta cheese with the addition of a yoghurt culture containing L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus (Kehagias et al., 1995). During recent years there has been a tendency to combine these thermophilic bacteria with mesophilic ones. The purpose of this study was to investigate the conditions under which micro-organisms that can contaminate Feta cheese brine as well as cultures used for the preparation of the cheese, can cause ropiness.
2. Materials and methods 2.1. Isolation and identification of the micro-organisms A rope-inducing micro-organism was isolated analyzing 15 samples of ropy Feta cheese brine taken randomly (Samaras, 1994). Another strain was accidentally taken from extreme viscous retail milk, stored at 41C for a long period of time. Both strains were purified by multiple propagation. The schematic key for tentative generic identification of foodborne bacteria introduced by Jay (2000) was followed for the strain identification. The former strain was identified as L. pseudoplantarum (ID 82%) by API 50 CHL. The latter one was estimated to belong to the genus Alcaligenes by performing the biochemical tests according to the Kersters and De Ley (1984). Both cultures (Lactobacillus and Alcaligenes) were grown overnight on MRS (Oxoid) agar and PCA (Oxoid) at 301C, respectively, before use. 2.2. Growth and development of ropiness in various media Whey, reconstituted skim milk and plate count agar (PCA) were used for ropiness development and determination. Nutrient broth was also used for growth measurement. The whey was obtained after Feta cheese curd making in our laboratory. It was sterilized by tyndallization at 621C, NaCl was added to obtain concentrations of 3%, 5%, 7%, 9%, and 11% (w/v) and transferred into 50 ml flasks. After L. pseudoplantarum was inoculated into the medium and the flasks were stored at 151C and 321C. Three replicated experiments were conducted and two samples were taken from each treatment after 0, 5, 10, 15, 20, 25, and 30 days of storage and analyzed. The pH of the whey was measured by using an ORION expandable ion Analyzer (EA 940). The viscosity was also measured by using a viscosimeter as described
elsewhere. The pH and the viscosity were used as indexes of growth and ropiness, respectively. In order to study the salt tolerance and the influence of temperature on Alcaligenes spp. the micro-organism was loop streaked on PCA containing 2%, 4%, 6%, and 8% NaCl. Plate dishes were incubated at 41C, 151C, and 321C for 72 h. The growth of the micro-organism was subjectively evaluated by using the scale according to the colony size: 0––no growth, 1––start growth, 2–– medium growth, 3––high growth, 4––very high growth and 5––extremely high growth. The ropiness development was also subjectively evaluated from colony sliminess as it is described in another paragraph below. In addition, Alcaligenes spp. was inoculated into approximately 50 ml of nutrient broth (Oxoid) and reconstituted skim milk with various NaCl concentrations (0%, 2%, 4%, 6%, and 8% w/v) in flasks. Incubation took place at 41C, 151C, and 321C, respectively. The growth in the nutrient broth was estimated by measuring transmittance of the inoculated medium by Spectronic 20 (Bausch & Lomb) at 550 nm. The ropiness development in skim milk was measured by a viscosimeter, as it is described in another paragraph below. 2.3. Development of ropiness in Feta cheese brine Feta cheese was prepared from pasteurized ewe’s milk in the laboratory according to the method of Glossiotis (1984). Four batches of Feta cheese were made in two trials. Yoghurt cultures, which are composed of L. delbrueckii subsp. bulgaricus and S. thermophilus are commonly used as starters for the manufacture of Feta. For the experiment needs the commercial yoghurt culture CH1 (Hansen, Copenhagen, Denmark) was used alone or in combination with another yoghurt culture V2 (Wiesby, Niebull, Germany) and the two bacteria isolates. The experimental design comprised four treatments as follows: (a) starter culture CH1 alone, (b) combination of two starter cultures CH1 and V2, (c) combination of starter culture CH1 and L. pseudoplantarum and (d) combination of starter culture CH1 and Alcaligenes spp. Approximately 1 kg of Feta cheese was placed in cans and the containers were filled with brine. The cans were closed using lids with an opening covered by a plastic stopper. Cheese was kept at 15–181C. During storage the pH of the brine was measured and ropiness was evaluated subjectively, as it is described in the next paragraph. 2.4. Evaluation of ropiness The evaluation and the measurement of ropiness are very hard tasks to perform. Ropiness system is unstable, any treatment could cause a collapse. Even more is not able to use an instrument in some instances, small
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particles in the system could interfere and give biased readings. It was a challenge to find out a method of ropiness measurement and the empirical one was developed. The ropiness of the whey and the reconstituted skim milk media was estimated by measuring the viscosity with a Brookfield LV viscosimeter (probe 1, revolution speed 60, measuring unit m Pa/s—milli Pascal per second). The viscosimeter could not be used for viscosity measurement of Feta brine because it contained small particles, which could interfere and give biased readings. Thus the ropiness development in Feta brine was evaluated subjectively by a method which was developed in our laboratory. A loop was inserted into the medium and was drawn out slowly. The formation of a thread and its length before breaking were taken into consideration for ropiness evaluation according to the following scale: 0––zero, 1––low, 2––moderate, 3–– high, 4––very high, and 5––extremely high. The method for evaluating ropiness of the colonies on PCA was based on the same principle and the same scale was also used. A loop was brought into contact with the colony and the formation of a thread was evaluated.
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spp. The morphological and physiological characteristics of the micro-organisms are presented in Table 1. 3.2. Growth and development of ropiness by L. pseudoplantarum in whey Generally, Feta cheese is manufactured under various conditions by quite a large number of firms. The cheese curd occurs at 341C, while the ripening process takes place at 16–181C. Since the ropiness occurs in the Feta brine, which mainly consists of whey, whey was used as a medium. The changes of pH and viscosity during incubation of L. pseudoplantarum in whey at 151C and 321C are shown in Fig. 1. The viscosity of both samples, the control and the inoculated, changed very little within the first 10 days of incubation at 151C and 321C, respectively. At the same time a pH decrease was observed in the inoculated sample indicating growth. This decrease of pH was even more pronounced in samples incubated at 321C. After 21 days of incubation a strong increase in viscosity was observed, indicating development of ropiness, only in the sample inoculated
Table 1 Morphological and physiological characteristics of L. pseudoplantarum and Alcaligenes spp. Characteristics
3.1. Morphological and physiological characteristics of the isolated micro-organisms Several experiments were carried out in various media under different conditions in order to collect information about the behavior and the properties of the two micro-organisms grown in various media under different conditions. Since L. pseudoplantarum was isolated from Feta cheese brine, whey was chosen as medium. Following the methodology described in Materials and Methods, the micro-organism isolated from ropy Feta brine was identified as L. pseudoplantarum (ID 82%) and the one isolated from ropy milk as Alcaligenes
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Motility Size Gram Catalase Anaerobic Spores Glucose fermentation Lactose fermentation Growth at 12–151C Litmus milk Gelatine hydrolysis
L. pseudoplantarum
Alcaligenes spp.
Small +
Medium +
+( ) +
Nd
a
, +b Slimy colonies Acidic Nd
Ropiness in milk Alkaline
Nd: not determined. a Aerobic conditions. b Anaerobic conditions.
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15 o C
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3. Results and discussion
2 0
5
10 15 20 25 30 Time (days)
0
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10 15 20 25 30 Time (days)
Fig. 1. Changes of pH and viscosity in whey during growth of L. pseudoplantarum at 151C and 321C: pH without inoculation (n), pH with inoculation (m), viscosity without inoculation (&), viscosity with inoculation (’).
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with L. pseudoplantarum at 151C. Contrarily, the viscosity did not increase in the samples incubated at 321C. On the other hand, ropiness development takes place the first days during ripening of Feta cheese at 15–181C, which was confirmed by the findings of this experiment. Usually, the finished product of Feta cheese contains approximately 3% NaCl. During processing dry salt is added to the cheese curd or the curd is put in brine of various concentrations, depending upon the producer. The NaCl concentration in the brine and in the curd varies until equilibrium has been achieved. This variation may favors ropiness development at a specific concentration. For this purpose six NaCl concentrations (0%, 3%, 5%, 7%, 9%, and 11%) were chosen, in order to find out which one is the optimum for ropiness development. The whey with 0% NaCl was not inoculated and used as control. In Fig. 2 the effect of NaCl concentration on pH and viscosity during growth
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of L. pseudoplantarum in whey at 151C is presented. The results clearly showed that this micro-organism developed ropiness by increasing the viscosity of the whey containing 7% NaCl. There was only a slight increase of viscosity in samples containing 5% and 3% NaCl and no increase in the samples with 9%, 11% salt and the control. The difference in the pH values with 3%, 5%, and 7% NaCl between inoculated and not inoculated samples is an indication of the growth of the microorganism, which occurred before the 20th day and maintained up to 40th day. On the other hand the pH value of the whey after taking Feta cheese curd is slightly below 5. Preliminary experiments have also showed development of ropiness in whey after the 20th day. At 9% and 11% NaCl concentrations the growth of the micro-organism was inhibited, since the pH remained constant and the viscosity did not change. The results of this study indicated that ropiness development in whey was affected by both temperature (151C) and
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Fig. 2. Effect of the NaCl concentration on pH and viscosity during growth of L. pseudoplantarum in whey at 151C: pH without inoculation (n), pH with inoculation (m), viscosity without inoculation (&), viscosity with inoculation (’). (A) 0% NaCl; (B) 3% NaCl; (C) 5% NaCl; (D) 7% NaCl; (E) 9% NaCl; and (F) 11% NaCl.
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the salt concentration (7% NaCl). Also, it occurred at a pH range of 4–4.5 (Figs. 2B–D). 3.3. Growth and development of ropiness by Alcaligenes spp. in solid and liquid media The effect of NaCl on the growth of Alcaligenes spp. on PCA and the ropy appearance of colonies is shown in Fig. 3. The micro-organism proliferated at all incubation temperatures (41C, 151C, and 321C) and only at NaCl concentration of 0% and 2%. However, ropiness was detected only at 41C and 151C. Feta is ripened initially at 16–181C, then it is kept at 41C until consumption. Therefore, during ripening and storage of Feta, any contamination of cheese or brine by Alcaligenes might lead to ropiness when NaCl concentration is not higher than 2%. The effect of NaCl concentration on the growth of Alcaligenes spp. in nutrient broth, expressed as transmittance, at various temperatures of 41C, 151C, and 321C is given in Fig. 4. This micro-organism proliferated at all three temperatures used and at a NaCl concentration of up to 4%. However, growth was also observed at 6% NaCl only at 151C which is an indication that at this temperature the conditions were more favorable for growth of the micro-organism. Preliminary studies indicated that ropiness was not developed at 151C and 321C (Fig. 3). Thereby the influence of NaCl concentration on viscosity during growth of Alcaligenes spp. in skim milk at 41C was studied. From Fig. 5, it can be seen that highest viscosity was measured when no NaCl was added to the milk. This finding comes in agreement with the data presented in Fig. 3 during growth of Alcaligenes spp. on PCA where ropiness was developed mainly at 0% NaCl and at 2%. The results of this study indicated that Alcaligenes spp. did not grow at NaCl concentrations higher than 6% (Fig. 4) and developed ropy colonies (Fig. 3) or ropiness in milk (Fig. 5) only when the NaCl
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concentration was up to 2% or 0%, respectively. However, since the micro-organism can grow at relatively low temperatures (41C), it will be interesting to investigate whether contamination of milk, cheese or brine by Alcaligenes spp. could influence the appearance of ropiness in brine solution.
3.4. Development of ropiness in Feta cheese brine prepared under various conditions In order to put in practice the findings described above, the two bacteria isolates were used in combination with commercial lactic acid starter cultures in order to make Feta cheese and study the development of ropiness. The changes in pH and the appearance of ropiness of Feta cheese brine are presented in Fig. 6. In the first five days, there was no increase in ropiness in brine in all samples. After eight days, ropiness increased gradually in the samples where CH1 culture was combined in cheese milk with each one of L. pseudoplantarum, Alcaligenes spp. and V2 culture. Ropiness increased further after the 8th day and was constantly high after the second week (Fig. 6). The sample in which CH1 culture was combined with V2 culture gave the highest ropiness (Fig. 6D), followed by the sample with CH1 culture+Alcaligenes spp. (Fig. 6A). In contrary, the sample of brine with CH1 culture alone showed no or very little increase of ropiness (Fig. 6C). From Fig. 6, it can be concluded, that the drop of pH followed a different pattern for the two pairs of samples: CH1 culture with V2 culture (C) and CH1 culture with Alcaligenes spp. (A) on the one hand, CH1 culture alone (C) and CH1 culture with L. pseudoplantarum (B) on the other. Under our experimental conditions, the pH drop alone did not seem to be the only factor in the development of ropiness (experiments C and B). The presence of the proper micro-organisms in the milk samples is essential too (experiments D and A).
Growth - Ropiness Intensity
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o
15 C
32 C
3 o 4 C
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No growth and no ropiness
1 0 0
2
4 NaCl %
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8
Fig. 3. Effect of NaCl concentration on growth and ropy appearance of colonies of Alcaligenes spp. on PCA at various temperatures; 41C, 141C, 321C: (&) growth, (’) ropiness.
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2 3 4 Time (days)
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6
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2 3 4 Time (days)
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Fig. 4. Effect of the NaCl concentration on the growth, expressed as Transmittance (% at 550 nm) of Alcaligenes spp. in nutrient broth at 41C(n), 151C(&) and 321C (J): (A) 0% NaCl; (B) 2% NaCl; (C) 4% NaCl; (D) 6% NaCl and (E) 8% NaCl.
21 Viscosity mPa.s -1
18 15 12 9 6 3 0 4
5
6
7 8 9 Time (days)
10
11
Fig. 5. Effect of the NaCl concentration: 0% (n), 2% (&), 4% (J), 6% ( * ), 8% (+) on viscosity during growth of Alcaligenes spp. in skim milk at 41C.
During recent years, there has been intensive interest in the bacteria producing ropiness in food, not only because of their effect on texture but also for the potential bioactive role of exopolysaccharides (Vemura
et al., 1998). However, information on the development of ropiness of cheeses in brine is limited (Chomakov, 1967; Samaras, 1994). This study revealed that a ropy yoghurt culture (V2) could produce ropiness in brine of Feta cheese under certain conditions (151C) which are quite different from those used for the manufacture of stirred yoghurt. The other one (CH1) did not cause ropiness in brine. Since the yoghurt bacteria (S. thermophilus and L. delbrueckii subsp. bulgaricus) are used as starters in Feta cheesemaking, it should be taken into consideration the selection of the no ropy strains, keeping always in mind that the ropy appearance of Feta cheese is not desirable. It is well known that ropy and not ropy strains exist in yoghurt starter cultures (Rawson and Marshall, 1997). The results of this study also showed that bacteria, which are inhabitants in dairy plants, like Alcaligenes and Lactobacillus can intensify ropiness when contaminating milk, brine or cheese. Alcaligenes spp. increased ropy appearance of brine at 151C but did not develop ropiness at the same temperature in salted milk. Our
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Ropiness
(A)
20
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Ropiness
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0 0
5
10 15 Time (days)
20
Fig. 6. Changes of pH (’) and viscosity (&) in the brine of Feta cheese by different cheese cultures and micro-organisms Alcaligenes spp. and L. pseudoplantarum at different periods of time: (A) CH1+Alcaligenes spp.; (B) CH1+L. pseudoplantarum; (C) CH1 culture and (D) CH1+V2 cultures).
results clearly showed that NaCl composition of the substrate as well as the temperature are both factors associated with the development of ropiness. It is essential to eliminate contaminants, which can cause ropiness by cleaning and disinfecting of the facilities of cheese plants.
References Cerning, J., 1990. Exocellular polysaccharides produced by lactic acid bacteria. FEMS Microbiol. Rev. 87, 113–130. Chomakov, C., 1967. Isolation of lactic acid bacteria causing ropiness of white cheese brine. Milchwissenschaft 22, 569–573. Driessen, F.M., Loones, A., 1992. Developments in the fermentation process (liquid, stirred and set fermented milks). Bull. Int. Dairy Fed. 277, 28–40. Duenas-Chasco, M.T., Rodriguez-Carvajal, M.A., Tejero-Mateo, P., Espartero, J.L., Irastorza-Iribas, A., Gil-Serrano, A.M., 1998. Structural analysis of the exopolysaccharides produced by Lactobacillus spp. G-77. Carbohydr. Res. 307, 125–133.
Glossiotis, C., 1984. Products from ewe milk. Proceedings of the Seminar in Dairy Science, Greek National Dairy Committee, pp. 233–256. Jay, J.M., 2000. Modern Food Microbiology. D. Van Nostrand, New York, USA, pp. 313–317. Kehagias, C., Koulouris, S., Samona, A., Malliou, S., Koumoutsos, G., 1995. Effect of various starters on the quality of cheese in brine. Food Microbiol. 12, 413–419. Kersters, K., De Ley, J., 1984. Genus Alcaligenes Castellani and Chalmers 1919, 936AL. In: Krieg, N.R., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, MD, USA, pp. 361–366. Rawson, H., Marshall, V.M., 1997. Effect of ropy strains of Lactobacillus delbrueckii spp. bulgaricus and Streptococcus thermophilus on rheology of stirred yogurt. Int. J. Food Sci. and Technol. 32, 213–220. Samaras, F., 1994. Factors affecting the ropiness development in Feta cheese brine. Proceedings of the Seminar in Dairy Science, Greek National Dairy Committee, pp. 145–155. Vemura, J., Itoh, T., Kaneko, T., Noda, K., 1998. Chemical characterization of exocellular polysaccharide from Lactobacillus delbrueckii subsp. Bulgaricu OLL1073R-1. Milchwissenschaft 53, 443–446.